Article

A neutrophil response linked to tumor control in

immunotherapy
Graphical abstract Authors
Jeremy Gungabeesoon,
Nicolas A. Gort-Freitas, Máté Kiss, ...,
Ralph Weissleder, Allon M. Klein,
Mikael J. Pittet

Correspondence
[email protected] (A.M.K.),
[email protected] (M.J.P.)

In brief
Successful cancer immunotherapy is
associated with high numbers of
neutrophils and expression of IRF1.

Highlights
d Neutrophils can acutely accumulate in tumors during
successful immunotherapy

d Therapy expands a distinct neutrophil state with an IFN-

stimulated gene signature

d The neutrophil response requires IRF1 and supports tumor

control

d Therapy-elicited neutrophil response in patients is

associated with better outcome

Gungabeesoon et al., 2023, Cell 186, 1448–1464

March 30, 2023 ª 2023 Elsevier Inc.
https://doi.org/10.1016/j.cell.2023.02.032 ll
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Article
A neutrophil response linked to tumor control
in immunotherapy
Jeremy Gungabeesoon,1,12 Nicolas A. Gort-Freitas,2,12 Máté Kiss,3,4,12 Evangelia Bolli,2,3,4 Marius Messemaker,1,5
Marie Siwicki,1 Mehdi Hicham,3,4 Ruben Bill,2,3,4 Peter Koch,1 Chiara Cianciaruso,2,3,4 Florent Duval,3,4
Christina Pfirschke,1 Michael Mazzola,6 Solange Peters,7,8 Krisztian Homicsko,4,9,10,11 Christopher Garris,1
Ralph Weissleder,1,2 Allon M. Klein,2,13,* and Mikael J. Pittet1,3,4,9,11,13,14,*
1Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA, USA
2Department of Systems Biology, Harvard Medical School, Boston, MA, USA
3Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland
4AGORA Cancer Research Center, Lausanne, Switzerland
5Division of Molecular Oncology and Immunology, Netherlands Cancer Institute, Amsterdam, the Netherlands
6Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA
7Service of Medical Oncology, Department of Oncology, CHUV, Lausanne, Switzerland
8Department of Oncology, University of Lausanne, Lausanne, Switzerland
9Ludwig Institute for Cancer Research, Lausanne, Switzerland
10Department of Oncology, CHUV, Lausanne, Switzerland
11Swiss Cancer Center Leman, Lausanne, Switzerland
12These authors contributed equally
13Senior author
14Lead contact

*Correspondence: [email protected] (A.M.K.), [email protected] (M.J.P.)

https://doi.org/10.1016/j.cell.2023.02.032

SUMMARY

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are
not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of
neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely
expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other
neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene
signature, also seen in human patients, and appeared essential for successful therapy, as loss of the inter-
feron-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil
response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12,
and IFNg. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated
with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in medi-
ating effective cancer therapy.

INTRODUCTION geneity at the level of their transcriptomes and surface protein

expression.20–24 This heterogeneity raises the question of
Neutrophils are the most abundant circulating leukocytes in the whether phenotypically distinct neutrophil states coexisting in
human body, and they accumulate in a wide range of cancer tumors could have different, potentially opposing, functional ac-
types.1–5 A large body of evidence from mouse models indicates tivities and whether specific anti-tumor states could be therapeu-
that tumor-infiltrating neutrophils can exhibit both tumor-pro- tically expanded. We recently showed that most neutrophil states
moting and anti-tumor functions.6 Promotion of cancer cell pro- identified in human lung tumors can also be found in mice, con-
liferation, metastasis, angiogenesis, and inhibition of anti-tumor firming that knowledge obtained about neutrophil heterogeneity
T cell responses have all been linked to neutrophils.7–12 Never- in mouse models is highly relevant for human disease.24 Notably,
theless, other studies have demonstrated their capacity to both human and mouse tumors harbor a neutrophil state charac-
directly kill cancer cells and stimulate anti-tumor immunity.13–18 terized by high expression of interferon-stimulated genes (ISGs),
Much of the biological mechanisms underlying the development yet the functional relevance of this state is not known.24
of these divergent functional states remain unknown.19 Given the potential of cancer immunotherapies to induce
Recent high-dimensional single-cell analyses have revealed durable clinical responses in some patients but not others,
that circulating and tumor-infiltrating neutrophils exhibit hetero- considerable efforts have been invested into understanding how

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successful immunotherapy changes the tumor microenvironment model in which tumor cells carrying the oncogenic G12D Kras
to favor tumor control. Single-cell omics studies revealed that mutation and lacking P53 are injected intravenously. The so-
treatments inducing successful anti-tumor T cell immunity can called KP tumors that develop in the lungs recapitulate key fea-
also have an indirect effect on the myeloid compartment. This tures of human lung adenocarcinomas and show resistance to
can involve the expansion of pro-inflammatory macrophages or treatment with immune checkpoint inhibitors.9,24,32–35 However,
dendritic cells (DCs) that express ISGs and other immunostimula- we found here that treatment with a CD40 agonist antibody led to
tory genes.25–28 Despite their abundance in many tumors, a significant reduction in tumor burden (Figures 1A and 1B).
neutrophils have been largely overlooked in these single-cell tran- This gave us an opportunity to study neutrophil responses in
scriptomics studies due to their inadvertent exclusion in standard the context of an immunotherapy capable of curbing lung tumor
sample preparation and analytical pipelines. Moreover, the life- progression. Specifically, we examined tumors early after initia-
span of neutrophils is only a few hours or days; therefore, an tion of treatment to identify potential changes in neutrophil abun-
immunotherapy-elicited neutrophil response may be missed if dance or phenotype that could contribute to early phases of the
tumors are examined late after treatment. It is therefore largely un- anti-tumor response. Here, 2 days after treatment, we found that
known whether successful immunotherapy could have an impact the number of neutrophils increased more than 2-fold in the
on the phenotype of neutrophils in the tumor microenvironment. lungs (Figure 1C).
Investigating treatment-induced reprogramming of neutrophils These data indicated that anti-CD40 (aCD40) treatment-
could also give us clues as to whether these cells could oppose induced control of KP tumors was associated with a neutrophil
or support tumor control upon therapy. response. We wished to establish whether a comparable
Our current knowledge about the impact of neutrophils on response could be observed in other tumors and immunother-
immunotherapy response mostly comes from mouse experi- apies. We therefore used the MC38 tumor model, which re-
ments in which all neutrophils were sought to be depleted during sponded both to aCD40 treatment (Figure 1D) and anti-PD-1
therapy.29–31 In light of the emerging evidence about the exis- (aPD-1) treatment (Figure S1A), as expected.36–38 Similar to
tence of diverse neutrophil states in tumors, it is conceivable our findings in the KP model, the number of neutrophils
that broad neutrophil depletion strategies eliminate both increased in MC38 tumors 2 days after treatment with aCD40
detrimental and beneficial neutrophil states. Hence, a deeper un- as seen by flow cytometry (Figure 1E) and independently by sin-
derstanding of the factors driving the acquisition of distinct gle-cell RNA sequencing (scRNA-seq) analysis of CD45+ cells
neutrophil states would allow for selective manipulation of (Figure 1F). Similarly, treatment of MC38 tumors with the
neutrophil subpopulations. This in turn would enable us to aPD-1 antibody led to an increase in neutrophil frequency as
gain a more nuanced picture of the role of neutrophils in seen by scRNA-seq (Figure 1G) and independently by flow cy-
immunotherapy. tometry (Figure S1B).
In the current study, we aim to address these knowledge gaps We next sought to define whether the neutrophil response
by examining how immunotherapy shapes the neutrophil compart- could be observed in the context of other therapies. To this
ment in mouse tumor models and how treatment-induced reprog- end, we treated KP tumor-bearing mice with three distinct thera-
ramming of neutrophils could influence tumor control. peutic approaches: paclitaxel combined with carboplatin (pac +
carbo), oxaliplatin combined with cyclophosphamide (oxa +
RESULTS cyc), or aPD-1 combined with anti-CTLA4 (aPD-1 + aCTLA4).
We have previously found that pac + carbo or aPD-1 + aCTLA4
Neutrophils accumulate in tumors in the context of treatment are largely ineffective in controlling KP lung tumor
successful therapy growth, whereas oxa + cyc treatment triggers immunogenic can-
To examine neutrophil responses after cancer therapy, we cer cell death leading to T cell-mediated tumor control.35 We
initially investigated an orthotopic mouse lung adenocarcinoma found that oxa + cyc treatment induced a neutrophil response,

Figure 1. Neutrophils accumulate in tumors in the context of successful therapy

(A) Representative images of hematoxylin and eosin-stained lung sections and quantification of KP lung tumor burden, 21 days after aCD40 treatment (n = 13
untreated, n = 15 aCD40-treated, data pooled from two independent experiments).
(B) Lung weight (proxy of tumor burden) of KP tumor-bearing mice, treated or not with aCD40 (n = 14 untreated, n = 15 aCD40-treated, data pooled from two
independent experiments). Dashed line indicates average lung weight in tumor-free mice.
(C) Flow cytometry-based quantification of neutrophils in KP tumors, 2 days after aCD40 treatment (n = 5 untreated, n = 10 aCD40-treated, data pooled from three
independent experiments). Representative dot plots of live CD45+ cells are shown.
(D) Tumor volume of aCD40-treated and untreated MC38 tumor-bearing mice, 9 days after treatment (n = 7 untreated, n = 8 aCD40-treated).
(E) Flow cytometry-based quantification of neutrophils in MC38 tumors, 2 days after aCD40 treatment (n = 8 untreated, n = 24 aCD40-treated, data pooled from
four independent experiments).
(F) scRNA-seq-based profiling of CD45+ cells in MC38 tumors, 2 days after aCD40 treatment (n = 2 per group). Abundance of major immune cell subsets is
quantified as fold change between aCD40-treated versus untreated conditions. Neutrophils are indicated in red in the UMAP.
(G) scRNA-seq-based profiling of CD45+ cells in MC38 tumors, 3 days after aPD-1 treatment (n = 2 per group). Abundance of major immune cell subsets is
quantified as fold change between aPD-1-treated versus untreated conditions. Neutrophils are indicated in red in the UMAP.
(H) Summary of assessed tumor models and treatments, showing quantification of tumor control and neutrophil accumulation. pac/carbo, paclitaxel/carboplatin;
oxa/cyc, oxaliplatin/cyclophosphamide. Table shows mean ± SEM.
Bar graphs show mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. *p < 0.05; **p < 0.01; ***p < 0.001.

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whereas pac + carbo or aPD-1 + aCTLA4 treatment did not A subset of Sellhi neutrophils expands in the context of
(Figures 1H and S1C–S1E). Taken together, these data suggest successful immunotherapy
that neutrophil accumulation can occur in different tumor types Considering the neutrophil states defined above, we found that
and is a common feature of treatments with the ability to control the total increase in neutrophil counts observed after aCD40
tumor growth (Figure 1H). treatment was attributed to only some of the states (Figure 3A).
aCD40 caused a greater than 10-fold increase in the abundance
States of immunotherapy-elicited neutrophils in KP of both N1a (Sellhi Ngphi) and N2 (Sellhi Cxcl10hi) neutrophils,
tumors whereas the number of N1b (Sellhi Lsthi), N4 (Siglecfhi Xbp1hi),
To define the identity of neutrophils that accumulate in tumors N5 (Siglecfhi Ccl3hi), and N6 (Siglecfhi Ngphi) cells remained un-
upon immunotherapy, we performed scRNA-seq combined changed. The N3 (Cxcl3hi) neutrophil state seemingly disap-
with multiplexed surface protein profiling on CD11b+ cells iso- peared; yet genes marking this state (e.g., Wfdc17, Tgm2, and
lated from the lungs of healthy mice, untreated KP tumor-bearing Gos2) did not vanish but became broadly expressed by N1b
mice, and aCD40-treated KP tumor-bearing mice (N = 42,007 (Sellhi Lsthi) and N2 (Sellhi Cxcl10hi) neutrophils (Figures S3A
cells total). and S3B). These observations were supported independently
We used our prior published work in mouse KP tumors24 as a by flow cytometry: aCD40 treatment led to an increase in tu-
reference to annotate cells and interpret changes in neutrophil mor-infiltrating SiglecFlo, but not SiglecFhi, neutrophils (Fig-
abundance and gene expression. A total of N = 30,468 cells ure 3B), confirming that immunotherapy-elicited neutrophils
were identified as neutrophils. In untreated KP tumors, neutro- are distinct from SiglecFhi tumor-promoting neutrophils. Of
phils exist in a continuum of states that we had previously parti- note, CD62L had insufficient discriminatory power for separating
tioned into six states (originally termed N1–N6). For the present these neutrophil subsets via flow cytometry and rather displayed
analysis we discerned an additional, seventh, neutrophil state a continuum of expression, possibly due to loss of the protein
by partitioning the N1 state into two states (Figures 2A and 2B; over extended tissue residence (Figure S3C). Nevertheless,
Table S1). We now refer to these as N1a and N1b to facilitate we could confirm treatment-induced expansion of SiglecFlo cells
cross-comparison between studies (Figures 2A, 2B, and S2A; with high CD62L expression (Figure S3C). Additionally, TotalSeq
Table S1). This was done to reduce errors in identifying ortholo- revealed that SiglecFlo neutrophils showed increased CD14
gous neutrophil states between untreated and immunotherapy- expression after aCD40 treatment (Figures S2C and S2D), which
treated conditions, and it also proved useful in precisely we confirmed by flow cytometry (Figure S3D).
describing the changes in heterogeneity that occurred after We wished to establish whether comparable changes in neu-
immunotherapy. trophils occur in other contexts and models of immunotherapy.
The seven neutrophil states that we observed could be group- To investigate this question, we first performed a meta-analysis
ed into three higher-level clusters, which we named Sellhi of scRNA-seq datasets from KP and MC38 tumors treated with
(comprising N1a, N1b, and N2), Cxcl3hi (comprising N3), and Si- aPD-1 or aCD40 to define orthologous states between these
glecfhi (comprising N4, N5, and N6) (Figures 2B, 2C, and S2B). experimental conditions (Figure 3C, left). We compared cell
Sellhi neutrophils were detected in all experimental conditions states obtained from different studies and experimental condi-
tested including healthy tissues, whereas Siglecfhi neutrophils tions by defining a reciprocal similarity score between states,
as well as Cxcl3hi neutrophils were only found in tumor-bearing analogous to recriprocal similarity commonly used to identify
tissues (Figure 2A), confirming previous findings.24 orthologous genes,41,42 and which we have previously em-
Using DNA-tagged TotalSeq antibodies,39 we evaluated ployed to identify orthologous DC states.43 This analysis re-
whether neutrophil immunophenotype was consistent with vealed that five of the transcriptional states we identified in
mRNA expression in the KP tumor model. As a control, the current study were conserved in KP and MC38 tumors as
TotalSeq revealed that all seven neutrophil states expressed well as in aCD40 and aPD-1 treatments (Figure 3C, right).
the canonical neutrophil marker Ly6G, even though Siglecfhi Notably, all Sellhi neutrophil states overall showed high
neutrophils expressed very low levels of Ly6g mRNA (Figure 2D). conservation of their transcriptome between aCD40-treated
We then examined the proteins CD62L and SiglecF, which are KP tumors and aCD40- or aPD-1-treated MC38 tumors. In
encoded by transcripts Sell and Siglecf, respectively. These pro- comparison, the Cxcl3hi state showed the poorest conservation
teins were expressed across the neutrophil states in the same both across tumors and between treatments, which is consis-
pattern as their transcripts in KP tumors, defining CD62Lhi/Si- tent with the lack of stability of this state upon treatment in
glecFlo (Sellhi), CD62Llo/SiglecFlo (Cxcl3hi), and CD62Llo/Si- KP lung tumors noted above.
glecFhi (SiglecFhi) clusters in the TotalSeq data (Figure 2D). We then examined changes in the abundances of orthologous
Significantly, we previously identified SiglecF expression to states (Figure 3D). Significantly, as with aCD40 treatment in KP
distinguish a neutrophil subpopulation with pro-tumor functions tumors, the scRNA-seq of MC38 tumors showed an increase
in mouse lung tumors.9,40 Thus, these results now link scRNA- in neutrophil abundance after both aCD40 and aPD-1 treatments
seq-defined states to previously defined neutrophil immunophe- in clusters orthologous to the KP tumor Sellhi clusters.
notypes. Examining the expression of 17 additional surface Altogether, these results indicate that several of the neutrophil
markers using TotalSeq did not identify markers or marker com- states we observe in lung tumors appear to represent stereo-
binations that were specific for a given neutrophil state among typed responses, as are the changes in neutrophils associated
the N1–N6 states (Figures S2C and S2D), but future high-dimen- with therapies that trigger effective anti-cancer adaptive
sional surface protein analyses may be able to do so. immunity.

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Figure 2. States of immunotherapy-elicited neutrophils in KP tumors

(A) scRNA-seq workflow, and uniform manifold approximation and projection (UMAP) visualization of neutrophil single-cell transcriptomes in lungs of healthy mice
(n = 2), KP tumor-bearing mice (n = 2), and aCD40-treated KP tumor-bearing mice (n = 2). A full list of marker genes for each neutrophil state is shown in Table S1.
(B) Enriched genes within neutrophil states in aCD40-treated KP tumor-bearing lungs shown in (A). Data underlying heatmap is shown in Table S1.
(C) mRNA expression of Ly6g and key markers used for low-resolution partitioning of neutrophil states into three higher-level clusters.
(D) Protein expression of corresponding surface proteins encoded by the marker genes shown in (C).

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Figure 3. A subset of Sellhi neutrophils expands in the context of successful immunotherapy

(A) Frequency of neutrophil states in KP tumors from aCD40-treated mice versus untreated mice, quantified as cells/mg tissue (n = 2 per group). Dotted lines
indicate 10-fold enrichment/depletion.
(B) Representative flow cytometry histogram of SiglecF expression by neutrophils in KP tumors 2 days after aCD40 treatment. Quantification of SiglecFlo and
SiglecFhi neutrophils in KP tumors of untreated or aCD40-treated mice (n = 5 untreated, n = 10 aCD40-treated, data pooled from three independent experiments).
Graphs show mean ± SEM. For comparisons between groups, Student’s two-tailed t test was used. *p < 0.05.
(C) Heatmap showing reciprocal similarity scores calculated by cross-comparing tumor neutrophil states from the scRNA-seq datasets listed in the table. Or-
thologous states are highlighted in yellow.
(D) Quantification of changes in abundance of orthologous Sellhi neutrophil states in KP and MC38 tumors after aCD40 or aPD-1 treatment, based on scRNA-seq
data shown in (C).

Neutrophils elicited by immunotherapy acquire an ISG cells (Figure 4B). By contrast, the genes associated with cyto-
signature toxic activity as well as a large set of ISGs were enriched in Sellhi
Having established that aCD40 treatment triggered intratumoral neutrophils (Figure 4C), and aCD40 treatment led to further in-
accumulation of Sellhi (Siglecflo) but not Siglecfhi (Selllo) neutro- duction of these genes in Sellhi cells (Figures 4D and 4E).
phils, we sought to understand how such changes might corre- To confirm the induction of an ISG phenotype by neutrophils
late with or affect successful anti-tumor responses. To explore upon aCD40 treatment, we used reporter mice for CXCL10, a
possible hypotheses, we asked whether the neutrophil subsets prototypical ISG.44 Flow cytometry analysis of CXCL10-BFP re-
differ in the expression of genes associated with pro- and anti- porter expression revealed that aCD40 treatment in KP tumors
tumoral activity, or whether therapy might modulate such genes indeed led to robust CXCL10 induction, specifically in tumor-
within the cell subsets. Using scRNA-seq data, we first examined infiltrating SiglecFlo neutrophils (Figure 4F). CXCL10 expression
the expression of genes previously linked to angiogenesis, extra- could be observed in 40% of SiglecFlo neutrophils and was
cellular matrix (ECM) remodeling, immunosuppression, tumor restricted to CD14+ cells (Figures S4A and S4B).
proliferation, and/or myeloid cell recruitment (Figure 4A). All of We examined data from murine MC38 tumors and human non-
these pro-tumor signatures showed higher expression in Si- small-cell lung cancer (NSCLC) to evaluate whether induction of
glecfhi neutrophils compared with Sellhi neutrophils (Figure 4A). this ISG response could be recapitulated in other contexts.
Interestingly, aCD40 treatment modulated genes associated Indeed, the increased ISG signature was also seen in Sellhi neu-
with tumor proliferation and myeloid cell recruitment in Siglecfhi trophils in the MC38 tumor model and was elevated in the same

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Figure 4. Neutrophils elicited by immunotherapy acquire an interferon-stimulated gene signature

(A) Expression of pro-tumor gene sets in Sellhi and Siglecfhi neutrophil states in untreated KP tumors.
(B) Expression of pro-tumor gene sets in Siglecfhi neutrophils in untreated and aCD40-treated KP tumors.
(C) Expression of anti-tumor gene sets in Sellhi and Siglecfhi neutrophil states in untreated KP tumors.
(D) Expression of anti-tumor gene sets in Sellhi neutrophils in untreated and aCD40-treated KP tumors.
(E) mRNA expression of the ISG Ifit3 in neutrophils in healthy, untreated tumor-bearing and aCD40-treated tumor-bearing lungs. Sellhi states are circled in blue.
Siglecfhi states are circled in red.
(F) Representative flow cytometry dot plots showing CXCL10-BFP and SiglecF expression by neutrophils in KP tumors, before and after aCD40 treatment in REX3
transgenic mice. Fold changes in relative abundance (% within neutrophils) of neutrophil subsets defined by CXCL10 and SiglecF in response to treatment are
plotted (n = 4 untreated, n = 8 aCD40-treated, data are pooled from two independent experiments).
(G) Heatmaps showing expression of ISGs by orthologous Sellhi neutrophil states in KP tumors (untreated or aCD40-treated) and MC38 tumors (untreated,
aCD40-treated, or aPD1-treated). In human NSCLC, the N1 neutrophil state is orthologous to mouse SellhiNgphi and SellhiLst1hi neutrophils, and the N2 state is
orthologous to SellhiCxcl10hi neutrophils.
(H) Flow cytometry histogram and quantification showing the change in CXCL10-BFP expression by neutrophils in MC38 tumors before and after aCD40
treatment in REX3 transgenic mice (n = 5 untreated, n = 7 aCD40-treated, data are pooled from two independent experiments).
(I) Proportion of CXCL10-BFP+ neutrophils in MC38 tumors with or without administration of neutralizing anti-IFNg antibody during aCD40 treatment (n = 4
untreated, n = 3 aCD40-treated, data pooled from two independent experiments).
Box and whiskers plots in (A)–(D) show 95% CI. Bar graphs in (F), (H), and (I) show mean ± SEM. For comparisons between two groups, Student’s two-tailed t test
was used. **p < 0.01; ****p < 0.0001.

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Figure 5. Immunotherapy-elicited neutrophils show a distinct phenotype and maturation state

(A) Proportion of CXCL10-BFP+ neutrophils in the blood and bone marrow of KP tumor-bearing mice with or without aCD40 treatment (n = 4 untreated, n = 5
aCD40-treated).
(B) ROS production in blood neutrophils of KP tumor-bearing mice with or without aCD40 treatment measured via CellROX fluorescence assay using flow cy-
tometry (n = 4 per group). MFI, median fluorescence intensity.
(C) Neutrotime score, indicating maturity, for each neutrophil state in aCD40-treated KP tumors. Box and whiskers plot shows 95% CI.
(D) Heatmap shows expression of individual early neutrotime genes across neutrophil states in aCD40-treated KP tumors.
(E) RNA velocity trajectories for neutrophil states in KP tumors of untreated or aCD40-treated mice.

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cell state in this model in response to both aCD40 and aPD-1 phils; the second ranged from N6 (Siglecfhi Ngphi) to N4 (Siglecfhi
immunotherapy (Figure 4G). Furthermore, among human neutro- Xbp1hi) neutrophils (Figures 5E and S5A). Thus, the RNA velocity
phils identified as analogous to the Sellhi populations,24 we data are also consistent with the possibility that neutrophils can
observed the same pattern in ISGs as among the murine subsets undergo two distinct state transition trajectories in tumors.
found in untreated KP tumors (Figure 4G). In addition, flow cy- Since the number of N1a (Sellhi Ngphi) neutrophils increased
tometry revealed an increase in CXCL10-BFP+ neutrophils in sharply after aCD40 treatment, we considered that this treat-
MC38 tumors after aCD40 treatment (Figure 4H). IFNg neutrali- ment recruited incompletely mature SiglecFlo neutrophils to the
zation reduced expression of CXCL10 in these neutrophils, sup- tumor. Accordingly, we observed reduced expression of
porting the notion that IFNg is a key factor in inducing ISGs maturity markers50,51 (CD101, CD11b, and Ly6G) on SiglecFlo
(Figure 4I). neutrophils, but not on SiglecFhi neutrophils, upon treatment
Taken together, these results show that aCD40 treatment in- (Figure 5F). Among SiglecFlo neutrophils, CD62Lhi cells ap-
duces the expression of ISGs in Sellhi (Siglecflo) neutrophils peared slightly less mature, compared with CD62Lint/lo cells (Fig-
and that ISG-expressing neutrophils are highly conserved ure S5B). Morphological analysis further confirmed appearance
across different tumor types and immunotherapies. of immature cells with lower nuclear segmentation within the
SiglecFlo population upon treatment. At the same time, the
Immunotherapy-elicited neutrophils show a distinct SiglecFhi population remained unaffected and showed a mature
phenotype and maturation state morphology with highly segmented nuclei (Figures 5G and
Neutrophils are typically short-lived, with a half-life of a few hours 5H).46,52 It has been recently reported that immature neutrophils
or days.6,45,46 Changes in the abundance and gene expression can be identified in tissues as MPOhiLy6Glo cells using immuno-
of neutrophils could therefore arise locally in the tumor, or they fluorescent staining of tissue sections.53 Indeed, we observed
may reflect changes during neutrophil maturation. Since neutro- increased infiltration of MPOhiLy6Glo cells into KP lung tumors
phils originate in the bone marrow and transit through the blood, following aCD40 treatment, while the density of MPOhiLy6Ghi
we sampled these sites for neutrophils after aCD40 treatment. cells remained the same (Figure S5C).
After treatment, we found that some neutrophils in the Finally, we asked whether stimuli other than aCD40 could
blood, and even the bone marrow, already showed increased trigger a similar neutrophil response. We found that LPS treat-
CXCL10-BFP expression upon therapy (Figure 5A). Circulating ment, but not type I/II interferon or poly(IC), induced appearance
neutrophils also showed increased production of reactive oxy- of CD14+CD101– neutrophils in the periphery, similar to aCD40
gen species (ROS) (Figure 5B). This suggested that in response treatment (Figure S5D). Taken together, these data demonstrate
to aCD40 treatment, neutrophils can begin acquiring features that aCD40-elicited neutrophils enter the tumor in a different
of the ISG phenotype before they reach the target site. state than SiglecFhi neutrophils: they already exhibit an interferon
This led us to ask whether ISG-expressing Sellhi neutrophils response and increased ROS production, they are less mature
stimulated by aCD40 might have a different origin than Siglecfhi than SiglecFhi neutrophils, and they resemble neutrophils that
neutrophils, which do not show a comparable response to emerge during systemic bacterial infections.54
treatment. Both Sellhi and Siglecfhi neutrophils contain a subset
enriched in Ngp, a transcript expressed during neutrophil matu- A neutrophil IRF1-mediated interferon response is
ration.47 It is therefore possible that N1a (Sellhi Ngphi) and N6 (Si- required for tumor control
glecfhi Ngphi) neutrophils are distinct precursors, which give rise Next, we asked whether neutrophils were required for tumor
to other Sellhi and Siglecfhi states, respectively. This hypothesis control in the context of aCD40 treatment. Therapy-elicited neu-
is consistent with two further observations. First, we examined trophils were able to induce IL-12 production in DCs and kill can-
the expression of a broader set of genes associated with early cer cells in vitro, suggesting their potential immunostimulatory
or immature neutrophils, defining a composite ‘‘neutrotime’’ and cytotoxic activity (Figures S6A and S6B). To study the role
gene expression score.47 We found that N1a (Sellhi Ngphi) and of therapy-elicited neutrophils in vivo, we used the MC38 tumor
N6 (Siglecfhi Ngphi) neutrophils displayed the lowest neutrotime model, in which aCD40 treatment triggered the highest increase
among the seven neutrophil states (Figures 5C and 5D). Thus, in neutrophil abundance among all the conditions examined (Fig-
these states expressed multiple transcripts associated with ure 1H). The neutrophils infiltrating MC38 tumors after aCD40
less mature cells. Second, we calculated ‘‘RNA velocities’’ to treatment showed the same increase in CD14 and loss of
identify trajectories on scRNA-seq uniform manifold approxima- CD101 as in the KP model (Figure 6A) and highly expressed
tion and projection (UMAP) plots.48,49 Applying this approach to CXCL10, a marker of the ISG response (Figures 4H and 6A).
our dataset (Figure 5E), two consistent trajectories were pre- We first considered non-specifically depleting neutrophils us-
dicted under all conditions examined. The first ranged from ing anti-Ly6G monoclonal antibodies—a method that is widely
N1a (Sellhi Ngphi) to N1b (Sellhi Lsthi) to N2 (Sellhi Cxcl10hi) neutro- used but recently has been reported to have limited efficacy,

(F) Representative histograms of CD101, CD11b, and Ly6G expression on distinct neutrophil subsets in KP tumors with or without aCD40 treatment, assessed by
flow cytometry.
(G) Representative images of hematoxylin and eosin-stained sorted neutrophils following cytospin.
(H) Quantification of nuclear lobes in different neutrophil subsets (n = 50–100 cells per condition).
Graphs show mean ± SEM in (A) and (B), and graph shows mean in (H). In (A) and (B), Student’s two-tailed t test was used; in (H), Mann-Whitney test was used.
*p < 0.05; **p < 0.01; ****p < 0.0001.

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particularly in depleting immature neutrophils.29 Indeed, we cells comprise a 50%–50% mixture of Irf1-proficient Csf3r
/

found that 28% of neutrophils remained in MC38 tumors after cells and Irf1-deficient cells, resulting in full IRF1 loss restricted
treatment with anti-Ly6G (Figures S6C and S6D). Furthermore, to neutrophils. Strikingly, mice with neutrophil-specific IRF1-
the cells persisting after anti-Ly6G treatment were less mature, deficiency failed to show tumor control following aCD40 treat-
indicated by their lower expression of CD101 and CXCR2 as ment, contrary to mice with wild-type neutrophils (Figure 6D).
well as their higher expression of CXCR4 (Figure S6E). As we Of note, type 1 conventional DCs and CD8+ T cells, two key re-
found that a large portion of immunotherapy-elicited neutrophils quirements of anti-tumor immunity upon aCD40 treatment, did
had decreased levels of Ly6G expression and were already less not show significantly different abundance in the tumors of
mature (Figures 5C–5H), anti-Ly6G treatment would likely not be WT/Csf3r
/
and Irf1
/
/Csf3r
/
mixed bone marrow chimeras
suitable to completely deplete these cells. (Figures S6G and S6H). Overall, we found that the emergence of
Instead, we searched for transcription factors that could play a CD14+CD101
neutrophils in the tumor upon treatment required
key role in the development of the immunotherapy-elicited the activity of the transcription factor IRF1, and preventing
neutrophil response and could be specifically targeted. We per- treatment-induced neutrophil response by neutrophil-specific
formed computational prediction of transcription factor activity, IRF1-deletion abrogated response to aCD40 immunotherapy.
based on highly enriched genes in aCD40-expanded neutrophil
states (N1a and N2) versus all other states. Among the top Therapy-elicited neutrophil accumulation in tumors
transcription factors predicted to be active in aCD40-expanded depends on key components of anti-tumor immunity
neutrophils, we found IRF1 to be one of the highest ranked and Given that ISG-responsive neutrophils were required for aCD40-
associated with the largest regulated gene set (Figure 6B). In mediated tumor control, we explored the mechanisms regulating
addition, IRF1 expression in neutrophils increased upon aCD40 their emergence in the tumor. We began by exploring the hypoth-
treatment (Figure S6F), and this transcription factor has been esis that the neutrophil response in tumors depends on compo-
described to regulate CXCL10 expression,55 one of the hall- nents that are key in the anti-cancer immune response. We and
marks of immunotherapy-elicited neutrophils (Figures 3A, 4F– others have previously found that anti-tumor immunity upon
4H, and 5A). These observations prompted us to examine aCD40 therapy relies on the presence of cDC1s, which require
whether deletion of Irf1 could prevent the emergence of the transcription factor BATF3 for their differentiation. IL-12, pro-
aCD40-elicited ISG-expressing neutrophils. As a proxy, we duced by DCs and other myeloid cells, is necessary for the acti-
again leveraged the observations that an increase in CD14 and vation of anti-tumor T cells, which in turn secrete IFNg to amplify
a reduction in CD101 expression in neutrophils occurred the anti-tumor immune response.36,58,59 Specifically, in aCD40-
following aCD40 treatment (Figure 6A). Interestingly, we found treated MC38 tumors, we found that IL-12 was produced almost
that aCD40 treatment in Irf1
/
mice failed to induce an increase exclusively by DCs and macrophages, whereas the IFNg-pro-
of CD14+CD101
neutrophils in MC38 tumors (Figure 6C). Thus, ducing cells were predominantly CD8+ T cells and natural killer
targeting IRF1 expression may be used to modulate immuno- cells (Figures S6I and S6J). CXCR3, the receptor for CXCL9/
therapy-elicited neutrophil responses in vivo. 10, has been reported as another key requirement for intratu-
Given these results, and to ensure that IRF1-deficiency is moral activation of T cells upon immunotherapy.60 We could
limited to neutrophils, we generated mixed bone marrow chi- indeed confirm high Cxcr3 expression primarily on T cells in
meras with 50% Csf3r
/
cells and 50% Irf1
/
cells. Csf3r
/
aCD40-treated MC38 tumors (Figure S6K).
cells are largely unable to differentiate into neutrophils but can To dissect the relevance of these components in the ISG
give rise to other lineages.17,56,57 Hence, in Csf3r
/
/Irf1
/
neutrophil response at the tumor site, we have employed either
mixed bone marrow chimeras, peripheral neutrophils originate transgenic mice or cytokine inhibitors. Interestingly, we found
only from Irf1 knockout bone marrow, whereas all other immune that Batf3
/
mice, Il12b
/
mice, wild-type mice treated with

Figure 6. A neutrophil IRF1-mediated interferon response is required for tumor control in mice, and a systemic therapy-induced neutrophil
response correlates with good outcome in patients
(A) Left: representative flow cytometry plots showing CD14, CD101, and CXCL10 expression in neutrophils in MC38 tumors with or without aCD40 treatment.
Right: quantification showing the change in expression of CD14 and CD101 on neutrophils in MC38 tumors upon aCD40 treatment (n = 8–12 untreated, n = 7–14
aCD40-treated, data pooled from three independent experiments). Graph shows mean ± SEM. MFI, median fluorescence intensity.
(B) Transcription factors predicted to be active in aCD40-expanded neutrophil states (N1a and N2), compared with all other states in KP tumors.
(C) Flow cytometry-based quantification of CD14+ CD101
and CD14
CD101+ neutrophils in MC38 tumors induced by aCD40 treatment, in the presence or
absence of Irf1 (n = 7 per group, data pooled from two independent experiments). Graph shows mean ± SEM. Student’s two-tailed t test. *p < 0.05.
(D) Schematic outlining the generation of bone marrow chimeras with Irf1-deficiency, specifically in neutrophils. MC38 tumor volumes and survival of Csf3r
/
/WT
(control), aCD40-treated Csf3r
/
/WT, and aCD40-treated Csf3r
/
/Irf1
/
bone marrow chimeras (n = 7–9 mice/group). Ordinary one-way ANOVA was used for
tumor growth curve, and log-rank (Mantel-Cox) test was used for the survival curve. *p < 0.05; **p < 0.01.
(E) Effect of Batf3 deletion, Il12b deletion, IFNg neutralization, or Cxcr3 deletion on the abundance of CD14+ CD101
and CD14
CD101+ neutrophils in MC38
tumors treated with aCD40 (n = 3–18 per group, data pooled from six independent experiments). Graph shows median and 95% confidence interval. For
comparisons between conditions, ordinary one-way ANOVA with Dunnett’s multiple comparisons test was used. *p < 0.05; **p < 0.01.
(F) Kaplan-Meier plot of progression-free survival of small-cell lung cancer patients treated with chemo-radiotherapy and ipilimumab + nivolumab immunotherapy
(n = 78), separated by baseline neutrophil to lymphocyte ratio (NLR).
(G) Kaplan-Meier plot of progression-free survival of small-cell lung cancer patients treated with chemo-radiotherapy and ipilimumab + nivolumab immuno-
therapy (n = 70), separated by NLR change post-chemo-radiotherapy versus baseline (NLR increase/NLR decrease: min. 10% change versus baseline). In (F) and
(G), univariate Cox regression was used.

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an IFNg-neutralizing antibody, or Cxcr3
/
mice all showed The results presented here also suggest that pro- and anti-tumor
impaired accumulation of CD14+CD101
neutrophils in MC38 neutrophils coexist within tumors and do not differentiate from
tumors after aCD40 treatment (Figure 6E). By contrast, each other, but rather they are likely to have distinct origins.
CD14
CD101+ neutrophils remained largely unaffected in the From a therapeutic perspective, it is possible that reprogram-
same experimental models. Overall, these results indicate that ming tumor-associated neutrophils may be less effective in con-
key components of anti-tumor immunity, including BATF3- trolling tumors than selectively increasing anti-tumor neutrophils
dependent DCs, IL-12 and IFNg production as well as the and/or depleting pro-tumor neutrophils. It will therefore be
CXCR3 chemokine receptor, are all required for the generation important to define the mechanisms that dictate the fate of
of the neutrophil response upon immunotherapy. neutrophil progenitors and how they can be harnessed for ther-
apeutic purposes.
Systemic neutrophil response in small-cell lung cancer Since anti-tumor neutrophils have high cytotoxic potential and
patients is associated with better outcome following expand systemically upon treatment, there must be regulatory
immunotherapy mechanisms that ensure that these cells perform their functions
Finally, we wanted to determine the relevance of a therapy-eli- only within the target sites. It is interesting to note that the
cited neutrophil response in human disease. To this end, we deployment of the neutrophilic response at the tumor site is
analyzed data from a clinical trial in small-cell lung cancer strictly dependent on IL-12, DCs, and IFNg, which are molecular
(NCT02046733).61 We focused on patients who received stan- and cellular factors present in tumors and are part of a positive
dard-of-care chemo-radiotherapy combined with ipilimumab feedback loop necessary for the local promotion of anti-tumor
(aCTLA4) and nivolumab (aPD-1) immune checkpoint inhibitors T cell responses.36 Accordingly, we did not observe a neutrophil
(n = 78). We examined the neutrophil to lymphocyte ratio (NLR) response in KP tumors following aPD-1 therapy, which fails to
in the peripheral blood, a widely used and technically robust in- induce anti-tumor T cell immunity in this model.35 By contrast,
dicator of neutrophil-biased hematopoiesis.62 High baseline aPD-1 elicited a neutrophil response in MC38 tumors in which
NLR (>2.5) in treatment-naive patients correlated with worse T cells can be activated by this treatment.36 It is also conceivable
outcome following adjuvant immunotherapy (Figure 6F), that T cell-mediated tumor cell killing and subsequent release of
compared with low NLR, in line with previous findings63 (p = danger signals contribute to neutrophil mobilization and infiltra-
0.0296, HR = 0.4712, 95% CI of HR: 0.2392–0.9282). To specif- tion into the tumor. Furthermore, IL-12+ DCs are found directly
ically investigate therapy-elicited neutrophil responses, we as- adjacent to blood vessels in the tumor stroma66 and should
sessed changes in NLR in response to chemo-radiotherapy. therefore be able to interact effectively with neutrophils entering
We compared the outcomes of patients showing NLR increase the tumor to allow them to exert their effector functions locally. It
upon therapy versus baseline (>10% increase, n = 54) with should also be noted that IL-12 and IFNg production occur in
patients showing NLR decrease (>10% decrease, n = 16). Inter- healthy tissue in the context of immunotherapy-related adverse
estingly, patients with therapy-induced increase in NLR experi- events, which triggers a tissue destructive neutrophil
enced significantly better progression-free survival following response.59 Taken together, these data suggest that IL-12 and
adjuvant immunotherapy than patients with decreased NLR IFNg signaling are required to promote cytotoxic neutrophil re-
(p = 0.0292, HR = 0.4635, 95% CI of HR: 0.2323–0.9251) (Fig- sponses and that these responses can occur in many different
ure 6G). Overall, these results indicate that a therapy-elicited tissues. Neutrophil expansion with an ISG signature has also
systemic neutrophil response can positively correlate with dis- been described after myocardial infarction or various infec-
ease outcome in lung cancer patients. tions.54,67–69 Therefore, ISG-expressing neutrophil states that
develop after immunotherapy could be analogous to neutrophils
DISCUSSION with tissue-destroying and immunostimulatory activity
described in other pathological contexts.
By detailing the complexity of neutrophils in the context of ther- It is likely that neutrophils stimulated by immunotherapy
apy in mice, this study reveals that the responses mediated by exhibit different anti-tumor functions. For example, previous
these cells are heterogeneous but stereotyped, and include work has indicated that neutrophil production of H2O270,71 or
states with critical anti-tumor effects. We believe these results granular enzymes, including neutrophil elastase13 and
are important because they reconcile previous findings that re- cathepsin-G,72 may have tumoricidal effects. Neutrophils may
vealed pro-tumor or anti-tumor effects for neutrophils when also support adaptive immunity, for example, by promoting anti-
these cells were studied as a single population.19 gen release through cancer cell killing. Also, some tumor-associ-
The transcription factor IRF1 has been described as an ated neutrophils upregulate costimulatory molecules and can
enhancer of the ISG response and a modulator of cytokine and cross-present antigens to CD8+ T cells.16,18,73 The high expres-
chemokine expression in both epithelial cells and DCs.55,64 The sion of CXCL10 by anti-tumor neutrophils could also promote
role of IRF1 in neutrophils remains less studied; however, the interactions with CXCR3-expressing T cells to drive anti-tumor
IRF1-binding motif is highly enriched in the open chromatin re- immunity. The importance of neutrophil-T cell interactions is
gions of granulocyte-monocyte progenitors after beta-glucan illustrated by the observation that enrichment of CD4+ PD-1+
treatment, and these progenitors give rise to neutrophils that T cells in granulocyte-dominated cell neighborhoods is associ-
have potent anti-tumor activity.65 These observations, along ated with a favorable prognosis in colorectal cancer.4 Also,
with the results of the present study, indicate that IRF1 may be some of the characteristics of immunotherapy-stimulated neu-
a critical upstream regulator of anti-tumor neutrophil production. trophils that we have defined here have been described as

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markers of anti-tumor neutrophils in previous studies. For B Flow cytometry and cell sorting
example, a subset of immunostimulatory neutrophils identified B Histology
in early human lung cancer lesions was characterized by CD14 B Quantification of neutrophils following different
expression and required IFNg for expansion.18 It is possible therapies
that CD14/TLR4 signaling directly contributes to neutrophil re- B Cytospin
programming because Myd88, downstream of TLR4, is required B Cytokine neutralization
for the acquisition of the anti-tumor neutrophil phenotype in B Anti-Ly6G treatment
mouse uterine carcinomas.74 Finally, we found that Sellhi neutro- B LPS, IFNb, IFNg, polyI:C treatments
phils in KP tumors expressed elevated levels of CXCL10 in B Bone marrow chimeras
response to immunotherapy, which is similar to tumoricidal neu- B In vitro co-cultures
trophils that can emerge after TGFb neutralization in mice.14,75 B Single-cell RNA sequencing and TotalSeq sample
Overall, our study demonstrates that neutrophils exhibit preparation from KP tumors and healthy control lungs
remarkable plasticity and can acquire an anti-tumor phenotype B Single-cell RNA sequencing sample preparation from
in response to immunotherapy. Although the treatment-induced MC38 tumors
neutrophil response may be short-lived, neutrophils could sup- B Single-cell RNA-sequencing and TotalSeq analysis
port the induction of a long-term adaptive immune response. B Analysis of clinical data from STIMULI trial
Therefore, cancer immunotherapy approaches that induce d QUANTIFICATION AND STATISTICAL ANALYSIS
anti-tumor T cell immunity in combination with therapies that
optimally engage, rather than deplete, anti-tumor neutrophils SUPPLEMENTAL INFORMATION
could lead to more durable tumor control after treatment.
Supplemental information can be found online at https://doi.org/10.1016/j.cell.
2023.02.032.
Limitations of the study
In this study, we first draw attention to a limitation of the mixed ACKNOWLEDGMENTS
bone marrow chimera technique used to assess the requirement
of IRF1 in neutrophils. In this experiment, it is assumed that We thank the Harvard Stem Cell Institute for help with FACS; the Single Cell
Csf3r
/
and Irf1
/
cells can engraft equally and that peripheral Core Facility at Harvard Medical School for help with scRNA-seq experiments;
the Biopolymers Facility at Harvard Medical School for sequencing; the MGH
neutrophils originate only from Irf1 knockout bone marrow,
Histopathology Research Core and Y. Iwamoto, as well as the EPFL Histology
whereas all other immune cells comprise a 50%–50% mixture Core Facility for processing, preparation, and staining of mouse histological
of Irf1-proficient Csf3r
/
cells and Irf1-deficient cells. Thus, it tissue specimens; G.J. Freeman for generously providing the aPD-1 antibody
is expected that up to 50% of non-neutrophil immune cells will reagent; U. Von Andrian, R. Nowarski, S. Pai, and members of the Pittet and
carry a loss of IRF1, and the remaining 50% of IRF1-proficient Weissleder laboratories for helpful discussions. We thank the patients who
cells will be sufficient to maintain a functional response. In future participated in the STIMULI trial and the staff at the ETOP Coordinating Office
and the ETOP Statistical Office for providing the clinical data. This work was
studies, the generation of conditional neutrophil-specific IRF1
supported in part by NIH grant R01-CA218579 (to A.M.K. and M.J.P.), NIH
knockouts should further define the function of this transcription grant P01-CA240239 (to M.J.P.), and the ISREC Foundation (to M.J.P.). J.G.
factor in the context of cancer immunotherapy. Second, the data and M.S. were supported in part by Landry Cancer Biology Research
presented in this study do not define the mechanisms by which Fellowships. R.B. was funded by a Postdoc.Mobility Fellowship and Return
neutrophils contribute to tumor control. The generation of Grant of the Swiss National Science Foundation (SNSF; P400PM_183852;
neutrophil-specific conditional knockouts of candidate genes P5R5PM_203164). M.K. was supported by the EMBO Postdoctoral Fellowship
(ALTF 662-2020) and the Human Frontier Science Program Postdoctoral
potentially involved in tumor control could help answer this ques-
Fellowship (LT000017/2021-L).
tion. Third, our human studies suggest the importance of revisit-
ing NLR as a dynamic readout with prognostic potential. This will AUTHOR CONTRIBUTIONS
require prospective investigations in a larger patient population.
J.G. and M.J.P. initiated the study. J.G., M.J.P., and M.K. designed experi-
STAR+METHODS ments. M.K. wrote the original draft. M.J.P., A.M.K., J.G., N.A.G.-F., and
M.K. prepared the figures and wrote the manuscript. J.G., M.K., M.S., R.B.,
P.K., C.C., E.B., C.P., M. Mazzola, M.H., and F.D. performed mouse experi-
Detailed methods are provided in the online version of this paper ments and related analyses. N.A.G.-F. carried out genomic data analysis. M.
and include the following: Messemaker designed the TotalSeq experiment and provided code for data
analysis. K.H. and S.P. provided human data. R.W. and C.G. provided input
d KEY RESOURCES TABLE for research design and interpretation. M.J.P. and A.M.K. designed analytical
d RESOURCE AVAILABILITY strategies and supervised experiments and data analysis.
B Lead contact
B Materials availability DECLARATION OF INTERESTS
B Data and code availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS M.J.P. has served as consultant for AstraZeneca, Elstar Therapeutics,
ImmuneOncia, KSQ Therapeutics, Merck, Siamab Therapeutics, Third Rock
B Cell lines
Ventures, and Tidal. R.W. has served as a consultant for Moderna, Lumicell,
B Mice Seer Biosciences, Earli, and Accure Health. The wife of R.B. is an employee
d METHOD DETAILS and shareholder of CSL Behring, and R.B. received a speaker’s fee from
B Mouse tumor models Janssen.

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Received: September 3, 2022 14. Fridlender, Z.G., Sun, J., Kim, S., Kapoor, V., Cheng, G., Ling, L., Worthen,
Revised: January 10, 2023 G.S., and Albelda, S.M. (2009). Polarization of tumor-associated neutro-
Accepted: February 24, 2023 phil phenotype by TGF-beta: ‘‘N1’’ versus ‘‘N2’’ TAN. Cancer Cell 16,
Published: March 30, 2023 183–194. https://doi.org/10.1016/j.ccr.2009.06.017.
15. Matlung, H.L., Babes, L., Zhao, X.W., Houdt, M. van, Treffers, L.W., Rees,
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Anti-mouse CD40 (Clone FGK4.5) BioXCell BE0016-2; RRID: AB_1107601
Anti-Mouse PD-1 (clone 29F.1A12) Gordon J. Freeman N/A
Anti-mouse CTLA-4 (clone 9D9) BioXCell BE0164; RRID: AB_10949609
Anti-mouse IFNg (clone XMG1.2) BioXCell BE0055; RRID: AB_1107694
Anti-mouse Ly6G (clone 1A8) BioXCell BP0075; RRID: AB_10312146
Anti-mouse CD45 (clone 30-F11) Biolegend 103126; RRID: AB_493535
Anti-mouse Ly6G (clone 1A8) Biolegend 127643; RRID: AB_2565971
Anti-mouse CD11b (clone M1/70) BD Biosciences 557657; RRID: AB_396772
Anti-mouse SiglecF (clone E50-2440) BD Biosciences 564514; RRID: AB_2738833
Anti-mouse CD14 (clone Sa14-2) Biolegend 123312; RRID: AB_940575
Anti-mouse CD101 (clone Moushi101) eBioscience 25-1011-82; RRID: AB_2573378
Anti-mouse CXCR4 (clone L276F12) Biolegend 146509; RRID: AB_2562786
Anti-mouse Ly6C (clone HK1.4) Biolegend 128014; RRID: AB_1732079
Anti-mouse F4/80 (clone BM8) Biolegend Cat# 123114, RRID:AB_893478
Anti-mouse CD11c (clone N418) Biolegend Cat# 117334, RRID:AB_2562415
Anti-mouse I-A/I-E (clone M5/114.15.2) Biolegend Cat# 107608, RRID:AB_313323
Anti-mouse CD172a (clone P84) Biolegend Cat#144021, RRID:AB_2650812
Anti-mouse CD62L (clone MEL-14) Biolegend Cat#104411, RRID:AB_313098
Anti-mouse CD3ε (clone 145-2C11) Biolegend Cat#100308, RRID:AB_312673
Anti-mouse CD19 (clone 1D3/CD19) Biolegend Cat#152407, RRID: AB_2629816
Anti-mouse NK1.1 (clone PK136) Biolegend Cat#108707, RRID: AB_313394
Anti-mouse CXCR2 (clone SA044G4) Biolegend Cat#149313, RRID: AB_2734210
Anti-mouse CD4 (clone RM4-5) BD Biosciences Cat# 553051, RRID:AB_398528
Anti-mouse CD8a (clone 53-6.7) BioLegend Cat# 100730, RRID:AB_493703
Purified rat anti-mouse Ly-6G Antibody Biolegend 127602; RRID: AB_1089180
Purified goat anti-mouse MPO Antibody Biotechne/R&D systems AF3667
Purified anti-mouse CD8a Antibody eBioscience 14-0808-82; RRID: AB_2572861
TotalSeq-A0013 anti-mouse Biolegend 128047; RRID: AB_2749961
Ly-6C Ab (clone HK1.4)
TotalSeq-A0431 anti-mouse CD170 Biolegend 155513; RRID: AB_2832540
(Siglec-F) Ab (clone S17007L)
TotalSeq-A0424 anti-mouse CD14 Biolegend 123333; RRID: AB_2800591
Ab (clone Sa14-2)
TotalSeq-A0444 anti-mouse CD184 Biolegend 146520; RRID: AB_2800682
(CXCR4) Ab (clone L276F12)
TotalSeq-A anti-mouse CXCR2 Ab Biolegend N/A
(clone SA044G4)
TotalSeq-A0105 anti-mouse CD115 Biolegend 135533; RRID: AB_2734198
(CSF-1R) Ab (clone AFS98)
TotalSeq-A0190 anti-mouse CD274 Biolegend 153604; RRID: AB_2783125
(B7-H1, PD-L1) Ab (clone MIH6)
TotalSeq-A0117 anti-mouse I-A/I-E Ab Biolegend 107653; RRID: AB_2750505
(clone M5/114.15.2)
TotalSeq-A0104 anti-mouse CD102 Biolegend 105613; RRID: AB_2734167
(ICAM-2) Ab (clone 3C4 (MIC2/4))
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
TotalSeq-A0074 anti-mouse CD54 Biolegend 116127; RRID: AB_2734177
(ICAM-1) Ab (clone YN1/1.7.4)
TotalSeq-A0557 anti-mouse CD38 Ab (clone 90) Biolegend 102733; RRID: AB_2750556
TotalSeq-A0595 anti-mouse CD11a Ab (clone M17/4) Biolegend 101125; RRID: AB_2783036
TotalSeq-A0112 anti-mouse CD62L Ab (clone MEL-14) Biolegend 104451; RRID: AB_2750364
TotalSeq-A0201 anti-mouse CD103 Ab (clone 2E7) Biolegend 121437; RRID: AB_2750349
TotalSeq-A0200 anti-mouse CD86 Ab (clone GL-1) Biolegend 105047; RRID: AB_2750348
TotalSeq-A0114 anti-mouse F4/80 Ab (clone BM8) Biolegend 123153; RRID: AB_2749986
TotalSeq-A0078 anti-mouse CD49d Ab (clone R1-2) Biolegend 103623; RRID: AB_2734159
TotalSeq-A0012 anti-mouse CD117 (c-kit) Ab (clone 2B8) Biolegend 105843; RRID: AB_2749960
TotalSeq-A0015 anti-mouse Ly-6G Ab (clone 1A8) Biolegend 127655; RRID: AB_2749962
TotalSeq-A0093 anti-mouse CD19 Ab (clone 6D5) Biolegend 115559; RRID: AB_2749981
TotalSeq-A0238 Rat IgG2a, k Isotype Ctrl Ab Biolegend 400571; RRID: N/A
(clone RTK2758)
TotalSeq-A0301 anti-mouse Hashtag 1 Ab (clone M1/42) Biolegend 155801; RRID: AB_2750032
TotalSeq-A0302 anti-mouse Hashtag 2 Ab (clone M1/42) Biolegend 155803; RRID: AB_2750033
TotalSeq-A0303 anti-mouse Hashtag 3 Ab (clone M1/42) Biolegend 155805; RRID: AB_2750034
TruStain fcX Anti-Mouse CD16/32 (clone 93) Biolegend 101319; RRID: AB_1574973
Rabbit Anti-Rat IgG Antibody, Biotinylated Vector Laboratories BA-4001; RRID: N/A
Anti-Ly-6G MicroBeads UltraPure, mouse Miltenyi Biotec 130-120-337; RRID: N/A
Donkey anti-goat Alexa 647 ThermoFisher Scientific A21447
Goat anti-rat Alexa568 ThermoFisher Scientific A11077
Chemicals, peptides, and recombinant proteins
Standard LPS, E. coli 0111:B4 Invivogen tlrl-eblps
Poly(I:C) HMW Invivogen tlrl-pic
Recombinant Murine IFN-g PeProtech 315-05
Recombinant Mouse IFN-b1 (carrier-free) Biolegend 581302
7-Aminoactinomycin D Sigma A9400-1MG
ACK lysis buffer Lonza 10-548E
Zombie Aqua Fixable Viability Kit Biolegend 423102
Zombie Green Fixable Viability Kit Biolegend 423111
Paclitaxel McKesson 769014
Carboplatin McKesson 724932
Oxaliplatin McKesson 1090455
Cyclophosphamide Sigma-Aldrich C0768-1G
Retrievagen A (pH6.0) BD Biosciences 550524
VECTASTAIN Elite ABC-HRP Kit, Peroxidase Vector Laboratories PK-6100
AEC+ Substrate-Chromogen Agilent K3469
Hematoxylin Solution, Harris Modified Sigma HHS32
Strepravidin DyLight 594 Vector Laboratories SA-5594
DAPI ThermoFisher Scientific D21490
Immpress HRP Ready-to-use Vector Laboratories MP-7444-15
FluoromountG Bioconcept 0100-01
Recombinant Mouse Flt3L Peprotech 550704
TLR7/8 agonist R848 Invivogen tlrl-r848
CellROX Green Flow Invitrogen C10492
Cytometry Assay Kit
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Critical commercial assays
Chromium Next GEM Chip G Single 10x Genomics Cat# 1000127
Cell Kit, 16 rxns
Chromium Next GEM Single Cell 3ʹ Kit v3.1, 4 rxns 10x Genomics Cat# 1000269
QuadroMACS Separator and Starting Kits Miltenyi Biotec Cat# 130-091-051
Deposited data
Raw Single Cell RNA Sequencing Data - CD11b+ This paper GEO: GSE224399
cells from mouse KP lung tumors +/- aCD40
immunotherapy and healthy lungs
Raw Single Cell RNA Sequencing Data - CD45+ This paper GEO: GSE224400
cells from mouse MC38 tumors +/- aCD40
immunotherapy
Raw Single Cell RNA Sequencing Data - CD45+ Garris et al.36 GEO: GSE112865
cells from mouse MC38 tumors +/- aPD1
immunotherapy
Single Cell RNA Sequencing Data (raw counts) - Zilionis et al.24 GEO: GSE127465
CD45+ cells from human lung tumors
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.edu/
of healthy mice (gene expression - CP10K) tools/springViewer_1_6_dev.html?
datasets/SPRING_private/
gungabeesoon22/all_cells_
healthy_gex/gex
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.
of healthy mice (surface protein expression - CLR) edu/tools/springViewer_1_6_
dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_healthy_adt/adt
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.
of KP1.9 tumor-bearing mice (gene expression - CP10K) edu/tools/springViewer_1_6_
dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_kp19_gex/gex
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.
of KP1.9 tumor-bearing mice (surface protein edu/tools/springViewer_1_6_
expression - CLR) dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_kp19_adt/adt
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.
of KP1.9 tumor-bearing mice after aCD40 edu/tools/springViewer_1_6_
immunotherapy (gene expression - CP10K) dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_acd40_gex/gex
Interactive browser of CD11b+ cells from lungs This paper https://kleintools.hms.harvard.
of KP1.9 tumor-bearing mice after aCD40 edu/tools/springViewer_1_6_
immunotherapy (surface protein expression - CLR) dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_acd40_adt/adt
Interactive browser of neutrophils from lungs of This paper https://kleintools.hms.harvard.
healthy mice (gene expression - CP10K) edu/tools/springViewer_1_6_
dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_healthy_gex/gex
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Interactive browser of neutrophils from lungs of This paper https://kleintools.hms.harvard.
healthy mice (surface protein expression - CLR) edu/tools/springViewer_1_6_
dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_healthy_adt/adt
Interactive browser of neutrophils from lungs of This paper https://kleintools.hms.harvard.
KP1.9 tumor-bearing mice (gene expression - CP10K) edu/tools/springViewer_1_6_
dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_kp19_gex/gex
Interactive browser of neutrophils from lungs of This paper https://kleintools.hms.harvard.
KP1.9 tumor-bearing mice (surface protein edu/tools/springViewer_1_6_
expression - CLR) dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_kp19_adt/adt
Interactive browser of neutrophils from lungs of KP1.9 This paper https://kleintools.hms.harvard.
tumor-bearing mice after aCD40 immunotherapy edu/tools/springViewer_1_6_
(gene expression - CP10K) dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_acd40_gex/gex
Interactive browser of neutrophils from lungs of KP1.9 This paper https://kleintools.hms.harvard.
tumor-bearing mice after aCD40 immunotherapy edu/tools/springViewer_1_6_
(surface protein expression - CLR) dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_acd40_adt/adt
Interactive browser of CD45+ cells from MC38 tumors This paper https://kleintools.hms.harvard.
either untreated or treated with aCD40 immunotherapy edu/tools/springViewer_1_6_
(gene expression - CP10K) dev.html?datasets/SPRING_
private/gungabeesoon22/all_
cells_mc38_pm_acd40/gex
Interactive browser of neutrophils from MC38 tumors This paper https://kleintools.hms.harvard.
either untreated or treated with aCD40 immunotherapy edu/tools/springViewer_1_6_
(gene expression - CP10K) dev.html?datasets/SPRING_
private/gungabeesoon22/
neutrophils_mc38_pm_acd40/gex
Experimental models: Cell lines
Murine MC38 colorectal carcinoma cell line Mark J. Smyth RRID: CVCL_B288
Murine MC38-H2B-GFP Ralph Weissleder N/A
Murine KP1.9 lung adenocarcinoma cell line derived Alfred Zippelius N/A
from lung tumor nodules of a C57BL/6KrasLSL-G12D/WT;
p53Flox/Flox mouse
Experimental models: Organisms/strains
Mouse: WT C57BL/6J The Jackson Laboratory Strain# 000664 RRID:IMSR_JAX:000664
Mouse: B6.129S(C)-Batf3tm1Kmm/J The Jackson Laboratory Strain #:013755 RRID:IMSR_JAX:013755
Mouse: B6.129S1-Il12btm1Jm/J The Jackson Laboratory Strain #:002693 RRID:IMSR_JAX:002693
Mouse: B6.129-Il12btm1Lky/J The Jackson Laboratory Strain# 006412 RRID:IMSR_JAX:006412
Mouse: B6.129X1(Cg)-Csf3rtm1Link/J The Jackson Laboratory Strain #:017838 RRID:IMSR_JAX:017838
Mouse: B6.129S2-Irf1tm1Mak/J The Jackson Laboratory Strain #:002762 RRID:IMSR_JAX:002762
Mouse: REX3 Transgenic Andrew D. Luster Groom et al.44
Mouse: KrasLSL-G12D/+;Trp53flox/flox Tyler Jacks DuPage et al.32
Mouse: B6.129S4-Ifngtm3.1Lky/J The Jackson Laboratory Strain#017581 RRID:IMSR_JAX:017581
Mouse: Cxcr3tm1Wwh Andrew D. Luster Hancock et al.76
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Software and algorithms
Python 3.8.13 Python Software foundation https://www.python.org
R 4.1.1 R Core https://www.r-project.org/
Scanpy 1.8.2 Wolf et al.77 https://github.com/scverse/scanpy
sctransform 0.3.2 Hafemeister and Satija78 https://github.com/satijalab/sctransform
Seaborn 0.11.2 Waskom79 https://seaborn.pydata.org/
Seurat 4.0.6 Stuart et al.80 https://github.com/satijalab/seurat/
releases/tag/v4.0.6
STARsolo 2.7 Dobin et al.81 https://github.com/alexdobin/STAR/
blob/master/docs/STARsolo.md
SPRING Weinreb et al.82 https://github.com/AllonKleinLab/
SPRING_dev
FlowJo v.10.8 FlowJo, LLC RRID: SCR_008520
Graphpad Prism v.9 GraphPad Prism RRID: SCR_002798
FIJI ImageJ Version 2.1.0/1.53c FIJI RRID: SCR_002285
QuPath v0.4.0 Digital Pathology Bankhead et al.83 RRID:SCR_018257
Code used for scRNAseq analyses This study https://github.com/AllonKleinLab/
ifn_neutrophils/tree/main/notebooks

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources or reagents should be directed to and will be fulfilled by the lead contact, Mikael J.
Pittet ([email protected]).

Materials availability
This study did not generate new unique reagents.

Data and code availability

d Single-cell RNA-seq data have been deposited at GEO and are publicly available as of the date of publication. Accession
numbers are listed in the key resources table. Microscopy and flow cytometry data reported in this paper will be shared by
the lead contact upon request.
d All original code has been deposited at GitHub and is publicly available as of the date of publication. The link is listed in the key
resources table.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Cell lines
KP1.9 cells, derived from lung tumor nodules of a male C57BL/6 KP mouse were obtained from Alfred Zippelius (University Hospital
Basel, Switzerland). KP1.9 cells were maintained in IMDM medium supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin. MC38 cells (obtained from Mark J. Smyth) and MC38-H2B-GFP cells (obtained from Ralph Weissleder), both derived
from a female mouse, were maintained in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin.

Mice
Animals were bred and housed under specific pathogen free conditions at the Massachusetts General Hospital and at the Agora Can-
cer Research Center. Experiments were approved by and were performed in accordance with the animal care and use committees of
MGH, University of Geneva and canton Vaud. C57BL6/J mice (Cat #000664), Batf3-/- (Cat #013755), Il12p40-/- (Cat #002693), IL-
12p40-IRES-eYFP (Cat #006412), Csf3r-/- (Cat #017838), Irf1-/- (Cat #002762), IFNg-IRES-eYFP (Cat #017581) were all obtained
from Jackson Laboratories. KrasLSL-G12D/+;Trp53flox/flox mice were from the lab of Tyler Jacks (MIT, Boston, USA) and maintained
in our facility. REX3-Tg and Cxcr3-/- mice were received from the lab of Andrew D. Luster (MGH, Boston, USA) and maintained in

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our facility. 7–14 week old mice were used for experiments. Male mice were used for experiments involving the KP1.9 tumor model.
Both male and female mice were used for all other experiments and experimental groups were sex-matched.

METHOD DETAILS

Mouse tumor models

KP lung tumors were induced by intravenous tail vein injection of 2.5 x 105 KP1.9 cells into male C57BL6/J mice, as described pre-
viously.9,35 KP1.9-derived tumors were allowed to grow for two weeks prior to aCD40 therapy. For assessing tumor control by
aCD40, on day 14 of tumor growth tumor-bearing mice were treated with 5 mg/kg of aCD40 (clone FGK4.5, BioXCell Cat
#BE0016-2) intraperitoneally. These mice were then euthanized between 33-35 days after tumor induction, and tumor burden
was assessed by measuring post-mortem lung weight and by histological analysis using hematoxylin and eosin (H&E) staining.
For experimental readouts assessing neutrophil responses, aCD40 was given two days before endpoint (typically day 33).
For chemotherapy or aPD1+aCTLA4 treatment experiments, KP lung tumors were induced by intratracheal (i.t.) delivery of
Adenovirus-Cre (AdCre) to KrasLSL-G12D/+;Trp53flox/flox mice, as described previously.9,35 Tumor-bearing mice were treated once a
week for 3 weeks, with intraperitoneal injections of 10 mg/kg of paclitaxel combined with 10 mg/kg of carboplatin, or 2.5 mg/kg
of oxaliplatin combined with 50 mg/kg of cyclophosphamide, as described previously.35 Treatments with a combination of aPD-1
(clone 29F.1A12, 200 mg/mouse, provided by Dr. G. J. Freeman) and aCTLA-4 (clone 9D9, 100 mg/mouse, BioXCell) were performed
as described previously.35 Mice were euthanized 3 days after the last treatment, and tumor burden was assessed by histological
analysis using hematoxylin and eosin (H&E) staining.
MC38 cells were implanted at 2 x 106 cells per tumor in the flank subcutaneously. Tumor-bearing mice were treated with 5 mg/kg of
aCD40 on day 7 of tumor growth. Tumor size was recorded over time with a digital caliper and tumor volumes were calculated as:


Volume = p 6 x length x width2

Experimental readouts assessing neutrophil responses were performed two days following aCD40 treatment. Experimental read-
outs assessing CD8+ T cells and DCs were performed seven days following aCD40 treatment. For aPD-1 treatments, tumor-bearing
mice were treated with 200 mg of aPD-1 when MC38 tumors reached approximately 75mm3, as described previously.36 Neutrophil
response readouts were performed 2 days after treatments.

Flow cytometry and cell sorting

KP tumor-bearing lungs were perfused post-mortem by PBS injection through the right ventricle of the heart. MC38 tumors and KP
tumor-bearing lungs were isolated and minced using surgical scissors, then digested with 0.2 mg/ml Collagenase I (Worthington) in
RPMI-1640 at 37C for 30 minutes shaking at 900 rpm. Digested tissues were then processed through a 40 mm cell strainer, centri-
fuged at 1500 RPM for 5 minutes, subjected to red blood cell lysis for 1 minute using ACK lysis buffer, and resuspended in PBS with
0.5% BSA for staining. For blood analyses, 5 ul blood was diluted in 1 ml of PBS with 2mM EDTA and 0.5% FBS. Red blood cells were
lysed using ACK lysis buffer for 5 minutes and resuspended in PBS with 0.5% BSA for staining. For bone marrow analyses, femurs
were harvested and bones were flushed using a 26-g needle with 0.5% BSA in PBS until bones appeared white. Harvested cells were
processed through a 40 mm cell strainer, subjected to ACK lysis for up to 5 minutes and resuspended in PBS with 0.5% BSA for stain-
ing. Cell suspensions were stained with Zombie Aqua or Zombie Green or 7-AAD (Biolegend) to exclude dead cells, incubated with Fc
Block TruStain FcX (Clone 93, Biolegend) in PBS with 0.5% BSA, and then stained with fluorochrome labeled antibodies (listed in the
key resources table). Cells were quantified using Precision Count Beads (Biolegend). ROS production was assessed using the
CellROX Green reagent (Invitrogen). Cells were resuspended in DMEM and incubated with the CellROX Green reagent for 30 min
at 37 C. After washing the cells with PBS, cells were stained for flow cytometry as described above. Samples were run on a BD
LSR II flow cytometer and analyzed using FlowJo software (Treestar). Cell sorting was performed using a BD FACS Aria II sorter.

Histology
KP tumor-bearing lungs were perfused post-mortem by PBS injection through the right ventricle of the heart. Then lungs were
excised and placed in ice cold PBS. Tissues were fixed in either 10% formalin overnight, then washed twice with PBS and placed
in 70% Ethanol or PBS until processing, or 4% paraformaldehyde and then cryoprotected in sucrose overnight and embedded in
OCT for freezing. For H&E, tissues were paraffin embedded, sectioned, and stained with Hematoxylin and Eosin at the MGH Histo-
pathology Research Core.
Paraffin-embedded sections were deparaffinized and rehydrated prior to immunohistochemical staining. Heat induced antigen
retrieval was performed using Retrievagen A (pH6.0) (550524, BD Biosciences), and the sections were permeabilized with 0.3%
Triton X-100 in PBS for 10 minutes at room temperature. After the sections were blocked with 4% normal rabbit serum in PBS for
1 hour at room temperature, a primary antibody, Ly6G (clone 1A8, BioLegend, 127602, 1:25) was incubated at 4 C overnight. A bio-
tinylated rabbit anti-rat IgG secondary antibody (Vector Laboratories, BA-4001, 1:100) was applied, and VECTASTAIN ABC-HRP kit
(Vector Laboratories, PK-6100) and AEC Substrate (Agilent, K3469) were used for the detection. Nuclei were counterstained with
Harris Hematoxylin (Sigma, HHS32) and all the images were captured by using a digital scanner NanoZoomer 2.0RS (Hamamatsu,

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Japan). For immunofluorescent staining, strepravidin DyLight 594 (Vector Laboratories, SA-5594, 1:600) was used after a biotinylated
rabbit anti-rat IgG and nuclei were stained with DAPI (ThermoFisher Scientific, D21490). The number of Ly6G+ cells was determined
manually and normalized to the area of the field of view.
Frozen lung tissues were permeabilized for 10 minutes in 0.1% Triton X-100 in PBS 1x. After 30 minutes blocking in 1% BSA in PBS,
primary Ly6G (clone 1A8, BioLegend, 127602, 1:100) and MPO (R&D systems, AF3667, 1:200) antibodies were applied on 10um sec-
tions and incubated overnight at 4 C. Secondary antibodies, donkey anti-goat Alexa 647 (ThermoFisher Scientific, A21447, 1:1000)
and goat anti-rat Alexa568 (ThermoFisher Scientific, A11077, 1:1000 ) were incubated sequentially for 45 minutes each. Sections
were counterstained with DAPI (ThermoFisher Scientific, D21490) and mounted with FluoromountG (Bioconcept, 0100-01). All the
images were captured by using a digital scanner Axioscan 7 (Zeiss).The numbers of MPOhiLy6Ghi and MPOhiLy6Glo cells were deter-
mined by manual counting in tumor areas and were normalized to the area of the field of view.
MC38 tumors were formalin fixed and paraffin embedded as described above. Sections were then stained using the Ventana
Discovery ULTRA automate (Roche Diagnostics). All steps were performed automatically with Ventana Solutions, unless otherwise
noted. The dewaxed and rehydrated paraffin sections were heat pre-treated using CC1 solution for 40 min at 95 C. Primary antibody
was applied and revealed with anti-rat Immpress HRP (Ready to use, Vector Laboratories) followed by incubation with Cy5 fluores-
cent tyramide. The primary antibody was rat anti-CD8 (clone 4SM15, ThermoFisher Scientific, 14-0808-82, 1:100) Sections were
counterstained with DAPI (ThermoFisher Scientific, D21490) and mounted with FluoromountG (Bioconcept, 0100-01). All the images
were captured by using a digital scanner Axioscan 7 (Zeiss). The fraction of CD8+ cells within all DAPI+ cells was determined in cross-
sections of entire tumors using an automated cell classifier in QuPath.

Quantification of neutrophils following different therapies

In Figure 1H, neutrophil quantification is shown based on flow cytometry (KP+aCD40; MC38+aCD40; MC38+aPD1) or immunoflu-
orescence staining of Ly6G in tissue sections (KP+aPD1/aCTLA4; KP+Oxa/Cyc; KP+Pac/Carbo).

Cytospin
SiglecFhi and SiglecFlo neutrophils were sorted from lung tissue of KP tumor-bearing mice with or without aCD40 treatment. Cyto-
spins were performed using a Shandon Cytospin 4 centrifuge (Thermo Fisher Scientific). In detail, 105 cells were centrifuged (700 rpm,
5 min) onto Tissue Path Superfrost Plus Gold microscope slides (ThermoFisher Scientific) and dried overnight at room temperature.
Cells were then fixed in 4% formaldehyde-buffered solution and stained with hematoxylin and eosin (H&E) using the ThermoScientific
Shandon Varistain Gemini ES Automated Slide Stainer. Slides were scanned using Axioscan 7 (Zeiss). The number of nuclear lobes
was counted manually on at least 50 cells per condition.

Cytokine neutralization
Neutralization of IFNg was performed by intraperitoneal injection of 1 mg of anti-IFNg (Clone XMG1.2, BioXCell Cat# BE0055) initially
on day 7 of tumor growth, with an additional 500 mg of anti-IFNg dosed on day 8, then mice were analysed on day 9.

Anti-Ly6G treatment
Anti-Ly6G (BioXCell Cat #BP0075) was administered at 500 mg / mouse on day 7 (–2h before aCD40) and boosted with 250 mg/mouse
on day 8, then mice were analysed on day 9.

LPS, IFNb, IFNg, polyI:C treatments

Mice were treated by intraperitoneal injection with 5 mg/kg of LPS (Invivogen, tlrl-eblps), 5 x 106 U/kg of recombinant mouse IFN-b1
(BioLegend, Cat# 581302), 7.5 x 105 U/kg of recombinant murine IFNg (Peprotech, Cat# 315-05) or 5 mg/kg of polyI:C (Invivogen, tlrl-
pic). Experimental readouts assessing neutrophil responses were performed two days following these treatments.

Bone marrow chimeras

C57BL6/J recipient mice (Cat #000664) were irradiated with a single dose of 1000 cGy using a cesium-137 irradiator. The next day,
bone marrow was harvested from donor mice, including WT C57BL6/J mice (Cat #000664), Csf3r-/- mice (Cat #017838), or Irf1-/- mice
(Cat #002762). Cells from each type of donor were counted manually. For 50:50 bone marrow chimeras, cells were mixed at a 1:1 ratio
before injection. Cells were injected retro-orbitally at 10-14 x 106 total cells / mouse in 200-400 mL volume, and mice were allowed to
reconstitute for 5.5-7.5 weeks.

In vitro co-cultures
Two days after aCD40 treatment, livers were excised and digested with 450 U/ml collagenase I, 125 U/ ml collagenase XI, 60 U/ml
DNase I, 60 U/ml hyaluronidase (Sigma Aldrich), and 20 mM HEPES buffer in PBS at 37 C for 20 minutes shaking at 900 rpm, as
described previously.59 Digested tissue was then processed through a 40 mm cell strainer, centrifuged at 1500 rpm for 5 minutes,
subjected to ACK red blood cell lysis, and resuspended in PBS with 0.5% BSA. Magnetic selection with anti-Ly-6G microbeads
(Miltenyi Biotec) was used to isolate neutrophils from the resulting cell suspension. Isolated neutrophils were co-cultured with
MC38-H2B-GFP tumor cells (40:1 neutrophil:cancer cell ratio). GFP+ tumor cells were quantified 24 hours later via microscopy

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(DeltaView). Co-culture of cancer cells with splenocytes from untreated mice was used as a negative control condition. For DC/neu-
trophil co-cultures, bone marrow cells were isolated from IL-12p40 reporter (IL12p40-eYFP) mice and cultured with 300 ng/mL Flt3L
(Peprotech) for 8-10 days to generate DCs. Neutrophils were co-cultured with DCs (10:1 neutrophil:DC ratio) for 24 hours, before
quantifying YFP+ cells via microscopy (DeltaVision). Treatment with TLR7/8 agonist R848 was used as a positive control condition.

Single-cell RNA sequencing and TotalSeq sample preparation from KP tumors and healthy control lungs
KP tumors were induced in C57BL6/J mice by iv. injection of KP1.9 cells, and allowed to grow for 31 days before treating, or not, with
aCD40. Two days after aCD40 treatment, the lungs of these mice were perfused, and tumor nodules were macroscopically dissected
from the lungs and digested as described above to generate single cell suspensions. Healthy lungs were processed similarly. Cells
were then stained with a combination of DNA-tagged TotalSeq-A antibodies and Hashtag antibodies (listed in the key resources ta-
ble), stained with a fluorochrome-labelled CD11b antibody for sorting, and labelled with 7-AAD to identify live cells. 7AAD-CD11b+
cells were sorted into PBS (no Ca or Mg) with 0.04% BSA, at a concentration of 1000-1500 cells/mL. After this, cells were processed
with Chromium Next GEM Single Cell 3’ Kit v3.1, 4 rxns (PN-1000269) before loading on a Chromium Next GEM Chip G Single Cell Kit,
16 rxns (PN-1000127). In collaboration with the Single Cell Core Facility at Harvard Medical School, standard steps were followed for
library preparation, quality control and amplification. Sequencing was performed in collaboration with the Biopolymers Facility at
Harvard Medical School, using the NovaSeqSP platform (1,600,000,000 reads, 20k reads/cell).

Single-cell RNA sequencing sample preparation from MC38 tumors

MC38 tumors were harvested 2 days after aCD40 treatment. Tissues were digested as described above to generate single cell sus-
pensions, cells were stained for CD45 and labeled with 7AAD (Sigma-Aldrich). 7AAD-CD45+ cells were sorted using a BD FACSAria
sorter. InDrops single cell RNA sequencing was performed as described previously.59,84,85 Briefly, a microfluidic device was used to
co-encapsulate individual cells and polyacrylamide beads carrying barcoding reverse transcription (RT) primers and lysis reagents
into 2-3 nl droplets, followed by primer release and RT at 50 C. After the RT reaction, droplets were broken, and the resulting bar-
coded cDNA was taken through the following sequencing library preparation steps 1) second strand synthesis, 2) in vitro transcription
providing linear amplification of the material, 3) fragmentation of the amplified RNA, 4) a second reverse transcription using random
hexamer primers bearing a universal PCR primer annealing site, and 5) indexing PCR, yielding a sequencing-ready library. DNA
sequences of primers used during the library preparation were described previously.59 Libraries were sequenced on the NextSeq
Illumina platform, paired-end mode, dual indexing.

Single-cell RNA-sequencing and TotalSeq analysis

scRNAseq read processing
For the KP tumor dataset, raw FASTQ files were processed by Cell Ranger 6.0.1 using mm10-2020 as a reference. Count matrices for
transcripts, captured antibodies, and multiplexing tags were simultaneously generated using cellranger multi with default parame-
ters. For the MC38 tumor dataset, the FASTQ files were processed using STARSolo.81
Data filtering and normalization
For the KP tumor dataset, cells expressing >5% mitochondrial transcripts or fewer than 300 total transcripts were excluded as low-
quality cells. Cells with transcript numbers above the 99th percentile within each experiment were considered potential doublets and
excluded.
The MC38 tumor dataset was processed as described previously.59 Specifically, library-specific thresholds on total counts
(ranging from 120 to 400 counts) and fraction of counts coming from mitochondrial genes (ranging from 12% to 15%) were manually
determined based on the empirical distributions of these magnitudes in each library.
For statistical analysis of both datasets, normalized counts per ten-thousand (CP10K) were used, except when indicated
otherwise.
Dimensionality reduction and visualization
Separate embeddings were created for immune cells derived from healthy lungs and KP tumors combined, and from aCD40-treated
KP tumors. Raw counts from healthy lung + KP tumor and aCD40-treated KP tumor were separately normalized through scTransform
v178 after removing genes present in fewer than 3 cells per dataset. PCA (n=50) was applied on the Pearson residuals from scTrans-
form. Neighbors-graphs (k=15) were constructed from the PC spaces of each dataset. 2D UMAP embeddings were generated from
each KNN graph. The MC38 tumor dataset was processed following the pipeline detailed in Siwicki et al.59
Identification of neutrophils
For the KP tumor dataset, a classifier was applied to the complete scRNAseq data set (including contaminating CD11b+ non-neutro-
phil cells) to define the major immune cell type identity of each transcriptome. The classifier was trained on scRNAseq data from
immune cells in KP lung tumors published earlier.24 Correct annotation of major cell types and separation of neutrophils from
non-neutrophil cell types was confirmed by inspection of the UMAP embedding and examination of cell type-specific marker
gene expression. In addition, Total-Seq confirmed specific expression of Ly6G protein on cells classified as neutrophils, but not
on cells annotated as non-neutrophils. In all analyses concerning neutrophil states, cells annotated as non-neutrophils were
excluded. Cells from clusters with a majority of non-neutrophil cells were also excluded regardless of their initial annotation in order
to prevent the inclusion of potential contaminants with ambiguous transcriptional identities.

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Cells from MC38 tumors were clustered using Scanpy’s implementation of the Leiden algorithm and manually annotated into
coarse immune subsets, including neutrophils, based on marker gene expression.
Identification of neutrophil states
A reference atlas of neutrophil states was constructed based on previously published scRNAseq data from KP tumors.24 The neutro-
phil population ‘‘N1’’ originally identified in Zilionis et al.24 represented a continuum of states whose variation proved important to
resolve in order to correctly define changes after aCD40 immunotherapy in the current study. Specifically, Ngphi cells within the
N1 state were enriched in many of the same genes as N6 cells and were directly connected to them in the nearest-neighbors graph.
For this reason, the ‘‘N1’’ cluster was partitioned by sub-clustering into two clusters, which defined N1a (SellhiNgphi) and N1b (Sell-
hi
Lst1hi) neutrophils. The remaining neutrophil populations (‘‘N2’’-‘‘N6’’) were left unchanged. Then, transcriptomes obtained in the
current study from healthy lung, KP tumor, aCD40-treated KP tumor were classified through the method reported previously24 using
the constructed atlas as reference. Because N4 and N6 formed a continuum, we denoised the annotations by reclassifying as N4 any
initially cell classified as N6 that neighbored at least one cell classified as N4. A single iteration of this denoising procedure was carried
out, on each dataset separately (healthy lung + KP tumor, aCD40-treated KP tumor). Similarly, the state identity of neutrophils from
MC38 tumors was inferred through classification.
Identification of marker genes for neutrophil states
For Figure 2B, Wilcoxon rank-sum test was used to identify differentially expressed genes in each neutrophil state in the
aCD40-treated KP tumor condition. Genes with an FDR above 0.01 and a log fold change below 0.25 were excluded. The reference
expression level (CP10Kref) was calculated for each gene, representing the second highest average expression among the neutrophil
subpopulations in the aCD40-treated condition. Candidate marker genes were then ranked by their fold change
 
CP10K + 1
log2
CP10Kref + 1

relative to the second highest expression level. The top 100 marker genes for each neutrophil state identified by this method are
shown in Figure 2B, and all genes that passed the initial filter are listed in Table S1.
For Figures S3A and S3B, Wilcoxon rank-sum test was used to identify genes differentially expressed in each neutrophil state in the
combined healthy lung + untreated KP tumor conditions. A series of filters was then applied to obtain the list of marker genes. Genes
with fold-changes smaller than 2, expressed in fewer than 10% of the in-group cells, or expressed in over 50% of out-group cells
were excluded. Remaining marker genes were then sorted by their normalized U statistic and the top 500 for each population
were considered. Lastly, genes appearing in the top 500 of more than one sub-population were discarded. Top marker genes per
neutrophil state identified using this method are shown in Figures S3A and S3B. The complete lists of marker genes for each neutro-
phil state in the healthy lung + untreated KP tumor dataset and in the aCD40-treated KP tumor dataset are shown in Table S1.
Quantifying the abundance of neutrophil states in untreated and aCD40-treated KP tumors
The number of neutrophils per mg of tissue corresponding to each neutrophil state (Figure 3A) was estimated by multiplying the
cross-replicate average fraction of each neutrophil state with the number of total neutrophils per milligram of tissue measured
through flow cytometry.
Processing of antibody-derived tag data
Raw counts derived from the antibody panel were CLR (centered log ratio) transformed as described by Stoeckius et al.39
Interactive SPRING viewer
The data and embeddings associated with each condition are available for interactive exploration through SPRING.82 Transcriptomic
data is shown as normalized counts (CP10K), and surface marker expression is expressed as CLR-transformed counts. Links cor-
responding to each embedding are listed under Deposited Data in the key resources table.
Quantifying the abundance of major immune subsets in untreated and treated MC38 tumors
For Figures 1F and 1G, we quantified the abundance of each major immune cell type in untreated, aCD40-treated, and aPD-1-treated
MC38 tumors as the fraction of all cells annotated as each cell type in each dataset. We report changes in abundance as the fold
change between the abundances of each cell type in the treated and untreated condition for each referenced dataset. More explicitly,
for each cell state i, the fold change FC(i) was computed as:
.
ðiÞ
ðiÞ ntreated Ntreated
ftreated
FCðiÞ = ðiÞ = .
funtreated nðiÞ
untreated Nuntreated

Where f is the fraction of the cell type, n is the number of cells annotated to cell state i, and N is the total number of cells sampled.
Evaluating cell state similarities across studies
Cells annotated as neutrophils from each study were classified using an immune atlas as reference (see: identification of neutrophil
states), leading to all cells receiving a label from the reference. Cells classified as anything but a neutrophil subset or as states rep-
resented by fewer than 10 cells in a condition were not considered for further analysis. Using each condition as a reference at a time,

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the probability of classification of each cell with respect to each state from the reference was computed. Reciprocal similarity scores
between each pair of states were computed as the harmonic mean of the average probabilities obtained by applying the classifier in
both directions, as described previously.24,43
Quantifying the abundance of Sellhi neutrophils in untreated and treated tumors across tumor models
The abundance of Sellhi neutrophils in each tumor model was determined by calculating the proportion of immune cells annotated as
N1a, N1b, or N2 (see: evaluating cell state similarities across studies) among the total number of immune cells present in each data-
set. The change in the percentage of Sellhi neutrophils between the treated and untreated conditions for each tumor model and treat-
ment is depicted in Figure 3D, where 0% represents no observed change. The untreated cells from each dataset were used as the
reference for comparison.
Calculating gene set scores
Gene set scores were computed as the average expression of the genes belonging to each gene set of interest set minus the average
expression of a control gene set, as described by Tirosh et al.86 and implemented in Scanpy. For these calculations, log-normalized
counts were standardized across all transcriptomes within each group of conditions (tumor-free + tumor-burdened, aCD40-treated).
The resulting Z-scores were employed as the measure of relative expression of each gene. The gene sets underlying Figures 4A–4D
are listed in Table S1.
The gene sets corresponding to angiogenesis, ECM remodeling, immunosuppression, tumor proliferation, and myeloid cell recruit-
ment were obtained from Engblom et al.9 The genes corresponding to neutrophil cytotoxicity were selected from the gene ontology
terms Neutrophil mediated cytotoxicity (GO:0070942) and Respiratory burst (GO:0045730). The genes correponding to neutrophil
degranulation were selected from the gene ontology term Neutrophil degranulation (GO:0043312). The genes used to define the
Interferon signaling scores were selected from the gene ontology terms Type I interferon-mediated signaling pathway
(GO:0060337) and Interferon Gamma Response (MSigDB HallMark M5913). The genes used to define the neutrotime scores corre-
spond to the early neutrotime genes from Grieshaber-Bouyer et al.47 and are listed individually in Figure 5D. For neutrotime scores,
signs of the scores obtained from Scanpy were flipped so that higher scores would reflect a higher degree of maturity.
RNA velocity
Velocyto49 was used under default parameters to generate splice-aware count matrices from the mapped reads from each library.
Unspliced and spliced counts were matched to the barcodes retained after filtering and annotated as neutrophils in each library.
Separate velocity embeddings were created for the healthy lung + KP tumor and the aCD40-treated KP tumor conditions. scVelo48
was used to compute connectivities and velocities in each group of conditions. For the cells from the healthy lung + KP tumor dataset,
all genes were employed in velocity calculations, and no subsequent filtering by R2 was performed. For cells from aCD40-treated KP
tumor, velocities were only computed for highly variable genes as determined by scVelo, and genes with a velocity R2 smaller than
0.01 were excluded from graph calculations. The stochastic model was employed in both cases. Velocity graphs and visualizations
were computed through scVelo based on pre-existing UMAP embeddings for each condition.
Transcription factor prediction
Prediction of active transcription factors was performed using the ChEA3 algorithm87 based on enriched genes in N1a and N2 neutro-
phil states compared to all other neutrophil states in anti-CD40-treated KP tumors. Predicted transcription factors were ranked
based on their average rank across all transcription factor-target gene libraries (mean rank).

Analysis of clinical data from STIMULI trial

We analyzed data from the 4–12 STIMULI clinical trial (NCT02046733) that was performed by the European Thoracic Oncology Plat-
form (ETOP, https://www.etop-eu.org) in patients with limited-disease small-cell lung cancer. All patients received standard-of-care
induction concomitant radio-chemotherapy (cis-/carboplatin + etoposide + thoracic radiotherapy) followed by consolidation therapy
either with ipilimumab and nivolumab or by standard-of-care observation.61 We focused on patients who received combination
immunotherapy as consolidation (n=78). The neutrophil-to-lymphocyte ratio (NLR) was calculated as the ratio of the absolute counts
(G/l) of neutrophils and lymphocytes in peripheral blood at enrolment (baseline NLR) and after radio-chemotherapy but before the first
dose of immunotherapy (post-therapy NLR). NLR change was calculated as a percentage change comparing post-therapy NLR
versus baseline NLR. From the 78 patients, 70 patients had available post-therapy NLR data for comparison with the baseline.
Four patients showed disease progression following radio-chemotherapy and were excluded from the consolidation part as per pro-
tocol. Four patients had missing post-radio-chemotherapy NLR data. Kaplan-Meier estimates of survival were analyzed using the
‘‘survival’’ (version 3.3-1) and ‘‘survminer’’ (version 0.4.9) packages in RStudio. Hazard ratios of survival were calculated using uni-
variate Cox regression in the ‘‘survival’’ R package.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analyses of data from mouse experiments were performed using GraphPad Prism, except for scRNAseq analyses for
which details are provided in the respective methods sections. Analysis of clinical data was performed in RStudio. Statistical param-
eters (sample size, P-value, statistical test) for all analyses are reported in the corresponding figure legends.

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Supplemental figures

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Figure S1. Tumor control and neutrophil abundance in MC38 tumors treated with aPD-1; neutrophil abundance in KP tumors treated with
different chemotherapy combinations or aPD-1/aCTLA4, related to Figure 1
(A) MC38 tumor volume of aPD-1-treated and untreated mice on day 14 of tumor growth, 7 days after treatment (n = 14 untreated, n = 12 aPD-1-treated).
(B) Flow cytometry-based quantification of neutrophils in MC38 tumors, 2 days after aPD-1 treatment (n = 11 untreated, n = 15 aPD-1-treated, data pooled from
two independent experiments).
(C) Immunohistochemistry-based quantification of neutrophils in KP tumors, 3 days after the last of 3 weekly treatments with oxa/cyc or pac/carbo (n = 5–21 fields
of view per group from 2 to 3 mice per group). pac/carbo, paclitaxel/carboplatin; oxa/cyc, oxaliplatin/cyclophosphamide.
(D) Representative microscopic images of Ly-6G+ cells (arrowheads) in KP lung tumors from untreated, oxa/cyc-treated or pac/carbo-treated mice.
(E) Left: representative immunofluorescence images of KP tumor-bearing lungs following different treatments. Right: abundance of neutrophils in KP tumor-
bearing lungs quantified by histology (n = 22–59 fields of view per group, from 2 to 3 mice per group). pac/carbo, paclitaxel/carboplatin; oxa/cyc, oxaliplatin/
cyclophosphamide.
Bar graphs show mean ± SEM. In (A) and (B), Student’s two-tailed t test; in (C) and (E): one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05;
**p < 0.01; ***p < 0.001; ****p < 0.0001.
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Figure S2. Relation between neutrophil states identified in Zilionis et al. and this study; discriminating mRNA markers and surface protein
profile of neutrophil states in KP tumors, related to Figure 2
(A) Table showing relation between the neutrophil states identified in the current study and in Zilionis et al.24
(B) mRNA expression of key markers used for high-resolution partitioning of neutrophil states into lower-level states.
(C) Heatmap showing protein expression of markers by neutrophil states in lungs of untreated KP tumor-bearing mice.
(D) Heatmap showing protein expression of markers by neutrophil states in lungs of aCD40-treated KP tumor-bearing mice.
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Figure S3. Top enriched genes across neutrophil states in KP tumors; expression profile of CD62L and SiglecF, expression of CD14 in KP
tumor neutrophils before and after aCD40 therapy, related to Figure 3
(A) Heatmap summarizing relative expression of top enriched genes across neutrophil states in tumor-free, KP tumor-bearing, and aCD40-treated KP tumor-
bearing lungs.
(B) Heatmap showing Pearson correlation between the average expression of top enriched genes among neutrophil states in untreated (left) and aCD40-treated
(right) KP tumor-bearing lungs.
(C) Top: representative flow cytometry plots showing CD62L and SiglecF expression on neutrophils infiltrating KP tumor-bearing lungs. Bottom: change in
abundance of distinct neutrophil subsets in KP tumor-bearing lungs upon aCD40 treatment compared with untreated controls (n = 5 per group). Graph shows
mean and SEM. Student’s two-tailed t test. *p < 0.05; ****p < 0.0001.
(D) Flow cytometry histogram and quantification showing the change in expression of CD14 on different neutrophil subsets in KP tumors before and after aCD40
treatment (n = 5 per group). MFI, median fluorescence intensity. Graph shows mean and SEM. Student’s two-tailed t test. *p < 0.05; ****p < 0.0001.
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Figure S4. Proportion of CXCL10+ cells and relation between CD14 and CXCL10 expression within SiglecFlo neutrophils after aCD40 therapy,
related to Figure 4
(A) Proportion of SiglecFlo neutrophils expressing CXCL10-BFP in KP tumor-bearing lungs 2 days after aCD40 treatment, determined by flow cytometry.
(B) Representative flow cytometry plot showing expression of CD14 and CXCL10-BFP in SiglecFlo neutrophils in KP tumor-bearing lungs 2 days after aCD40
treatment.
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Figure S5. Detailed graphs of RNA velocity vectors; maturation status of neutrophil subsets defined by CD62L/SiglecF; histological analysis
of MPOhi tumor neutrophils; effect of different stimuli on neutrophil CD14/CD101 expression, related to Figure 5
(A) RNA velocity vector field grid overlaid on UMAP embeddings for tumor-free + KP tumor-bearing (left) and aCD40-treated mice (right).
(B) Representative histograms of CD101, CD11b, and Ly6G expression on distinct neutrophil subsets assessed by flow cytometry in aCD40-treated KP tumors.
(C) Left: representative immunofluorescence image of KP tumor-bearing lung 2 days following aCD40-treatment. Representative examples of MPOhiLy6Glo and
MPOhiLy6Ghi neutrophils are shown in higher magnification. Right: abundance of MPOhiLy6Glo and MPOhiLy6Ghi neutrophils in tumors quantified by histology (n =
8–10 regions of interest per group, from 4 to 5 animals per group). Student’s two-tailed t test. **p < 0.01.
(D) Flow cytometry histograms showing the changes in CD14 and CD101 expression that occur after treatment with LPS, IFNb, IFNg, or poly(IC) (n = 2–3 un-
treated, n = 3–4 aCD40-treated).
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Figure S6. In vitro functional assays with therapy-elicited neutrophils; results from Ly6G-mediated neutrophil depletion; Irf1 expression in
tumor neutrophils; tumor CD8+ T cell and cDC1 abundance in mixed bone marrow chimeras; cell types expressing IL-12, IFNg, and Cxcr3 in
tumors, related to Figure 6
(A) Quantification of MC38-GFP+ tumor cells following 24-h co-culture with neutrophils from aCD40-treated mice or splenocytes from untreated mice (n = 13
fields of view per condition). Graph shows mean and SEM. One-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; ****p < 0.0001.
(B) Quantification of IL-12-YFP+ dendritic cells (DCs) following 24-h co-culture with neutrophils from aCD40-treated mice or treatment with TLR7/8 agonist ligand
(R848) (n = 6–12 fields of view per condition). Graph shows mean and SEM. One-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05; ***p < 0.001.
(C) Flow cytometry plot of MC38 tumor showing the gate used for identifying neutrophils. CD11b+Ly6G+ cells are highlighted in red.
(D) Frequency of neutrophils (gated as shown in C) within live cells in MC38 tumors, in aCD40-treated mice with or without aLy6G treatment, normalized to the
aCD40-treated group (n = 6–7 per group). Graph shows mean + SEM.
(E) Median fluorescence intensity of surface markers on circulating neutrophils in aCD40-treated mice with or without aLy6G treatment, normalized to the aCD40-
treated group (n = 6–7 per group). Graphs show mean and SEM. Student’s two-tailed t test. ***p < 0.001; ****p < 0.0001.
(F) Irf1 expression by neutrophil states in untreated KP tumor-bearing and aCD40-treated KP tumor-bearing tissues.
(G) Quantification of CD8+ cells in MC38 tumor sections (n = 3–4 tumors per group). The fraction of CD8+ cells within all DAPI+ cells was determined in cross-
sections of entire tumors using an automated cell classifier in QuPath. Graph shows mean and SEM. One-way ANOVA with Tukey’s multiple comparisons test.
(H) Quantification type 1 conventional dendritic cells (cDC1s; CD45+F4/80
CD11c+MHCII+CD172
) in MC38 tumors by flow cytometry (n = 4–5 tumors per
group). Graph shows mean and SEM. One-way ANOVA with Tukey’s multiple comparisons test.
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(I) Proportions of F4/80
CD11c+MHCII+ dendritic cells (DCs), F4/80+ macrophages and other cells within IL-12-YFP+ cells in MC38 tumors 2 days after aCD40
treatment, assessed by flow cytometry (n = 7). Graph shows mean and SEM.
(J) Proportions of CD4+ T cells, CD8+ T cells, NK1.1+ NK cells and other cells within IFNg-YFP+ cells in MC38 tumors 2 days after aCD40 treatment, assessed by
flow cytometry (n = 4). Graph shows mean and SEM.
(K) Cxcr3 expression based on scRNA-seq in major immune cell types in MC38 tumors treated with aCD40 (n = 2).