A Phase I Study of Donor Regulatory T Cells As Treatment for Steroid Dependent/Refractory Chronic Graft Versus Host Disease
Blood, Dec 2, 2016
Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant ... more Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant morbidity and mortality, limiting quality of life and survival in the allogeneic transplant survivor. CD4+CD25+CD127dimFoxP3+ regulatory T cells (Treg) are a naturally occurring cell population isolatable in the peripheral blood (PB) with key immune regulatory function. Treg have been shown to protect against GVHD in a number of murine models and in patients. Further, clinical correlations have been observed between the presence of CGVHD and a decreased frequency of Treg post allogeneic hematopoietic cell transplant (HCT) as well as the use of low dose IL-2 resulting in clinical responses with an associated increase in Treg. Based on these observations we embarked on a phase I safety and tolerability trial of the treatment of steroid-refractory or dependent CGVHD with infusion of donor Treg in matched related donor recipients. Methods: Eligible patients were ³ 18 years of age with steroid refractory or dependent CGVHD with a matched related HCT donor. All patients were on systemic immunosuppression at the time of enrollment. Donor derived Treg were enriched from a non-mobilized PB apheresis product over 2 days of apheresis. The fresh apheresis product underwent enrichment by CD25+ immunomagnetic selection on the CliniMACS¨ Cell Selection System (Miltenyi Biotec) and Treg were purified by high-speed flow cytometry and gated on the CD4+CD127dim population. The functional activity of the Treg product was assessed via a mixed lymphocyte reaction (MLR). The trial dosing was through a 3+3 dose escalation design with three Treg dose cohorts: 1 x 10E5 Treg/kg, 5 x 10E5 Treg cells/kg and 1.5 x 10E6 Treg cells/kg. Results: Ten patients were enrolled and received donor Treg cell infusions. Isolation of highly purified Treg was successful from all donors. A median of 1.9x10E8 (range 0.9-2.78x10E8) Treg were collected with a purity that ranged from 87.1-99.3% FoxP3+ (Table 1). Function of the Treg enriched apheresis product confirmed Treg suppressive activity with a median suppression of 69.1% (48.7-77.2%) at a ratio of responder cells:CD4+/CD8+:Treg cells of 1:2:1, with sustained suppression to a ratio of 1:2:0.1. All 10 pateints were treated with three patients treated on all three dose cohorts and an additional patient was treated on the third cohort due to the planned cell dose being not reached with the third patient-donor pair. There were no adverse toxicities with the Treg infusion and no incidence of acute GVHD or acute exacerbation of CGVHD. The ratio of Treg/CD4+ cells in the patients' PB ranged from 2-4.4% at the time of the infusion and remained stable <3 months post infusion. Six of 10 patients have had stable to improved CGVHD with one of these patients having had a flare of CGVHD in the setting of a lapse in immunosuppressive medication and response with reinstitution of immunosuppression. Four patients have had unresponsive or progressive disease requiring the addition of new immunosuppressive agents <2 months post Treg cell infusion. All enrolled patients remain alive at a follow up of >150 days post Treg cell infusion. Summary: This study is the first clinical experience utilizing highly purified donor derived Treg isolated through clinical scale high speed cell sorting. Donor Treg cell therapy for the treatment of steroid refractory or dependent CGVHD at doses of 1.5x10E5-1.5x10E6 Treg/kg were administered without toxicity including absence of AGVHD or exacerbation of CGVHD. The Treg dose of 1.5 x 10E6/kg of recipient weight may be the maximum feasible dose attainable without ex vivo Treg cell expansion. Treg mediated suppression in the MLR documented the in vitro suppressive capability of the Treg cells with investigations ongoing to characterize the functionality of the Treg cells infused. Although this trial was designed to describe the feasibility, safety and tolerability of Treg cell infusions in patients with active CGVHD, the preliminary outcomes of clinical responses and survival are encouraging and comparable to other therapies for steroid refractory/dependent CGVHD with only a single infusion of cells. Further strategies such as multiple infusions or the addition of low dose IL-2 could be considered to attempt to augment the clinical efficacy of Treg based cellular immunotherapy. Disclosures Meyer: Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties.
High-Throughput VDJ Sequencing Is Superior to Quantitative PCR and Flow Cytometry for the Quantification of Minimal Residual Disease In Chronic Lymphocytic Leukemia After Hematopoietic Cell Transplantation
Blood, Nov 19, 2010
Abstract 1290 Background: The primary cause of poor outcome following allogeneic hematopoietic ce... more Abstract 1290 Background: The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention such as chemotherapy or donor lymphocyte infusion to prevent clinical relapse. Validated methods for MRD assessment include allele-specific oligonucleotide quantitative PCR (ASO Q-PCR) and flow cytometry (FC). ASO Q-PCR affords high-sensitivity, but is laborious, time-intensive, and not widely available due to its dependence on patient-specific primers. FC is widely available, but is expensive and has lower sensitivity. Boyd et al. (Sci Transl Med 2009;1:12ra23) demonstrated the feasibility of high-throughput sequencing (HTS) of the immunoglobulin heavy chain variable region (IgVH) VDJ segment for quantifying CLL MRD using ASO Q-PCR as a reference standard. In the current study, we compare HTS, ASO Q-PCR, and FC for tracking a group of CLL patients at a series of time points following HCT. Methods: We performed HTS using BIOMED-2 (Van Dongen et al., Leukemia 2003;17:2257) framework 1 (FR1) and 2 (FR2) consensus VH primers with a consensus JH primer. PCR products were sequenced on a 454 pyrosequencer. We tested the sensitivity of both primer sets by creating serial dilutions of a FACS-sorted CLL population into a donor leukapheresis product with known B cell composition such that the CLL cells were as low as 10e-5cells. For our comparisons of HTS, ASO Q-PCR, and FC, we tested a total of 29 samples from 6 CLL patients at up to 10 different time points following HCT. Results: Although all 6 patients were found to have unmutated IgVH using conventional analysis, we discovered single amino acid sequence variants in up to 17% of CLL-related sequences using HTS. We verified that these changes were not the result of technical artifact by detecting the same oligoclonal species at several time points. For our analyses, we thus defined the “CLL clone” as the dominant sequence plus those sequences of the same CDR3 length that differed by up to one amino acid. In a normal donor, HTS with FR1 and FR2 primers respectively amplified 93.7% and 91.7% of expected V-J combinations (R2 = 0.92), indicating broad repertoire coverage. When purified CLL cells were serially diluted to 10e-5 in a normal donor population, we detected the CLL clone with both primer sets when we sequenced the IgVH VDJ with 150,000 reads, confirming 10e-5 sensitivity. ASO Q-PCR demonstrated the same level of sensitivity in this dilution series. In order to compare FC to the DNA-based techniques, we transformed FC data to represent it as CLL cells per microgram of total genomic DNA per cell. FC under-represented MRD levels by up to 80% in comparison to HTS and ASO Q-PCR. The PCR-based techniques were in accord when residual disease was low (R2 = 0.88). At high disease burdens, the dynamic range of HTS quantification is limited by the number of sequence reads dedicated to those specimens; however, HTS data permits analysis of CLL clones per total VDJ sequences which appears to be useful for clinical interpretation in such scenarios. Finally, HTS revealed VDJ repertoire reconstitution and hypermutation kinetics. In 3 patients who received post-HCT Rituximab (RTX) for GVHD prophylaxis, 6.0 +/− 0.6% of sequences were hypermutated at day +365 versus 17.8 +/− 3.0% in those who did not receive RTX (p = 0.005), confirming expected delays in immune reconstitution following post-HCT RTX therapy. Conclusions: HTS exhibits 10e-5 sensitivity for CLL MRD assessment, which is equivalent to ASO Q-PCR and superior to FC. Further validation using material from 35 CLL patients previously evaluated by ASO Q-PCR is underway. We anticipate HTS will broaden the availability of MRD assessment by providing high sensitivity using generally applicable consensus primers. HTS also has the benefits of detecting somatic mutations and revealing rich data regarding VDJ repertoire reconstitution. Furthermore, HTS of B and T cell immunoreceptor genes using consensus primer sets has the potential to provide MRD assessment of all lymphoid malignancies following HCT. Disclosures: No relevant conflicts of interest to declare.
H-Y antigens are a group of minor histocompatibility antigens encoded on the Y-chromosome with ho... more H-Y antigens are a group of minor histocompatibility antigens encoded on the Y-chromosome with homologous H-X antigens on the X-chromosome. The disparate regions of the H-Y antigens are highly immunogenic and play an important role in understanding human alloimmunity. In this review, we investigate the history of H-Y antigen discovery along with their critical contributions in transplantation and pregnancy. In hematopoietic cell transplantation, male recipients with female donors who become seropositive for B-cell responses as H-Y antibodies following transplantation have increased rates of chronic graft-versus-host disease and decreased rates of relapse. Conversely, female patients who receive male kidney allografts are more likely than other gender combinations to develop H-Y antibodies and reject their allografts. Finally, in the setting of pregnancy, mothers who initially gave birth to boys are more likely to have subsequent pregnancy complications, including miscarriages, in association with H-Y antibody development. H-Y antigens continue to serve as a model for alloimmunity in new clinical scenarios. Our development of more sensitive antibody detection and next-generation DNA sequencing promises to further advance our understanding and better predict the clinical consequences of alloimmunity.
Rituximab Infusion Two Months after Nonmyeloablative Transplantation Maintains B-Cell Disease Control with Minimal GVHD
Blood, Nov 16, 2006
B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of... more B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of Ab production against minor histocompatibility Ags with chronic GVHD, and rituximab efficacy in chronic GVHD. We hypothesize that anti-B cell therapy beginning 56 days after nonmyeloablative total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) transplantation reduces chronic GVHD. Cognizant of possible GVL loss with B cell depletion, we treated patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) because any loss of GVL may be offset by rituximab’s direct anti-B cell tumor efficacy. Patients were conditioned with TLI (80 cGy in 10 fractions, d-11 to d-1) and ATG (1.5mg/kg/day, d-11 to d-7, total 7.5mg/kg). PBSC were infused on day 0. Primary GVHD prophylaxis was MMF from day 0 until d28 for MRD, d100 for URD and cyclosporine (CSA) tapered off by 6 months. To diminish donor B cell alloimmunity, rituximab (375 mg/m2) was infused on days 56, 63, 70, and 77 post-transplant. Fourteen patients have received HCT, and 11 (median age 57, range 46–65 yrs) have received four rituximab infusions (5 fludarabine-refractory, unmutated immunoglobulin heavy-chain variable region gene (IgVH) CLL; 6 MCL). Median follow-up is 290 days (108–381 days). No infusion related toxicities occurred. Transient leukopenia in all patients and frank neutropenia in one patient was noted as early as 30 days post-rituximab. There was one graft loss at day 50 in a patient with MCL progression. Only 3/11 patients had peripheral blood CD3 donor chimerism >95% before rituximab infusion day 56, but 6/7 patients achieved full donor chimerism…
Phase I Experience with a Bi-Specific CAR Targeting CD19 and CD22 in Adults with B-Cell Malignancies
Blood, Nov 29, 2018
Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymp... more Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymphoblastic leukemia (ALL) and >40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of the CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest cl... more Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye-dilution assay for tracking cell proliferative history to mass cytometry, and uncouple division, time and regulatory protein expression in single naïve human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins that are controlled predominantly by division state or time, and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naïve T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK/ITK inhibitor administered before activation, to direct differentiation toward a T SCM-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Immune Phenotypic Quantification of Axicabtagene Ciloleucel and Brexucabtagene Autoleucel Demonstrates Lymphoma Histology Specific Differences in CAR19 Expansion and Toxicity
Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (... more Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4 + Helios + CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (T Reg) cells. Validation cohort analysis upheld the link between higher CAR T Reg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR T Reg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans.
Higher Rates of Severe Infection and Persistent Cytopenias in Long-Term CAR19 Responders Than after Autologous HCT: A Single Institution Study of 139 Subjects
Biology of Blood and Marrow Transplantation, Feb 1, 2012
units homozygous for the CCR5-delta32 allele will provide an improved probability of finding an a... more units homozygous for the CCR5-delta32 allele will provide an improved probability of finding an appropriately HLA-matched donor for a patient with HIV infection in need of a HCT. Methods: CCR5 genotype analysis is performed on DNA extracted from cord blood using a PCR based assay. Biomathematicians at the NMDP developed estimates regarding the probability of providing an adequately HLA matched cord blood unit. Results: We have tested about 10,000 cryopreserved cord blood units and have identified 81 homozygous CCR5-delta32 units. Testing an additional 25,000 cord blood units from Caucasians is expected to increase the special inventory to about 300 units which is projected to provide for Caucasians an adequately HLA matched cord blood unit with an adequate cell dose for 73.61% of pediatric patients and 27.92% of adults. The initial population for transplantation is patients in need of a HCT for a hematologic malignancy or other indication, and who are also infected with HIV, although some selected patients with AIDS who have no other illness may also ultimately be considered for transplantation. Conclusions: Cord blood transplantation has a unique role in providing for long term control or possible cure of HIV infection. Disclosure: No relevant conflicts of interest to declare.
Prophylactic Rituximab after Reduced Intensity Conditioning Transplantation Results in Low Chronic Gvhd
Blood, Nov 16, 2008
Background: B cells are implicated in the pathophysiology of chronic GVHD. We hypothesize that pr... more Background: B cells are implicated in the pathophysiology of chronic GVHD. We hypothesize that prophylactic anti-B cell therapy delivered two months after reduced intensity conditioning (RIC) transplantation would prevent or reduce chronic GVHD incidence from the historical 50%. Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) were the target diseases as they are B cell malignancies with clear allogeneic GVL benefit. Methods: CLL and MCL patients were conditioned with total lymphoid irradiation (TLI) 80 cGy in 10 fractions, d-11 to d-1 and anti-thymocyte globulin (ATG) 1.5mg/kg/day, d-11 to d-7 (total 7.5mg/kg). PBPC were infused on day 0. Primary GVHD prophylaxis was cyclosporine (CSA) on d-3 with taper by 6 months, and MMF from day 0 until d28 for related donors, d100 for unrelated donors. Rituximab (375 mg/m2/week ×4) was infused on days 56, 63, 70, and 77 post-transplant. Results: 36 patients accrued to the study (median age 57, range 31–66 yrs), with 34 patients completing the 4 rituximab infusions. All 22 CLL patients were high risk (fludarabinerefractory, unmutated VH-IgG, or P53 deletion). The 14 MCL patients included 4 patients in PR and 10 patients in CR status at transplant. Median follow-up is 20 months. Twenty patients had sibling donors; 16, unrelated donors. Median CD34 cell dose was 7.5 CD34/kg. All patients had donor cell engraftment except for one patient who had graft failure with stable autologous recovery. Full donor chimerism (PB CD3>95%) was achieved in 14 out of 31 patients (45%) by day 90. However, all but 5 patients had achieved full donor chimerism at 1 year. The incidence of grade 2–4 acute GVHD was 6%. The incidence of chronic GVHD was 18%. Day 100 NRM was 0% and 1-year NRM was 3%. Ten relapses have occurred (5 CLL, 5 MCL). Estimated FFP and OS at 2 years for CLL patients is 82%(CI +/−16%) and 73% (CI +/−33%), respectively; for MCL patients, 64% and 68%, respectively Full donor chimerism was associated with persistent disease remission. Twelve of 19 VHIg mutated CLL patients have achieved minimal residual disease (MRD) by quantitative allele-specific oligonucleotide-IgH PCR (ASO-Q-PCR). DLI was given to 5 CLL patients and 1 MCL who had relapsed and had not achieved full donor chimerism.. There were no infusional toxicities with rituximab. Transient rituximab related neutropenia occurred in 10 patients d100–150. Post-transplant infections included influenza B, RSV, fungal sinusitis, pseudomonas, klebsiella infection, VZV reactivation, and one PTLD before day 56 rituximab. All recovered Of 22 patients at risk for CMV reactivation, 10 reactivated ( range 4 to 56 days post-HCT). Therefore, rituximab did not contribute to the CMV reactivation or increase infection incidence. Conclusion: Prophylactic rituximab infusion post RIC transplantation is well tolerated, provides safe donor B cell depletion without detrimental effect on engraftment or infection incidence, and is associated with a low incidence of chronic GVHD while maintaining GVL. A randomized trial of rituximab prophylaxis after allogeneic HCT is warranted.
Characterization of immunoglobulin E plasma cells that are elevated in the upper airway mucosa of nonatopic patients with chronic rhinosinusitis without nasal polyps
International Forum of Allergy & Rhinology, Feb 16, 2016
The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhi... more The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhinosinusitis (CRS) are poorly understood. Previous studies have shown that B cells and immunoglobulin E (IgE) levels are elevated in the nasal tissue of patients with atopic chronic rhinosinusitis without nasal polyps (CRSsNP). However, less is known regarding B cell subsets and IgE-producing plasmablasts in nonatopic CRSsNP patients. Human blood and ethmoid sinus mucosa samples were analyzed from control (n = 6) and nonatopic CRSsNP (n = 11) patients. Tissue samples were evaluated using high-dimensional flow cytometry. A population of IgE antibody secreting cells is significantly increased in situ within inflamed nasal tissue of nonatopic CRSsNP subjects when compared to control nasal tissue and the circulating peripheral blood (p < 0.05). This IgE plasma cell population displays ∼90% cell surface Ig lambda light chain, is mitotically active (Ki-67(+) ), and displays intracellular IgE expression. The predominant B cell population expressing IgE are plasmablasts (CD38(high) , CD138(-) ) not typically found in the blood or peripheral tissue of these patients. The nasal mucosa from nonatopic CRSsNP patients demonstrate a significant regional spike in resident in situ IgE plasmablast cells not seen in control nasal tissue or peripheral blood from the same patient. The restricted expression of Ig lambda light chain in this mitotically active IgE plasmablast population supports the hypothesis of aberrant B cell proliferation in the context of CRS. These findings suggest the presence of a unique regional immune microenvironment for B cell priming and/or selection within chronically inflamed airway tissues.
A Phase I Study of Donor Regulatory T Cells As Treatment for Steroid Dependent/Refractory Chronic Graft Versus Host Disease
Blood, Dec 2, 2016
Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant ... more Background: Chronic graft versus host disease (CGVHD) remains a notable cause of post transplant morbidity and mortality, limiting quality of life and survival in the allogeneic transplant survivor. CD4+CD25+CD127dimFoxP3+ regulatory T cells (Treg) are a naturally occurring cell population isolatable in the peripheral blood (PB) with key immune regulatory function. Treg have been shown to protect against GVHD in a number of murine models and in patients. Further, clinical correlations have been observed between the presence of CGVHD and a decreased frequency of Treg post allogeneic hematopoietic cell transplant (HCT) as well as the use of low dose IL-2 resulting in clinical responses with an associated increase in Treg. Based on these observations we embarked on a phase I safety and tolerability trial of the treatment of steroid-refractory or dependent CGVHD with infusion of donor Treg in matched related donor recipients. Methods: Eligible patients were ³ 18 years of age with steroid refractory or dependent CGVHD with a matched related HCT donor. All patients were on systemic immunosuppression at the time of enrollment. Donor derived Treg were enriched from a non-mobilized PB apheresis product over 2 days of apheresis. The fresh apheresis product underwent enrichment by CD25+ immunomagnetic selection on the CliniMACS¨ Cell Selection System (Miltenyi Biotec) and Treg were purified by high-speed flow cytometry and gated on the CD4+CD127dim population. The functional activity of the Treg product was assessed via a mixed lymphocyte reaction (MLR). The trial dosing was through a 3+3 dose escalation design with three Treg dose cohorts: 1 x 10E5 Treg/kg, 5 x 10E5 Treg cells/kg and 1.5 x 10E6 Treg cells/kg. Results: Ten patients were enrolled and received donor Treg cell infusions. Isolation of highly purified Treg was successful from all donors. A median of 1.9x10E8 (range 0.9-2.78x10E8) Treg were collected with a purity that ranged from 87.1-99.3% FoxP3+ (Table 1). Function of the Treg enriched apheresis product confirmed Treg suppressive activity with a median suppression of 69.1% (48.7-77.2%) at a ratio of responder cells:CD4+/CD8+:Treg cells of 1:2:1, with sustained suppression to a ratio of 1:2:0.1. All 10 pateints were treated with three patients treated on all three dose cohorts and an additional patient was treated on the third cohort due to the planned cell dose being not reached with the third patient-donor pair. There were no adverse toxicities with the Treg infusion and no incidence of acute GVHD or acute exacerbation of CGVHD. The ratio of Treg/CD4+ cells in the patients' PB ranged from 2-4.4% at the time of the infusion and remained stable <3 months post infusion. Six of 10 patients have had stable to improved CGVHD with one of these patients having had a flare of CGVHD in the setting of a lapse in immunosuppressive medication and response with reinstitution of immunosuppression. Four patients have had unresponsive or progressive disease requiring the addition of new immunosuppressive agents <2 months post Treg cell infusion. All enrolled patients remain alive at a follow up of >150 days post Treg cell infusion. Summary: This study is the first clinical experience utilizing highly purified donor derived Treg isolated through clinical scale high speed cell sorting. Donor Treg cell therapy for the treatment of steroid refractory or dependent CGVHD at doses of 1.5x10E5-1.5x10E6 Treg/kg were administered without toxicity including absence of AGVHD or exacerbation of CGVHD. The Treg dose of 1.5 x 10E6/kg of recipient weight may be the maximum feasible dose attainable without ex vivo Treg cell expansion. Treg mediated suppression in the MLR documented the in vitro suppressive capability of the Treg cells with investigations ongoing to characterize the functionality of the Treg cells infused. Although this trial was designed to describe the feasibility, safety and tolerability of Treg cell infusions in patients with active CGVHD, the preliminary outcomes of clinical responses and survival are encouraging and comparable to other therapies for steroid refractory/dependent CGVHD with only a single infusion of cells. Further strategies such as multiple infusions or the addition of low dose IL-2 could be considered to attempt to augment the clinical efficacy of Treg based cellular immunotherapy. Disclosures Meyer: Stanford University: Patents & Royalties. Negrin:Stanford University: Patents & Royalties.
High-Throughput VDJ Sequencing Is Superior to Quantitative PCR and Flow Cytometry for the Quantification of Minimal Residual Disease In Chronic Lymphocytic Leukemia After Hematopoietic Cell Transplantation
Blood, Nov 19, 2010
Abstract 1290 Background: The primary cause of poor outcome following allogeneic hematopoietic ce... more Abstract 1290 Background: The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention such as chemotherapy or donor lymphocyte infusion to prevent clinical relapse. Validated methods for MRD assessment include allele-specific oligonucleotide quantitative PCR (ASO Q-PCR) and flow cytometry (FC). ASO Q-PCR affords high-sensitivity, but is laborious, time-intensive, and not widely available due to its dependence on patient-specific primers. FC is widely available, but is expensive and has lower sensitivity. Boyd et al. (Sci Transl Med 2009;1:12ra23) demonstrated the feasibility of high-throughput sequencing (HTS) of the immunoglobulin heavy chain variable region (IgVH) VDJ segment for quantifying CLL MRD using ASO Q-PCR as a reference standard. In the current study, we compare HTS, ASO Q-PCR, and FC for tracking a group of CLL patients at a series of time points following HCT. Methods: We performed HTS using BIOMED-2 (Van Dongen et al., Leukemia 2003;17:2257) framework 1 (FR1) and 2 (FR2) consensus VH primers with a consensus JH primer. PCR products were sequenced on a 454 pyrosequencer. We tested the sensitivity of both primer sets by creating serial dilutions of a FACS-sorted CLL population into a donor leukapheresis product with known B cell composition such that the CLL cells were as low as 10e-5cells. For our comparisons of HTS, ASO Q-PCR, and FC, we tested a total of 29 samples from 6 CLL patients at up to 10 different time points following HCT. Results: Although all 6 patients were found to have unmutated IgVH using conventional analysis, we discovered single amino acid sequence variants in up to 17% of CLL-related sequences using HTS. We verified that these changes were not the result of technical artifact by detecting the same oligoclonal species at several time points. For our analyses, we thus defined the “CLL clone” as the dominant sequence plus those sequences of the same CDR3 length that differed by up to one amino acid. In a normal donor, HTS with FR1 and FR2 primers respectively amplified 93.7% and 91.7% of expected V-J combinations (R2 = 0.92), indicating broad repertoire coverage. When purified CLL cells were serially diluted to 10e-5 in a normal donor population, we detected the CLL clone with both primer sets when we sequenced the IgVH VDJ with 150,000 reads, confirming 10e-5 sensitivity. ASO Q-PCR demonstrated the same level of sensitivity in this dilution series. In order to compare FC to the DNA-based techniques, we transformed FC data to represent it as CLL cells per microgram of total genomic DNA per cell. FC under-represented MRD levels by up to 80% in comparison to HTS and ASO Q-PCR. The PCR-based techniques were in accord when residual disease was low (R2 = 0.88). At high disease burdens, the dynamic range of HTS quantification is limited by the number of sequence reads dedicated to those specimens; however, HTS data permits analysis of CLL clones per total VDJ sequences which appears to be useful for clinical interpretation in such scenarios. Finally, HTS revealed VDJ repertoire reconstitution and hypermutation kinetics. In 3 patients who received post-HCT Rituximab (RTX) for GVHD prophylaxis, 6.0 +/− 0.6% of sequences were hypermutated at day +365 versus 17.8 +/− 3.0% in those who did not receive RTX (p = 0.005), confirming expected delays in immune reconstitution following post-HCT RTX therapy. Conclusions: HTS exhibits 10e-5 sensitivity for CLL MRD assessment, which is equivalent to ASO Q-PCR and superior to FC. Further validation using material from 35 CLL patients previously evaluated by ASO Q-PCR is underway. We anticipate HTS will broaden the availability of MRD assessment by providing high sensitivity using generally applicable consensus primers. HTS also has the benefits of detecting somatic mutations and revealing rich data regarding VDJ repertoire reconstitution. Furthermore, HTS of B and T cell immunoreceptor genes using consensus primer sets has the potential to provide MRD assessment of all lymphoid malignancies following HCT. Disclosures: No relevant conflicts of interest to declare.
H-Y antigens are a group of minor histocompatibility antigens encoded on the Y-chromosome with ho... more H-Y antigens are a group of minor histocompatibility antigens encoded on the Y-chromosome with homologous H-X antigens on the X-chromosome. The disparate regions of the H-Y antigens are highly immunogenic and play an important role in understanding human alloimmunity. In this review, we investigate the history of H-Y antigen discovery along with their critical contributions in transplantation and pregnancy. In hematopoietic cell transplantation, male recipients with female donors who become seropositive for B-cell responses as H-Y antibodies following transplantation have increased rates of chronic graft-versus-host disease and decreased rates of relapse. Conversely, female patients who receive male kidney allografts are more likely than other gender combinations to develop H-Y antibodies and reject their allografts. Finally, in the setting of pregnancy, mothers who initially gave birth to boys are more likely to have subsequent pregnancy complications, including miscarriages, in association with H-Y antibody development. H-Y antigens continue to serve as a model for alloimmunity in new clinical scenarios. Our development of more sensitive antibody detection and next-generation DNA sequencing promises to further advance our understanding and better predict the clinical consequences of alloimmunity.
Rituximab Infusion Two Months after Nonmyeloablative Transplantation Maintains B-Cell Disease Control with Minimal GVHD
Blood, Nov 16, 2006
B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of... more B cells are implicated in the pathophysiology of chronic GVHD, as evidenced by the association of Ab production against minor histocompatibility Ags with chronic GVHD, and rituximab efficacy in chronic GVHD. We hypothesize that anti-B cell therapy beginning 56 days after nonmyeloablative total lymphoid irradiation (TLI) and anti-thymocyte globulin (ATG) transplantation reduces chronic GVHD. Cognizant of possible GVL loss with B cell depletion, we treated patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) because any loss of GVL may be offset by rituximab’s direct anti-B cell tumor efficacy. Patients were conditioned with TLI (80 cGy in 10 fractions, d-11 to d-1) and ATG (1.5mg/kg/day, d-11 to d-7, total 7.5mg/kg). PBSC were infused on day 0. Primary GVHD prophylaxis was MMF from day 0 until d28 for MRD, d100 for URD and cyclosporine (CSA) tapered off by 6 months. To diminish donor B cell alloimmunity, rituximab (375 mg/m2) was infused on days 56, 63, 70, and 77 post-transplant. Fourteen patients have received HCT, and 11 (median age 57, range 46–65 yrs) have received four rituximab infusions (5 fludarabine-refractory, unmutated immunoglobulin heavy-chain variable region gene (IgVH) CLL; 6 MCL). Median follow-up is 290 days (108–381 days). No infusion related toxicities occurred. Transient leukopenia in all patients and frank neutropenia in one patient was noted as early as 30 days post-rituximab. There was one graft loss at day 50 in a patient with MCL progression. Only 3/11 patients had peripheral blood CD3 donor chimerism >95% before rituximab infusion day 56, but 6/7 patients achieved full donor chimerism…
Phase I Experience with a Bi-Specific CAR Targeting CD19 and CD22 in Adults with B-Cell Malignancies
Blood, Nov 29, 2018
Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymp... more Autologous CD19 directed CAR T-cell therapy has response rates of >70% in adult acute lymphoblastic leukemia (ALL) and >40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of the CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest cl... more Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye-dilution assay for tracking cell proliferative history to mass cytometry, and uncouple division, time and regulatory protein expression in single naïve human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins that are controlled predominantly by division state or time, and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naïve T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK/ITK inhibitor administered before activation, to direct differentiation toward a T SCM-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Immune Phenotypic Quantification of Axicabtagene Ciloleucel and Brexucabtagene Autoleucel Demonstrates Lymphoma Histology Specific Differences in CAR19 Expansion and Toxicity
Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (... more Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4 + Helios + CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (T Reg) cells. Validation cohort analysis upheld the link between higher CAR T Reg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR T Reg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans.
Higher Rates of Severe Infection and Persistent Cytopenias in Long-Term CAR19 Responders Than after Autologous HCT: A Single Institution Study of 139 Subjects
Biology of Blood and Marrow Transplantation, Feb 1, 2012
units homozygous for the CCR5-delta32 allele will provide an improved probability of finding an a... more units homozygous for the CCR5-delta32 allele will provide an improved probability of finding an appropriately HLA-matched donor for a patient with HIV infection in need of a HCT. Methods: CCR5 genotype analysis is performed on DNA extracted from cord blood using a PCR based assay. Biomathematicians at the NMDP developed estimates regarding the probability of providing an adequately HLA matched cord blood unit. Results: We have tested about 10,000 cryopreserved cord blood units and have identified 81 homozygous CCR5-delta32 units. Testing an additional 25,000 cord blood units from Caucasians is expected to increase the special inventory to about 300 units which is projected to provide for Caucasians an adequately HLA matched cord blood unit with an adequate cell dose for 73.61% of pediatric patients and 27.92% of adults. The initial population for transplantation is patients in need of a HCT for a hematologic malignancy or other indication, and who are also infected with HIV, although some selected patients with AIDS who have no other illness may also ultimately be considered for transplantation. Conclusions: Cord blood transplantation has a unique role in providing for long term control or possible cure of HIV infection. Disclosure: No relevant conflicts of interest to declare.
Prophylactic Rituximab after Reduced Intensity Conditioning Transplantation Results in Low Chronic Gvhd
Blood, Nov 16, 2008
Background: B cells are implicated in the pathophysiology of chronic GVHD. We hypothesize that pr... more Background: B cells are implicated in the pathophysiology of chronic GVHD. We hypothesize that prophylactic anti-B cell therapy delivered two months after reduced intensity conditioning (RIC) transplantation would prevent or reduce chronic GVHD incidence from the historical 50%. Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) were the target diseases as they are B cell malignancies with clear allogeneic GVL benefit. Methods: CLL and MCL patients were conditioned with total lymphoid irradiation (TLI) 80 cGy in 10 fractions, d-11 to d-1 and anti-thymocyte globulin (ATG) 1.5mg/kg/day, d-11 to d-7 (total 7.5mg/kg). PBPC were infused on day 0. Primary GVHD prophylaxis was cyclosporine (CSA) on d-3 with taper by 6 months, and MMF from day 0 until d28 for related donors, d100 for unrelated donors. Rituximab (375 mg/m2/week ×4) was infused on days 56, 63, 70, and 77 post-transplant. Results: 36 patients accrued to the study (median age 57, range 31–66 yrs), with 34 patients completing the 4 rituximab infusions. All 22 CLL patients were high risk (fludarabinerefractory, unmutated VH-IgG, or P53 deletion). The 14 MCL patients included 4 patients in PR and 10 patients in CR status at transplant. Median follow-up is 20 months. Twenty patients had sibling donors; 16, unrelated donors. Median CD34 cell dose was 7.5 CD34/kg. All patients had donor cell engraftment except for one patient who had graft failure with stable autologous recovery. Full donor chimerism (PB CD3>95%) was achieved in 14 out of 31 patients (45%) by day 90. However, all but 5 patients had achieved full donor chimerism at 1 year. The incidence of grade 2–4 acute GVHD was 6%. The incidence of chronic GVHD was 18%. Day 100 NRM was 0% and 1-year NRM was 3%. Ten relapses have occurred (5 CLL, 5 MCL). Estimated FFP and OS at 2 years for CLL patients is 82%(CI +/−16%) and 73% (CI +/−33%), respectively; for MCL patients, 64% and 68%, respectively Full donor chimerism was associated with persistent disease remission. Twelve of 19 VHIg mutated CLL patients have achieved minimal residual disease (MRD) by quantitative allele-specific oligonucleotide-IgH PCR (ASO-Q-PCR). DLI was given to 5 CLL patients and 1 MCL who had relapsed and had not achieved full donor chimerism.. There were no infusional toxicities with rituximab. Transient rituximab related neutropenia occurred in 10 patients d100–150. Post-transplant infections included influenza B, RSV, fungal sinusitis, pseudomonas, klebsiella infection, VZV reactivation, and one PTLD before day 56 rituximab. All recovered Of 22 patients at risk for CMV reactivation, 10 reactivated ( range 4 to 56 days post-HCT). Therefore, rituximab did not contribute to the CMV reactivation or increase infection incidence. Conclusion: Prophylactic rituximab infusion post RIC transplantation is well tolerated, provides safe donor B cell depletion without detrimental effect on engraftment or infection incidence, and is associated with a low incidence of chronic GVHD while maintaining GVL. A randomized trial of rituximab prophylaxis after allogeneic HCT is warranted.
Characterization of immunoglobulin E plasma cells that are elevated in the upper airway mucosa of nonatopic patients with chronic rhinosinusitis without nasal polyps
International Forum of Allergy & Rhinology, Feb 16, 2016
The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhi... more The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhinosinusitis (CRS) are poorly understood. Previous studies have shown that B cells and immunoglobulin E (IgE) levels are elevated in the nasal tissue of patients with atopic chronic rhinosinusitis without nasal polyps (CRSsNP). However, less is known regarding B cell subsets and IgE-producing plasmablasts in nonatopic CRSsNP patients. Human blood and ethmoid sinus mucosa samples were analyzed from control (n = 6) and nonatopic CRSsNP (n = 11) patients. Tissue samples were evaluated using high-dimensional flow cytometry. A population of IgE antibody secreting cells is significantly increased in situ within inflamed nasal tissue of nonatopic CRSsNP subjects when compared to control nasal tissue and the circulating peripheral blood (p < 0.05). This IgE plasma cell population displays ∼90% cell surface Ig lambda light chain, is mitotically active (Ki-67(+) ), and displays intracellular IgE expression. The predominant B cell population expressing IgE are plasmablasts (CD38(high) , CD138(-) ) not typically found in the blood or peripheral tissue of these patients. The nasal mucosa from nonatopic CRSsNP patients demonstrate a significant regional spike in resident in situ IgE plasmablast cells not seen in control nasal tissue or peripheral blood from the same patient. The restricted expression of Ig lambda light chain in this mitotically active IgE plasmablast population supports the hypothesis of aberrant B cell proliferation in the context of CRS. These findings suggest the presence of a unique regional immune microenvironment for B cell priming and/or selection within chronically inflamed airway tissues.
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