Naive B Cells Research Articles

The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells.

B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen--activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high -frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions.

Molecular Features of Germinal Cell Derived B-Cell Lymphomas Using miRNA Signatures

Background:B-cell development is a complex biological process that initiates in bone marrow and matures in lymph nodes. These hierarchical stages are tightly regulated by transcription factors and microRNAs (miRNAs). B-cells are prone to genetic alteration during these maturation stages due to DNA breaks required for B-cell receptor re-arrangement and additional changes that occur during the germinal center (GC)-reaction for class switch recombination and somatic hypermutation. The unwarranted genomic changes resulting from these processes can lead to B-cell malignancies with varied clinical and pathological characteristics. Of the lymphoma subtypes derived from mature B-cells, a subset of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large cell lymphoma (PMBL), follicular lymphoma (FL) and Burkitt lymphoma (BL) are putatively derived from GC B-cells, with varied morphological, pathological and clinical characteristics. These tumor are characterized by distinct genetic abnormalities,[ t(14;18) in GCB-DLCBL, and FL or t(8;14)] in BL, and mutational profile. We postulate miRNA expression signatures, may also explain the wide-ranging clinico-pathological characteristics of these GC B- cell derived tumors.Methods:We performed a meta-analysis of miRNA profiles of DLBCL, BL, PBML, FL and EBV+DLBCL performed by us or others. (Iqbal 2015; 2012; Leich 2011; Andrade 2014). We also included lymphomas derived from naïve B-cells (MCL, SLL) and post GC B-cells (activated B cells like ABC-DLBCL), EBV (+) DLBCL, normal B-cell subsets and cell lines of distinct B-cell lineages. The majority of the cases have been characterized by gene expression profiling (GEP) with corresponding pathological clinical characteristics in above studies. The data was obtained using Taqman® human microRNA array (Applied Biosystems, CA) containing 380 miRNAs and analyzed using BRB-array Tools.ResultsIn our initial miRNA expression profile of 249 lymphomas including GCB-DLBCL (n=34), ABC-DLBCL(n= 29), PMBL(n= 9), BL(n= 33), FL (n= 32), EBV+DLBCL (n=8); MCL(n=30) and SLL(n=12)showed distinct hierarchical clusters enriched with GEP defined subgroup, with few interspersed cases from other subgroups suggesting that miRNAs can complement and delineate GEP defined molecular subgroups, an observation consistent with earlier findings (Iqbal 2015).When we examined miRNAs, that are specifically associated with GC B- derived lymphomas, we observed a subset of miRNA including miR-146a, miR-142-3p, miR-17, miR19b, and miR106a were present at higher levels (top 5% in abundance by CT values) in GC B lymphomas. Of these, somatic mutations in miR142 have been recently reported in GC derived lymphomas (Alyssa, Leukemia 2016). These miRNAs also represented the most abundant miRNAs in centroblast, suggesting lineage specificity of miRNA signature in these lymphomas. However, miR222 highly expressed in centroblast was not expressed at higher levels in GCB-DLBCL and BL. A number of miRNAs specifically abundant in distinct lymphomas included (BL: miR-20a, miR-16), (GCB-DLBCL: miR-223, miR-24) and (FL: miR222). MiR-150 was highly expressed in NHLs putatively derived from naive B-cells like MCL or SLL, but absent from GC-derived NHLs. Consistent with this, miR-150 was present in naive B-cells, but not in centroblast, suggesting that miR-150 may have discrete function during early B-cell differentiation and late maturation stages.Within GC derived lymphoma, we examined miRNA profiles in clinically aggressive (BL); indolent lymphomas (FL) and intermediate aggressive (GCB-DLBCL) and their association with normal B-cell subsets, and indeed a number of miRNAs (ex. miR-150) were associated with indolent lymphomas, whereas a subset of miRNAs was present in aggressive lymphomas (ex. miR-16), suggesting these miRNAs may contribute the distinct clinical characteristics. Though we have identified unique miRNA profiles in these GC derived NHLs, the target genes of these miRNAs remains to be identified.Conclusion:We identified microRNAs that were differentially expressed between GC-derived DLBCL and other lymphomas, which may help explain distinguishing features of these lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Expression of the SOX11 Oncogene in Mantle Cell Lymphoma Is Associated with Activation and De Novo 3D Looping of a Distant Enhancer Element

IntroductionSOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2.AimsWe aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions:(i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs?(ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells?(iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues?MethodsWe have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium.ResultsHiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines.By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs.By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases.When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis.ConclusionsWe provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. DisclosuresNo relevant conflicts of interest to declare.

CLL Stereotyped IGHV-D-J Rearrangements Can Be Detected Throughout Normal B-Cell Developmental Stages in Aged People When Using Ultra-Deep, Next Generation Sequencing Techniques

B cells originate in the bone marrow where precursors pass through a series of highly regulated processes to generate a functional B-cell receptor (BCR). IGHV-D-J and IGLV-J rearrangements are achieved via a combinatorial process with associated amino acid additions/deletions at recombination sites leading to an immense variety of structurally diverse BCRs. Due to the large number of possibilities, potentially harmful BCRs recognizing self-antigens can be generated. In the early stages of B cell development a number of distinct selection mechanisms eliminate higher affinity auto-reactive B cells.Chronic lymphocytic leukemia (CLL) is a CD5+ B cell malignancy. Besides having a unique phenotype not always assignable to a defined B-cell subset, many CLL B cells express auto-reactive BCRs; those that do not can often be shown to have derived from self-reactive clones. In addition, certain CLL patients express virtually identical IGHV-D-J rearrangements that can be organized into categories called "stereotyped BCRs". To date, stereotyped BCRs have not been conclusively shown to exist in the normal B-cell repertoire. This might be due to strict autoreactive censoring that eliminates stereotyped BCRs during B-cell ontogeny and/or because the number of IGHV-D-J rearrangements sequenced from individuals has not been sufficient to detect infrequent Igs. Therefore, using ultradeep, next generation IGHV-D-J sequencing we searched for CLL-specific stereotyped BCR rearrangements in the early phases of normal B cell maturation, before all mechanisms of selection are complete.We sorted several B-cell populations from bone marrow samples obtained from healthy patients undergoing hip replacement without an autoimmunity or chronic infection history. The MiSeq platform was used to determine the IGHV-D-J repertoire of pro-, pre-, immature, transitional, naive, and memory B cells, and of plasmablasts. Sequences were analyzed using the pRESTO bioinformatic tool and R. Using VDJ rearrangement and CDR3 length criteria we focused on sequences potentially similar to those in CLL. Sequences were submitted to an online tool (ARReST/Assign subset) to be attributed to a particular CLL stereotyped subset based on a degree of certitude. We identified 156 sequences belonging to defined CLL stereotyped subsets with varying levels of similarity (extreme to low), most residing in early/antigen-independent compartments such as pre-B cells and immature, transitional, and naive B cells.When stereotyped rearrangements of all degrees of certainty were considered, the greatest number of CLL-like stereotypes were found among the most immature B-cell populations, with frequency decreasing with advanced maturation (e.g., 0.04% in pro-B cells vs. 0.028 in naive and 0.01% in memory B cells). This is consistent with the notion that auto-reactive stereotyped rearrangements are eliminated from the normal repertoire. However, when using only those IGHV-D-J rearrangements with extreme or very high certitude, CLL-like stereotyped sequences were found at the highest frequency in the naive B-cell subset (0.02% vs 0.0058% in immature and 0.007 in pre-B cells). In addition, we identified at least one sequence for a majority of the most frequent 18 stereotyped subsets, although the proportional representation of each subset in our data is different from that seen in CLL. Specifically, the highest scoring recurrent subsets in our analysis were subsets 3 (61% of total CLL sequences), 5 (9.5%) and 64B (7%), which are not the most common in the known CLL cohort. Rather, we found only a few sequences belonging to the most common subsets such as subsets 1 and 2 (4.5% and 2% of the total CLL sequences), and none belonging to subset 4, although this subset is always isotype class-switched and IGHV-mutated and hence would not be expected to be found in the developing or naive repertoire.Finding a significant presence of CLL-like stereotyped sequences when using ultra-deep DNA sequencing techniques in early phases of B-cell development and in the naive B-cell fraction suggests that CLL-like B cells are present among normal B cell populations albeit at very low frequencies. Moreover the presence of only certain of the most prevalent stereotypes such as subset 3 and 5, respectively the 5th and 6th biggest subsets, suggests these clones might not exhibit high auto-reactive affinity allowing them to more readily escape mechanisms of selection. DisclosuresStamatopoulos:Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Newly-Generated Regulatory B- and T-Cells Are Associated with Clinical Improvement and Reversal of Dermal Fibrosis in Systemic Sclerosis Patients after Autologous Hematopoietic Stem Cell Transplantation

Background: Autologous Hematopoietic Stem Cell Transplantation (AHSCT) has been shown more effective to treat severe forms of systemic sclerosis (SSc) than conventional immunosuppressive therapy.However, despite advances in clinical management, therapeutic mechanisms of AHSCT still need to be more completely understood. Furthermore, identification of biomarkers of therapeutic response may help to refine current clinical protocols and improve future therapeutic goals.Objective:To investigate immunological mechanisms associated with the therapeutic efficacy of AHSCT.Methods: Thirty-one patients with progressive SSc, refractory to first line therapy, were selected for AHSCT. Follow-up included clinical assessment of general health, skin thickness (modified Rodnan's Skin Score, mRSS), lung function (Forced Vital Capacity, FVC), levels of anti-Scl70 autoantibodies and C-reactive protein (CRP). Dermal fibrosis was evaluated by PicroSirius (PS), hematoxylin/eosin (HE) and Masson's-Trichrome (MT). Sixteen additional SSc patients under standard treatment were enrolled as controls. PBMCs were collected before and every 6 months, until 36 months post-AHSCT. Thymic function was measured by RT-qPCR quantification of β- and signal joint (sj)-T-cell receptor excision circles (sjTREC) and intra-thymic T-cell division (n) was calculated by the formula: n= LOG(sjTREC/βTREC)/LOG2. B cell replication history was quantified by coding-joint (Cj) and sj-kappa-deleting recombination excision circles (sjKREC), and B-cell divisions in the peripheral blood (N) were calculated by the formula: N= LOG(Cj/sjKREC)/LOG2. Telomere length was evaluated by multiplex RT-qPCR. CD19+CD24hiCD38hi Bregs, CD19+CD27-IgD+ naive, CD19+CD38lowIgD+ Bm2, CD3+CD4+CD31+CD45RA+ recent thymic emigrants (RTE), CD8+CD28-CD57+ exhausted and CD4+CD25hiFoxP3+ (GITR+/CTLA-4+) Tregs were quantified by FACS. IL-10-producing Tregs were quantified after 24h stimulation with anti-CD3/CD28 Dynabeads. IL-10-producing Breg counts were assessed after 24h stimulation with CpG and CD40L. Skin biopsies were stained for IL-10 expression.Results: Rodnan's score decreased at 6 months, remaining lower than baseline until 36 months after AHSCT. FVC values stabilized and anti-Scl-70 autoantibody titers and C-reactive protein (CRP) levels decreased after AHSCT. PS, HE and MT skin biopsy staining evidenced decreased collagen deposition. Immune reconstitution analyses evidenced that thymic function was significantly reduced at 6 months post-AHSCT, and higher than baseline at 24 months, never changing intrathymic T-cell division rates and strongly correlating with thymic-derived RTE exportation. Moreover, at 6 months post-AHSCT, increased numbers of exhausted T-cells correlated with reduced telomere T/S ratio, indicating that while thymic function is suppressed, early after AHSCT, the immune reconstitution is based on homeostatic proliferation of residual cells. Regulatory T-cells increased at 12 months post-transplantation, correlating with sjTREC values, with higher CD45RA expression and IL-10 production than at baseline. Bone marrow output of naive B-cells increased from 12 until 36 months post-AHSCT, resulting in reduced B-cell division in the periphery, as shown bypersistent increase of conventional naive as well as of non-conventional Bm2 naive B-cell counts. Finally, regulatory B-cell counts increased transiently from 6 to 12 months after AHSCT, presenting higher IL-10 production than at baseline. Skin biopsies evidenced higher IL-10 expression at 6 and 12 months post-transplantation as compared to baseline. Six transplanted patients presented disease reactivation with subsequent increase in Rodnan's score and/or worsening of lung function after AHSCT. These patients presented lower FoxP3, GITR and CTLA-4 expressions at 12 months post-transplantation. Regulatory B-cell counts after AHSCT were also lower in relapsing than in non-relapsing patients.Conclusions: Our results suggest that increased counts of newly-generated regulatory B- and T-cell after AHSCT are associated with clinical improvement in SSc patients. Clinical remission may be associated with amelioration of the immunoregulatory network and restoration of self-tolerance promoted by the procedure. DisclosuresNo relevant conflicts of interest to declare.

Rapid B-Cell Recovery Is Predictive of Graft-Versus-Host Disease-Free Survival, Few Infectious Events, and Good Quality-of-Life after Cord Blood Transplantation Using Myeloablative Regimen without Antithymocyte Globulin

Background:The severity of complications of transplantation is governed mainly by the status of immune reconstitution. Compared to other stem cell sources, cord blood transplantation (CBT) is characterized by an impairment of early T cell immune reconstitution but rapid B-cell neogenesis. In this study, we investigated the kinetics of early B-cell immune reconstitution and its relation with clinical outcome after CBT using myeloablative regimens without antithymocyte globulin (ATG).Methods:(1) Between July 2011 and Feb. 2016, 199 patients with malignant hematological diseases who received an unrelated CBT at Anhui provincial hospital were included in this study. The patients received myeloablative conditioning without ATG and cyclosporin A (CSA) and mycophenolate mofetile as graft-versus-host disease (GVHD) prophylaxis in CBT protocols. (2) Dynamic monitoring of blood cells and lymphocyte recovery after CBT. (3)Lymphocyte subsets were assessed by flow-cytometry after fluorochrome-conjugated monoclonal antibodies for T-cells (CD3, CD4, and CD8), B-cells (CD19), and NK-cells (CD56, CD16). Immunoglobulin levels were measured by immunoturbidimetry assay. These analyses were done approximately at 1-6, 9, 12, 18 and 24 months post-transplantation.(4)The relationship between immune reconstitution and clinical outcome was analyzed.Results: (1) From 1 month after CBT, the absolute monocyte count (AMC) and absolute lymphocyte count (ALC) sustained to increase until month 18, the median ALC, CD3+-cell, CD3+CD4+-cell, and CD3+CD8+-cell counts achieved normal at month 2, 3, 5, and 2, respectively. The median NK-cell counts achieved normal at month 1 after CBT. (2) Early B-cell recovery was delayed with gradual increase in the number of cells, starting around month 3 after CBT. The median CD19+-cell count at days 30, 60 and 90 was 1/µl, 2/µl, and 7/µl, respectively. The median CD19+-cell count reached normal at month 5, the counts continue to increase beyond the normal range until month 18 post CBT. The median serum IgM, IgG and IgA levels reached the normal range at month 3, 4, and 9 after CBT, respectively. (3)Thirty-one cases died of severe infection and the median time of death from infection was 91 days after CBT (range, 31-413 days). By assessment of the rate of readmission within 30 days of hospital discharge, the patients with lower counts of CD19+ population after CBT has been associated with a higher readmission rates because of infectious episodes and GVHD. Onset of B-cell robust recovery ,which characterized by CD19+-cell count greater than 45/µl at month 3, 90/µl at month 4, and 280/µl at month 6, the patients’ subsequently readmission-rate was decreased significantly. The 30-day readmission rate was 6.9%,11% and 24% after CBT when the patients achieved robust B-cell recovery at month 3, 4, and 6, respectively.Assessment of B-cell counts within 83 graft samples, 32 of 83 patients had robust B cell recovery at month 3 who received comparable total B-cell and naive B-cell numbers in the grafts. The patients with or without rapid B-cell recovery showing a similar relapse rate (18.56% vs 15.34%, respectively). The 3-year probabilities of overall survival (OS) and disease-free survival (DFS) were 59%, respectively. Conclusion: Patients with rapid B-cell recovery are less likely to develop post-transplant GVHD, infections, and readmitted to hospital. The CD19+ B-cell counts may serve as useful predictor of GVHD-free survival, the 30-day readmission rate and quality-of-life after CBT. DisclosuresNo relevant conflicts of interest to declare.

Opposite Effects of Bruton Tyrosine Kinase (BTK) Inhibitor Therapy with Ibrutinib and FCR Chemo-Immunotherapy on the Normal B Lymphocyte Repertoire in Patients with Chronic Lymphocytic Leukemia

Disease-inherent and treatment-related immune dysfunction remain leading causes for morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). The advent of kinase inhibitors that target B cell receptor (BCR) signaling, which lack myelo- and T lymphocyte toxicity, raised hopes that these new agents may be less immunosuppressive and allow for better immune reconstitution when compared to chemo-immunotherapy (CIT). The effects of the BTK inhibitor ibrutinib or CIT with fludarabine, cyclophosphamide and rituximab (FCR) on the normal B cell repertoire have not been well characterized. Here, we used state-of-the-art immunosequencing technology to investigate how ibrutinib treatment affects the regeneration of non-malignant B-cells when compared to patients treated with FCR.Clinical data on infection rates and immunoglobulin levels was analyzed from 40 CLL patients treated with ibrutinib (median number of two pre-treatments) or frontline CIT with FCR at MD Anderson Cancer Center. In a representative subset of 20 patients, flow cytometry and next generation sequencing (NGS) of the immunoglobulin heavy chain (IGH) gene locus was used to monitor non-malignant B-cell immune reconstitution for 24 months after start of treatment with ibrutinib or FCR.Comparison of ibrutinib treatment with CIT revealed that immunoglobulin levels remained stable and relatively low in both cohorts, except for an increase in IgA during ibrutinib treatment, as previously reported. NGS results showed that ibrutinib treatment significantly decreased the non-malignant B-cells count after 24 months of treatment, while the counts were quantitatively stable in the FCR cohort. Next, we determined the dynamics of non-malignant B-cell immune repertoire composition over treatment. Based on the mutational status of the V gene, non-malignant B-cells were classified as IGH hypermutated (<98% identity to the corresponding germline V gene, corresponding to antigen-experienced B-cells) or IGH unmutated (≥98% identity to the corresponding germline V gene, corresponding to antigen-naïve B-cells). Before treatment initiation, the mean percentage of antigen-experienced B-cells did not significantly differ between the groups (ibrutinib 39%, FCR 48%). After 24 months, a significant decrease of antigen-experienced B-cells was observed in the FCR cohort, while the ratio of antigen-experienced and antigen-naïve B-cells remained unchanged in ibrutinib treated patients (ibrutinib 39%, FCR 22%, p=0.01). Analysis of the IGH clonotype repertoire using the Shannon-Wiener and the inverse Simpson diversity indices confirmed these results, showing that the non-malignant IGH repertoire was composed of balanced numbers of antigen-experienced and antigen-naïve medium sized clones before treatment initiation in both cohorts. In line with the IGH repertoire shift towards antigen-naïve B-cells in FCR treated patients, the medium-sized clones disappeared after treatment, with large numbers of small-sized unmutated clones dominating after 24 months (p<0.0001). In ibrutinib treated patients, the repertoire diversity remained stable throughout the course of treatment.Taken together, our data indicate that continuous treatment with ibrutinib preserves preexisting (partially antigen-experienced) B-cells but impairs de-novo generation of naive B-cells. In contrast, FCR leads to a deletion of memory B-cells but also a subsequent substantial renewal of the B-cell repertoire. Both patterns may differentially affect immune-competence towards infections. DisclosuresBokemeyer:Karyopharm: Research Funding. Jain:Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Genentech: Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria, Research Funding; BMS: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding. Wierda:Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Genentech: Research Funding. Burger:Pharmacyclics: Research Funding.

Functional disparities among BCL-2 members in tonsillar and leukemic B-cell subsets assessed by BH3-mimetic profiling

For successful treatment of malignant B-cells it is crucial to understand intrinsic survival requirements in relation to their normal progenitors. Long-lived humoral immunity as well as most B-cell malignancies, originate in the germinal center (GC). Murine GC B-cells depend on pro-survival protein MCL-1, but not BCL-XL. In contrast, naive and memory B-cells depend on BCL-2, but not BCL-XL or MCL-1. For human B-cell subsets, the functional relationships among BCL-2 members are unclear, and also if and how they shift after malignant transformation. We here dissect these aspects in human tonsil and primary leukemia (CLL) cells by single and combined treatment with novel, highly specific BH3-mimetics. We found that MCL-1 expression in GC B-cells is regulated post-translationally and its importance is highlighted by preferential binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced and binds solely to weak sensitizer BIK, potentially explaining why BCL-XL is not required for GC B-cell survival. Using novel BH3-mimetics, we found that naive and memory B-cells depend on BCL-2, GC cells predominantly on MCL-1, whereas plasma cells need both BCL-XL and MCL-1 for survival. CLL cells switch from highly sensitive for BCL-2 inhibition to resistant after CD40-stimulation. However, combined inhibition of BCL-2, plus BCL-XL or MCL-1 effectively kills these cells, thus exposing a weakness that may be therapeutically useful. These general principles offer important clues for designing treatment strategies for B-cell malignancies.

IL-10+ regulatory B cells are enriched in cord blood and may protect against cGVHD after cord blood transplantation

AbstractCord blood (CB) offers a number of advantages over other sources of hematopoietic stem cells, including a lower rate of chronic graft-versus-host disease (cGVHD) in the presence of increased HLA disparity. Recent research in experimental models of autoimmunity and in patients with autoimmune or alloimmune disorders has identified a functional group of interleukin-10 (IL-10)-producing regulatory B cells (Bregs) that negatively regulate T-cell immune responses. At present, however, there is no consensus on the phenotypic signature of Bregs, and their prevalence and functional characteristics in CB remain unclear. Here, we demonstrate that CB contains an abundance of B cells with immunoregulatory function. Bregs were identified in both the naive and transitional B-cell compartments and suppressed T-cell proliferation and effector function through IL-10 production as well as cell-to-cell contact involving CTLA-4. We further show that the suppressive capacity of CB-derived Bregs can be potentiated through CD40L signaling, suggesting that inflammatory environments may induce their function. Finally, there was robust recovery of IL-10–producing Bregs in patients after CB transplantation, to higher frequencies and absolute numbers than seen in the peripheral blood of healthy donors or in patients before transplant. The reconstituting Bregs showed strong in vitro suppressive activity against allogeneic CD4+ T cells, but were deficient in patients with cGVHD. Together, these findings identify a rich source of Bregs and suggest a protective role for CB-derived Bregs against cGVHD development in CB recipients. This advance could propel the development of Breg-based strategies to prevent or ameliorate this posttransplant complication.

A Public Database of Memory and Naive B-Cell Receptor Sequences.

The vast diversity of B-cell receptors (BCR) and secreted antibodies enables the recognition of, and response to, a wide range of epitopes, but this diversity has also limited our understanding of humoral immunity. We present a public database of more than 37 million unique BCR sequences from three healthy adult donors that is many fold deeper than any existing resource, together with a set of online tools designed to facilitate the visualization and analysis of the annotated data. We estimate the clonal diversity of the naive and memory B-cell repertoires of healthy individuals, and provide a set of examples that illustrate the utility of the database, including several views of the basic properties of immunoglobulin heavy chain sequences, such as rearrangement length, subunit usage, and somatic hypermutation positions and dynamics.

Disturbed canonical nuclear factor of κ light chain signaling in B cells of patients with common variable immunodeficiency

Most patients with common variable immunodeficiency (CVID) present with severely reduced switched memory B-cell counts, and some display an increase of CD21low B-cell counts (CVID 21low), whereas others do not (CVID 21norm). Altered B-cell receptor (BCR) signaling might contribute to the defective memory formation observed in patients with CVID. We sought to investigate canonical nuclear factor of κ light chain (NF-κB) signaling in Bcells from patients with CVID as a central pathway in B-cell differentiation. Degradation of inhibitor of κBα (IκBα) and p65 phosphorylation, nuclear translocation of p65, and regulation of target genes and cell function were investigated after different modes of B-cell stimulation. BCR-mediated canonical NF-κB signaling was impaired in all mature naive CVID-derived Bcells. This impairment was more profound in naive Bcells from CVID 21low patients than CVID 21norm patients and most pronounced in CD21low Bcells. The signaling defect translated into reduced induction of Bcl-xL and IκBα, 2 bona fide target genes of the canonical NF-κB pathway. CD40 ligand- and Toll-like receptor 9-mediated signaling were less strongly altered. Signaling in CD21low Bcells but not CD21+ Bcells of patients with HIV was similarly affected. Combined with the previous description of disturbed Ca2+ signaling, the discovery of NF-κB signaling defects, especially in CVID 21low patients, suggests a broad underlying signaling defect affecting especially BCR-derived signals. Given the immune phenotype of monogenic defects affecting Ca2+ and NF-κB signaling, the latter is more likely to contribute to the humoral deficiency. The strongly disturbed BCR signaling of CD21low Bcells is characteristic for this cell type and independent of the underlying disease.

T-helper signals restore B-cell receptor signaling in autoreactive anergic B cells by upregulating CD45 phosphatase activity

We recently identified a human B-cell population that is naturally autoreactive and tolerized by functional anergy (BND cells). We sought to identify the molecular mechanism of how anergic autoreactive BND cells escape functional anergy and whether this process is altered in patients with lupus. Isolated peripheral blood naive and BND cells were cultured with various stimuli, and their activation status was determined by using an intracellular Ca(2+) mobilization assay. Lyn kinase and Syk activities were assessed by using phospho-flow analysis. CD45 phosphatase activity was determined by using a novel flow-based assay, which takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid, an analog of phosphotyrosine, which can be incorporated into peptides. Real-time quantitative PCR was used to quantitate LYN, SYK, and CD45 mRNA. T-helper signals reversed the state of anergy, allowing BND cells to fully respond to antigenic stimulation by restoring signaling through the B-cell receptor (BCR). The mechanism was dependent on increased activity of the tyrosine phosphatase CD45 and CD45-dependent activation of Lyn and Syk. CD45 phosphatase activity was increased by T-cell help both in BND and naive Bcells. Furthermore, we found that BND cells obtained from patients with systemic lupus erythematosus exhibited increased CD45 activity and BCR-signaling capacity, thus being less tolerized than BND cells from healthy control subjects. Our findings suggest that CD45 is a key regulator of BCR-signaling thresholds mediated by T-cell help. This raises the possibility that BND cells could represent precursors of autoantibody-secreting plasma cells and suggests a role for these autoreactive Bcells in contributing to autoimmunity if not properly controlled.

(517) - Loss of Correlation Between Naive and Memory Class-Switched B-Cells in Heart Recipients with Acute Cellular Rejection

(517) - Loss of Correlation Between Naive and Memory Class-Switched B-Cells in Heart Recipients with Acute Cellular Rejection

Mammary-tumor-educated B cells acquire LAP/TGF-β and PD-L1 expression and suppress anti-tumor immune responses.

B lymphocytes play a role in inhibiting the immune response against certain tumors, but the underlying mechanisms are poorly understood. EMT-6 mammary tumors grow well in wild-type (WT) mice but show reduced growth in B-cell-deficient μ(-/-) BALB/c mice (BCDM). WT mice demonstrate extensive B-cell infiltration into the tumor bed, reduced CD8(+) T cell and CD49(+) NK cell infiltration, and markedly reduced cytolytic T-cell response relative to BCDM. Expression of LAP/TGF-β1, CD80, CD86 and PD-L1 is significantly increased in tumor-infiltrating B cells (TIL-B) relative to splenic B cells. LAP/TGF-β1 expression on TIL-B progressively increased from 5.4±1.7% on day 8 to 43.1±6.1% by day 21 post tumor implantation. Co-culture of EMT-6 tumor cells with Naive-B cells ex vivo generated B cells (EMT6-B) with a similar immunophenotype to TIL-B. Purified TIL-B, or in-vitro-generated EMT6-B suppressed CD4(+), CD8(+) and CD4(+)CD25(-) T-cell proliferation, and Th1 cytokine secretion, and also suppressed purified NK-cell proliferation in response to IL-15, compared to naive splenic B cells. Acquired B regulatory function required direct tumor cell: B-cell contact, and was partially reversed by antibody to TGF-β or PD-L1, leading to tumor rejection in vivo B-cell acquisition of a suppressive phenotype following tumor infiltration may result in profound inhibition of T-cell anti-tumor responses.

The Effect of BAFF Inhibition on Autoreactive B-Cell Selection in Murine Systemic Lupus Erythematosus.

The goal of this study was to determine how B-cell-activating factor of the TNF family (BAFF) availability influences selection of the autoreactive B-cell repertoire in NZB/W and NZW/BXSB lupus-prone mice bearing the site-directed heavy-chain transgene 3H9 that encodes for anti-dsDNA and anti-cardiolipin (CL) autoantibodies. We used a bone marrow chimera system in which autoreactive 3H9 transgenic B cells were allowed to mature in competition with wild-type cells and could be identified by green fluorescent protein. The light-chain repertoire associated with the 3H9 heavy chain in naive and antigen-activated B-cell subsets was assessed using single-cell polymerase chain reaction. We found that deletion of autoreactive transgenic B cells occurred in the bone marrow of both strains regardless of BAFF availability, and there were only modest and physiologically non-relevant effects on the naive B-cell repertoire. BAFF inhibition had different effects on selection of the germinal center repertoire in the two strains. In the NZW/BXSB strain, BAFF inhibition phenocopied the loss of one TLR7 allele in that it influenced the selection of 3H9-encoded autoreactive B cells in the germinal center but did not prevent somatic mutation. In the NZB/W strain, BAFF inhibition did not alter the selection of 3H9-encoded B cells in the germinal center, but it influenced selection of a subset of germinal center cells into the plasma cell compartment. Our data underscore the complexity of regulation of the autoreactive B-cell repertoire by BAFF and may help to explain the heterogeneity of responses observed after BAFF inhibition in humans.

Impaired Response to Influenza Vaccination in AML Patients Post-Chemotherapy Associated with a Highly Atypical B-Cell Profile

The immunotherapy of cancer is arguably the most promising therapy under development, and vaccination against cancer antigens is a promising strategy to stimulate adaptive immune responses against malignant clones. However, the ability of patients treated with chemotherapy to respond appropriately to immune challenge may be suboptimal. This study was designed to determine the functional capacity of the immune system in adult acute myeloid leukemia (AML) patients who have completed chemotherapy and are potential candidates for immunotherapy. We used the response to influenza vaccination as a surrogate for the health of the immune system in AML patients in a complete remission (CR) post-chemotherapy.Ten adult AML patients in CR after completion of intensive chemotherapy were recruited to the clinical protocol J1293. They were on average 37 weeks post-treatment (range 4-148) when receiving the 2012-2013 inactivated seasonal influenza vaccine. Peripheral blood samples were collected at baseline and 30 days post-vaccination. Ten age and sex matched healthy donors (HD) served as baseline controls. Serological response to vaccination was assessed via microneutralization assays; multi-parameter flow cytometry was used to characterize lymphocyte subsets. ELISPOT assays were used to evaluate lymphocyte function, microarrays were used to assess gene expression, and deep sequencing of the B-cell receptor heavy chain (IGH) was performed to determine expansion and clonality of B-cells. Wilcoxon rank-sum tests were used to assess statistical significance.Only 2/10 (20%) patients seroconverted (AML responders, or AML-R) with a four-fold increase or greater in influenza-specific antibody. One responder was 148 weeks post-treatment; the other had acute promyelocytic leukemia (APL) and was 4 weeks post-treatment. Deep immunophenotyping revealed no striking differences in T-cell compartments between AML at baseline and HD, indicating rapid T-cell recovery after chemotherapy. In contrast, we observed a highly atypical B-cell profile. AML non-responders (AML-NR) at day 0 versus HD had significantly reduced frequencies of mature IgA+ (4.5% vs. 11%) and IgG+ (2.4% vs. 5.9%) B-cells (as a percentage of CD19+CD20+). Further dissection exposed markedly higher frequencies of CD10+CD27- transitional B-cells (36% vs. 16%, p<0.005) and dramatically fewer memory (resting, tissue-like, activated) B-cells (5% vs. 25%, p<0.005) (as a percentage of CD19+CD20+) in AML-NR at day 0 versus HD. There were no significant changes in any B-cell population at day 30 over baseline in any patient. Examining frequencies of transitional, naive, and memory B-cells in AML patients at day 0 when ranked by time since treatment showed a decrease in transitional B-cells with a corresponding increase in naive B-cells over time but no concurrent increase in memory B-cell frequencies (Fig 1). These data suggest B-cell deficiencies of several types: loss of the memory B-cell compartment due to chemotherapy, a subsequent excess of transitional B-cells, and a lack of naive B-cell development into specific, class-switched effectors of the antibody response, which together likely lead to humoral immune incompetence.Interestingly, functional T-cell assays revealed that of 5 evaluable patients, including the 2 AML-R and 3 AML-NR, 5/5 (100%) patients had an increase in influenza-specific cytokine production (1.24 - 4.40x higher on day 30 over baseline), suggesting a functional T-cell response even with deficient influenza-specific antibody production. Supervised clustering of microarray data identified many upregulated genes in AML-NR related to apoptosis, BCR, IL2, IL-4, IL-8, and IL-12 signaling pathways, indicative of developing B-cells. IGH sequencing demonstrated AML-NR had greater variability in CDR3 length than seen in HD, consistent with an antigen inexperienced B-cell repertoire.These data suggest that while some aspects of cellular immunity recover comparatively quickly, the humoral immune system is incompletely reconstituted in the year following intensive cytotoxic chemotherapy for AML. Abnormal frequencies of transitional and memory B-cells may explain the poor response to vaccination often seen in patients after chemotherapy. Furthermore, the uncoupled recovery of B-cell and T-cell immune capacity observed here might have implications for the success of immunotherapies based on vaccination. [Display omitted] DisclosuresNoonan:Celgene: Speakers Bureau. Borrello:Celgene: Research Funding.

Somatic Mutation and Isotype Switch Events Outside Igv Gene Loci Provide New Evidence for a Role of Antigenic Challenge in Mantle Cell Lymphoma Etiology

Mantle cell lymphoma (MCL) represents 5-10% of B-cell lymphomas and generally exhibits an aggressive clinical behavior. t(11;14) translocation, the genetic hallmark and early initiating event of MCL pathogenesis, lead to the constitutive CCND1 expression and consequently to cell-cycle deregulation. However, t(11;14) is also present in the blood from rare healthy individuals indicating that alone the t(11;14) is not sufficientfor malignant transformation. It has long been assumed in MCL that t(11;14)+ cells were actually carried by circulating naive and antigen-inexperienced B-cells. Yet, several reports, including long-term persistence of t(11;14)+ cells in lymphoma free-patients, biased immunoglobulin heavy variable chain gene (IgVH) repertoire and evidence of subset of cases with mutated IgVH genes led us to question this model and further investigate the role of antigenic challenge in MCL etiologic origin.As a first approach, we sought to determine the class-switch recombination (CSR) status of t(11;14)+ cells in MCL patients. CSR, as somatic hypermutation (SHM), are diversification mechanisms of the B cell receptor induced upon antigen encounter in the germinal center (GC) reaction, and absent in naive cells. Evidence for ongoing switch events in MCL has been previously reported with detection of mature switch transcripts, however in MCL cells, sIgM(D) expression remains clearly evident suggesting that effective deletional switch events occur infrequently on the functional allele. To characterize CSR in t(11;14)+ cells, we thus took advantage of their unique BCL1/IGH translocation signature assuming that BCL1/IGH fusions do not prevent CSR to occur on the downstream constant region of the IGH locus. Using 2 long-range PCR assays designed to amplify unswitched BCL1/Sµ and switched BCL1/Sγ regions, DNA from 30 MCL patients differing by their IgVH status were tested. Of the MCL cases, we confirmed that 7 of 30 (23%) were unmutated (UM, 100% germline homology) and 23 of 30 (77%) were mutated (M), considering as mutated all cases exhibiting any level of SHM (ranging from minimal: >0.3% to high rate: 7.0%). Although mutated BCRs were common in most MCL cases, we found that only 1 out of 30 MCL cases underwent CSR to IgG on the non-productive, translocated allele. Accordingly, this case carried a mutated IgVH gene with 97.5% germline identity in line with a GC/antigen-experienced signature of the tumor cells. Contrary to SHM events of the IgVH gene region, CSR deletional events appeared very infrequent in the bulk of MCL cells both on the functional and translocated allele, contrasting from findings in other GC-derived non-Hodgkin lymphomas where the same selective pressure to maintain a sIgM is accompanied by deletional switch events on the non-functional allele. To get more insights into a role of antigenic drive in MCL and AID targeting outside the VH gene loci, we next evaluated the clonal evolution of t(11;14)+ cells from 11 MCL cases (2 UM and 9 M) by analyzing SHM in the Sμ region on the translocated allele using sequencing of LR-PCR amplicons (with 2 subclones/patient). We found that 63% (7 out of 11) of BCL1/IGH clones display significant levels of SHM in the Sµ together with intra-Sµ indels (2/11) compatible with off-target AID-mediated events linked to chronic(?) antigen encounter. In addition, we found a good correlation between mutated BCR and the SHM levels in Sμ regions for a given patient. Importantly, in one instance subclones from a MCL case displayed intraclonal diversification (despite the fact that the expected rate of SHM is much lower in the Sµ region than in the IgVH region), indicative of an ongoing AID activity at least in a subset of MCL cases. In conclusion, we found that CSR events, although detectable in some MCL cases, do not appear to participate predominantly or to be selected during MCL lymphomagenesis. By contrast, analysis of SHM profiles both on the functional and the non-functional translocated allele provide evidence for an active AID activity in MCL cells inside and outside V-gene loci, highlighting the propensity of some t(11;14)+ clones to acquire a complementary oncogenic hit over time as a result of recurrent antigen challenge. DisclosuresNo relevant conflicts of interest to declare.

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.

Disseminated Peritoneal Mantle Cell Lymphoma Mimicking Peritonitis

Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell lymphoma that represents 7% of non-Hodgkin cases, composed of CD5-positive naive B-cell of mantle zone surrounding germinal center follicles. MCL is clinically challenging to manage, with an ominous prognosis. MCL involves lymph nodes, spleen, bone marrow, and gastrointestinal tract organs. MCL involvement of peritoneal …

Systemic Sclerosis Patients Present Alterations in the Expression of Molecules Involved in B-Cell Regulation.

The activation threshold of B cells is tightly regulated by an array of inhibitory and activator receptors in such a way that disturbances in their expression can lead to the appearance of autoimmunity. The aim of this study was to evaluate the expression of activating and inhibitory molecules involved in the modulation of B cell functions in transitional, naive, and memory B-cell subpopulations from systemic sclerosis patients. To achieve this, blood samples were drawn from 31 systemic sclerosis patients and 53 healthy individuals. Surface expression of CD86, MHC II, CD19, CD21, CD40, CD22, Siglec 10, CD35, and FcγRIIB was determined by flow cytometry. IL-10 production was evaluated by intracellular flow cytometry from isolated B cells. Soluble IL-6 and IL-10 levels were measured by ELISA from supernatants of stimulated B cells. Systemic sclerosis patients exhibit an increased frequency of transitional and naive B cells related to memory B cells compared with healthy controls. Transitional and naive B cells from patients express higher levels of CD86 and FcγRIIB than healthy donors. Also, B cells from patients show high expression of CD19 and CD40, whereas memory cells from systemic sclerosis patients show reduced expression of CD35. CD19 and CD35 expression levels associate with different autoantibody profiles. IL-10+ B cells and secreted levels of IL-10 were markedly reduced in patients. In conclusion, systemic sclerosis patients show alterations in the expression of molecules involved in B-cell regulation. These abnormalities may be determinant in the B-cell hyperactivation observed in systemic sclerosis.