scholarly journals Low Numbers of CD27- IgD- 'Double Negative' Senescent B-Cells Early after Reduced Intensity T Cell Depleted Haematopoietic Stem Cell Transplantation Are Predictive of Subsequent Chronic GvHD

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4529-4529
Author(s):  
Francesca Kinsella ◽  
Harriet Protheroe ◽  
Hayden Pearce ◽  
Charlotte F Inman ◽  
Suzy A Eldershaw ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chronic Gvhd ◽  
Subsequent Development ◽  
Haematopoietic Stem Cell ◽  
Cell Repertoire ◽  
Graft Versus Host ◽  
Post Transplant ◽  
B Cell Repertoire ◽  
B Cell Subsets

Chronic graft versus host disease (cGvHD) is an important complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). It is now believed that B cells play a major role in the development of cGvHD as evidenced by the efficacy of B-cell directed therapies, and the disturbances in B cell subsets found in patients. This is characterised by a relative lack of naive B cells and increase in CD27+ (antigen experienced) mature B cells that can mediate host directed responses. Despite this the cellular mechanisms underlying the pathogenesis of cGvHD remain unclear. We investigated the phenotype and function of CD19+ B-cell subsets in a cohort of 46 patients undergoing reduced intensity-conditioned allo-HSCT with alemtuzumab. 76% of patients had myeloid disease and 24% lymphoid disease. All patients received PBMC grafts, fludarabine and melphalan conditioning and 10mg/day alemtuzumab from day -5 for 5 days. Overall survival at 3 years was 65% and relapse free survival was 62%. 16% patients experienced acute GvHD (grade 2+) and 29% developed chronic GvHD. B cell subsets were examined at 6 weeks post-transplant and CD27-IgD- 'double negative' (DN) B-cells were seen to dominate the B cell repertoire (mean 55% of B-cells). DN B-cells are an ill-defined memory B cell subset associated with immune senescence, auto-immune inflammatory conditions and cancer but their role in allo-HSCT has not been described. The frequency of DN B-cells decreased over time, as the naive B cell compartment regenerated, but still represented 31% of B cells at 1 year. However the mean number of DN B cells remained remarkably stable (0.033x109/L at 6 weeks v 0.037x109/L at 1 year). 14% of DN B-cells early post-transplant were transitional (CD24high CD38high) whilst 10% were plasma cells (CD24- CD38high). The remaining 76% had increased CD86 expression compared to memory B-cells (16% v 9.6%; *p=0.039) and lower levels of surface immunoglobulin. DN B-cells were less functional than other B cell subsets with decreased proportions of cells producing IL-2 (20%), IL-6 (17%), IFN-γ (6.2%), and IL-10 (6.2%) compared to naive or memory B cells. They also demonstrated reduced proliferation to in vitro stimulation (Ki-67 5.3%). Importantly, the proportion of DN B cells early post transplant was then studied in relation to transplant outcome and a lower frequency of cells was found to be correlated with subsequent development of cGvHD (38% v 62%; *p=0.04). These data show that senescent CD27-IgD- DN B-cells dominate the B cell repertoire in the early period after lymphocyte-depleted allo-HSCT. Furthermore, they are significantly reduced in patients who subsequently develop cGvHD. These data suggest that early B cell senescence can directly regulate the subsequent development of chronic graft versus host disease and indicate that the composition of the B cell repertoire at an early time-point post-transplant may be used to predict subsequent clinical outcome. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.

1987 ◽  
Vol 165 (2) ◽  
pp. 340-358 ◽  
Author(s):  
L W Duran ◽  
E S Metcalf
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lipid A ◽  
Predominant Role ◽  
Cell Repertoire ◽  
Cell Responses ◽  
B Cell Repertoire ◽  
Cell Clones ◽  
B Cell Subsets ◽  
Synthetic Antigens

In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.

scholarly journals AB0146 DRUG DEPENDENT ALTERATIONS IN B-CELL REPERTOIRE IN SYSTEMIC LUPUS ERYTHEMATOSUS PATIENTS WITH LOW DISEASE ACTIVITY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1373.2-1373
Author(s):  
S. Zenz ◽  
B. Dreo ◽  
B. Prietl ◽  
S. Kofler ◽  
H. Sourij ◽  
...  
Keyword(s):  
B Cells ◽  
Disease Activity ◽  
B Cell ◽  
Lupus Erythematosus ◽  
Cell Repertoire ◽  
Low Disease Activity ◽  
B Cell Repertoire ◽  
Drug Dependent ◽  
B Cell Subsets

Background:B-cells play a major role in the pathogenesis and perpetuation of the immune response in systemic lupus erythematosus (SLE). So far, B-cell subtypes have been studied well, but the precise mechanisms of the B-cell alterations during disease activity and during remission, depending on different medication, are still unclear.Objectives:The aim of our study was to investigate the drug dependent alterations in the B-cell repertoire of SLE patients with low disease activity (SLEDAI – 2K ≤4).Methods:Peripheral blood samples from 39 patients suffering from SLE (mean±SD; age 43±13 years, 87.2% females, disease duration 11.1±7 years) were drawn over 2 years. All SLE patients were in remission or low disease activity (median±SE, SLEDAI of 2.0±1.5). B-cells were characterized using CD19, CD20, CD5, CD27 antibodies and were grouped in naïve (IgD+27-), non-switched memory (IgD+, CD27+), memory (IgD-,CD27+), B1 (CD5+27-) and MBL-like (CD5++) B-cells. A quantitative flow cytometric bead-based assay (QuantiBRITE PE kit from Becton Dickinson) was used for the estimation of CD19 antibodies bound per cell. Further, CD38 and CD86 antibodies were used to characterize the B-cell subsets. All cytometric measurements were performed using a standardized BD LSR Fortessa platform. After 3 years of follow-up, patients’ data about disease activity and current medication were obtained.Results:22 SLE patients were treated with hydroxychloroquine (85.8%) and 19 patients received mycophenolate mofetil (MMF; n=14; 54.6%) or azathioprine (AZA; n= 5; 19.5 %). 5 patients were treated with other DMARDs. Independently of hydroxychloroquine and/or MMF, no significant differences were seen in naïve, non-switched memory, post-switched memory, plasma blasts, B1- or MBL-like B-cells. Patients treated with AZA had significantly lower naïve B-cells (mean±SD, 39.3±6.7vs. 73.1±19.3 %; p = 0.028), but had significantly higher IgD-post switched B-cells (31.2±9.1 vs.12.5 ±9.2 %; p = 0.028, respectively) compared with no AZA-treatment. Interestingly, activated B-cells (5.5±1.5 vs. 1.8±1.1%; p = 0.009) were significantly higher in AZA-treated. After 3 years of follow-up, almost all patients were in remission (median±SE, SLEDAI of 2.0±2.0), except of 3 patients with a SLEDAI of ≥ 6. Interestingly, those patients had at baseline, statistically higher naïve B-cells (p = 0.041) and lower B1-like B-cells (p =0.020) compared with patients with low disease activity.Conclusion:Our results suggest that independently of hydroxychloroquine and/or MMF treatment, all patients with low disease activity had similar normal B-cell subsets. Interestingly, in the small group of patients who were treated with AZA, a reduced regeneration of B-cells was shown. Patients with higher disease and high naïve B-cells showed an increased disease activity after three years.Acknowledgments:The research was performed in “CBmed” and funded by the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien.Disclosure of Interests:None declared

scholarly journals Efficient Peripheral Clonal Elimination of B Lymphocytes in MRL/lpr Mice Bearing Autoantibody Transgenes

1998 ◽  
Vol 188 (5) ◽  
pp. 909-917 ◽  
Author(s):  
Jennifer A. Kench ◽  
David M. Russell ◽  
David Nemazee
Keyword(s):  
B Cells ◽  
B Cell ◽  
Genetic Background ◽  
Cell Tolerance ◽  
Cell Repertoire ◽  
B Cell Tolerance ◽  
B Cell Repertoire ◽  
Nuclear Antigens ◽  
Large Numbers ◽  
Liver And Kidney

Peripheral B cell tolerance was studied in mice of the autoimmune-prone, Fas-deficient MRL/ lpr.H-2d genetic background by introducing a transgene that directs expression of membrane-bound H-2Kb antigen to liver and kidney (MT-Kb) and a second transgene encoding antibody reactive with this antigen (3-83μδ, anti-Kk,b). Control immunoglobulin transgenic (Ig-Tg) MRL/lpr.H-2d mice lacking the Kb antigen had large numbers of splenic and lymph node B cells bearing the transgene-encoded specificity, whereas B cells of the double transgenic (Dbl-Tg) MRL/lpr.H-2d mice were deleted as efficiently as in Dbl-Tg mice of a nonautoimmune B10.D2 genetic background. In spite of the severely restricted peripheral B cell repertoire of the Ig-Tg MRL/lpr.H-2d mice, and notwithstanding deletion of the autospecific B cell population in the Dbl-Tg MRL/lpr.H-2d mice, both types of mice developed lymphoproliferation and exhibited elevated levels of IgG anti-chromatin autoantibodies. Interestingly, Dbl-Tg MRL/lpr.H-2d mice had a shorter lifespan than Ig-Tg MRL/lpr.H-2d mice, apparently as an indirect result of their relative B cell lymphopenia. These data suggest that in MRL/lpr mice peripheral B cell tolerance is not globally defective, but that certain B cells with receptors specific for nuclear antigens are regulated differently than are cells reactive to membrane autoantigens.

Autoreactive B-cell repertoire in mice with chronic graft versus host disease

1988 ◽  
Vol 25 (11) ◽  
pp. 1217-1222 ◽  
Author(s):  
Anton G. Rolink ◽  
Philipp Thalmann ◽  
Cristoph Berger ◽  
Thaddäus Radaszkiewicz ◽  
Fritz Melchers
Keyword(s):  
B Cell ◽  
Graft Versus Host Disease ◽  
Cell Repertoire ◽  
Host Disease ◽  
Graft Versus Host ◽  
B Cell Repertoire

scholarly journals High-frequency representation of a single VH gene in the expressed human B cell repertoire.

1993 ◽  
Vol 177 (2) ◽  
pp. 409-418 ◽  
Author(s):  
A K Stewart ◽  
C Huang ◽  
B D Stollar ◽  
R S Schwartz
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Frequency ◽  
Gene Segment ◽  
Normal Subjects ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
V Genes ◽  
Vh Gene ◽  
Vh Genes

Idiotype (Id) 16/6 marks a variable (V) region structure that occurs frequently in the human immunoglobulin repertoire. The basis of the Id has been traced to a germline heavy chain gene segment, VH18/2 (VH26). To pursue the molecular basis for the frequency of Id 16/6, we have analyzed polymerase chain reaction-generated C mu, C gamma, and VH3 family V gene libraries derived from the circulating and tonsillar B cells of four normal individuals and from the B cells of two patients with active systemic lupus erythematosus (SLE). The frequency of VH18/2 in these libraries was compared with three control VH genes, VH56P1, VH21/28, and VHA57. Plaque lifts from C mu and C gamma VH cDNA libraries were screened with gene-specific oligonucleotide probes. The frequency of VH18/2 ranged from 4 to 10% of JH+ plaques (two of five times that of control VH genes). In four VH3 family-specific libraries derived from rearranged DNA, VH18/2 represented 19-33% of VH3+ plaques. Hybridizing VH18/2 plaques were 98-100% homologous to the germline VH gene; mutations when present were often in framework 3. Extensive variation was seen in the complementarity determining region 3 sequences of these rearranged V genes. The high frequency of VH18/2 expression in the B cell repertoire was confirmed by sequencing randomly picked JH+ plaques. In two patients with active SLE the frequency of use of VH18/2 was not greater than that observed in normal subjects. These results show that VH18/2 is overrepresented in the B cell repertoire of normal subjects and suggest that the immune repertoire may be dominated by relatively few V genes.

Effect of age on the expressed B cell repertoire: role of B cell subsets

1993 ◽  
Vol 5 (9) ◽  
pp. 1035-1039 ◽  
Author(s):  
A. Hu ◽  
D. Ehleiter ◽  
A. Ben-Yehuda ◽  
R. Schwab ◽  
C. Russo ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
B Cell Subsets ◽  
Effect Of Age

scholarly journals Shared HIV envelope-specific B cell clonotypes induced by a pox-protein vaccine regimen

2021 ◽  
Author(s):  
Kristen W. Cohen ◽  
Lamar Ballweber-Fleming ◽  
Michael Duff ◽  
Rachael E. Whaley ◽  
Aaron Seese ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Critical Role ◽  
Memory B Cells ◽  
Protein Vaccine ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Specific Igg

An effective HIV-1 vaccine will likely induce potent, broad neutralizing antibodies. No candidate vaccines have elicited these responses presumably because they fail to activate human B cell precursors that can affinity mature to generate broad neutralizing antibodies. To identify the B cell clonotypes that are elicited, we conducted in-depth analyses of the envelope-specific B cell repertoire in recipients of ALVAC-HIV vector (vCP2438) and bivalent subtype C gp120 protein (HVTN100). We observed high frequencies of envelope-specific IgG+ memory B cells with restricted immunogenetic diversity, relative to non-vaccine induced memory B cells, with preferential expansions of distinct variable genes but limited accumulation of mutations. Many envelope-specific clonotypes were shared across vaccinees, but did not overlap with the envelope-negative memory repertoire, within and across subjects. Single-cell sequencing of envelope-specific IgG+ memory B cells often revealed VH1-2*02 and VK3-20 sequence co-expression and in one case, contained a 5 amino acid CDRL3, the canonical signature of VRC01-class antibodies, confirming that these B cells are extremely rare but detectable. Our study provides evidence that immunogens play a critical role in selecting and restricting the responding B cell repertoire and supports the rational design of HIV vaccines targeting specific B cell lineages for induction of broadly-reactive neutralizing antibodies.

scholarly journals 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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4402-4402
Author(s):  
Simon Schliffke ◽  
Mariela Sivina ◽  
Ekaterina Kim ◽  
Benjamin Thiele ◽  
Nuray Akyüz ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Treatment Initiation ◽  
Lymphocytic Leukemia ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Btk Inhibitor ◽  
V Gene ◽  
Immunoglobulin Levels

Abstract 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. Disclosures Bokemeyer: 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.

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