AB0144 STRATIFICATION OF PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME BY MEASURING B-CELL HEALTH

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1372-1373
Author(s):  
G. M. Verstappen ◽  
J. C. Tempany ◽  
H. Cheon ◽  
A. Farchione ◽  
S. Downie-Doyle ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
B Cell Differentiation ◽  
Research Support ◽  
Differentiation Capacity ◽  
Cell Responses ◽  
Healthy Donors

Background:Primary Sjögren’s syndrome (pSS) is a heterogeneous immune disorder with broad clinical phenotypes that can arise from a large number of genetic, hormonal, and environmental causes. B-cell hyperactivity is considered to be a pathogenic hallmark of pSS. However, whether B-cell hyperactivity in pSS patients is a result of polygenic, B cell-intrinsic factors, extrinsic factors, or both, is unclear. Despite controversies about the efficacy of rituximab, new B-cell targeting therapies are under investigation with promising early results. However, for such therapies to be successful, the etiology of B-cell hyperactivity in pSS needs to be clarified at the individual patient level.Objectives:To measure naïve B-cell function in pSS patients and healthy donors using quantitative immunology.Methods:We have developed standardised, quantitative functional assays of B-cell responses that measure division, death, differentiation and isotype switching, to reveal the innate programming of B cells in response to T-independent and dependent stimuli. This novel pipeline to measure B-cell health was developed to reveal the sum total of polygenic defects and underlying B-cell dysfunction at an individual level. For the current study, 25 pSS patients, fulfilling 2016 ACR-EULAR criteria, and 15 age-and gender-matched healthy donors were recruited. Standardized quantitative assays were used to directly measure B cell division, death and differentiation in response to T cell-independent (anti-Ig + CpG) and T-cell dependent (CD40L + IL-21) stimuli. Naïve B cells (IgD+CD27-) were sorted from peripheral blood mononuclear cells and were labeled with Cell Trace Violet at day 0 to track cell division until day 6. B cell differentiation was measured at day 5.Results:Application of our standardized assays, and accompanying parametric models, allowed us to study B cell-intrinsic defects in pSS patients to a range of stimuli. Strikingly, we demonstrated a hyperresponse of naïve B cells to combined B cell receptor (BCR) and Toll-like receptor (TLR)-9 stimulation in pSS patients. This hyperresponse was revealed by an increased mean division number (MDN) at day 5 in pSS patients compared with healthy donors (p=0.021). A higher MDN in pSS patients was observed at the cohort level and was likely attributed to an increased division burst (division destiny) time. The MDN upon BCR/TLR-9 stimulation correlated with serum IgG levels (rs=0.52; p=0.011). No difference in MDN of naïve B cells after T cell-dependent stimulation was observed between pSS patients and healthy donors. B cell differentiation capacity (e.g., plasmablast formation and isotype switching) after T cell-dependent stimulation was also assessed. At the cohort level, no difference in differentiation capacity between groups was observed, although some pSS patients showed higher plasmablast frequencies than healthy donors.Conclusion:Here, we demonstrate defects in B-cell responses both at the cohort level, as well as individual signatures of defective responses. Personalized profiles of B cell health in pSS patients reveal a group of hyperresponsive patients, specifically to combined BCR/TLR stimulation. These patients may benefit most from B-cell targeted therapies. Future studies will address whether profiles of B cell health might serve additional roles, such as prediction of disease trajectories, and thus accelerate early intervention and access to precision therapies.Disclosure of Interests:Gwenny M. Verstappen: None declared, Jessica Catherine Tempany: None declared, HoChan Cheon: None declared, Anthony Farchione: None declared, Sarah Downie-Doyle: None declared, Maureen Rischmueller Consultant of: Abbvie, Bristol-Meyer-Squibb, Celgene, Glaxo Smith Kline, Hospira, Janssen Cilag, MSD, Novartis, Pfizer, Roche, Sanofi, UCB, Ken R. Duffy: None declared, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Philip D. Hodgkin Grant/research support from: Medimmune, Vanessa L. Bryant Grant/research support from: CSL

CD154:CD40 Co-Stimulatory Blockade: A Specific Approach To Prevent Sensitization to Donor Alloantigens.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2246-2246
Author(s):  
Hong Xu ◽  
Jun Yan ◽  
Suzanne T. Ildstad
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Skin Grafting ◽  
Dominant Factor ◽  
Skin Grafts ◽  
B Cell Differentiation ◽  
Cell Responses ◽  
B Cell Responses

Abstract Introduction: Recipient sensitization is one of the most critical problems facing clinical transplantation. Patients with sickle cell disease are presensitized to HLA-alloantigens from chronic transfusion therapy. These allosensitized recipients experience a higher failure rate in engraftment and require more intense conditioning. A method to prevent sensitization would have a significant impact on transplant outcomes in these recipients. We have identified that B-cell responses and humoral immunity are the dominant factor in sensitization to MHC alloantigens. T-cell-dependent B-cell responses require physical cell:cell contacts that drive the different steps of B-cell differentiation to memory B-cells or to Ab-producing cells. Several signal pathways have been identified that are important for initiation of B-cell differentiation, including costimulation through the CD28/B7 or TNF/TNFR families. Here, we examined whether blocking the costimulatory interaction between T- and B-cells with anti-CD154, anti-OX40L, or anti-ICOS during exposure to alloantigen would prevent allosensitization. Methods: B6 (H-2b) mice received allogeneic BALB/c (H-2d) skin grafts on day 0 and were treated with anti-CD154 (day 0 and day +3), anti-OX40L (day 0 to +4), or anti-ICOS (day +4, 6, 8 and 10). Anti-donor specific antibodies were measured by flow cytometry cross-match assay 4 weeks after skin grafting and reported as mean fluorescence intensity (MFI). Results: Recipients treated with anti-CD154, anti-OX40L, or anti-ICOS rejected BALB/c skin grafts with a time course similar to normal B6 controls (12.7±2.4 days). The anti-donor antibody titer in mice treated with anti-CD154 (5.9±3.9) was slightly, but not significantly (P>0.05) higher than for naïve mice (3.8±0.6). Levels of allospecific Ab were markedly higher in mice treated with mAb anti-ICOS (123.6±38.1; P<0.001) and anti-OX40L (80.6±23.3; P<0.0001) compared with naïve controls or anti-CD154 treated animals. The Ab level in mice treated with mAb anti-ICOS was similar to sensitized controls (skin grafting only without mAb treatment; MFI: 132.6±38.5; P=0.69). The anti-donor Ab in mice treated with mAb anti-OX40L was significantly lower (P=0.02) compared to sensitized controls, but the difference was much more significant when compared with naïve controls. To evaluate sensitization in vivo, mice were conditioned with 950 cGy and transplanted with 15×106 of BALB/c BM cells 5–7 weeks after skin grafting. As expected, engraftment did not occur in sensitized control mice (no Ab treatment). Engraftment also did not occur in mice treated with anti-ICOS. Only 16.7% of mice engrafted with anti-OX40L treatment. In contrast, 75.0% of mice engrafted with anti-CD154 treatment. Conclusion: Blocking molecular interactions between CD40/CD154 abrogated the generation of antibody against donor antigens. The CD154 blockade effect on Ab production was specific and not a general feature of co-stimulatory blockade, as blockade of ICOS or OX40L did not completely inhibit Ab generation. These data suggest that the engagement of B-cell molecule CD40 and its ligand CD154 plays a central role in T-cell-dependent B-cell responses. These findings could have a significant impact on management of sensitized recipients in the clinic. Figure Figure

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

scholarly journals MiR-17-92 Is Required for the Pathogenicity of T and B Cells in Chronic Gvhd

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4535-4535
Author(s):  
Yongxia Wu ◽  
Steven D Schutt ◽  
Ryan P Flynn ◽  
Mengmeng Zhang ◽  
Hung D Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Cd4 T Cell ◽  
B Cell Differentiation ◽  
T And B Cells ◽  
And Function

Abstract Chronic graft-versus-host disease (cGVHD) remains to be a major cause of mortality and morbidity after allogeneic hematopoietic cell transplantation (allo-HCT). cGVHD is characterized as autoimmune-like fibrosis and antibody production, mediated by pathogenic T and B cells. Through producing pro-inflammatory cytokines, CD4 T cells are the driving force of cGVHD. Donor B cells augment the pathogenesis of cGVHD not only by acting as antigen-presenting cells (APCs) and promoting CD4 T-cell expansion and survival, but also by producing autoantibodies. microRNA (miR)-17-92 has been shown to regulate T-cell immunity including allogeneic, anti-viral, and anti-tumor responses. Recently, miR-17-92 was found to act together with Bcl-6 to promote the differentiation of Follicular help T (Tfh) cells. Furthermore, B-cell deficiency of miR-17-92 impairs IgG2c production. Since Tfh differentiation and antibody production are required for the development of cGVHD, we hypothesize that miR-17-92 contributes to the pathogenesis of cGVHD by promoting pathogenic T- and B-cell responses. By using Cre-loxp system, we generated B6 mice with conditional deficiency of miR-17-92 in T cells (CD4cre), B cells (CD19cre), or both (CD4CD19cre). aGVHD to cGVHD transition model (B6 to BALB/c) was utilized to test the effects of individual and combinational deficiency of miR-17-92 in T and/or B cells in the development of cGVHD. BALB/c mice were lethally irradiated and transferred with splenocytes plus BM cells derived from CD4cre, CD19cre or CD4CD19cre miR-17-92flox/flox B6 mice. WT B6 (Cre- miR-17-92flox/flox) mice were used as control donors. A significantly reduction of GVHD mortality was observed only in the recipients with CD4CD19cre grafts, but not with CD4cre or CD19cre grafts. Deficiency of miR-17-92 in donor T or B cells indeed improved the clinical manifestation of cGVHD, but the deficiency in both T and B cells showed further improvement, indicating the additive role of miR-17-92 in T and B cells in the pathogenesis of cGVHD. Mechanistically, deficiency of miR-17-92 in T cells resulted in the reduction of Tfh generation (Fig. A), germinal center (GC) B-cell and plasma cell differentiation, and the expression of MHC-II and CD86 on donor B cells in recipient spleens. Furthermore, deficiency of miR-17-92 in B cells significantly reduced the levels of total IgG and IgG2c in recipient serum (Fig. A). These data suggest that miR-17-92 contributes to both T- and B-cell differentiation and function, which is required for the development of cGVHD. To extend our findings, we used a bronchiolitis obliterans cGVHD model (B6 to B10.BR). Recipient mice were pre-conditioned and received either BM alone from WT or CD19cre B6 mice, or BM plus purified T cells from WT or CD4cre B6 mice. Deficiency of miR-17-92 in T cells or BM-derived B cells resulted in significant improvement in pulmonary functions in recipient mice, as demonstrated by a decrease in resistance and elastance and an increase in compliance (Fig. B). Consistently, we found that miR-17-92 promoted Tfh and GC B-cell differentiation (Fig. B), while inhibiting differentiation of T follicular regulatory cells in recipient spleens 60 days after allo-HCT. For translational purpose, we tested whether inhibition of miR-17-92 could ameliorate cGVHD using locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, key members in this microRNA cluster. In a SLE cGVHD model (DBA2 to BALB/c), administration of anti-miR-17, but not anti-miR-19, significantly suppressed the incidence of proteinuria and the severity of clinical manifestation by inhibiting donor splenocyte expansion, expression of costimulatory molecules on donor B cells, and differentiation of GC B cells and plasma cells (Fig. C). In addition, systemic delivery of anti-miR-17 significantly improved skin cGVHD by restraining IL-17 producing CD4 T-cell infiltration in skin-draining lymph nodes in a scleroderma-cGVHD model (B10.D2 to BALB/c). Taken together, the current work reveals that miR-17-92 is required for T- and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the control of cGVHD after allo-HCT. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Conserved Gammaherpesvirus Protein Kinase Selectively Promotes Irrelevant B Cell Responses

2019 ◽  
Vol 93 (8) ◽  
Author(s):  
Eric J. Darrah ◽  
Christopher N. Jondle ◽  
Kaitlin E. Johnson ◽  
Gang Xin ◽  
Philip T. Lange ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Protein Kinase ◽  
B Cell ◽  
Germinal Center ◽  
Unique Feature ◽  
Latent Infection ◽  
B Cell Differentiation ◽  
Cell Responses ◽  
B Cell Responses

ABSTRACTGammaherpesviruses are ubiquitous pathogens that are associated with B cell lymphomas. In the early stages of chronic infection, these viruses infect naive B cells and subsequently usurp the B cell differentiation process through the germinal center response to ensure latent infection of long-lived memory B cells. A unique feature of early gammaherpesvirus chronic infection is a robust differentiation of irrelevant, virus-nonspecific B cells with reactivities against self-antigens and antigens of other species. In contrast, protective, virus-specific humoral responses do not reach peak levels until a much later time. While several host factors are known to either promote or selectively restrict gammaherpesvirus-driven germinal center response, viral mechanisms that contribute to the irrelevant B cell response have not been defined. In this report we show that the expression and the enzymatic activity of the gammaherpesvirus-encoded conserved protein kinase selectively facilitates the irrelevant, but not virus-specific, B cell responses. Further, we show that lack of interleukin-1 (IL-1) receptor attenuates gammaherpesvirus-driven B cell differentiation and viral reactivation. Because germinal center B cells are thought to be the target of malignant transformation during gammaherpesvirus-driven lymphomagenesis, identification of host and viral factors that promote germinal center responses during gammaherpesvirus infection may offer an insight into the mechanism of gammaherpesvirus pathogenesis.IMPORTANCEGammaherpesviruses are ubiquitous cancer-associated pathogens that usurp the B cell differentiation process to establish life-long latent infection in memory B cells. A unique feature of early gammaherpesvirus infection is the robust increase in differentiation of B cells that are not specific for viral antigens and instead encode antibodies that react with self-antigens and antigens of other species. Viral mechanisms that are involved in driving such irrelevant B cell differentiation are not known. Here, we show that gammaherpesvirus-encoded conserved protein kinase and host IL-1 signaling promote irrelevant B cell responses and gammaherpesvirus-driven germinal center responses, with the latter thought to be the target of viral transformation.

scholarly journals Autoreactive T cells preferentially drive differentiation of non-responsive memory B cells at the expense of germinal center maintenance

2018 ◽  
Author(s):  
Rajiv W Jain ◽  
Kate A Parham ◽  
Yodit Tesfagiorgis ◽  
Heather C Craig ◽  
Emiliano Romanchik ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Fate ◽  
Germinal Center ◽  
Memory B Cells ◽  
B Cell Differentiation ◽  
Cell Fate Decisions ◽  
The Status

AbstractB cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG), and compare them the response to a standard model foreign antigen (NP-haptenated ovalbumin, NPOVA). Both antigens generated productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response the status of the cognate T cell partner drove preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation was largely independent of T cell influence. Interestingly, memory B cells formed in the MOG GC were unresponsive to secondary challenge and this could not be overcome with T cell help.

scholarly journals T cell-derived B cell differentiation factor(s). Effect on the isotype switch of murine B cells.

1982 ◽  
Vol 155 (3) ◽  
pp. 734-748 ◽  
Author(s):  
P C Isakson ◽  
E Puré ◽  
E S Vitetta ◽  
P H Krammer
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Interleukin 2 ◽  
B Cell Differentiation ◽  
Differentiation Factor ◽  
T Cell Lines ◽  
Cell Differentiation Factor

Culturing BALB/c B cells for 6 d at low cell density in the presence of lipopolysaccharide (LPS) results in the appearance of a small number of IgG plaque-forming cells (PFC). The addition of supernatants from concanavalin A (Con A)-induced alloreactive (AKR anti-B6) long-term T cell lines (PK 7.1.1a and 7.1.2) or a T cell hybridoma (FS7-6.18) to LPS-treated B cells resulted in a marked increase in IgG PFC (3--10-fold higher than in cultures treated with LPS alone. The number of induced IgG PFC was not affected by removing IgG-bearing cells on the fluorescence-activated cell sorter, indicating that T cell-derived B cell differentiation factor enhances isotype switching of sIgG- cells, rather than selecting and expanding pre-existing subpopulations of sIgG+ cells. We also investigated the subclass of IgG produced in the absence or presence of T cell factors and found that PK 7.1.1a, PK 7.1.2, and FS7-6.18 supernatants selectively increased IgG1 production. Several other T cell supernatants containing a variety of lymphokines had no effect, suggesting that PK 7.1.1a, PK 7.1.2, and FS7-6.18 lines produce factor(s) that can specifically enhance the recovery of IgG secreting cells in culture in the presence of LPS. These factors, which we have termed B cell differentiation factors, are different from interleukin 1, interleukin 2, T cell-replacing factor, colony-stimulating factor, macrophage-activating factor, and immune interferon. Our results suggest that soluble factors produced by T cell lines and hybridomas can markedly influence both the class and subclass of Ig produced by B cells.

scholarly journals Optimized Protocols for in Vitro T Cell-Dependent and T Cell-Independent Activation for B Cell Differentiation Studies Using Limited Cells

2021 ◽  
Author(s):  
Casper Marsman ◽  
Dorit Verhoeven ◽  
Jana Koers ◽  
Theo Rispens ◽  
Anja ten Brinke ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Plasma Cells ◽  
Cell Formation ◽  
B Cell Differentiation ◽  
Differentiation Potential ◽  
Human B Cell

Background/methods: For mechanistic studies, in vitro human B cell differentiation and generation of plasma cells are invaluable techniques. However, the heterogeneity of both T cell-dependent (TD) and T cell-independent (TI) stimuli and the disparity of culture conditions used in existing protocols makes interpretation of results challenging. The aim of the present study was to achieve the most optimal B cell differentiation conditions using isolated CD19+ B cells and PBMC cultures. We addressed multiple seeding densities, different durations of culturing and various combinations of TD stimuli and TI stimuli including B cell receptor (BCR) triggering. B cell expansion, proliferation and differentiation was analyzed after 6 and 9 days by measuring B cell proliferation and expansion, plasmablast and plasma cell formation and immunoglobulin (Ig) secretion. In addition, these conditions were extrapolated using cryopreserved cells and differentiation potential was compared. Results: This study demonstrates improved differentiation efficiency after 9 days of culturing for both B cell and PBMC cultures using CD40L and IL-21 as TD stimuli and 6 days for CpG and IL-2 as TI stimuli. We arrived at optimized protocols requiring 2500 and 25.000 B cells per culture well for TD and TI assays, respectively. The results of the PBMC cultures were highly comparable to the B cell cultures, which allows dismissal of additional B cell isolation steps prior to culturing. In these optimized TD conditions, the addition of anti-BCR showed little effect on phenotypic B cell differentiation, however it interferes with Ig secretion measurements. Addition of IL-4 to the TD stimuli showed significantly lower Ig secretion. The addition of BAFF to optimized TI conditions showed enhanced B cell differentiation and Ig secretion in B cell but not in PBMC cultures. With this approach, efficient B cell differentiation and Ig secretion was accomplished when starting from fresh or cryopreserved samples. Conclusion: Our methodology demonstrates optimized TD and TI stimulation protocols for more indepth analysis of B cell differentiation in primary human B cell and PBMC cultures while requiring low amounts of B cells, making them ideally suited for future clinical and research studies on B cell differentiation of patient samples from different cohorts of B cell-mediated diseases.

Enhanced B-Cell Differentiation and Increased Production of Antibodies Targeting Surface Membrane Antigens in Patients with Chronic Graft-Versus-Host Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 670-670
Author(s):  
Kathy S. Wang ◽  
Corey S. Cutler ◽  
Sarah Nikiforow ◽  
Vincent T. Ho ◽  
John Koreth ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
High Dose ◽  
B Cell Differentiation ◽  
Advisory Committees ◽  
High Dose Steroid ◽  
Healthy Donors

The mechanisms of chronic graft-versus-host disease (cGVHD) are complex and involve multiple elements of the immune system. Previous studies have indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. T cells that facilitate B-cell differentiation, such as T Follicular Helper cells (Tfh), were also shown to exhibit enhanced function in patients with cGVHD. IL-21, a cytokine produced by Tfh cells plays an important role in the B-cell differentiation process. Activation of IL-21R expressed on B cells promotes B-cell differentiation into plasmablasts and plasma cells. To examine the responsiveness of B cells to this Tfh cytokine and antibody production in the setting of cGVHD, we analyzed B cells from 107 patients after allogeneic HSCT: 31 with no cGVHD, 44 with persistent stable cGVHD, and 22 patients with cGVHD receiving high-dose steroid therapy. Samples were collected from 9 months on after transplant. We also analyzed samples from 32 healthy donors as controls. The total CD19+CD20+ B-cell numbers in patients with stable cGVHD were comparable to those of healthy donors and slightly higher than normal in patients without cGVHD, suggesting normal recovery of B cells in these patients by the time the samples were collected. In patients with cGVHD, the number of IL-21R expressing B cells (CD19+CD20+IL21R+) was significantly increased compared to patients without cGVHD (p=0.046) or healthy donors (p=0.0036). The number of plasmablasts (CD19+CD20-CD38Hi) in patients with cGVHD was also significantly higher than in healthy donors (p=0.0079). Plasmablasts levels were higher in patients with cGVHD compared to those without cGVHD, but this difference was not statistically significant (p=0.3702). High-dose steroid therapy reduced both the number of IL-21R positive B cells and the number of plasmablasts, but neither reduction was statistically significant. We also examined IL-21 levels in plasma using multiplex cytokine bead arrays. Levels of circulating IL-21 were significantly higher in patients with cGVHD compared with healthy donors or patients without cGVHD. Increased levels of IL-21 and increased numbers of IL-21R+ B cells suggest that both homeostatic stimulation and inherent B-cell susceptibility promote B-cell differentiation in patients with cGVHD. Previous studies have shown that male transplant patients who received sex-mismatched grafts developed significantly higher levels of anti-H-Y IgG antibodies and the development of H-Y antibodies is highly correlated with development of cGVHD. To examine the impact of the observed enhanced B-cell differentiation on production of allo- and autoantibodies, we developed a fluorescent-linked immuno-assay to detect antibodies capable of targeting cell-surface proteins. Membrane protein extracts were prepared from a human skin fibroblast cell line (Detroit 551) and coated onto 96-well plates. Plasma samples from the above cohort of patients and healthy donors were incubated on the plate, and IgG antibodies reactive with plate-bound antigens were detected with fluorescence-conjugated anti-human IgG. Compared with patients who did not develop cGVHD and healthy donors, patients who developed cGVHD showed significantly higher levels of IgG reactive against cell-surface antigens (cGVHD vs HD, p=0.0124; cGVHD vs no GVHD, p=0.0027). High-dose steroid therapy significantly reduced the level of these antibodies (cGVHD vs cGVHD-T, p=0.0009). We also examined the presence of these antibodies at various times following HSCT. While patients who did not develop cGVHD had persistently low levels of these antibodies over a 3-year period, patients who developed cGVHD during this time had persistently high levels of these antibodies. These results indicate that in patients who developed cGVHD after transplant, B-cell differentiation and the production of antibodies that target living cells were enhanced. These data further support a role for B cells and antibodies in the development of cGVHD. Further characterization of the specificity of these antibodies will enhance our understanding of the role played by donor B cells in the development of cGVHD and provide new potential targets for therapy. Disclosures Koreth: prometheus labs inc: Research Funding; kadmon corp: Membership on an entity's Board of Directors or advisory committees; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; amgen inc: Consultancy; LLS: Research Funding; millennium pharmaceuticals: Research Funding. Armand:Roche: Research Funding; Pfizer: Research Funding; Infinity Pharmaceuticals: Consultancy; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Soiffer:GentiumSpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Soluble FAS Ligand directs human memory B cells towards antibody-secreting cells

2020 ◽  
Author(s):  
Saskia D. van Asten ◽  
Peter-Paul Unger ◽  
Casper Marsman ◽  
Sophie Bliss ◽  
Tineke Jorritsma ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Fas Ligand ◽  
Memory B Cells ◽  
B Cell Differentiation ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Antibody Secreting Cells

AbstractDifferentiation of antigen-specific B cells into class-switched, high affinity antibody-secreting cells provides protection against invading pathogens but is undesired when antibodies target self-tissues in autoimmunity, beneficial non-self blood transfusion products or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired antibody formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 antibodies, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive antibodies, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Sign in / Sign up

Export Citation Format

Share Document