Donor-specific Antibody Responses Research Articles

Marginal zone B cells are required for optimal humoral responses to allograft

Antibody-mediated rejection (AMR) is among the leading causes of graft failure in solid organ transplantation. However, AMR treatment options are limited by incomplete understanding the mechanisms underlying de novo donor specific antibody (DSA) generation. The development of pathogenic isotype-switched DSA in response to transplanted allografts is typically attributed to follicular B cells undergoing germinal center reaction whereas the contribution of other B cell subsets have not been previously addressed. The current study investigated the role of recipient marginal zone (MZ) B cells in DSA responses using a mouse models of heart and renal allotransplantation. MZ B cells rapidly differentiate into antibody-secreting cells in response to allotransplantation. Despite selective depletion of FO B cells in heart allograft recipients, MZ B cells are sufficient for T-dependent IgM and early IgG DSA production. Furthermore, the presence of intact MZ B cell subset is required to support generation of pathogenic isotype-switched DSA in renal allograft recipients containing donor-reactive memory helper T cells. These findings are the first demonstration for the role of MZ B cells in humoral alloimmune responses following solid organ transplantation and identify MZ B cells as a potential therapeutic target for minimizing de novo DSA production and AMR in transplant recipients.

CTLA4-Ig alleviates the allogeneic immune responses against insulin-producing cells in a murine model of cell transplantation.

The adoptive transfer of insulin-producing cells (IPCs) is one of the promising treatments for insulin-dependent diabetes mellitus. While the use of allogeneic cell resources is inevitable in the case of a series of patients, alloimmune responses are a major barrier ahead of the successful implementation of allogeneic therapeutic cells. This study is aimed at evaluating the potential of CTLA4-Ig, as an approved immunomodulatory biologic, in protecting the IPCs against allogeneic immune responses. The C57BL/6 and BALB/c mice were used to establish a murine model of allogeneic cell transplantation. The mouse bone-marrow-derived mesenchymal stem cells were in vitro differentiated into IPCs, and the in vitro as well as the in vivo immune responses against IPCs were evaluated in the presence and absence of CTLA4-Ig. The allogeneic IPCs induced the in vitro activation of CD4+ T-cells, IFN-γ release, and the proliferation of lymphocytes, which all were controlled by CTLA4-Ig. Upon in vivo transfer of IPC into an allogeneic host, the splenic CD4+ and CD8+ T-cells exhibited a significant activation, and there was a significant donor-specific antibody response. Either of the mentioned cellular and humoral responses were modulated by a CTLA4-Ig regimen. This regimen also reduced the infiltration of CD3+ T-cells into the IPC injection site along with the improved overall survival of diabetic mice. CTLA4-Ig could be a complementary therapy for improving the efficacy of allogeneic IPC therapy through modulating the cellular and humoral responses that can lead to prolonged durability of IPCs within an allogeneic host.

Taking the A(llorecognition) train: connecting passenger T cells to DSA

Refers to: Charmetant X, Chen C-C, Hamada S, et al. Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation. Sci Transl Med. 2022;14:eabg1046.

Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

MO942: Regulatory T and B Cell Responses are Equally Compromised During Antibody-Mediated Rejection of Kidney Allografts

Abstract BACKGROUND AND AIMS Although considerable advances have been made in understanding the cellular effector mechanisms responsible for donor-specific antibody (DSA) generation leading to antibody-mediated rejection (ABMR), the identification of cellular regulators of such immune responses is lacking. METHOD In a cohort of 96 kidney transplant recipients, we used high-dimensional flow cytometry to concomitantly profile and track the two major subsets of regulatory lymphocytes in the blood: T regulatory (TREG) and transitional B (TrB) cells. Additionally, we established co-culture assays to address their respective capacities to suppress antibody responses in vitro. RESULTS TREG and TrB were potent suppressors of T follicular helper-mediated B cell differentiation into plasmablast and antibody generation. While TREG and TrB were both durably expanded in patients who did not develop DSA post-transplant, patients who manifested DSA and progressed to ABMR displayed a marked and persistent numerical reduction in TREG and TrB cells. Strikingly, specific cell clusters expressing the transcription factor T-bet were selectively depleted in both TREG and TrB compartments in patients with ABMR. Importantly, the coordinated loss of these T-bet + CXCR5+ TREG and T-bet+CD21–TrB cell clusters was correlated with increased and inflammatory DSA responses, more extensive microvascular inflammation and a higher rate of kidney allograft loss. CONCLUSION Our study identified coordinated and persistent defects in the regulatory T and B cell responses in patients undergoing ABMR, which may contribute to their loss of humoral immune regulation and thus warranting timely therapeutic interventions to replenish and sustain TREG and TrB cells in these patients.

Concomitant loss of regulatory T and B cells is a distinguishing immune feature of antibody-mediated rejection in kidney transplantation

Although considerable advances have been made in understanding the cellular effector mechanisms responsible for donor-specific antibody generation leading to antibody-mediated rejection (ABMR), the identification of cellular regulators of such immune responses is lacking. To clarify this, we used high dimensional flow cytometry to concomitantly profile and track the two major subsets of regulatory lymphocytes in blood: T regulatory (TREG) and transitional B cells in a cohort of 96 kidney transplant recipients. Additionally, we established co-culture assays to address their respective capacity to suppress antibody responses invitro. TREG and transitional B cells were found to be potent suppressors of T follicular helper-mediated B-cell differentiation into plasmablast and antibody generation. TREG and transitional B cells were both durably expanded in patients who did not develop donor-specific antibody post-transplant. However, patients who manifested donor-specific antibody and progressed to ABMR displayed a marked and persistent numerical reduction in TREG and transitional B cells. Strikingly, specific cell clusters expressing the transcription factor T-bet were selectively depleted in both TREG and transitional B-cell compartments in patients with ABMR. Importantly, the coordinated loss of these T-bet+CXCR5+TREG and T-bet+CD21- transitional B-cell clusters was correlated with increased and inflammatory donor specific antibody responses, more extensive microvascular inflammation and a higher rate of kidney allograft loss. Thus, our study identified coordinated and persistent defects in regulatory T- and B-cell responses in patients undergoing ABMR, which may contribute to their loss of humoral immune regulation, and warrant timely therapeutic interventions to replenish and sustain TREG and transitional B cells in these patients.

Reversing donor-specific antibody responses and antibody-mediated rejection with bortezomib and belatacept in mice and kidney transplant recipients.

Active antibody-mediated rejection (AMR) is a potentially devastating complication and consistently effective treatment remains elusive. We hypothesized that the reversal of acute AMR requires rapid elimination of antibody-secreting plasma cells (PC) with a proteasome inhibitor, bortezomib, followed by the sustained inhibition of PC generation with CTLA4-Ig or belatacept (B/B). We show in mice that B/B therapy selectively depleted mature PC producing donor-specific antibodies (DSA) and reduced DSA, when administered after primary and secondary DSA responses had been established. A pilot investigation was initiated to treat six consecutive patients with active AMR with B/B. Compassionate use of this regimen was initiated for the first patient who developed early, severe acute AMR that did not respond to steroids, plasmapheresis, and intravenous immunoglobulin after his third kidney transplant. B/B treatment resulted in a rapid reversal of AMR, leading us to treat five additional patients who also resolved their acute AMR episode and had sustained disappearance of circulating DSA for ≤30months. This study provides a proof-of-principle demonstration that mouse models can identify mechanistically rational therapies for the clinic. Follow-up investigations with a more stringent clinical design are warranted to test whether B/B improves on the standard of care for the treatment of acute AMR.

Blockage of Interleukin-6 Pathway Using Therapeutic Anti-IL6 vs. Anti-IL6R Has Differential Effects on Alloantibody Suppression and IL-6 Homeostasis

Alloantibody-mediated rejection (AMR) and failure of desensitization in highly sensitized patients remain significant problems in transplantation. Interleukin 6, a pleomorphic cytokine with important roles in immune regulation and inflammation, is a potential target for new therapeutic strategies in combating AMR. Suppression of donor specific antibody (DSA) by IL6 system blockage was studied using an anti-IL6 receptor (anti-IL6R) and an anti-IL6 antibody in a mouse model of allosensitization by skin allograft. Both anti-IL6R and anti-IL6 treatments significantly suppressed serum amyloid A (SAA), an acute phase reactant induced by IL-6 (p<0.01 vs. control) following skin transplantation. De novo DSA, including both IgM and IgG were significantly reduced by anti-IL6R (p<0.01 vs. control group) and by a 3-dose regimen of anti-IL6 (p<0.05). Interestingly, an intensified dosing regimen of anti-IL6 (15-dose group) consisting of 15 daily IP injections had no effect (p>0.05) in DSA responses despite robust SAA inhibition. Serum IL-6 concentrations in the 15-dose group significantly increased by 2.6-fold at day 1(p<0.01 vs. control), 24-fold at day 7(p<0.01), and 188-fold (p<0.01) at day 14, indicating profound disturbance in IL-6 homeostasis. In contrast, 3-dose anti-IL6 treatment and anti-IL6R treatment, which exhibited evidence of DSA suppression exhibited a modest accumulation of IL-6 and soluble IL-6R in the blood. In recall alloantibody responses to a 2nd skin allograft, anti-IL6R treatment was able to significantly attenuate DSA IgG responses (p<0.05 vs. control). Anti-IL6 treatment, which exhibited a weighty increase in blood IL6, however, failed to suppress DSA. Targeting of IL-6 pathway with pharmaceutic antibodies is an applicable approach to allogeneic desensitization in transplantation. To achieve a safe and effective antibody suppression, we are obligated further investigation to determine the root cause of IL-6 accumulation in the blood and the mechanism(s) by which anti-IL6 antibodies induce disturbance in homeostasis of IL6 and soluble IL6R.

Regulatory CD8 T cells that recognize Qa-1 expressed by CD4 T-helper cells inhibit rejection of heart allografts.

Induction of longstanding immunologic tolerance is essential for survival of transplanted organs and tissues. Despite recent advances in immunosuppression protocols, allograft damage inflicted by antibody specific for donor organs continues to represent a major obstacle to graft survival. Here we report that activation of regulatory CD8 T cells (CD8 Treg) that recognize the Qa-1 class Ib major histocompatibility complex (MHC), a mouse homolog of human leukocyte antigen-E (HLA-E), inhibits antibody-mediated immune rejection of heart allografts. We analyzed this response using a mouse model that harbors a point mutation in the class Ib MHC molecule Qa-1, which disrupts Qa-1 binding to the T cell receptor (TCR)-CD8 complex and impairs the CD8 Treg response. Despite administration of cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig), Qa-1 mutant mice developed robust donor-specific antibody responses and accelerated heart graft rejection. We show that these allo-antibody responses reflect diminished Qa-1-restricted CD8 Treg-mediated suppression of host follicular helper T cell-dependent antibody production. These findings underscore the critical contribution of this Qa-1/HLA-E-dependent regulatory pathway to maintenance of transplanted organs and suggest therapeutic approaches to ameliorate allograft rejection.

Reducing Donor-specific Antibody During Acute Rejection Diminishes Long-term Renal Allograft Loss: Comparison of Early and Late Rejection

Reduction in donor-specific antibody (DSA) has been associated with improved renal allograft survival after antibody-mediated rejection (AMR). These observations have not been separately analyzed for early and late AMR and mixed acute rejection (MAR). The purpose of this study was to evaluate long-term responses to proteasome inhibitor-based therapy for 4 rejection phenotypes and to determine factors that predict allograft survival. Retrospective cohort study evaluating renal transplant recipients with first AMR episodes treated with proteasome inhibitor-based therapy from January 2005 to July 2015. A total of 108 patients were included in the analysis. Immunodominant DSA reduction at 14 days differed significantly (early AMR 79.6%, early MAR 54.7%, late AMR 23.4%, late MAR 21.1%, P < 0.001). Death-censored graft survival (DCGS) differed at 3 years postrejection (early AMR 88.3% versus early MAR 77.8% versus late AMR 56.7% versus late MAR 54.9%, P = 0.02). Multivariate analysis revealed that immunodominant DSA reduction > 50% at 14 days was associated with improved DCGS (odds ratio, 0.12, 95% CI, 0.02-0.52, P = 0.01). In summary, significant differences exist across rejection phenotypes with respect to histological and DSA responses. The data suggest that DSA reduction may be associated with improved DCGS in both early and late AMR.

The pathology of solid organ xenotransplantation.

The use of genetically modified pigs has resulted in prolonged xenograft organ survival, overcoming the initial barriers that lead to hyperacute rejection and immediate loss of the graft. The purpose of the present review is to revisit the xenogeneic response and the pathologic changes in the xenograft organ in the context of recent publications of large animal studies that highlight existing challenges. Transgenic modifications that have included complement regulatory proteins and coagulation regulatory proteins have prolonged xenograft survival in pig to nonhuman primate kidneys, livers, and hearts. Modifications of immunosuppressive regimens such as the addition of mTOR inhibition and costimulatory blockade have also led to better outcomes. Antibody-mediated rejection and thrombotic microangiopathy persist as primary challenges to the field and require further systematic exploration. The efforts to overcome the natural antibody response to xenoantigens are largely sufficient. There is great opportunity for designing immunosuppression protocols and for detecting early coagulopathies, complement activation, and donor-specific antibody response. With graft survival prolongation, there is also a greater need to understand mechanisms and to enhance diagnostic tools for pathologic evaluation.

OR21 Detection of de novo donor specific antibody response by next generation sequencing B cell receptor

OR21 Detection of de novo donor specific antibody response by next generation sequencing B cell receptor

Cell Kinetics of Blood Circulating CD38+/CD138+ Plasma Cells During the Development of De Novo Donor Specific Antibody Responses

Emerging of donor specific antibodies (DSA) following transplantation is an indicator of allogenic sensitization and a predisposing factor for antibody mediated rejection. Though various methods of alloantibody detection exist, a timely and accurate test to identify allo-sensitization has not been clinically available. We report here, a study of blood CD38+/CD138+ plasma cells during the development of DSA responses in an attempt to develop a new tool for early diagnosis of allo-sensitization.

CD4+ T Cell Help Is Mandatory for Naive and Memory Donor-Specific Antibody Responses: Impact of Therapeutic Immunosuppression.

Antibody-mediated rejection is currently the leading cause of transplant failure. Prevailing dogma predicts that B cells differentiate into anti-donor-specific antibody (DSA)-producing plasma cells only with the help of CD4+ T cells. Yet, previous studies have shown that dependence on helper T cells decreases when high amounts of protein antigen are recruited to the spleen, two conditions potentially met by organ transplantation. This could explain why a significant proportion of transplant recipients develop DSA despite therapeutic immunosuppression. Using murine models, we confirmed that heart transplantation, but not skin grafting, is associated with accumulation of a high quantity of alloantigens in recipients’ spleen. Nevertheless, neither naive nor memory DSA responses could be observed after transplantation of an allogeneic heart into recipients genetically deficient for CD4+ T cells. These findings suggest that DSA generation rather result from insufficient blockade of the helper function of CD4+ T cells by therapeutic immunosuppression. To test this second theory, different subsets of circulating T cells: CD8+, CD4+, and T follicular helper [CD4+CXCDR5+, T follicular helper cells (Tfh)], were analyzed in 9 healthy controls and 22 renal recipients. In line with our hypothesis, we observed that triple maintenance immunosuppression (CNI + MMF + steroids) efficiently blocked activation-induced upregulation of CD25 on CD8+, but not on CD4+ T cells. Although the level of expression of CD40L and ICOS was lower on activated Tfh of immunosuppressed patients, the percentage of CD40L-expressing Tfh was the same than control patients, as was Tfh production of IL21. Induction therapy with antithymocyte globulin (ATG) resulted in prolonged depletion of Tfh and reduction of CD4+ T cells number with depleting monoclonal antibody in murine model resulted in exponential decrease in DSA titers. Furthermore, induction with ATG also had long-term beneficial influence on Tfh function after immune reconstitution. We conclude that CD4+ T cell help is mandatory for naive and memory DSA responses, making Tfh cells attractive targets for improving the prevention of DSA generation and to prolong allograft survival. Waiting for innovative treatments to be translated into the clinical field ATG induction seems to currently offer the best clinical prospect to achieve this goal.

Immunological characterization of de novo and recall alloantibody suppression by CTLA4Ig in a mouse model of allosensitization

It is well known that CTLA4Ig inhibits allogenic T-cell activation in transplantation. The immunological features and mechanisms associated with alloantibody suppression by CTLA4Ig, however, are poorly understood. Here, we used a mouse model of allosensitization to evaluate the efficacy of CTLA4Ig (abatacept) in suppression of donor-specific antibody (DSA) during de novo and recall alloantibody responses. We found that abatacept inhibited de novo DSA IgM and IgG responses to HLA-A2 expressing skin grafts. Abatacept administered during primary T cell priming also reduced recall IgG responses induced by re-immunization. Suppression of de novo DSA responses by abatacept is associated with a reduction in splenic expression of the germinal center activation marker GL7 and a reduction of CD4+PD1+CXCR5+ follicular T helper (Tfh) subset in splenic lymphocytes detected by flow cytometry. The efficacy of abatacept on recall DSA suppression is moderate. In vitro experiments demonstrated that abatacept inhibited DSA IgG secretion by CD138+ plasma cells isolated from allograft recipients. Additional experiments using an IgG1 secreting mouse hybridoma cell line showed that abatacept binds to CD80 expressed on these cells with subsequent inhibition of cell proliferation and reduction in IgG ELISpot formation. In conclusion, CTLA4Ig is a potent suppressor of de novo DSA responses and also affects recall responses. The data suggests modification of recall DSA responses is due to a direct suppressive effect on plasma cells.

Anti-huCD20 Antibody Therapy for Antibody-Mediated Rejection of Renal Allografts in a Mouse Model

We have reported that B6.CCR5(-/-) mice reject renal allografts with high serum donor-specific antibody (DSA) titers and marked C4d deposition in grafts, features consistent with antibody-mediated rejection (AMR). B6.huCD20/CCR5(-/-) mice, where human CD20 expression is restricted to B cells, rejected A/J renal allografts by day 26 posttransplant with DSA first detected in serum on day 5 posttransplant and increased thereafter. Recipient treatment with anti-huCD20 mAb prior to the transplant and weekly up to 7 weeks posttransplant promoted long-term allograft survival (>100 days) with low DSA titers. To investigate the effect of B cell depletion at the time serum DSA was first detected, recipients were treated with anti-huCD20 mAb on days 5, 8, and 12 posttransplant. This regimen significantly reduced DSA titers and graft inflammation on day 15 posttransplant and prolonged allograft survival >60 days. However, DSA returned to the titers observed in control treated recipients by day 30 posttransplant and histological analyses on day 60 posttransplant indicated severe interstitial fibrosis. These results indicate that anti-huCD20 mAb had the greatest effect as a prophylactic treatment and that the distinct kinetics of DSA responses accounts for acute renal allograft failure versus the development of fibrosis.

Achieving Incompatible Transplantation through Desensitization: Current Perspectives and Future Directions

The application of life-saving transplantation is severely limited by the shortage of organs, and histoincompatibility. To increase transplant rates in sensitized patients, new protocols for HLA and blood type incompatible (ABOi) desensitization have emerged. These approaches require significant desensitization using intravenous immunoglobulin, rituximab and plasma exchange. In addition, the development of donor-specific antibody responses post transplant is the major cause of allograft failure with return to dialysis. This increases patient morbidity/mortality and cost. Immunotherapeutic agents used for desensitization evolved from drug development in oncology and autoimmune diseases. Currently, there is a renaissance in development of novel drugs likely to improve antibody reduction in transplantation. These include agents that inactivate IgG molecules, anticytokine antibodies, costimulatory molecule blockade, anticomplement agents and therapies aimed at the plasma cell.

Successful Desensitization With Combination of Costimulation Blockade and Bortezomib Via Regulating Plasma Cells and Follicular Helper T Cells in Sensitized Rhesus Model.

[Background] Preformed donor specific antibody (DSA) affects a significant population of sensitized transplant patients, portending a poorer prognosis in graft survival. We investigated the effect of proteasome inhibitor and costimulation blockades (COB) on DSA responses and plasma cells in pre-sensitized rhesus macaques in order to develop an effective desensitization regimen. [Method] All recipients received a fully mismatched skin graft. Grafts were all rejected within 2 weeks without treatment. Animals received Bortezomib alone (N=3, 1.33mg/m2), Belatacept/2C10R4 (N=3, 20mg/kg), or combined Belatacept/2C10R4/Bortezomib (N=3) twice a week for 4 weeks upon stabilization of their DSA levels. T and B cell phenotypes and DSA were assessed with flow cytometry. Bone marrow plasma cells were evaluated by ELISPOT. Sensitized animals received kidney transplantation from the same donor as their skin graft with or without desensitization in the presence of induction/maintenance immunuosuppression (Basiliximab/Prograf/Cellcept). [Results] The combination of COB and bortezomib showed the most efficient reduction of BM plasma cells compared to Bortezomib alone and Belatacept/2C10R4 treatment (9.6±1.2 vs. 25.6±12.2 and 45.8±0.6%; p<0.05). Triple drug treatment also showed the greatest reduction of serum DSA. Isotype switched IgG+ B cells were reduced by combined treatment that could reflect Tfh cell activity. Finally, we confirmed that LN follicular helper T cells (Tfh; PD-1hiICOShiCD4+) were reduced with triple treatment.Figure: No Caption available.Renal allografts without desensitization showed accelerated rejection (N=5, MST=3.6±2.7). In contrast, desensitization with triple treatment has resulted in no signs of rejection on POD 22. [Conclusion] Desensitization with Bortezomib alone moderately reduces plasma cells but induces germinal center responses reflected by Tfh cell populations. However, targeting costimulation signals via Belatacept (CTLA4-Ig) and 2C10R4 (anti-CD40L) in conjunction with Bortezomib profoundly reduced Tfh cell populations, plasma cell number, and serum DSA.

Characterization of Donor Specific Antibody Response Following Liver Transplantation in Rats.

Characterization of Donor Specific Antibody Response Following Liver Transplantation in Rats.

Selective CD28 Blockade Exhibits Superior Suppression of Donor-Specific Antibody Relative to CTLA4Ig.

Costimulation blockade in the form of CTLA4Ig is known to attenuate donor-specific antibody responses. However, CTLA4Ig indiscriminately blocks both the CD28 costimulatory and CTLA4 co-inhibitory pathways. While follicular helper T (TFH) cell development and germinal center (GC) formation are CD28-dependent, CTLA4 has been shown to play a critical role in suppressing the provision of T cell help for GC B cell function and immunoglobulin production. Thus, selective CD28 blockade has potential to inhibit GC development while garnering the inhibitory effects of intact CTLA4 signaling on T cell-dependent antibody responses. To test this possibility, we used a T cell receptor transgenic murine skin allograft model to compare a novel CD28-specific domain antibody (dAb) to CTLA4Ig. The selective CD28 dAb resulted in superior skin allograft survival in comparison to CTLA4Ig (MST >100d vs. 34d). Importantly, animals treated with the anti-CD28 dAb experienced a greater reduction in donor-specific IgG as compared to those treated with CTLA4Ig. Flow cytometric analysis of donor-reactive CD4+Thy1.1+ T cells from draining lymph nodes revealed increased expression of CTLA4 on TFH cells (CXCR5+GL7+/-) compared to naïve and non-TFH cells (CXCR5-GL7-), with the greatest level of CTLA4 expression on maximally polarized GC TFH cells (CXCR5+GL7+). Analysis of donor-specific CD4+Thy1.1+ T cells also revealed a greater reduction in the frequency and absolute number of TFH cells (CXCR5+GL7+/-) with selective CD28 blockade as compared to CTLA4Ig. Additionally, we observed a reciprocal increase in the frequency and absolute number of naïve and non-TFH cells (CXCR5-GL7-) that was greatest with CD28 dAb treatment. These findings suggest that selective CD28 blockade in the presence of intact CTLA4 signaling has a greater inhibitory effect on T cell dependent antibody responses than CTLA4Ig. DISCLOSURE:Suchard, S.: Employee, Bristol-Myers Squibb. Nadler, S.: Employee, Bristol-Myers Squibb.