The role of CTLA-4 in induction and maintenance of peripheral T cell tolerance

T cell receptor engagement and the B7-CD28 / CTLA-4 signaling pathways play critical roles in T cell activation and regulation. CD28 engagement results in T cell activation, differentiation and survival while CTLA-4 signals block IL-2 production, cell cycle progression and T cell differentiation. We explored the role of CTLA-4 in peripheral tolerance induced by intravenous administration of ethylene carbodiimide-fixed, antigen-coupled splenocytes in the PLP139 - 151-induced relapsing experimental autoimmune encephalomyelitis system. Tolerance induction with PLP139 - 151-coupled splenocytes correlates with low B7 expression on the fixed antigen-presenting cells, conditions that would favor CTLA-4-mediated inhibition. Administration of CTLA-4Ig or anti-CTLA-4 concomitant with the 'tolerogenic' stimulus, however, failed to reverse tolerance induction. In contrast, blocking CTLA-4 at the time of secondary 'immunogenic' encounter with antigen reversed the tolerant state. These findings indicate that CTLA-4 is required to maintain the unresponsive state of the tolerized T cells upon antigenic stimulation under inflammatory conditions and, therefore, have important implications for therapeutic regulation of autoimmune disease.     

Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses

Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.     

The B7/CD28 costimulatory family in autoimmunity

Summary: Host defense is dependent on the appropriate induction of immune responses. A central concept in immunology is the ability of the immune system to differentiate foreign from self‐antigens. The failure of the immune response to recognize foreign pathogens can result in infection and disease in the host. The inappropriate response of the immune system to self‐antigens is equally problematic, leading to autoimmune disease. Central and peripheral tolerance mechanisms control self‐reactive T‐cell responses and protect peripheral tissues from autoimmune attack. This review examines the roles of B7/CD28 family members, which can augment or antagonize T‐cell receptor signaling, in the regulation of central and peripheral T‐cell tolerance. We also discuss how B7/CD28 pathways influence both T‐cell‐intrinsic and ‐extrinsic mechanisms of regulation.     

Differential requirements for co-stimulatory signals from B7 family members by resting versus recently activated memory T cells towards soluble recall antigens

The interaction between CD28 on T cells with CD80 (B7-1) andCD86 (B7-2) on APCs is considered to be of critical importancefor primary T cell activation both in vivo and in vitro. Therelative importance of this co-stimulatory signal in memoryT cell activation is, however, less clear, and was thereforestudied by in vitro experiments on T cell responses to solublerecall antigens using peripheral blood mononuclear cells orT cell clones. Our data demonstrate that B7-2 represents themajor co-stimulatory signal for the activation of resting peripheralblood memory T cells with recall antigens, as evidenced by theeffects of anti-B7-1 and anti-B7-2 on T cell proliferation aswell as on IL-2 and INF-y production. Since CTLA-4-lg and anti-CD28Fab fragments had similar inhibitory effects to the combinationof anti-B7-1 plus anti-B7-2, the involvement of a third co-stimulatoryCD28/CTLA-4 ligand is unlikely. Despite the strong effects ofB7-blocking agents, a variable fraction of the memory T cellswas resistant to inhibition. Moreover, T cell clones or in vitropreactivated T cells could efficiently be restimulated by solubleantigens on autologous APCs in the absence of B7-1 or B7-2 co-stimulation.These data show that most memory T cells that are freshly isolatedfrom the blood are still dependent on CD28 triggering for theiractivation. However, recently activated T cells can apparentlybypass the requirement for B7 and use other co-stimulatory signalsfor reactivation, a finding with important implications forthe development of immunosuppressive strategies.     

Involvement of CTLA-4 in T-cell anergy induced by staphylococcal enterotoxin A in vitro

Superantigens, like staphylococcal enterotoxin A (SEA), induce a strong proliferative response followed by clonal deletion of a substantial portion of defined V(beta) T-cells. The remaining cells display in vitro anergy. Anergy is a major mechanism to ensure antigen-specific tolerance in T-lymphocyte in the adult. Co-stimulatory molecules B7-1 (CD80)/B7-2 (CD86) and their counter-receptors CD28/CTLA-4 play pivotal roles in T-cell activation and immune regulation. While increasing data further suggested a role for CTLA-4 in regulating T-cell tolerance in vivo, the mechanism by which CTLA-4 influences T-lymphocyte tolerance is unclear. In the present study, we established an in vitro anergy model using superantigen SEA as the anergizing agents and examined CD3, CD28 and CTLA-4 expression of anergic T-cells in response to SEA rechallenge. It is found that anergic T-cell fails to produce the autocrine growth factor interleukin-2 (IL-2) upon stimulation, and addition of exogenous IL-2 can reverse the anergic state. Both TCR/CD3 complex and CD28 expression is not reduced in anergic cells during whole immune response, but the expression of CTLA-4 on the cell surface is enhanced dramatically in the late stages of an immune response. Using CTLA-4/B7-blocking agent, we found T-cell anergy was aborted and anergic T-cells restored the ability to proliferate and produce IL-2, suggesting that CTLA-4 may play a critical role in the induction of T-cell anergy.     

CD28/CTLA-4/B7 and CD40/CD40L costimulation and activation of regulatory T cells

Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.     

B7-CD28 interaction is a late acting co-stimulatory signal for human T cell responses

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered as the most important co-stimulatory signal for T cell activation. APC in a resting condition express either no or only low levels of B7 molecules. These are up-regulated as a result of interactions with activated T cells, thus suggesting that B7-CD28 interaction is not required at initiation of T cell activation. To study this issue, we blocked B7-CD28 interaction at various time points after in vitro stimulation of peripheral blood T cells with allogeneic monocytes. Epstein-Barr virus-transformed B cells or soluble antigens. We observed that T cell proliferation and IL-2 production were inhibited by B7- blocking agents (CTLA-4-Ig or anti-B7 mAb) almost to the same degree when added either at initiation of culture or 24 h later. B7-blocking agents still resulted in significant inhibition of allogeneic T cell activation when added after 48 h. Furthermore, when CTLA-4-Ig was added at the start of an allogeneic T cell stimulation, addition of anti-CD28 mAb after 24 h of culture nearly fully restored T cell proliferation to control levels. Finally, we demonstrate that delayed addition of B7- blocking agents together with cyclosporin A 1 day after the onset of culture of T cells with allogeneic B cells is highly efficient to induce energy as evaluated by lack of proliferation, cytotoxic T lymphocyte reactivity and IFN-gamma or IL-5 production upon alloantigen rechallenge. Taken together, our data can explain why B7 expression on APC is not required at the time of initial APC-T cell contact, and suggest that the effect of the CD28 signal indeed consists in prolonging IL-2 production and amplifying T cell responses, rather than in providing a critical co-stimulatory signal at the time of initial TCR triggering.      

Phenotypic analysis of CTLA-4 and CD28 expression during transient peptide-induced T cell activation in vivo.

The T cell co-stimulatory receptors CD28 and CTLA-4 appear to have opposite effects on T cell activation, mediating augmentation and inhibition of T cell responses respectively. Since these two receptors use the same ligands, CD80 (B7-1) and CD86 (B7-2), the co-ordinate timing of CD28 and CTLA-4 expression has a major impact on the regulation of immune responses. While the kinetics of co-stimulatory molecules have been established for T cell stimulation in vitro, little is known about CD28 and CTLA-4 expression in response to T cell activation in vivo. In this study we have investigated the kinetics of CD28 and CTLA-4 expression upon CD4(+) T cell activation in response to soluble peptide in vivo. Using mice transgenic for a T cell receptor specific for the I-Au-restricted N-terminal peptide of myelin basic protein MBP Ac1-9, we show maximal up-regulation of both CD28 and CTLA-4 2 days after peptide administration. CTLA-4 expression correlated positively with early activation markers on the same cells and was high on blast cells. Administration of peptide analogs with higher affinity for I-Au MHC class II revealed a higher increase in CTLA-4 than in CD28 expression in response to improved TCR ligation. Further, a small population of CD4(+) T cells expressing CTLA-4, CD25 and CD45RBlow was identified in mice that had not been treated with specific peptide. The implications of these observations for immune regulation are discussed.     

Role of the CTLA-4 receptor in t cell activation and immunity

Costimulatory molecules of the B7 family regulate the activation of T lymphocytes. T cell activation is promoted by binding of B7 molecules to CD28 and inhibited by binding to CTLA-4 (CD152). The balance between positive signals through CD28 and negative signals through CTLA-4 is critical for the fate of the T cell and is subject to tight regulation. Recent in vitro and in vivo studies have significantly advanced our understanding of the function of the CTLA-4 receptor. The results of these experiments suggest that CTLA-4 is critical for the induction of self-tolerance, and that it may have distinct signaling functions in resting and activated T cells. In resting T cells, CTLA-4 crosslinking leads to cell-cycle arrest, whereas in activated T cells, CTLA-4 crosslinking induces apoptosis. In this article, we will review the physiologic functions of the CTLA-4 receptor.     

Regulation of CD28 expression on CD8+ T cells by CTLA-4

CD28 and CTLA-4 are the critical costimulatory receptors that predominantly determine the outcome of T cell stimulation, with CD28 promoting positive costimulation and CTLA-4 inducing inhibitory signals. Blockage of the B7-CD28/CTLA-4 pathway leads to transplantation tolerance. However, the exact mechanism of the inhibitory function of CTLA-4 remains elusive. Here, we investigated the influence of CTLA-4 expression on CD28 using CTLA-4-transfected Jurkat T cells as well as primary T cells. Up-regulation of CTLA-4 induced abrogation of IL-2 production, indicating an anergic phenotype of CTLA-4(high) T cells. Besides the negative signaling function of CTLA-4, we show for the first time that CTLA-4 expression promotes the down-regulation of CD28 on the T cell surface as a result of enhanced internalization and degradation of CD28. These data suggest that apart from the established competition for B7.1 and B7.2 by CTLA-4, inhibition of T cells by CTLA-4 might be additionally explained by reduction of CD28 on the cell surface, which might impede T cell response to stimulation. Our data provide a previously unrecognized mechanism for T cell regulation.     

B7-h2 is a costimulatory ligand for CD28 in human

CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T?cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for?the costimulation of human T?cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27(kip1), and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.     

CTLA-4 and autoimmunity: New insights into the dual regulator of tolerance

Cytotoxic T-Lymphocye Antigen 4 (CTLA-4) or CD152 is an inhibitory molecule that plays a critical role in maintenance of tolerance to self-antigens. CTLA-4 is structurally as well as functionally related to CD28, since it shares 31% of homology and binds the B7 family molecules CD80 and CD86 with higher affinity. Nevertheless, CTLA-4 has opposing effects on T cell activation and current evidence shows that its inhibitory role goes beyond the ligand-binding interaction. CTLA-4 competes with CD28 in binding to B7, interacts within the immunological synapsis elements and with clathrin adaptor proteins and tyrosine phosphatases through its cytoplasmic domain to regulate cell trafficking and to set the activation threshold within T cells. Moreover, we have learned from the knock out model that CTLA-4 plays a key role in regulatory T cells and in central tolerance. Because of its importance in maintenance of peripheral tolerance, CTLA-4 has been implicated in several autoimmune diseases, such as systemic lupus erythematosus. Multiple single-nucleotide polymorphisms have been located to human Ctla-4 gene, and their association with autoimmune disease is still a matter of controversy. Despite the promising results of abatacept or CTLA-4-Ig in rheumatoid arthritis and murine lupus nephritis, more clinical randomized trials and standardization of outcomes are needed to prove its efficacy and safety in human lupus nephritis.     

Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production.

Although CD28 triggering provides an important co-stimulatory signal to T cells, blocking the CD80/CD86 - CD28 interaction with CTLA-4lg fusion protein is not sufficient for tolerance induction in vivo or in vitro. According to more recent data, interruption of the CD40 - CD154 interaction might complement the effect of CTLA-4lg and induce graft acceptance. We studied the effects of a blocking anti-CD40 monoclonal antibody (mAb) and/or blocking anti-CD80/anti-CD86 mAb in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with allogeneic PBMC. T cells activated by alloantigens in the presence of anti-CD80, anti-CD86 and anti-CD40 entered a state of alloantigen-specific non-responsiveness as evidenced upon restimulation by lack of proliferation, cytotoxic activity, and IL-2, IL-5 and IL-13 production. IFN-gamma production during restimulation was less than in the control cultures, while the production of IL-10 was enhanced. Addition of recombinant IL-2 during the restimulation rescued alloantigen-specific activity. We conclude that the simultaneous blocking of the CD40 - CD154 and CD80/CD86 - CD28 interaction during allogeneic T cell activation induces T cell anergy. Since anergic cells induced by this treatment still produce high levels of IL-10, the latter could contribute to modulation of antigen-presenting cell activity and to bystander suppression of residual reactive T cells.     

Superantigen responses and co-stimulation: CD28 and CTLA-4 have opposing effects on T cell expansion in vitro and in vivo

Co-stimulation via the CD28/CTLA-4 system appears critical forT cell proliferation to peptide antigens presented in associationwith MHC. In this study, we examine the roles of CD28 and CTLA-4in the response of murine T cells to the superantigen staphylococcalenterotoxin B (SEB). In vitro, antibodies against B7-1/B7-2or Fab fragments of anti-CD28 antibodies significantly inhibitthe response of splenocytes to SEB. Conversely, Fab fragmentsof anti-CTLA-4 antibodies augment the proliferative response.Further, addition of blocking antibodies directed against B7-1/B7-2augment proliferation co-stimulated by intact anti-CD28 antibodies.These data support the hypothesis that CD28 and CTLA-4 exertopposing effects upon early T cell activation. In vivo, Intactanti-CD28 antibodies and non-stimulatory Fab fragments of anti-CD28appear to have similar inhibitory effects upon the expansionof V_ß8⁺ T cells. In contrast, both intact and Fabfragments of anti-CTLA-4 appear to amplify this expansion. Weconclude that the SEB response is significantly augmented byCD28-derived signaling and this in turn may be attenuated bysignals through CTLA-4.     

CTLA-4 promotes Foxp3 induction and regulatory T cell accumulation in the intestinal lamina propria

Thymic induction of CD4⁺Foxp3⁺ regulatory T (Treg) cells relies on CD28 costimulation and high-affinity T-cell receptor (TCR) signals, whereas Foxp3 (forkhead box P3) induction on activated peripheral CD4⁺ T cells is inhibited by these signals. Accordingly, the inhibitory molecule CTLA-4 (cytotoxic T-lymphocyte antigen 4) promoted, but was not essential for CD4⁺ T-cell Foxp3 induction in vitro. We show that CTLA-4-deficient cells are equivalent to wild-type cells in the thymic induction of Foxp3 and maintenance of Foxp3 populations in the spleen and mesenteric lymph nodes, but their accumulation in the colon, where Treg cells specific for commensal bacteria accumulate, is impaired. In a T cell–transfer model of colitis, the two known CTLA-4 ligands, B7-1 and B7-2, had largely redundant roles in inducing inflammation and promoting Treg cell function. However, B7-2 proved more efficient than B7-1 in inducing Foxp3 in vitro and in vivo. Our data reveal an unappreciated role for CTLA-4 in establishing the Foxp3⁺ compartment in the intestine.     

T cells are the main cell type expressing B7-1 and B7-2 in the central nervous system during acute, relapsing and chronic experimental autoimmune encephalomyelitis.

T cell co-stimulation through the CD28 receptor on T cells is critical to the induction of experimental autoimmune encephalomyelitis (EAE). In this study, expression of the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86), as well as the receptors CD28 and CTLA-4, were quantitated in central nervous system (CNS) tissues from mice at various stages of EAE. Immunohistochemistry and flow cytometry of CNS-infiltrating cells revealed a high percentage of infiltrating T cells expressing B7-1 and B7-2 during acute, chronic and relapsing EAE. Of the infiltrating cells 10-20% were CTLA-4(+), most of which were CD4(+) T cells. B7-1 and B7-2 expression within the CNS during active EAE might increase the potential for local activation of autoimmune T cells; however, the high level of expression of B7 molecules may also provide a mechanism for the autoregulation of activated CTLA-4(+) T cells.     

Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways

Summary: Classically, the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4) and B7 families of cell surface molecules regulate complex signaling pathways that profoundly affect T-cell responses. The recent identification and characterization of additional CD28 and B7 family members including programmed death-1 (PD-1), programmed death ligand-1 (PD-L1) (B7-H1), and PD-L2 (B7-DC) has added to the complexity and greater appreciation of how surface molecules control T-cell activation and peripheral tolerance. CD28/B7 interactions mediate co-stimulation and significantly enhance peripheral T-cell responses. CTLA-4, in contrast, interacting with the same B7 molecules, results in decreased T-lymphocyte activity and regulates the immune response. Similarly, PD-1 interactions with PD-L1 and PD-L2 downmodulate T-cell immune responses. Despite these similarities, the regulatory roles of the CTLA-4 and PD-1 pathways are distinct. This may be due, at least in part, to the differential expression patterns of the CTLA-4 and PD-1 ligands both temporally and spatially. This article examines the role of CTLA-4 and PD-1 in limiting autoreactivity and establishing peripheral self-tolerance with the hypothesis that CTLA-4 signals are required early in the lymph node during initiation of an immune response and PD-1 pathways act late at the tissue sites to limit T-cell activity.     

Negative co-receptors on lymphocytes

The past year has seen significant advances in our understanding of critical roles of negative immunoregulatory signals delivered through the B7-CD28 superfamily in regulating T cell activation and tolerance. Structural data on CTLA-4 have provided novel insights into the inhibitory functions of CTLA-4. Initial characterization of the PD-1-PD-1-ligand pathway has revealed that this pathway can downregulate TCR- and CD28-mediated signals. Recent studies indicate that ICOS exerts distinct effects at different phases of an immune response: ICOS can inhibit as well as stimulate T cell responses.     

FcR cross-linking on monocytes results in impaired T cell stimulatory capacity

Presentation of antigen to T lymphocytes without the appropriateco-stlmulatory signals results in a state of antigen-specificunresponsiveness. Despite the presumed importance of the B7-CD28interaction for the initiation and maintenance of T cell-mediatedimmune responses, relatively few studies have addressed theregulation of B7 expression. We have studied the expressionof the CD80 (B7-1) and B7-2 molecules on peripheral blood monocytesfollowing different activation signals, and it was demonstratedthat not only IFN-, but also granulocyte macrophage colony stimulatingfactor can induce CD80 expression on monocytes. In addition,we found that cross linking of FcR on monocytes strongly inhibitsthe up-regulation of CD80 and B7-2, with as a functional consequencethat the capacity to function as antigen presenting cells (APC)and to.stimulate T cell activation is severely impaired. Whencultures were prepared In 96-well plates coated with human IgG,stimulation of T cells with allogenelc monocytes resulted onlyin modest T cell proliferation and no detectable IL-2 secretionas compared with untreated culture plates or plates coated withFab fragments of human IgG. Under these conditions cross-linkingof CD28 on the T cells with specific mAb completely revertedthe inhibitory effect observed after culture on IgG-coated plates.Furthermore, FcR cross-linking on monocytes strongly Inhibitedthe capacity of monocytes to Induce a specific memory T cellresponse to viral, bacterial and fungal antigens, whereas thetreatment did not impair the capacity of the T cells to respondto pokeweed mitogen, phytohemagglutinin and concanavalin A.We conclude that after FcR cross-linking, the impaired APC functionis most likely due to the inability of monocytes to providethe essential costimulatory signals to the T cells via the B7–CD28/CTLA-4interaction.     

Differential expression of costimulatory molecules in chronic inflammatory periodontal disease tissue

Although B cell activation and subsequent immunoglobulin production are the immunopathological features of chronic inflammatory periodontal disease, in situ expression of costimulatory molecules in humoral immunity has not been investigated. In the present study we examined the expression of CD40, CD40 ligand (CD40L), CD80, CD86, CD28 and cytolytic T lymphocyte-associated antigen-4 (CTLA-4) on lymphocytes immunohistochemically. Cryostat sections were prepared from the gingival tissue samples of 14 patients with moderate to advanced adult periodontitis. In vitro kinetics of the expression of CD40L and CTLA-4 by peripheral blood T cells and that of CD80 and CD86 by peripheral blood B cells were also investigated by flow cytometry. Positive percentage expression of CD40L, CD28 and CTLA-4, and CD40, CD80 and CD86 was calculated for the number of CD3⁺ and CD19⁺ cells, respectively. Flow cytometric analysis demonstrated that the expression of CD40L and CTLA-4 on T cells, and CD80 and CD86 on B cells of peripheral blood was up-regulated upon activation. While most T cells and B cells expressed CD28, and CD80 and CD86, respectively, in gingival tissues, the expression of CD40L and CTLA-4 was lower but highly variable between specimens. Furthermore, these two molecules seemed to be expressed reciprocally in the lesion. As both CD40L and CTLA-4 expression are induced transiently by stimulation, variability in the expression of the molecules may reflect immunological activities and participation in the regulation of B cell activation of the lesion.