The coinhibitory receptor CTLA-4 controls B cell responses by modulating T follicular helper, T follicular regulatory, and T regulatory cells.

The receptor CTLA-4 has been implicated in controlling B cell responses, but the mechanisms by which CTLA-4 regulates antibody production are not known. Here we showed deletion of CTLA-4 in adult mice increased Tfh and Tfr cell numbers and augmented B cell responses. In the effector phase, loss of CTLA-4 on Tfh cells resulted in heightened B cell responses, whereas loss of CTLA-4 on Tfr cells resulted in defective suppression of antigen-specific antibody responses. We also found that non-Tfr Treg cells could suppress B cell responses through CTLA-4 and that Treg and/or Tfr cells might downregulate B7-2 on B cells outside germinal centers as a means of suppression. Within the germinal center, however, Tfr cells potently suppress B cells through CTLA-4, but with a mechanism independent of altering B7-1 or B7-2. Thus, we identify multifaceted regulatory roles for CTLA-4 in Tfh, Tfr, and Treg cells, which together control humoral immunity.

The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells.

The inducible costimulatory molecule ICOS has been suggested to be important in the development of interleukin 17 (IL-17)-producing helper T cells (T(H)-17 cells) and of follicular helper T cells (T(FH) cells). Here we show that ICOS-deficient mice had no defect in T(H)-17 differentiation but had fewer T(H)-17 cells after IL-23 stimulation and fewer T(FH) cells. We also show that T(FH) cells produced IL-17 and that T(FH) cells in ICOS-deficient mice were defective in IL-17 production. Both T(H)-17 and T(FH) cells had higher expression of the transcription factor c-Maf. Genetic loss of c-Maf resulted in a defect in IL-21 production and fewer T(H)-17 and T(FH) cells. Thus our data suggest that ICOS-induced c-Maf regulates IL-21 production that in turn regulates the expansion of T(H)-17 and T(FH) cells.

Overexpression of the Ctla-4 isoform lacking exons 2 and 3 causes autoimmunity.

CTLA-4 is a potent inhibitor of T cell activation, primarily upon binding to its costimulatory ligands (B7.1 and B7.2) expressed on APCs. However, variants of CTLA-4 can also function independently of B7 molecules. 1/4CTLA-4 is a highly conserved isoform encoded by exons 1 and 4 of the Ctla4 gene that lacks the ligand-binding and the transmembrane domains, and as yet, its function is not known. To investigate the function of 1/4CTLA-4, we generated transgenic (Tg) mice overexpressing this variant. Cytokine production by 1/4CTLA-4 Tg T cells was elevated compared with wild type T cells. The frequency of CD44(high) memory T cells in 1/4CTLA-4 Tg mice was increased, and as the mice aged, the frequency further increased. 1/4CTLA-4 Tg mice >1 y old had increased expression of T cell activation markers and developed spontaneous autoimmunity, including elevated production of autoantibodies. In contrast with young 1/4CTLA-4 Tg mice, aged 1/4CTLA-4 Tg mice had elevated frequencies of Foxp3(+) regulatory T cells, but the regulatory T cells from these mice were not able to inhibit colitis development. Collectively, these data suggest that the function of the 1/4CTLA-4 isoform is distinct from that of CTLA-4 in that it enhances T cell activation and promotes autoimmunity rather than inhibiting immune responses.

Dendritic cell expression of the transcription factor T-bet regulates mast cell progenitor homing to mucosal tissue.

The transcription factor T-bet was identified in CD4(+) T cells, and it controls interferon gamma production and T helper type 1 cell differentiation. T-bet is expressed in certain other leukocytes, and we recently showed (Lord, G.M., R.M. Rao, H. Choe, B.M. Sullivan, A.H. Lichtman, F.W. Luscinskas, and L.H. Glimcher. 2005. Blood. 106:3432-3439) that it regulates T cell trafficking. We examined whether T-bet influences homing of mast cell progenitors (MCp) to peripheral tissues. Surprisingly, we found that MCp homing to the lung or small intestine in T-bet(-/-) mice is reduced. This is reproduced in adhesion studies using bone marrow-derived MCs (BMMCs) from T-bet(-/-) mice, which showed diminished adhesion to mucosal addresin cellular adhesion molecule-1 (MAdCAM-1) and vascular cell adhesion molecule-1 (VCAM-1), endothelial ligands required for MCp intestinal homing. MCp, their precursors, and BMMCs do not express T-bet, suggesting that T-bet plays an indirect role in homing. However, adoptive transfer experiments revealed that T-bet expression by BM cells is required for MCp homing to the intestine. Furthermore, transfer of WT BM-derived dendritic cells (DCs) to T-bet(-/-) mice restores normal MCp intestinal homing in vivo and MCp adhesion to MAdCAM-1 and VCAM-1 in vitro. Nonetheless, T-bet(-/-) mice respond vigorously to intestinal infection with Trichinella spiralis, eliminating a role for T-bet in MC recruitment to sites of infection and their activation and function. Therefore, remarkably, T-bet expression by DCs indirectly controls MCp homing to mucosal tissues.

The programmed death-1 ligand 1:B7-1 pathway restrains diabetogenic effector T cells in vivo.

Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.

The novel costimulatory programmed death ligand 1/B7.1 pathway is functional in inhibiting alloimmune responses in vivo.

The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1-B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-γ-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-γ production from alloreactive CD4(+) T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.

Tolerogenicity is not an absolute property of a dendritic cell.

Pharmacological modulation is known to temper the immune capacity of DC, enhancing the notion that modulated Ag-bearing DC might be used therapeutically to induce tolerance. We have investigated phenotypic features shared by such DC, and queried their potential to tolerize in different settings. Immature, IL-10, TGF-beta and 1alpha,25-dihydroxyvitamin D(3)-modulated BMDC all induced tolerance to male skin in female TCR transgenic A1.RAG mice, and the modulated DC also tolerized after exposure to the TLR4-ligand LPS. Transcript profiling revealed that this was achieved despite retaining much of the normal LPS-maturation response. No shared tolerance-associated transcripts could be identified. Equivalent BMDC could not tolerize in Marilyn TCR-transgenic mice. Simultaneous presentation of both A1.RAG and Marilyn peptide-Ag (Dby-H2E(k) and Dby-H2A(b)) on immature (C57BL/6JxCBA/Ca) F1 BMDC also only achieved tolerance in A1.RAG mice. Both strains registered Ag, but Foxp3(+) Treg were only induced in A1.RAG mice. In contrast, Marilyn T cells showed greater proliferation and an inflammatory bias, in response to Ag presented by immature F1 BMDC in vitro. In summary, while pharmacological agents can skew DC to reinforce their immature tolerogenic phenotype, the outcome of presentation is ultimately an integrated response including T-cell-intrinsic components that can over-ride for immune activation.

Overexpression of PD-1 on T cells promotes tolerance in cardiac transplantation via ICOS-dependent mechanisms.

The programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is a potent inhibitory pathway involved in immune regulation and is a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 on T cells (PD-1 Tg) promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade with CTLA-4-Ig. PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II-mismatched cardiac transplant model, whereas the overexpression still allowed the generation of an effective immune response against an influenza A virus. Notably, Tregs from PD-1 Tg mice were required for tolerance induction and presented greater ICOS expression than those from WT mice. The survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.

The Fully human anti-CD47 antibody SRF231 exerts dual-mechanism antitumor activity via engagement of the activating receptor CD32a.

BACKGROUND: CD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as 'macrophage checkpoint' inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4 anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here. METHODS: SRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment. RESULTS: SRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231. CONCLUSION: SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a.

Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4(+)Foxp3(-) (T conv) and regulatory Foxp3(+) (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells.

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.

Induction of regulatory T cells and dominant tolerance by dendritic cells incapable of full activation.

Transplants tolerated through a process known as infectious tolerance evoke continuous recruitment of regulatory T (Treg) cells that are necessary to maintain the unresponsive state. This state is maintained long-term and requires continuous Ag exposure. It is not known, however, whether infectious tolerance operates through sustained recruitment of pre-existing regulatory cells, induction of regulatory cells, or both. Using mice deficient in natural Treg cells, we show here that quiescent donor dendritic cells (DC) laden with histocompatibility Ag can induce Treg cells de novo that mediate transplantation tolerance. In contrast, fully activated DC fail to do so. These findings suggest that DC incapable of delivering full activation signals to naive T cells may favor their polarization toward a regulatory phenotype. Furthermore, they suggest a role for quiescent endogenous DC in the maintenance of the tolerant state.