B7 Blockade Research Articles

Nivolumab in unresectable advanced, recurrent or metastatic esophageal squamous cell carcinoma.

Esophageal cancer (EC) is the eighth most common cancer worldwide. In view ofbiology and anatomical restrictions, multimodality treatment strategies have been developed for EC. However, the prognosis of patients with advanced EC remains especially poor. Immunotherapy, such as PD-1/PD-L1 and CTLA-4/B7 blockade, has emerged as a potent treatment for many types of cancer and has been approved in many countries. Based on the results of the ATTRACTION-3 trial, nivolumab, an anti-PD-1 monoclonal antibody, was approved by the US FDAfor patients with platinum-resistant, unresectable, recurrentor metastatic esophageal squamous cell carcinoma. The CheckMate 648 trial demonstrated that the combination of nivolumab with platinum-based fluoropyrimidine chemotherapy and combination immunotherapy with nivolumab and ipilimumab, an anti-CTLA-4 monoclonal antibody, showed a survival benefit in patients with advanced esophageal squamous cell carcinomacompared with doublet chemotherapy. This review focuses on nivolumab-containing treatments for patients with advanced esophageal squamous cell carcinoma.

Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance.

Despite significant clinical advancement with the use of immune checkpoint blockade (ICB) in non-small cell lung cancer (NSCLC) there are still a major subset of patients that develop adaptive/acquired resistance. Understanding resistance mechanisms to ICB is critical to developing new therapeutic strategies and improving patient survival. The dynamic nature of the tumor microenvironment and the mutational load driving tumor immunogenicity limit the efficacy to ICB. Recent studies indicate that myeloid cells are drivers of ICB resistance. In this study we sought to understand which immune cells were contributing to resistance and if we could modify them in a way to improve response to ICB therapy. Our results show that combination anti-PD-1/CTLA-4 produces an initial antitumor effect with evidence of an activated immune response. Upon extended treatment with anti-PD-1/CTLA-4 acquired resistance developed with an increase of the immunosuppressive populations, including T-regulatory cells, neutrophils and monocytes. Addition of anti-Ly6C blocking antibody to anti-PD-1/CTLA-4 was capable of completely reversing treatment resistance and restoring CD8 T cell activity in multiple KP lung cancer models and in the autochthonous lung cancer KrasLSL-G12D/p53fl/fl model. We found that there were higher classical Ly6C+ monocytes in anti-PD-1/CTLA-4 combination resistant tumors. B7 blockade illustrated the importance of dendritic cells for treatment efficacy of anti-Ly6C/PD-1/CTLA-4. We further determined that classical Ly6C+ monocytes in anti-PD-1/CTLA-4 resistant tumors are trafficked into the tumor via IFN-γ and the CCL2-CCR2 axis. Mechanistically we found that classical monocytes from ICB resistant tumors were unable to differentiate into antigen presenting cells and instead differentiated into immunosuppressive M2 macrophages or myeloid-derived suppressor cells (MDSC). Classical Ly6C+ monocytes from ICB resistant tumors had a decrease in both Flt3 and PU.1 expression that prevented differentiation into dendritic cells/macrophages. Therapeutically we found that addition of anti-Ly6C to the combination of anti-PD-1/CTLA-4 was capable of complete tumor eradication. Classical Ly6C+ monocytes differentiate into immunosuppressive cells, while blockade of classical monocytes drives dendritic cell differentiation/maturation to reinvigorate the anti-tumor T cell response. These findings support that immunotherapy resistance is associated with infiltrating monocytes and that controlling the differentiation process of monocytes can enhance the therapeutic potential of ICB.

Modulation of Xenogeneic T-cell Proliferation by B7 and mTOR Blockade of T Cells and Porcine Endothelial Cells.

Activation of porcine endothelial cells (PECs) is the mechanistic centerpiece of xenograft rejection. This study sought to characterize the immuno-phenotype of human T cells in response to PECs and to explore the immuno-modulation of B7 and mammalian target of rapamycin blockade of T cells and/or PECs during xeno-responses. Rapid memory T-cell (TM) responses to PECs were assessed by an intracellular cytokine staining. T-cell proliferation to PEC with or without belatacept or rapamycin was evaluated by a mixed lymphocyte-endothelial cell reaction (MLER). Additionally, rapamycin-pretreated PECs were used in MLER. Cell phenotypes were analyzed by flow cytometry. Tumor necrosis factor-α/interferon-γ producers were detected in CD8+ cells stimulated by human endothelium but not PECs. MLER showed proliferation of CD4+ and CD8+ cells with predominantly memory subsets. Purified memory and naive cells proliferated following PEC stimulation with an increased frequency of TM in PEC-stimulated naive cells. Proliferating cells upregulated programmed cell death-1 (PD-1) and CD2 expression. Belatacept partially inhibited T-cell proliferation with reduced CD2 expression and frequency of the CD8+CD2highCD28- subset. Rapamycin dramatically inhibited PEC-induced T-cell proliferation, and rapamycin-preconditioned PECs failed to induce T-cell proliferation. PD-1 blockade did not restore T-cell proliferation to rapamycin-preconditioned PECs. Humans lack rapid TM-mediated responses to PECs but induce T-cell proliferative responses characterized largely as TM with increasing CD2 and PD-1 expression. B7-CD28 and mammalian target of rapamycin blockade of T cells exhibit dramatic inhibitory effects in altering xeno-proliferating cells. Rapamycin alters PEC xeno-immunogenicity leading to inhibition of xeno-specific T-cell proliferation independent of PD-1-PD ligand interaction.

The role of B7-1 in proteinuria of glomerular origin

The damage and loss of podocytes is a primary hallmark of nephrotic syndrome. In the pursuit of targetable molecules that are involved in podocyte pathophysiology, some studies have identified B7-1 (also known as CD80) as a potential biomarker. Furthermore, B7-1 blockade has been proposed as a podocyte-specific treatment for patients with nephrotic syndrome who have limited therapeutic options, such as those with focal segmental glomerulosclerosis, minimal change disease, diabetic nephropathy and lupus nephritis. In this Perspectives article, we describe and compare supporting and contradicting data on the role of podocyte B7-1 in the pathogenesis of various podocytopathies. Moreover, we highlight crucial issues that should be addressed urgently - such as standardization of sample processing time, material conservation and antibody usage in immunohistochemical protocols - as a clinical trial that is investigating the efficacy of B7-1 blockade in treatment-resistant nephrotic syndrome is ongoing.

Selective CD28 Inhibition Modulates Alloimmunity and Cardiac Allograft Vasculopathy in Anti-CD154-Treated Monkeys.

Selective CD28 inhibition is actively pursued as an alternative to B7 blockade using cytotoxic T lymphocyte antigen 4 Ig based on the hypothesis that the checkpoint immune regulators cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 will induce tolerogenic immune signals. We previously showed that blocking CD28 using a monovalent nonactivating reagent (single-chain anti-CD28 Fv fragment linked to alpha-1 antitrypsin [sc28AT]) synergizes with calcineurin inhibitors in nonhuman primate (NHP) kidney and heart transplantation. Here, we explored the efficacy of combining a 3-week "induction" sc28AT treatment with prolonged CD154 blockade. Cynomolgus monkey heterotopic cardiac allograft recipients received sc28AT (10 mg/kg, d0-20, n = 3), hu5C8 (10-30 mg/kg, d0-84, n = 4), or combination (n = 6). Graft survival was monitored by telemetry. Protocol biopsies and graft explants were analyzed for International Society of Heart and Lung Transplantation acute rejection grade and cardiac allograft vasculopathy score. Alloantibody, T-cell phenotype and regulatory T cells were analyzed by flow cytometry. Immunochemistry and gene expression (NanoString) characterized intra-graft cellular infiltration. Relative to modest prolongation of median graft survival time with sc28AT alone (34 days), hu5C8 (133 days), and sc28AT + hu5C8 (141 days) prolonged survival to a similar extent. CD28 blockade at induction, added to hu5C8, significantly attenuated the severity of acute rejection and cardiac allograft vasculopathy during the first 3 months after transplantation relative to hu5C8 alone. These findings were associated with decreased proportions of circulating CD8 and CD3CD28 T cells, and modulation of inflammatory gene expression within allografts. Induction with sc28AT promotes early cardiac allograft protection in hu5C8-treated NHPs. These results support further investigation of prolonged selective CD28 inhibition with CD40/CD154 blockade in NHP transplants.

Another Piece of the Puzzle of Podocyte B7-1 Expression: Lupus Nephritis

Background/Aims: Lupus nephritis (LN) is a frequent complication and a major predictor of poor prognosis of systemic lupus erythematosus. Immune complex deposition and T cell infiltration are crucial events in LN pathogenesis. B7-1 (CD80), normally expressed by antigen-presenting cells, is one of the major co-stimulators of T-cell activation through the binding with its counter-receptors CD28 and cytotoxic T-lymphocyte antigen-4. Unexpectedly, B7-1 induction was described at the podocyte level in patients affected by different renal diseases, including LN. These observations suggested a novel exciting function for B7-1 as a biomarker of podocyte injury, and hence that B7-1 inhibitory drugs could serve as podocyte-targeted treatment of intractable renal diseases. However, subsequent studies hardly questioned the reliability of B7-1 detection assays and the therapeutic efficacy of B7-1 blockade in proteinuric patients, casting doubts on B7-1 expression by podocytes. Here, we thoroughly investigated whether B7-1 was indeed expressed by podocytes in LN, before even considering employing B7-1 blockade in patients with severe manifestations of LN and unfavourable prognosis. Methods: Applying different immunohistochemical assays with 4 primary antibodies, we analysed kidney biopsies from 42 LN patients at different stages of the disease, and from NZB/NZW mice, an LN model. Results: B7-1 was not induced in podocytes in human and murine LN; instead its expression was confined to infiltrating inflammatory cells. Conclusion: B7-1 is not expressed by podocytes in LN. A renoprotective effect of B7-1 blockade in LN patients cannot be ruled out but, if confirmed, cannot be the result of an effect on podocyte B7-1.

CD28/CTLA-4/B7 costimulatory pathway blockade affects regulatory T-cell function in autoimmunity.

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA-4 on effector T (Teff) cells, its immune-modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA-4Ig at day 7 and 9 after immunization, when myelin-reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL-17 and IFN-γ. Importantly, CTLA-4Ig treatment resulted in a transient reduction of Ki67 and CTLA-4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.

Selective Blockade of CD28/B7/CTLA4 Pathway With Monovalent Anti-CD28 Versus Targeting of B7 With Belatacept, in Kidney Allograft in Non-Human Primate, After CNI Weaning.

Targeting the CD28/B7/CTLA-4 costimulation pathway with the fusion protein CTLA4-Ig (Belatacept) in association with mycofenolate mofetil (MMF), is a less toxic alternative to calcineurin inhibitors (CNI). However, B7-specific blocking strategies inhibit CTLA-4 signals crucial to the function of regulatory T cells (Tregs). We previously showed in a model of kidney allograft in non-human primate (NHP) that a monovalent antibody fragment against CD28, which selectively blocks CD28 without affecting CTLA4, increased intragraft and peripheral blood Tregs and delayed acute rejection as single therapy and synergized with CNI to prevent efficiently acute rejection. In the current study, we performed a comparison of FR104, a monovalent pegylated Fab'anti-CD28 antagonist antibody with B7 blockade in kidney allograft in the baboon. Biologics were used de novo together with an initial 1 month treatment of low dose tacrolimus, weaned thereafter between months 1 and 2. After 2 months, therefore, recipients were under monotherapy with their biologics. In case of acute rejection, animals were treated with bolus of steroids. In the Belatacept group (n=3), two recipients developed severe acute rejection before or during CNI weaning (day 15 and 43) and bolus of steroid were ineffective. Histological examination confirmed rejection (Banff classification IIB: i3t2v2 and i3t3v2). The third recipient was lost on day 70 for pyelonephritis. In FR104-treated baboons (n=2), one animal developed an acute rejection episode during the CNI weaning period, but steroids bolus was able to reverse this episode. These two first FR104-treated recipients still have a stable kidney function 6 and 8 months post-transplantation. Interestingly, on the 1 and 3-months protocol biopsies, both FR104-treated recipients displayed an interstitial infiltration. Immuno-phenotyping analyses, emphasizing on peripheral Treg count (CD4+CD127lowCD25+) by flow cytometry, did not reveal any difference between the two biologics. These results suggest that the selective blockade of CD28 was efficient to allow reducing immunosuppression and maintaining recipients under this single therapy. Additional experiments are necessary to confirm the data and to further understand the mechanisms of action, in particular Tregs quantification in the biopsy. DISCLOSURES:Poirier, N.: Employee, Effimune SAS. Vanhove, B.: Stockholder, Effimune SAS.

Generation of BTNL8+ Inducible Tregs By Allogeneic Human Regulatory Macrophages Is IDO- and B7-Dependent.

Purpose: This study identifies the phenotypic and functional changes that human T cells undergo when exposed to regulatory macrophages. Methods: Human regulatory macrophages (M regs) represent a unique and stable state of macrophage activation, characterized by their ability to suppress activated T cells. M regs arise from CD14+ monocytes over a 6-day culture period in the presence of M-CSF, human serum, and a final 24-hour pulse of IFN-γ. In preclinical models, pre-transplant administration of donor-derived M regs prolonged allograft survival. This graft-protective effect initially depended upon metabolically-competent cells, but persisted beyond the lifespan of the M regs, implying regulation of recipient T cells by M regs. Here, mechanisms by which human M regs influence allogeneic T cell responses in vitro are examined. Results: Phenotypic and functional changes imposed by human M regs on allogeneic T cells were investigated by flow cytometry, whole-genome gene expression profiling, and secondary suppression assays. After exposure to M regs, CD4+ T cells were induced to express FoxP3, CD25, CTLA4, OX40, GITR, ICOS, EBI3, and BTNL8. Such FoxP3+ T cells generated in M reg cocultures suppressed polyclonal T cell proliferation and inhibited TNFα-induced dendritic cell maturation. The Treg-specific demethylated region (TSDR) of the FOXP3 gene in M reg-cocultured CD4+ T cells was 5% demethylated. The M reg-induced regulatory T cell (Treg) phenotype was not alloantigen-dependent, but was contact-dependent, and could be abolished by B7 blockade with CTLA4-Ig or by indoleamine 2,3-dioxygenase (IDO) inhibition with 1 mM 1-L-methyltryptophan. Tacrolimus and rapamycin did not affect FoxP3+ T cell development. Conclusions: M regs drive an antigen non-specific, B7- and contact-dependent induction of activated induced Tregs through a mechanism partially dependent on IDO. This finding supports the concept that pre-transplant treatment of recipients with donor-derived M regs engenders a pro-tolerogenic state and hints at a feedforward mechanism by which it could be perpetuated. Clinical applications of M reg immunosuppressive therapy are currently being pursued within The ONE Study framework.

Cutting Edge: Pseudomonas aeruginosa Abolishes Established Lung Transplant Tolerance by Stimulating B7 Expression on Neutrophils

The mechanisms that link bacterial infection to solid organ rejection remain unclear. In this study, we show that following the establishment of lung allograft acceptance in mice, Pseudomonas aeruginosa airway infection induces a G-CSF-dependent neutrophilia that stimulates acute rejection. Graft-infiltrating neutrophils sharply upregulate the B7 molecules CD80 and CD86, but they do not express CD40 or MHC class II in response to P. aeruginosa infection. Neutrophil B7 promotes naive CD4(+) T cell activation and intragraft IL-2(+), IFN-γ(+), and IL-17(+) T lymphocyte accumulation. Intravital two-photon microscopy reveals direct interactions between neutrophils and CD4(+) T cells within pulmonary allografts. Importantly, lung rejection in P. aeruginosa-infected recipients is triggered by CD80/86 on neutrophils and can be prevented by B7 blockade without affecting clearance of this pathogen. These data show that neutrophils enhance T cell activation through B7 trans-costimulation and suggest that inhibiting neutrophil-mediated alloimmunity can be accomplished without compromising bacterial immune surveillance.

Roles of CD28, CTLA4, and Inducible Costimulator in Acute Graft-versus-Host Disease in Mice

T cells deficient for CD28 have reduced ability to expand and survive, but still cause graft-versus-host disease (GVHD). Inducible costimulator (ICOS), a member of the CD28 family, is expressed on antigen-activated T cells and plays unique roles in T cell activation and effector function. We hypothesized that ICOS contributes to the development of GVHD in the absence of B7:CD28/CTLA4 costimulation. In this study, we evaluated the roles of CD28, CTLA4, and ICOS in the pathogenesis of acute GVHD after myeloablative allogeneic bone marrow transplantation. Unexpectedly, we found that blocking CD28 and CTLA4 signals using the clinically relevant reagent CTLA4-Ig increases the severity of GVHD mediated by CD4(+) T cells, and that such treatment does not add any benefit to the blockade of ICOS. In contrast, selectively blocking CD28 and ICOS, but not CTLA4, prevents GVHD more effectively than blocking either CD28 or ICOS alone. Taken together, these results indicate that CD28 and ICOS are synergistic in promoting GVHD, whereas the CTLA4 signal is required for T cell tolerance regardless of ICOS signaling. Thus, blocking CD28 and ICOS while sparing CTLA4 represents a promising approach for abrogating pathogenic T cell responses after allogeneic bone marrow transplantation.

B7 Blockade Alters the Balance between Regulatory T Cells and Tumor-reactive T Cells for Immunotherapy of Cancer

In prostate cancer-bearing host, regulatory T (Treg) cells restrain activity of tumor antigen-specific T cells. Because B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers. The anti-B7-1/B7-2 monoclonal antibodies were administered to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice. Here, we showed that short-term administration of anti-B7-1/B7-2 monoclonal antibodies in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed T antigen-specific CTL effectors in anti-B7-treated but not control IgG-treated TRAMP mice. Transient blockade of B7-1/B7-2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.

Indoleamine 2,3-dioxygenase (IDO) modulates immunological tolerance to cardiac allografts

Objectives: Negative or inhibitory costimulatory pathways regulate T cell activation and play a role in peripheral tolerance. The concept that Indoleamine 2, 3-dioxygenase (IDO) expressing dendritic cells (DCs) are able to suppress T-cell responses and promote tolerance is a relatively new paradigm in immunology. This study aimed to investigate the interplay of IDO and CD28:B7 and/or CD40-CD154 co-stimulatory pathway blockade in a fully mismatched allograft model.

Lentiviral gene transfer of CTLA4 generates B cells with reduced costimulatory properties

Peripheral T-cell (TC) tolerance can be induced by tolerogenic antigen-presenting cell (APC). A prerequisite is the reduction or blockade of B7 of APC. Besides dendritic cell, B cells can be used as APC. Here, we show the generation B cells with reduced B7 expression by lentiviral transduction of endoplasmic reticulum (ER)-directed CTLA4. Vectors coding for the human CTL4-Ig were used for the human B-cell line Raji. Transduction efficiency was over 90% (MOI = 3). For the murine B-cell line A20 and for primary mouse B cells, murine CTLA4 was used. We show that B cells with reduced B7 expression reduce the antigen (Ag) specific TC proliferation in vitro. B cells expressing an ER-directed CTLA4 may reduce Ag-specific immune responses.

Requirements for Co-Stimulation in T-Cell Dependent and T-Cell Independent Re-Stimulation of FVIII-Specific Memory B Cells

Memory B cells specific for factor VIII (FVIII) are critical for maintaining FVIII inhibitors in patients with hemophilia A. They are precursors of anti-FVIII antibody-producing plasma cells and are highly efficient antigen-presenting cells for the activation of T cells. The eradication of FVIII-specific memory B cells will be a prerequisite for any successful new approach to induce immune tolerance in patients with FVIII inhibitors. Little is known about the regulation of these cells. Previously we showed that ligands for toll-like receptors (TLR) 7 and 9 are able to re-stimulate FVIII-specific memory B cells in the absence of T-cell help. However, alternative “helper cells” such as dendritic cells are essential for providing help to memory B cells under such conditions. Based on these findings, we asked which co-stimulatory interactions are required for the restimulation of memory B cells in the presence of dendritic cells and ligands for TLR and whether these co-stimulatory interactions are the same as those required for the restimulation of memory B cells in the presence of activated T cells. We used spleen cells from hemophilic mice treated with human FVIII to generate highly purified populations of memory B cells, CD4+ T cells and dendritic cells. The required purity was achieved by a combination of magnetic bead separation and fluorescence-activated cell sorting. The memory B cell compartment was specified by the expression of CD19 together with IgG and the absence of surface IgM and IgD. Memory B cells were cultured in the presence of FVIII to stimulate their differentiation into anti-FVIII antibody-producing plasma cells. Different combinations of CD4+ T cells, ligands for TLR 7 and 9 and dendritic cells were added to the memory-B-cell cultures. Blocking antibodies and competitor proteins were used to specify the co-stimulatory interactions required for the re-stimulation of memory B cells in the presence of either CD4+ T cells or dendritic cells and ligands for TLR 7 and 9. Our results demonstrate that the blockade of B7-1 and B7-2 as well as the blockade of CD40L inhibit the re-stimulation of FVIII-specific memory B cells and their differentiation into anti-FVIII antibody-producing plasma cells in the presence of T-cell help. Similar requirements apply for the re-stimulation of memory B cells in the presence of dendritic cells and ligands for TLR 7 or 9. Dendritic cells in the absence of ligands for TLR are not able to provide help for the re-stimulation of memory B cells, which indicates that dendritic cells need to be activated. Furthermore, ligands for TLR 7 or 9 were not able to re-stimulate memory B cells in the complete absence of dendritic cells. Based on these results we conclude that dendritic cells activated by ligands for TLR 7 or 9 can substitute for activated CD4+ T cells in providing co-stimulatory help for memory-B-cell re-stimulation. CD40-CD40L interactions seem to be the most important co-stimulatory interactions for the re-stimulation of memory B cells, not only in the presence of activated CD4+ T cells but also in the presence of ligands for TLR and dendritic cells.

A Novel Alloantigen-Specific CD8+PD1+ Regulatory T Cell Induced by ICOS-B7h Blockade In Vivo

Delayed ICOS-B7h signal blockade promotes significant prolongation of cardiac allograft survival in wild-type but not in CD8-deficient C57BL/6 recipients of fully MHC-mismatched BALB/c heart allografts, suggesting the possible generation of CD8(+) regulatory T cells in vivo. We now show that the administration of a blocking anti-ICOS mAb results in the generation of regulatory CD8(+) T cells. These cells can transfer protection and prolong the survival of donor-specific BALB/c, but not third party C3H, heart grafts in CD8-deficient C57BL/6 recipients. This is unique to ICOS-B7h blockade, because B7 blockade by CTLA4-Ig prolongs graft survival in CD8-deficient mice and does not result in the generation of regulatory CD8(+) T cells. Those cells localize to the graft, produce both IFN-gamma and IL-4 after allostimulation in vitro, prohibit the expansion of alloreactive CD4(+) T cells, and appear to mediate a Th2 switch of recipient CD4(+) T cells after adoptive transfer in vivo. Finally, these cells are not confined to the CD28-negative population but express programmed death 1, a molecule required for their regulatory function in vivo. CD8(+)PD1(+) T cells suppress alloreactive CD4(+) T cells but do not inhibit the functions by alloreactive CD8(+) T cells in vitro. These results describe a novel allospecific regulatory CD8(+)PD1(+) T cell induced by ICOS-B7h blockade in vivo.

CD28:B7 interaction is necessary for the protective effect of T cell vaccination in EAE

The mechanisms of T cell vaccination (TCV) are still unclear, especially the molecular interactions for recognition of autoreactive T cells by the immune system. Here we investigated the role of CD28:B7 interaction in TCV-induced protection in the murine EAE model. We demonstrate that there is increased expression of both B7-1 and B7-2 on autoreactive Th1 cells compared to Th2 cells. Blockade of B7 on the vaccinating autoreactive T cell surface or blockade of CD28 in recipient mice reduced the protective effect of TCV. Furthermore, we showed that TCV significantly inhibited Ag-specific CD4 and CD8 T cell proliferation and decreased Ag-specific IFN-gamma production by CD4 T cells in mice undergoing TCV, and blocking of B7 on the surface of vaccinating T cells reduced this inhibition on Ag-specific CD4 and CD8 T cell proliferation, more significantly on Ag-specific CD4 T cell proliferation. These data indicated that B7 expression on autoreactive T cells is necessary for the recognition of autoreactive T cells by the immune system and subsequent protection from EAE in mice undergoing TCV.

IL‐18 stimulates IL‐13‐mediated IFN‐γ‐sensitive host resistance in vivo

IL-4 and IL-13 are up-regulated during in vivo responses to many nematode parasites, but increasing evidence suggests that increases in IL-13 can also occur independently of the IL-4-dominant Th2 response. Blocking B7 after Trichuris muris inoculation inhibits resistance and IL-4 elevations, instead resulting in an IFN-gamma-dominant response associated with susceptibility. However, blocking IFN-gamma under these conditions restores IL-13-dependent resistance. In this study, we examined the mechanism of IL-13 up-regulation and associated protection during this in vivo immune response. CD4+ T cells and DX5+ TCR- cells were identified as the major producers of IL-13, and the DX5+ TCR- cells were phenotyped as NK cells, since they expressed CD11b, IL-2Rbeta and Ly49C but not c-kit or Fc epsilonRI. NK cell-derived IL-13 elevations were T cell-dependent, as CD4+ T cell depletion blocked IL-13 production by mesenteric lymph node cells and induced susceptibility. IL-13 expression was increased independently of IL-12; however, blocking IL-18 function inhibited IL-13 production and increased susceptibility. These results indicate that CD4+ T cells and NK cells are the major sources of IL-13 during the in vivo Th1 response induced by B7 blockade and that under these conditions, IL-18 is specifically required for the in vivo up-regulation of IL-13 production and associated host protection.

CTLA-4 blockade augments human T lymphocyte-mediated suppression of lung tumor xenografts in SCID mice.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

Dysregulated B7-1 and B7-2 Expression on Nonobese Diabetic Mouse B Cells Is Associated with Increased T Cell Costimulation and the Development of Insulitis

Little is known about the pathogenic role of B cell dysfunction in T cell-mediated autoimmune disease. We previously reported that B cell hyper-responsiveness, resistance to apoptosis, and accumulation in islets occur during the onset of insulitis, but not in type 1 diabetes (T1D), in NOD mice. In this study we extended these studies to further determine how islet-infiltrated B cells contribute to this inflammatory insulitis. We demonstrate the presence of an increased percentage of B7-1(+) and a decreased percentage of B7-2(+) B cells in the spleen of autoimmune disease-prone NOD and nonobese diabetes-resistant mice compared with the spleen of nonautoimmune disease-prone C57BL/6 and BALB/c mice. An age-dependent differential expression of B7-1 and B7-2 was associated with the development of insulitis and CD4(+)CD25(+) T cell deficiency in autoimmune disease-prone mice. Whereas BCR and LPS stimulation increased B7-2 expression on B cells from autoimmune disease-prone and nonautoimmune disease-prone mice, LPS-induced B7-1 expression was higher on NOD than C57BL/6 B cells. Interestingly, increased expression of B7-1 and B7-2 was found on islet-infiltrated B cells, and this increase was associated with enhanced T cell costimulation. Islet-infiltrated B cells were shown to be a source of TNF-alpha production in islets. B7 blockade of BCR-stimulated NOD B cells by anti-B7-1 and anti-B7-2 mAbs during coadoptive transfer with diabetogenic T cells into NOD.scid mice protected these recipients from T1D. These results suggest that increased B7-1 and B7-2 expression on islet-infiltrated NOD B cells is associated with increased T cell costimulation and the development of inflammatory insulitis in NOD mice.