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Autoimmune diseases are diseases in which the regulatory mechanisms of the immune response are disturbed. As a result, the body loses self-tolerance. Since one of the main regulatory mechanisms of the immune response is the CTLA4-CD80/86 axis, this hypothesis suggests that autoimmune diseases potentially share a similar molecular basis of pathogenesis. Hence, investigating the CTLA4-CD80/86 axis may be helpful in finding an appropriate treatment strategy. Therefore, this study aims to investigate the molecular basis of the CTLA4-CD80/86 axis in the regulation of the immune response, and then its role in developing some autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. As well, the main therapeutic strategies affecting the CTLA4-CD80/86 axis have been summarized to highlight the importance of this axis in management of autoimmune diseases., (© 2023 John Wiley & Sons Ltd.)

Foxp3-expressing CD4 + CD25 + regulatory T cells (Tregs) constitutively and highly express the immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), whose Treg-specific deficiency causes severe systemic autoimmunity. As a key mechanism of Treg-mediated suppression, Treg-expressed CTLA-4 down-regulates the expression of CD80/CD86 costimulatory molecules on antigen-presenting cells (APCs). Here, we show that Treg-expressed CTLA-4 facilitated Treg-APC conjugation and immune synapse formation. The immune synapses thus formed provided a stable platform whereby Tregs were able to deplete CD80/CD86 molecules on APCs by extracting them via CTLA-4-dependent trogocytosis. The depletion occurred even with Tregs solely expressing a mutant CTLA-4 form lacking the cytoplasmic portion required for its endocytosis. The CTLA-4-dependent trogocytosis of CD80/CD86 also accelerated in vitro and in vivo passive transfer of other membrane proteins and lipid molecules from APCs to Tregs without their significant reduction on the APC surface. Furthermore, CD80 down-regulation or blockade by Treg-expressed membrane CTLA-4 or soluble CTLA-4-immunoglobulin (CTLA-4-Ig), respectively, disrupted cis-CD80/programmed death ligand-1 (PD-L1) heterodimers and increased free PD-L1 on dendritic cells (DCs), expanding a phenotypically distinct population of CD80 lo free PD-L1 hi DCs. Thus, Tregs are able to inhibit the T cell stimulatory activity of APCs by reducing their CD80/CD86 expression via CTLA-4-dependent trogocytosis. This CD80/CD86 reduction on APCs is able to exert dual suppressive effects on T cell immune responses by limiting CD80/CD86 costimulation to naïve T cells and by increasing free PD-L1 available for the inhibition of programmed death-1 (PD-1)-expressing effector T cells. Blockade of CTLA-4 and PD-1/PD-L1 in combination may therefore synergistically hinder Treg-mediated immune suppression, thereby effectively enhancing immune responses, including tumor immunity., Competing Interests: Competing interest statement: Authors S.S., J.B.W., and the reviewer M.K.L. contributed independently as coauthors to guideline mega-studies, which were published as A. Cossarizza et al., Eur. J. Immunol. 47, 1584–1797 (2017); A. Fuchs et al., Front. Immunol. 8, 1–15 (2018); and A. Cossarizza et al., Eur. J. Immunol. 49, 1457–1973 (2019).

Certain cytotoxic chemotherapeutic drugs are immunogenic, stimulating tumor immunity through mechanisms that are not completely understood. Here we show how the DNA-damaging drug cisplatin modulates tumor immunity. At the maximum tolerated dose (MTD), cisplatin cured 50% of mice with established murine TC-1 or C3 tumors, which are preclinical models of human papillomavirus (HPV)-associated cancer. Notably, the curative benefit of cisplatin relied entirely upon induction of tumor-specific CD8 + T cells. Mechanistic investigations showed that cisplatin stimulated tumor infiltration of inflammatory antigen-presenting cells (APC) expressing relatively higher levels of the T-cell costimulatory ligands CD70, CD80, and CD86. Cell death triggered by cisplatin was associated with the release of at least 19 proteins in the tumor environment that could act as damage-associated molecular patterns and upregulate costimulatory molecules, either alone or in concert, but the responsible proteins remain unknown. Essentially, the curative effect of cisplatin was abrogated in mice lacking expression of CD80 and CD86 on APCs. Furthermore, cisplatin treatment was improved by CTLA-4 blockade, which increases the availability of CD80/86 to bind to CD28. In contrast, there was no effect of CD27 stimulation, which replaces CD70 interaction. At the cisplatin MTD, cure rates could also be increased by vaccination with synthetic long peptides, whereas cures could also be achieved at similar rates at 80% of the MTD with reduced side effects. Our findings reveal an essential basis for the immunogenic properties of cisplatin, which are mediated by the induction of costimulatory signals for CD8 + T-cell-dependent tumor destruction. Cancer Res; 76(20); 6017-29. ©2016 AACR., (©2016 American Association for Cancer Research.)

Type 2 diabetes mellitus (T2D) is a metabolic disease that is strongly tied to obesity and often preceded by insulin resistance (IR). It has been established that chronic inflammation of hypertrophic adipose tissue depots in obese individuals leads to obesity-associated IR and is mediated by cells of the innate immune system, particularly macrophages. More recently, cells of the adaptive immune system, B and T lymphocytes, have also emerged as important regulators of glucose homeostasis, raising the intriguing possibility that antigen-driven immune responses play a role in disease. In this review, we critically evaluate the roles that various B and T cell subsets play in IR, and then we examine the data suggesting that antigen-driven mechanisms, such as antigen presentation and costimulation, may drive the activity of these lymphocytes.

Memory B cells (MBCs) are long-lived sources of rapid, isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2, independently of isotype, identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely, CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence, the differentiation and regeneration of MBCs are compartmentalized.

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy., (Copyright © 2014 by the American Society of Nephrology.)

Background: Atherosclerosis is based on a chronic inflammatory process including the innate and adaptive immune response. Costimulatory molecules and their receptors provide decisive signals for antigen-specific cell activation. The contribution of B7-related pathways to atherosclerosis has hardly been explored., Methods: In the present study, we investigated the contribution of B7-1 to inflammation and tissue injury in the human plaque microenvironment in order to identify possible target structures of future therapeutic agents ex vivo and in vitro., Results: Carotid artery plaque stimulation with lipopolysaccharides (LPS) could be significantly inhibited by RhuDex(®), a specific inhibitor of the costimulatory molecule B7-1 ex vivo (P<0.001). Coculture of antigen-presenting cells with T-cells demonstrated that the inhibitory effects of RhuDex(®) derived from reduced T-cell activation. In addition, incubation of monocytes/macrophages with LPS and RhuDex(®) resulted in an inhibitory negative feedback on antigen-presenting cells. Signaling pathways affected by RhuDex(®) seem to be nuclear transcription factor kappa B, activator protein-1, and extracellular signal-regulated kinase 1/2., Conclusion: The present data support B7-1 alone as an important costimulatory molecule in the context of LPS-mediated inflammation in atherosclerotic lesions. Due to its marked inhibitory effects, RhuDex(®) may be a useful therapy to modulate the inflammatory milieu in atherosclerosis.

A key pathophysiologic role for activated T-cells in mediating adipose inflammation and insulin resistance (IR) has been recently postulated. However, mechanisms underlying their activation are poorly understood. In this study, we demonstrated a previously unrecognized homeostatic role for the costimulatory B7 molecules (CD80 and CD86) in preventing adipose inflammation. Instead of promoting inflammation, which was found in many other disease conditions, B7 costimulation reduced adipose inflammation by maintaining regulatory T-cell (Treg) numbers in adipose tissue. In both humans and mice, expression of CD80 and CD86 was negatively correlated with the degree of IR and adipose tissue macrophage infiltration. Decreased B7 expression in obesity appeared to directly impair Treg proliferation and function that lead to excessive proinflammatory macrophages and the development of IR. CD80/CD86 double knockout (B7 KO) mice had enhanced adipose macrophage inflammation and IR under both high-fat and normal diet conditions, accompanied by reduced Treg development and proliferation. Adoptive transfer of Tregs reversed IR and adipose inflammation in B7 KO mice. Our results suggest an essential role for B7 in maintaining Tregs and adipose homeostasis and may have important implications for therapies that target costimulation in type 2 diabetes.

Although T cell activation has been classically described to require distinct, positive stimulation signals that include B7-1 (CD80) and B7-2 (CD86) costimulation, overriding suppression signals that avert immune-mediated host injury are equally important. How these opposing stimulation and suppression signals work together remains incompletely defined. Our recent studies demonstrate that CD8 Teff activation in response to cognate peptide stimulation is actively suppressed by the Foxp3(+) subset of CD4 cells, called Tregs. Here, we show that the elimination of Treg suppression does not bypass the requirement for positive B7-1/B7-2 costimulation. The expansion, IFN-γ cytokine production, cytolytic, and protective features of antigen-specific CD8 T cells stimulated with purified cognate peptide in Treg-ablated mice were each neutralized effectively by CTLA-4-Ig that blocks B7-1/B7-2. In turn, given the efficiency whereby CTLA-4-Ig overrides the effects of Treg ablation, the role of Foxp3(+) cell-intrinsic CTLA-4 in mitigating CD8 Teff activation was also investigated. With the use of mixed chimera mice that contain CTLA-4-deficient Tregs exclusively after the ablation of WT Foxp3(+) cells, a critical role for Treg CTLA-4 in suppressing the expansion, cytokine production, cytotoxicity, and protective features of peptide-stimulated CD8 T cells is revealed. Thus, the activation of protective CD8 T cells requires positive B7-1/B7-2 costimulation even when suppression by Tregs and in particular, Treg-intrinsic CTLA-4 is circumvented.

Background: Despite extensive research, the mechanism of immature dendritic cells (DCs) induced immune hyporesponsiveness remains incompletely understood., Methods: Recipient DCs from C3H mouse bone marrow cells were incubated with donor antigen from splenic lymphocytes of C57BL/6 mouse; these DCs were transfected with CD80/86 specific siRNA using lentiviral vectors. Flow cytometry was used to evaluate expression of CD80/86 on the antigen-pulsed recipient DCs. Immune regulatory activity was examined by mixed lymphocyte reaction, in which irradiated DCs were cultured with C3H spleen T cells. After the reaction, interleukin (IL)-2, IL-4, IL-10, and interferon (INF)-γ levels of mixed lymphocyte reaction culture supernatant were measured by enzyme-linked immunosorbent assay. The apoptotic T lymphocytes were identified by Annexin V and CD3 staining., Results: There was a significant inhibition of CD80/86 expression in DCs transfected with CD80/86 lentiviral vectors compared with the control groups (P < 0.05), indicating the specificity of RNA interference. Enzyme-linked immunosorbent assay results showed a significant reduction of INF-γ, IL-2 and IL-10 in the CD80/86 lentivirus transfected group compared to the control groups (P < 0.05). There was no significant difference in IL-4 levels between the groups (P > 0.05). We also showed that CD80/86 low DCs loaded with alloantigen (1) stimulated low T cell proliferative responses via the indirect recognition pathway and (2) enhanced apoptotic activity (P < 0.05) in co-cultured T cells., Conclusions: Lentiviral vector transfection can effectively and specifically knock down target genes in DCs. The CD80/86 low DCs may show tolerogenic activity via induction of T-cell apoptosis, thereby modulating the activity of recipient-derived DCs. The use of this approach may potentially be clinically applicable.

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.

Elevated levels of intracellular cyclic adenosine monophosphate (cAMP) in naturally occurring T regulatory (nTreg) cells play a key role in nTreg-cell-mediated suppression. Upon contact with nTreg cells, cAMP is transferred from nTreg cells into activated target CD4(+) T cells and/or antigen-presenting cells (APCs) via gap junctions to suppress CD4(+) T-cell function. cAMP facilitates the expression and nuclear function of a potent transcriptional inhibitor, inducible cAMP early repressor (ICER), resulting in ICER-mediated suppression of interleukin-2 (IL-2). Furthermore, ICER inhibits transcription of nuclear factor of activated T cell c1/α (NFATc1/α) and forms inhibitory complexes with preexisting NFATc1/c2, thereby inhibiting NFAT-driven transcription, including that of IL-2. In addition to its suppressive effects mediated via ICER, cAMP can also modulate the levels of surface-expressed cytotoxic T lymphocyte antigen-4 (CTLA-4) and its cognate B7 ligands on conventional CD4(+) T cells and/or APCs, fine-tuning suppression. These cAMP-driven nTreg-cell suppression mechanisms are the focus of this review., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 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.

Minimal change disease is the most common nephrotic syndrome in children. Although the etiology of minimal change disease remains to be elucidated, it has been postulated that it is the result of a circulating T-cell factor that causes podocyte cytoskeleton disorganization leading to increased glomerular capillary permeability and/or changes in glomerular basement membrane heparan sulfate glycosaminoglycans resulting in proteinuria. Minimal change disease has been associated with allergies and Hodgkin disease. Consistent with these associations, a role for interleukin-13 with minimal change disease has been proposed. Furthermore, studies evaluating podocytes also have evolved. Recently, increased expression of CD80 (also termed B7-1) on podocytes was identified as a mechanism for proteinuria. CD80 is inhibited by binding to CTLA-4, which is expressed on regulatory T cells. Recently, we showed that urinary CD80 is increased in minimal change disease patients and limited studies have suggested that it is not commonly present in the urine of patients with other glomerular diseases. Interleukin-13 or microbial products via Toll-like receptors could be factors that induce CD80 expression on podocytes. CTLA-4 appears to regulate CD80 expression in podocytes, and to be altered in minimal change disease patients. These findings lead us to suggest that proteinuria in minimal change disease is caused by persistent CD80 expression in podocytes, possibly initiated by stimulation of these cells by antigens or cytokines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Regulatory functions for B lymphocytes have been reported in murine models of autoimmune diseases in which B-cell deficient mice were shown to exhibit exacerbated disease. The B cells responsible for the immune regulations were identified as a subpopulation of interleukin 10-secreting cells. However, the mechanism of induction and the characteristics of regulatory B cells in humans have been hardly studied. This study reports that regulation of T cell responses can be induced by B cells following CD40-dependent cognate interaction. T cell proliferation and cytokine production were differentially regulated. Thus, CD40-induced regulatory B cells partially inhibited T cell proliferation following CD40 interaction without requirement of soluble factor. In contrast, modulation of Th1 differentiation resulted from CD80- and CD86-dependent interactions and from IL-10 production. The suppressive effects were mediated by CD19(high)IgD+CD38(high)CD24(high)CD5(high) B cells and appeared to be indirect, through the induction of regulatory T cells as indicated by the appearance of Foxp3+CD4+CD25+T cells. These data suggest that activation signals from T cells initiate regulatory properties in B cells that modulate T cell responses involving regulatory T cells. Finally, studies in autoimmune patients revealed that regulation of T cell proliferation was defective in systemic lupus erythematosus but efficient in other diseases. Restoration of efficient B-cell regulatory activity could provide innovative B-cell based treatment of autoimmune diseases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell-mediated destruction of insulin-producing β cells, and viral infections can prevent the onset of disease. Invariant natural killer T cells (iNKT cells) exert a regulatory role in T1D by inhibiting autoimmune T cell responses. As iNKT cell-plasmacytoid dendritic cell (pDC) cooperation controls viral replication in the pancreatic islets, we investigated whether this cellular cross talk could interfere with T1D development during viral infection. Using both virus-induced and spontaneous mouse models of T1D, we show that upon viral infection, iNKT cells induce TGF-β-producing pDCs in the pancreatic lymph nodes (LNs). These tolerogenic pDCs convert naive anti-islet T cells into Foxp3(+) CD4(+) regulatory T cells (T reg cells) in pancreatic LNs. T reg cells are then recruited into the pancreatic islets where they produce TGF-β, which dampens the activity of viral- and islet-specific CD8(+) T cells, thereby preventing T1D development in both T1D models. These findings reveal a crucial cooperation between iNKT cells, pDCs, and T reg cells for prevention of T1D by viral infection.

Objective: To investigate the role of CD86(high) marginal zone (MZ) precursor B cells in type I interferon (IFN)-induced T cell-dependent responses in autoimmune BXD2 mice., Methods: Confocal microscopic imaging was used to determine the location of plasmacytoid dendritic cells (DCs), MZ precursor B cells, and CD4 T cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Immunohistochemical staining was used to determine IgG(bright) cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Enzyme-linked immunosorbent assay was used to determine serum levels of IFNα and autoantibodies, and 4-hydroxy-3-nitrophenylacetyl hapten (NP)-chicken γ-globulin (CGG) (NP-CGG)- or NP-Ficoll-induced anti-NP2 antibody titers. Real-time quantitative polymerase chain reaction was used to determine the levels of type I IFN transcripts. T cell proliferation was measured using (3) H-thymidine. The expression of CD86 and CD80 was determined by fluorescence-activated cell sorting analysis., Results: The deletion of type I IFN receptor abrogated the development of IgG(bright) cells and suppressed a T cell-dependent antibody response. Type I IFN signaling was associated with the expression of CD86, but not CD80, on follicular, MZ, and MZ precursor B cells. However, MZ precursor B cells demonstrated the highest expression of CD86 and the highest capacity for T cell costimulation with intact type I IFN receptor. This effect was blocked by an antibody that neutralizes CD86. In IFN receptor-intact BXD2 mouse spleens, MZ precursor B cells clustered at the T cell-B cell border. CD86 deletion suppressed germinal center formation, autoantibody production, and development of autoimmune diseases in BXD2 mice., Conclusion: Type I IFN can promote autoimmune responses in BXD2 mice through up-regulation of CD86(high) expression on MZ precursor B cells and trafficking of MZ precursor B cells to the T cell-B cell border to provide costimulation of CD4 T cells., (Copyright © 2011 by the American College of Rheumatology.)

Objectives: Costimulatory signals are essential for T-cell activation and hence play a very important role in antitumor immunity. B7 and 4-1BBL which belongs to tumor necrosis factor (TNF) family provide costimulatory interaction for T-cell activation and function. This study investigated the role of B7 and 4-1BBL in the amplification of tumor immunity by transduction of the B7-1, B7-2 and 4-1BBL into mouse hepatocellular carcinoma cell line H22., Methods: The tumorigenicity of H22 variants expressing either B7-1, B7-2 (H22/B7-1/B7-2) or 4-1BBL was compared with an H22 variant expressing B7-1, B7-2 and 4-1BBL (H22/B7-1/B7-2/4-1BBL). The study next investigated whether the combination of B7-1/B7-2 and 4-1BBL cell injection induced cytotoxic T lymphocyte (CTL) response and IL-2/IFN-γ secretion. The immune mechanisms underlying this combination treatment were then analyzed., Results: Syngeneic BALB/c mice injected with H22/B7-1/B7-2/4-1BBL cells that expressed elevated levels of B7-1, B7-2 and 4-1BBL showed a tumor development frequency of 50% compared with 100% in mice injected with the H22 parental line, H22/neo, H22/B7-1/B7-2 and H22/4-1BBL. Mice inoculated with H22 tumor cells expressing B7-1, B7-2 and 4-1BBL developed a strong cytotoxic T lymphocyte response and long-term immunity against wild-type tumor, suggesting a synergistic effect between the B7 and 4-1BBL costimulatory pathways. Results showed that H22/B7-1/B7-2/4-1BBL tumor vaccines probably protect the infiltrating lymphocytes from apoptosis and induce NF-κB activation to improve T-cell-mediated antitumor response., Conclusions: In this study, the antitumor consequences of using B7-1, B7-2 and 4-1BBL gene transfer have demonstrated the therapeutic potential of gene therapy approach for hepatocellular carcinoma.

Programmed death ligand-1 (PD-L1) plays a critical role in T-cell regulatory function. Here, we report a newly discovered effect of PD-L1 on angiogenesis. We demonstrate that PD-L1 and its receptor CD80, but not PD-1, are expressed by primary murine lung and heart vascular endothelial cells and the miscrovascular endothelial cell line (MS1) at both the mRNA and protein levels in vitro. The inhibition of PD-L1 or CD80 expression in MS1 cells, by small-interfering RNA transfection, led to a significant up-regulation of vascular endothelial growth factor receptor 2 expression and cell proliferation levels in MS1 cells. Furthermore, MS1 cells were found to have a significantly lower proliferation and vascular endothelial growth factor receptor 2 expression levels when they were co-cultured with PD-L1-expressing normal corneal epithelial cells, as compared to MS1 cells co-cultured with PD-L1(-/-) corneal epithelial cells. In a suture-induced corneal angiogenesis model, we observed a significantly higher level of angiogenic response in PD-L1(-/-) knockout mice as compared to wild-type mice, although there was no significant difference in the expression of inflammatory cytokines (interleukin-1α, interleukin-1β, or tumor necrosis factor-α) or the infiltration of innate immune cells (neutrophils and macrophages) between the two groups. We conclude that the expression of PD-L1 in both vascular endothelial cells and corneal epithelial cells regulates corneal angiogenesis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen-presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC-mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4-Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long-term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1-deficient endothelium hearts had significant higher frequency of IFN-γ-producing alloreactive cells as well as higher frequency of CD8(+) effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance., (©2011 The Authors Journal compilation©2011 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Aim: To establish a ubiquitously expressed PD-L1 transgenic mouse model and evaluate its recovery of motor function after spinal cord injury., Methods: Clone and sequence the complete mouse PD-L1 cDNA and construct the pCAG-PD-L1 transgenic vector by inserting the PD-L1 cDNA into the pCAGGS vector. The PD-L1 transgenic (TgPD-L1) mice were established by pronuclear micro-injection with fertilized eggs from C57BL/6 mice and the genotypes were confirmed by PCR with tail genomic DNA. The expression of PD-L1 on T and B lymphocytes from mouse spleen were detected by flow cytometry. The expression of PD-L1 in peripheral tissues was displayed by immunohistochemistry. The expression level of PD-L1 in spinal tissue was evaluated by RT-PCR. The recovery of motor function was analyzed by Basso-Beattie-Bresnahan(BBB) locomotion testing system at 3, 7, 14, 21, 28 and 35 day after spinal severe crush with forceps in mice., Results: Three lines of TgPD-L1 mice in C57BL/6 background were generated and the exogenous PD-L1 gene can be heritable steadily to offsprings. PD-L1 was highly exppressed in spinal tissue, peripheral tissues, T and B lymphocytes using RT-PCR, immunohistochemistry and flow cytometry respectively in TgPD-L1 mice. The BBB scores were obviously higher at 21 day post-injury in TgPD-L1 than those of in WT mice (P<0.05)., Conclusion: The TgPD-L1 mice whose background are C57BL/6 were established successfully and high expression level of PD-L1 in tissues promotes locomotion recovery after spinal cord injury in TgPD-L1 mice.

CMV establishes a lifelong persistent infection, and viral immune-modulating strategies are important in facilitating this. A particularly diverse CD8 T cell response develops as a result of this host-virus détente, with the CMV-specific memory T cell pool displaying unique functions and phenotypes. To gain insight into the factors that regulate CMV-specific CD8 T cell responses, we examined the influence of the B7-CD28 costimulatory pathway on magnitude, kinetics, and phenotype. Initial expansion of mouse CMV-specific CD8 T cells that establish stable memory pools was severely lower in mice lacking B7-CD28 signaling, and the resulting memory levels also remained reduced during persistent/latent infection. In contrast, expansion of CD8 T cells that undergo memory inflation during chronic infection was less affected in the absence of B7-CD28 costimulatory signals, eventually reaching the levels seen in wild-type mice at later times. Regardless of their differential requirements for B7-CD28 signals, both stable and inflationary memory T cell populations showed normal cytotoxic capacity. These results reveal that B7-CD28 costimulation differentially regulates the magnitude and kinetics of the multifaceted CD8 T cell response that develops during CMV infection.

Foxp3(+) regulatory T (Treg) cells include thymic-derived natural Treg and conventional T-derived adaptive Treg cells. Both are proposed to play important roles in downregulating inflammatory immune responses. However, the mechanisms of Treg expansion in inflammatory environments remain unclear. In this study, we report that, in an autoimmune-like graft-versus-host disease model of DBA/2 (H-2(d)) donor to BALB/c (H-2(d)) recipients, donor Treg cells in the recipients predominantly originated from expansion of natural Treg cells and few originated from adaptive Treg cells. In vivo neutralization of IFN-γ resulted in a marked reduction of donor natural Treg expansion and exacerbation of graft-versus-host disease, which was associated with downregulation of host APC expression of B7H1. Furthermore, host APC expression of B7H1 was shown to augment donor Treg survival and expansion. Finally, donor Treg interactions with host APCs via B7.1/B7H1 but not PD-1/B7H1 were demonstrated to be critical in augmenting donor Treg survival and expansion. These studies have revealed a new immune regulation loop consisting of T cell-derived IFN-γ, B7H1 expression by APCs, and B7.1 expression by Treg cells.

Asthma is the result of chronic airway inflammation associated predominantly with CD4+ cells, eosinophils, mast cells, and basophils. Several T-cells subsets, including NKT cells, play a critical role in orchestrating the inflammation in the airways predominantly, by secreting interleukin-4 and interleukin-13. Recently, programmed death-1 (PD-1) with its ligands, programmed death ligand B7H1 (PD-L1) and B7DC (PD-L2), was shown to regulate T-cell activation and tolerance. PD-1 has been characterized as a negative regulator of conventional CD4+T cells. In addition, the relative roles of PD-L1 and PD-L2 in regulating the activation and function of T cells have recently been characterized. Recent studies have demonstrated that PD-L1 and PD-L2 have important but opposing roles in modulating and polarizing T-cell functions in airway hyperreactivity. Whereas the severity of asthma is greatly enhanced in absence of PD-L2, PD-L1 deficiency resulted in reduced airway hyperresponsiveness and only minimal inflammation. This observation is partially because of the polarization of NKT cells in PD-L1- and PD-L2-deficient mice. This review will discuss the recent literature regarding the role of PD-L1 and PD-L2 in allergic disease and asthma. Current understanding of the role of PD ligands in allergic asthma gives impetus to the development of novel therapeutic approaches., (© 2010 John Wiley & Sons A/S.)

Although CD4 T cells are required for host resistance to Mycobacterium tuberculosis, they may also contribute to pathology. In this study, we examine the role of the inhibitory receptor PD-1 and its ligand PD-L1 during M. tuberculosis infection. After aerosol exposure, PD-1 knockout (KO) mice develop high numbers of M. tuberculosis-specific CD4 T cells but display markedly increased susceptibility to infection. Importantly, we show that CD4 T cells themselves drive the increased bacterial loads and pathology seen in infected PD-1 KO mice, and PD-1 deficiency in CD4 T cells is sufficient to trigger early mortality. PD-L1 KO mice also display enhanced albeit less severe susceptibility, indicating that T cells are regulated by multiple PD ligands during M. tuberculosis infection. M. tuberculosis-specific CD8 T cell responses were normal in PD-1 KO mice, and CD8 T cells only had a minor contribution to the exacerbated disease in the M. tuberculosis-infected PD-1 KO and PD-L1 KO mice. Thus, in the absence of the PD-1 pathway, M. tuberculosis benefits from CD4 T cell responses, and host resistance requires inhibition by PD-1 to prevent T cell-driven exacerbation of the infection.

Antagonism of CXCR4 disrupts the interaction between the CXCR4 receptor on hematopoietic stem cells (HSCs) and the CXCL12 expressed by stromal cells in the bone marrow, which subsequently results in the shedding of HSCs to the periphery. Because of their profound immunomodulatory effects, HSCs have emerged as a promising therapeutic strategy for autoimmune disorders. We sought to investigate the immunomodulatory role of mobilized autologous HSCs, via target of the CXCR4-CXL12 axis, to promote engraftment of islet cell transplantation. Islets from BALB/c mice were transplanted beneath the kidney capsule of hyperglycemic C57BL/6 mice, and treatment of recipients with CXCR4 antagonist resulted in mobilization of HSCs and in prolongation of islet graft survival. Addition of rapamycin to anti-CXCR4 therapy further promoted HSC mobilization and islet allograft survival, inducing a robust and transferable host hyporesponsiveness, while administration of an ACK2 (anti-CD117) mAb halted CXCR4 antagonist-mediated HSC release and restored allograft rejection. Mobilized HSCs were shown to express high levels of the negative costimulatory molecule programmed death ligand 1 (PD-L1), and HSCs extracted from wild-type mice, but not from PD-L1 knockout mice, suppressed the in vitro alloimmune response. Moreover, HSC mobilization in PD-L1 knockout mice failed to prolong islet allograft survival. Targeting the CXCR4-CXCL12 axis thus mobilizes autologous HSCs and promotes long-term survival of islet allografts via a PD-L1-mediated mechanism.

Background: The pathogenesis of hypertension remains poorly understood, and treatment is often unsuccessful. Recent evidence suggests that the adaptive immune response plays an important role in this disease. Various hypertensive stimuli cause T-cell activation and infiltration into target organs such as the vessel and the kidney, which promotes vascular dysfunction and blood pressure elevation. Classically, T-cell activation requires T-cell receptor ligation and costimulation. The latter often involves interaction between B7 ligands (CD80 and CD86) on antigen-presenting cells with the T-cell coreceptor CD28. This study was therefore performed to examine the role of this pathway in hypertension., Methods and Results: Angiotensin II-induced hypertension increased the presence of activated (CD86(+)) dendritic cells in secondary lymphatic tissues. Blockade of B7-dependent costimulation with CTLA4-Ig reduced both angiotensin II- and deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Activation of circulating T cells, T-cell cytokine production, and vascular T-cell accumulation caused by these hypertensive stimuli were abrogated by CTLA4-Ig. Furthermore, in mice lacking B7 ligands, angiotensin II caused minimal blood pressure elevation and vascular inflammation, and these effects were restored by transplantation with wild-type bone marrow., Conclusions: T-cell costimulation via B7 ligands is essential for development of experimental hypertension, and inhibition of this process could have therapeutic benefit in the treatment of this disease.

Unlabelled: Liver fibrosis is mediated by the transformation of hepatic stellate cells (HSC) from a quiescent to an activated state. To understand the role of HSC in liver immunity, we investigated the effect of this transition on T cell stimulation in vitro. Unlike quiescent HSC, activated HSC did not induce proliferation of antigen-specific T cells. Phenotypic analysis of quiescent and activated HSC revealed that activated HSC expressed the coinhibitory molecule B7-H4. Silencing B7-H4 by small interfering RNA (siRNA) in activated HSC restored the ability of T cells to proliferate, differentiate, and regain effector recall responses. Furthermore, expression of B7-H4 on HSC inhibits early T cell activation and addition of exogenous interleukin (IL)-2 reversed the T cell anergy induced by activated HSC., Conclusion: These studies reveal a novel role for activated HSC in the attenuation of intrahepatic T cell responses by way of expression of the coinhibitory molecule B7-H4, and may provide fundamental insight into intrahepatic immunity during liver fibrogenesis., (Copyright © 2010 American Association for the Study of Liver Diseases.)

We previously reported that after exposure to inflammatory cytokines, such as IL-17 and IFN-γ, RPE cells express increased amounts of suppressor of cytokine signaling, leading to general suppression of the inflammatory response. Here, we demonstrate that RPE cells expressed increased levels of PD-L1 in response to IL-17, IFN-γ, or Poly I:C. These PD-L1(hi) RPE cells inhibited the pathogenic activities of IRBP-specific T cells, which usually induced uveitis when injected into naïve mice (EAU). The suppressed pathogenicity of these uveitogenic T cells after exposure to PD-L1(hi) RPE cells could be partially reversed by anti-PD-L1 antibodies. Nevertheless, IRBP-specific T cells pre-exposed to PD-L1(hi) RPE cells displayed substantial suppressor activity, which strongly inhibited the activation of fresh IRBP-Teffs in response to subsequent antigenic challenge and when transferred into naïve mice, inhibited the induction of EAU by IRBP-Teff transfer. These findings suggest that inflammatory cytokine-triggered up-regulation of PD-L1 on RPE constitutes a critical factor for inducing infiltrated uveitogenic T cells with regulatory activities, which may accelerate the natural resolution of T cell-mediated intraocular inflammation.

Purpose: Inducible costimulator (ICOS) is an important costimulatory molecule involved in T-cell activation. In this study, the role of ICOS in the pathogenesis of uveitis in Behçet's disease (BD) was investigated., Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from BD patients with uveitis in the active or remission phase and in healthy subjects. Total RNA was isolated from PMBCs, and mRNA expression was analyzed on an oligonucleotide microarray. ICOS expression on CD4(+) T cells was determined by flow cytometry, and the functional costimulatory effect of ICOS/B7RP-1 interaction was assessed on stimulation with concanavalin A (conA) or IRBP in the presence or absence of anti-ICOS mAb., Results: As the result of microarray analysis, ICOS in PBMCs showed the greatest difference in expression in BD patients with uveitis compared with healthy control subjects. ICOS expression on CD4(+) T cells in BD patients with uveitis was significantly higher than that in healthy individuals, both before and after conA stimulation. Among the BD patients, ICOS expression on CD4(+) T cells was significantly higher in those with active uveitis than in those with remitted uveitis. Blockade of ICOS/B7-related protein-1 (B7RP-1) interaction by anti-ICOS mAb significantly decreased IFN-γ, IL-17, and TNF-α production by PBMCs when stimulated with conA or IRBP in BD with active uveitis., Conclusions: High ICOS expression in BD patients with uveitis contributed to the upregulation of IFN-γ, IL-17, and TNF-α production, suggesting that abnormal ICOS costimulation may play an immunopathologic role in the pathogenesis of uveitis in BD.

B7-H3, a new member of the B7 superfamily, acts as both a T cell costimulator and coinhibitor, and thus plays a key role in the regulation of T cell-mediated immune responses. However, it is unclear whether B7-H3 is involved in the innate immune monocyte/macrophage-mediated inflammatory response. In this paper, we show that, although B7-H3 alone failed to stimulate proinflammatory cytokine release from murine macrophages, it strongly augmented both LPS- and bacterial lipoprotein-induced NF-kappaB activation and inflammatory response. This occurred in both a TLR4- and TLR2-dependent manner. Blockage of B7-H3 in vivo attenuated LPS-induced proinflammatory cytokine release and endotoxic shock-related lethality. Furthermore, we found that patients diagnosed with sepsis, in contrast to healthy individuals, exhibited significant levels of raised plasma soluble B7-H3 (sB7-H3) and that this level correlated with the clinical outcome and levels of plasma TNF-alpha and IL-6. In addition, a putative receptor for B7-H3 was detected on monocytes and peritoneal macrophages from septic patients but not on monocytes from healthy donors. Stimulation of human monocytes with LPS and inflammatory cytokines led to a substantial release of sB7-H3. Taken together, our data indicate that significantly elevated plasma sB7-H3 in septic patients may predict a poor outcome. Furthermore, we demonstrate that B7-H3 functions as a costimulator of innate immunity by augmenting proinflammatory cytokine release from bacterial cell wall product-stimulated monocytes/macrophages and may contribute positively to the development of sepsis.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

Zymosan is a complex fungal component shown to be capable of both promoting and suppressing the development of autoimmune disorders in mice. In this study, we show that a single injection of zymosan just prior to diabetes onset can significantly delay the progression of disease in NOD mice. Zymosan treatment of NOD mice induced the production of biologically active TGF-beta from cells infiltrating the pancreas and was associated with expansion of programmed cell death 1 ligand 1(+)TGF-beta(+) macrophages and Foxp3(+) regulatory T cells in vivo. Neutralization of either TGF-beta or programmed cell death 1 ligand 1 abrogated the protective effects of zymosan. Zymosan acted through TLR2 as well as ERK and p38 MAPK to induce macrophage secretion of TGF-beta and promotion of Foxp3(+) regulatory T cells in vitro and in vivo.

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti-B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were re-exposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance.

Th cells that produce IL-17 (Th17 cells) are a distinct subset of Th cells implicated in several autoimmune diseases. Although CD28-B7 interaction has been shown to be involved in Th17 differentiation in vitro, the role of CTLA-4 in controlling Th17 development is completely unknown. We report in this paper that blocking the CTLA-4-B7 interaction potentiates Th17 cell differentiation in vitro and in vivo. Furthermore, blocking CTLA-4-B7 interaction in vivo confers the susceptibility of experimental autoimmune myocarditis to CD28(-/-) mice or increases the severity of experimental autoimmune myocarditis in wild-type mice. The enhanced disease susceptibility is mediated by heightened Th17 responses. With these results, we are the first to demonstrate that CTLA-4-B7 interaction inhibits Th17 differentiation in vitro and in vivo and suppresses Th17-mediated autoimmunity.

Lymphocytic choriomeningitis virus (LCMV)-specific CD8(+) T cell responses are considered to be independent of CD28-B7 costimulation. However, the LCMV-specific response has never been evaluated in B7.1/B7.2(-/-) mice. For this reason, we decided to study the T cell response in B7.1/B7.2(-/-) mice infected with two different strains of LCMV, one (Traub strain) typically causing low-grade chronic infection, and another (Armstrong clone 53b) displaying very limited capacity for establishing chronic infection. Using Traub virus we found that most B7.1/B7.2(-/-) mice were unable to rid themselves of the infection. Chronic infection was associated with a perturbed CD8(+) T cell epitope hierarchy, as well as with the accumulation of cells expressing markers of terminal differentiation and being unable to respond optimally to Ag restimulation. Examination of matched CD28(-/-) mice revealed a similar albeit less pronounced pattern of CD8(+) T cell dysfunction despite lack of virus persistence. Finally, analysis of B7.1/B7.2(-/-) mice infected with Armstrong virus revealed a scenario quite similar to that in Traub infected CD28(-/-) mice; that is, the mice displayed evidence of T cell dysfunction, but no chronic infection. Taken together, these results indicate that B7 costimulation is required for induction and maintenance of LCMV-specific CD8(+) T cell memory, irrespective of the LCMV strain used for priming. However, the erosion of CD8(+) T cell memory in B7.1/B7.2(-/-) mice was more pronounced in association with chronic infection. Finally, virus-specific T cell memory was more impaired in the absence of B7 molecules than in the absence of the CD28 receptor, supporting earlier data suggesting the existence of additional stimulatory receptors for B7.

PURPOSE. Given that dry eye disease (DED) is associated with T cell-mediated inflammation of the ocular surface and that PD-L1 is an important negative or inhibitory regulator of immune responses constitutively expressed at high levels by corneal epithelial cells, the authors studied the expression and function of PD-L1 in DED. METHODS. Dry eye was induced in untreated wild-type mice, PD-L1(-/-) mice, and wild-type mice treated with anti-PD-L1 antibody by exposing these mice to a desiccating environment in the controlled environment chamber modified with subcutaneous administration of scopolamine. Real-time PCR was used to quantify the expression of chemokine gene transcript levels of multiple CC and CXC chemokine ligands and receptors. Epifluorescence microscopy was used to evaluate corneal infiltration of CD3(+) T cells after immunohistochemical staining. RESULTS. The increased expression of specific chemokine ligands and receptors in PD-L1(-/-) corneas of normal mice is associated with significant increases in T-cell homing into these corneas. Similar, and more enhanced, increases in T-cell infiltration were observed in PD-L1(-/-) DED mice or DED mice treated with anti-PD-L1 antibody compared with controls. In addition, the authors found significantly decreased expression of PD-L1 by corneal epithelial cells in DED and significantly increased corneal fluorescein staining score with PD-L1 functional blockade using anti-PD-L1 antibody. CONCLUSIONS. Downregulation of corneal epithelial PD-L1 amplifies dry eye-associated corneal inflammation and epitheliopathy by increasing the expression of chemokine ligands and receptors that promote T-cell homing to the ocular surface.

T cell responses are compromised in the elderly. The B7-CD28 family receptors are critical in the regulation of immune responses. We evaluated whether the B7-family and CD28-family receptors were differentially expressed in dendritic cells, macrophages, and CD4(+) and CD8(+) T cells from young and old mice, which could contribute to the immune dysfunction in the old. Although most of the receptors were equally expressed in all cells, >85% of the old naive CD8(+) T cells expressed B7-H1 compared with 25% in the young. Considering that B7-H1 negatively regulates immune responses, we hypothesized that expression of B7-H1 would downregulate the function of old CD8(+) T cells. Old CD8(+) T cells showed reduced ability to proliferate, but blockade of B7-H1 restored the proliferative capacity of old CD8(+) T cells to a level similar to young CD8(+) T cells. In vivo blockade of B7-H1 restored antitumor responses against the B7-H1(-) BM-185-enhanced GFP tumor, such that old animals responded with the same efficiency as young mice. Our data also indicate that old CD8(+) T cells express lower levels of TCR compared with young CD8(+) T cells. However, following antigenic stimulation in the presence of B7-H1 blockade, the levels of TCR expression were restored in old CD8(+) T cells, which correlated with stronger T cell activation. These studies demonstrated that expression of B7-H1 in old CD8(+) T cells impairs the proper activation of these cells and that blockade of B7-H1 could be critical to optimally stimulate a CD8 T cell response in the old.

Keratinocytes (KCs) may play important roles for maintenance of peripheral tolerance in the upper layers of the skin. Coinhibitory signals mediated via the programmed death (PD)-1 and its ligand B7-H1 (PD-L1/CD274) are crucial for the downregulation of T cell immune responses and for the maintenance of peripheral tolerance. In this study, to investigate the role of KC-expressed B7-H1 in the regulation of T cell immune responses, we generated transgenic (tg) mice overexpressing B7-H1 under the control of keratin 14 (K14) promoter (K14-B7-H1 tg). K14-B7-H1 tg mice displayed impaired contact hypersensitivity (CH) responses to primary and secondary hapten challenges. The K14-B7-H1 tg mice did not exhibit substantial impairment of cutaneous dendritic cell migration after sensitization and of hapten-specific proliferation and IFN-gamma production of CD4(+) and CD8(+) T cells in the draining lymph nodes, suggesting that overexpression of B7-H1 on KCs did not affect the induction phase of the CH response. The systemic or s.c. injection of hapten-sensitized T cells into the K14-B7-H1 tg mice did not efficiently induce the CH response. IFN-gamma expression and apoptosis of KCs in the challenged ears were impaired in K14-B7-H1 tg mice. IFN-gamma production by presensitized CD8(+) T cells stimulated with hapten-pulsed KCs was markedly impaired for the KCs obtained from the K14-B7-H1 tg mice but was restored by the addition of an anti-B7-H1 mAb. These results suggest that KC-associated B7-H1 directly downregulates the effector function of CD8(+) T cells by associating with PD-1 at local inflammatory sites and that it plays a role in peripheral T cell tolerance against exogenous Ags.

During chronic viral infection, persistent exposure to viral Ags leads to the overexpression of multiple inhibitory cell-surface receptors that cause CD8(+) T cell exhaustion. The severity of exhaustion correlates directly with the level of infection and the number and intensity of inhibitory receptors expressed, and it correlates inversely with the ability to respond to the blockade of inhibitory pathways. Friend virus (FV) is a murine retrovirus complex that induces acute high-level viremia, followed by persistent infection and leukemia development, when inoculated into immunocompetent adult mice. In this article, we provide conclusive evidence that FV infection results in the generation of virus-specific effector CD8(+) T cells that are terminally exhausted. Acute FV-induced disease is characterized by a rapid increase in the number of virus-infected erythroblasts, leading to massive splenomegaly. Most of the expanded erythroblasts strongly express programmed death ligand-1 and MHC class I, thereby creating a highly tolerogenic environment. Consequently, FV-specific effector CD8(+) T cells uniformly express multiple inhibitory receptors, such as programmed cell death 1 (PD-1), T cell Ig domain and mucin domain 3 (Tim-3), lymphocyte activation gene-3, and CTLA-4, rapidly become nonresponsive to restimulation and are no longer reinvigorated by combined in vivo blockade of PD-1 and Tim-3 during the memory phase. However, combined blockade of PD-1 and Tim-3 during the priming/differentiation phase rescued FV-specific CD8(+) T cells from becoming terminally exhausted, resulting in improved CD8(+) T cell functionality and virus control. These results highlight FV's unique ability to evade virus-specific CD8(+) T cell responses and the importance of an early prophylactic approach for preventing terminal exhaustion of CD8(+) T cells.

Under steady-state conditions, B7-1 is present as a mixed population of noncovalent dimers and monomers on the cell surface. In this study, we examined the physiological significance of this unique dimer-monomer equilibrium state of B7-1. We demonstrate that altering B7-1 to create a uniformly covalent dimeric state results in enhanced CD28-mediated formation of T cell-APC conjugates. The enhanced T cell-APC conjugate formation correlates with persistent concentration of signaling molecules PKC- and lck at the immunological synapse. In contrast, T cell acquisition of B7-1 from APCs, an event that occurs as a consequence of CD28 engagement with B7-1/B7-2 and is thought to play a role in the dissociation of T cell-APC conjugates, is highly reduced when B7-1 is present in the covalently dimeric state. The ability of covalently dimeric and wild type B7-1 to costimulate Ag-specific T cell proliferation was also assessed. In contrast to the enhanced ability of dimeric B7-1 to support conjugate formation and early parameters of T cell signaling, sensitivity to competitive inhibition by soluble CTLA-4-Ig indicated that the covalent dimeric form of B7-1 is less efficient in costimulating T cell proliferation. These findings suggest a novel model in which optimal T cell costimulatory function of B7-1 requires high-avidity CD28 engagement by dimeric B7-1, followed by dissociation of these noncovalent B7-1 dimers, facilitating downregulation of CD28 and internalization of B7-1. These events regulate signaling through TCR/CD28 to maximize T cell activation to proliferation.

The caliber and magnitude of T cell responses are regulated by costimulatory molecules following the engagement of TCRs and MHC molecules. B7-DC has the highest homology with B7-H1 in the B7 family, and both of them bind an immunoregulatory molecule, programmed death 1. Previous studies have demonstrated that B7-DC stimulates T cell proliferation and CTL generation, which sharply contrasts the inhibitory role of B7-H1. Th2 cytokines prompt B7-DC expression, which in turn enhances Th1 responses. In this study, we used an intestinal nematode, Nippostrongylus brasiliensis, to induce strong Th2 responses and to evaluate B7-DC function under Th2-polarizing conditions in vivo. By either blocking B7-DC expression during N. brasiliensis infection or by examining N. brasiliensis-infected B7-DC knockout mice, we observed enhanced eosinophilia, the overproduction of serum IgE, and increased Th2 cytokine production along with decreased Th1 cytokine production (particularly IFN-gamma production), indicating that B7-DC inhibits Th2 responses. Our results further demonstrate that the inhibition of Th2 responses by B7-DC occurs independently of programmed death 1 but conceivably acts through an as yet unknown alternative receptor that enhances Th1 responses. Although the deficiency of B7-DC expression that enhanced the production of IL-13 paradoxically resulted in better protection against N. brasiliensis infection, our results show that B7-DC plays an important role in bolstering a robust Th1 response that is required for effective antiviral and anticancer immunity, even under a strong Th2-polarizing environment induced by N. brasiliensis infection.

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

Both the programmed death (PD) 1-PD-ligand (PD-L) pathway and regulatory T (T reg) cells are instrumental to the maintenance of peripheral tolerance. We demonstrate that PD-L1 has a pivotal role in regulating induced T reg (iT reg) cell development and sustaining iT reg cell function. PD-L1(-/-) antigen-presenting cells minimally convert naive CD4 T cells to iT reg cells, showing the essential role of PD-L1 for iT reg cell induction. PD-L1-coated beads induce iT reg cells in vitro, indicating that PD-L1 itself regulates iT reg cell development. Furthermore, PD-L1 enhances and sustains Foxp3 expression and the suppressive function of iT reg cells. The obligatory role for PD-L1 in controlling iT reg cell development and function in vivo is illustrated by a marked reduction in iT reg cell conversion and rapid onset of a fatal inflammatory phenotype in PD-L1(-/-)PD-L2(-/-) Rag(-/-) recipients of naive CD4 T cells. PD-L1 iT reg cell development is mediated through the down-regulation of phospho-Akt, mTOR, S6, and ERK2 and concomitant with the up-regulation of PTEN, all key signaling molecules which are critical for iT reg cell development. Thus, PD-L1 can inhibit T cell responses by promoting both the induction and maintenance of iT reg cells. These studies define a novel mechanism for iT reg cell development and function, as well as a new strategy for controlling T reg cell plasticity.

Tumors have evolved multiple mechanisms to evade immune destruction. One of these is expression of T cell inhibitory ligands such as programmed death-ligand 1 (PD-L1; B7-H1). In this study, we show that PD-L1 is highly expressed on mesothelioma tumor cells and within the tumor stroma. However, PD-L1 blockade only marginally affected tumor growth and was associated with the emergence of activated programmed death-1(+) ICOS(+) CD4 T cells in tumor-draining lymph nodes, whereas few activated CD8 T cells were present. Full activation of antitumor CD8 T cells, characterized as programmed death-1(+) ICOS(+) Ki-67(+) and displaying CTL activity, was only observed when CD4 T cells were depleted, suggesting that a population of suppressive CD4 T cells exists. ICOS(+) foxp3(+) regulatory T cells were found to be regulated through PD-L1, identifying one potentially suppressive CD4 T cell population. Thus, PD-L1 blockade activates antitumor CD8 T cell most potently in the absence of CD4 T cells. These findings have implications for the development of PD-L1-based therapies.

B cell responses are regulated by Ag recognition, costimulatory signals provided by interaction with helper T cells, and by innate signals. We recently provided evidence for a link between the effects of innate and costimulatory signals on B cells during influenza virus infection, by demonstrating that most B cells in the regional lymph nodes of the respiratory tract enhance surface expression of the costimulator B7-2 (CD86) within 24-48 h following infection via a type I IFNR-dependent mechanisms, a finding we are confirming here. While the role of B7-1/2 for helper T cell activation is well documented, its role in direct B cell regulation is poorly understood. Here, our in vivo studies with mixed bone marrow irradiation chimeric mice, lacking B7-1/2 only on B cells, demonstrated that B7-1/2 expression is crucial for induction of maximal local, but to a lesser extent systemic, IgG Ab responses following influenza virus infection. In contrast to mice that completely lack B7-1/2 expression, loss of B7-1/2 on B cells alone did not significantly affect germinal center formation or the extent of CD4(+) T cell activation and IFN-gamma secretion. Instead, our in vitro studies identify a dramatic effect of B7-2 engagement on IgG, but not IgM secretion by already class-switched B cells. Concomitantly, B7-2 engagement induced expression of X-box binding protein 1 (XBP-1) and spliced XBP1, evidence for increased protein synthesis by these cells. Taken together, these results identify direct signaling through B7-1/2 as a potent regulator of IgG secretion by previously activated B cells.