Treg-expressed CTLA-4 depletes CD80/CD86 by trogocytosis, releasing free PD-L1 on antigen-presenting cells.

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 CD80lo free PD-L1hi 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.

Tumor Eradication by Cisplatin Is Sustained by CD80/86-Mediated Costimulation of CD8+ T Cells.

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.

CD80 and PD-L2 define functionally distinct memory B cell subsets that are independent of antibody isotype.

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.

Role of podocyte B7-1 in diabetic nephropathy.

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.

T-cell costimulation protects obesity-induced adipose inflammation and insulin resistance.

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.

B7-1/B7-2 blockade overrides the activation of protective CD8 T cells stimulated in the absence of Foxp3+ regulatory T cells.

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.

Cyclic AMP underpins suppression by regulatory T cells.

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.

Minimal change disease: a CD80 podocytopathy?

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.

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

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

[Establishment of PD-L1 transgenic mouse model and recovery of the motion after spinal cord injury].

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.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

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.

Differential B7-CD28 costimulatory requirements for stable and inflationary mouse cytomegalovirus-specific memory CD8 T cell populations.

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.

A novel function for programmed death ligand-1 regulation of angiogenesis.

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-1ß, 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.

Viral infection prevents diabetes by inducing regulatory T cells through NKT cell-plasmacytoid dendritic cell interplay.

Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell-mediated destruction of insulin-producing ß 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-ß-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-ß, 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.

Human T cells induce their own regulation through activation of B cells.

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.

Host APCs augment in vivo expansion of donor natural regulatory T cells via B7H1/B7.1 in allogeneic recipients.

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.

Triple expression of B7-1, B7-2 and 4-1BBL enhanced antitumor immune response against mouse H22 hepatocellular carcinoma.

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.

CD4 T cells promote rather than control tuberculosis in the absence of PD-1-mediated inhibition.

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.

Targeting the CXCR4-CXCL12 axis mobilizes autologous hematopoietic stem cells and prolongs islet allograft survival via programmed death ligand 1.

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.

PD-L1(hi) retinal pigment epithelium (RPE) cells elicited by inflammatory cytokines induce regulatory activity in uveitogenic T cells.

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.