PD-1 agonism by anti-CD80 inhibits T cell activation and alleviates autoimmunity.

Targeted blockade of the checkpoint molecule programmed cell death 1 (PD-1) can activate tumor-specific T cells to destroy tumors, whereas targeted potentiation of PD-1 is expected to suppress autoreactive T cells and alleviate autoimmune diseases. However, the development of methods to potentiate PD-1 remains challenging. Here we succeeded in eliciting PD-1 function by targeting the cis-PD-L1-CD80 duplex, formed by binding of CD80 to the PD-1 ligand PD-L1, that attenuates PD-L1-PD-1 binding and abrogates PD-1 function. By generating anti-CD80 antibodies that detach CD80 from the cis-PD-L1-CD80 duplex and enable PD-L1 to engage PD-1 in the presence of CD80, we demonstrate that the targeted dissociation of cis-PD-L1-CD80 duplex elicits PD-1 function in the condition where PD-1 function is otherwise restricted. We demonstrate using murine models that the removal of PD-1 restriction is effective in alleviating autoimmune disease symptoms. Our findings establish a method to potentiate PD-1 function and propose the removal of restraining mechanisms as an efficient strategy to potentiate the function of inhibitory molecules.

Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.

LAG-3 inhibits the activation of CD4+ T cells that recognize stable pMHCII through its conformation-dependent recognition of pMHCII.

The success of tumor immunotherapy targeting the inhibitory co-receptors PD-1 and CTLA-4 has indicated that many other co-receptors might be potential druggable targets, despite limited information about their functional differences. Here we identified a unique target selectivity for the inhibitory co-receptor LAG-3 that was intrinsic to its immunoregulatory roles. Although LAG-3 has been reported to recognize major histocompatibility complex (MHC) class II, it did not recognize MHC class II universally; instead, we found that it selectively recognized stable complexes of peptide and MHC class II (pMHCII). LAG-3 did not directly interfere with interactions between the co-receptor CD4 and MHC class II or between the T cell antigen receptor and MHC class II. Instead, LAG-3 preferentially suppressed T cells responsive to stable pMHCII by transducing inhibitory signals via its intracellular region. Thus, LAG-3 might function more selectively than previously thought and thereby maintain tolerance to dominant autoantigens.

PD-1 Imposes Qualitative Control of Cellular Transcriptomes in Response to T Cell Activation.

Targeted blockade of programmed cell death 1 (PD-1), an immune-checkpoint receptor that inhibits T cell activation, provides clinical benefits in various cancers. However, how PD-1 modulates gene expression in T cells remains enigmatic. Here we investigated how PD-1 affects transcriptome changes induced by T cell receptor (TCR) activation. Intriguingly, we identified a huge variance in PD-1 sensitivity among TCR-inducible genes. When we quantified the half maximal effective concentration (EC50) as the relationship between change in gene expression and TCR signal strength, we found that genes associated with survival and proliferation were efficiently expressed upon TCR activation and resistant to PD-1-mediated inhibition. Conversely, genes encoding cytokines and effector molecules were expressed less efficiently and sensitive to PD-1-mediated inhibition. We further demonstrated that transcription factor binding motifs and CpG frequency in the promoter region affect EC50 and thus the PD-1 sensitivity of genes. Our findings explain how PD-1, dependent on the TCR signal strength, calibrates cellular transcriptomes to shape functional properties of T cell populations.

PD-1 preferentially inhibits the activation of low-affinity T cells.

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

T-cell-intrinsic and -extrinsic regulation of PD-1 function.

Cancer immunotherapies that target PD-1 (programmed cell death 1) aim to destroy tumors by activating tumor-specific T cells that are otherwise inactivated by PD-1. Although these therapies have significantly improved the outcomes of patients with diverse cancer types and have revolutionized cancer treatment, only a limited proportion of patients benefits from the therapies currently. Therefore, there is a continued need to decipher the complex biology of PD-1 to improve therapeutic efficacies as well as to prevent immune-related adverse events. Especially, the spaciotemporal context in which PD-1 functions and the properties of T cells that are restrained by PD-1 are only vaguely understood. We have recently revealed that PD-1 function is strictly restricted at the activation phase of T-cell responses by the cis-interactions of PD-L1 and CD80 on antigen-presenting cells, which is critical for the induction of optimal T-cell responses. We also found that the sensitivity to the effects of PD-1 in T cells is essentially determined by T-cell-intrinsic factors. In T cells bearing T-cell antigen-receptors (TCRs) with lower affinity to antigenic peptides, PD-1 inhibits the expression of TCR-inducible genes more efficiently; thereby PD-1 preferentially suppresses low-affinity T cells. Thus, PD-1 function is coordinately regulated by various T-cell-intrinsic and -extrinsic factors that alter the responsiveness of T cells and the availability of PD-1 ligands. Precise and deeper understanding of the regulatory mechanisms of PD-1 is expected to facilitate the rational development of effective and safe immunotherapies.

Atypical motifs in the cytoplasmic region of the inhibitory immune co-receptor LAG-3 inhibit T cell activation.

T cell activation is tightly regulated by both stimulatory and inhibitory co-receptors and has been a focus in the development of interventions for managing cancer or autoimmune diseases. Targeting the inhibitory co-receptors programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) has successfully eradicated tumors but induced immune-related adverse events in humans and mice. The beneficial and adverse effects of targeting these co-receptors highlight their importance in cancer immunity and also autoimmunity. Although the therapeutic potencies of other inhibitory co-receptors are under extensive investigation, their inhibitory mechanisms and their functional differences are not well understood. Here we analyzed the inhibitory mechanisms of lymphocyte activation gene-3 (LAG-3), another inhibitory co-receptor, by using an in vitro T cell activation system and a high-affinity anti-LAG-3 antibody that strongly interferes with the binding of LAG-3 to its ligand. We found that the expression level of LAG-3 strongly correlates with the inhibitory function of LAG-3, suggesting that LAG-3 functions as a rheostat rather than as a breaker of T cell activation. By evaluating the inhibitory capacities of various LAG-3 variants relative to their expression levels, we found that LAG-3 transduces two independent inhibitory signals through an FXXL motif in the membrane-proximal region and the C-terminal EX repeat. These motifs have not been reported previously for inhibitory co-receptors, suggesting that LAG-3 inhibits T cell activation through a nonredundant inhibitory mechanisms along with the other inhibitory co-receptors. Our findings provide a rationale for combinatorial targeting of LAG-3 and the other inhibitory co-receptors to improve cancer immunotherapy.

Glucocorticoids potentiate the inhibitory capacity of programmed cell death 1 by up-regulating its expression on T cells.

The inhibitory co-receptor programmed cell death 1 (PD-1, Pdcd1) plays critical roles in the regulation of autoimmunity, anticancer immunity, and immunity against infections. Immunotherapies targeting PD-1 have revolutionized cancer management and instigated various trials of improved cancer immunotherapies. Moreover, extensive trials are underway to potentiate PD-1 function to suppress harmful immune responses. Here we found that both natural and synthetic glucocorticoids (GCs) up-regulate PD-1 on T cells without altering the expression levels of other co-receptors and cell surface molecules. GC-induced up-regulation of PD-1 depended on transactivation of PD-1 transcription mediated through the glucocorticoid receptor. We further found that a GC response element 2525 bp upstream of the transcription start site of Pdcd1 is responsible for GC-mediated transactivation. We also observed that in vivo administration of GCs significantly up-regulates PD-1 expression on tumor-infiltrating T cells. By analyzing T cells differing in PD-1 expression, we directly demonstrated that the amount of PD-1 on the cell surface correlates with its inhibitory effect. Accordingly, GCs potentiated the capacity of PD-1 to inhibit T cell activation, suggesting that this PD-1-mediated inhibition contributes, at least in part, to the anti-inflammatory and immunosuppressive effects of GCs. In light of the critical roles of PD-1 in the regulation of autoimmunity, we expect that the potentiation of PD-1 activity may offer a promising therapeutic strategy for managing inflammatory and autoimmune diseases. Our current findings provide a rationale for strategies seeking to enhance the inhibitory effect of PD-1 by increasing its expression level.

PD-1 efficiently inhibits T cell activation even in the presence of co-stimulation through CD27 and GITR.

Cancer immunotherapies targeting programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 revolutionized cancer treatment and instigated various trials to develop new cancer immunotherapies with higher therapeutic efficacy. Agonistic Abs against tumor necrosis factor receptor super family (TNFRSF) molecules are highly expected due to their high potential to enhance survival, proliferation, and effector function of T cells. To date, agonistic antibodies (Abs) against CD27, GITR, OX40, and 4-1BB have been reported to increase the efficacy of anti-PD-1 therapy in animal models and clinical trials of these combinatorial therapies are underway. However, the mechanisms how agonistic Abs against TNFRSF molecules potentiate anti-PD-1 therapy are not well understood. Here we examined the potency of PD-1 to inhibit the antigen-dependent activation of T cells in the presence of co-stimulation through CD27 and GITR by using in vitro and ex vivo co-culture systems of T cells and antigen presenting cells. The cytokine secretion from T cells upon antigen stimulation was strongly augmented by the engagement of CD27 or GITR with their corresponding ligands. Remarkably, PD-1 efficiently inhibited the activation of T cells even in the presence of co-stimulation through CD27 or GITR. Accordingly, cytokine secretion was synergistically augmented when PD-1 blockade was combined with triggering of CD27 or GITR. These results indicate that the triggering of TNFRSF molecules and PD-1 blockade can act on the same individual cells simultaneously to augment the magnitude of T cell activation, providing the rationale for the combinatorial usage of agonistic Abs against TNFRSF molecules and blocking Abs against PD-1 or PD-L1.

PD-1 aborts the activation trajectory of autoreactive CD8+ T cells to prohibit their acquisition of effector functions.

Anti-PD-1 therapy can induce eradication of tumors and immune-related adverse events (irAEs) in humans and model animals. However, how anti-PD-1 therapy modifies cellular phenotypes of CD8+ T cells to destroy tumors and damage self-tissues remains to be clarified. Here we performed single cell mRNA expression profiling of autoreactive CD8+ T cells under or beyond PD-1 suppression in target tissues and reconstructed their activation trajectory. Autoreactive CD8+ T cells went through four activation phases and PD-1 strongly attenuated the transition from the second- to the third-phase, where effector functions were acquired. Shifts in cluster composition of autoreactive CD8+ T cells markedly reflected the severity of autoimmunity. In addition, genes up-regulated along the activation-trajectory in autoimmunity were highly expressed in responders of melanoma patients in anti-PD-1 therapy, suggesting that tumor-specific T cells need to be activated in a similar trajectory to destroy tumors in human patients upon PD-1 blockade. These findings reveal that PD-1 blockade facilitates the activation trajectory of CD8+ T cells to boost their effector functions. Targeted manipulation of the trajectory could lead to new therapeutic opportunities.

DCIR maintains bone homeostasis by regulating IFN-γ production in T cells.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor mainly expressed in DCs. Dcir (-/-) mice spontaneously develop autoimmune enthesitis and ankylosis accompanied by fibrocartilage proliferation and ectopic ossification. However, the mechanisms of new bone/cartilage formation in Dcir (-/-) mice remain to be elucidated. In this study, we show that DCIR maintains bone homeostasis by regulating IFN-γ production under pathophysiological conditions. DCIR deficiency increased bone volume in femurs and caused aberrant ossification in joints, whereas these symptoms were abolished in Rag2(-/-)Dcir(-/-) mice. IFN-γ-producing T cells accumulated in lymph nodes and joints of Dcir(-/-) mice, and purified Dcir(-/-) DCs enhanced IFN-γ(+) T cell differentiation. The ankylotic changes and bone volume increase were suppressed in the absence of IFN-γ. Thus, IFN-γ is a positive chondrogenic and osteoblastogenic factor, and DCIR is a crucial regulator of bone metabolism; consequently, both factors are potential targets for therapies directed against bone metabolic diseases.

CTRP3 plays an important role in the development of collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease exhibited most commonly in joints. We found that the expression of C1qtnf3, which encodes C1q/TNF-related protein 3 (CTRP3), was highly increased in two mouse RA models with different etiology. To elucidate the pathogenic roles of CTRP3 in the development of arthritis, we generated C1qtnf3(-/-) mice and examined the development of collagen-induced arthritis in these mice. We found that the incidence and severity score was higher in C1qtnf3(-/-) mice compared with wild-type (WT) mice. Histopathology of the joints was also more severe in C1qtnf3(-/-) mice. The levels of antibodies against type II collagen and pro-inflammatory cytokine mRNAs in C1qtnf3(-/-) mice were higher than WT mice. These observations indicate that CTRP3 plays an important role in the development of autoimmune arthritis, suggesting CTRP3 as a possible medicine to treat RA.

DCIR suppresses osteoclastic proliferation and resorption by downregulating M-CSF and RANKL signaling.

Dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lectin receptor that acts as a negative regulator in the immune system and bone metabolism. We previously revealed that DCIR deficiency enhanced osteoclastogenesis and antigen presentation of dendritic cells, and that asialo-biantennary N-glycan (NA2) functions as a ligand for DCIR. NA2 binding to DCIR suppressed murine and human osteoclastogenesis that occurs in the presence of M-CSF and RANKL. The DCIR-NA2 axis, therefore, plays an important role in regulating osteoclastogenesis in both mice and humans, although the underlying mechanisms remain unclear. Here we found that Dcir -/- bone marrow-derived macrophages (BMMs) exhibited greater proliferative and differentiation responses to M-CSF and RANKL, respectively, than wild-type (WT) BMMs. Moreover, Dcir -/- osteoclasts (OCs) increased resorptive activity and cell fusion more significantly than WT OCs. DCIR deficiency affects gene expression patterns in OCs, and we found that the expression of neuraminidase 4 was increased in Dcir -/- OCs. Furthermore, DCIR-NA2 interaction in WT BMMs, but not Dcir -/- BMMs, decreased Akt phosphorylation in response to M-CSF and RANKL. These data suggest that DCIR regulates osteoclastogenesis by downregulating M-CSF and RANKL signaling, and that DCIR-mediated signaling may contribute to the terminal modification of oligosaccharides by controlling the expression of glycosylation enzymes.

The C-type lectin receptor Clec1A plays an important role in the development of experimental autoimmune encephalomyelitis by enhancing antigen presenting ability of dendritic cells and inducing inflammatory cytokine IL-17.

Clec1A, a member of C-type lectin receptor family, has a carbohydrate recognition domain in its extracellular region, but no known signaling motif in the cytoplasmic domain. Clec1a is highly expressed in endothelial cells and weakly in dendritic cells. Although this molecule was reported to play an important role in the host defense against Aspergillus fumigatus by recognizing 1,8-dihydroxynaphthalene-melanin on the fungal surface, the roles of this molecule in un-infected animals remain to be elucidated. In this study, we found that Clec1a-/- mice develop milder symptoms upon induction of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The maximum disease score was significantly lower, and demyelination and inflammation of the spinal cord were much milder in Clec1a-/- mice compared to wild-type mice. No abnormality was detected in the immune cell composition in the draining lymph nodes and spleen on day 10 and 16 after EAE induction. Recall memory T cell proliferation after restimulation with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in vitro was decreased in Clec1a-/- mice, and antigen presenting ability of Clec1a-/- dendritic cells was impaired. Interestingly, RNA-Seq and RT-qPCR analyses clearly showed that the expression of inflammatory cytokines including Il17a, Il6 and Il1b was greatly decreased in Clec1a-/- mice after induction of EAE, suggesting that this reduced cytokine production is responsible for the amelioration of EAE in Clec1a-/- mice. These observations suggest a novel function of Clec1A in the immune system.

Interaction between periostin and BMP-1 promotes proteolytic activation of lysyl oxidase.

Intra- and intermolecular covalent cross-linking between collagen fibrils, catalyzed by lysyl oxidase (LOX), determines the mechanical properties of connective tissues; however, mechanisms that regulate the collagen cross-linking according to tissue specificity are not well understood. Here we show that periostin, a secretory protein in the dense connective tissues, promotes the activation of LOX. Previous studies showed that periostin null mice exhibit reduced collagen cross-linking in their femurs, periosteum, infarcted myocardium, and tendons. Presently, we showed that active LOX protein, formed by cleavage of its propeptide by bone morphogenetic protein-1 (BMP-1), was decreased in calvarial osteoblast cells derived from periostin null mice. Overexpression of periostin promoted the proteolytic cleavage of the propeptide, which increased the amount of active LOX protein. The results of co-immunoprecipitation and solid phase binding assays revealed that periostin interacted with BMP-1. Furthermore, this interaction probably resulted in enhanced deposition of BMP-1 on the extracellular matrix, suggesting that this enhanced deposition would lead to cleavage of the propeptide of LOX. Thus, we demonstrated that periostin supported BMP-1-mediated proteolytic activation of LOX on the extracellular matrix, which promoted collagen cross-linking.

DCIR and its ligand asialo-biantennary N-glycan regulate DC function and osteoclastogenesis.

Dendritic cell immunoreceptor (DCIR) is a C-type lectin receptor with a carbohydrate recognition domain and an immunoreceptor tyrosine-based inhibitory motif. Previously, we showed that Dcir-/- mice spontaneously develop autoimmune enthesitis and sialadenitis, and also develop metabolic bone abnormalities. However, the ligands for DCIR functionality remain to be elucidated. Here we showed that DCIR is expressed on osteoclasts and DCs and binds to an asialo-biantennary N-glycan(s) (NA2) on bone cells and myeloid cells. Osteoclastogenesis was enhanced in Dcir-/- cells, and NA2 inhibited osteoclastogenesis. Neuraminidase treatment, which exposes excess NA2 by removing the terminal sialic acid of N-glycans, suppressed osteoclastogenesis and DC function. Neuraminidase treatment of mice ameliorated collagen-induced arthritis and experimental autoimmune encephalomyelitis in a DCIR-dependent manner, due to suppression of antigen presentation by DCs. These results suggest that DCIR activity is regulated by the modification of the terminal sialylation of biantennary N-glycans, and this interaction is important for the control of both autoimmune and bone metabolic diseases.

TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens.

TARM1 is a member of the leukocyte immunoglobulin-like receptor family and stimulates macrophages and neutrophils in vitro by associating with FcRγ. However, the function of this molecule in the regulation of the immune system is unclear. Here, we show that Tarm1 expression is elevated in the joints of rheumatoid arthritis mouse models, and the development of collagen-induced arthritis (CIA) is suppressed in Tarm1-/- mice. T cell priming against type 2 collagen is suppressed in Tarm1-/- mice and antigen-presenting ability of GM-CSF-induced dendritic cells (GM-DCs) from Tarm1-/- mouse bone marrow cells is impaired. We show that type 2 collagen is a functional ligand for TARM1 on GM-DCs and promotes DC maturation. Furthermore, soluble TARM1-Fc and TARM1-Flag inhibit DC maturation and administration of TARM1-Fc blocks the progression of CIA in mice. These results indicate that TARM1 is an important stimulating factor of dendritic cell maturation and could be a good target for the treatment of autoimmune diseases.

LAG-3: from molecular functions to clinical applications.

To prevent the destruction of tissues owing to excessive and/or inappropriate immune responses, immune cells are under strict check by various regulatory mechanisms at multiple points. Inhibitory coreceptors, including programmed cell death 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4), serve as critical checkpoints in restricting immune responses against self-tissues and tumor cells. Immune checkpoint inhibitors that block PD-1 and CTLA-4 pathways significantly improved the outcomes of patients with diverse cancer types and have revolutionized cancer treatment. However, response rates to such therapies are rather limited, and immune-related adverse events are also observed in a substantial patient population, leading to the urgent need for novel therapeutics with higher efficacy and lower toxicity. In addition to PD-1 and CTLA-4, a variety of stimulatory and inhibitory coreceptors are involved in the regulation of T cell activation. Such coreceptors are listed as potential drug targets, and the competition to develop novel immunotherapies targeting these coreceptors has been very fierce. Among such coreceptors, lymphocyte activation gene-3 (LAG-3) is expected as the foremost target next to PD-1 in the development of cancer therapy, and multiple clinical trials testing the efficacy of LAG-3-targeted therapy are underway. LAG-3 is a type I transmembrane protein with structural similarities to CD4. Accumulating evidence indicates that LAG-3 is an inhibitory coreceptor and plays pivotal roles in autoimmunity, tumor immunity, and anti-infection immunity. In this review, we summarize the current understanding of LAG-3, ranging from its discovery to clinical application.

PD-1 Primarily Targets TCR Signal in the Inhibition of Functional T Cell Activation.

Cancer-immunotherapy targeting programmed cell death 1 (PD-1) activates tumor-specific T cells and provides clinical benefits in various cancers. However, the molecular basis of PD-1 function is still enigmatic. Especially, it is unclear which signaling pathway PD-1 primarily targets. Besides, the capacity of PD-1 to inhibit the T cell receptor (TCR)-dependent activation of T cells in the presence of co-stimulation is also controversial. Here we used co-culture systems of T cells and antigen-presenting cells with targeted deletion and overexpression of co-receptors and ligands and examined the inhibitory potency of PD-1 against T cell activation upon TCR stimulation with CD28 and ICOS co-stimulation. As an unambiguous criterion of T cell activation, we used the acquisition of cytokine production capacity, which represents one of the most important functions of T cells. PD-1 inhibited functional T cell activation upon TCR stimulation in the absence as well as in the presence of CD28 co-stimulation, indicating that PD-1 can directly inhibit TCR signal. Notably, CD28 co-stimulation rather attenuated the efficiency of PD-1 in inhibiting TCR-dependent functional T cell activation. In addition, PD-1 inhibited TCR-dependent functional T cell activation with ICOS co-stimulation as efficiently as that with CD28 co-stimulation. Furthermore, we found that the maintenance of antigen-induced follicular helper T (TFH) cells that required ICOS co-stimulation was persistently restrained by PD-1 in vivo. These findings indicate that PD-1 primarily targets TCR signal in the inhibition of functional T cell activation. Thus, PD-1 functions as the rheostat of T cell activation rather than an inhibitor of a specific stimulatory co-receptor.

Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses.

Targeted blockade of PD-1 with immune checkpoint inhibitors can activate T cells to destroy tumors. PD-1 is believed to function mainly at the effector, but not in the activation, phase of T cell responses, yet how PD-1 function is restricted at the activation stage is currently unknown. Here we demonstrate that CD80 interacts with PD-L1 in cis on antigen-presenting cells (APCs) to disrupt PD-L1/PD-1 binding. Subsequently, PD-L1 cannot engage PD-1 to inhibit T cell activation when APCs express substantial amounts of CD80. In knock-in mice in which cis-PD-L1/CD80 interactions do not occur, tumor immunity and autoimmune responses were greatly attenuated by PD-1. These findings indicate that CD80 on APCs limits the PD-1 coinhibitory signal, while promoting CD28-mediated costimulation, and highlight critical components for induction of optimal immune responses.