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.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

CD80 on skin stem cells promotes local expansion of regulatory T cells upon injury to orchestrate repair within an inflammatory environment.

Following tissue damage, epithelial stem cells (SCs) are mobilized to enter the wound, where they confront harsh inflammatory environments that can impede their ability to repair the injury. Here, we investigated the mechanisms that protect skin SCs within this inflammatory environment. Characterization of gene expression profiles of hair follicle SCs (HFSCs) that migrated into the wound site revealed activation of an immune-modulatory program, including expression of CD80, major histocompatibility complex class II (MHCII), and CXC motif chemokine ligand 5 (CXCL5). Deletion of CD80 in HFSCs impaired re-epithelialization, reduced accumulation of peripherally generated Treg (pTreg) cells, and increased infiltration of neutrophils in wounded skin. Importantly, similar wound healing defects were also observed in mice lacking pTreg cells. Our findings suggest that upon skin injury, HFSCs establish a temporary protective network by promoting local expansion of Treg cells, thereby enabling re-epithelialization while still kindling inflammation outside this niche until the barrier is restored.

The B7:CD28 family and friends: Unraveling coinhibitory interactions.

Immune responses must be tightly regulated to ensure both optimal protective immunity and tolerance. Costimulatory pathways within the B7:CD28 family provide essential signals for optimal T cell activation and clonal expansion. They provide crucial inhibitory signals that maintain immune homeostasis, control resolution of inflammation, regulate host defense, and promote tolerance to prevent autoimmunity. Tumors and chronic pathogens can exploit these pathways to evade eradication by the immune system. Advances in understanding B7:CD28 pathways have ushered in a new era of immunotherapy with effective drugs to treat cancer, autoimmune diseases, infectious diseases, and transplant rejection. Here, we discuss current understanding of the mechanisms underlying the coinhibitory functions of CTLA-4, PD-1, PD-L1:B7-1 and PD-L2:RGMb interactions and less studied B7 family members, including HHLA2, VISTA, BTNL2, and BTN3A1, as well as their overlapping and unique roles in regulating immune responses, and the therapeutic potential of these insights.

cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

Diversification of human plasmacytoid predendritic cells in response to a single stimulus.

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses.

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

Early developing B cells undergo negative selection by central nervous system-specific antigens in the meninges.

Self-reactive B cell progenitors are eliminated through central tolerance checkpoints, a process thought to be restricted to the bone marrow in mammals. Here, we identified a consecutive trajectory of B cell development in the meninges of mice and non-human primates. The meningeal B cells were located predominantly at the dural sinuses, where endothelial cells expressed essential niche factors to support B cell development. Parabiosis experiments together with lineage tracing showed that meningeal developing B cells were replenished continuously from hematopoietic stem cell (HSC)-derived progenitors via a circulation-independent route. Autoreactive immature B cells that recognized myelin oligodendrocyte glycoprotein (MOG), a central nervous system-specific antigen, were eliminated specifically from the meninges. Furthermore, genetic deletion of the Mog gene restored the self-reactive B cell population in the meninges. These findings identify the meninges as a distinct reservoir for B cell development, allowing in situ negative selection to ensure a locally non-self-reactive immune repertoire.

Quorum Regulation via Nested Antagonistic Feedback Circuits Mediated by the Receptors CD28 and CTLA-4 Confers Robustness to T Cell Population Dynamics.

T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics-akin to bacterial quorum regulation-is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8+ T cells to collectively regulate the balance between proliferation and apoptosis. Mechanistically, T cell expressed CD80 and CD86 interacted with the receptors CD28 and CTLA-4 on neighboring T cells; these interactions fed two nested antagonistic feedback circuits that regulated interleukin 2 production in a manner dependent on T cell density as confirmed by in vivo modulation of this network. Thus, CD8+ T cell-population-intrinsic mechanisms regulate cellular behavior, thereby promoting robustness of population dynamics.

Dendritic cells direct circadian anti-tumour immune responses.

The process of cancer immunosurveillance is a mechanism of tumour suppression that can protect the host from cancer development throughout its lifetime1,2. However, it is unknown whether the effectiveness of cancer immunosurveillance fluctuates over a single day. Here we demonstrate that the initial time of day of tumour engraftment dictates the ensuing tumour size across mouse cancer models. Using immunodeficient mice as well as mice lacking lineage-specific circadian functions, we show that dendritic cells (DCs) and CD8+ T cells exert circadian anti-tumour functions that control melanoma volume. Specifically, we find that rhythmic trafficking of DCs to the tumour draining lymph node governs a circadian response of tumour-antigen-specific CD8+ T cells that is dependent on the circadian expression of the co-stimulatory molecule CD80. As a consequence, cancer immunotherapy is more effective when synchronized with DC functions, shows circadian outcomes in mice and suggests similar effects in humans. These data demonstrate that the circadian rhythms of anti-tumour immune components are not only critical for controlling tumour size but can also be of therapeutic relevance.

Active eosinophils regulate host defence and immune responses in colitis.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

Dimers Aren't Forever: CD80 Breaks up with PD-L1.

Targeting the CTLA-4 and PD-1 "checkpoints" is an effective treatment for a number of cancers. In this issue of Immunity, Hui et al. reveal that interaction between a CTLA-4 ligand, CD80, and its counterpart in the PD-1 pathway, PD-L1, affects both PD-1 and CTLA-4 function, raising new questions about the biological effects of using checkpoint inhibitors alone and in combination.

Liver Is a Generative Site for the B Cell Response to Ehrlichia muris.

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.

PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways.

Combined immunotherapy targeting the immune checkpoint receptors cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1), or CTLA-4 and the PD-1 ligand (PD-L1) exhibits superior anti-tumor responses compared with single-agent therapy. Here, we examined the molecular basis for this synergy. Using reconstitution assays with fluorescence readouts, we found that PD-L1 and the CTLA-4 ligand CD80 heterodimerize in cis but not trans. Quantitative biochemistry and cell biology assays revealed that PD-L1:CD80 cis-heterodimerization inhibited both PD-L1:PD-1 and CD80:CTLA-4 interactions through distinct mechanisms but preserved the ability of CD80 to activate the T cell co-stimulatory receptor CD28. Furthermore, PD-L1 expression on antigen-presenting cells (APCs) prevented CTLA-4-mediated trans-endocytosis of CD80. Atezolizumab (anti-PD-L1), but not anti-PD-1, reduced cell surface expression of CD80 on APCs, and this effect was negated by co-blockade of CTLA-4 with ipilimumab (anti-CTLA-4). Thus, PD-L1 exerts an immunostimulatory effect by repressing the CTLA-4 axis; this has implications to the synergy of anti-PD-L1 and anti-CTLA-4 combination therapy.

The CTLA-4 immune checkpoint protein regulates PD-L1:PD-1 interaction via transendocytosis of its ligand CD80.

CTLA-4 and PD-1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD-L1, has been shown to block PD-L1/PD-1 binding and to prevent PD-L1 inhibitory functions. Since CTLA-4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD-L1 interaction. We find that transendocytosis of CD80 results in a time-dependent recovery of PD-L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD-L1 partner remains on the plasma membrane of the antigen-presenting cell (APC). CTLA-4 interactions with CD80 do not appear to be inhibited by PD-L1, but efficient removal of CD80 requires an intact CTLA-4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA-4 binding to CD80/PD-L1 complexes. These data are consistent with CTLA-4 acting as modulator of PD-L1:PD-1 interactions via control of CD80.

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.

Blockade of trans PD-L1 interaction with CD80 augments antitumor immunity.

PD-L1 has two receptors: PD-1 and CD80. Previous reports assumed that PD-L1 and CD80 interacted in trans, but recent reports showed that only cis PD-L1/CD80 interactions existed, and prevention of cis PD-L1/CD80 interactions on antigen-presenting cells (APCs) reduced antitumor immunity via augmenting PD-L1/PD-1 and CD80/CTLA4 interactions between T and APCs. Here, using tumor-bearing mice capable of cis and trans or trans only PD-L1/CD80 interactions, we show that trans PD-L1/CD80 interactions do exist between tumor and T cells, and the effects of trans PD-L1/CD80 interactions require tumor cell expression of MHC-I and T cell expression of CD28. The blockade of PD-L1/CD80 interactions in mice with both cis and trans interactions or with only trans interactions augments antitumor immunity by expanding IFN-γ-producing CD8+ T cells and IFN-γ-dependent NOS2-expressing tumor-associated macrophages. Our studies indicate that although cis and trans PD-L1/CD80 interactions may have opposite effects on antitumor immunity, the net effect of blocking PD-L1/CD80 interactions in vivo augments CD8+ T cell-mediated antitumor immunity.

Absence of CD80 reduces HSV-1 replication in the eye and delays reactivation but not latency levels.

Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80-/- mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80-/- mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.

Dendritic Cells Coordinate the Development and Homeostasis of Organ-Specific Regulatory T Cells.

Although antigen recognition mediated by the T cell receptor (TCR) influences many facets of Foxp3(+) regulatory T (Treg) cell biology, including development and function, the cell types that present antigen to Treg cells in vivo remain largely undefined. By tracking a clonal population of Aire-dependent, prostate-specific Treg cells in mice, we demonstrated an essential role for dendritic cells (DCs) in regulating organ-specific Treg cell biology. We have shown that the thymic development of prostate-specific Treg cells required antigen presentation by DCs. Moreover, Batf3-dependent CD8α(+) DCs were dispensable for the development of this clonotype and had negligible impact on the polyclonal Treg cell repertoire. In the periphery, CCR7-dependent migratory DCs coordinated the activation of organ-specific Treg cells in the prostate-draining lymph nodes. Our results demonstrate that the development and peripheral regulation of organ-specific Treg cells are dependent on antigen presentation by DCs, implicating DCs as key mediators of organ-specific immune tolerance.