The aim of this study was to examine whether short hairpin RNA (shRNA) expressing lentiviral particles targeting B7-H1 infection could result in B7-H1 knockdown on dendritic cells (DCs) and to investigate whether B7-H1 silencing could augment the immune function of DCs and further elicit a more potent anti-tumor immune effect against bladder cancer cells in vitro.

B7-H1 and B7-H3, two members of the B7 family that are thought to regulate T-cell activation, are expressed in human non-small cell lung cancer (NSCLC). However, their prognostic significance is poorly understood. In the present study we reported that B7-H1 and B7-H3 were expressed in 96/128 (72.7%) and 89/128 (69.5%) samples, respectively. B7-H1 and B7-H3 expression and the number of infiltrating T-cell intracellular antigen-1+ and interferon-γ+ cells in NSCLC tissues were significantly higher than those in the adjacent tissues (p<0.01). High B7-H1 or B7-H3 expression was associated with lymph node metastasis and TNM stage (p<0.05, respectively). Sex, TNM stage, B7-H1, B7-H3, and T-cell intracellular antigen-1 expression remained significant prognostic factors after adjusting for other prognostic factors in a multivariate Cox proportional hazards regression model. In vitro studies revealed that knockdown of B7-H3 on tumor cells enhanced T-cell growth and interferon-γ secretion when stimulated by anti-CD3 and anti-CD28 monoclonal antibodies. Interferon-γ reduced CXCR4 expression on cancer cells and inhibited the CXCL12-induced cell migration.B7-H1 and B7-H3 are independent predictors of poorer survival in patients with NSCLC. Interference of the signal pathways of these negative regulatory molecules might be a new strategy for treating NSCLC.

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and are associated with immune suppression. Here, we described high level of expression of B7-H1 (CD274), PD-1 (CD279) and CTLA4 (CD152) by Gr-1(+)CD11b(+) MDSCs obtained from both ascites and spleens of mice bearing the 1D8 ovarian carcinoma, whereas B7-DC (CD273), CD40 and CD86 were absent. In contrast, B7-H1, PD-1 and CTLA-4 expression was not detected on Gr-1(+)CD11b(+) cells from naive mice. Expression of B7-H1 by Gr-1(+)CD11b(+) cells from naive mice could be induced by co-culture with 1D8 ovarian carcinoma cells. Gr-1(+)CD11b(+) cells derived from 1D8 tumor-bearing mice markedly suppressed antigen-specific immune responses, whereas Gr-1(+)CD11b(+) cells from naive mice did not. siRNA-mediated knockdown of B7-H1 in Gr-1(+)CD11b(+) cells of 1D8 tumor-bearing mice alleviated suppression of antigen-specific immune responses. Suppression of antigen-specific immune responses via B7-H1 on Gr-1(+)CD11b(+) myeloid cells was mediated by CD4(+)CD25(+) Foxp3(+) T regulatory cells and required PD-1. Antibody blockade of either B7-H1 or PD-1 retarded the growth of 1D8 tumor in mice. This suggests that expression of B7-H1 on Gr-1(+)CD11b(+) myeloid cells triggered by the 1D8 mouse model of ovarian carcinoma suppresses antigen-specific immunity via interaction with PD-1 on CD4(+)CD25(+) Foxp3(+) regulatory T cells.

Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8(+) T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8(+) T cells are required for the development of protective immunity. However, antigen-specific CD8(+) T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8(+) T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8(+) T cell responses. Here we show that L. donovani parasites evade CD8(+) T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8(+) T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8(+) T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL.

Protective T‑cell immunity against cancer and infections is dependent on the generation of a durable effector and memory T‑cell pool. Studies from cancer and chronic infections reveal that B7-H1 (PD-L1) engagement with its receptor PD-1 promotes apoptosis of effector T cells. It is not clear how B7-H1 regulates T‑cell apoptosis and the subsequent impact of B7-H1 on the generation of memory T cells. In immunized B7-H1-deficient mice, we detected an increased expansion of effector CD8(+) T cells and a delayed T‑cell contraction followed by the emergence of a protective CD8(+) T‑cell memory capable of completely rejecting tumor metastases in the lung. Intracellular staining revealed that antigen-primed CD8(+) T cells in B7-H1-deficient mice express lower levels of the pro-apoptotic molecule Bim. The engagement of activated CD8(+) T cells by a plate-bound B7-H1 fusion protein led to the upregulation of Bim and increased cell death. Assays based on blocking antibodies determined that both PD-1 and CD80 are involved in the B7-H1-mediated regulation of Bim in activated CD8(+) T cells. Our results suggest that B7-H1 may negatively regulate CD8(+) T‑cell memory by enhancing the depletion of effector CD8(+) T cells through the upregulation of Bim. Our findings may provide a new strategy for targeting B7-H1 signaling in effector CD8(+) T cells to achieve protective antitumor memory responses.

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.

Antigen-presenting cells (APC) are considered to play a critical role in promoting the (re)activation of potentially autoreactive T cells in multiple sclerosis (MS), an inflammatory demyelinating disorder of the central nervous system (CNS). B7-H1 (PD-L1) is a novel member of the B7 family proteins which exert costimulatory and immune regulatory functions. Here we characterize the expression and functional activity of B7-H1 expressed on monocytes and dendritic cells (DC) of healthy donors and MS patients. B7-H1 is constitutively expressed on monocytes and differentially matured DC, but not on B cells. IFN-beta, the principle immune modulatory agent used for the treatment of MS, strongly enhances B7-H1 expression on monocytes and semi-matured DC, but not B cells, in vitro. Importantly, B7-H1 expressed on APC strongly inhibits autologous CD4 T-cell activation. Neutralization of B7-H1 on monocytes or differentially matured monocyte-derived DC markedly increases the secretion of the pro-inflammatory cytokines, IFN-gamma and IL-2, T-cell proliferation, and the expression of T-cell activation markers. B7-H1 exhibits strong inhibitory effects when expressed on monocytes, immature or semi-mature DC, but less so when expressed on fully matured DC. B7-H1-dependent immune inhibition is in part mediated by CD4/CD25+ regulatory T cells. There is no difference in the baseline expression levels of monocytic B7-H1 between untreated MS patients and healthy donors. However, both groups show a significant concentration-dependent up-regulation of B7-H1 mRNA and protein in response to IFN-beta in vitro. Serial measurements of B7-H1 mRNA in MS patients before and 6 months after initiation of IFN-beta therapy corroborated the relevance of these results in vivo: Nine of nine patients showed a significant increase in B7-H1 mRNA levels after 6 months of IFN-beta therapy (median 1.04 vs. 8.78; p<0.05, two-sided t-test). Accordingly, protein expression of B7-H1 on monocytes was up-regulated after 24 h of IFN-beta application. In summary, B7-H1 expressed on APC acts as a strong inhibitor of autologous CD4 T-cell activation and may thus contribute to the maintenance of peripheral immune tolerance. IFN-beta up-regulates B7-H1 in vitro and in MS patients in vivo and might represent a novel mechanism how IFN-beta acts as a negative modulator on APC T-cell interactions in the periphery.

The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.

The application of chimeric antigen receptor (CAR)-T cell therapy in patients with advanced solid tumors remains a significant challenge. Simultaneously targeting antigen and the solid tumor microenvironment are two major factors that greatly impact CAR-T cell therapy outcomes. In this study, we engineered CAR-T cells to specifically target B7-H3, a protein commonly found in solid human tumors, using a single-chain variable fragment (scFv) derived from an anti-B7-H3 monoclonal antibody. We tested the antitumor activity of B7-H3 CAR-T cells in mouse models with solid human tumors and determined that B7-H3 CAR-T cells exhibited potent antitumor activity against B7-H3+ tumor cells in vitro and in vivo. In addition, PD-1 decoy receptors were engineered to include extracellular PD-1 fused to the intracellular stimulatory domain of either CD28 or IL-7 receptor, respectively, which were then introduced into B7-H3 CAR-T cells. As a result, these newly modified, superior CAR-T cells exhibited more persistent antitumor activity in B7-H3+/B7-H1+ tumors in vivo. Our findings indicate that B7-H3 specific CAR-T cells have the potential to treat multiple types of advanced solid tumors.

In mammals, T cell activation requires specific recognition of the peptide-MHC complex by the TcR and co-stimulatory signals. Important co-stimulatory receptors expressed by T cells are the molecules of the CD28 family, that regulate T cell activation, proliferation and tolerance. These receptors recognize B7s and B7-homologous (B7H) molecules that are typically expressed by the antigen presenting cells. In teleost fish, typical T cell responses have been described and the TcR, MHC and CD28/CTLA4 genes have been characterized. In contrast, the members of the B7 gene family have only been described in mammals and birds and have yet to be addressed in lower vertebrates. To learn more about the evolution of components guiding T cell activation in vertebrates, we performed a systematic genomic survey for the B7 co-stimulatory and co-inhibitory IgSF receptors in lower vertebrates with an emphasis on teleost fish. Our search identified fish sequences that are orthologous to B7, B7-H1/B7-DC, B7-H3 and B7-H4 as defined by sequence identity, phylogeny and combinations of short or long-range syntenic relationships. However, we were unable to identify clear orthologs for B7-H2 (CD275, ICOS ligand) in bony fish, which correlates with our prior inability to find ICOS in fish. Interestingly, our results indicate that teleost fish possess a single B7.1/B7.2 (CD80/86) molecule that likely interacts with CD28/CTLA4 as the ligand-binding regions seem to be conserved in both partners. Overall, our analyses implies that gene duplication (and loss) have shaped a molecular repertoire of B7-like molecules that was recruited for the refinement of T cell activation during the evolution of the vertebrates.

Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.

B7-DC, one of the recently described B7 family members, has the capacity to inhibit T cell responses via engagement of the immunoreceptor tyrosine-based inhibitory motif-containing inhibitory PD-1 receptor as well as enhance responses via an as yet unidentified costimulatory receptor. B7-DC is highly homologous to a coinhibitory B7 family member, B7-H1, which also binds PD-1. It is currently unclear which B7-DC function-costimulation or inhibition-predominates in vivo. To study in vivo functions of B7-DC, we evaluated immune responses in B7-DC knockout (KO) mice. Although not eliminated, interferon-gamma (IFN-gamma) production by CD4 T cells and IFN-gamma-dependent humoral responses were reduced in B7-DC KO mice relative to wild type mice. Antigen-specific CD8 T cell responses and cytotoxic T lymphocyte (CTL) activity were also diminished in B7-DC KO mice. Hepatic tumors grew more quickly in B7-DC KO mice, associated with a decrease in intrahepatic tumor-specific CD8 T cells. These results highlight the contrasting in vivo roles of B7-DC and B7-H1 and indicate that B7-DC functions as a tuning molecule, selectively augmenting T helper 1 and CTL responses.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy characterized by the presence of prominent infiltration of lymphocytes, including natural killer (NK) cells. Although NK cells can directly target EBV-infected tumor cells without restriction by the MHC, EBV-positive (EBV+) NPC cells often develop resistance mechanisms that allow them to evade immune surveillance by NK cells. Elucidating the mechanisms involved in EBV-induced NK-cell dysfunction will contribute to the design of novel NK cell-based immunotherapies to treat NPC. Herein, we confirmed that the cytotoxic function of NK cells was impaired in EBV+ NPC tissues and found that EBV infection-induced expression of B7-H3 in NPC negatively correlated with NK-cell function. The inhibitory effect of EBV+ tumor expression of B7-H3 on NK-cell function was clarified in vitro and in vivo. Mechanistically, activation of the PI3K/AKT/mTOR signaling pathway via EBV latent membrane protein 1 (LMP1) was responsible for EBV infection-induced upregulation of B7-H3 expression. In an NPC xenograft mouse model with adoptive transfer of primary NK cells, deletion of B7-H3 on tumor cells in combination with anti-PD-L1 treatment restored NK cell-mediated antitumor activity and significantly improved the antitumor efficacy of NK cells. On the basis of our findings, we conclude that EBV infection can inhibit NK cell-mediated antitumor function by inducing upregulation of B7-H3 expression and provide a rationale for NK cell-based immunotherapies in combination of PD-L1 blockade and overcoming the immunosuppression of B7-H3 to treat EBV-associated NPC.

The molecular mechanisms whereby CD8+ T cells become "exhausted" in the tumor microenvironment remain unclear. Programmed death ligand-1 (PD-L1) is upregulated on tumor cells and PD-1-PD-L1 blockade has significant efficacy in human tumors; however, most patients do not respond, suggesting additional mechanisms underlying T cell exhaustion. B7 superfamily member 1 (B7S1), also called B7-H4, B7x, or VTCN1, negatively regulates T cell activation. Here we show increased B7S1 expression on myeloid cells from human hepatocellular carcinoma correlated with CD8+ T cell dysfunction. B7S1 inhibition suppressed development of murine tumors. Putative B7S1 receptor was co-expressed with PD-1 but not T cell immunoglobulin and mucin-domain containing-3 (Tim-3) at an activated state of early tumor-infiltrating CD8+ T cells, and B7S1 promoted T cell exhaustion, possibly through Eomes overexpression. Combinatorial blockade of B7S1 and PD-1 synergistically enhanced anti-tumor immune responses. Collectively, B7S1 initiates dysfunction of tumor-infiltrating CD8+ T cells and may be targeted for cancer immunotherapy.

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.

Pregnancy necessitates physiological exposure, and often re-exposure, to foreign fetal alloantigens. The consequences after pregnancy are highly varied, with evidence of both alloimmunization and expanded tolerance phenotypes. We show that pregnancy primes the accumulation of fetal-specific maternal CD8+ T cells and their persistence as an activated memory pool after parturition. Cytolysis and the potential for robust secondary expansion occurs with antigen re-encounter in non-reproductive contexts. Comparatively, CD8+ T cell functional exhaustion associated with increased PD-1 and LAG-3 expression occurs with fetal antigen re-stimulation during subsequent pregnancy. PD-L1/LAG-3 neutralization unleashes the activation of fetal-specific CD8+ T cells, causing fetal wastage selectively during secondary but not primary pregnancy. Thus, CD8+ T cells with fetal alloantigen specificity persist in mothers after pregnancy, and protection against fetal wastage in subsequent pregnancies is maintained by their unique susceptibility to functional exhaustion. Together, distinct mechanisms whereby fetal tolerance is maintained during primary compared with subsequent pregnancies are demonstrated.

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4+ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4+ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

The immunological synapse allows antigen-presenting cells (APCs) to convey a wide array of functionally distinct signals to T cells, which ultimately shape the immune response. The relative effect of stimulatory and inhibitory signals is influenced by the activation state of the APC, which is determined by an interplay between signal transduction and metabolic pathways. While pathways downstream of toll-like receptors rely on glycolytic metabolism for the proper expression of inflammatory mediators, little is known about the metabolic dependencies of other critical signals such as interferon gamma (IFNγ). Using CRISPR-Cas9, we performed a series of genome-wide knockout screens in murine macrophages to identify the regulators of IFNγ-inducible T cell stimulatory or inhibitory proteins MHCII, CD40, and PD-L1. Our multiscreen approach enabled us to identify novel pathways that preferentially control functionally distinct proteins. Further integration of these screening data implicated complex I of the mitochondrial respiratory chain in the expression of all three markers, and by extension the IFNγ signaling pathway. We report that the IFNγ response requires mitochondrial respiration, and APCs are unable to activate T cells upon genetic or chemical inhibition of complex I. These findings suggest a dichotomous metabolic dependency between IFNγ and toll-like receptor signaling, implicating mitochondrial function as a fulcrum of innate immunity.

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.