Dong, Haidong

Abstract We investigated the influence of PD-1 expression on the systemic antitumor response (abscopal effect) induced by stereotactic ablative radiotherapy (SABR) in preclinical melanoma and renal cell carcinoma models. We compared the SABR-induced antitumor response in PD-1–expressing wild-type (WT) and PD-1–deficient knockout (KO) mice and found that PD-1 expression compromises the survival of tumor-bearing mice treated with SABR. None of the PD-1 WT mice survived beyond 25 days, whereas 20% of the PD-1 KO mice survived beyond 40 days. Similarly, PD-1–blocking antibody in WT mice was able to recapitulate SABR-induced antitumor responses observed in PD-1 KO mice and led to increased survival. The combination of SABR plus PD-1 blockade induced near complete regression of the irradiated primary tumor (synergistic effect), as opposed to SABR alone or SABR plus control antibody. The combination of SABR plus PD-1 blockade therapy elicited a 66% reduction in size of nonirradiated, secondary tumors outside the SABR radiation field (abscopal effect). The observed abscopal effect was tumor specific and was not dependent on tumor histology or host genetic background. The CD11ahigh CD8+ T-cell phenotype identifies a tumor-reactive population, which was associated in frequency and function with a SABR-induced antitumor immune response in PD-1 KO mice. We conclude that SABR induces an abscopal tumor-specific immune response in both the irradiated and nonirradiated tumors, which is potentiated by PD-1 blockade. The combination of SABR and PD-1 blockade has the potential to translate into a potent immunotherapy strategy in the management of patients with metastatic cancer. Cancer Immunol Res; 3(6); 610–9. ©2015 AACR.

Abstract Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.

Abstract An immunoinhibitory role of B7 homologue 1 (B7-H1) expressed by non-T cells has been established; however, the function of B7-H1 expressed by T cells is not clear. Peak expression of B7-H1 on Ag-primed CD8 T cells was observed during the contraction phase of an immune response. Unexpectedly, B7-H1 blockade at this stage reduced the numbers of effector CD8 T cells, suggesting B7-H1 blocking Ab may disturb an unknown function of B7-H1 expressed by CD8 T cells. To exclusively examine the role of B7-H1 expressed by T cells, we introduced B7-H1 deficiency into TCR transgenic (OT-1) mice. Naive B7-H1–deficient CD8 T cells proliferated normally following Ag stimulation; however, once activated, they underwent more robust contraction in vivo and more apoptosis in vitro. In addition, B7-H1–deficient CD8 T cells were more sensitive to Ca-dependent and Fas ligand-dependent killing by cytotoxic T lymphocytes. Activation-induced Bcl-xL expression was lower in activated B7-H1–deficient CD8 T cells, whereas Bcl-2 and Bim expression were comparable to the wild type. Transfer of effector B7-H1–deficient CD8 T cells failed to suppress tumor growth in vivo. Thus, upregulation of B7-H1 on primed T cells helps effector T cells survive the contraction phase and consequently generate optimal protective immunity.

Abstract Purpose: Release of inhibitory coregulatory proteins into the circulation may represent one mechanism by which tumors thwart immune responses. Our objective was to determine whether soluble B7-H1 (sB7-H1) levels in patients with clear cell renal cell carcinoma (ccRCC) are associated with pathologic features and patient outcome. Experimental Design: We developed an ELISA for quantification of sB7-H1 in biological fluids. Biochemical confirmation of the measured analyte as sB7-H1 was done by protein microsequencing using supernates from tumor cell lines. Biological activity of sB7-H1 was assessed in vitro utilizing T-cell apoptosis assays. We tested sB7-H1 levels in the sera from 172 ccRCC patients and correlated sB7-H1 levels with pathologic features and patient outcome. Results: sB7-H1 was detected in the cell supernatants of some B7-H1–positive tumor cell lines. Protein sequencing established that the measured sB7-H1 retained its receptor-binding domain and could deliver proapoptotic signals to T cells. Higher preoperative sB7-H1 levels were associated with larger tumors (P < 0.001), tumors of advanced stage (P = 0.017) and grade (P = 0.044), and tumors with necrosis (P = 0.003). A doubling of sB7-H1 levels was associated with a 41% increased risk of death (P = 0.010). Conclusion: Our observations suggest that sB7-H1 may be detected in the sera of ccRCC patients and that sB7-H1 may systemically impair host immunity, thereby fostering cancer progression and subsequent poor clinical outcome. Clin Cancer Res; 17(7); 1915–23. ©2011 AACR.

Abstract Agonists of TLR have been explored as vaccine adjuvants for tumor immunotherapy. However, their immunological consequences are not fully understood. Although TLR signaling increases the functional potential of dendritic cells (DCs) for priming T cells, coinduction of potentially negative immunoregulatory capacities may impair effector T cell generation. We examined the expression and function of B7 family costimulatory molecules on DCs after activation with the TLR3 agonist, polyinosinic:polycytidylic acid. We demonstrated that polyinosinic:polycytidylic acid consistently up-regulated both B7-2 and B7-H1 molecules on resident, migratory DCs from spleen and lymph nodes. Depletion or blockade of B7-H1 on activated DCs increased the magnitude of effector CD8 T cell expansion. DC-based or protein-based tumor vaccines, in combination with B7-H1 blockade, induced strong effector CD8 T cell responses, resulting in protective immunity against newly established tumors. Our studies suggest that TLR3 signaling has the potential to up-regulate both positive and negative coregulatory molecules on APCs. Selective blockade of negative regulatory molecules in combination with TLR3 agonist may be an effective strategy for increasing the efficacy of tumor vaccines.

AbstractStromal elements present within the tumor microenvironment may suppress host immunity and promote the growth of malignant lymphocytes in B cell–derived non-Hodgkin lymphoma (NHL). In contrast, little is known about the microenvironment's role in T cell–derived NHL. B7-H1 (PD-L1, CD274), a member of the B7 family of costimulatory/coinhibitory ligands expressed by both malignant cells and stromal cells within the tumor microenvironment, has emerged as an important immune modulator capable of suppressing host immunity. Therefore, B7-H1 expression and function were analyzed in cutaneous and peripheral T-cell NHL. B7-H1 was expressed by tumor cells, monocytes, and monocyte-derived cells within the tumor microenvironment in T-cell NHL and was found to inhibit T-cell proliferation and promote the induction of FoxP3+ regulatory T cells. Collectively, the data presented provide the first evidence implicating B7-H1 in the suppression of host immunity in T-cell lymphoproliferative disorders and suggest that the targeting of B7-H1 may represent a novel therapeutic approach.

Abstract Purpose: B7-H1 is expressed by clinically aggressive forms of renal cell carcinoma (RCC) and predicts adverse outcome. B7-H1 is known to impair host immunity via interaction with the Programmed Death-1 (PD-1) receptor, which is expressed by activated T cells. Levels of immune cells expressing PD-1 (PD-1+) in clinical RCC tumors have not been evaluated. Thus, we tested whether immune cell PD-1 expression is observed within aggressive RCC tumors. Experimental Design: Between 2000 and 2003, 267 patients underwent nephrectomy at our institution for clear cell RCC and had fresh-frozen tissue available for review. These RCC specimens were immunostained using anti–PD-1 (clone MIH4) and outcome analyses were conducted. Results: Mononuclear immune cell infiltration was observed in 136 (50.9%) specimens. PD-1+ immune cells were present in 77 of these 136 (56.6%) tumors. In contrast, RCC tumor cells did not express PD-1. Patients with PD-1+ immune cells were significantly more likely to harbor B7-H1+ tumor cells (P < 0.001), larger tumors (P = 0.001), and tumors of higher nuclear grade (P = 0.001). Likewise, intratumoral PD-1+ immune cells were associated with advanced tumor-node-metastasis stage (P = 0.005), coagulative tumor necrosis (P = 0.027), and sarcomatoid differentiation (P = 0.008). With a median follow-up of 2.9 years, 52 patients died from RCC. Univariately, patients with PD-1+ immune cells were at significant risk of cancer-specific death compared with PD-1− patients (risk ratio, 2.24; P = 0.004). Conclusions: Levels of immune cells expressing PD-1 were increased in patients with high-risk RCC tumors. Interactions between immune cell PD-1 and B7-H1 may promote cancer progression by contributing to immune dysfunction in patients with RCC.

Abstract B7-H1 participates in T-cell costimulation functioning as a negative regulator of immunity. Recent observations suggest that B7-H1 is expressed by renal cell carcinoma (RCC) tumor cells and is associated with poor prognosis. However, outcome analyses have been restricted to patients with fresh-frozen tissue and limited follow-up. We report the clinical effect of B7-H1 in RCC patients with a median of 10 years of follow-up. Between 1990 and 1994, 306 patients underwent nephrectomy for clear cell RCC and had paraffin tissue available for review. We did immunohistochemistry with anti-B7-H1 and conducted outcome analyses. Among the 306 patients, 73 (23.9%) harbored tumors with B7-H1 expression. Patients with tumor B7-H1 were at a significantly increased risk of both death from RCC [risk ratio (RR), 3.92; P < 0.001] and overall mortality (RR, 2.37; P < 0.001). The 5-year cancer-specific survival rates were 41.9% and 82.9% for patients with and without tumor B7-H1, respectively. In a multivariate model, tumor B7-H1 remained associated with cancer-specific death even after adjusting for tumor-node-metastasis stage, grade, and performance status (RR, 2.00; P = 0.003). In the subset of 268 patients with localized RCC, tumor B7-H1 was significantly associated with metastatic cancer progression (RR, 3.46; P < 0.001) and death from RCC (RR, 4.13; P < 0.001) even after adjusting for stage, grade, and performance status (RR, 1.78, P = 0.036). RCC patients with tumor B7-H1 are at significant risk of rapid cancer progression and accelerated rates of mortality. B7-H1 may function as a key determinant in RCC, abrogating immune responses directed against this immunogenic tumor. (Cancer Res 2006; 66(7): 3381-5)

Expression of B7-H1, a costimulating glycoprotein in the B7 family, is normally restricted to macrophage-lineage cells, providing a potential costimulatory signal source for regulation of T cell activation. In contrast, aberrant expression of B7-H1 by tumor cells has been implicated in impairment of T cell function and survival, resulting in defective host antitumoral immunity. The relationship between tumor-associated B7-H1 and clinical cancer progression is unknown. Herein, we report B7-H1 expression by both renal cell carcinoma (RCC) tumors of the kidney and RCC tumor-infiltrating lymphocytes. In addition, our analysis of 196 clinical specimens reveals that patients harboring high intratumoral expression levels of B7-H1, contributed by tumor cells alone, lymphocytes alone, or tumor and/or lymphocytes combined, exhibit aggressive tumors and are at markedly increased risk of death from RCC. In fact, patients with high tumor and/or lymphocyte B7-H1 levels are 4.5 times more likely to die from their cancer than patients exhibiting low levels of B7-H1 expression (risk ratio 4.53; 95% confidence interval 1.94–10.56; P < 0.001.) Thus, our study suggests a previously undescribed mechanism whereby RCC may impair host immunity to foster tumor progression. B7-H1 may prove useful as a prognostic variable for RCC patients both pre- and posttreatment. In addition, B7-H1 may represent a promising target to facilitate more favorable responses in patients who require immunotherapy for treatment of advanced RCC.

B7-H1 and B7-DC are ligands for PD-1, a receptor implicated in negative regulation of T and B cell functions. These ligands, however, also costimulate T cell responses. It remains elusive whether or not costimulation is mediated through PD-1. By comparative molecular modeling and site-directed mutagenesis, we found that nonconserved residues between these ligands on the A′GFCC′C′′ face mediate interaction with PD-1. This indicates significant structural heterogeneity of the interactions between PD-1 and its ligands. Importantly, ligand mutants with abolished PD-1 binding capacity could still costimulate proliferation and cytokine production of T cells from normal and PD-1–deficient mice. Our results reveal unique binding characteristics of B7-H1 and B7-DC and provide direct evidence for an independent costimulatory receptor other than PD-1.