Significance of B7-H1 Overexpression in Kidney Cancer

B7-H1 is a cell surface glycoprotein belonging to the B7 family of costimulatory molecules. Constitutive protein expression is restricted to a fraction of macrophage-lineage cells, although B7-H1 can be induced on activated T lymphocytes. In addition, some human tumor cells can acquire the ability to aberrantly express B7-H1. In vitro studies demonstrate that B7-H1, expressed by tumor cells or activated lymphocytes, impairs T-cell function and survival and enhances apoptosis of activated tumor-specific T cells. Consistent with this, in vivo monoclonal antibody blockade of B7-H1 has been shown to potentiate antitumor responses in several murine cancer models. Thus, tumor-associated B7-H1 has recently garnered much attention as a potential inhibitor of host antitumor immunity. We describe herein the published investigations looking at the role of B7-H1 in renal cell carcinoma (RCC). Clinical observations demonstrate that B7-H1 is aberrantly expressed in primary and metastatic RCC. The group from Mayo Clinic recently performed immunohistochemistry on fresh-frozen and paraffin-embedded nephrectomy specimens. All patients had clear-cell RCC, and pathologic evaluation was performed by a single urologic pathologist. Their results demonstrated that B7-H1, expressed by tumor cells or lymphocytes, is associated with aggressive pathologic features, including TNM stage, nuclear grade, tumor size, and coagulative necrosis. With a median clinical follow-up of 11 years, patients with tumor B7-H1 were at significant risk of disease progression, cancer-specific death, and overall mortality even after multivariate analyses. Five-year cancer-specific survival rates were 42% and 83% for patients with and without tumor B7-H1, respectively. The basis for these associations could relate to the recognized ability of B7-H1 to inhibit antitumor T-cell–mediated immunity. Based on the current literature, B7-H1 is an independent predictor of prognosis in RCC and represents a promising target for immune manipulation in this refractory tumor.     

Costimulatory molecule B7-H1 in primary and metastatic clear cell renal cell carcinoma

BACKGROUND: Cancer cell expression of costimulatory molecule B7-H1 has been implicated as a potent inhibitor of T-cell-mediated antitumoral immunity. The authors recently reported that B7-H1 is aberrantly expressed in primary renal cell carcinoma (RCC). Blockade of B7-H1, as demonstrated in several murine cancer models, now represents a promising therapeutic target in RCC. However, the potential expression of B7-H1 in metastatic RCC has not been investigated. In the current study, the authors updated their primary RCC results with additional follow-up and investigated the potential role of B7-H1 in metastatic RCC. METHODS: Between 2000 and 2004, 196 patients underwent nephrectomy and 26 patients had resection of RCC metastases for clear cell RCC. Immunohistochemical analysis was performed on tumor cryosections using a B7-H1 monoclonal antibody (clone 5H1). A urologic pathologist quantified the percentage of B7-H1-positive tumor cells and lymphocytes. RESULTS: Variable levels of B7-H1 were expressed on primary RCC tumor cells (n = 130 [66.3%]) and primary tumor-infiltrating lymphocytes (n = 115 [58.7%]). Patients with high expression of B7-H1 on primary tumor cells and/or lymphocytes were significantly more likely to die of RCC compared with patients with low B7-H1 expression (risk ratio [RR] = 4.17; 95% confidence interval [95% CI], 1.97-8.84; P < 0.001) and this risk persisted in multivariate analysis after adjusting for the Mayo Clinic stage, size, grade, and necrosis score (RR = 2.63; 96% CI, 1.23-5.64; P = 0.013). Of the 26 metastatic specimens, cancer cell and lymphocyte B7-H1 expression were demonstrated in 17 (65.4%) and 18 (69.2%) specimens, respectively. In total, 14 (54.3%) metastatic specimens had high aggregate B7-H1 levels compared with 44.4% in primary RCC specimens. CONCLUSIONS: Patients with RCC with high B7-H1 expression were significantly more likely to die even after multivariate analysis. The authors also demonstrated that a high percentage of RCC metastases similarly harbored B7-H1. The authors surmised that B7-H1 blockade may augment current immunotherapy, including patients treated for metastases after cytoreductive nephrectomy.     

Tumor B7-H1 is associated with poor prognosis in renal cell carcinoma patients with long-term follow-up

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.     

PD-1 is expressed by tumor-infiltrating immune cells and is associated with poor outcome for patients with renal cell carcinoma.

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.     

Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma

PURPOSE: Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC. EXPERIMENTAL DESIGN: Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using chi2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models. RESULTS: B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029). CONCLUSIONS: Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.     

B7-H3 ligand expression by prostate cancer: a novel marker of prognosis and potential target for therapy

B7 coregulatory ligands can be aberrantly expressed in human disease. In the context of cancer, these ligands may act as antigen-specific inhibitors of T-cell-mediated antitumoral immunity. We recently reported that B7-H1 expression by carcinomas of the kidney and bladder portends aggressive disease and diminished survival. The expression of these proteins in prostate cancer, however, has not been investigated. We evaluated B7-H3 and B7-H1 protein expression in the pathologic specimens of 338 men treated for clinically localized prostate cancer between 1995 and 1998 with radical retropubic prostatectomy. Expression levels of B7-H3 in prostate cancer were correlated with pathologic indicators of aggressive cancer as well as clinical outcome. We report that B7-H3 is uniformly and aberrantly expressed by adenocarcinomas of the prostate, high-grade prostatic intraepithelial neoplasia, and four prostate cancer cell lines, whereas B7-H1 is rarely expressed. B7-H3 is expressed by benign prostatic epithelia, although at a more reduced level relative to neoplastic tissue. Increasing levels of B7-H3 intensity correlate with worsening clinicopathologic features of prostate cancer. Marked B7-H3 intensity, present in 67 (19.8%) specimens, confers a >4-fold increased risk of cancer progression after surgery (risk ratio, 4.42; P < 0.001). A survey of normal tissues revealed that B7-H3 is expressed within the liver, urothelium, and fetal kidney. In summary, B7-H3 is aberrantly expressed in all prostate cancers and represents an independent predictor of cancer progression following surgery. Moreover, B7-H3 encompasses a novel diagnostic and potential therapeutic target for the clinical management of prostate cancer and, perhaps, other malignancies as well.     

Effect of TLR4 and B7-H1 on Immune Escape of Urothelial Bladder Cancer and its Clinical Significance

Background/Aim: Toll-like receptor 4 (TLR4) and B7-H1, both normally expressed restricted to immune cells,are found to be aberrantly expressed in a majority of human tumors and may play important roles in regulationof tumor immunity. It has been shown that urothelial bladder cancer (UBC) patients can manifest tumoralimmune escape which may be a potential critical factor in tumor pathogenesis and progression. However, so far,the mechanisms of UBC-related immune escape have not been clarified. The aim of this study was to investigatethe effect of TLR4 and B7-H1 on immune escape of UBC. Methods: Bladder cancer T24 cells were pre-incubatedwith LPS and co-cultured with tumor specific CTLs. CTL cytotoxicity and apoptosis rates were measured by MTTassay and flow cytometry, respectively. The effects of an ERK inhibitor on B7-H1 expression and CTL cytotoxicityagainst T24 cells were also evaluated. In addition, TLR4, B7-H1 and PD-1 protein expression was analyzed byimmunohistochemistry in 60 UBC specimens and 10 normal urothelia. Results: TLR4 activation protected T24cells from CTL killing via B7-H1 overexpression. However PD98059, an inhibitor of ERK, enhanced CTL killingof T24 cells by reducing B7-H1 expression. TLR4 expression was generally decreased in UBC specimens, whileB7-H1 and PD-1 were greatly overexpressed. Moreover, expression of both B7-H1 and PD-1 was significantlyassociated with UICC stage and WHO grade classification. Conclusions: TLR4 and B7-H1 may contribute toimmune escape of UBC. Targeting B7-H1 or the ERK pathway may offer new immunotherapy strategies forbladder cancer.     

Blockade of B7-H1 with sPD-1 improves immunity against murine hepatocarcinoma

BACKGROUND: The B7-H1/PD-1 pathway has been demonstrated to be involved in tumor evasion. In a previous study, we constructed a eukaryotic expression plasmid (pPD-1A), which expresses soluble PD-1 (sPD-1). In this study, the question of whether or not the blockade of B7-H1 with sPD-1 in vivo and vitro can improve antitumor immunity was investigated. MATERIALS AND METHODS: The proliferation of lymphocytes activated by dendritic cells (DCs), which were treated with sPD-1 in vitro, was detected with MTT colorimetry. Mice inoculated with H22 cells were treated by intramuscular injection with pPD-1A. The mRNA expression was analyzed with RT-PCR. RESULTS: The early activation of lymphocytes in vitro was partly improved by sPD-1 blockade. The growth of H22 cells was inhibited significantly after pPD-1A administration. The mRNA expression of 4-1BB, B7.1, IFN-gamma and TNF-alpha of lymphocytes was up-regulated and that of OX40 and IL-10 was down-regulated after pPD-1A administration. CONCLUSION: Blockade of the PD-1/B7-H1 pathway with sPD-1 may be a promising strategy for immunotherapy for hepatocarcinoma. Both cytokines and co-stimulatory molecules of lymphocytes could be regulated by sPD-1 blockade.     

Clinical significance of B7-H1 (PD-L1) expression in human acute leukemia

B7-H1 (B7 homologue 1, PD-L1), expressed on the surface of tumor cells or antigen-presenting cells in the tumor microenvironment, can suppress antitumor immune reaction, promote tumor growth and help tumor cells to escape the immune response. To investigate the correlations between B7-H1 expression and acute leukemia patients' immunotherapeutic efficacies and prognoses, we detected the expression of B7-H1 by immunohistochemistry and Real-time quantitative PCR and monitored the immunotherapeutic efficacies and prognosis in 60 acute leukemia patients, in which 14 cases of acute monocyte leukemia(M5) patients received active immunotherapy. The levels of mRNA and protein expression of B7-H1 changed synchronously.B7-H1 was expressed in human acute leukemia cells, especially in M5. The expressions of B7-H1 were significantly higher in the relapse patients than in the de novo patients and after immunotherapy than before immunotherapy. Significant correlations existed between the expressions of B7-H1 and the M5 patients' immunological reactions and immunotherapeutic efficacies.B7-H1 negative patients had better immunotherapeutic efficacies than the positive patients who were prone to have a severe complication of pulmonary infection. Multivariate analysis indicated that B7-H1 status was an independent prognostic factor for M5 patients. In conclusion, B7-H1 is highly expressed on leukemia cells of M5 patients, can significantly affect the immunotherapeutic efficacies of M5 patients and is a novel prognostic marker for M5.     

Relationship between B7-H4, regulatory T cells, and patient outcome in human ovarian carcinoma

B7-H4 is a recently identified B7 family member. We previously showed that ovarian tumor and associated macrophages expressed B7-H4; tumor B7-H4+ macrophages and CD4+CD25+FOXP3+ regulatory T cells (Treg cells) suppressed tumor-associated antigen-specific T-cell immunity. To determine the pathologic relationship between B7-H4, macrophages, and Treg cells in the tumor environment, in addition to Treg cell numbers, we quantified B7-H4 expression in the tumor and tumor-associated macrophages in 103 patients with ovarian carcinoma. We observed that the intensity of B7-H4 expression in macrophages was significantly correlated with Treg cell numbers in the tumor. Further, both Treg cells and macrophage B7-H4, but not tumor B7-H4, were negatively associated with patient outcome. Tumor Treg cells enabled macrophages to spontaneously produce interleukin (IL)-10 and IL-6. Tumor macrophages stimulated B7-H4 expression in an autocrine manner through IL-10 and IL-6. Our previous work showed that tumor-associated macrophages spontaneously produced chemokine CCL22 to mediate Treg cell trafficking into tumor, and Treg cells induced B7-H4 on antigen-presenting cells (APC) including macrophages. Altogether, our data support the concept that there is a mechanistic interaction between Treg cells and macrophage, and that Treg cells may convey the suppressive activity to APCs through B7-H4 induction in human ovarian cancer.     

T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival

PURPOSE: Aberrant expression of T-cell coregulatory molecules has been investigated as a mechanism by which certain cancers may evade host immune surveillance. We evaluated expression of the T-cell coregulators B7-H1, B7-H3, and PD-1 in urothelial cell carcinoma (UCC) of the bladder. EXPERIMENTAL DESIGN: Immunohistochemistry for B7-H1, B7-H3, and PD-1 was done on paraffin-embedded sections from 318 consecutive patients with UCC who underwent radical cystectomy. Expression was correlated with clinicopathologic outcomes and postoperative survival. RESULTS: B7-H3 was widely expressed in UCC, as 222 of 314 (70.7%) tumors showed positive staining. Expression of B7-H3 in UCC was significantly increased compared with adjacent, nontumor urothelium, as a median of 70% of tumor cells expressed B7-H3, compared with 20% of cells in nontumor specimens (P < 0.001). The increase in B7-H3 expression was independent of tumor stage (P = 0.13). Expression of B7-H1 by UCC tumors (P < 0.001) and PD-1 by tumor-infiltrating lymphocytes (P = 0.012) were significantly associated with increased pathologic stage. Patients who had received intravesical bacillus Calmette-Guerin before cystectomy tended to show increased expression of B7-H3 (P = 0.023) and PD-1 (P = 0.071) but were less likely to express B7-H1 (P = 0.027). Moreover, for the subset of patients with organ-confined disease (n = 167), B7-H1 expression independently predicted all-cause mortality after cystectomy (hazard ratio, 3.18; 95% confidence interval, 1.74-5.79; P < 0.001). CONCLUSIONS: B7-H3 is highly expressed in UCC across tumor stages, whereas B7-H1 and PD-1 expression are associated with advanced disease. B7-H1 expression predicts mortality after cystectomy for patients with organ-confined tumors. These molecules may represent novel diagnostic or prognostic markers, as well as therapeutic targets, for patients with UCC.     

TLR4 signaling induces B7-H1 expression through MAPK pathways in bladder cancer cells

TLR4 (Toll-like receptor 4) and B7-H1, which were known to be restricted to immune cells in the past, were found to be aberrantly expressed in a majority of tumor cells, facilitating tumor evasion from immune surveillance. Our study demonstrated that activation of TLR4 signaling in bladder cancer cells up-regulated B7-H1 expression. Furthermore, this regulation was significantly attenuated by ERK or JNK inhibitor. Our results elucidated the molecule mechanism of regulation of B7-H1 expression through TLR4 signaling and may suggest new strategies of down-regulating the cancer-associated B7-H1 expression for bladder cancer treatment.     

The HIF-1α hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immunotherapy

The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by positron emission tomographic (PET) imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) of transplanted colon carcinomas, melanomas, and spontaneous breast adenocarcinomas are CD137 (4-1BB)-positive, as opposed to their counterparts in tumor-draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Importantly, hypoxia does not upregulate CD137 in hypoxia-inducible factor (HIF)-1α-knockout T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating and co-transferred HIF-1α-sufficient T lymphocytes. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies to the tumor tissue. As a result, low-dose intratumoral injections avoid liver inflammation, achieve antitumor systemic effects, and permit synergistic therapeutic effects with PD-L1/B7-H1 blockade. SIGNIFICANCE: CD137 (4-1BB) is an important molecular target to augment antitumor immunity. Hypoxia in the tumor microenvironment as sensed by the HIF-1α system increases expression of CD137 on tumor-infiltrating lymphocytes that thereby become selectively responsive to the immunotherapeutic effects of anti-CD137 agonist monoclonal antibodies as those used in ongoing clinical trials.     

共刺激分子B7-H3在骨肉瘤组织中的表达及临床意义

背景与目的：B7-H3是近年来新发现的协同刺激分子B7家族成员,但目前其在骨肿瘤中的表达及作用机制尚不明确。该研究旨在通过检测人骨肉瘤中B7-H3分子的表达,分析其与患者临床病理因素以及术后生存时间之间的关系。方法：采用免疫组织化学法检测61例人骨肉瘤组织、对应癌旁组织及良性骨肿瘤组织中B7-H3分子的表达以及肿瘤浸润T淋巴细胞的浸润程度。结果：骨肉瘤组织中B7-H3分子表达的阳性率为91.8(56/61),而B7-H3在癌旁及骨纤维结构发育不良组织中几乎不表达。B7-H3分子在骨软骨瘤中表达率为56.8%,但染色强度明显弱于骨肉瘤组织。B7-H3的表达与患者的Ennecking分期、是否发生肺转移之间的差异有统计学意义(P<0.05),与肿瘤组织中CD8⁺T淋巴细胞浸润程度呈负相关(P<0.05),与患者预后呈负相关(P<0.05)。结论：B7-H3在人骨肉瘤中组织异常高表达,并与肿瘤的进展、患者的预后密切相关;B7-H3可能参与了骨肉瘤微环境中的CD8⁺T细胞功能的调节,促使肿瘤细胞逃避免疫监视。     

HSP70 vaccine in combination with gene therapy with plasmid DNA encoding sPD-1 overcomes immune resistance and suppresses the progression of pulmonary metastatic melanoma

Many tumor immunotherapy efforts are focused on the generation of strong T-cell response against tumor antigens. However, strong T-cell response does not always coincide with tumor rejection, for which upregulated expression of immunoinhibitory molecules may be responsible. In this study, the treatment with heat shock protein 70 (HSP70) vaccine induced an infiltration of T cells into the tumor site as well as the expression of IFN-gamma and IL-2, and delayed lung metastases of tumor, but the tumor progression nonetheless occur finally. We demonstrated that B7-H1 expressed by residual tumor cells was responsible for the resistance of tumor to the therapy with HSP70 vaccine. Blockade of B7-H1 by i.v. injection pPD-1A, a plasmid encoding the extracellular domain of PD-1 (sPD-1), could reverse this resistance and enhance the therapeutic efficacy. To complement these findings, we investigated the gene expression of tumor-infiltrating lymphocytes (TILs) by Real-time PCR analysis, which revealed that the expression of TH1 cytokines IFN-gamma and IL-2 by TIL in the mice treated with HSP70 vaccine in combination with sPD-1 was increased and the expression of negative regulatory molecules IL-10, TGF-beta and foxp3 was decreased, demonstrating that multifunctional properties afforded by the combination therapy can effectively overcome tumor resistance and promote effective antitumor immunity. The in vivo transfection with pPD-1A could be performed as infrequently as once a week and still produce a significant antitumor effect. These findings suggest that the treatment with HSP70 vaccine followed by blockade of tumor-B7-H1 with sPD-1 may provide a promising approach for tumor immunotherapy.     

The B7 Family Member B7-H6: a New Bane of Tumor

B7-H6 is a ligand of NKp30, which is an activating receptor of natural killer (NK) cells. High expression of B7-H6 is found in certain types of tumor cells, such as lymphoma, leukemia and gastric carcinoma. The expression of B7-H6 can be induced by inflammatory stress in healthy cells. The expression of B7-H6 is significantly correlated with distant metastasis status and post-operative prognosis in cancer patients. The effectiveness of B7-H6 modified antitumor immunotherapy strategies had been verified in tumor-bearing mice, which opened a new door to targeted therapy. In this review, we will focus on the recent development on the roles of B7-H6 in tumor immunity, as well as mechanisms involved in the regulation of B7-H6 expression.     

B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression.

PURPOSE: B7-H1/PD-L1 (B7-H1) and B7-DC/PD-L2 (B7-DC) are ligands for the receptor PD-1, which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of B7-H1 and B7-DC in tumor specimens of non-small cell lung cancer and their relationships with clinicopathological variables and postoperative survival. Furthermore, we examined the correlation between B7-H1 expression on tumor cells and the number of tumor-infiltrating lymphocytes (TILs) or PD-1 expression on TILs. EXPERIMENTAL DESIGN: The expression of B7-H1 and B7-DC in 52 surgically resected specimens of non-small cell lung cancer was evaluated immunohistochemically. RESULTS: Expression of B7-H1 and B7-DC was focally observed in all non-small cell lung cancer tumor specimens. No relationship was found between the expression of B7-H1 or B7-DC and clinicopathological variables or postoperative survival. However, in the same sections evaluated, significantly fewer TILs were identified in B7-H1-positive tumor regions than in B7-H1-negative tumor regions in a subset of five patients (P = 0.01). Moreover, the percentage of TILs expressing PD-1 was significantly lower in B7-H1-positive tumor regions than in B7-H1-negative tumor regions (P = 0.02). CONCLUSIONS: The expression of B7-H1 on tumor cells in local areas reciprocally correlated with the number of TILs, and this may contribute to negative regulation in antitumor immune responses in non-small cell lung cancer.     

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c⁺CD8⁺ T cells. The CD11c⁺CD8⁺ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c⁺CD8⁺ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.     

B7-H1 is correlated with malignancy-grade gliomas but is not expressed exclusively on tumor stem-like cells

Human glioblastoma is well known for its capacity to interfere with effective antitumor immune responses. B7-H1 is the third member of the B7 family that plays important roles in tumor immune evasion. Recent studies have shown that brain tumor stem-like cells (TSCs) contribute to tumorigenesis and radioresistance. However, the relationship between B7-H1 and the clinical behavior of brain TSCs remains unclear. In the present study, we report that B7-H1 is correlated with the malignancy grade of astrocyte tumors. B7-H1 was significantly upregulated at the growing edge of the tumors. Immunostaining and flow cytometric analysis indicate that B7-H1 was expressed primarily by Ki67-negative tumor cells. In vitro, tumors cultured under medium favoring the growth of neural stem cells were able to form spheres, along with expression of neural stem/progenitor cell markers. These cells were able to differentiate into different neural lineages when cultured in differentiation medium, indicating that these cells have TSC characteristics. We also found that B7-H1 was expressed, but not exclusively on CD133-positive stem cells. Interestingly, we found that CD133-negative tumor cells also had the capacity to form brain tumors. Our data establish a correlation between the expression of the negative costimulatory molecule B7-H1 and the malignancy grade of human gliomas, suggesting that B7-H1 may be a novel tumor marker and target for therapy, although it is not expressed exclusively on brain TSCs.     

Tumor-induced immune suppression of in vivo effector T-cell priming is mediated by the B7-H1/PD-1 axis and transforming growth factor beta

We have generated effector T cells from tumor-draining lymph nodes (TDLN) that are efficacious in adoptive immunotherapy. We now examine the effect of concomitant tumors on the generation of effector T cells. We inoculated methylcholanthrene (MCA) 205 in the flanks of normal mice and mice bearing MCA 205 lung metastases. TDLN cells from these mice were activated and expanded in vitro, and adoptively transferred to mice bearing lung metastases. Effector T cells generated from TDLN in mice with only flank tumor mediated potent antitumor activity. However, antitumor efficacy of the effector T cells generated from TDLN in mice with pre-existent lung tumor (cTDLN) was reduced. Phenotyping studies showed that dendritic cells in cTDLN expressed higher levels of B7-H1, whereas cTDLN T cells expressed higher levels of PD-1. The levels of IFNgamma were reduced, and the levels of CD4(+)Foxp3(+) regulatory T cells were increased in cTDLN versus TDLN. The in vitro activation of cTDLN was increased by blocking B7-H1 or transforming growth factor (TGF)-beta. Importantly, we found a synergistic up-regulation of IFNgamma with simultaneous blockade of B7-H1 and TGF-beta that was much greater than observed with TDLN. In vitro activation of cTDLN with anti-B7-H1 and anti-TGF-beta and in vivo administration of these antibodies after adoptive transfer resulted in the abrogation of the suppression associated with cTDLN. These results show a major role for the B7-H1/PD-1 axis and TGF-beta as synergistic suppressive mechanisms in cTDLN. Our data have clinical relevance in the generation of effector T cells in the tumor-bearing host.