Urothelial Carcinoma of Bladder is the second most common urological malignancy associated with high mortality and morbidity. Initial step in management of NMIBC is transurethral resection followed by intravesical chemotherapy or BCG therapy, while some patients experience early recurrence despite the therapy and eventually undergo cystectomy. Meanwhile, there have been no major advancements in treatment of MIBC patients, with neoadjuvant chemotherapy followed by radical cystectomy as mainstay of treatment [3, 4, 24].

Over the years, immunotherapy has been established as new pillar of cancer care. There is plethora of inhibitory pathways hardwired into immune system that are crucial for maintaining physiological immune responses. Targeting of “PD-1/PD-L1” axis has exhibited unprecedented clinical efficacy in several cancers including UCB [7, 25]. However, these achievements are obscured by low response rates as proportion of patients do not respond to treatment or remain largely refractory to these therapies. Consequently, there is still considerable debate with regards to PD-L1 expression in UCB which is still not defined. Some studies have reported PD-L1 overexpression to be associated with poor prognosis while some others have shown no correlation of PD-L1 with the clinical outcomes in UCB [26, 27]. Therefore, additional strategies need to be identified and tested to improve efficacy of cancer immunotherapy.

As T cells are the main effector cells in the arsenal of immune cells to fight against cancer cells, tumor tries to dampen their effect by abnormally expressing multiple coinhibitory molecules to escape immune invasion [5]. Studies have shown involvement of B7-H4 in tumor immune escape by cellular and humoral immunity and inducing T cell apoptosis. B7-H4 upon binding to its putative receptor on T cell reprograms CD4+ and CD8+ T cell by curbing the release of antitumor cytokines and CD8+ T cell cytotoxicity against tumors [28]. High expression of B7-H4 has been reported in various solid tumors and is also associated with worse patient survival [28, 29]. Till date, there is no study to comprehensively evaluate the expression of B7-H4 in corroboration with PD-L1 in UCB.

In the present study, we characterized the expression of B7-H4 and PD-L1 on T cells by surface staining using flow cytometry in PBMC of UCB patients and matched healthy controls. An elevated frequency of CD4+T and CD8+T cells were observed in UCB patients relative to healthy volunteers. B7-H4 and PD-L1 were observed to be significantly increased on T cell population in UCB patients. The peripheral expression of PD-L1 and B7-H4 has been earlier reported on immune cells in other malignancy but has never been evaluated in UCB [28,29,30]. Importantly, the frequency of B7-H4+CD8 T cells was significantly greater than B7-H4+CD4 T cells in circulation of UCB patients. CD8+B7-H4+ and CD8+PD-L1+cells exhibited a statistically significant increase in MIBC patients when compared to NMIBC patients. The expression of B7-H4 and PD-L1 in periphery of UCB patients is indicative of upregulation of immune inhibitory molecules in the periphery might be associated with anti-inflammatory response and exhibits a correlation with disease severity.

We further assessed the relative gene expression of B7-H4 and PD-L1 in tumor and adjacent normal tissue and was observed to be significantly enhanced in bladder cancer tissue. Of note, a positive trend with disease progression was observed wherein their expression was significantly elevated in MIBC patients in comparison to NMIBC patients. In agreement with our results, a single study in UCB reported an increased expression of B7-H4 in the tumor tissue in comparison to normal [31]. To further support these results, the tissue localization of B7-H4 and PD-L1 were examined by immunohistochemistry wherein B7-H4 and PD-L1 were highly expressed in bladder tumor tissue than in normal bladder tissue. Positive expression of B7-H4+ tumor cells in UCB and other solid tumors have been previously reported [31,32,33]. This overall shows that B7-H4 is known to overexpressed in tumor tissue and associated with poor prognosis in cancer.

The transcript, and frequency of B7-H4 exhibited a positive association of significant strength with histological grade and muscle invasion showcasing a positive trend with disease severity and clinical significance, suggesting its involvement in tumor progression which might be through impairment of host T cell-mediated immunity. A study by Zang et.al. has reported similar observation showcasing positive association of B7-H4 with clinical parameters in prostate cancer [34].

A vital question for checkpoint inhibition therapy is the relative contribution of immune checkpoints leading to synergistic effect of suppressing T cell responses. Previously in HCC mouse model, the synergistic effect of blocking PD-1 and B7-S1 has shown to reinvigorate CTLs mediated tumor immunity.

Therefore, to assess the therapeutic potential of B7-H4 in UCB, in-vitro blocking experiments were performed and T cell activity was evaluated of UCB patients. Upon co-culture of PBMC with high grade bladder cancer cell line T24, we observed an escalation in granzyme B and IFN-γ of CD-8+ T cells and IFN-γ of CD-4 T cells. Combinatorial blocking of B7-H4 and PD-L1 significantly restored granzyme B and IFN-γ producing capacity of CD8+ T cells and IFN-γ levels of CD4+ T cells of UCB patients. While no difference in T cell activity was observed between NMIBC and MIBC patients which could be attributed to small sample size. This is the first study in bladder cancer to use combinatorial blocking of B7-H4 and PD-L1 to assess the T cell activity on bladder cancer cells. A single study on immune checkpoint blockade by Liu et al. has previously reported that blocking B7-H4 alone in bladder cancer cell line led to an increased CD-8+ T cell activity is in concordance with our findings [35].

In conclusion, our data showcase that B7-H4 and PD-L1 expression was higher in CD8+ T cells of UCB patients compared to healthy controls in circulation, highlighting the plausible role of immune checkpoints in downregulating CD8+ T cell effector function. Also, B7-H4 displayed an elevated molecular expression in bladder tumor. Upon stratifying patients into MIBC and NMIBC their expression was increasing with disease severity and might be suggesting that cancer cells are constantly trying to escape the immune evasion by CD8+ T cells. To explore the therapeutic potential, in-vitro blocking of B7-H4 and PD-L1 were performed which displayed that functionality of T cell from UCB patients could be restored by the combinatorial blocking of B7-H4 and PD-L1. The current study highlights B7-H4 as a novel target showing a trend similar to PD-L1. Taken together, our results provide a rationale for the co-blockade of B7-H4 and PD-L1 in UCB patients for better treatment in future after validation.