Clinical Significance of combinatorial blockade of novel immune checkpoint B7-H4 with PD-L1 in Urothelial Carcinoma of Bladder: A rational therapeutic approach

doi:10.21203/rs.3.rs-2186489/v1

Purpose: Urothelial carcinoma of bladder (UBC) is complex disease with high mortality and recurrence rates. Current standard regimens have exhibited anti-tumor activity however, proportion of patients are non-responsive or ineligible to receive such treatments, which stresses upon identifying new effective options for treating UBC. Immune checkpoints have emerged as potential class of therapeutics to be tested in UBC treatment.

Methods: 40 UBC patients and 30 healthy controls were recruited from whom blood was taken. Histopathologically proven tumors and adjacent normal tissue were obtained from patients. The frequency of T cells and immune checkpoints (B7-H4 and PD-L1) was assessed in circulation. Molecular expression of immune checkpoints has been studied in tumors and normal tissue. In-vitroblocking of B7-H4 alone and in combination with PD-L1 was performed to test for T cell functionality. Finally, these immune checkpoints were correlated with clinico-pathological parameters.

Results: Elevated expression of B7-H4 and PD-L1 were observed in CD8 T cells of UBC patients. Relative mRNA expression and immunohistochemistry displayed upregulation in bladder tumor tissue. Co-blockade enhanced IFN-gand granzyme B of CD-8 T cells. Also, B7-H4 was significantly associated with clinico-pathological parameters.

Conclusion: Increased expression of B7-H4 along with PD-L1 in periphery and tumor of UBC patients highlights involvement of B7-H4 in disease progression, showcasing elevated expression with disease progression. Combinatorial blocking enhanced functional T cell immune response in UBC patients. Also, positive association of B7-H4 with clinico-pathological parameters showcase it as potential therapeutic target for treatment of UBC patient in future after further validation.

Urothelial Carcinoma of Bladder

Muscle invasive/and Non-Muscle Invasive bladder cancer

Immune checkpoints

B7-H4

PD-L1

Immunotherapy

Urinary bladder cancer is the 10th most commonly diagnosed cancer and is associated with a high recurrence rate (Sung et al., 2021). Urothelial carcinoma of bladder (UBC) presents the primary histological subtype among 90% of all bladder cancer cases (Magers et al., 2019). At presentation, approximately 75% of cases are non-muscle invasive or superficially invasive bladder cancer (with TNM stage Ta – T1). While rest 25% are muscle invasive bladder cancer (MIBC) present with detrusor muscle invasion (T2 stage and higher). They are usually high grade and have a propensity to metastasize to distant sites. The current regime for treating NMIBC involves tumor resection followed by intravesical chemotherapy instillation or BCG. While for MIBC, multimodal treatment involving radical cystectomy with neoadjuvant chemotherapy is present. The current standard of care therapies has shown a high response rate among UBC patients but survival outcomes with these regimens remain poor (De Santis et al., 2012; von der Maase et al., 2005). Therefore, there is a need to identify new approaches to break this therapeutic stalemate.

When a normal cell undergoes neoplastic transformation, it is able to break the constant immunosurveillance of the host immune system via diverse mechanisms among which includes T cell dysfunction. T cells tend to undergo a state of exhaustion which is demarcated by progressive diminution of various effector functions. Negative regulatory signals arising due to increased and sustained expression of immunosuppressive checkpoints are one of the main mechanisms for effector T-cell dysfunction in tumor microenvironment (Rotte et al., 2018). Monoclonal antibodies targeting T-cell inhibitory checkpoints have emerged as a promising class of immunotherapeutics in selectively targeting the pathways that depress the natural immune response by eliciting an anti-tumor immune response. Two immune checkpoints: programmed death-1 (PD-1) and its ligands (PD-L1) and CTLA-4 have been targeted and have shown clinical benefits across tumors of various anatomical sites including UBC (Plimack et al., 2017; Rosenberg et al., 2016). By expressing PD-L1, cancer cells can evade the immune system by suppressing T cell-mediated immune response and maintaining an immune-suppressive tumor microenvironment and therefore use it for its advantage (Rotte et al., 2018).

There has been a paradigm shift for UBC treatment modality with FDA approval of PD-L1 blockers (Avelumab, Atezolizumab, and Durvalumab) and PD-1 blockers (Pembrolizumab and Nivolumab) and are now gaining accelerated usage into clinics for the treatment of advanced stage bladder cancer patients (Sanli et al., 2017; Siefker-Radtke and Curti, 2018). Although these immune checkpoint inhibitors have made great strides in the treatment of advanced cancer patients with anti-tumor activity and a manageable safety profile still response rates are low in most cases with only a fraction of patients benefitting from this therapy (Powles et al., 2018). A major obstacle against the development of effective anti-cancer immunotherapy stems from inadequate stimulation of tumor-specific T cells or T cell exhaustion. Increasing evidence reveals that exhausted T cells in the tumor microenvironment express several checkpoints and that blockade of a single checkpoint may not be sufficient to elicit an effective immune response (Rotte et al., 2018).

A novel immune checkpoint B7-H4 (B7S1), a member of B7 family shares 18% amino acid identity with human PD-L1. It is a T cell co-inhibitory molecule and has a putative receptor on T cell to downregulate its functional activity (“B7 family checkpoint regulators in immune regulation and disease: Trends in Immunology,” n.d.). While initially found to be present on professional antigen-presenting cells, its expression is now evident on cancer cell. B7-H4 is known to be associated with poor prognosis and decreased survival in various solid tumors (Arigami et al., 2011; Cheng et al., 2009; Sun et al., 2012). Li et.al. showed that targeted genetic deletion of B7-H4 in an orthotopic model of liver cancer is associated with increased CD8⁺ T cell tumor infiltration with decreased markers of exhaustion and exhibited an elevated anti-tumor efficacy (Li et al., 2018). Therefore, inhibition of B7-H4 may be an alternative strategy to reinvigorate tumor-specific T cell responses.

Limited reports are available exploring the role of B7-H4 in UBC. To date, there is no report on exploiting immune checkpoint B7-H4 in combination with PD-L1 in NMIBC and MIBC patients. The diagnostic and therapeutic potential of various molecules has been previously assessed by our group including T cells in UBC (Anand et al., 2019; Chugh et al., 2013; Khandelwal et al., 2020; Satyam et al., 2011; Singh et al., 2021). Therefore, in this maiden study, we aim to investigate the molecular expression of B7-H4 and PD-L1 in UBC patients. To achieve this hypothesis, the immunophenotyping of B7-H4 and PD-L1 was performed, followed by molecular expression (mRNA and protein) of these immune checkpoints in study subjects followed by correlation with clinical parameters. Lastly, the therapeutic potential was assessed by in-vitro blocking of B7-H4 in combination with PD-L1 in UBC cells.

Sample recruitment

This study has been approved by Institutional Human Ethics Committee (IEC-685/06.09.2019) before its commencement with written consent obtained from all the study subjects. 40 UBC patients undergoing radical cystectomy or TURBT were recruited from the Department of Urology, AIIMS, New Delhi, India. Histopathologically confirmed tumor (n = 40) and adjacent normal tissue (n = 22) of the same patients were obtained from these patients. Besides, 10 healthy bladder tissue were obtained from patients with Benign Prostate Hyperplasia (BPH). Histological grading was done as high and low grade tumors as per WHO guidelines (Magers et al., 2019). Tissue samples were stored in RNA later (Invitrogen) for RNA isolation and in 10% formalin for immunohistochemistry. 10 ml blood sample was collected from the same UBC patients and thirty age and sex-matched healthy controls in sterile endotoxin-free vacutainers with EDTA as an anticoagulant for isolation of Peripheral Blood Mononuclear Cells.

PBMC isolation

PBMCs were isolated by Ficoll-Histopaque (HighMedia) density gradient centrifugation. The remaining red blood cells were subjected to lysis using RBC lysis buffer (Thermo-Fisher Scientific) followed by a wash with PBS. Viable cells were counted with an inverted microscope using trypan blue.

Flowcytometry and Immunophenotyping

10⁶ cells in PBS were incubated with LIVE/DEAD fixable dead stain kit (Invitrogen) to exclude dead cells for 30 min at 4⁰C prior to antibody staining and were washed. For multi-colour flow cytometry, cells were subjected to surface staining at 4⁰C for 30 min in dark in staining buffer fluorochrome tagged primary antibodies given in Supplementary Table-1. Flow cytometry was performed using LSR II instrument (BD PharMingen) on FACS Diva software. Flow cytometric analysis was performed according to standard protocols. Data were analysed using Flowjo (Tree Star). Negative gating was based on a fluorescence minus one (FMO) strategy as described in Supplementary Fig. 1.

Quantitative real time PCR

The tissue sample stored in RNA later were snap frozen in liquid nitrogen and then were crushed using Diethyl pyrocarbonate (DEPC) treated mortar pestle. RNA was extracted using Trizol® (Sigma, USA) and quantified using Nanodrop™ spectrophotometer (ThermoFisher, USA) to check for purity at 260/280 and 260/230 ratios. After DNase treatment, 1µg of RNA was used to synthesize cDNA using M-MuLV Reverse Transcriptase (200 units/µl, Fermentas). The cDNA synthesized were used as template to study gene amplification on the Bio-Rad CFX96 Touch™ Real-Time PCR Detection System, USA using SYBR Green dye (Bio-Rad). The relative mRNA expression of B7-H4 and PD-L1 were studied and b-actin was used as reference gene. The primer sequence for PD-L1, B7-H4 and b-actin are given in Supplementary Table-2. qPCR was performed in triplicates. The 2^−δCt method was applied to estimate relative mRNA expression where Ct values of the given genes were normalized to that of b-actin and compared with their respective controls.

Immunohistochemistry

Tissue section were kept in 10% formalin and were paraffin embedded to form the blocks. The paraffin embedded tissue blocks were sectioned using microtome to 5mm on poly-L-Lysine coated slides. The slides containing the specimens were deparaffinized with xylene and hydrated with alcohol series and then 3.0% H₂O₂ was applied to block endogenous peroxidase activity. Antigen retrieval was carried out in a citrate buffer (Thermo Scientific, Heat Induced Epitope Retrieval) at 120°C with microwave for 20 minutes. The sections were incubated at 25°C for 75 minutes with primary antibodies listed in Supplementary Table-3 for overnight followed by three successive washes with PBST buffer. Biotinylated anti-mouse IgG (PK‐6102; Vector Laboratories) and biotinylated goat anti‐rabbit IgG (PK‐4001; Vector Laboratories) were used as secondary antibodies. To stain the secondary antibodies VECTASTAIN Elite ABC kit was used according to the manufacturer's protocol followed by diaminobenzidine (DAB) staining using kit (SK‐4100; Vector Laboratories). Finally, the sections were then counter stained with Mayer's haematoxylin. Tissue expression was quantified using the immunoreactive score wherein total score was assigned as intensity score + area score with 0 being the lowest and 7 being the highest intensity score. The scoring was done as previously reported (Qian et al., 2011).

Cell culture and Blocking Assay

T24 cells were cultured in RPMI media (Hi media) supplemented with 10% FBS and 1% penicillin/streptomycin at 37ºC with 5% CO₂. For Co-culture experiment 2x10⁵ T24 cells were cultured in RPMI complete media with 2x10⁵ PBMC at different concentration of either anti-B7-H4, anti-PD-L1 or both and were co-cultured for 24hrs. Following which, the cytokine producing activity and cytotoxicity of T cell was assessed by evaluating the percentage of granzyme B + CD8 T cells and IFN-g producing CD4 and CD8 T cells were estimated using flow cytometry. The antibodies used are shown in Supplementary Table-2.

Statistical analysis

Statistical assessment was performed using GraphPad Prism (GraphPad Software, San Diego, CA) and Stata 12. Data was represented as median(range) or mean ± SD. Significance was determined by Mann–Whitney U test to compare tumor and adjacent normal tissue. Student’s t test was used for immunophenotyping between patient and healthy controls. Kruskal Wallis test was employed for analysis having more than two groups. To assess the level of association of these immune checkpoints with clinico-pathological parameters, Wilcoxon-rank sum test or Student’s t test was employed. Correlation was done using Spearman’s correlation coefficient. The significance was defined at p < 0.05 with confidence interval of 95%.

Characteristics of study subjects:

Forty UBC patients were recruited from whom histopathologically proven tumor tissue and adjacent normal tissue were obtained. Besides, 30 healthy controls were recruited for comparison. Patient demographic data is depicted in Table-1. According to histological grading, the majority of patients were high grade (29), and rest were low grade (11). Upon stratifying the patients, based upon muscle invasion, 22 patients were categorized as NMIBC and 18 as MIBC. On pathological tumor staging (pT) according to WHO staging of 2016, UBC patients were categorized as pT0, pT1, pT2, and pT3 stages (Magers et al., 2019).

Elevated frequencies of T cells subsets and immune checkpoints in the circulation of UBC patients:

Flow cytometric analysis of B7-H4 and PD-L1 in UBC patients has been shown in Fig. 1A significantly increased frequency of CD8 and CD4 T cells was observed in peripheral circulation of UBC patients compared with healthy individuals (p < 0.0001). The frequency of B7-H4 and PD-L1 was significantly higher among CD8 and CD4 T cells of UBC patients in contrast to healthy volunteers (p < 0.0001). Upon comparison of B7-H4 and PD-L1 expression on CD8 T cells and CD4 T cells of the same patients, CD8 T cells displayed a significantly elevated expression of B7-H4 in comparison to CD4 T cells of UBC patients (p < 0.0001). While there was not any significant difference in PD-L1 expression [Fig. 2 (A-H); Table-2].

The frequency of B7-H4 and PD-L1 was also assessed between MIBC and NMIBC patients wherein a significantly elevated expression was observed in MIBC patients for both B7-H4 and PD-L1 (p < 0.0001) which depicts the plausible involvement of these immune checkpoints in disease severity [Fig. 2 (I-J)].

Comparison of B7-H4 and PD-L1 protein expression was evaluated on CD4 + T cell and CD8 + T cell in UBC patients wherein 100% positivity was observed for both immune checkpoints in these T cell populations as summarized in Table-3(a). CD8 + B7-H4 + T cell positivity was observed to be between 2.8–14.4% calculated as percentage change and showcases an elevation with muscle invasion of tumor in UBC patients. Similarly, CD8 + PD-L1 + T cells positivity ranged between 7.5–23.7%.

Upregulated relative mRNA expression of Immune checkpoints in bladder cancer tissue:

To assess the mRNA levels, q-PCR was performed on cDNA extracted from tumor tissue and adjacent normal tissue of UBC patients.

The relative mRNA expression of B7-H4 was found to be significantly elevated in tumor tissue compared to normal adjacent tissue (p = 0.0007). PD-L1 exhibited a heightened expression in tumor tissue than its normal counterpart (p = 0.004). When the expression was evaluated between non-muscle invasive and muscle-invasive bladder cancer patients, B7-H4 expression was observed to be significantly elevated in MIBC than in NMIBC patients (p < 0.0001). Similarly, PD-L1 showed a significant increase in muscle invasive bladder cancer patients (p = 0.001) [Fig. 3 (A-D)]

Also, comparison of B7-H4 and PD-L1 positive expression in tumor tissue of UBC patients has been summarized in Table-3(a). Positivity of B7-H4 and PD-L1 was 81% and 66% respectively in bladder tumor tissue of UBC patients.

Increased tissue localization of B7-H4 and PD-L1 in bladder tumor assessed by Immunohistochemistry:

Immunohistochemistry was performed to check for expression of B7-H4 and PD-L1 in tumor tissue of UBC patients and an elevated expression of B7-H4 and PD-L1 in tumor tissue in comparison to healthy bladder tissue was observed [Fig. 4]. B7-H4 exhibited a strong expression on the membrane and cytoplasm of bladder tissue whilst a faint expression was observed in healthy bladder tissue. Overall, MIBC tissue displayed a significantly elevated expression of B7-H4 (p < 0.001). PD-L1 staining was observed in tumor tissue of UBC patients and a weak to negative staining was observed in normal bladder tissue showcasing an increased expression in MIBC patients (p < 0.01). The proportion of MIBC patients exhibited an immunopositivity for B7-H4 in 7/10 (70%) which was higher than the positivity displayed by NMIBC patients in 2/10 (20%). Of the 20 patients, B7-H4 accounted for (9/20) 45% positivity. While PD-L1 showed positivity in 6/10 MIBC patients (60%) and 2/10 NMIBC patients (20%). Overall, 8/20 (40%) UBC patients showed PD-L1 positivity.

T cells from UBC patients are efficiently reinvigorated by co-blockade of B7-H4 and PD-L1:

To assess the in-vitro effects, we investigated the effects of the blockade of B7-H4 or PD-L1 and the co-blockade of B7-H4 and PD-L1. To achieve this, PBMCs were isolated from UBC patients and co-cultured with T24 bladder cancer cell line. The basal level expression of B7-H4 and PD-L1 by T24 bladder cancer cell line is shown in Supplementary Fig. 2. We used concentrations of 0.5µl and 1.0µl of blocking antibody and examined the cytotoxic markers of CD8 + T cell i.e., granzyme B and IFN-γ in CD8 + T and CD4 + T cell. Blockade of B7-H4 significantly increased the production of granzyme B and IFN-γ in CD8 + cell and CD4 + T cells at concentration of 1.0µl. Similar results were observed for PD-L1 at concentration of 1ul [Fig. 5]. This indicates an increase in inflammatory and cytotoxic nature of T cells against cancer. Importantly, co-blockade of B7-H4 and PD-L further significantly enhanced the capacity of CD8 + cells to produce IFN-γ and granzyme B against cancer cells (p < 0.001) [Supplementary Fig. 3]. Similarly, CD4 + T cells showed significantly increased expression of IFN-γ (p < 0.001) upon combinatorial blockade. Patients were categorized into NMIBC and MIBC and were examined for the effect of combinatorial blockade of PBMC on the UBC cell line wherein no significant difference was observed. Therefore, these results suggest combinatorial blockade approach can be used to achieve an effective anti-tumor response in these patients [Fig. 5].

Association of B7-H4 and PD-L1 with clinico-pathological parameters:

To assess the clinical significance of B7-H4 and PD-L1, their association with clinico-pathological parameters: Grade and Muscle Invasion has been depicted in Table-4(a-b). Firstly, the frequency of B7-H4 and PD-L1 in circulation of UBC patients were tested for their association with histological grade and muscle invasion. Both B7-H4 (p < 0.0001) and PD-L1(p < 0.0002) exhibited a statistically significant association with grade. A similar trend was observed for muscle invasion (p < 0.0001) as well. Next, the transcript levels of B7-H4 and PD-L1 in urothelial carcinoma tissue B7-H4 (p < 0.004) showed a significant association with grade while no association for observed for PD-L1. A statistically significant association was observed for both B7-H4 and PD-L1(p < 0.0001) with muscle invasion.

Correlation Analysis:

Total B7-H4 exhibited a positive correlation with PD-L1 in PBMC of UBC patients (p < 0.02). Also, the tissue levels of B7-H4 showed a significant correlation with PD-L1(p < 0.004). Next, the B7-H4 + CD4 + T cells and B7-H4 + CD8 + T cells also showed positive correlation (p < 0.01) in PBMC of UBC patients while no such trend was observed for PD-L1. We also observed a significant correlation between B7-H4 + CD8 + T cells and PD-L1 + CD 8 + T cells in circulation (p < 0.0001). Lastly, the tissue and PBMC levels of B7-H4 depicted a significantly positive correlation (p < 0.002) [Table-5].

Urothelial carcinoma of Bladder is the second most common urological malignancy associated with high mortality and morbidity. The initial step in the 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 the treatment of MIBC patients, with neoadjuvant chemotherapy followed by radical cystectomy as the mainstay of treatment (De Santis et al., 2012; von der Maase et al., 2005; Zhang et al., 2016).

Over the years, immunotherapy has been established as new pillar of cancer care. There is a plethora of inhibitory pathways hardwired into immune system that are crucial for maintaining physiological immune responses. The targeting of “PD-1/PD-L1” axis has exhibited unprecedented clinical efficacy in several cancers including UBC (Blackburn et al., 2008; Rosenberg et al., 2016). 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. Therefore, additional strategies need to be identified and tested to improve the 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 (Rotte et al., 2018). Studies have shown the 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 them by either inhibiting CD4 T cell proliferation or the T cell division cycle. It also reprograms the CD8 T cell by curbing the release of antitumor cytokines and CD8 T cell cytotoxicity against tumors (26). A high expression of B7-H4 has been reported in various solid tumors and is also associated with worse patient survival (Matsunaga et al., 2011; Smith et al., 2014).

Till date, there is no study to comprehensively evaluate the expression of B7-H4 in corroboration with PD-L1 in UBC. Therefore, in this maiden study, we want to investigate the basal level expression of B7-H4 along with PD-L1 in UBC to broaden the therapeutic repertoire for better treatment of UBC patients.

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 UBC patients and matched healthy controls. An elevated frequency of CD4 + T and CD8 + T cells were observed in UBC patients relative to healthy volunteers. B7-H4 and PD-L1 were observed to be significantly increased on T cell population in UBC patients. These results are in concordance with previous report in other cancer (Donahue et al., 2017; Matsunaga et al., 2011). Importantly, the frequency of B7-H4 + peripheral blood CD8 T cells was significantly greater than CD4 T cells in UBC patients. Also, the frequency of CD 8 + B7-H4 + and CD 8 + PD-L1 + cells exhibited a statistically significant increase in muscle invasive bladder cancer patients when compared to non-muscle invasive bladder cancer patients. This is a maiden study showcasing the frequency of B7-H4 in periphery of UBC patients and is indicative of upregulation of immune inhibitory molecules B7-H4 and PD-L1 in the periphery which might be associated with anti-inflammatory response and exhibits a correlation with disease severity.

To know the presence of these molecules in the tissue, relative gene expression of B7-H4 and PD-L1 were investigated 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 muscle-invasive bladder tumors in comparison to non-muscle invasive bladder tumors. In agreement with our results, a single study in UBC has shown similar results (Fan et al., 2014). 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. A positive expression of B7-H4 + tumor cells in UBC and other solid tumors have been previously reported (Ding et al., 2021; Fan et al., 2014, p. 7; Tringler et al., 2006).

To assess the clinical significance of these immune checkpoints, the transcript, and frequency of B7-H4 and PD-L1 were assessed for their association with histological grade and muscle invasion. B7-H4 exhibited a positive association of significant strength with histological grade and muscle invasion showcasing a positive trend with disease severity 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 a similar observation showcasing a positive association of B7-H4 with clinical parameters in prostate cancer (Zang et al., 2007).

A vital question for checkpoint inhibition therapy is the relative contribution of immune checkpoints leading to the synergistic effect of suppressing the effector phase of T cell responses. Therefore, to assess the therapeutic potential of these immune checkpoints in UBC, in-vitro blocking experiments were performed and evaluated T cells in periphery. Upon co-coculture of PBMC with high grade bladder cancer cell line T24, we observed an escalation in the cytotoxicity of CD-8 and CD-4 T cells with an increase in granzyme B and IFN-g levels. The combinatorial blocking of B7-H4 and PD-L1 significantly restored the granzyme B and IFN-g producing capacity of CD8 T cells and IFN-g levels of CD4 T cells of UBC patients. While no difference in cytotoxicity of T cells was observed between NMIBC and MIBC patients which could be attributed to the small sample size (n = 3) in each group. This is the first study in bladder cancer to use combinatorial blocking of B7-H4 and PD-L1 to assess the cytotoxicity of T cells 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-8T cell cytotoxicity is in concordance with our findings (LIU et al., 2014). The present study suggests that altering B7-H4 expression alone or in combination with PD-L1 may enhance T cell cytotoxicity to UBC cells and promote a functional T cell immune response which further highlights the efficacy of the combinatorial blocking approach.

Overall, our data showcase that B7-H4 and PD-L1 expression was higher in T cells of UBC patients compared to healthy controls in peripheral blood. Notably, the frequency of B7-H4 was higher on CD8 T cells than on CD4 T cells of UBC patients, which highlights the plausible role of immune checkpoints in downregulating CD8 T cell effector function. Also, B7-H4 expression was highly elevated in bladder tumor tissue. Upon stratifying patients with muscle invasive and non- muscle invasive bladder cancer 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. Further to explore its therapeutic potential, in-vitro blocking of B7-H4 and PD-L1 were performed which displayed that functionality of T cell from UBC patients could be restored by the combinatorial blocking of B7-H4 and PD-L1. This maiden 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 UBC patients for better treatment in future after validation.

Author contributions:

PS and AS₁ conceived and designed the study. PS, BN and SK₁ provided patient sample;

DR, AS₂ and SK₂performed experiments, acquired and analysed data, did statistical analyses, drafted the manuscript; PS, AS₁ and BN edited the final manuscript. [AS₁ : Alpana Sharma; AS₂: Aishwarya Singh; SK₁: Seema Kaushal; SK₂: Santosh Kurra]

Conflict of Interest:

The authors declare no conflict of interest.

Funding:

The authors would like to thank All India Institute of Medical Sciences (AIIMS), New Delhi, India (Grant no. E8-734/A-734/2019/RS) for providing financial assistance to carry out the present study.

Ethical Approval:

This research involving human patients and healthy individuals had provided written informed consent. This study has been approved by Institutional Human Ethics Committee of All India Institute of Medical Sciences, New Delhi, India (IEC-685/06.09.2019) prior to its commencement.

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Table 1: Baseline characteristics of UBC patients and control subjects

PATIENTS

Total Number (n)

40

Male/Female

37/3

Age (Years)

Range

59±7

(35-65)

Histological Grade

High Grade

Low Grade

29

11

Muscle Invasion

Muscle invasive

Non-muscle Invasive

18

22

pT Stage

pTa

pT1

pT2

pT3

pT4

9

13

8

6

4

CONTROLS

Total Number (n)

30

Male/Female

21/9

Age (Years)

Range

47 ± 11

(32-58)

Patients were staged as per the AJCC 2016 pathological staging system.

pT Stage: Pathological Tumor Stage

Values are represented as mean ± SD

Table 2: The frequency of T cell subsets and immune checkpoints in circulation of healthy controls and UBC patients

Variable	Healthy control (n=30)	Patients (n=40)
CD3+ CD8+ T cell	33.07±3.26	42.14±1.94
CD3+ CD4+ T cell	36.31±1.75	42.76±2.32
CD8+ B7-H4+ T Cells	24.38±1.14	37.16±3.39
CD4+ B7-H4+ T Cells	10.32±1.97	16.71±4.31
CD8+ PDL1+ T cells	26.39±5.3	39.83±3.88
CD4+ PDL1+ T cells	29.25±2.6	38.69±2.09

Values are presented as Mean±SD

Table 3(a): B7-H4 and PD-L1 positive expression in UBC patients in circulation and tumor tissue

	B7-H4 expression		PD-L1 expression
	Negative	Positive	Negative	Positive
¹protein expression in PBMC
CD4+ T cells	-	100% (2.8-14.4)	-	100% (4.5-14.75)
CD8+ T cells	-	100% (8.7-24.6)	-	100% (7.5-23.7)

²mRNA expression in tumor tissue	18.18%	18/22=81.8%	33%	14/22=63.63%

B7-H4 and PD-L1 positivity was calculated by : 1. Comparing the mean of matched healthy controls with the basal expression in UBC patients. Represented as percentage change with respect to control; 2. By comparing the relative mRNA expression of tumor tissue and adjacent normal tissue of UBC patients.

Table 3(b): B7-H4 and PD-L1 positive expression in bladder tumor tissue and healthy bladder tissue

	B7-H4			PD-L1
	Cases	Positive	Negative	Cases	Positive	Negative
Controls	10	0	10	10	0	10
NMIBC	10	2 (20%)	8	10	2 (20%)	8
MIBC	10	7 (70%)	3	10	6 (60%)	4
Low grade	5	0	5	5	0	5
High grade	15	9 (60%)	6	15	8 (53%)	7
Total UBC	20	9 (45%)	11	20	8 (40%)	12

Immunohistochemical expression of B7-H4 and PD-L1 in healthy bladder tissue, tumor bladder of NMIBC and MIBC patients. Scoring was done with respect to staining intensity with 0= least staining and 3= intense staining) and the proportion/area of stained cells (0 to 4, 0:<10% area stained; 4+: more than 70% cells stained) were semi-quantitatively determined. A combined score of ≥6 was considered to indicate overexpression/ positive cases or defined as the cut-of value for positive cases.

Table 4(a): The level of significance of frequency of B7-H4 and PD-L1 in correlation with clinico- pathological features of UBC patients

	B7-H4	PD-L1
GRADE
Low Grade (n=11)	31.24±1.82	29.95±1.95
High Grade (n=29)	37.03± 3.35	33.9±2.98
p	0.0001	0.0002
MUSCLE INVASION
Non-Muscle Invasive (n=22)	32.64±3.14	30.96±2.5
Muscle Invasive (n=18)	38.85±1.25	35.08± 2.6
p	0.0001	0.0001

[ B7-H4 and PD-L1 in PBMC are calculated as % CD3+ T cell] Values are represented as mean ± SD. Bold value

denotes significance.

Table 4(b): The association of relative mRNA expression of B7-H4 and PD-L1 in tumor tissue with the clinico-pathological features of UBC patients

Median (Range)(X10^-4)
	B7-H4	PD-L1
GRADE
Low Grade (n=11)	4.478 (.825- 7.858)	10.649 (.781-29.04)
High Grade (n=29)	7.2 (.925- 48.09 )	19.12 (1.412-63.15)
p	0.0041	0.2369
MUSCLE INVASION
Non-Muscle Invasive (n=22)	4.478 (.825-7.858)	7.806 (.781-29.04)
Muscle Invasive (n=18)	34.48 (5.87-48.09)	24.38 (3.66-63.15)
p	0.0001	0.0001

[B7-H4 and PD-L1 in tumor tissue calculated as total tumor tissue] Values are represented as Median(Range). Bold value denotes significance.

Table 5: Spearman correlation analysis of immune checkpoints in UBC patients

	*Variables to be correlated*
	r = Spearman Coefficient
Variable	B7-H4 and PD-L1	p
a) PBMC^*	0.3511	(0.026)
b) Tissue^#	0.4451	(0.004)

Variable	CD4+T cell and CD8+ T Cell (In PBMC)	p
a) B7-H4	0.4451	(0.01)
b) PD-L1	-0.065	(0.69)

Variable	B7-H4 and PD-L1 (In PBMC)	p
a) CD4+ T cell	0.06	(0.7)
b) CD8+ T cell	0.92	(0.0001)

Variable	Tissue^# and PBMC^*	p
a) B7-H4	0.5551	(0.0002)
b) PD-L1	0.1805	(0.2650

Bold value denotes significant correlation. [ *: B7-H4 and PD-L1 in PBMC are calculated as % CD3+ T cell; #: B7-H4 and PD-L1 in tumor tissue calculated as total tumor tissue]

No competing interests reported.

SupplementaryMaterial.docx

Clinical Significance of combinatorial blockade of novel immune checkpoint B7-H4 with PD-L1 in Urothelial Carcinoma of Bladder: A rational therapeutic approach

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Abstract

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1. Introduction

2. Materials And Methods

3. Results

4. Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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