PD-L1 downregulation by carbonic anhydrase IX immunotherapy prompts immune checkpoint blockade in renal cell carcinoma

Abstract

• CAIX-targeting immunotherapies decrease PD-L1 expression in ccRCC cell lines. • PI3K/Akt signaling pathways are involved in PD-L1 decrease after CAIX immunotherapy. • CAIX CAR T cell therapy induces immune checkpoint blockade in ccRCC in vivo. The carbonic anhydrase IX (CAIX) expression occurs in most cases of clear cell renal cell carcinoma (ccRCC). This tumor type is characterized by an immunosuppressive microenvironment, where approximately one-fourth of patients overexpress the programmed cell death ligand-1 (PD-L1), significantly increasing their risk of death. Herein, we present a secondary effect of CAIX inhibition using monoclonal antibodies (mAbs) and CAR T cells, leading to PD-L1 downregulation in ccRCC and in vivo immune checkpoint blockade . We identified a positive correlation between CAIX and PD-L1 expression in ccRCC cell lines using in silico RNA-seq data analysis, prompting us to perform fluorescence-activated cell sorting of SKRC52 ccRCC cell subpopulations based on their positive or negative expression of CAIX and PD-L1. After two weeks in culture, the cell population selected to be negative for CAIX and positive for PD-L1 became negative for CAIX and PD-L1. To explore the phenomenon, CAIX blockade was performed using two anti-CAIX monoclonal antibodies (mAbs) in multiple doses in two CAIX+ PD-L1+ clear cell renal cell carcinoma (ccRCC) cell lines. The expression of CAIX and PD-L1 decreased after treatment with the mAbs, and the PI3K/Akt signaling pathway is involved in this modulation. CAIX-targeted CAR T cells in vivo also reduced PD-L1 expression, resulting in superior CD3 infiltration and granzyme B expression, which decreased T cell exhaustion. Our findings demonstrate that CAIX-targeted immunotherapies can induce an indirect immune checkpoint blockade by PD-L1 downregulation. Renal Cell Cancer (RCC) Cells Interacting with T Cells Before and After Anti-Carbonic Anhydrase IX (CAIX) Immunotherapies. A. In the untreated state, RCC cells expressing CAIX and programmed cell death ligand-1 (PD-L1) activate immune checkpoints on T cells, leading to increased expression of PD-1, TIM-3, CD39, and LAG-3. This process results in decreased T cell effector functions, supporting tumor progression. B. After treatment with anti-CAIX antibodies or CAR T cells, RCC cells exhibit reduced expression of CAIX and PD-L1. This action results in immune checkpoint blockade in T cells, characterized by reduced expression of T cell exhaustion markers PD-1, TIM-3, CD39, and LAG-3 and improved T cell effector functions, thereby mitigating tumor development.