Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

Abstract

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.

Fig. 1

P2X5 gene expression in solid tumor cells. P2X5 mRNA expression was determined by real-time quantitative RT-PCR in 42 solid tumor cell lines. Cell lines from the following tumor types were analyzed: renal cell carcinoma (RCC; n = 11), melanoma (n = 4), colorectal carcinoma (n = 7), brain cancer (n = 10), and breast cancer (n = 10). Expression is shown relative to the P2X5 expression measured in reference B-cell line JVM-2, which is susceptible to lysis by LRH-1-specific CTL. The housekeeping gene HMBS was used for normalization. Solid tumor cell lines with P2X5 expression higher than 0.4 were considered positive and potential targets for LRH-1-specific CTL, based on previous expression and recognition studies. This arbitrary threshold is indicated with a dashed line

Fig. 2

Cytotoxicity against solid tumor cell lines was determined after incubation with LRH-1-specific CTL (filled up triangle), HLA-B7-specific CTL (filled inverted triangle; positive control) or medium only (filled diamond). Survival of unstimulated and cytokine-stimulated target cells in the absence or presence of CTL at an E:T ratio of 3:1 is shown of an LRH-1 ⁺ and LRH-1 ⁻ EBV-LCL (a) and of LRH-1⁺ brain tumor cell line DAOY (b). P2X5 mRNA expression is shown between parentheses. Data are depicted as mean ± SD of triplicate wells. c Microscopic analysis of cytokine-stimulated DAOY cells at 40 h of co-culture with CTL or medium

Fig. 3

Cytotoxicity against LRH-1 ⁺ melanoma cell line BLM (a) and LRH-1 ⁻ melanoma cell line FM3 (b) was determined after incubation with LRH-1-specific CTL (filled up triangle), HLA-B7-specific CTL (filled inverted triangle; positive control) or medium only (filled diamond). Survival of unstimulated and cytokine-stimulated target cells in the absence or presence of CTL at an E:T ratio of 3:1 is shown. P2X5 mRNA expression is shown between parentheses. Data are depicted as mean ± SD of triplicate wells

Fig. 4

Cytotoxicity against LRH-1 ⁺ RCC cell lines SKRC33 (a) and SKRC18 (b) was determined after incubation with LRH-1-specific CTL (filled up triangle), HLA-B7-specific CTL (filled inverted triangle; positive control) or medium only (filled diamond). Survival of unstimulated and cytokine-stimulated target cells in the absence or presence of CTL at an E:T ratio of 3:1 is shown. P2X5 mRNA expression is shown between parentheses. Data are depicted as mean ± SD of triplicate wells. c LRH-1-peptide loading resulted in improved lysis of SKRC18

Fig. 5

a The expression of adhesion and MHC molecules in epithelial cancer cell lines. Surface expression of HLA-B7, CD54 (ICAM-1) and CD58 (LFA-3) was determined by flow cytometry. Prior to analysis cells were cultured for 4 days in the absence or presence of inflammatory cytokines IFNγ and TNFα. Data are depicted as fold induction of the mean fluorescence intensity (MFI) upon cytokine stimulation. b Cytokine stimulation of cancer cells results in improved degranulation of CTL. Anti-CD107-antibody was added to the co-cultures of flow cytometry-based cytotoxicity assays. Degranulation of LRH-1-specific CTL was measured after 1 day of co-culture with solid tumor cell lines and control EBV-LCL. Data are depicted as mean ± SD of triplicate measurements