Recognition of prostate and breast tumor cells by helper T lymphocytes specific for a prostate and breast tumor-associated antigen, TARP

Abstract

Purpose: T cell-based immunotherapy via the in vitro or in vivo expansion of prostate tumor-associated antigen (TAA)-specific T lymphocytes is one of the most promising therapeutic approaches to treat prostate cancer. T-cell alternate reading frame protein (TARP) is a mitochondrial protein that is specifically expressed in prostate epithelial cells. We have done experiments aimed at identifying helper T lymphocyte (HTL) epitopes for TARP for the design of T cell-based immunotherapy for prostate cancer.

Experimental design: Dendritic cells from normal donors were pulsed with synthetic peptides derived from TARP, which were predicted to serve as HTL epitopes. These dendritic cells were used to stimulate CD4(+) T cells in vitro to trigger HTL responses against TARP. T-cell responses to these peptides were also studied with lymphocytes from prostate cancer patients.

Results: The two peptides, TARP(1-14) and TARP(14-27), were shown to elicit effective in vitro HTL responses using lymphocytes from both normal volunteers and prostate cancer patients. Peptide TARP(1-14)-reactive HTLs were found restricted by HLA-DR53 and could recognize naturally processed protein antigen derived from tumor cells, which was presented by autologous dendritic cells. Most significantly, stimulation with peptide TARP(14-27) generated four HTL lines restricted by HLA-DR1, HLA-DR9, HLA-DR13, and HLA-DR15, some of which capable of recognizing naturally processed antigens presented by dendritic cell or directly by TARP-positive tumor cells.

Conclusions: Our results show that TARP constitutes a TAA that can be recognized by tumor-reactive HTL. The newly described TARP epitopes could be used to optimize and improve T-cell epitope-based immunotherapy against prostate and other tumors expressing TARP.

Fig. 1

HLA-DR53-restricted HTL responses to peptide TARP_1-14. An HTL line was selected from an HLA-DR4/DR9, HLA-DR53 normal male individual by weekly stimulation of CD4⁺ Tcells with peptide and APCas described in Materials and Methods. A, using autologous PBMCas APC, the T-cell response to peptide TARP_1-14 was inhibited by anti – HLA-DR mAb L243 but not by anti -HLA class I mAb W6/32 (both used at 10 μg/mL). B, when mouse fibroblast (L cells) transfected with HLA-DR genes were used as APC, it became evident that the HTL line recognized peptide TARP_1-14 (used at 3 μg/mL) in the context of HLA-DR53. Columns, triplicate determinations; bars, SD. Columns without bars had SD<10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 2

HTL responses to peptideTARP_14-27. HTL lines were selected from (A) an HLA-DR1/DR15 normal male individual (K18) and from (B)anHLA-DR9/DR14 normal male individual (TNG4) and HTL clones were selected from (C) an HLA-DR9/DR13 normal male individual (9H12) and from (D) an HLA-DR4/DR15 normal male individual (6E15) by weekly stimulation of CD4⁺ Tcells with peptide and APCas described in Materials and Methods.When autologous PBMC were used as APC, all HTL responses to peptide TARP_14-27 were inhibited by anti – HLA-DR mAb L243 but not by anti -HLA class I mAbW6/32 (both at used10 μg/mL).When mouse L-cell fibroblasts transfected with HLA-DR genes were used as APC, HTL line K18 recognized the peptide in the context of HLA-DR1 allele (A), HTL line TNG4 recognized the peptide in the context of HLA-DR9 allele (B), HTL clone 9H12 recognized the peptide in the context of HLA-DR13 allele (C), and HTL clone 6E5 recognized the peptide in the context of HLA-DR15 allele (D). Columns, triplicate determinations; bars, SD. Columns without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 3

Peptide dose-response curves were done to estimate the avidity of these HTL for peptide TARP_1-14 (A)and TARP_14-27 (B-E) using autologous PBMCs and dendritic cells (DC) as APC. Points, triplicate determinations; bars, SD. Points without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 4

Recognition of naturally processed antigen by the HLA-DR53 -restricted,TARP_1-14-specific HTL line. A, when autologous DC were used as APC, the peptide-reactive HTL were able to respond to various amounts of cell lysates derived from LNCaP and SKBr3 tumor cells expressing TARP but not to TARP-negative PC3 tumor cells. B, recognition of tumor lysates via autologous dendritic cell (DC) was inhibited by anti – HLA-DR mAb L243 at 10 μg/mL. Triplicate determinations; bars, SD. Columns and symbols without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 5

Recognition of naturally processed antigen by TARP_14-27-reactive HTL. The HTL derived from different four donors were able to recognize various amounts of TARP-positive tumor cell lysates (LNCaP, SKBr3, and MCF7) but not TARP-negative PC3 tumor cells by autologous dendritic cells (A-D). Points, triplicate determinations; bars, SD. Points without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 6

Indirect recognition of peptide TARP_14-27 via exogenous antigen presentation by autologous dendritic cells (DC). The HTLs derived from different four donors were able to recognize the TARP-positive tumor cell lysates (LNCaP, SKBr3, and MCF7) but not TARP-negative PC3 tumor cell lysate by autologous dendritic cells and antigen recognition in all cases was inhibited by anti – HLA-DR mAb L243 at 10 μg/mL (A-D). Columns, triplicate determinations; bars, SD. Columns without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 7

Direct recognition of MHCII/TARP_14-27 on tumor cells by HTL. A, cell surface expression of HLA-DR molecules on tumor cells.Tumor cell lines (SKBr3, PC3, and MCF7) were cultured with IFN-γ (500 units/mL) for 48 hours to enhance MHCantigen expression. The expression of HLA-DR molecules on tumor cells was evaluated by staining with anti – HLA-DR (L243) mAb conjugated with fluorescein isothiocyanate (grey line, open histograms). Staining with the isotype negative control (black-filled histograms). B, HLA-DR13 -restricted HTL clone produced IFN-γ as the result of recognizing antigen on HLA-DR13+ TARP+ SKBr3 tumor cells presenting naturally processed epitope. C, similarly, the HLA-DR15 -restricted HTL clone also produced IFN, as the result of recognizing antigen on HLA-DR15+ TARP+ MCF7 tumor cells. In both cases, production of IFN-γ by the HTL clones was inhibited by anti – HLA-DR mAb L243 at10 μg/mL. Columns, triplicate determinations; bars, SD. Columns without bars had SD < 10% the value of the mean. Representative of at least two to three experiments that were done with the same samples.

Fig. 8

T-cell responses to peptideTARP_1-14 and TARP_14-27 in patients with prostate cancer. PBMCs were isolated from seven prostate cancer patients and were stimulated with peptides as described in Materials and Methods. The peptide-stimulated PBMCs derived from patients 1-7 (A-G) produced lymphokines (IFN-γ and/or GM-CSF), and production of lymphokines by PBMCs was inhibited by adding anti – HLA-DR mAb L243 at 10 μg/mL during culture. Column, triplicate determinations; bars, SD. Columns without bars had SD < 10% the value of the mean. ND, not determined.