Limited Immunogenicity of an HLA-A*03:01-restricted Epitope of Erv-k-env in Non-hiv-1 Settings: Implications for Adoptive Cell Therapy in Cancer

Abstract

Repetitive elements (REs) are often expressed at higher levels in tumor cells than normal cells, implicating these genomic regions as an untapped pool of tumor-associated antigens. In ovarian cancer (OC), protein from the RE ERV-K is frequently expressed by tumor cells. Here we determined whether the targeting of a previously identified immunogenic epitope in the envelope gene (env) of ERV-K resulted in target antigen specificity in non-HIV-1 settings. We found that transducing healthy donor T cells with an ERV-K-Env-specific T cell receptor construct resulted in antigen specificity only when co-cultured with HLA-A*03:01 B lymphoblastoid cells. Furthermore, these transduced T cells were not specific for HLA-A*03:01 + OC cells nor for the cognate peptide in HLA-matched systems from multiple healthy donors. These data suggest that the ERV-K-Env epitope recognized by this T cell receptor is of low immunogenicity and has limited potential as a T cell target for OC.

Keywords: Endogenous retroviruses; Immunotherapy; Repetitive elements; T cell receptor; Tumor immunology.

Figure 1. OM9.2 T cells did not respond to HLA-A*03:01 OC cells more than untransduced T cells.

A. Diagram of the ELISpot assay design used in B-C. B. Representative images of developed wells for the level of background IFN-γ secretion (media), IFN-γ secretion in the presence of ES-2 cells (1:1 ES-2), and positive control of IFN-γ (PHA) for the ELISpot assay quantified in C. Number of spots in each well are indicated to the bottom right of each image. Images shown are for OM9.2 T cells. C. Number of IFN-γspots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: media = background level of IFN-γ secretion from T cells, actin = negative control, various effector:target ratios of T cells (effectors) with HLA-A*03:01 OC cell lines (targets), PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated in duplicate or triplicate as cell numbers allowed for n = 3 donors. Error bars represent SEM of all data points for all donors. The Wilcoxon rank sum test was used to compare the number of IFN-γ spots between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons are in Tables S1 and S2. *p < 0.05, **p < 0.005, ***p < 0.0005. ELISpot = Enzyme-linked immunosorbent spot; PHA = phytohemagglutinin; OC = ovarian cancer; SEM = standard error of the mean.

Figure 2. OM9.2 T cells were not activated by free ERV-K-Env peptide.

A. Diagram of the ELISpot assay design used in B-D. B. Representative images of developed wells for the level of background IFN-γ secretion (media) and positive control of IFN-γ(PHA) for the ELISpot assays quantified in C-D. Number of spots in each well are indicated to the bottom right of each image. Images shown are for OM9.2 T cells. C-D. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: media = background level of IFN-γ secretion from T cells, actin = negative control, ERV-K-Env = ERV-K-Env peptide, pp65 = HLA-B*35 off-target control, PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated with the indicated peptide concentrations in duplicate or triplicate as cell numbers allowed at 200 ng for n = 2 donors (C) or at 10 μg for n = 6 donors (D). Error bars represent SEM of all data points for all donors. The Wilcoxon rank sum test was used to compare the number of IFN-γ spots between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons are in Tables S1 and S2. *p < 0.05, **p < 0.005, ***p < 0.0005. ELISpot = Enzyme-linked immunosorbent spot; PHA = phytohemagglutinin; SEM = standard error of the mean.

Figure 3. OM9.2 T cells were not activated by the ERV-K-Env peptide when presented by peptide-pulsed APCs.

A, E, H. Diagrams of the ELISpot assay designs used in B-D, F-G, and I-J, respectively. B, F, I. Representative images of developed wells for OM9.2 T cell levels of background IFN-γ secretion (media) and IFN-γ secretion in the presence of unpulsed APCs and ERV-K-Env-pulsed APCs for the ELISpot assays quantified in C-D, G, and J, respectively. Spot numbers are indicated to the bottom right of each image. C-D, G, J. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: Media = background level of IFN-γ secretion from T cells, Raji/PHA blast/DC = background level of IFN-γ secretion from T cells co-cultured with unpulsed APCs, actin-pulsed Raji/PHA blast/DC = negative control-pulsed APCs, ERV-K-Env-pulsed Raji/PHA blast/DC = ERV-K-Env-pulsed APCs, pp65-pulsed Raji/PHA blast/DC = HLA-B*35 off-target control pulsed APCs, PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated with the indicated peptide concentrations in duplicate or triplicate as cell numbers allowed for n = 1 donor. Error bars represent SD of all data points for one donor. The Wilcoxon rank sum test was used to compare the number of IFN-γ spots between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons are in Tables S1 and S2. *p < 0.05, **p < 0.005, ***p < 0.0005. APCs = antigen presenting cells; ELISpot = Enzyme-linked immunosorbent spot; PHA = phytohemagglutinin; SD = standard deviation.

Figure 4. OM9.2 T cells were activated by a high concentration of peptide-pulsed B LCLs.

A. Diagram of the ELISpot assay design used in B-C, and E. B. Representative images of developed wells for OM9.2 T cell levels of background IFN-γsecretion (media) and IFN-γ secretion co-cultured with unpulsed and peptide-pulsed OM9-derived B LCLs for the ELISpot assays quantified C and E. Spot numbers are indicated to the bottom right of each image. C., E. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: Media = background level of IFN-γ secretion from T cells, B LCLs = background level of IFN-γ secretion from T cells co-cultured with unpulsed B LCLs, actin-pulsed B LCLs = negative control-pulsed B LCLs, ERV-K-Env-pulsed B LCLs, pp65-pulsed B LCLs= HLA-B*35 off-target control pulsed B LCLs, PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated with the indicated peptide concentrations in duplicate or triplicate as cell numbers allowed for n = 2 donors (C) or n = 3 donors (E). The Wilcoxon rank sum test was used to compare the number of IFN-γ spots between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons are in Tables S1 and S2. *p < 0.05, **p < 0.005, ***p < 0.0005. D. Flow cytometry plots of intracellular IFN-γ and TNF-α from indicated cell types after 6 hours of co-culture. Cells were plated in a single well for one technical replicate. Gating strategy in Figure S4C. ELISpot = Enzyme-linked immunosorbent spot; B LCL = B lymphoblastoid cell line; PHA = phytohemagglutinin; SEM = standard error of the mean.

Figure 5. OM9.2 T cells were activated by a high concentration of peptide-pulsed B LCLs derived from another donor.

A. Diagram of the ELISpot assay design used in B-C. B. Representative images of developed wells of OM9.2 T cell levels of background IFN-γ secretion (media) and IFN-γ secretion in the presence of unpulsed non-OM9 B LCLs and ERV-K-Env-pulsed non-OM9 B LCLs for the ELISpot assays quantified in C. Number of spots in each well are indicated to the bottom right of each image. C. Number of IFN-γ spots per well normalized to the positive control (expressed as % of PHA) of untransduced T cells (black) and OM9.2 T cells (red) for the indicated conditions: Media = background level of IFN-γ secretion from T cells plated alone, Non-OM9 B LCLs = background level of IFN-γ secretion from T cells in the presence of unpulsed non-OM9 B LCLs,actin-pulsed non-OM9 B LCLs = negative control pulsed non-OM9 B LCLs, ERV-K-Env-pulsed non-OM9 B LCLs, PHA = positive control. Each donor is indicated by a different symbol. ELISpot conditions were plated with the indicated peptide concentrations in duplicate or triplicate as cell numbers allowed for n = 2 donors. Error bars represent SEM of all data points for all donors. The Wilcoxon rank sum test was used to compare the number of IFN-γ spots between untransduced and transduced T cells (indicated by *). The Kruskal Wallis test was used to compare the number of IFN-γ spots between each antigen condition for untransduced or transduced cells. Any Kruskal Wallis test that had p < 0.05 was followed by post-hoc pairwise Wilcoxon rank sum tests and adjusted for multiple comparisons (indicated by #). All statistical comparisons are in Tables S1 and S2. *p < 0.05, **p < 0.005, ***p < 0.0005. ELISpot = Enzyme-linked immunosorbent spot; B LCL = B lymphoblastoid cell line; PHA = phytohemagglutinin; SEM = standard error of the mean.