Repertoire enhancement with adoptively transferred female lymphocytes controls the growth of pre-implanted murine prostate cancer

Abstract

Background: In prostate cancer, genes encoding androgen-regulated, Y-chromosome-encoded, and tissue-specific antigens may all be overexpressed. In the adult male host, however, most high affinity T cells targeting these potential tumor rejection antigens will be removed during negative selection. In contrast, the female mature T-cell repertoire should contain abundant precursors capable of recognizing these classes of prostate cancer antigens and mediating effective anti-tumor immune responses.

Methodology/principal findings: We find that syngeneic TRAMP-C2 prostatic adenocarcinoma cells are spontaneously rejected in female hosts. Adoptive transfer of naïve female lymphocytes to irradiated male hosts bearing pre-implanted TRAMP-C2 tumor cells slows tumor growth and mediates tumor rejection in some animals. The success of this adoptive transfer was dependent on the transfer of female CD4 T cells and independent of the presence of CD25-expressing regulatory T cells in the transferred lymphocytes. We identify in female CD4 T cells stimulated with TRAMP-C2 a dominant MHC II-restricted response to the Y-chromosome antigen DBY. Furthermore, CD8 T cell responses in female lymphocytes to the immunodominant MHC I-restricted antigen SPAS-1 are markedly increased compared to male mice. Finally, we find no exacerbation of graft-versus-host disease in either syngeneic or minor-antigen mismatched allogeneic lymphocyte adoptive transfer models by using female into male versus male into male cells.

Conclusions/significance: This study shows that adoptively transferred female lymphocytes, particularly CD4 T cells, can control the outgrowth of pre-implanted prostatic adenocarcinoma cells. This approach does not significantly worsen graft-versus-host responses suggesting it may be viable in the clinic. Further, enhancing the available immune repertoire with female-derived T cells may provide an excellent pool of prostate cancer reactive T cells for further augmentation by combination with either vaccination or immune regulatory blockade strategies.

Figure 1. Female mice reject TRAMP-C2 prostate cancers while male mice develop tolerance following puberty.

A) TRAMP-C2 tumor cells grow progressively in adult male mice, with a slower kinetic but complete penetrance in pre-pubescent male mice, and grow briefly but then are rejected in adult female mice. n = 5 mice/group, one of 2 experiments with similar results. B) Adult female mice were transplanted with female, male, or a 1∶1 mixture of female and male T-cell depleted bone marrow and 10 weeks after transplantation challenged with TRAMP-C2 tumors. n = 5 mice/group, results of a single experiment. C) Preventing onset of puberty in male mice can mediate delayed growth of TRAMP-C2 tumors. Adult male mice surgically castrated before onset of puberty (4 weeks) show delayed growth of TRAMP-C2, while castration 1 week prior to tumor challenge (7 weeks) had no effect on tumor growth. n = 10 mice/group, tumor growth curve and area under curve normalized to median value for group 3 from each experiment. Combined results of 2 experiments.

Figure 2. Adoptive transfer of female lymphocytes into male mice challenged with TRAMP-C2 controls tumor growth.

A) Adult male mice were myeloablated via irradiation and transplanted with female T-cell depleted bone marrow followed by challenge with TRAMP-C2 tumors 10 weeks after transplantation. n = 5 mice/group, results of a single experiment. B) Adult male mice were sublethally irradiated with 6 Gy and challenged intradermally with 1×10⁶ TRAMP-C2. The following day, mice received a donor lymphocyte infusion (DLI) of 3×10⁷ splenocytes i.v. n = 10 mice/group, one of 3 experiments with similar results. C) Adoptively-transferred CD25+ regulatory T cells do not mediate increased tumor growth seen following male DLI. Mice were irradiated and tumor-challenged as in B, and received splenocytes that were CD25-depleted using magnetic beads where indicated. n = 10 mice/group, results of a single experiment. D) Female CD4 T cells mediate delayed growth of TRAMP-C2 tumors. Mice were irradiated and tumor-challenged as in B, and then received 1.5×10⁷ splenocytes that were in vivo CD4-depleted, mixed with 1.5×10⁷ splenocytes that were CD8-depleted, of either male or female origin. n = 10 mice/group, one of 2 experiments with similar results.

Figure 3. Female lymphocytes strongly recognize the CD4 antigen DBY and the CD8 antigen SPAS-1.

A) Immunization of females with TRAMP-GMCSF cells produces augmented CD4 responses against DBY and CD8 responses against SPAS-1. Male and female B6 mice were injected intradermally with 1×10⁶ irradiated TRAMP-GMCSF at days −9, −6, and −3 prior to sacrifice. CD4+ and CD8+ splenocytes were purified with magnetic beads and restimulated with CD11c+ female splenic DCs pulsed with the indicated peptide, or alternatively were stimulated with anti-CD3ε/anti-CD28 beads, or with phorbol myristate acetate (PMA) and ionomycin. Supernatant concentrations of IFN-γ and other cytokines were assayed by BD cytometric bead array. Results of a single experiment. B) DNA vaccination with SPAS-1 produces immune responses in male mice adoptively transferred with female lymphocytes. Male B6 mice were given 6 Gy irradiation followed by male or female adoptive lymphocyte transfer. Mice were vaccinated by gene gun with SPAS-1-expressing DNA plasmids on days 1, 7, and 13 following adoptive transfer. On day 19, splenocytes were incubated with or without the SPAS-1 and DBY peptides, and production of IFN-γ by CD4 and CD8 T cells was evaluated by intracellular flow cytometry. Results of a single experiment.

Figure 4. Adoptively transferred female lymphocytes do not produce increased graft-versus-host disease.

Male B6 mice were given 6 Gy irradiation followed by an i.v. infusion of 3×10⁷ splenocytes, either male or female, from syngeneic B6 or allogeneic LP donors. For comparison, female B6 mice were lethally irradiated (11 Gy) and transplanted with 5×10⁶ T-cell depleted bone marrow cells and 2×10⁶ purified splenic T cells from MHC-mismatched B10.BR mice (H2k into H2b). Mice were evaluated on the day indicated for histological evidence of GVHD in the liver, small intestine, and large intestine. Results of a single experiment.