Here we report that adoptively transferred naïve female lymphocytes slow the growth of and, in some cases, reject pre-implanted TRAMP-C2 prostatic adenocarcinoma cells in sublethally irradiated male mice. A similar finding has also been recently reported by Yi et. al, who also used TRAMP-C2 cells 
. They identified T cells as mediators of anti-prostate cancer immune responses in female mice and found that adoptive transfer of vaccinated female lymphocytes can lead to control of growth of TRAMP-C2 tumors in male hosts. Our study supports their initial findings and expands on them considerably by identifying mechanisms by which the female immune system mounts superior responses to prostate cancer. We demonstrate that female CD4 T cells are essential mediators of this effect, particularly those specific for the immunodominant epitope from the male antigen DBY. While Yi et. al found that female mice immunized with TRAMP-C2 developed some T-cell responses against normal prostate tissue as well as the prostate antigen STEAP, our data demonstrate that responses to STEAP are relatively minor in comparison with responses to the spontaneously arising immunodominant tumor antigen mutated SPAS-1, which was not assayed for in the previous study. Our results indicate that CD4 T cell responses against DBY, which are unique to female immune repertoires, can markedly augment CD8 T cell responses against a spontaneously arising mutant tumor antigen such as SPAS-1. CD4 T cell responses to DBY appear to be particularly robust in the setting of I-Ab MHC II restriction; T cells derived from mice bearing a transgenic T-cell receptor specific for the same DBY epitope have been previously shown capable of mounting responses against TRAMP-C2 tumors in RAG knockout mice 
. We found that female lymphocytes in a male host were also able to mount superior responses to vaccination against SPAS-1, in the setting of concurrently developing spontaneous responses to DBY.
It is possible that CD4 T cells specific for DBY can supply help for CD8 T cells that mediate GVHD as well as GVT. However, by utilizing a model with sublethal irradiation followed by lymphocyte transfer without co-transfer of hematopoietic stem cells, we were able to markedly minimize graft-versus-host disease effects. Male mice treated with large doses of female lymphocytes had no clinical evidence for graft-versus-host disease and only very mild pathological evidence. At the same time, however, this approach may have allowed the establishment of tolerance to male and prostate antigens recognized by transferred female-derived T cells, potentially limiting the efficacy of this strategy and resulting in less mice fully rejecting their tumors.
Many of the recent advances in tumor immunotherapy have focused on either vaccination strategies or blockade of immune regulatory mechanisms. Two recent immune strategies that obtained FDA approval include vaccination against sipuleucel-T for metastatic CaP 
, and antibody-mediated blockade of the T-cell negative co-stimulatory receptor CTLA-4 for the treatment of metastatic melanoma 
. The goal of vaccination strategies are to generate new anti-tumor T cell responses, while immune regulatory blockade strategies seek to release endogenous anti-tumor immune responses that are generated spontaneously but restrained and ineffective 
. While both approaches show promise, clinical data show that most patients eventually still succumb to progression of their cancer. Combinations of immune approaches may be the most promising. In this study, we identify a third approach, namely enhancement of the immune repertoire by adoptively transferring sex-mismatched lymphocytes with improved ability to recognize tumor antigens. Repertoire enhancement has the potential to dynamically synergize with both tumor vaccination and immune regulatory blockade strategies.
For metastatic castration-resistant prostate cancer, a disease which continues to have a poor prognosis, we show that female lymphocytes, particularly CD4 T cells, adoptively transferred into prostate-cancer challenged male mice can mediate substantial anti-tumor effects. In terms of safety, this approach appears clinically viable as we observed no substantial GVHD in either syngeneic or MHC-matched, minor mismatched allogeneic female to male lymphocyte transfers. The data presented here supports further evaluation of this approach to prostate cancer immunotherapy and its potential to synergize with other supportive immunotherapeutic approaches.