Generation of mammaglobin-A-specific CD4 T cells and identification of candidate CD4 epitopes for breast cancer vaccine strategies

Background: Mammaglobin-A (MGB) is a breast cancer-associated antigen that is an attractive target for immune intervention. MGB has been shown to induce a specific CD8 T cell response in breast cancer patients, but little is known about a possible MGB-specific CD4 T cell response. Methods: Peripheral blood-derived CD4⁺CD25^- T cells were stimulated in vitro with MGB-pulsed antigen-presenting cells (APC). The MGB and human leukocyte antigen (HLA) class II specificity of the CD4 T cell lines was confirmed by cytokine release following restimulation with autologous and allogenic APC pulsed with MGB from different sources. Candidate HLA class II-restricted epitopes were identified by computer algorithm and validated in cytokine release assays. Results: MGB-specific CD4 T cells were successfully generated in cultures from six of seven donors. Restimulation of MGB-specific CD4 T cells with MGB-pulsed APC induced significantly higher levels of interferon (IFN)-γ release than APC pulsed with an irrelevant protein (P = 0.0004). Cultures from five of seven donors showed a pure Th1 type response as evidenced by the absence of interleukin (IL)-4. MGB-specific CD4 T cells recognized both recombinant and naturally processed MGB presented by APC. This recognition was HLA class II-restricted, as HLA-DR mismatched APC were not recognized. MGB-specific CD4 T cells from three of four donors recognized MGB-derived, HLA class II-restricted peptides pulsed onto APC. Conclusions: We have successfully generated MGB-specific CD4 T cell cultures and identified candidate MGB HLA class II epitopes. These studies should facilitate study of the CD4 T cell response to MGB, and the development and monitoring of vaccine strategies targeting this unique antigen.