Comparing CD8⁺ T cell repertoires in non-tolerant FVB/N and tolerant HER-2/neu transgenic mice

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The success of cancer immunotherapy will depend on the development of vaccine approaches that can overcome multiple mechanisms of T cell tolerance to tumor-associated antigens (TAAs). Such antigens are often present at low levels in normal tissue. One example is the transmembrane tyrosine kinase Her/2-neu (neu), which is overexpressed in 30-40% of breast cancers. Patients that develop neu-expressing breast cancers can mount T cell responses, but these responses are usually incapable of controlling their cancer. We are using the Her-2/neu (neu-N) transgenic mouse model to dissect the mechanisms of CD8⁺ T cell tolerance using neu as a model antigen. These FVB/N derived mice express rat neu cDNA under the mammary-specific MMTV promoter, and develop spontaneous neu-expressing mammary tumors that are difficult to eradicate with neu-targeted vaccines. We have previously shown that these mice exhibit evidence of immune tolerance to neu-expressing tumors after vaccination that is similar to what is observed in patients. Vaccination of the parental FVB/N mice uncovers T cells specific for the immunodominant CD8⁺ T cell epitope, RNEU_420-429. In contrast, vaccination of neu-N mice reveals CD8⁺ T cells directed against the whole neu protein but not against RNEU_420-429. CD4⁺CD25⁺ T regulatory cell (T reg) depletion prior to vaccination can uncover RNEU_420-429-specific T cells in about 20% of neu-N mice that are capable of tumor eradication. Assessment of Vβ regions demonstrated that the neu-directed FVB/N T cell repertoire is relatively small, with a predominance of Vβ4 T cells. In contrast, the neu-directed neu-N T cell repertoire is polyclonal, with an absence of Vβ4 usage except after T reg depletion. In order to better understand these differences in CD8⁺ T cell responses between vaccinated FVB/N and neu-N mice, we performed a full protein screen using a 15-mer peptide library. To accomplish this, 15-mer peptides overlapping by 10 amino acids were synthesized and assessed for recognition by vaccine induced lymphocytes from either the FVB/N or neu-N mice. Using this approach, 6 additional epitopes recognized by FVB/N derived lymphocytes and 35 additional epitopes recognized by neu-N derived lymphocytes were identified. Experiments are underway to understand how these T cell repertoires change in vaccinated tumor bearing mice, with and without the depletion of T regs. These studies will provide new information on the diversity of the T cell repertoire under conditions of immune tolerance and should ultimately lead to a better understanding of the conditions required to effectively activate the complete T cell repertoire for improved cancer immunotherapy.