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CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. The goal of this project was to characterize CD4+ T lymphocytes infiltrating primary human breast tumors (TIL), analyze T cell signaling pathways influenced by the tumor microenvironment, correlate differential gene expression with the presence of a specific CD4+ T cell subset(s) and identify a profile predictive of positive patient outcome. Affymetrix U133 Plus 2.0 arrays were used to analyze CD4+ T cells freshly isolated from the primary tumor, axillary lymph node (LN) and blood (PB) of patients with ER+ and ER- invasive breast carcinoma in the discovery set (10 patients). Microarrays, flow cytometry and/or qRT-PCR were used to extend and confirm the data in CD4+ TIL isolated from >60 primary breast tumors as well as purified CD4+ T cells from healthy donors PB treated with supernatant (SN) derived from fresh breast tumor tissue. Assessment of the relative distribution of infiltrating mononuclear cells revealed that the CD4+ T cell subset was consistently the principal component. We detected gene expression changes in the CD4+ TIL compared to patient's LN and PB counterparts that reflected altered cellular signaling pathways, including significant suppression of the TCR/CD3 complex and downstream signaling molecules along with a highly restricted pattern of Th cytokine and chemokine expression. A comparison with publically available Th profiling microarray data revealed that the TIL have characteristics of an activated memory effector CD4+ T cell phenotype, with all the major subsets (Th1, Th2, Th17, Treg, Tfh) represented. Although very few differences were detected in a direct comparison between CD4+ TIL from ER+ and ER- tumors, important changes in TIL (including a Th subset skew and differential cytokine expression) from extensive versus minimally infiltrated tumors were observed, with those containing an extensive CD4+ T cell infiltrate associated with a better clinical outcome. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight. These experiments revealed that the rested TIL reverse expression in a number of critical cytokine and signaling genes. PB CD4+ T cells from healthy donors treated with primary tumor SN also displayed significant changes in gene expression, gaining >75% similarity to freshly isolated CD4+ TIL. These experiments further found that activation of CD4+ T cells in conjunction with tumor SN treatment rapidly suppressed stimulation-induced changes in gene expression. Together our data suggest that although activated CD4+ T cells infiltrating breast tumors are quickly suppressed, tumors that recruit higher levels of CD4+ T cells, even in the face of rapid immunosuppression, produce a better memory T cell pool that is ultimately capable of maintaining effective anti-tumor immunity in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3541. doi:1538-7445.AM2012-3541