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6 Background: Limited data in cancer patients have suggested that chemotherapy and radiation impact local and systemic immune cell populations. Radiation therapy (RT) is known to deplete circulating lymphocytes but is thought to increase local antigen presentation. The dynamics of these competing effects on the kinetics of intratumoral infiltration and expansion of activated and immunoregulatory T cells are unknown. Methods: We prospectively evaluated intratumoral immune infiltration during fractionated RT using multi-spectral flow cytometry. Cervical brushings were obtained from 14 patients before (baseline) and during RT (week 1, 3 and 5). Cells collected from the cervical brushings were stained with a 16-color panel of antibodies that included markers to identify T cell and dendritic cell subsets with activation and suppressor molecules. Changes in immune cell subsets at different time points were evaluated and calculated using matched-pair analysis with Wilcoxon rank sum test. Results: CD3+ T cells declined over the first week of treatment (28% of CD3 at baseline, vs. 14.8% at week 1, p = 0.0273). The percentage of CD3+ cells subsequently increased at 3 weeks (25.6%) and 5 weeks (37.8%). Both CD8+ and CD4+ T cells underwent a decline at week 1 followed by expansion at week 3 and 5. Percentages of regulatory T cells (CD4+Foxp3+) showed a similar trend of reduction and further expansion but did not reach significance. The percentage of CD8+ T cells expressing the T cell activation marker CD69 and the cytotoxic protease Granzyme B (GrzB) continuously increased over time (CD69+: 11.8%, 27.7%, 38.7%, 57.5%, and GrzB+: 23.9%, 53.2%, 48.1%, 58.2%). While the percentage of dendritic cells (CD11c+ CD11b+) was stable during treatment, the subset of activated dendritic cells expressing CD86 increased at week 1 and subsequently declined (week 1, 19.1% vs week 5, 9.8%, p = 0.0642). Conclusions: Activated CD8+ effector T cells expand in the cervix during radiation therapy. Moreover, in the first week of treatment, CD8+ T cells contract while dendritic cells undergo activation suggesting this may be a critical time to intervene to maximize anti-tumor immunity.