Background: Immunotherapy (IO) is a new treatment option for advanced microsatellite unstable colorectal cancer. Radiotherapy is an integral part of neoadjuvant treatment for locally advanced rectal adenocarcinoma and has been shown to increase tumor PD-L1 expression and tumor infiltrating lymphocytes (TIL). These changes in the tumor microenvironment enhance the response to IO. We report herein on expression of PD-L1 and CD-8 + TIL pre and post neoadjuvant chemoradiotherapy (CRT) in rectal adenocarcinoma. Expression of TIM-3, LAG-3, IDO1, OX40, IFN-ɤ, IL-17 and CXCL9 will also be reported. Methods: We retrospectively evaluated 39 patients with rectal adenocarcinoma from 2007-2016 treated with neoadjuvant CRT using 5FU based therapy. Pre and post CRT tissue samples were stained with VENTANA PD-L1 (SP263) rabbit monoclonal antibody to quantify PD-L1 expression. CD8 + TIL were recorded over one high power field (40x objective) in the area of densest infiltrate. Additional biomarkers are being assayed with RT-PCR. Relevant clinical endpoints of local relapse, distant metastasis, and survival were also collected. Results: Median age was 60 years (range 32-87). Median follow up was 16.7 months (range 2.6 -120.1). Median duration from completion of CRT to resection was 73 days (range 44 - 315). Among the pre-CRT cases 7.7% expressed PD-L1 versus 20.5% post-CRT (OR 3.1, p = 0.11). Median number of CD8+ TIL in the pre-CRT biopsies was 49. In post-CRT specimens, 82.1% cases had TIL > 50 (p = 0.002). PD-L1 expression did not correlate with pathological complete response, local or distant failure. Radiation dose/fractionation, time from CRT completion to resection, and CD8+ TIL did not correlate with increase in PD-L1 expression. Conclusions: Neoadjuvant CRT for rectal adenocarcinoma increased PD-L1 expression and CD8+ TIL which are known biomarkers for IO response. These data suggest a novel role for IO in neoadjuvant treatment of rectal cancer.