Background: Immunotherapy (IO) in combination with chemoradiotherapy (CRT) in the neoadjuvant treatment for locally advanced rectal adenocarcinoma (RAC) is an area of active clinical research. There are no well-defined biomarkers in RAC predicting response to neoadjuvant therapy. Increase in tumor PD-L1 expression and tumor infiltrating lymphocytes (TIL) has been observed after neoadjuvant CRT which potentially enhances response to IO. We report herein expression of PD-L1 and CD8+ TIL and other biomarkers of immune activation including CXCL9, TIM3 (HAVCR2), IDO1, IFNG, IL17RE, LAG3 and OX40 (TNFRSF4) pre and post neoadjuvant CRT in RAC. Methods: We retrospectively evaluated 38 RAC patients 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 (hpf) (40x objective) in the area of densest infiltrate. Additional biomarkers CXCL9, TIM3 (HAVCR2), IDO1, IFNG, IL17RE, LAG3 and OX40 (TNFRSF4) were assayed with RT-PCR. Independent sample and paired sample t-tests were used for statistical analysis of difference in biomarker expression. Relevant clinical endpoints including local relapse, distant metastases and survival were 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). PD-L1 expression increased from 10% to 23% in pre-CRT versus post-CRT patients. CD8+ TIL increased in 30/39 (83%) patients with an average increase from 66.48 to 129.20 CD8+ TIL/hpf in the pre-CRT versus post-CRT setting. Using RT-PCR, a statistically significant increase in the expression of CXCL9, IFNG and OX40 (TNFRSF4) was found pre-CRT versus post-CRT using paired samples t-test. Conclusions: Neoadjuvant CRT for RAC increased PD-L1 expression and CD8+ TIL along with an increased expression of CXCL9, IFNG and OX40 (TNFRSF4). These data suggest a novel role for IO in neoadjuvant treatment of rectal cancer. Clinical outcome data on these patients will be presented.