Introduction: The purpose of this study is to correlate T cell subset abundance with the accumulation and proteolysis of versican (VCAN) in colorectal cancer (CRC). Background: In CRC, VCAN accumulation is associated with the exclusion of CD8+ tumor infiltrating lymphocytes (TILs) and its proteolysis, into versikine (Vkine), correlates with increased TIL abundance. Here we additionally investigate for changes in CD4+ T cells, T-regulatory cells (Tregs), and T-helper-1 cells (TH1s) in association with the presence of VCAN and Vkine. Methods: VCAN, Vkine, TIL, and T cell abundance were assessed via immunohistochemistry or immunofluorescence in 455 human CRC samples. VCAN and Vkine were scored using an intensity binning system ranging from 0-3+ and T cells were counted per high power field (HPF). Tumors were designated as high (2 or 3+) or low (0 or 1) for both VCAN and Vkine. Cores designated VCAN low and Vkine high are considered VCAN proteolytic predominant (VPP) and all other combinations are considered VCAN proteolytic weak (VPW). CD4 subsets were visualized by colocalizing CD4 with FOXP3 for Tregs and Tbet for TH1s using dual immunofluorescence. Findings: High VCAN accumulation correlated with low TIL abundance. VCAN high cancers had 6 CD8+ TILs/HPF while VCAN low cases had 20 CD8+ TILs/HPF (p<0.001). VCAN proteolysis correlated with increased CD8+ TIL abundance. VPP samples had 46 CD8+ TILs/HPF and VPW ones had 6 CD8+ TILs/HPF (p<0.001). When evaluating total CD4+ TIL abundance, no association was found with either VCAN accumulation or proteolysis. When evaluating CD4 subset abundance, VCAN accumulation had no predictive value for TH1 numbers, but VCAN proteolysis correlated with increased TH1 abundance. VPP cancers had 25 TH1s/HPF and VPW cancers had 9 TH1s/HPF (p=0.02). Neither VCAN accumulation nor proteolysis was predictive of Treg abundance. Accordingly, VCAN high cores had a lower TIL: Treg ratio than VCAN low cores, with VCAN high samples having a CD8+ TIL: Treg ratio of 0.4:1 and VCAN low samples having a ratio of 1:1 (p<0.001). VPP cases showed an elevated TIL: Treg ratio compared to VPW CRCs, with VPP cancers having a CD8+ TIL: Treg ratio of 2:1 and VPW having a ratio of 0.4:1 (p=0.001). Conclusions: Here we demonstrate that VCAN accumulation within the CRC TME is associated with fewer CD8+ TILs and a lower TIL: Treg ratio. Conversely, high levels of VCAN proteolysis correlated with increased CD8+ TIL and TH1 abundance, and a greater TIL: Treg ratio. This indicates that VCAN accumulation and proteolysis differentially predict the abundance of different T cell subtypes and polarization states which could be important for immunotherapy responses. Citation Format: Sean G Kraus, Anna Field, Derek Buckalew, Cheri Pasch, Dustin Deming. Versican accumulation and proteolysis differentially predict T cell subset abundance within the colorectal cancer tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B34.