Because CD137 expression by T-cells is activation-dependent, the capture of CD137pos T-cells from healthy donor blood can be utilized as a sensitive approach for rapid identification and isolation of rare circulating antigen-specific CD8+ T-cells, though this requires stimulation with defined viral or tumor antigen(23). encouraging clinical results reported(12C14). These results support the further pursuit and optimization of TIL-based immunotherapy for these and other cancers, 11-cis-Vaccenyl acetate although identification and expansion of natural tumor-reactive TILs for therapy still represents a challenge. Following antigen-induced stimulation, human T-cells undergo dynamic functional and phenotypic changes, including upregulated surface expression of multiple activation-associated molecules, including CD25, CD69, CD38 and others. This provides the opportunity to identify and isolate antigen-specific T-cells through antibody binding of the upregulated determinant and subsequent enrichment by 11-cis-Vaccenyl acetate magnetic separation or fluorescence-activated cell sorting (FACS). CD137 (4-1BB, TNFSFR9) is a TNFR-family member with costimulatory function that was originally identified as an inducible molecule expressed on activated mouse and human CD8+ and CD4+ T-cells(15C17). CD137 signaling regulates T-cell proliferation and survival, particularly within the T-cell memory pool(18C20), can upregulate Bcl-XL anti-apoptotic protein expression(21) and supports CD8+ T-cell expansion(19, 22). Because CD137 expression by T-cells is activation-dependent, the capture 11-cis-Vaccenyl acetate of CD137pos T-cells from healthy donor blood can be utilized as a sensitive approach for rapid identification and isolation of rare circulating antigen-specific CD8+ T-cells, though this requires stimulation with defined viral or tumor antigen(23). CD137pos cytomegalovirus, Epstein-Barr virus, influenza or human adenovirus-specific T-cells can be isolated from donor blood by a similar strategy(24). This strategy can facilitate identification of new immunogenic epitopes from pre-defined antigens(23), however its great potential has not been fully explored. To isolate and study naturally-occurring tumor-reactive T-cells, tumors represent a more promising reservoir than blood. An increased relative frequency of defined tumor antigen-specific T-cells reside in tumor(25), and although tumor cells present defined tumor antigens, whole-exome sequencing in various solid tumors has revealed that tumor cells harbor a vast, heterogeneous array of 11-cis-Vaccenyl acetate patient-specific mutated antigens that can be recognized by TILs with tumor-rejecting capability(26). Unlike in blood, naturally-occurring tumor-reactive TILs may express activation-associated molecules as a product of direct interaction with tumor cells(27), thus eliminating the need for stimulation with defined antigens to reveal this subset. Based upon these findings, we hypothesized that CD137, an activation-induced T-cell agonist, may play a role in the biology of tumor-reactive T-cells in solid human cancers. Here, we explored the immunobiology of CD137 in spontaneous immune responses against human cancer. Further, we investigated the application of a rapid, tumor-based CD137 isolation approach for selective enrichment of activated tumor-reactive TILs with potent anti-tumor function and using optimized conditions of short-term culture in the presence of homeostatic cytokines and cancer cells. Results Naturally-occurring CD137 expression by TILs and TALs of ovarian cancer To evaluate CD137 expression T-cells from human cancer, baseline CD137 surface expression was evaluated by flow cytometry on T-cells derived directly from either enzyme-digested solid tumor (TILs), ascites (TALs) or peripheral blood from patients 11-cis-Vaccenyl acetate with ovarian cancer. CD137 expression level was significantly higher on TILs than on CD3+ T-cells from blood (0.2%0.054, n=6, p<0.0057; Fig. 1A, B). TILs expressed CD137 at variable levels among samples (range of 0.9%C20%, mean= 7.8%5.7; n=12; Fig. 1B). The frequency of CD137pos TALs in ascites (1.82%1.04, n=13) was also significantly higher than in blood (p<0.0016), but lower than in solid tumor (p<0.0012). Thus, a hierarchy exists in CD137 expression, with highest frequencies detected in intratumoral T-cells, followed by T-cells in loose association with tumor and, lastly, by blood T-cells not directly interacting with tumor cells. CD137 was expressed by both CD4+ and CD8+ T-cell subsets from solid or IFNB1 ascites tumor, with greater frequencies detected in TILs (Fig. 1C). There was no difference in the frequency of CD137pos cells between the CD4+ or CD8+ T-cell subsets in either TIL or TAL (p>0.05; Fig. 1C). Thus, patients with ovarian cancer naturally harbor T-cells with an activated CD137pos phenotype, preferentially in tumor sites. Open in a separate window Figure 1 Naturally occurring CD137+ T cells exist in human ovarian cancerCD137 expression on TILs, TALs and PBMC from patient samples. All samples were stained with anti-CD3, CD8 and CD137 antibodies, gated on viable cells (7-AAD-) and examined by flow cytometry. (A) CD137 staining on the CD3+ T cell population compared to the.