Tumor-infiltrating lymphocytes in colorectal tumors display a diversity of T cell receptor sequences that differ from the T cells in adjacent mucosal tissue

Abstract

Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor-infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison with the TCRs from adjacent healthy mucosal tissue. In parallel, we captured TIL counts using standard immunohistochemistry. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent mucosal tissue, and the number of shared clones is not dependent on distance between the samples. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.

Fig. 1

Comparison of TCR diversity, colorectal tumor versus mucosal tissue. The range of TCR diversity (as measured by the number of unique TCR clones identified) is presented for 15 paired mucosal and tumor tissue samples. Values are given in quartiles, from min to max. There is no significant difference between the number of unique TCR clones identified in the two tissue hypes; however, the variance in TCR diversity is significantly higher in tumor than in mucosal tissue (p = 0.04 by a Fligner–Killeen test)

Fig. 2

Comparison of TCR distribution entropy, colorectal tumor versus mucosal tissue. Above is a comparison of Shannon entropy, normalized based on the number of unique TCR clones detected, between 15 mucosal and 15 tumor samples. The mean normalized entropy value is reported for each tissue type, with error bars representing one standard deviation. A two-tailed Wilcoxon signed-rank test (a nonparametric equivalent of the paired t test) demonstrates a significant trend toward lower entropy values (i.e., a more clonal TCR frequency distribution) in tumor samples (p = 0.01)

Fig. 3

TCRβ sequence properties in tumor and mucosal tissue samples. a A comparison of TRBV gene segment usage in tumor and mucosal tissues (N = 15 samples of each tissue type). Each TRBV gene is graphed according to its frequency of usage in tumor and mucosal samples. TRBV gene usage is quite similar in the two tissue types. b A comparison of TRBJ gene segment usage in tumor and mucosal tissues. Each TRBV gene is graphed according to its frequency of usage in tumor and mucosal samples. TRBJ gene usage is very similar in both tissues. c A comparison of CDR3 region lengths between tumor and mucosal tissue samples. The proportion of TCR sequences with CDR3 regions of each length are presented in a histogram for each tissue type. CDR3 region length is similar in tumor and mucosal tissue samples

Fig. 4

Relationship between spatial distance and TCR repertoire overlap. Above, we present each of 13 samples for which paired tumor and mucosal tissue samples were available along with information about the distance between the tumor site and the healthy mucosal tissue sample. The line connecting each pair of samples has a length proportional to the spatial distance between the two samples, and a width proportional to the proportion of TCR sequencing reads belonging to TCR clones shared in both samples. Samples are sorted by (distance/TCR overlaps). There is no significant evidence of a relationship between the distance between tumor and nearby healthy tissue and the degree of TCR repertoire overlap (Pearson’s r = 0.16)