Characteristics of Tumor Infiltrating Lymphocyte and Circulating Lymphocyte Repertoires in Pancreatic Cancer by the Sequencing of T Cell Receptors

Abstract

Pancreatic cancer has a poor prognosis and few effective treatments. The failure of treatment is partially due to the high heterogeneity of cancer cells within the tumor. T cells target and kill cancer cells by the specific recognition of cancer-associated antigens. In this study, T cells from primary tumor and blood of sixteen patients with pancreatic cancer were characterized by deep sequencing. T cells from blood of another eight healthy volunteers were also studied as controls. By analyzing the complementary determining region 3 (CDR3) gene sequence, we found no significant differences in the T cell receptor (TCR) repertoires between patients and healthy controls. Types and length of CDR3 were similar among groups. However, two clusters of patients were identified according to the degree of CDR3 overlap within tumor sample group. In addition, clonotypes with low frequencies were found in significantly higher numbers in primary pancreatic tumors compared to blood samples from patients and healthy controls. This study is the first to characterize the TCR repertoires of pancreatic cancers in both primary tumors and matched blood samples. The results imply that specific types of pancreatic cancer share potentially important immunological characteristics.

Figure 1. Types and numbers of complementary determining region 3 (CDR3) in each group.

Types and numbers of CDR3 were similar among groups; however, levels of diversity seemed higher in tissue and blood samples from patients compared to blood samples from healthy controls.

Figure 2. The top-20 expressed gene of the V gene segments in each group.

No significant differences were detected between groups. The TRBV20-1 and TRBV2 variants were the most frequent in most samples.

Figure 3. The VJ pairs compared in tumor tissue and blood samples from the 16 patients.

Wilcoxon rank sum tests were performed to assess each VJ pair, and P < 0.001 was considered as significant. The TRBV9:TRBJ2-1 and TRBV20-1:TRBJ1-6 were found significantly higher.

Figure 4. Diversity of TCR repertoires was similar among groups.

Inverse Simpson’s diversity index was used to assess the diversity of TCR repertoires, and no significant differences were found among these groups.

Figure 5. Complementary determining region 3 (CDR3) length usage difference was not detected in groups.

The median lengths were 42 base pairs and normal distribution was found in all three groups.

Figure 6. Overlap of TCR repertoires in patients.

(a) The similarity of TCR repertoires from the 16 patients and 8 healthy controls was analyzed. Healthy individuals formed an independent cluster. Tumor tissue and blood samples from patients were mixed together but some clusters were formed. (b) Fussy heat map showed two clusters in tumor tissue and blood samples from the patients.

Figure 7. Differences in different clonotypes in the three groups.

T cell clonotypes were categorized by different frequencies. (a) Tumor tissue samples showed significantly lower levels of medium frequency (0.001%–0.01%) clonotype compared to blood samples from the patients and healthy controls. (b) Tumor tissue samples showed significantly lower levels of medium frequency (<0.001%) clonotype compared to blood samples from the patients and healthy controls. ****P < 0.0001, *****P < 10⁻⁵, **********P < 10⁻¹⁰.