Characterization of T cell repertoire of blood, tumor, and ascites in ovarian cancer patients using next generation sequencing

Abstract

Tumor-infiltrating lymphocytes (TILs) play an important role in regulating the host immune response and are one of key factors in defining tumor microenvironment. Some studies have indicated that T cell infiltration in malignant ascites is associated with clinical outcome, but few studies have performed detailed characterization of T cell diversity or clonality in malignant effusions. We have applied a next generation sequencing method to characterize T cell repertoire of a set of primary cancers, ascites, and blood from 12 ovarian cancer patients and also analyzed the T cell subtype populations in malignant fluids from 3 ovarian cancer patients. We observed enrichment of certain T cells in tumors and ascites, but most of the enriched T cell receptor (TCR) sequences in tumors and ascites were not common. Moreover, we analyzed TCR sequences of T cell subtypes (CD4⁺, CD8⁺, and regulatory T cells) isolated from malignant effusions and also found clonal expansion of certain T cell populations, but the TCR sequences were almost mutually exclusive among the three subgroups. Although functional studies of clonally expanded T cell populations are definitely required, our approach offers a detailed characterization of T cell immune microenvironment in tumors and ascites that might differently affect antitumor immune response.

Keywords: T cell receptor; malignant ascites; next generation sequencing; ovarian cancer; tumor immunity.

Figure 1.

The clonality of T lymphocytes in tumor, ascites, and blood of 12 ovarian cancer patients. (A) The distribution of the unique CDR3 sequences detected in TCR-α (TRA) and TCR-β (TRB). Each pie graph was colored automatically by the Excel program according to frequency ranks and therefore the same color dose not represent an identical CDR3 sequence. Gray color indicates portion of clonotypes less than 1% frequency. (B) The diversity indexs for TCR-α and TCR-β were calculated by the inverse Simpson's index (1/Ds) formulation.

Figure 2.

Comparison of T cell repertoires in tumor tissues and ascites. (A) Heatmaps show frequencies of 20 dominant TCR clonotypes selected from tumor tisseus (T) and ascites (A). The vertical axis indicates each of unique CDR3 sequences. Common CDR3s are presented only once. The scale bar represents their frequency. CDR3 sequences with more than 10% of frequency were presented with the squares of red color and indicated by * (10% ˜ 30%), # (30% ˜ 50%), and ++ (more than 50%). (B) The proportion of common CDR3 between tumor tissues and ascites is graphed.

Figure 3.

Expression of CD8 and FOXP3 in tumor tisseus and ascites. Expression raitos of CD8/CD4 and FOXP3/CD4 in 12 ovarican cancer patients are presented. Quantity of each transcript was analyzed by real-time PCR and normalized by an internal control, GAPDH.

Figure 4.

Isolation of T cell subpopulations from malignant effusions. (A) The lymphocytes were gated from live cells collected from malignant effusion in P201 by flow cytometry. Three different T cell subtypes were sorted using a combination of fluorescent-conjugated monoclonal antibodies for CD3⁺, CD4⁺, CD8⁺, and CD25⁺, as indicated on X- and Y-axes. (B) The proportion of T cell subtypes in malignant effusions from three ovarian cancer patients, P201, P202, and P203.

Figure 5.

The TCR clonality in subpopulations of T cells. The pie charts illustrate distribution of unique CDR3 sequences detected in TRA and TRB. The same color between pie charts does not represent a same CDR3 sequence. Gray color indicates portion of clonotypes less than 1% frequency.

Figure 6.

Characteristics of TCR repertoires in different T cell subtypes. Heatmaps show frequencies of 20 dominant TCR clonotypes selected from each subtype of T cells in malignant effusion or T cells in blood. The vertical axis indicates each of unique CDR3 sequences. Common CDR3s are presented only once. The scale bar represents frequency of each unique CDR3 sequence. The squares of red color represent CDR3 clones with more than 5% of frequency, and indicated by * (5% ˜ 15%), # (15% ˜ 25%), and ++ (more than 25%).