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. 2021 May 27;43(1):226-239.
doi: 10.3390/cimb43010019.

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Affiliations

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Hye Won Lee et al. Curr Issues Mol Biol. .

Abstract

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, "exhausted" T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.

Keywords: adoptive immunotherapy; anti-tumor immunity; gamma-delta T cells; renal cell carcinoma; tumor microenvironment; tumor-infiltrating lymphocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of γδ T cells within renal cell carcinoma (RCC) and patients’ blood. Experimental schemes: Tumor-infiltrating and blood lymphocytes were analyzed as indicated (TN, T naïve; TCM, central memory T cell; TEM, effector memory T cell). The cytotoxic capacity of each of the sorted γδ T cell populations was assessed for their cytotoxic capacity against Caki-1 and ACHN RCC cell lines.
Figure 2
Figure 2
Analysis of peripheral blood (PB) and tumor-infiltrating lymphocytes (TILs). (A) PB and TILs were analyzed for γδ TCR and CD3. (B) CD3high γδ T cells from peripheral blood lymphocytes (PBLs) and TILs and CD3low γδ T cells from TILs were analyzed for CD4 and CD8, as well as for FAS and CD28. ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Figure 3
Figure 3
Identification of intra-tumoral CD3low γδ T cells. (A) Peripheral blood lymphocytes (PBLs) and TIL γδ T cells were assessed for the expression of Vδ1 and Vδ2 chains, while gated Vδ1 and Vδ2 γδ T cells were analyzed for the expression of Vγ9 chain. The level of the CD3 expression is shown for Vγ9-δ1+, Vγ9+-δ1+, and Vγ9+-δ2+ T cells. (B) Gated Vγ9+-δ1+ and Vγ9+-δ2+ T cells in PBLs or TILs were analyzed for FAS and CD28 to assess their differentiation statuses.
Figure 4
Figure 4
Immune modulation and cytotoxic activity. (A) The proportions of dead cells were shown for the cytotoxicity assays using two RCC cell lines and sorted γδ T cell populations as mean ± SD. (B) Vγ9+-δ1+ TILs and Vγ9+-δ2+ peripheral blood lymphocytes (PBLs) were compared for the expression of co-stimulatory molecules (ICOS and CD27), immune checkpoint receptors (PD-1, TIGIT, LAG3, and TIM-3) and CD45RA. Shaded histograms show the staining of the negative controls. Values from all patients are shown as dot graphs with mean ± SD and statistical significances. (C) Cytotoxic activity of intra-tumoral Vγ9δ1 T cells.
Figure 5
Figure 5
Cytokine secretion by Vγ9+-δ1+ or Vγ9+-δ2+ cells. Lymphocytes from peripheral blood (PBL) or tumor (TIL) stimulated with or without phorbol myristate acetate (PMA) and ionomycin for 4 hours were stained with Abs against indicated cytokines; (A) IL-17 and IFN-γ, (B) IL-2 and IL-10, and (C) IL-4. The cytokine expression was shown for the gated Vγ9+-δ1+ or Vγ9+-δ2+ cells.
Figure 6
Figure 6
Analysis of immune gene signatures and TCR repertoire for 524 Kidney Renal Clear Cell Carcinoma patients from The Cancer Genome Atlas database. (A) Scatter plots of Gene Set Enrichment Analysis enrichment scores between γδ T cell and other immune gene signatures. (B) Percentage of samples in which Vγ9Vδ1 clone was detected using RNA-seq data. (C) The relative proportion of clones with the combination of Vγ and Jγ segments (left) and combination of Vδ and Jδ segments combination (right) in tumor samples.

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