Biased usage of BV gene families of T-cell receptors of WT1 (Wilms' tumor gene)-specific CD8+ T cells in patients with myeloid malignancies

Abstract

WT1 (Wilms' tumor gene 1) protein is a potent pan-tumor-associated antigen (TAA) and WT1-specific cytotoxic T lymphocytes (WT1 tetramer(+) CD8(+) T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single-cell level comparative analysis of T-cell receptor beta-chain variable region (TCR-BV) gene families of a total of 1242 spontaneously induced WT1 tetramer(+) CD8(+) T cells in HLA-A*2402(+) patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR-BV gene families of individual TAA-specific CD8(+) T cells at single-cell level. Usage analysis using single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1 tetramer(+) CD8(+) T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR-BV gene families indicated that the usage frequencies of TCR-BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1-specific immune response.

Figure 1

Increase in WT1 (Wilms’ tumor gene 1) tetramer⁺ CD8⁺ T cells with more maturated phenotypes in patients. (a) Representative profiles of flow cytometric analysis using WT1 tetramer. The numbers at the upper‐right corners in dot plots of lineage antigens CD4, 14, 16, 19, 33, 34 and 56‐negative and CD3‐, CD8‐positive gate represent the ratio of WT1 tetramer⁺ CD8⁺ T cells to total CD8⁺ T cells. (b) Ratios of WT1 tetramer⁺ CD8⁺ T cells to total CD8⁺ T cells in peripheral blood mononuclear cells (PBMC)s of healthy donors (HDs) (n = 5) and acute myeloid leukemia (AML) (n = 4), and myelodysplastic syndrome (MDS) (n = 4) patients. Bars represent medians; n.s., not significant. (c) Frequencies of four differentiation subsets of WT1 tetramer⁺ CD8⁺ T cells. White, hatched and black columns represent HDs, AML, and MDS patients, respectively. Statistical significance between HDs and AML, and HDs and MDS is indicated by asterisks (*P < 0.05).

Figure 2

Usage frequencies of T‐cell receptor β‐chain variable region (TCR‐BV) gene families in WT1 (Wilms’ tumor gene 1) tetramer⁺ CD8⁺ T cells. Usage frequencies (%) of each member of the BV gene family in WT1 tetramer⁺ CD8⁺ T cells of five healthy donors (HDs) and four acute myeloid leukemia (AML) and four myelodysplastic syndrome (MDS) patients. Black and white columns represent the TCR‐BV gene families with usage frequencies above and below mean values + 1SD, respectively.

Figure 3

Usage frequencies of T‐cell receptor β‐chain variable region (TCR‐BV) gene families with the biased usage. Ratios of healthy donors (HDs) and patients with biased usage of the indicated TCR‐BV gene families to the total number of HDs and patients examined, respectively.

Figure 4

Statistical comparison between healthy donors (HDs) and patients of usage frequencies of individual T‐cell receptor β‐chain variable region (TCR‐BV) gene families in WT1 (Wilms’ tumor gene 1) tetramer⁺ CD8⁺T cells. Statistical significance was assessed by one‐factor ANOVA or Kruskal–Wallis test. n.s., not significant.