Surface NKG2C Identifies Differentiated αβT-Cell Clones Expanded in Peripheral Blood

Abstract

T cells that express CD56 in peripheral blood of healthy humans represent a heterogeneous and poorly studied subset. In this work, we analyzed this subset for NKG2C expression. In both CD56⁺ and CD56^- subsets most of the NKG2C⁺ T cells had a phenotype of highly differentiated CD8⁺ TEMRA cells. The CD56⁺NKG2C⁺ T cells also expressed a number of NK cell receptors, such as NKG2D, CD16, KIR2DL2/DL3, and maturation marker CD57 more often than the CD56^-NKG2C⁺CD3⁺ cells. TCR β-chain repertoire of the CD3⁺CD56⁺NKG2C⁺ cell fraction was limited by the prevalence of one or several clonotypes which can be found within the most abundant clonotypes in total or CD8⁺ T cell fraction TCRβ repertoire. Thus, NKG2C expression in highly differentiated CD56⁺ T cells was associated with the most expanded αβ T cell clones. NKG2C⁺ T cells produced almost no IFN-γ in response to stimulation with HCMV pp65-derived peptides. This may be partially due to the high content of CD45RA⁺CD57⁺ cells in the fraction. CD3⁺NKG2C⁺ cells showed signs of activation, and the frequency of this T-cell subset in HCMV-positive individuals was positively correlated with the frequency of NKG2C⁺ NK cells that may imply a coordinated in a certain extent development of the NKG2C⁺ T and NK cell subsets under HCMV infection.

Keywords: NKG2C; NKT-like cells; T cell differentiation; TCR repertoire; cytomegalovirus infection.

Figure 1

Gating strategy for the assessment of the distribution of NKG2C⁺ cells in CD56⁻ and CD56⁺ (NKT-like) T cell subsets and in NK cells. Experimental sample and fluorescence minus one (FMO) control lacking NKG2C-antibody are presented.

Figure 2

TCR-beta repertoire structure. (A) Repertoire structure of the CD3⁺CD56⁺NKG2C⁺ fraction. Ranking of clonotypes was performed in the cell fraction isolated by fluorescence-activated cell sorting. Top 10 clonotypes identified in samples for donors are shown proportionally of their percentage in the fraction. (B) The top clonotypes of the CD3⁺CD56⁺NKG2C⁺ fraction matched in total TCR-beta repertoire sequenced from PBMC sample.

Figure 3

Phenotypic analysis of NKG2C⁺ cells. (A) Gating strategy for the assessment of the distribution of various surface markers in NKG2C⁺ cells in CD56⁻ and CD56⁺ αβ T cell subsets. As an example, NKG2D expression level in the T cell fractions is presented; antibody panel: CD3-PE-Cy7, CD56-APC, γδTCR-Brilliant Violet 421, NKG2C-AF488, NKG2D-PE. (B) Analysis of CD8 expression in CD56⁺NKG2C⁺ αβ T cells. Representative staining is presented. (C) Analysis of CD8 expression in CD56⁺NKG2C⁺ T cells of the donor 2.

Figure 4

Phenotypic analysis of T cell subsets. (A) NKG2C+ cell frequencies analyzed in CD3+CD56+/−CD8+/− cell subsets (n = 7). (B) Expression of CD45-RA, CD45-RO, CCR7 (CD197), CD27, CD28, CD57, CD16, NKG2D, KIR2DL2/DL3, NKG2A, CD161, CD69, HLA-DR and γδTCR on NKG2C⁻ and NKG2C⁺ CD56⁻/⁺ T cells (n = 8, 9, 9, 9, 5, 7, 8, 7, 8, 7, 5, 8, 8 and 8, respectively. (B) NKG2C+ in CD8^+/−CD56^+/− T cell subsets (n = 7). Means ± SEM are presented (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Mann-Whitney U test).

Figure 5

Production of IFN-γ in NKG2C⁻ and NKG2C⁺CD8⁺ T cells. (A) Representative staining of cells from one of the donors is presented. CMV-specific IFN-γ response was induced using pp65 peptide set (PepTivator, Miltenyi Biotec). Staphylococcal enterotoxin B was used as a positive control. (B) Summary of data on IFN-γ production accessed in CD56⁻NKG2C⁻, CD56⁻NKG2C⁺, CD56⁺NKG2C⁻, CD56⁺NKG2C⁺ cells from the CD3⁺CD8⁺ T cell subset. Means ± SEM of three independent experiments are presented. *p < 0.05.

Figure 6

IFN-γ production induced by SEB (positive control) or pp65 peptide set in CD8⁺ T cells. (A) IFN-γ production levels in CD8⁺ and more differentiated CD8⁺CD57⁺CD45RA⁺ T cell subsets. (B) IFN-γ production levels in CD8⁺NKG2C⁺ T cell subsets and in more differentiated CD8⁺CD57⁺CD45RA⁺NKG2C⁺ T cells. Percentages of CD57⁺CD45RA⁺ cells in CD8⁺ and CD8⁺NKG2C⁺ cell fractions are indicated. Representative staining is presented.

Figure 7

Cytotoxic activity of NKG2C⁻/⁺ CD56⁻/⁺ T cells estimated by surface expression of LAMP-1 in degranulation assay. (A) Natural cytotoxicity of T cells measured against K562 target cells. T cell subsets isolated from PBMC by cell sorting were stimulated overnight with IL-2 (500 U/ml). Percentage of LAMP-1–positive T cells after incubation with target cells was measured. Means ± SEM of 6 independent experiments and histograms of one of the experiments are presented. (B) Antibody-dependent cell-mediated cytotoxicity of T cells measured against Rituximab-coated C1R target cells. Means ± SEM for 5 independent experiments and histograms of one of the experiments are presented. *p < 0.05.