Distinctive CD26 Expression on CD4 T-Cell Subsets

Abstract

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome's sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (T_CM) cells while CD26high expression is present in effector Th1, Th2, Th17, and T_EM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.

Keywords: DPP4; T cell memory; T helper polarization; soluble CD26.

Figure 1

Cell-surface CD45R0 and CD26 in the CD4 T cells. (A) Representative (n = 11) flow cytometry dot-plot showing lymphocytes gated physically on FSC and CD4 (controls are shown in Supplemental Figure S1). (B) Dot-plots showing the differential expression of CD45R0 and CD26 in the lymphocyte region gated in A: CD4+ CD45R0low/ − CD26+ (naïve T cells; red square); and effector/memory CD4+ CD45R0+ CD26− (CD26neg; black square, mean ± SD 47.5 ± 12.0% of CD45R0+ ; range 33–72.2%), CD4+ CD45R0+ CD26+ (intermediate; grey square) and CD4+ CD45R0+ CD26++ (CD26high; dotted black square, 18.9 ±6.7% of CD45R0+ ; range 5–28.5%). (C) Matching of CD45R0+ cells (mean of % values ± SD, range 29.5–59.2%) and CD26+ (range 59.2–86.9%) CD4 lymphocytes in each healthy donor (n = 11). (D) Analysis of correlation between percentages of CD45R0+ and CD26+ in CD4 lymphocytes (Pearson’s correlation).

Figure 2

Phenotypes of central (CM) and effector/memory (EM) in CD4 T cell subsets defined with CD26 and CD45R0. Surface CD27, CD62L and CCR7 positivity frequencies in the four CD4⁺ T cell subsets defined by cell surface CD45R0 and CD26. Lymphocytes were gated using the same strategy shown in Figure 1, and for each marker of T_EM and T_CM subsets, its panel shows the respective frequencies in the respective gated region (the three CD45R0 with CD26high, + or neg, and the R0-, mean of % values ± SD respectively: CD27+, 76.8 ± 10, 83.1 ± 8.3, 85.3 ± 10.7, 94.5 ± 8.2, n = 11; CD62L +, 40.4 ± 17.1, 63.6 ± 10.2, 76.5 ± 8.4, 96.5 ± 2, n = 11; CCR7 + , 11 ± 9.4, 23.7 ± 10.5, 21.7 ± 13.3, 53.5 ± 13.3, n = 6). * p < 0.001, ** p < 0.01, *** p < 0.05.

Figure 3

Markers of pre-effector programs in CD4 T cell subsets defined with CD26 and CD45R0. Cell-surface CXCR5 and CCR4 positivity frequencies in the four CD4⁺ T cell subsets defined by surface CD45R0 and CD26 expression. Lymphocytes were gated using the same strategy shown in Figure 1, and for each marker of T_EM and T_CM subsets, its panel shows the respective frequencies in the respective gated region (the three CD45R0 with CD26high, + or neg, and the R0-, CXCR5: 5.6 ± 5.5, 19.9 ± 7.8, 23.4 ± 9.1, 0.6 ± .8, n = 7; CCR4: 31.4 ± 8.4, 51.5 ± 7.7, 65.2 ± 7.3, 6.6 ± 4.3, n = 11). * p < 0.001, ** p < 0.01, *** p < 0.05.

Figure 4

Markers of effector programs in CD4 T cell subsets defined with CD26 and CD45R0. Cell surface CXCR3 and CCR5 positivity frequencies in the four CD4+ T cell subsets defined by surface CD45R0 and CD26 expression. Lymphocytes were gated using the same strategy shown in Figure 1, and for each marker of T_EM and T_CM subsets, its panel shows the respective frequencies in the respective gated region (the three CD45R0 with CD26high, + or neg, and the R0-, CXCR3: 81.5 ± 6.9, 58.9 ± 4.6, 60.6 ± 10.6, 5 ± 2.4, n = 11; CCR5: 29.8 ± 10.9, 11.7 ± 7.6, 21.1 ± 15.8, 1.7 ± 1, n = 10). * p < 0.001, ** p < 0.01, *** p < 0.05.

Figure 5

Cell-surface CD26 in T helper 17 and 22 cell subsets defined by CD4, CCR6 (CD196), CD161 and CCR4 (CD194). (A) Representative flow cytometry (n = 4) dot-plots showing lymphocyte gating strategy for CD4, CCR6, and CD161 vs. CCR4 for Th17 cells (above) and CCR10 vs. CCR4 for Th22 cells (below). (B) In Th17 cells, the expression of CD26 in the CCR4+ cells is low and the CD26high cells (if any) are CCR4- (the square). (C) Most Th22 cells are CCR4+ and CD26- (the square) or CD26low.

Figure 6

Cell surface CD45R0 and CD26 staining in CD4 T cells activated in-vitro from naïve cells under different polarization conditions (controls are shown in Supplemental Figure S6): Th0 (no polarization), Th1, Th2 and Th17. Numbers in each quadrant correspond to the respective percentages. The dot plots are representative from one out of 4 different experiments. For comparison, MFI data from CD26 staining are shown in the bottom for each condition (as the range of intensities from one experiment to another was somewhat high, the means were not calculated).

Figure 7

Each bar shows the mean ± SD of ng mL⁻¹/3 × 10⁶ cells cultured for 3 days in the following conditions: Th0 (no polarization), Th1, Th2, and Th17 (n = 4 or more for each condition).