Murine Regulatory CD4+ T Cells Are Increased in Leukemic Spleens and Show High Co-Expression of Co-Regulatory Molecules CD39, CD73, PD1, and TIGIT

Abstract

Comprehensive characterization of AML-associated T cells during disease progression is essential to identify relevant immune escape mechanisms and new immunotherapeutic approaches. Investigating the processes that lead to an immunosuppressive environment under progression of AML is difficult in humans, because by the time of diagnosis the disease is often progressed far beyond the initial stages. Therefore, to investigate T-cell phenotypes during progression a C57BL/6 mouse model was used. The CD3⁺ T cells were characterized by performing multiparametric flow analyses at different time points (day 0 = healthy mice, day 7, day 14, and day 21). The study revealed that the spleen is highly infiltrated by reg CD4⁺ T cells at day 21 of AML progression. These spleen-infiltrating reg CD4⁺ T cells mainly showed an effector memory differentiation with high expression and co-expression of the checkpoint molecules TIGIT, PD-1, OX40, and the two ectoenzymes CD39 and CD73. Substantial expression of the checkpoint molecules was restricted to the central memory and effector memory compartments. Furthermore, functional evaluation of TIGIT was performed. Blocking TIGIT resulted in a significantly increased lysis of C1498 AML cells in cocultures with AML-primed CD3⁺ T cells. Together these data confirm that the expression of the checkpoint receptor TIGIT is relevant for dysfunction of AML-associated T cells and, thus, represents a suitable target for future immunotherapeutic approaches.

Keywords: CD39; CD73; PD-1; T cells; TIGIT; antitumor immunity; checkpoint molecules; murine AML; regulatory CD4+ T cells; spleen.

Figure 1

Multiparametric flow cytometry (MFC) of the spleen-derived CD3⁺ T cells was performed on splenocytes from healthy mice (d0, n = 4) and leukemic mice at different time points after transplantation (d7, n = 4; d14, n = 4; and d21, n = 6): (A) exemplary gating strategy on FACS plots; (B) frequencies of CD4⁺, CD8⁺, and reg CD4⁺ T cells at different time points in relation to their parent population (CD3⁺ cells for CD4⁺ and CD8⁺ T cells; CD4⁺ T cells for reg CD4⁺ T cells); (C) exemplary illustration of the fluorescence intensity for the different immune checkpoint (IC) molecules against fluorescence minus one control (FMO). The vertical line indicates fluorescence level of FMO; (D) summary data of IC expression on CD4⁺ T cells, CD8⁺ T cells, and reg CD4⁺ T cells. Frequencies are displayed with the median. p-values were obtained by the Kruskal–Wallis test or the ordinary one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 2

(A) Exemplary gating strategy for the T-cell subgroups NA = CD62L⁺CD44⁻; Tem = CD62L⁻CD44⁺; Tcm = CD62L⁺CD44⁺; and DN (double-negative) = CD62L⁻CD44⁻ subpopulations; (B) exemplary distribution patterns of the immune checkpoint expression on the different T-cell subpopulations; (C) summary data of immune checkpoint expression on T-cell subgroups. Frequencies are displayed with the median. p-values were obtained by the Kruskal–Wallis test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 3

(A) Exemplary gating strategy for the co-expression data; (B) summary data of TIGIT/PD-1 and CD39/CD73 co-expression; (C) SPICE analysis of five co-regulatory receptors at different time points and on different T-cell subsets, and the overlapping arcs illustrate co-expression for these molecules; (D) gating strategy and summary data of the quadruple expression of TIGIT/PD-1/CD39/CD73. Frequencies are displayed with the median. p-values were obtained by the Kruskal–Wallis test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns = not significant.

Figure 4

(A) Illustration of ex vivo cytotoxicity assay methodology in the healthy donor (HD) cohort; (B) quantity of dead tumor cells compared between the isotype and aTIGIT groups in the HD cohort; (C) illustration of ex vivo cytotoxicity assay methodology in the leukemia cohort; (D) quantity of dead tumor cells compared between the isotype and aTIGIT groups in the leukemia cohort. Frequencies are displayed with the median. p-values were obtained by the unpaired t test. *** p < 0.001 and ns = not significant. Illustrations were created with BioRender.com.