Increased Frequency of TOX⁺ CD39⁺ TIGIT⁺ CD73^- CD8⁺ T Cells in Patients with Newly Diagnosed AML

Immune checkpoint therapy has revolutionized the treatment of patients with cancer. Checkpoint receptors and their ligands play an important role in T cell activation and exhaustion and are currently the focus in understanding the antitumoral immune responses. In patients with acute myeloid leukemia (AML) limited data have been published that comprehensively describe the expression of checkpoint receptors on different T cell subsets.

We performed multicolor flow cytometry on peripheral blood mononuclear cells (PB, PBMCs) from patients with newly diagnosed AML (n=20) and PBMCs from age matched healthy donors (HDs; n=12), focusing on differentiation, the clinically actionable exhaustion receptor T cell immunoglobulin and ITIM domain (TIGIT), the two ectoenzymes ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73). Our studies included also analysis of interleukin-7 receptor-α (CD127) and the intracellular expression of the transcription factor T cell factor 1 (TCF-1). Both markers are known to be expressed in long living memory CD8⁺ T cells and harboring the ability for self-renewal. The thymocyte selection-associated high mobility group box protein (TOX) was also analyzed, since this molecule has recently been described as regulator of CD8⁺ T cell exhaustion.

Comparison of PB from patients with newly diagnosed AML vs. HDs revealed that the frequency of CD8⁺, CD4con (CD4⁺CD127⁺CD25^-) and CD4reg (CD4⁺CD127^-CD25⁺) T cells was similar among both groups. However, the frequency of CD8⁺ EMRA T cells (CCR7^- CD45RO^- CD8⁺ CD3⁺) was increased in PB from patients with AML compared to PB from HDs (39,05 ± 4,38 vs. 14,29 ± 3,83; p<0,05).

TIGIT, CD39 and CD73 emerged as checkpoints of interest on CD8⁺ T cells. The frequency of TIGIT⁺ CD8⁺ T cells and CD39⁺ CD8⁺ T cells in PBs from patients with newly diagnosed AML was increased compared with that in HDs (42,60 ± 4,67 vs. 20,36 ± 3,68; p=0,00 and 6,54 ± 2,05 vs. 1,31 ± 0,32; p=0,05). Whereas reduced frequency of CD73⁺ CD8⁺ T cells occurred in PB from patients with AML vs. HD (41,35 ± 4,75 vs. 66,18 ± 7,28; p=0,01). Analysis between TIGIT and CD73 expression showed inverse correlation between both targets in AML (r=-0,53; p=0,01). The frequency of the TIGIT⁺ CD73^- CD8⁺ T cell population was increased in AML (36,85 ± 5,17, vs. 16,23 ± 5,09 ;p=0,01). This increased frequency of TIGIT⁺ CD73^- cells in AML was related to EM (CCR7^- CD45RO⁺ CD8⁺ CD3⁺)T cells (42,89 ± 4,78 vs. 25,27 ± 3,39; p=0,01) and EMRA CD8⁺ T cells (56,01 ± 5,68 vs. 36,54 ± 4,85; p=0,03). Moreover, CD39 was aberrantly expressed on this population: we observed an increased frequency of CD39⁺ TIGIT⁺ CD73^- CD8⁺ T cells in PB from patients with newly diagnosed AML compared to PB from HDs (13,47 ± 4,03 vs. 3,03 ± 1,21; p=0,02).

Next we focused on CD127 and TCF-1 which are involved in creating long living antigen independent memory CD8⁺ T cells and ability to self-renewal while producing differentiated effector cells. Comparing expression of CD127 and TCF-1 on CD39⁺ TIGIT⁺ CD73^- CD8⁺ T cells showed a significantly decreased frequency of CD127 (17,73 ± 2,16, vs. 30,72 ± 7,12; p=0,04) and TCF-1 (14,67 ± 2,90 vs. 39,74 ± 9,32; p=0,03) in PB from patients with newly diagnosed AML compared to HDs. Expression of TIGIT and TCF-1 inversely correlated in AML (r =-0,87; p<0,05).

To further evaluate the exhaustion status of TIGIT⁺ CD73^- CD8⁺ T cells we examined the expression of TOX, recently described as one of the key regulators governing CD8⁺ T cell exhaustion, the frequency of TOX⁺ cells was increased in AML (50,57 ± 8,21 vs. 22,14 ± 4,86; p=0,01). Analysis of co-expression showed that the TOX⁺ CD39⁺ TIGIT⁺ CD73^- CD8⁺ population was significantly increased in PB from patients with newly diagnosed AML compared to their counterparts in PB from HDs (21,81 ± 3,14 vs. 3,84 ± 1,10; p=0,04).

In summary, we could show that in PB from patients with newly diagnosed AML an aberrant cell population of CD39⁺ TIGIT⁺ CD73^- CD8⁺ T cells is prevalent in contrast to the PB from HDs. In this cell population we observed elevated expression of TOX which has recently described as one of the key regulators governing CD8⁺ T cell exhaustion. In contrast downregulation of CD127 and TCF-1 was found in these cells. These data might contribute to CD8⁺ T cell exhaustion in AML and support further functional analysis to investigate the relevance of combinatorial inhibition of TIGIT and CD39.