CD73+ leukemia-reactive CD8 T cells assessed by flow cytometry. a Wilcoxon matched-pairs signed rank test for unpaired and paired data, respectively. Comparisons of patient characteristics were analyzed using Fishers exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). To evaluate correlation, Pearsons correlation coefficients were used. For all analyses, values less than 0.05 were considered statistically significant. Results CD73 is highly expressed on CD8 T cells in peripheral blood from AML patients To determine which cell components express CD73 in a patient with AML, peripheral blood mononuclear cells (PBMCs) were assessed for CD73 expression by flow cytometry gated on markers for blast (CD45int, low SSC), Treg (CD45hiCD4+CD25+), CD4, CD8, NK (CD45hiCD3?CD56+), monocytes (CD45hiCD11b+CD14hi/low), and dendritic cells (DCs; CD45hiCD3?CD19?CD56?CD14?HLA-DR+). In contrast to the findings in previous studies of solid tumors that primary tumor or tumor cell lines highly express CD73, we observed minimal CD73 expression on AML blasts (mean frequency 4.75??6.21, Fig.?1a, b), whereas the majority of patients express significant level of CD73 on their CD8 T cells (mean frequency 22.26??13.79, Fig.?1a, b). There was moderate CD73 expression on monocytes and DCs, while low expression was detected on Treg, CD4 T cells, and NK cells (Fig.?1a, b). This data suggests the involvement of CD73 in CD8 T cell response in AML. Open in a separate window Fig. 1 CD73 is downregulated on CD8 T cells in patients with newly diagnosed AML compared with healthy controls and patients with complete remission (CR). Flow cytometry analysis of CD73 expression was performed on PBMCs collected from AML patients at the initial diagnosis (values were obtained by the unpaired test or Mann-Whitney test. ***values were obtained by unpaired test. d Representative flow data (left) and plots (right) showing expression of CD73 on CD8 T cells from AML patients at initial diagnosis and complete remission. values were obtained Rabbit Polyclonal to ECM1 by paired test Low CD73 expression on CD8 T cells associates with high leukemia burden We then focused our study on CD8 T cells and compared the expression of CD73 in AML (values were obtained by Kruskal-Wallis test followed by Dunns multiple comparisons test. *values were obtained by paired test (PD-1, TIGIT, LAG-3) or Wilcoxon matched-pairs signed rank test (2B4, CD160). fCj Correlative analysis of CD73 and expression of above receptors are shown. Pearsons test was used to test for correlations Since T cell exhaustion is a consequence of over-activation of T cells caused by high antigenic stimulation, we also analyzed the activation status of CD73? CD8 T cells by measuring HLA-DR expression. We observed a significantly higher expression of HLA-DR in CD73? CD8 T cells compared with that in CD73+ CD8 T cells (Fig.?4a). Consistently, the expression of CD73 was inversely correlated with that of HLA-DR (values were obtained by Wilcoxon matched-pairs signed rank test. b Correlative analysis of CD73 and HLA-DR is shown. Pearsons test was used to test for correlations. c Intracellular expression of EOMES on CD73? and CD73+ CD8+ T cells from AML patients (values were obtained by paired test Furthermore, we examined the expression of Eomesodermin (Eomes), a key transcription factor governing CD8+ T cell exhaustion. It has been demonstrated that Eomeshi CD8 T cells are terminally exhausted and not able to be reinvigorated by PD-1 blockade. Intracellular Eomes was assessed on PBMCs from patients with newly diagnosed AML. We observed significantly higher expression of Eomes in CD73? CD8 T cells than in CD73+ cells (values were obtained by paired test (IL-2, IFN-) or Wilcoxon matched-pairs signed rank test (TNF-). b CD8 T cells were purified from PBMC of HLA-A*0201 AML patients at initial diagnosis..This data suggests the involvement of CD73 in CD8 T cell response in AML. Open in a separate window Fig. on CD8 T cells in peripheral blood from AML patients To determine which cell components express CD73 in a patient with AML, peripheral blood mononuclear cells (PBMCs) were assessed for CD73 expression by flow cytometry gated on markers for blast (CD45int, low SSC), Treg (CD45hiCD4+CD25+), CD4, CD8, NK (CD45hiCD3?CD56+), monocytes (CD45hiCD11b+CD14hi/low), and dendritic cells (DCs; CD45hiCD3?CD19?CD56?CD14?HLA-DR+). In contrast to the findings in previous studies of solid tumors that primary tumor or tumor cell lines highly express CD73, we observed minimal CD73 expression on AML blasts (mean frequency 4.75??6.21, Fig.?1a, b), whereas the majority of patients express significant level of CD73 on their CD8 T cells (mean frequency 22.26??13.79, Fig.?1a, b). There was moderate CD73 expression on monocytes and DCs, while low expression was detected on Treg, CD4 T cells, and NK cells (Fig.?1a, ARQ-092 (Miransertib) b). This data suggests the involvement of CD73 in CD8 T cell response in AML. Open in a separate window Fig. 1 CD73 is downregulated on CD8 T cells in patients with newly diagnosed AML compared with healthy controls and patients with complete remission (CR). Flow cytometry analysis of CD73 expression was performed on PBMCs collected from AML patients at the initial diagnosis (values were obtained by the unpaired test or Mann-Whitney ARQ-092 (Miransertib) test. ***values were obtained by unpaired test. d Representative flow data (left) and plots (right) showing expression of CD73 on CD8 T cells from AML patients at initial diagnosis and complete remission. values were obtained by paired test Low CD73 expression on CD8 T cells associates with high leukemia burden We then focused our study on CD8 T cells and compared the expression of CD73 in AML (values were obtained by Kruskal-Wallis test followed by Dunns multiple comparisons test. *values were obtained by paired test (PD-1, TIGIT, LAG-3) or Wilcoxon matched-pairs signed rank test (2B4, CD160). fCj Correlative analysis of CD73 and expression of above receptors are shown. Pearsons test was used to test for correlations Since T cell exhaustion is a consequence of over-activation of T cells caused by high antigenic stimulation, we also analyzed the activation status of CD73? CD8 T cells by measuring HLA-DR expression. We observed a significantly higher expression of HLA-DR in CD73? CD8 T cells compared with that in CD73+ CD8 T cells (Fig.?4a). Consistently, the expression of CD73 was inversely correlated with that of HLA-DR (ideals were acquired by Wilcoxon matched-pairs authorized rank test. b Correlative analysis of CD73 and HLA-DR is definitely shown. Pearsons test was used to test for correlations. c Intracellular manifestation of EOMES on CD73? and CD73+ CD8+ T cells from AML individuals (values were acquired by paired test Furthermore, we examined the manifestation of Eomesodermin (Eomes), a key transcription factor governing CD8+ T cell exhaustion. It has been shown that Eomeshi CD8 T cells are terminally worn out ARQ-092 (Miransertib) and not able to become reinvigorated by PD-1 blockade. Intracellular Eomes was assessed on PBMCs from individuals with newly diagnosed AML. We observed significantly higher manifestation of Eomes in CD73? CD8 T cells than in CD73+ cells (ideals were acquired by paired test (IL-2, IFN-) or Wilcoxon matched-pairs authorized rank test (TNF-). b CD8 T cells were purified from PBMC of HLA-A*0201 AML individuals at initial analysis. Then, they were co-cultured with T2 cells (used as antigen-presenting cells) that were pulsed with HLA-A*0201-binding WT-1126-134 peptide for 6?days. Then, cells were collected and intracellular staining was performed. Shown are the manifestation of IL-2, TNF-, and IFN- in CD73? vs. CD73+ leukemia-reactive CD8 T cells assessed by circulation cytometry. In the remaining are representative circulation cytometry data. In the right are the statistical summary plots; each dot shows one patient. ideals were acquired by combined College students test or Wilcoxon signed-rank test. c, d Manifestation of CD95 (c) and Annexin V (d) on CD73? and CD73+ CD8 T cells from AML individuals (values were acquired by paired test Susceptibility to apoptosis is also a hallmark for practical status of T cells. CD73? CD8 T cells from AML individuals showed a pattern of higher susceptibility to apoptosis manifested by significantly higher manifestation of CD95 manifestation ( em P /em ? ?0.0001, Fig.?5c). Interestingly, manifestation of Annexin V was similar between CD73? and CD73+ CD8 T cells ( em P /em ?=?0.8725, Fig.?5d). Collectively, our findings demonstrate that in AML.