Expression of CD200 on AML blasts directly suppresses memory T-cell function

Previous studies have shown that immunosuppression in acute myeloid leukemia (AML) is associated with changes in the adaptive immune compartment. Such changes include the suppression of memory T-cell function 1 and the suppression of Th1 cytokine (TNFα, IL-2 and IFNγ)-producing cells. 2 A suppressed immune response in AML is associated with a worse patient outcome and increased risk of relapse, 3 as well as increased risk of infection impairing patient recovery. 4 The over-expression of the immunosuppressive ligand CD200 is also associated with an increased risk of relapse in AML (hazard ratio 1.7); an observation consistent with a hypothesis in which CD200 inhibits clearance of residual disease. 5 , 6 As memory T-cell responses are central for tumor immunosurveillance and contribute to prolonged molecular remission, 7 we carried out this study to establish how these responses were affected in AML patients over-expressing CD200 ( Supplemental Table S1 ). We initially investigated whether CD200 expression on AML blasts influenced CD8 + T-cell cytotoxic potential and the frequency of TNFα-, IL-2- and IFNγ-producing CD4 + /CD8 + memory T-cells ( Supplementary Materials and Methods and Supplemental Figure S1 for gating strategy). Using CD107a as a marker of cytotoxic function, AML cells were activated with PMA/ionomycin. We show that the frequency of CD107a + CD8 + memory T-cells was significantly reduced by ∼ 50% for CD200 hi patients when compared with CD200 lo AML, demonstrating that cytotoxic memory T-cell activity was compromised in CD200 hi patients ( Figure 1a ). Furthermore, the frequencies of TNFα-, IL-2- and IFNγ-producing CD4 + memory cells were also reduced by ∼ 50% for CD200 hi patients when compared with CD200 lo AML ( Figure 1b ), significantly so in the case of IL-2 and IFNγ. Interestingly, CD200 lo patients displayed a higher IFNγ response, not only with respect to CD200 hi patients but also in comparison to healthy donors, suggesting a role for this cytokine in AML, which is attenuated by CD200. No difference was observed for TNFα-, IL-2- and IFNγ-producing CD8 + memory cells between CD200 hi , CD200 lo and healthy donors (data not shown). CD200 has also been reported to mediate suppression of the Th1 response in chronic lymphocytic leukemia as well as solid tumors, 8 , 9 suggesting that CD200-mediated Th1 suppression is a central mechanism in cancer immunomodulation. Figure 1 Cytotoxic T-cell response and Th1 memory/recall response in CD200 hi and CD200 lo AML patients. AML patient cytotoxic and intracellular Th1 cytokine memory T-cell responses were measured by flow cytometry following PMA/ionomycin stimulation (for full methods and flow cytometric gating strategies see Supplementary Materials and Methods and Supplemental Figure S1 ). ( a ) Summary data illustrating a significant difference in CD107a + CD8 + memory T-cells between CD200 hi and CD200 lo AML patients. ( b ) The production of TNFα, IL-2 or IFNγ for CD200 hi , CD200 lo and healthy donors for CD4 + memory T-cells. ( c ) Pie charts summarizing the proportion of CD4 + and CD8 + memory cells capable of producing one (1=TNFα, IL-2 or IFNγ), two (2=TNFα/Il-2, TNFα/IFNγ or IFNγ/IL-2) or three (3=TNFα/IL-2/IFNγ) cytokines simultaneously for CD200 hi or CD200 lo (refer to Supplemental Figure S1 for Boolean analysis). The ability of T-cells from CD200 hi and CD200 lo AML patients to mount recall responses to common microbial antigens (PPP, Supplementary Materials and Methods ) was assessed by IFNγ ELISPOT. ( d ) Representative ELISPOT wells for CD200 hi and CD200 lo patients. ( e ) Average spots per well for CD200 hi and CD200 lo patients. AML patient data represents mean±1 s.d., n =9 for CD200 hi and n =12 for CD200 lo . † P <0.05, analyzed by one-tailed unpaired t -test; * P <0.05, analyzed by one-way ANOVA with Tukey’s multiple comparison test. Full size image