Increased CD200 Expression in Acute Myeloid Leukemia Is Linked with an Increased Frequency of Foxp3 Þ Regulatory T Cells

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Chem Biol 2011; 18: 314–323. Constitutive JAK3 activation induces lymphoproliferative syndromes in murine 15 Changelian PS, Moshinsky D, Kuhn CF, Flanagan ME, Munchhof MJ, Harris TM et al. bone marrow transplantation models. Blood 2009; 113: 2746–2754. The specificity of JAK3 kinase inhibitors. Blood 2008; 111: 2155–2157. OPEN Increased CD200 expression in acute myeloid leukemia is linked with an increased frequency of FoxP3 þ regulatory T cells Leukemia (2012) 26, 2146–2148; doi:10.1038/leu.2012.75 that CD200 expression on AML blasts promotes Treg formation. We also examined whether CD200 expression was differentially expressed on AML blasts with a putative leukemic stem cell phenotype (CD34 þ CD38 À ),7 but could find no evidence for this CD200 is a type-1a transmembrane cell-surface glycoprotein that within the six samples examined (Supplementary Figure S2). is normally expressed in critical tissues such as the central nervous Given that the depletion of Tregs has been shown to improve system and testis, as well as certain leukocytes, including T and B T-cell-mediated therapy, as well as improving antitumor activity lymphocytes, where its role is to promote peripheral tolerance in leukemia patients that are in complete remission;8,9 we and protect immune privileged sites.1 CD200 has no known investigated whether Tregs from CD200hi patients were intracellular signaling motif, but induces immunosuppression functionally immunosuppressive. We examined the ability of through engagement with CD200R, a cell-surface receptor purified Tregs to suppress naı¨ve responder T-cell (CD4 þ CD25 À ) homolog, which is expressed on leukocytes of myeloid lineage, proliferation following CD3/CD28 co-stimulation (refer to including mast-cells, macrophages, basophils, dendritic cells as Supplementary Materials and Methods). The data demonstrate well as certain T-cell populations.2 CD200, which is frequently that Tregs isolated from CD200hi AML patients were capable of overexpressed in acute myeloid leukemia (AML) patient blasts and suppressing T-cell proliferation at responder to Treg ratios of 41 associated with a worse outcome,3 has the potential to induce the to 0.01 (Figure 1c). Reciprocal analysis of Tregs from CD200lo formation of CD4 þ CD25 þþFoxP3 þ regulatory T cells (Tregs),4 patients could not be carried out because of the extremely low a subset of immunosuppressive T cells that are linked with a frequencies of these cells in CD200lo patients (Figure 1d). In fact, poor prognosis in AML.5 Most importantly, Tregs have been Treg frequencies in CD200lo AML patients were uniformly lower documented to suppress the anti-leukemia response in vitro from than in healthy donor controls, suggesting that CD200 has an AML patients,6 suggesting that these cells have an important influence on Treg induction in this context. role in regulating AML patient tumor immunity. We therefore Since mouse models have suggested that CD200-induced investigated whether CD200 protein expression on human AML Tregs are likely to mediate Th1 immunosuppression,10 a cytokine blasts was associated with an increased frequency of Treg cells in response with a prognostic link in leukemia,11 we measured the AML using a cohort of 40 AML patients (at the point of diagnosis Th1 cytokine response (TNFa, IL2 and IFNg production) in before to any treatment; Supplementary Table S1 for patient CD200hi patients before and after Treg depletion. Representative demographic). AML blast CD200 protein expression and patient flow cytometric plots confirmed Treg depletion using magnetic Treg frequency was analyzed by flow cytometry (for full gating separation (Figure 2a). However, removal of Tregs alone was insuffi- strategy, refer to Supplementary Figure S1). The data show that cient to significantly improve the Th1 cytokine response as detected CD200 blast expression level correlated significantly with the by intracellular cytokine staining (Figure 2b). Elsewhere we report that, frequency of Treg cells (Figure 1a). This association was found to in addition to its known influence on Treg production, CD200 is also be independent of white blood cell count (Figure 1b), suggesting directly immunosuppressive through engagement with CD200R Accepted article preview online 20 March 2012; advance online publication, 13 April 2012 Leukemia (2012) 2142 – 2158 & 2012 Macmillan Publishers Limited Letters to the Editor 2147 Figure 2. Assessment of AML patient Treg depletion in recovering the Th1 response in CD200hi patients. Tregs cells were depleted from CD200hi AML patients by magnetic separation. (a) Representative flow cytometric data depicting T cells (i) before depletion of Tregs (CD3 þ CD4 þ CD25 þþFoxP3 þ ) and (ii) following Treg depletion from a CD200hi AML patient. AML cells were stimulated with PMA/ionomycin for 6 h at 37 1C before intracellular Th1 cytokine staining was performed. Flow cytometry was used to assess Th1 cytokine levels within the CD45 þþCD3 þ CD4 þ fraction. (b) Summary data showing the Th1 response from CD200hi AML patients±Treg cells (n ¼ 6). link with CD200 expression has not previously been established. Our data also indicate that while Tregs in these patients may be functionally immunosuppressive, they may only become influential following cytoreduction. Indeed, several studies have identified that increased frequencies of Tregs are associated with Figure 1. Assessment of AML blast CD200 expression with respect to 15,16 Treg frequency and function. Treg cells from 40 AML patients at diagnosis relapse in myeloid malignancy. Furthermore, phase-I were identified by flow cytometry based on the frequency of CD3 þ studies using anti-CD200 monoclonal antibody immunotherapy CD4 þ CD25 þþ T cells that were 495% FoxP3 þ (refer to Supplementary have shown that blocking CD200 is sufficient to reduce the Figure S1). (a) Linear regression analysis comparing AML blast CD200 Treg frequency in chronic lymphocytic leukemia patients,17 expression level with the frequency of Tregs from 40 diagnostic AML illustrating that blocking CD200 may be therapeutically samples. (b) Correlation of AML blast frequency with Treg cell frequency. advantageous in AML. To assess the suppressive function, Tregs and naı¨ve T cells (CD4 þ CD25 À ) were isolated from CD200hi AML patients by magnetic separation and added together at increasing ratios before CD3/CD28 co-stimulation. CONFLICT OF INTEREST Suppression was measured by the ability of Tregs to inhibit naı¨ve The authors declare no conflict of interest. CD4 þ T-cell proliferation (refer to Supplementary Materials and Methods). (c) Suppression of naı¨ve CD4 þ T-cell proliferation by AML Treg cells at indicated Treg to responder ratios (data represent ACKNOWLEDGEMENTS mean±1 s.d., n ¼ 4). (d) Treg cell frequency stratified with respect to AML blast CD200 expression. *Po0.05 and **Po0.01 analyzed by This work was funded by Leukemia and Lymphoma Research UK. We thank one-way analysis of variance with Tukey’s multiple comparison test. Gareth Betts (Cardiff University) for his technical assistance in analyzing Tregs. We are grateful to the patients for access to material enrolled in the NCRI clinical trials.

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