Effector CD4⁺CD45RA⁻CD25^brightFoxp3^bright Regulatory T Cell (eTreg) Distribution Is Significantly Impaired in Chronic Myelomonocytic Leukemia (CMML) and Correlates with TET 2 Mutational Status.

Naturally occurring CD4⁺CD45RA⁺CD25^highFoxp3⁺ regulatory T cells (rTregs) and in particular the highly suppressive effector CD4⁺CD45RA⁻CD25^brightFoxp3^bright Tregs (eTregs) are critical to limit impaired immune responses. On the other hand, in advanced stages of cancers, increased numbers of Tregs are found in the tumor microenvironment, where they probably suppress antitumor immune responses. Likewise, expansion of Tregs in the bone marrow (BM) and peripheral blood (PB) has been shown in high risk MDS and AML (Kordasti, Blood, 2007). CMML, the most frequent MDS/MPN, displays both MDS and MPN features, has heterogeneous but generally poor prognosis and is characterized by a large number of somatic gene mutations, the most common being those of ASXL1 and TET2 genes. While ASXL1 mutations confer poor prognosis (Gelsi-Boyer, Br J Haematol, 2010) the prognostic impact of TET2 mutations is more controversial. Here we report that distribution of eTreg is impaired among patients with CMML and that high eTREG counts are associated with TET2 mutations.

Between February 2011 and July 2012, 33 CMML patients and 25 healthy controls were studied. PBMC were purified from blood samples on Ficoll-Hypaque gradients to determine populations of resting CD4⁺CD45RA⁺FoxP3⁺CD25⁺ Tregs (rTregs) and effector CD4⁺CD45RA⁻CD25^brightFoxp3^bright Tregs (eTregs) by flow cytometry. For CMML patients DNA was extracted from total BM nucleated cells or PB CD14⁺monocytes obtained by immunomagnetic selection and subjected to PCR analysis for target genes.

Median age of the 33 patients analyzed was 75 years (range 47–88), M/F: 20/13. Twenty nine pts (87%) had CMML 1 and 4 (13%) had CMML 2. Median WBC count was 8.4 G/l (range 3.1–39.4), median blood monocytes 1.9 G/l (range 1–9.3), and median BM blasts 5% (range 2–22). Twenty four (73%) pts had WBC<13G/l and 9 (27%) WBC≥13G/l. Eight (23%) pts had splenomegaly (SMG) and 2 (6%) had extra medullary disease (EMD) (skin involvement exclusively). Abnormal karyotype was found in 11 (33%) pts, including 3 isolated +8, 3 isolated -Y, but no −7 or complex abnormality. In 29 pts screened for gene mutations, 16 (55%) TET2, 10 (35%) SRSF2, 7 (24%) ASXL1, 3 (10%) JAK2, 3 (10%) SF3B1, 3 (10%) ZRSR2, 3 (10%) CBL, 2 (6%) IDH2, 2 (6%) RAS, 2 (6%) RUNX1, 1 (3%) FLT3, and 1 (3%) DNMT3a mutations were found. No pt received cytoreductive treatment before Treg analysis.

Overall median numbers of eTregs (1.97% (range 0–11.8%) vs 1.81% (0.4–3.76%), p= 0.96) and rTregs (0.79% (0.1–9.32%) vs 0.94% (0.15–2.33%), p=0.75) did not significantly differ in CMML pts and controls. Nevertheless, distribution of eTregs was significantly impaired among CMML pts compared to controls with 9 pts vs 1 control having <1% eTreg and 9 pts vs 2 controls having >3% eTreg (Table 1). Interestingly, eTegs were significantly increased among the 16 pts with TET2 mutations (2.95% (0.82–11.8%) vs 1% (0–4.8%), p=0.0057), potentially increased in SRSF2 mutated pts (3.05% (0–11.8%) vs 1.86% (0.3–5.71%) p=0.12) and similar in pts carrying ASXL1 mutations (2.23% (0–5.33%) vs 2.03% (0.3–11.8%), p=0.82). Impairment of eTreg distribution was not correlated with WBC count, PB monocytosis, Hb, plt count, BM blast %, karyotype, SMG, EMD and survival. By contrast, rTregs were distributed in CMML patients as in controls.

Our analysis shows for the first time significantly impaired distribution of activated Tregs in CMML pts, and particularly in CMML with TET2 mutation, without association to specific prognostic factors or survival. As clonal TET2 mutations also occur in lymphocytes of MDS and CMML, our results may suggest the existence of a control loop connecting myeloid and regulatory T cell lineages, which needs further investigation. Treg differentiation and function are influenced by Foxp3 promoter methylation. Therefore generalized hypomethylation secondary to loss of TET2 function in stem cells could result in Treg expansion, which in turn could impact myeloid differentiation and/or function. In any case, Treg expansion may participate in the state of immunosuppression associated to a specific group of CMML.

Fenaux:Novartis: Honoraria, Research Funding.