Possible Role of Impaired Erk1,2 Phosphorilation and Increased sIL2r Alpha Plasma Levels in the Reduced Frequency of Circulating T Regulatory Cells of Patients with Primary Myelofibrosis

Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cells, characterized by an increased number of circulating CD34+ cells. Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. A subset of patients display immune-related abnormalities that suggest an (auto)immune pathogenesis. In addition, it has been reported a significant association between personal history of a broad span of autoimmune diseases and subsequent risk of myeloproliferative neoplasm (Kristinsson SY Haematologica 2010; 95:1216-1220).

CD4+ T cells maintain cancer surveillance and immune tolerance. Chronic

inflammation has been proposed as a driver of clonal evolution in myeloproliferative neoplasms (MPN) (Hasselbalch HC et al. Leuk Res 2013;37:214-20), suggesting that T cells play an important role in their pathogenesis. A recent paper by Keohane C et al (BJH 2015; doi 10.1111/bjh 13519) shows that peripheral blood (PB) T regulatory cells (Tregs) are reduced in MPN patients compared to healthy subjects (CTRLs) and that this decrease is even more pronounced following JAKi therapy (Massa M et al Leukemia 2014;28:449-51).

Unpublished data from our group further indicate a significant (p<0.001) decrease of PB Tregs in PMF (n=193, CD4+CD25highCD127low/- FOXP3+ cells expressed as % of CD4+ cells, median 0.87, 0-10.2) versus CTRLs (n=16, median 2.09, 0.54-6.5). Based on the distribution of Tregs in CTRLs, we established a cut-off point of 1.0% to define an actual reduction of Tregs.

Study design. To investigate the possible factors favouring a decrease of Tregs, we examined in patients with PMF and <1% Tregs (n=15), and in CTRLs (n=10): 1) the percentage of PB CD4+, CD4+CD25+, and CD4+CD25++CD127low/- cells expressing membrane Annexin V at time of sampling as a measure of spontaneous apoptosis 2) the phosphorylation of Stat1, Stat3, Stat5, Stat6, Erk1,2 and p38-MAPK determined by using flow-based intracellular staining (BD Biosciences) at baseline and following stimulation with the appropriate cytokine in CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells 3) the levels of circulating soluble IL2 receptor alpha (sIL2rα) in 34 patients with PMF and Tregs <1% and 11 CTRLs by ELISA (R&D), due to the relevance of IL2 in the generation, function, and survival of Tregs and data reported by Tefferi A et al (J Clin Onc 2011; 29:1356-63).

Results.

1) The percentages of PB CD4+, CD4+CD25+, or CD4+CD25++CD127low/- cells expressing membrane Annexin V were comparable in patients with PMF and CTRLs (data not shown).

2) The phosphorilation of Stat1, Stat3, Stat5, Stat6, and p38-MAPK (expressed as mean fluorescence intensity: MFI) on CD4+, CD4+CD25-, CD4+CD25+, and CD4+CD25++FOXP3+ cells before and after stimulation with IFNα, IL6, IL4, or PMA was comparable in patients and CTRLs. The Erk1,2 phosphorilation was comparable in patients and CTRLs at baseline condition, while in patients with PMF Erk1,2 phosphorilation following stimulation with PMA was significantly lower in both CD4+CD25+ cells (p=0.039; median MFI 5.4, range 0.3-40) and CD4+CD25++FOXP3+ cells (p=0.01; median MFI 4.3, range 1.4-9.3) than in CTRLs (CD4+CD25+ cells median MFI 8.1, range 4.9-20; CD4+CD25++FOXP3+ cells median MFI 7.3, 3.7-10.8).

3) sIL2rα plasma levels were higher (p=0.01) in patients with PMF (1658 pg/ml, range 476-8968) than in CTRLs (1040 pg/ml, 482-1719); the sIL2rα plasma levels were significantly (R=-0.35, p=0.039) inversely correlated with the percentages of circulating Tregs. No significant correlation was found with the presence (n=13)/absence (n=14) of JAK2V617F mutation.

Conclusions. These results indicate that in patients with PMF Tregs present an untuned Erk signaling; moreover, sIL2rα plasma levels are higher in patients than in CTRLs, and significantly inversely correlated with Treg percentages. These findings may partly explain the reduced frequency of Tregs in PMF since Erk signaling is pivotal in T cell differentiation, regulation of T cell homeostasis, and defects in Erk expression have been associated with autoimmunity (Chiung-Fang Chang J Immunol 2012; 189:721-731). Moreover, increased levels of sIL2rα may impact on IL-2-mediated functions and alterate the optimal activation of Erk1/2 that is IL-2 dependent (Lequn Li, Blood. 2005;106:3068-3073),