Reduced Frequency of CD4+CD25++CD127low/-FOXP3+ Regulatory T Cells in Patients with Primary Myelofibrosis

Abstract

Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cell, categorized among the Ph-negative myeloproliferative neoplasms (MPNs). 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. However, imbalanced immune responses that lead to chronic inflammation has been hypothesized to play an important role in the pathogenesis of the disease (Hasselbalch HC et al. Leuk Res 2013;37:214-20). Study design. In the attempt to explain the mechanism of immune dysregulation, and to explore how it could influences the disease phenotype, we examined the frequency of circulating CD4+CD25++CD127low/-FOXP3+ regulatory T cells (Tregs), as measured by cytofluorimetric technique, in 193 consecutive patients with PMF and 16 healthy controls (HCs). Results. The percentage of Treg cells between PMF patients and HCs was significantly different [median, 0.87% (range 0% to 10.2%) vs. 2.09% (range 0.54% to 6.5%); Mann-Whitney U test, P<0.001]. Based on the distribution of Tregs in HCs, we established a cut-off point of 1.0% for comparing different groups. By using this cut-off, 106 PMF patients (54.9%) had reduced values of Tregs. We also compared the patients who were on hydroxyurea at the time of study with those patients who were not on the drug. There were no differences between the two groups. Considering the whole population of patients, there was no difference in Treg frequency according to sex, age, spleen size, platelet count, circulating CD34+ cells frequency, degree of bone marrow fibrosis, IWG prognostic score. However, marginally significant association between lower frequency of Tregs and lower value of hemoglobin was evidenced (r=0.14; P=0.06). By contrast, significant differences in Tregs percentage was revealed among the different disease genotypes: Tregs were lower in patients with CALR or MPL mutation than in those with JAK2V617F mutation [median, 0.72% (range 0% to 6.7%), vs. 1% (range 0% to 10.2%); Kruskal Wallis ANOVA, P=0.01)]. Unmutated JAK2V617F genotype was the only significant factor for lower frequency of Tregs in peripheral blood at a linear regression model considering the level of hemoglobin as a covariate. In patients with a JAK2V617F genotype (n=111), no significant association was shown between the percentage of Tregs and hematological or clinical parameters. By contrast, significant difference was found between patients with JAK2V617F allele burden greater or lower than 50%. In patients with low-V617F (<50% allele burden), the median Tregs frequency was 1.11% (range 0.01% to 3.7%) while in patients with high-V617F (≥50% allele burden), the median Tregs frequency was 1.93% (range 0-10.2%) (P=0.05). In patients with JAK2V617F unmutated genotype (n=50), a strong association was shown between Tregs and hemoglobin, documenting that patients with lower frequency of Tregs had the most severe anemia (r=0.60; P<0.001). In these patients, a Tregs percentage lower than 1% predicted a hemoglobin lower than 100 g/L with a positive predictive value of 37%, and a negative predictive value of 100%; thus, no patients with a hemoglobin lower than 10 g/dl had a value of Tregs higher than 1%. Tregs were also inversely correlated with age (R= -0.47; P=0.001), and a lower percentage of Tregs was associated with higher value of white blood cells (r=-0.42; P=0.03), higher value of circulating CD34+ cells (R=-0.43; P=0.03), and lower platelet count (R= 0.33; P=0.034). Conclusions. Collectively, these results indicate that reduced frequency of circulating Tregs represents a hallmark of PMF. Since Tregs play an important role in modulating innate and adaptive immune responses, low Tregs frequency is pivotal for the inflammatory/autoimmune unbalancing of the disease. We documented that the frequency of circulating Tregs was modulated by the disease genotype, having patients with JAK2V617F mutation and high allele burden the highest frequency of Tregs. In patients with unmutated JAK2V617F genotype, the severity of Treg depletion was associated with the severity of the disease. The greatest influence of Treg reduction was exerted on the severity of anemia. These findings support the development of new strategies to increase the number of Tregs in highly symptomatic PMF patients. DisclosuresNo relevant conflicts of interest to declare.