Correlation Between IL10 and CD8⁺ Cells in MDS Patients Adds New Insight Into the Role of the Immune System in This Disease

Abstract 3836

Low-risk MDS is characterized by increased apoptosis in the bone marrow and autoimmune disease-like profile, whereas advanced MDS is distinguished by immune evasion, lower apoptosis and secondary DNA damage, facilitating progress into acute leukemia. This evidence, together with immunosupressive therapeutics, indicates a role for the immune system in the progression of early MDS to the advanced stage. Thus, new insights related to the immune system are important to better understand the pathogenesis of MDS. The aim of this study was to investigate the absolute lymphocyte count, the frequency of peripheral CD4⁺ and CD8⁺ and their ratio in healthy controls and MDS patients, after age-adjusted analysis. These data were correlated with biological parameters, FOXP3, and also with expressions of the anti-inflammatory cytokines IL10, TGFß1, and CTLA4. A total of 29 samples from healthy donors were used as controls (median age=39 years; range 28–60) and 49 patients with a diagnosis of MDS (median age=67 years; range, 27–89; WHO: RCUD=09, RCMD=23, RARS=08, RAEB-1=07, RAEB-2=02) were included in the study. The study was approved by the ethics committee and informed written consent was provided. CD3⁺CD4⁺ and CD3⁺CD8⁺ cell frequencies were determined by flow-cytometry. FOXP3, IL10, TGFß1 and CTLA4 expression levels from peripheral blood CD3⁺ cells were determined by q-PCR. Linear regression analysis and 2-tailed Spearman's correlation coefficient were used for statistical analyses. Correlation of CD3⁺CD4⁺ and CD3⁺CD8⁺ cell frequencies with clinical data (age, sex, hemoglobin, leukocyte, granulocyte, platelet, number cytopenias and dysplasias, percentage of blast in BM, karyotype, and transfusion dependency) was performed by a univariate and multivariate regression model. We observed a significant decrease in absolute lymphocyte counts in the MDS group, when compared to controls, after adjusting for age (P=0.002). On the other hand, the age-adjusted percentages of T cell subsets were significantly higher in high-risk MDS WHO, for CD3⁺CD4⁺ frequency (P=0.02), and in low-risk, for CD3⁺CD8⁺ frequency (P=0.04), both compared with the control group. The CD4:CD8 ratio was significantly higher in the high-risk group, when compared to the low-risk group (P=0.03). Univariate and multivariate analysis, showed that advanced age correlated with decreased CD3⁺CD8⁺ frequency. Transfusion dependency correlated positively with CD3⁺CD4⁺ frequency. FOXP3 expression correlated positively with IL10, TGFß1 and CTLA4 expressions. FOXP3 expression was significantly lower in the low-risk MDS group compared to controls, and demonstrated similar levels to those of the control group in the high-risk group. The same expression pattern was observed for IL10 transcripts. Interestingly, IL10 transcripts correlated negatively with the percentage of CD3⁺CD8⁺cells (P=0.02; r=-0.35). There were no significant differences in TGFß1 and CTLA4 expressions. Several studies have shown that lymphocytes are generally not involved in the MDS malignant clone. Hence, our results showing alterated absolute lymphocyte count and T cell frequencies support the hypothesis of immune abnormalities in MDS. The higher CD3⁺CD8⁺ frequency in low-risk MDS and its correlation with age are in agreement with the literature. Moreover, there was an increased CD3⁺CD4⁺ cell frequency in high-risk MDS, followed by a positive correlation of these cells with transfusion dependency. Therefore, the increased CD4:CD8 ratio observed in the high-risk group could be a consequence of CD4⁺ cell activation due to transfusion dependency, which was present in 44.4% of the high-risk patients. These findings may explain, in part, why studies of T cell subsets in MDS have been so contradictory when transfusion dependency is not taken into account. A profile of autoimmune disease is described during early stage MDS, as illustrated by the low number of Tregs, and confirmed herein by the lower FOXP3 expression. Although Tregs play a role in the immune system deregulation in MDS patients, via interleukin secretion, this has not yet been proven. This is the first report to show an inverse pattern of CD8⁺ cell frequency with IL10 expression in MDS patients, suggesting the role of Treg in the immune system deregulation in this disease, via IL10 secretion.