Azacitidine Improves the CD4+ T-Cell Repertoire in Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia with Multilineage Displasia

Introduction and aims: Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia are known to display several immunological abnormalities. Azacitidine represents a therapeutic options for these disorders and, beside the well known effects on bone marrow precursors, has been demonstrated to potentially influence T-cell polarization. The aim of this study is to monitor the kinetic of the T-cell receptor (TCR) repertoire during Azacitidine treatment in order to explore its potential ability, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these patients.

Materials and methods: Our study consisted in a flow cytometric and spectratyping analysis performed on the peripheral blood of 11 patients (5 with MDS and 6 with AML with multilineage dysplasia) and 30 normal controls. Each patient was evaluated at baseline and then every 3 cycles of Azacitidine. The flow cytometry analysis was based on a panel of 24 beta variable (BV) family-specific antibodies. A BV expansion was defined as any value of BV family expression higher than the mean + 3 standard deviations calculated in controls. The profile of the third complementarity-determining-region (CDR3) in separated helper and cytotoxic T-cells was then analyzed by spectratyping. After immunomagnetic CD4+/CD8+ cell separation, RNA extraction and reverse transcriptase PCR, CDR3 fragment analysis was performed through capillary electrophoresis. Spectratyping evaluation was carried out by determining the percentage of Gaussian, skewed and oligoclonal BV subfamilies.

Results: Our patients had a median of 3 assessments during their treatment with Azacitidine. On flow cytometry at baseline, in CD4+ cells 5 patients did not show any lymphocyte expansion while 5 of them showed a single BV expansion. One of these expansions disappeared after 3 cycles while 4 were stable during treatment. When reassessed during therapy, 2 of the patients showed each the appearance of 3 new BV expansions, some of which however disappeared at the following evaluations. Overall CD4+ expansions during the all period of study were 11 (5 at baseline and 6 emerged during treatment) and their size ranged from 5.1% in BV 13.6 to 23.9% in BV 11. Within the CD8+ subset, at baseline 7 out of 10 patients showed at least one T-cell expansion. In details 3 patients showed a single expansion while 4 of them displayed 2 different BV expansions. Of these 11 baseline expansions 7 were stable during treatment, while 4 of them quickly disappeared. Remarkably, one of these expansions which had disappeared in a patient in remission reappeared at disease relapse. Six patients showed the appearance of a single BV expansion during treatment, which once again was usually transient. Overall CD8+ expansions during the all period of evaluation were 17 (11 at baseline and 6 emerged later) and their size ranged from 2.5% in BV 5.3 to 50.1% in BV 3.

Noteworthy, when analyzed by spectratyping during their treatment our patients showed significant changes in their CDR3 profiles, which were much more evident in helper T lymphocytes. In fact, the frequency of BV showing a skewed CDR3 profile was significantly decreased from baseline to the following evaluations in the CD4+ subset (mean 81.45% vs. 70.17%, p= 0.004). This pattern was even more pronounced in patients responding to Azacitidine (mean 89.60% vs. 61.47%, p= 0.002). Also in the CD8+ subset a trend towards a reduction in the frequency of skewed CDR3 profiles (mean 99.27% vs. 98.74%, p= 0.01) was demonstrated. In the patient with the longest follow up it was possible to observe a dramatic decrease in the degree of CD4+ CDR3 skewing from 91% at baseline to 22% at 18 months, when he was still responding to Azacitidine.

Conclusions: Our findings firstly confirmed in our patients an overall derangement of the TCR repertoire. However this pattern seems to gradually improve during Azacitidine treatment, as witnessed by the disappearance of some BV expansions observed on flow cytometry but much more by the progressive restoration of the CDR3 diversity detected by spectratyping, especially within the CD4+ subset. Therefore our data suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of patients with MDS and AML with multilineage dysplasia.