Poster Session II: Bone marrow failure syndromes incl PNH - Biology - translational research
The significance of T-cell oligoclonal expansion in patients with aplastic anemia (AA) is actively studied at the present time, because oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may confirm the immune nature of the disease.
Determination of T cell subpopulation composition, dominant TCR-Vβ subclones and the expression of PD-1 and PD-L1 in T-cell repertoires using flow cytometry in AA patients.
31 AA patients (pts) with median age of 31 (19-71), m/f ratio 1:1,2 were divided in 2 groups: pts with newly diagnosed (ND) AA (n = 23), and non-response pts (NR) for different lines of IST (n = 8). We used flow cytometry method for the study. Analysis was performed on BD FACS Canto II, using commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these pts. We performed analysis of BM samples from healthy donors as a control group (n = 20). The maximal value each of the 24 subclones (for T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclones exceeded this threshold. We identified different T-cell subpopulations in all 2 groups of AA and healthy donors: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software.
Almost all patients in the two groups had immunodominant clonotypes. We found out that all pts in 2 groups had immunodominant clonotypes among TCL cells, but no clonotypes was detected in 7 pts (30%) in ND group and in 3 pts (37%) in NR group among Th cells. We identified the most common clonotypes in comparison with healthy donors - Vβ17 among the Th cells and Vβ11, Vβ14 among the TCL cells. In ND group Vβ17 was highly expanded in 6 (26%) pts among Th, and Vβ11 and Vβ14 - in 8 (34%) out of 23 pts among TCL. In NR group the most frequent was Vβ11 and Vβ13.1 clone in TCL - in 3 (37%) pts out of 8 pts. In NR group in overall clonal expansion was less frequent than in ND groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in two groups (ND, NR) compared to healthy donors. In NR group percentage of NK-T cells, CD4+ memory cells and CD4+CD25+ cells were higher than in control group (p < 0,024), but percentage of naive CD4+ cells and CD4+ and CD8+ cells co-expressing PD-L1 was lower than in control group (p < 0.019). ND patients had more CD4+ and CD8+ effectors and CD4+ memory cells than healthy donors, and percentage of naive CD4+ and CD8+ cells and CD4+PD-L1+ cells was lower (p < 0,02). ND patients had lower number of NK-T cells in comparison to NR patients (p = 0,03).
In our study we found immunodominant clonotypes in all groups of patients with AA compared with healthy donors, which may confirm the immune character of the disease. We also found statistically significant differences in the subpopulation composition of T cells in all groups of patients with AA. Dynamic observation of changes in the most common clonotypes in AA pts during treatment can provide suitable therapy tactics.