Characterization of CD34+ Cells from Patients with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) Using a t-Distributed Stochastic Neighbor Embedding (t-SNE) Protocol.

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were "leukemia-related" containing a great proportion of CD34+/CD38- hematopoietic stem cells (HSCs) or CD34+ cells with a strong co-expression of CD45RA/CD123, respectively. CD34+ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference.

Myelodysplastic syndrome patients display alterations in their immune status reflected by increased PD-L1-expressing stem cells and highly dynamic exhausted T-cell frequencies.

Little data are available for the expression of immune checkpoint (IC) molecules within myelodysplastic syndrome (MDS). Here, we report increased PD-L1+ CD34+ CD38- and PD-L1+ CD34+ CD38+ stem cell frequencies within MDS patients compared to stem cell recipients in remission. Additionally, we observed exceedingly similar PD1+ and Tim-3+ T-cell frequencies between acute myeloid leukaemia (AML) and MDS samples that were elevated compared to patients in remission. Furthermore, we found highly dynamic Tim-3+ and PD1+ T-cell frequencies within serial samples of relapsing MDS with excess blasts (MDS-EB II) patients, correlating with further disease markers. These findings support the idea of a potential successful implementation of IC inhibitor treatment in suitable MDS patients.