High-dimensional profiling of T- and NK-cell frequencies and unsupervised clustering analyses to identify immune dysfunction phenotypes in MDS patients
Myelodysplastic syndrome (MDS) is a heterogenous disease, presenting itself as dysplasia of the bone marrow resulting in (pan)cytopenia of the blood. For higher-risk patients, allogeneic stem cell transplantation (alloSCT) is the only curative treatment modality, which can be attributed to powerful donor-derived anti-tumor immune responses. However, many high-risk patients are unfit to receive alloSCT due to their high age (average over 65) and frailty. Instead, these patients receive hypomethylating agents, of which the response rate is only ~30%. This emphasizes the medical need for adjuvant (immuno)therapies to prevent disease progression and improve overall survival. Here, we investigated T and natural killer (NK) cell frequencies, phenotype and functionality in MDS patients, correlating with disease status (lower versus higher risk). Insights into the level of immune (dys)function in MDS will provide directions for adjuvant therapeutic strategies to boost tumor-reactive T and NK cells.
Using high-dimensional flow cytometry, we investigated the immune composition in bone marrow of 10 healthy donors (HD) and 30 MDS patients. We investigated general leukocyte composition, T and NK cell subsets, co-inhibitory and co-stimulatory molecule expression profiles in depth. Moreover, we assessed T cell (poly)functionality in response to PMA/ionomycin stimulation followed by intra-cellular cytokine staining and flow cytometry analysis, separating results for the different helper and cytotoxic effector/memory T cell subsets. Using unsupervised automated clustering analyses (i.e. FlowSOM) we examined differences in (abundance of) T and NK cell subsets in our HD and MDS patients and between MDS risk-groups. Within these subsets, expression patterns were analyzed in depth. Notably, these findings were subsequently confirmed using manual-gating analysis.
Our results corroborate with literature, displaying higher levels of regulatory T cells within the CD4+ T cell compartment in higher-risk MDS and lower abundance of naïve-like cytotoxic T cells in all MDS groups compared to HD. Within the CD8+ T cells, an increase in subpopulations reflecting terminal differentiation (CD45RA+CCR7-KLRG1+), exhaustion (PD-1+, TIGIT+) and senescence (CD57+) was observed in the MDS patients versus healthy controls. Similarly, NK cell subsets displaying activating receptors (i.e. NKp30+, DNAM+, NKG2A+) were decreased in higher-risk MDS. Interestingly, upon polyclonal stimulation cytotoxic and helper T cells from MDS patients displayed poly-functionality, increased degranulation potential and increased IL-2, IFNγ and TNFα production.
By integrating phenotypic profiles with functional data and expanding our existing data set we acquire insight into the level and complexity of T and NK cell (dys)function in MDS. We expect to identify immunotherapeutic strategies that could be applied to improve anti-tumor T and NK cell responses in MDS patients.