Single-cell analysis of bone marrow CD8+ T cells in Myeloid Neoplasms predicts response to treatment with Azacitidine

Treatment with hypomethylating agents (HMA), and specifically azacitidine (AZA), is the standard of care for patients with higher-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) that are not eligible to receive intensive chemotherapy. Despite research efforts, it is not possible to predict response to treatment with HMA. In this study, we aimed to identify immune cell signatures in the bone marrow (BM) associated with treatment outcomes. By employing mass cytometry, we performed an in-depth immunophenotypic analysis in BM samples deriving from patients with myeloid neoplasms prior to treatment initiation. We identified an increased pre-treatment frequency of a CD8+ T cell subset, characterized as CD57+CXCR3+CCR7-CD45RA+, in patients with MDS and AML who did not respond to treatment with AZA, compared to responders. Furthermore, an increased baseline frequency (>29%) of CD57+CXCR3+CD8+ T cells was correlated with poor overall survival. We further engaged scRNA-seq to assess the transcriptional profile of BM CD8+ T cells from treatment-naïve patients with MDS and AML, to identify molecular signatures in CD8+ T subpopulations associated with favorable outcomes. Response to treatment was positively associated with enrichment of IFN-mediated pathways coupled with enhanced cytotoxic signature, whereas enrichment of the TGF-β signaling pathway was observed in cell clusters from non-responders. Together, this study identified a specific CD57+CXCR3+ CD8+ T cell population with predictive value in patients with MDS and AML treated with AZA and characterized molecular signatures in CD8+ T cells linked to cytokine signaling that were associated with treatment outcomes. Key-points ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the HFRI Research Projects to Support Faculty Members & Researchers and Procure High-Value Research Equipment grant (HFRI FM17 452) and the General Secretariat for Research and Technology Management and Implementation Authority for Research, Technological Development, and Innovation Actions (MIA RTDI) (grant T2EDK 02288, MDS TARGET). TA and NEP are supported by the ERC under the European Union Horizon 2020 research and innovation program (grant agreement no 947975 to T. Alissafi) and from the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the 2nd Call for H.F.R.I. Research Projects to support Post-Doctoral Researchers (Project Number: 166 to T. Alissafi) and the Sub-action 1. Funding New Researchers RRF: Basic Research Financing (Horizontal support for all Sciences) (Project number: 15014 to T. Alissafi). IPK is funded by the Academy of Medical Sciences (R2429101) and Rosetrees Trust (R2449101). TC is supported by the Deutsche Forschungsgemeinschaft (TRR332, project B4). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by the Ethics Committee of the University Hospital of Alexandroupolis, under the reference number (877/23-10-2019). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The authors state that all data supporting this study are available in the main text or the supplementary material. The raw scRNA-sequencing data for this study have been deposited in the NCBI Gene Expression Omnibus repository and are accessible through accession number GSE250077. All other raw data can be provided by the authors upon reasonable request.