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Background: Mesenchymal stem (stromal) cells (MSCs) constitute an emerging therapeutic strategy for several human diseases. MSCs may be a promising treatment for Rheumatoid Arthritis (RA), potentially acting to “reset” the immune system and/or induce immune tolerance in a manner that would resemble biologically a “pre-RA” state [1, 2]. We performed a Phase 1 randomized double blind randomized double blind controlled study of patients who had sero-positive RA (presence of rheumatoid factor and/or antibodies to citrullinated peptides) for less than two years and had inadequate response to methotrexate (NCT03186417). Eight patients received a single MSC infusion; five patients received 2M/kg, and three patients received 4M/kg allogeneic MSCs. Two patients received placebo infusion. We have previously reported the safety of allogeneic MSCs in early RA following single infusion of bone marrow-derived allogeneic MSCs as well as improvement in patient reported outcomes using Patient Reported Outcomes Measurement System (PROMIS) scales for physical function, fatigue and pain interference. Disease activity score 28 with CRP (DAS28CRP) decreased from a mean of 4.8 at screening to 3.6 at week four post-infusion. We have previously hypothesized that increases in T-regulatory (Treg) cell number and/or function would mediate response to therapeutic MSCs in early RA.
Objectives: We sought to define a transcriptomic signature of response to MSC infusion in RA by comparing the peripheral blood T-regulatory (Treg) cell transcriptomes of those patients who had drop in DAS28CRPs that constituted a “good EULAR response” (responders) to those who received MSCs but had a more modest response or who did not improve at all (non-responders). This signature will provide a set of potential biomarkers of response to therapy, as well as biological insight into the potential mechanisms of underlying the successful induction of response in responders as well as factors that may be inhibiting the improvement of disease in non-responders.
Methods: Tregs were enriched using a Treg isolation kit (Cat # 18063 STEMCELLTechnologies). Single cell RNA (scRNA) sequencing was performed on approximately 10,000 cells per sample using the Chromium Next GEM Single Cell 3′ Kit v3.1 (10X Genomics) and sequenced on an Illumina NovaSeq X (paired-end, 100 cycles, 40k reads/cell). Preprocessing was performed using cellranger software v4.0.0 (10X Genomics) with aligned reads to the GRCh38 reference genome. Advanced statistical and bioinformatic analysis was performed with a custom pipeline based on Seurat v4.1.1 (R programming language v4.1.1).
Results: Using scRNA seq we first compared Treg genes that were differentially regulated post-treatment compared to pre-treatment in MSC responders but not in non-responders. Multiple contrasts were performed to define signatures associated with response to MSC infusion including a double contrast of post vs pre-infusion differential gene expression in responders compared to post vs pre-infusion in non-responders. Genes that were upregulated post-infusion in MSC responders included H3F3B, S100A9, FKBP5 and IL7R while those that were upregulated in non-responders included RGS1, BAZ1A and IRF1.
Conclusion: We identified numerous DEGs that modulate the inflammatory response that were differentially regulated and include cytokines and chemokines in MSC-responders. Differential regulation of multiple histone genes like H3F3B suggests that there may also be dynamic epigenetic features of response to MSC therapy. Furthermore, enrichment of a large set of translational regulators, aminoacyl-tRNA synthetases (aaRSs), was associated with response to infusion. These data suggest that early on in RA, regulatory T-cell gene expression can be tuned to have a transcriptomic gene profile that favors immune tolerance related genes. We hypothesize that infusion of MSCs induces regulatory T-cell genes that have a secretagogue and cell surface molecules that help tune dendritic cells (DCs) from an inflammatory phenotype to an anti-inflammatory profile. DCs may in turn help to reduce activation of macrophages and lymphocytes and result in attenuation of clinical synovitis.
REFERENCES: [1] Verstappen M, van der Helm-van Mil AHM. Sustained DMARD-free remission in rheumatoid arthritis - about concepts and moving towards practice. Joint Bone Spine. 2022 Nov;89(6):105418. doi: 10.1016/j.jbspin.2022.105418. Epub 2022 May 27. PMID: 35636705; PMCID: PMC7615888.
[2] Zaripova LN, Midgley A, Christmas SE, Beresford MW, Pain C, Baildam EM, Oldershaw RA. Mesenchymal Stem Cells in the Pathogenesis and Therapy of Autoimmune and Autoinflammatory Diseases. Int J Mol Sci. 2023 Nov 7;24(22):16040. doi: 10.3390/ijms242216040. PMID: 38003230; PMCID: PMC10671211.
Acknowledgements: Drs. Breitman and Cameron contributed equally to this work. The authors gratefully acknowledge the contributions of Arnold Caplan and Elizabeth Mellins whose untimely deaths preceded submission of this abstract. This project was made possible with funding from NIH/NIAMS AR069226 to and was supported by the Clinical and Translational Science Collaborative of Northern Ohio which is funded by the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health, UM1TR004528. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We gratefully acknowledge the CWRU David and Virginia Baldwin Fund for support.
Disclosure of Interests: Maya Breitman: None declared, Cheryl Cameron: None declared, Tracey Bonfield: None declared, Jane Reese: None declared, Emma Barnboym: None declared, Maricela Haghiac: None declared, Steven Lewis: None declared, Hillard Lazarus: None declared, Noor Hussein: None declared, Donald Anthony: None declared, Mark Cameron: None declared, Brian Richardson: None declared, Nora Singer Novartis, Pfizer.
© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (