ABSTRACT Mesenchymal stem/stromal cell (MSC) therapy holds great promise as both a therapeutic option and as a biofactory, as cells produce therapeutic proteins to augment their efficacy in disease treatment. This study investigates the therapeutic effects and the mechanistic insights of alpha-1 antitrypsin overexpressing MSCs (AAT-MSCs) in diabetes prevention and treatment. A single infusion of AAT-MSCs not only delayed diabetes onset but reversed new-onset type 1 diabetes (T1D) in the nonobese diabetic (NOD) mice. Using single-cell RNA sequencing, flow cytometry, and functional analyses, we characterized the impact of AAT-MSCs on immune cells, particularly CD4 + and CD8 + T cells, in pancreatic lymph nodes (PLNs) and islets of NOD mice. AAT-MSCs enhanced the immunosuppressive function and the communication of regulatory T cells (Tregs) with other immune cells while reducing the numbers of T helper 1 (Th1) cells and CD8 + cytotoxic T cells. In vitro experiments further confirmed the capacity of AAT-MSCs to promote the proliferation of Tregs, which consequently fostered an exhausted phenotype in CD8 + T cells, thereby facilitating β cell survival and potentially aiding in diabetes remission. Thus, our findings underscore the significant protective effects of AAT-MSCs, delineate their novel mechanistic insight on recipient immune cells, and provide evidence for the clinical application of AAT-MSCs in treating T1D.

Nonobese diabetic (NOD) mice are a widely used animal model to study mechanisms leading to autoimmune diabetes. A gluten-free diet reduces and delays the incidence of diabetes in NOD mice, but the underlying mechanisms remain largely unknown. In this study, we performed single-cell transcriptomic and flow cytometry analysis of T cells and innate lymphocytes in the spleen and pancreatic lymph nodes of NOD mice fed a gluten-free or standard diet. We observed that the gluten-free diet did not induce a substantial alteration in the abundance or phenotype of any lymphocyte subset that would directly explain its protective effect against diabetes. However, the gluten-free diet induced subtle changes in the differentiation of subsets with previously proposed protective roles in diabetes development, such as Tregs, activated γδT cells, and NKT cells. Globally, the gluten-free diet paradoxically promoted activation and effector differentiation across multiple subpopulations and induced genes regulated by IL-2, IL-7, and IL-15. In contrast, the standard diet induced type I interferon-responsive genes. Overall, the gluten-free diet might prevent diabetes in NOD mice by inducing small-scale changes in multiple cell types rather than acting on a specific lymphocyte subset.
© 2025 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.

CXCR4 cell surface expression is critical for the homing of T regulatory (Treg) cells to the bone marrow (BM). We hypothesize that CXCR4 enrichment on Tregs cell surface may abbreviate their transit time to reach BM. Umbilical cord-blood CD25+ Tregs underwent CXCR4 dual enrichment and ex vivo expansion using the CRANE process to generate CXCR4-enriched Tregs (TregCXCR4) cells, which showed a faster migration across the Transwell membrane toward CXCL12/stromal cell-derived factor 1α (SDF1α) at 15, 30, and 60 min, when compared to unmanipulated Tregcontrol cells (p < 0.0001). TregCXCR4 exhibited preferential homing to BM in vivo at 12 and 24 h. Metacluster analysis of BM showed a decrease in CD8+ and an increase in CD39 and CD73 and CXCR5 when compared to Tregcontrol. TregCXCR4 decreased plasma TGF-β1/β2 and IFN-γ levels. When compared to control, TregCXCR4 cells decreased in CD8+ T cell, IFN-γ, and TNF-α expression in BM. We conclude that TregCXCR4 show enhanced migration toward CXCL12/SDF1α and a preferential homing to BM resulting in resolution of inflammation.
© 2024 The Author(s).

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.
© 2024. The Author(s), under exclusive licence to Springer Nature Limited.

Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.
© 2024 The Author(s).