Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103- counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.
© 2024. The Author(s).

The serine/threonine-specific Moloney murine leukemia virus (PIM) kinase family (i.e., PIM1, PIM2, and PIM3) has been extensively studied in tumorigenesis. PIM kinases are downstream of several cytokine signaling pathways that drive immune-mediated diseases. Uncontrolled T helper 17 (Th17) cell activation has been associated with the pathogenesis of autoimmunity. However, the detailed molecular function of PIMs in human Th17 cell regulation has yet to be studied. In the present study, we comprehensively investigated how the three PIMs simultaneously alter transcriptional gene regulation during early human Th17 cell differentiation. By combining PIM triple knockdown with bulk and scRNA-seq approaches, we found that PIM deficiency promotes the early expression of key Th17-related genes while suppressing Th1-lineage genes. Further, PIMs modulate Th cell signaling, potentially via STAT1 and STAT3. Overall, our study highlights the inhibitory role of PIMs in human Th17 cell differentiation, thereby suggesting their association with autoimmune phenotypes.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their in vivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8+ T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma.
Crown Copyright © 2022.

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.
© 2021. The Author(s).

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.