Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323-339, to assess mTEC antigen presentation to CD4+ T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.
©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.

Oncoprotein AML1-ETO (AE) derived from t(8;21)(q22;q22) translocation is typically present in a portion of French-American-British-M2 subtype of acute myeloid leukemia (AML). Although these patients have relatively favorable prognoses, substantial numbers of them would relapse after conventional therapy. Here, we explored whether reinforcing the endogenous differentiation potential of t(8;21) AML cells would diminish the associated malignancy. In doing so, we noticed an expansion of immature erythroid blasts featured in both AML1-ETO9a (AE9a) and AE plus c-KIT (N822K) (AK) murine leukemic models. Interestingly, in the AE9a murine model, a spontaneous step-wise erythroid differentiation path, as characterized by the differential expression of CD43/c-Kit and the upregulation of several key erythroid transcription factors (TFs), accompanied the decline or loss of leukemia-initiating potential. Notably, overexpression of one of the key erythroid TFs, Ldb1, potently disrupted the repopulation of AE9a leukemic cells in vivo, suggesting a new promising intervention strategy of t(8;21) AML through enforcing their erythroid differentiation.
© 2022 Federation of American Societies for Experimental Biology.

The cytokine interleukin-3 (IL-3) acts on early hematopoietic precursor cells. In humans, Treg cells secrete IL-3 and repress inflammatory cells except for basophils. The present study aims to elucidate the contribution of IL-3 in the development and the course of allergic asthma. We therefore analyzed the secretion of IL-3 in PBMCs and total blood cells in two cohorts of pre-school children with and without asthma. In a murine model of allergic asthma, we analyzed the phenotype of IL-3-/- mice compared to wild-type mice. PBMCs from asthmatic children showed increased IL-3 secretion, which directly correlated with improved lung function. IL-3-/- asthmatic mice showed increased asthmatic traits. Moreover, IL-3-deficient mice had a defect in T regulatory cells in the lung. In conclusion, IL-3 downregulation was found associated with more severe allergic asthma in pre-school children. Consistently, targeting IL-3 resulted in an induced pathophysiological response in a murine model of allergic asthma.
© 2022 The Author(s).

Cells exist within complex milieus of communicating factors, such as cytokines, that combine to generate context-specific responses, yet nearly all knowledge about the function of each cytokine and the signaling propagated downstream of their recognition is based on the response to individual cytokines. Here, we found that regulatory T cells (Tregs) integrate concurrent signaling initiated by IL-2 and IL-4 to generate a response divergent from the sum of the two pathways in isolation. IL-4 stimulation of STAT6 phosphorylation was blocked by IL-2, while IL-2 and IL-4 synergized to enhance STAT5 phosphorylation, IL-10 production, and the selective proliferation of IL-10-producing Tregs, leading to increased inhibition of conventional T cell activation and the reversal of asthma and multiple sclerosis in mice. These data define a mechanism of combinatorial cytokine signaling and lay the foundation upon which to better understand the origins of cytokine pleiotropy while informing improved the clinical use of cytokines.
© 2021, Zhou et al.

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin-induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin-induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti-inflammatory phenotype (M2), and reduced the antigen-presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B-cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self-maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B-cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion.
© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.