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- Naive and stem cell memory T cell subset recovery reveals opposing reconstitution patterns in CD4 and CD8 T cells in chronic graft vs. host diseasePublication . Soares, Maria Vieira; Azevedo, Rita I.; Ferreira, Ines; Bucar, Sara; Ribeiro, Ana C.; Vieira, Ana I. S.; Pereira, Paulo N G; Ribeiro, Ruy M.; Ligeiro, Dario; Alho, Ana Cristina; Sampaio Soares, António; Camacho, Nádia; Martins, Carlos; Lourenço, Fernanda; Moreno, Raul; Ritz, Jerome; Lacerda, JoãoThe success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of hematological malignancies remains hampered by life-threatening chronic graft vs. host disease (cGVHD). Although multifactorial in nature, cGVHD has been associated with imbalances between effector and regulatory T cells (Treg). To further elucidate this issue, we performed a prospective analysis of patients undergoing unrelated donor allo-HSCT after a reduced intensity conditioning (RIC) regimen containing anti-thymocyte globulin (ATG) and the same GVHD prophylaxis, at a single institution. We studied T cell subset homeostasis over a 24-month follow-up after HSCT in a comparative analysis of patients with and without cGVHD. We also quantified naive and memory T cell subsets, proliferation and expression of the apoptosis-related proteins Bcl-2 and CD95. Finally, we assessed thymic function by T cell receptor excision circle (TREC) quantification and T cell receptor (TCR) diversity by TCRVβ spectratyping. While the total number of conventional CD4 (Tcon) and CD8 T cells was similar between patient groups, Treg were decreased in cGVHD patients. Interestingly, we also observed divergent patterns of Naive and Stem Cell Memory (SCM) subset recovery in Treg and Tcon compared to CD8. Patients with cGVHD showed impaired recovery of Naive and SCM Tcon and Treg, but significantly increased frequencies and absolute numbers of Naive and SCM were observed in the CD8 pool. Markedly increased EMRA CD8 T cells were also noted in cGVHD. Taken together, these results suggest that Naive, SCM and EMRA CD8 play a role in the emergence of cGHVD. Reduced Naive and recent thymic emigrant Tcon and Treg in cGVHD was likely due to impaired thymic output, as it was accompanied by decreased CD4 TREC and TCR diversity. On the other hand, CD8 TCR diversity was similar between patient groups. Furthermore, no correlation was observed between CD8 TREC content and Naive CD8 numbers, suggesting limited thymic production of Naive CD8 T cells in patients after transplant, especially in those developing cGVHD. The mechanisms behind the opposing patterns of CD4 and CD8 subset cell recovery in cGVHD remain elusive, but may be linked to thymic damage associated with the conditioning regimen and/or acute GVHD.
- Light-entrained and brain-tuned circadian circuits regulate ILC3s and gut homeostasisPublication . Godinho-Silva, Cristina; Domingues, Rita G.; Rendas, Miguel; Raposo, Bruno; Ribeiro, Hélder; da Silva, Joaquim Alves; Vieira, Ana I. S.; Costa, Rui M; Barbosa-Morais, Nuno; Carvalho, Tânia; Veiga-Fernandes, HenriqueGroup 3 innate lymphoid cells (ILC3s) are major regulators of inflammation, infection, microbiota composition and metabolism1. ILC3s and neuronal cells have been shown to interact at discrete mucosal locations to steer mucosal defence2,3. Nevertheless, it is unclear whether neuroimmune circuits operate at an organismal level, integrating extrinsic environmental signals to orchestrate ILC3 responses. Here we show that light-entrained and brain-tuned circadian circuits regulate enteric ILC3s, intestinal homeostasis, gut defence and host lipid metabolism in mice. We found that enteric ILC3s display circadian expression of clock genes and ILC3-related transcription factors. ILC3-autonomous ablation of the circadian regulator Arntl led to disrupted gut ILC3 homeostasis, impaired epithelial reactivity, a deregulated microbiome, increased susceptibility to bowel infection and disrupted lipid metabolism. Loss of ILC3-intrinsic Arntl shaped the gut 'postcode receptors' of ILC3s. Strikingly, light-dark cycles, feeding rhythms and microbial cues differentially regulated ILC3 clocks, with light signals being the major entraining cues of ILC3s. Accordingly, surgically or genetically induced deregulation of brain rhythmicity led to disrupted circadian ILC3 oscillations, a deregulated microbiome and altered lipid metabolism. Our work reveals a circadian circuitry that translates environmental light cues into enteric ILC3s, shaping intestinal health, metabolism and organismal homeostasis.
- Suppression by allogeneic-specific regulatory T cells is dependent on the degree of HLA compatibilityPublication . Bianchi, Joana; Vieira, Ana I. S.; Ligeiro, Dário; Azevedo, Rita I.; Lacerda, JoãoRegulatory T cell (Treg) infusion for graft-versus-host disease treatment has been increasingly investigated. However, polyclonal Treg may suppress the desired graft-versus-leukemia effect. Although allogeneic-specific (allo-specific) Treg may provide a more-targeted graft-versus-host disease treatment, there is the need to develop easily translatable expansion protocols and to better characterize their specificity and mechanisms of suppression. In this article, we provide a robust protocol for human allo-specific Treg expansion and characterize their phenotype, potency, and specificity of suppression by testing different expansion conditions and suppression assay milieus. We found that higher concentrations of IL-2 during expansion with allogeneic APC yielded allo-specific Treg that were more-potent suppressors and displayed a more activated phenotype. Although responses to the same APC present during expansion were the most suppressed, responses to third-party APC partially matched to the expansion APC were still significantly more suppressed than responses to fully mismatched APC. Furthermore, suppression of responses to the expansion APC was strictly contact dependent, whereas suppression of responses to mismatched APC was partially independent of contact. Finally, distinct subsets in fresh and expanded Treg could be described using multidimensional visualization techniques. We propose that allo-specific Treg are HLA specific and that the mechanisms of suppression elicited depend on their compatibility with the stimulators.
- Mesenchymal stromal cells induce regulatory T cells via epigenetic conversion of human conventional CD4 T cells in vitroPublication . Azevedo, Rita I.; Minskaia, Ekaterina; Fernandes-Platzgummer, Ana; Vieira, Ana I. S.; da Silva, Cláudia Lobato; Cabral, Joaquim M.S.; Lacerda, JoãoRegulatory T cells (Treg) play a critical role in immune tolerance. The scarcity of Treg therapy clinical trials in humans has been largely due to the difficulty in obtaining sufficient Treg numbers. We performed a preclinical investigation on the potential of mesenchymal stromal cells (MSCs) to expand Treg in vitro to support future clinical trials. Human peripheral blood mononuclear cells from healthy donors were cocultured with allogeneic bone marrow-derived MSCs expanded under xenogeneic-free conditions. Our data show an increase in the counts and frequency of CD4+ CD25high Foxp3+ CD127low Treg cells (4- and 6-fold, respectively) after a 14-day coculture. However, natural Treg do not proliferate in coculture with MSCs. When purified conventional CD4 T cells (Tcon) are cocultured with MSCs, only cells that acquire a Treg-like phenotype proliferate. These MSC-induced Treg-like cells also resemble Treg functionally, since they suppress autologous Tcon proliferation. Importantly, the DNA methylation profile of MSC-induced Treg-like cells more closely resembles that of natural Treg than of Tcon, indicating that this population is stable. The expression of PD-1 is higher in Treg-like cells than in Tcon, whereas the frequency of PDL-1 increases in MSCs after coculture. TGF-β levels are also significantly increased MSC cocultures. Overall, our data suggest that Treg enrichment by MSCs results from Tcon conversion into Treg-like cells, rather than to expansion of natural Treg, possibly through mechanisms involving TGF-β and/or PD-1/PDL-1 expression. This MSC-induced Treg population closely resembles natural Treg in terms of phenotype, suppressive ability, and methylation profile.
- Molecular markers distinguishing T Cell subtypes with TSDR strand-bias methylationPublication . Minskaia, Ekaterina; Saraiva, Bárbara C.; Soares, Maria Vieira; Azevedo, Rita I.; Ribeiro, Ruy M.; Kumar, Saumya; Vieira, Ana I. S.; Lacerda, JoãoHuman regulatory CD4+CD25+FOXP3+ T cells (Treg) play important roles in the maintenance of self-tolerance and immune homeostasis in various disease settings and are also involved in the suppression of effective immune responses. These cells are heterogeneous in phenotype and function, and the ability to reliably distinguish between various FOXP3-expressing subpopulations can affect the development of successful therapies. This study demonstrates that hypomethylated CpG sites, present in four regions of the FOXP3 locus, CAMTA1 and FUT7 gene regions, can be used to distinguish several subsets of Treg from conventional CD4+ T lymphocytes (Tcon) in donors of both genders. We describe a previously unreported strand-bias hemimethylation pattern in FOXP3 promoter and TSDR in donors of both genders, with the coding strand being demethylated within promoter and methylated within TSDR in all CD4+ lymphocyte subtypes, whereas the template strand follows the previously described pattern of methylation with both regions being more demethylated in Treg subtypes and mostly methylated in Tcon. This strand-specific approach within the TSDR may prove to be instrumental in correctly defining Treg subsets in health and in disease.