CXCR5 and TIM-3 expressions define distinct exhausted T cell subsets in experimental cutaneous infection with Leishmania mexicana.

T-cell exhaustion is a key stage in chronic infections since it limits immunopathology, but also hinders the elimination of pathogens. Exhausted T (Tex) cells encompass dynamic subsets, including progenitor cells that sustain long-term immunity through their memory/stem like properties, and terminally-differentiated cells, resembling the so-called Tex cells. The presence of Tex cells in chronic leishmaniasis has been reported in humans and murine models, yet their heterogeneity remains unexplored. Using flow cytometry, we identified Tex cells subtypes based on PD-1, CXCR5 and TIM-3 expressions in draining lymph nodes (dLNs) and lesion sites of C57BL/6 mice infected with L. mexicana at 30-, 60- and 90-days post-infection. We showed that infected mice developed a chronic infection characterized by non-healing lesions with a high parasite load and impaired Th1/Th2 cytokine production. Throughout the infection, PD-1+ cells were observed in dLNs, in addition to an enhanced expression of PD-1 in both CD4+ and CD8+ T lymphocytes. We demonstrated that CD4+ and CD8+ T cells were subdivided into PD-1+CXCR5+TIM-3- (CXCR5+), PD-1+CXCR5+TIM-3+ (CXCR5+TIM-3+), and PD-1+CXCR5-TIM-3+ (TIM-3+) subsets. CXCR5+ Tex cells were detected in dLNs during the whole course of the infection, whereas TIM-3+ cells were predominantly localized in the infection sites at day 90. CXCR5+TIM-3+ cells only increased at 30 and 60 days of infection in dLNs, whereas no increase was observed in the lesions. Phenotypic analysis revealed that CXCR5+ cells expressed significantly higher levels of CCR7 and lower levels of CX3CR1, PD-1, TIM-3, and CD39 compared to the TIM-3+ subset. CXCR5+TIM-3+ cells expressed the highest levels of all exhaustion-associated markers and of CX3CR1. In agreement with a less exhausted phenotype, the frequency of proliferating Ki-67 and IFN-γ expressing cells was significantly higher in the CXCR5+ subset within both CD4+ and CD8+ T cells compared to their respective TIM-3+ subsets, whereas CD8+CXCR5+TIM-3+ and CD8+TIM-3+ subsets showed an enhanced frequency of degranulating CD107a+ cells. In summary, we identified a novel, less-differentiated CXCR5+ Tex subset in experimental cutaneous leishmaniasis caused by L. mexicana. Targeting these cells through immune checkpoint inhibitors such as anti-PD-1 or anti PD-L1 might improve the current treatment for patients with the chronic forms of leishmaniasis.

Caracterización morfológica, génica y protéica en la diferenciación de células madre embrionarias de ratón a células pancreáticas tempranas / Morphological, genetic and protein characterization in the differentiation of embryonic stem cells to early pancreatic cells

Debido al auge de la medicina regenerativa, las Células Madre (SC) representan una fuente de reemplazo celular para cualquier tejido, decidiendo emprender este trabajo de investigación con el objetivo de diferenciar células madre embrionarias de ratón (mESC) a células pancreáticas tempranas, realizando su caracterización génica y morfológica. Primeramente se cultivaron y arrestaron en su ciclo celular fibroblastos embrionarios de ratón (MEF) con mitomicina, posteriormente se expandieron las mESC y se sometieron a un protocolo de diferenciación de 21 días hacía células pancreáticas tempranas, evaluándose durante la diferenciación su morfología y expresión relativa de los genes sox-17, pdx-1, ins-1 e ins-2, determinando además la producción de las proteínas insulina y glucagón mediante inmunocitoquímica y citometría de flujo. Se obtuvieron cuerpos embrionarios (EBs) a partir de mESC, con características morfológicas diferentes de acuerdo a su diferenciación, los cuales expresaron genes de la línea germinal endodérmica (sox-17 y pdx-1) a los días 0, 11 y 17 de diferenciación, gen inductor del desarrollo embrionario pancreático (pdx-1) al día 11 de diferenciación y, genes de expresión pancreática (ins-1 e ins-2) a los días 17 y 21 de diferenciación. Finalmente se detectó la producción de proteínas insulina y glucagón en los EBs al día 21 de diferenciación. Se logró diferenciar mESC. El análisis morfológico evidenció cúmulos celulares tridimensionales correspondientes a EBs. Con el análisis de los patrones de expresión génica, se distinguieron inicialmente células con características genéticas de endodermo y posteriormente a partir del día 17 células pancreáticas tempranas, las cuales al día 21 de diferenciación expresaron las proteínas insulina y glucagón...

Due to the boom in regenerative medicine, Stem Cells (SC) represent a source of cell replacement to any tissue, we decided to undertake this research with the objective of differentiating mouse embryonic stem cells (mESC) to early pancreatic cells, developing their genetic and morphological characterization. Initially Mouse embryonic fibroblasts (MEF) were grown and arrested in their cell cycle with mitomycin, subsequently mouse embryonic SC (mESC) were expanded and subjected in to a pancreatic cell differentiation protocol of 21 days. During differentiation, morphology and the relative expression of sox-17, pdx-1, Ins-1 and Ins-2 genes were assessed, also the production of insulin and glucagon proteins was determinated by fluorescence microscopy and flow cytometry. Embryoid bodies (EBs) were obtained from mESC, with different morphological characteristics according to their differentiation, which expressed endodermal germ line genes (sox-17 y pdx-1) at days 0, 11 and 17 of differentiation, an inductor gene of embryonic pancreas development (pdx-1) was detected at day 11 of differentiation. Pancreas genes (ins-1 e ins-2) were expressed at day 17 and 21 of differentiation. Finally the production of insulin and glucagon proteins was detected on the EBS at day 21 of differentiation. In conclusion, the mESC differentiation was achieved. The morphological analysis evidenced three-dimensional cell clusters corresponding to EBs. Analysis of the gene expression patterns in the differentiation process, cells initially showed genetic characteristics of endoderm and thereafter from day 17 of differentiation characteristics of early pancreatic cells which by day 21 of differentiation expressed insulin and glucagon proteins...