Chemokine receptors on human regulatory T cells during cutaneous leishmaniasis.

The aim of this work was to define the population of regulatory T cells (Tregs) which are circulating in the blood of Leishmania infected individuals clinically displaying a lesion (active disease-AD) and sub-clinical (SC) ones. We have individually collected blood samples, processed the PBMC and stained with fluorochrome-conjugated antibodies against CD3, CD4, Foxp3, CD25, CTLA-4, Ki-67, CCR4, CCR5, and CCR7. Cells were analyzed by flow cytometry. Our results suggest that CD25 and CTLA-4 are upregulated in Tregs of AD patients when compared to SC and uninfected (UN) controls. Moreover, Tregs proliferate upon infection based on Ki-67 nuclear antigen staining. Finally, we have observed that these Tregs of SC and AD patients upregulate CCR4, but not CCR5 and CCR7. There is an increase in the number of circulating Tregs in the blood of Leishmania infected individuals. These cells are potentially more suppressive based on the increased upregulation of CD25 and CTLA-4 during clinical infection (AD) when compared to SC infection. Tregs of both SC and AD cohorts are proliferating and express CCR4, which potentially guide them to the skin, but do not upregulate CCR5 and CCR7.

Blood neutrophils from children with COVID-19 exhibit both inflammatory and anti-inflammatory markers.

BACKGROUND: Perhaps reflecting that children with COVID-19 rarely exhibit severe respiratory symptoms and often remain asymptomatic, little attention has been paid to explore the immune response in pediatric COVID-19. Here, we analyzed the phenotype and function of circulating neutrophils from children with COVID-19. METHODS: An observational study including 182 children with COVID-19, 21 children with multisystem inflammatory syndrome (MIS-C), and 40 healthy children was performed in Buenos Aires, Argentina. Neutrophil phenotype was analyzed by flow cytometry in blood samples. Cytokine production, plasma levels of IgG antibodies directed to the spike protein of SARS-CoV-2 and citrullinated histone H3 were measured by ELISA. Cell-free DNA was quantified by fluorometry. FINDINGS: Compared with healthy controls, neutrophils from children with COVID-19 showed a lower expression of CD11b, CD66b, and L-selectin but a higher expression of the activation markers HLA-DR, CD64 and PECAM-1 and the inhibitory receptors LAIR-1 and PD-L1. No differences in the production of cytokines and NETs were observed. Interestingly, the expression of CD64 in neutrophils and the serum concentration of IgG antibodies directed to the spike protein of SARS-CoV-2 distinguished asymptomatic from mild and moderate COVID-19. INTERPRETATION: Acute lung injury is a prominent feature of severe COVID-19 in adults. A low expression of adhesion molecules together with a high expression of inhibitory receptors in neutrophils from children with COVID-19 might prevent tissue infiltration by neutrophils preserving lung function. FUNDING: This study was supported by the Ministry of Science and Technology (National Agency for Scientific and Technological Promotion, IP-COVID-19-0277 and PMO BID PICT 2018-2548), and University of Buenos Aires from Argentina (20020170100573BA).

Unraveling the Role of Immune Checkpoints in Leishmaniasis.

Leishmaniasis are Neglected Tropical Diseases affecting millions of people every year in at least 98 countries and is one of the major unsolved world health issues. Leishmania is a parasitic protozoa which are transmitted by infected sandflies and in the host they mainly infect macrophages. Immunity elicited against those parasites is complex and immune checkpoints play a key role regulating its function. T cell receptors and their respective ligands, such as PD-1, CTLA-4, CD200, CD40, OX40, HVEM, LIGHT, 2B4 and TIM-3 have been characterized for their role in regulating adaptive immunity against different pathogens. However, the exact role those receptors perform during Leishmania infections remains to be better determined. This article addresses the key role immune checkpoints play during Leishmania infections, the limiting factors and translational implications.

An Animal Model of Acute and Chronic Chagas Disease With the Reticulotropic Y Strain of Trypanosoma cruzi That Depicts the Multifunctionality and Dysfunctionality of T Cells.

Chagas disease (ChD), a complex and persistent parasitosis caused by Trypanosoma cruzi, represents a natural model of chronic infection, in which some people exhibit cardiac or digestive complications that can result in death 20-40 years after the initial infection. Nonetheless, due to unknown mechanisms, some T. cruzi-infected individuals remain asymptomatic throughout their lives. Actually, no vaccine is available to prevent ChD, and treatments for chronic ChD patients are controversial. Chronically T. cruzi-infected individuals exhibit a deterioration of T cell function, an exhaustion state characterized by poor cytokine production and increased inhibitory receptor co-expression, suggesting that these changes are potentially related to ChD progression. Moreover, an effective anti-parasitic treatment appears to reverse this state and improve the T cell response. Taking into account these findings, the functionality state of T cells might provide a potential correlate of protection to detect individuals who will or will not develop the severe forms of ChD. Consequently, we investigated the T cell response, analyzed by flow cytometry with two multicolor immunofluorescence panels, to assess cytokines/cytotoxic molecules and the expression of inhibitory receptors, in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi-infected mice. Acute ChD induced a high antigen-specific multifunctional T cell response by producing IFN-γ, TNF-α, IL-2, granzyme B, and perforin; and a high frequency of T cells co-expressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional antigen-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) co-expressed on T cells. Taken together, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression. Thus, these results constitute a model that will facilitate an in-depth search for immune markers and correlates of protection, as well as long-term studies of new immunotherapy strategies for ChD.

CD8+ T Cell Response to Trypanosoma cruzi Antigens during Chronic Chagas Disease.

Flow cytometry is a valuable technique in cellular immunology that allows evaluating effective parameters of the immune response associated with CD8+ T cells. During Chagas disease, infection caused by Trypanosoma cruzi parasite, similar to other intracellular infectious agents, antigen-specific CD8+ T cells are essential for controlling the infection. However, CD8+ T cell response is only partially effective in some chronic Chagas disease patients. Thus, characterization and phenotyping of T. cruzi-specific CD8+ T cells are of great importance during chronic Chagas disease.

Expression of inhibitory receptors on CD4+ and CD8+ T cells from healthy Colombian donors / Expresión de receptores Inhibidores en linfocitos T CD4+ y CD8+ de donantes sanos Colombianos / Expressão de receptores inibitórios sobre linfócitos T CD4+ e CD8+ de doadores saudáveis Colombianos

T cell activation involves positive cellular signals that promote effector functions and negative signals that contribute to the regulation of these responses. These regulatory signals are generated upon activation of receptors on T cells that include CD160, 2B4, Programmed Death-1 and CTLA-4. Objective. To evaluate the expression of inhibitory receptors like CD160, 2B4, Programmed Death-1 and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. Materials and methods. Peripheral blood mononuclear cells from 30 healthy donors from Bogotá (Colombia) were obtained via Ficoll-Hypaque density gradient and cells were stained with specific conjugated antibodies previously titrated. Results. The CD160, 2B4, and Programmed Death-1 inhibitory markers were detected on CD4+ T cells with expression levels of 0.35%, 1.04%, and 1.35%, respectively. On CD8+ T cells, these markers were expressed at higher levels: 16%, 8.97%, and 4.3%, respectively. In contrast to the other receptors, CTLA-4 frequency of expression showed no significant difference between CD4+ (1.56%) and CD8+ (1.53%) T cells. Frequency of CD160/2B4 and CTLA-4/ Programmed Death-1 coexpression was 0.18% and 0.09% on CD4+ cells, and 4.02% and 0.2% on CD8+ T cells. Conclusions. This is the first report showing the frequency of inhibitory receptors such as CD160, 2B4, Programmed Death-1, and CTLA-4 on CD4+ and CD8+ T cells from healthy Colombian donors. Our findings serve as a baseline for the analysis and comparison of these receptors in Colombian populations with different disease conditions.

La activación de células T involucra señales positivas que promueven funciones efectoras y señales negativas que contribuyen a la regulación de la respuesta inmune. Estas actividades regulatorias son generadas por la activación de receptores de las células T e incluyen moléculas como CD160, 2B4, PD-1 y CTLA-4. Objetivo. Evaluar la expresión de los receptores inhibitorios CD160, 2B4, PD-1 y CTLA-4 en células T CD4+ y CD8+ de donantes sanos colombianos. Materiales y métodos. Se obtuvieron células mononucleares de sangre periférica de 30 donantes sanos provenientes de Bogotá (Colombia) mediante gradiente de densidad por Ficoll-Hypaque, las células fueron marcadas con anticuerpos conjugados a fluorocromos previamente titulados. Resultados. CD160, 2B4, y PD-1 presentaron porcentajes de expresión en células T CD4+ de 0.35%, 1.04% y 1.35% respectivamente. En células T CD8+ estos receptores mostraron niveles de expresión más altos con porcentajes de 16.3%, 8.97% y 4.3% respectivamente. A diferencia de los otros receptores, CTLA-4 no mostró diferencias entre células T CD4+ (1.56%) y CD8+ (1.53%). Los porcentajes de co-expresión de CD160/2B4 y CTLA-4/PD-1 en células T CD4+ fueron de 0.18% y 0.09%, en células T CD8+ se observaron porcentajes de expresión de 4.02% y 0.2%. Conclusión. Este es el primer reporte que muestra la frecuencia de expresión de receptores inhibitorios tales como CD160, 2B4, PD-1 y CTLA-4 en células T CD4+ y CD8+ de donantes sanos colombianos. Estos hallazgos representan una línea de base para el análisis y la comparación de estos receptores en la población colombiana con diferentes enfermedades.

A ativação de células T envolve sinais positivos que promovem funções efetoras e sinais negativos que contribuem para a regulação da resposta imune. Estas atividades reguladoras são geradas através da ativação de receptores de células T e incluem moléculas tais como CD160, 2B4, PD-1 e CTLA-4. Objetivo. Avaliar a expressão de receptores inibitórios CD160 e 2B4, PD-1 e CTLA-4 em células T CD4+ e CD8+ de doadores saudáveis colombianos. Materiais e métodos. Foram obtidas células mononucleares do sangue periférico de 30 doadores saudáveis provenientes de Bogotá (Colômbia) a partir de gradiente de densidade por Ficoll-Hypaque, as células foram marcadas com anticorpos conjugados com fluorocromos previamente titulados. Resultados. CD160, 2B4, e PD-1 mostraram percentagens de expressão em células T CD4+ de 0,35%, 1,04% e 1,35%, respectivamente. Em células T CD8+ estes receptores mostraram níveis de expressão mais elevados com percentagens de 16,3%, 8,97% e 4,3% respectivamente. Ao contrário de outros receptores, CTLA-4 não apresentou diferenças entre células T CD4+ (1,56%) e CD8+ (1,53%). As percentagens de co-expressão de CD160/2B4 e CTLA-4/PD-1 em células T CD4+ foram de 0,18% e 0,09%; em células T CD8+ foram observadas percentagens de expressão de 4,02% e 0,2%. Conclusão. Este é o primeiro relatório que apresenta a frequência de expressão de receptores inibitórios tais como CD160, 2B4, PD-1 e CTLA-4 em células T CD4+ e CD8+ de doadores saudáveis colombianos. Estes resultados representam uma linha de base para análise e comparação desses receptores na população colombiana com diferentes doenças.