Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8⁺ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8⁺ T cells are required for the development of protective immunity. However, antigen-specific CD8⁺ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8⁺ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8⁺ T cell responses. Here we show that L. donovani parasites evade CD8⁺ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8⁺ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8⁺ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL. The protozoan parasite Leishmania donovani is the cause of visceral leishmaniasis, a chronic disease that currently affects 12 million people worldwide. We are interested in understanding the immune mechanisms that can control infection. Preliminary studies suggested that CD8⁺ T cells can kill parasites and limit disease; however, studying these important killer cells has been hindered, because we do not know what parasite molecules they recognize. To overcome this, we engineered parasites to express ovalbumin. Since many tools exist to track and measure immune cells targeted at ovalbumin, we can now track the specific CD8⁺ T cell responses that develop upon infection with Leishmania. We found that Leishmania initially induced CD8⁺ T cells to divide and produce molecules such as IFN-gamma that may help them to kill parasites. However, the CD8⁺ T cells rapidly lost their effector function and died off as infection progressed. More encouragingly, though, we were able to recover some CD8⁺ T cell function by blocking immune inhibitory molecules that are induced by parasite infection. The recovered T cells killed parasites and controlled infection. These results are important as they could be exploited for the design of new therapeutic vaccine strategies aimed at inducing protective CD8⁺ T cells.

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.

Inflammatory bowel disease (IBD) is a chronic disease of unknown etiology in which genetic factors contribute to development of disease. Single nucleotide polymorphisms (SNPs) in multidrug resistance 1 (MDR1) gene encoding transporter P-glycoprotein have been associated with IBD, but their role in disease susceptibility remains unclear. Therefore, the aim of this study was to investigate the association of three MDR1 polymorphisms, C1236T (rs1128503), G2677T/A (rs2032582) and C3435T (rs1045642), with Serbian IBD patients. A total of 206 IBD patients, 107 Crohn's disease (CD) and 99 ulcerative colitis (UC), and 255 healthy controls were included in the study. All subjects were genotyped using TaqMan SNP genotyping assays. Comparisons between the groups were performed using the Pearson Chi-square test. False discovery rate according to Benjamini-Hochberg procedure was applied to adjust for multiple comparisons. Carriers of T allele of all three MDR1 SNPs were more common in UC patients compared to healthy controls, suggesting predisposing role of T allele of these SNPs in UC pathogenesis. Consistently, TT genotype of C1236T and TTT haplotype were also found more frequently in UC patients. On the other hand, C allele and CC genotype of C1236T and C3435T, as well as G allele and GG genotype of G2677T/A were more frequent in healthy subjects, implying protective role of these variants in UC. Likewise, CGC haplotype and CGC/CGC diplotype were more frequent in controls. Contrary to UC, no statistical difference was observed between CD patients and controls in any of the SNPs analyzed. MDR1 gene variants and haplotypes were associated with UC in Serbian IBD patients, further supporting their potential role in susceptibility to UC.

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1β were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.