Expression of Inhibitory Receptors on T and NK Cells Defines Immunological Phenotypes of HCV Patients with Advanced Liver Fibrosis

Chronic HCV can result in advanced liver disease, including cirrhosis. Patients with advanced fibrosis experience poor clinical outcomes and increased risk for hepatocellular carcinoma (HCC). These outcomes are, in part, a consequence of immune dysfunction. Increased inhibitory receptor and Galectin-9 (GAL-9) expression is a possible mechanism promoting lymphocyte dysfunction. In this study, we measured the expression of inhibitory receptors and GAL-9 on T/NK cells of patients with chronic HCV with no to moderate fibrosis (F0-F2) and advanced fibrosis (F3-F4). To analyze their co-expression, we employed t-SNE analysis. Notably, we found that F3-F4 patients had higher frequencies of >3 inhibitory receptor co-expression on NK cells. Moreover, F3-F4 patients manifest a higher frequency of NK cells co-expressing TIGIT and TIM-3, and CD4/NK cells co-expressing LAG-3 and GAL-9. In conclusion, we identified phenotypes of immune dysregulation that could explain the increased susceptibility to infection and HCC in patients with chronic HCV with advanced fibrosis.Virology; Immunolog

Deficiency of Leishmania phosphoglycans influences the magnitude but does not affect the quality of secondary (memory) anti-Leishmania immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8(+) T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals

Regulatory T cells enhance susceptibility to experimental Trypanosoma congolense infection independent of mouse genetic background.

BACKGROUND: BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines. METHODOLOGY/FINDINGS: BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10(3)T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25(+) T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4(+)CD25(+)Foxp3(+) T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25(+) cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25(+) T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines. CONCLUSION: Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice

CD8⁺ T cells Are Preferentially Activated during Primary Low Dose <i>Leishmania major</i> Infection but Are Completely Dispensable during Secondary Anti-<i>Leishmania</i> Immunity

<div><p>We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose <i>Leishmania major</i> infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose <i>L. major</i> infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-<i>Leishmania</i> immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-<i>Leishmania</i> immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during <i>in vitro</i> and <i>in vivo</i> recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary <i>L. major</i> challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-<i>Leishmania</i> immunity following low dose infection, they are completely dispensable during secondary immunity.</p></div

<i>lpg2− L. major</i> mediated protection is not associated with a strong DTH response.

<p>C57BL/6 mice infected with WT and <i>lpg2</i>− <i>L. major</i> (>16 wks) were challenge with 5 million WT parasites in their contralateral footpad and delayed DTH response was measured 72 hr post-challenge (<b>A</b>)<b>.</b> After 3 wk post challenge, mice were sacrificed and parasite burden was determined (<b>B</b>)<b>.</b> In some experiments, CD3⁺ T cells were purified from spleens of WT or <i>lpg2-L. major</i>-infected mice and adoptively transferred into naïve mice that were then challenged with virulent <i>L. major</i>. Three weeks after challenge, mice were sacrificed to determine parasite burden (<b>C</b>). Data presented are representative of 4 (A and B) and 2 independent experiments (n = 3–5 mice per group) with similar results.</p

High and low dose infections preferentially expand CD4⁺ and CD8⁺ T cells, respectively.

<p>C57BL/6 mice were infected with low (1×10³) or high (2×10⁶) dose <i>L. major</i> promastigotes in their right hind footpad, sacrificed after 7 days and the draining lymph-node cells were labelled with CFSE dye and co-cultured with <i>L. major</i>-infected bone marrow-derived dendritic cells (BMDC) at a DC: lymph node cell ratio of 1∶100. After 4 days, the percentages of proliferating (CFSE^lo) CD4⁺ and CD8⁺ T cells (A and B) were assessed by flow cytometry. (A) is a representative histogram plots of individual animals while B represents the mean +/− SE of proliferating cells of all the animals (6 mice) within the group. Proliferating (CFSE^lo) IFN-γ (upper panels) and TNF (lower panels) -producing CD4⁺ (C and D) and CD8⁺ (E and F) T cells were also determined by flow cytometry. C and E are representative dot plots of individual animals within the group while D and F represent the mean +/− SE of proliferating cytokine-producing cells of all the animals (6 mice) within the group. The lymph nodes draining the infected feet were collected and digested with collagenase and the cells were then stained with different fluorochrome-conjugated antibodies against different DC subsets and the absolute numbers (upper panel) and percentage (lower panel) of CD11c⁺MHCII⁺ (G) and CD11c⁺CD103⁺CD8α⁺ (H) dendritic cells were determined by flow cytometry. Live cells were first gated on CD11c⁺ cells and further analyzed for MHC class II, CD103 and CD8α expression. Results are representative of 2 independent experiments (n = 6 mice/group) with similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p

T cells from the spleens of WT and <i>lpg2−</i> infected C57BL/6 mice proliferate and produce IFN-γ in response to <i>L. major</i>–infected DCs.

<p>C57BL/6 mice were infected with WT and <i>lpg2− L. major</i> and the kinetics of lesion development and progression was monitored for over 16 weeks (<b>A</b>). At 16 weeks after infection, infected mice were sacrificed and parasite burden was determined by limiting dilution (<b>B</b>). Spleen cells were restimulated <i>in vitro</i> with SLA for 72 hr and the frequency of IFN-γ-producing cells was determined by flow cytometry (<b>C</b>). The culture supernatant fluids were assessed for IFN-γ (<b>D</b>) and IL-10 (<b>E</b>) by ELISA. In some experiments, CFSE-labeled purified T cells purified from infected mice were co-cultured for 5 days with <i>L. major</i>–infected BMDCs (T:BMDC = 100∶1), stained for surface expression of CD4 and CD8 and intracellularly for IFN-γ and analyzed by flow cytometry. Shown are the percentages of cells that proliferated i.e. diluted CFSE dye (<b>F</b>) and produce IFN-γ (<b>G</b>). Data are presented are representative of 2 independent experiments with similar results.</p