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. 2014 Jan 15;209(2):290-9.
doi: 10.1093/infdis/jit401. Epub 2013 Aug 6.

CD8 T cell exhaustion in human visceral leishmaniasis

Shalini Gautam  1 , Rajiv Kumar, Neetu Singh, Abhishek Kumar Singh, Madhukar Rai, David Sacks, Shyam Sundar, Susanne Nylén
Affiliations

CD8 T cell exhaustion in human visceral leishmaniasis

Shalini Gautam et al. J Infect Dis. .

Abstract

Little is known about CD8 T cells in human visceral leishmaniasis (VL) and it is unclear if these cells have a protective, pathological and/or suppressive function. In experimental VL CD8 T cells have been shown to contribute to parasite control and play an important role in vaccine-generated immunity. To better understand the role of CD8 T cells in human VL, we examined molecules associated with anergy and cytotoxic T lymphocytes (CTL) in peripheral blood mononuclear cells (PBMC) and splenic aspirates (SA), and in CD8 cells derived from these tissues. Gene and surface marker expression suggest that splenic CD8 cell predominantly display an anergic phenotype, whereas CD8-PBMC have features of both anergic cells and CTLs. CD8 cells contribute to the baseline IFNγ levels in whole blood (WB) and SA cultures, but not to the Leishmania induced IFNγ release that is revealed using WB cultures. Blockade of CTLA-4 or PD1 had no effect on IFNγ production or parasite survival in SA cultures. Following cure, CD8 T cells contribute to the Leishmania induced IFNγ production observed in Leishmania stimulated cell cultures. We suggest CD8 T cells are driven to anergy/exhaustion in human VL, which affect their ability to contribute to protective immune responses.

Keywords: CD8 T cell; CTLA-4; IL-10; PBMC; PD1; Visceral leishmaniasis; spleen.

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Figures

Figure 1.
Figure 1.
SA and PBMC express mRNAs differently. Relative mRNA expression of anergy and CTL associated genes in tissue samples from controls and VL patients pre- and 3 weeks post-treatment (A) whole SA and Splenic MNC (HOD) and (B) VL and EC PBMC. EC were not analyzed for perforin, granzyme A (GrzA) or TRAIL mRNA. C, Comparison of mRNA expression in splenic aspirates and PBMC pre-treatment. The relative amounts of RNA shown are not comparable between different graphs. Pre- and post-treatment samples were from the same donors and compared using students' paired t-test. EC/HOD and VL groups were compared using students' t-test. Non-parametric tests (Mann–Whitney U test or Wilcoxon matched pair test) were used when normality test failed. Significance differences between groups where n ≥ 6, are indicated with P-values. Each symbol represents one sample. Different donors/samples may be presented in different graphs.
Figure 2.
Figure 2.
VL CD8 cells express high levels of both IL-10 and IFNγ mRNA. mRNA expression in VL and EC PBMC subsets isolated sequentially by MACS beads as indicated. Significant differences are indicated by P-values for IL-10 and IFNγ. Foxp3 expression was significantly higher in CD25+ cells compared to all other subsets (P = .001). CD3 ε expression was significantly higher in all positively selected cell subsets (CD25, CD8 and CD3) compared to whole PBMC and depleted cells (P < .05). Each symbol represents one donor. Comparison between VL and EC were done using t-test and between cell populations originating from the same donors using a paired t-test. Non-parametric tests (Mann–Whitney U test or Wilcoxon matched pair test) were used when normality test failed.
Figure 3.
Figure 3.
Gene expression in SA and PBMC CD8 cells is suggestive of anergic/exhausted cells. A, Relative gene expression, as indicated in figure, in MACS purified CD8 cells from PBMC and spleen of patient and controls. B, Comparison of relative gene expression in paired pre-treatment SA and PBMC CD8 cells. Samples analyzed for different transcripts are not necessarily from the same donors, thus only samples within the same graph can be compared. Significance differences between groups where n ≥ 6, are indicated with P-values. Each symbol represents one sample. Paired samples are indicated with a solid line. All groups were compared using Mann–Whitney U test. The pre-post treatment groups contain both paired and unpaired samples. P-values in graphs show test for unpaired samples. Wilcoxon match paired test was used for comparison of paired PBMC and SA CD8 cells.
Figure 4.
Figure 4.
VL CD8T cells have elevated surface expression of anergy associated proteins. Surface expressions of (A) CTLA-4 and PD1 (B) Perforin, Granzyme A and Granulysin and (C) iNKRs. Significant differences between groups are indicated with P-values in graphs. Different donors/samples may be presented in different graphs. Comparison was made using student's t-test. Pre- and post-treatment samples were not paired.
Figure 5.
Figure 5.
CD8 effector responses are lacking in active VL. A, Background levels of IFNγ in 3-day cultures of whole splenic aspirate (SA) or SA depleted of CD8 cells (SA-CD8) (B) Background or SLA induced levels of IFNγ and IL-10 in 24 hours whole blood (WB) cultures depleted or not of CD8 cells (WB-CD8). SLA values shown are net responses with background values subtracted. C, Parasite growth following CD8 depletion. D, Expression of LAMP1 in different cell populations as indicated on x-axis in unstimulated (left) and SLA (center) or SEB (right) stimulated cultures. Statistical comparison was done using 1-way ANOVA followed by comparison between VL and EC groups using Mann–Whitney U test and Wilcoxon matched pair test for comparison between stimulations (P values indicated).
Figure 6.
Figure 6.
CD8 cells contribute to antigen specific IFNγ production following clinical cure (6 months post-treatment). A, Frequency of IFNγ CD8T cells out of total (CD3+) T cell population following 24 hours stimulation with or without SLA as determined by intra cellular staining and FACS analysis. BFA was added to the cultures for the last 8 hours. B, Effect of pan-HLA-I blockade on IFNγ detection in supernatants from SLA stimulated whole blood following 24 hours of culture, comparison was done with isotype control stimulated samples (C) Effect of CD8 depletion on IFNγ detection in supernatants from SLA stimulated WBA. Net values (IFNγ in stimulated minus IFNγ in unstimulated samples) are shown in figure B & C. Statistical significances using student's paired t-test are indicated by P-values in figures.
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References

    1. Kaye PM, Cooke A, Lund T, Wattie M, Blackwell JM. Altered course of visceral leishmaniasis in mice expressing transgenic I-E molecules. Eur J Immunol. 1992;22:357–64. - PubMed
    1. Stager S, Smith DF, Kaye PM. Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol. 2000;165:7064–71. - PubMed
    1. Stern JJ, Oca MJ, Rubin BY, Anderson SL, Murray HW. Role of L3T4+ and LyT-2+ cells in experimental visceral leishmaniasis. J Immunol. 1988;140:3971–7. - PubMed
    1. Tsagozis P, Karagouni E, Dotsika E. CD8(+) T cells with parasite-specific cytotoxic activity and a Tc1 profile of cytokine and chemokine secretion develop in experimental visceral leishmaniasis. Parasite Immunol. 2003;25:569–79. - PubMed
    1. Tsagozis P, Karagouni E, Dotsika E. Function of CD8+ T lymphocytes in a self-curing mouse model of visceral leishmaniasis. Parasitol Int. 2005;54:139–46. - PubMed

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