Fig 1.
Chronic L. donovani infection leads to accumulation of CD4+ T cells in the spleen.
(A) Splenic parasite burden was determined by real time RT-PCR of L. donovani 18s mRNA at 0, 7, 14, 21, and 28 days post-infection). (B) The frequency of CD4+ splenic T cells from uninfected and 28-day infected hamsters was determined by flow cytometry. Total lymphocytes were gated based on FSC and SSC. Shown is the frequency of CD4+ lymphocytes in total spleen cell population (n = 4–6 hamsters) *p<0.05.
Table 1.
Upregulated genes related to T cell activation and function a.
Fig 2.
Splenic expression of markers of CD4+ T cell subpopulations over the course of chronic L. donovani infection.
RNA was isolated from spleen tissue from uninfected controls (C) and hamsters infected for 7, 14, 21 and 28 days. Gene expression for (A) Th1 (B) Th2 and (C) Treg cells was determined by real time RT-PCR. Fold change was calculated relative to basal gene expression in uninfected baby hamster kidney (BHK) cell line. Figures are representative of at least 3 independent experiments with 3–6 animals per experiment. *p<0.05, **p<0.01, ****p<0.0001. Th1-associated genes indicated by the color red, Th2-associated genes indicated by the color green, Treg-associated genes indicated by the color purple.
Fig 3.
Splenic CD4+ T cell expression of markers of T cell subpopulations over the course of chronic L. donovani infection.
CD4+ T cells were isolated from spleen tissue from uninfected (Un) or 28-day infected (Inf) hamsters by positive selection. (A) The post-separation purity of CD3+CD4+ T cells was >90% in multiple independent experiments. (B-E) RNA was isolated from the purified splenic CD4+ T cell population and mRNA expression of markers of Th1 (B, C), Th2 (D), and Treg cells (E) was determined by real time RT-PCR. Results are expressed as a relative fold-increase between experimental samples and uninfected BHK cells. Shown is the mean and SEM of a single experiment representative of 2 independent experiments from 6 hamsters per group. Expression of (B) T-bet and (E) Foxp3 was verified in CD4+ splenocytes by flow cytometry. Data is representative of at least 2 independent experiments. *p<0.05, **p<0.01. Th1-associated genes indicated by the color red, Th2-associated genes indicated by the color green, Treg-associated genes indicated by the color purple, Th2/Treg-associated genes indicated by the color black, Th1/Th2-associated genes indicated by the color blue.
Fig 4.
Chemokine receptor and ligand mRNA expression in hamster spleen tissue over the course of chronic L. donovani infection.
mRNA expression of type 1, type 2 and regulatory type chemokine ligands and their receptors was determined by real time RT-PCR in spleen tissue from uninfected controls (C) or hamsters infected for 7, 14, 21 and 28 days. Results are expressed as a relative fold-increase between experimental samples and uninfected BHK cells. Shown is the mean and SEM of a single experiment representative of 2 independent experiments from 3–6 hamsters per time point. *p<0.05; **p<0.01, ***p<0.001. Th1-associated genes indicated by the color red, Th2/Treg-associated genes indicated by the color black, Th1/Th2-associated genes indicated by the color blue.
Fig 5.
Chemokine ligand mRNA expression in splenic macrophages from chronically infected hamsters.
Splenic macrophages were isolated from uninfected (Un) or 28-day infected (Inf) hamsters. mRNA expression of type 1, type 2 and regulatory type chemokine ligands was determined by real time RT-PCR. Results are expressed as a relative fold-increase compared to uninfected BHK cells. Shown is the mean and SEM of a single experiment representative of 2 independent experiments from 4 hamsters per time point. Student’s t-test was performed to compare control uninfected (C) and chronically 28 day infected (28dpi) samples. *p<0.05. Th1-associated chemokines indicated by the color red, Th1/Th2-associated chemokines indicated by the color blue, Th2/Treg-associated chemokines indicated by the color gray.
Fig 6.
Control of intracellular parasite burden by IFNγ-LPS and splenic CD4+ T cell-mediated macrophage activation.
(A) Hamster bone marrow-derived macrophages were primed with IFNγ before being triggered with LPS and infected with L. donovani. Parasite burden and macrophage activation were determined by measuring mRNA expression (real time RT-PCR) of Leishmania 18s, iNOS and Arg1, respectively. (B) Bone marrow-derived macrophages from uninfected hamsters were infected in vitro with luciferase-transfected L. donovani promastigotes and co-cultured with CD4+ T cells purified from uninfected or 28-day infected hamsters for 48 hours. The intracellular parasite burden in CD4+ T cell-macrophage co-cultures was determined using relative luminescent unit values and is presented as the percent increase or decrease from the baseline (1 hr) parasite burden (100%) for each group. (C) iNOS and Arg1 mRNA expression in CD4+ T cell-macrophage co-cultures was determined by real time RT-PCR at baseline (1 hr) and 48 hours later. Results are expressed as a relative fold change in comparison to the initial time point of each treatment group. All data shown is representative of at least 2 independent experiments with 6 replicates per group. ND = Not Detected. **p<0.01, ***p<0.001, ****p<0.0001.
Fig 7.
Increased splenic expression of inhibitory markers in chronic VL.
mRNA expression for inhibitory markers was determined by real time RT-PCR for hamster (A) total spleen tissue (B) splenic CD4+ T cells and (C) splenic macrophages. Results are expressed as relative fold-increase over uninfected BHK cells. Shown is the mean and SEM of a single experiment representative of 2 independent experiments from 3–6 hamsters per time point. *p<0.05; **p<0.01.
Fig 8.
Ex vivo blockade of PD-L2 reduces parasite burden.
Spleen cells from 28-day chronically infected hamsters were cultured with αPD-L2 antibody or isotype control for 48 hours. The parasite burden (18s mRNA expression) and expression of iNOS, IFNγ, Arg1 and IL-10 were determined by real time RT-PCR. Results are expressed as a relative fold-increase of the αPD-L2 antibody or isotype control groups compared to infected splenocytes at 0 hours. Shown is the mean and SEM of two independent experiments with 6 replicates per group.