Fig 1.
CXCR3 chemokine receptor is highly expressed on specific CD8+ and activated CD4+ T cell surfaces as well as the CXCR3 ligands in the heart of infected mice.
C57BL/6 mice were infected with 1x104 blood forms of the Y strain of Trypanosoma cruzi. a-The bar graph indicates the frequency of VNHRFTLV specific CD8+ T cells and the representative dot-plot graphs show the frequency of specific CD8+ T cells in the spleen. The histogram showed CXCR3 expression on specific CD8+ T in the spleen of naïve and infected mice. b-The bar graph indicates the frequency of VNHRFTLV specific CD8+ T cells and the representative dot-plot graphs show the frequency of specific CD8+ T cells in lymph node. The histogram showed CXCR3 expression on specific CD8+ T in the lymph node of naïve and infected mice 15 days post infection. The grey line represents the naïve animals while the red line represents the infected ones. c-The graphs represent MFI of CXCR3 in specific CD8+ and (d) activated CD4+ T cells. CXCR3 expression was evaluated on days 5, 10, 15, 20 and 30 post infection (dpi) in the spleen and lymph node. e-Relative expression of CXCL10 and (f) CXCL9 chemokines in naïve and infected mice hearts 15 days post infection. Data are mean ± SD and are representative of one independent experiment with n = 4.
Fig 2.
CXCR3 receptor is important to parasitemia and survival of C57BL/6 infected with Trypanosoma cruzi.
a-Experimental scheme showing the model used in this study. Both Isotype control and anti-CXCR3 antibody treated groups were infected with 1x104 blood trypomastigotes of the Y strain of T. cruzi. On the same day of infection, anti-CXCR3 group was treated with 250 μg of anti-CXCR3 antibody while the isotype control group was treated with the same concentration of anti-IgG of Rat antibody. The treatment was performed every 48 hours until 15 days after infection by intraperitoneal route. b-Graph of parasitemia showed in linear scale of Isotype Control and anti-CXCR3 groups. c-The Kaplan–Meier curves for survival of Isotype control and anti-CXCR3 groups. The statistical differences between the groups were compared using the log-rank test (p = 0.0091). Data are mean ± SD and are representative of 3 independent experiments with n = 4. *values are significantly different between isotype Control and anti-CXCR3 groups in indicated days (*p>0.001). The statistical analysis was performed using the Student’s t-test.
Fig 3.
Anti-CXCR3 group decreased number of specific CD8+ T cells as well as polyfunctionality after infection with T. cruzi.
a-Representative dot-plot graphs of each group show the frequency of specific CD8+ T cells (in Q2 quadrant) in the spleen and the bar graph represents the mean of specific CD8+ T cells frequency on day 15 after infection. Those cells were labeled with H2Kb-VNHRFTLV multimer. b-The bar graph with absolute numbers of specific CD8+ T cells in the spleen on day 15 after infection. c-Histograms represent the gate on specific CD8+ T cells positive for BrdU; the red line represents one mouse from the naïve group while the blue one refers to the T. cruzi infected mice (Isotype control and anti-CXCR3 groups, respectively). d-Percentage of specific CD8+ T cells positive for BrdU (thymidine analogues) in the spleen. The in vivo proliferation assay was performed by BrdU administration (5mg/mouse). e-Percentage of specific CD8+ T cells in the spleen positive for Annexin V. f-The histogram represents the gate for CFSELow and CFSEhigh (target cells) population in the spleen from naïve, Isotype control and anti-CXCR3 groups on day 12 post infection (12 dpi) and the bar graph represents the cytotoxicity percentage of specific CD8+ T cells from naïve, Isotype control and anti-CXCR3 groups. g-The percentage of cytotoxicity of specific CD8+ T cells from the spleen on day 15 post infection. h-The Mean Intensity of Fluorescence (MFI) of granzyme B on specific CD8+ T cells surface. i-Number of IFN-γ producing cells quantified by ELISPOT assay. j-Quantity of cytokines (pg/mL) from splenocytes supernatants. CBA mouse Th1/Th2/Th17 cytokine kit was used. k-Pie graphs show the percentage of specific CD8+ T cells that performed each combination as shown in legends (3, 2 or 1 function). The polyfunctionality of specific CD8+ T cells was performed after ICS staining and Boolean analyses. Data are mean ± SD and represent 3 independent experiments with n = 3. *P<0.05 comparing noninfected controls (naïve), Isotype control and anti-CXCR3 groups. # P<0.01 comparing Isotype control and anti-CXCR3 groups.
Fig 4.
CXCR3 contributes to specific CD8+ T cells activation.
The immunophenotyping of specific CD8+ T cells was performed in the spleen. We evaluated the expression of markers associated with activation, homing and memory. a-The histogram graphs represent each molecule analyzed from naïve (grey line), Isotype Control (red line) and anti-CXCR3 (blue line) groups. b-The bar graphs show MFI of each molecule analyzed in specific CD8+ T cells from naïve, Isotype Control and anti-CXCR3 groups. Data are mean ± SD and represent 2 independent experiments with n = 3. *P<0.05 comparing noninfected controls (naïve), Isotype Control and anti-CXCR3 groups. #P < .01 comparing Isotype control and anti-CXCR3 groups. The statistical analysis was performed using One-way ANOVA, followed by Tukey post-hoc test.
Fig 5.
CXCR3 guides CD8+ T cells migration into T. cruzi infected heart.
a-Immunohistochemistry staining of hearts from naïve, Isotype Control and anti-CXCR3 groups; CD8+ T cells were labeled in green. b-The relative expression of CD8+ T cells in the heart of Isotype Control and anti-CXCR3 groups. c-The dot-plot graphs represent the frequency of specific CD8+ T cells (pool of 3 mice/group) in the heart of Isotype Control and anti-CXCR3 groups. d-Histological section from infected hearts; the white arrows indicate the amastigote nests. e-Number of amastigote nests from Isotype Control and anti-CXCR3 groups counted in 50 fields. f-The parasite burden quantification by qPCR from infected hearts. The qPCR was performed from 50 ng of the heart’s DNA. Data are mean ± SD and represent 2 independent experiments. #P < .01 comparing Isotype control and anti-CXCR3 groups. The statistical analysis was performed using Student’s t-test.
Fig 6.
Plasmacytoid dendritic cells (pDCs) produced CXCL10 chemokine and interacted in vivo with CD8+ T cells during T. cruzi infection.
To analyze which antigen-presenting cells (APCs) produced CXCR3 ligands, a REX3 linage mouse (CXCL10-BFP and CXCL9-RFP reporter) was infected on days 4, 9, 12, and 20 and we analyzed the number of MO-DCs, pDCs and chemokine production. a-The graphs represent the number of MO-DCs and pDCs on days 0, 4, 9, 12, and 20 after infection. b-The MO-DCs were gated as F4/80+CD11b+ and DC-SIGN+MHC-II+; the graph represents the percentage of MO-DCs-producing chemokines—CXCL9 and/or CXCL10. c-The pDCs were gated as CD11clow and pDCA-1+ and the graph represents the percentage of pDCs producing chemokines—CXCL9 and/or CXCL10. d-Image Flow Cytometry of chimera mice that received IFN-γ GFP, CXCL9-RFP and CXCL10-BFP cells; the double cells were gated using DRAQ5 labeling. e-Image Flow Cytometry of REX3 infected mice shows the markers used as well as the interaction between CXCL10 pDC-producing cells and CD8+ T cells. f-Image Flow Cytometry of REX3 infected mice shows the markers used as well as the interaction between CXCL10 MO-DC-producing cells and CD8+ T cells. g-The number of pDCs and MO-DCs interacting with CD8+ T cells after T. cruzi infection. Data are mean ± SD and represent one experiment with n = 3. *indicates statistical differences between the number of pDCs and MO-DCs interacting with CD8+ T cells (*P = 0.001515, statistical analysis was performed using Student’s t-test).
Fig 7.
The number of plasmacytoid dendritic cells (pDCs) decreased after anti-CXCR3 treatment and T. cruzi infection.
The number of pDCs after anti-CXCR3 treatment was quantified on day 12 after infection in the spleen and lymph node. a-The dot-plots represent the gate strategy used to measure the frequency of pDCs in the spleen and the bar graph represents the number of pDCs in the spleen of naïve, Isotype control and anti-CXCR3 groups. b-The MFI of CXCR3 receptor on pDCs surface in the spleen of naïve, Isotype control and anti-CXCR3 groups. c-The dot-plots represent the gate strategy used to measure the frequency of pDCs in the lymph node and the bar graph represents the number of pDCs in the lymph node. d-The MFI of CXCR3 receptor on pDCs surface on the lymph node of naïve, Isotype control and anti-CXCR3 groups. Data are mean ± SD and represent one experiment with n = 3. *P<0.05 comparing noninfected controls (naïve), Isotype control and anti-CXCR3 groups. #P < .01 comparing Isotype control and anti-CXCR3 groups.