(A) 10⁵ OT-I CD8⁺ T cells were adoptively transferred into congenic mice prior to infection with either wild type (LV9) or ovalbumin-transgenic (PINK) L. donovani amastigotes. Graph represents the average number±se of OT-I CD8⁺ T cells found in the spleen of each individual mouse during the course of infection. (B) Congenic mice received 10⁴ OT-I CD8⁺ T cells prior to infection with 2×10⁷ PINK amastigotes. Graph represents the average number±se of parasites found in the spleen of each individual mouse. Parasite load was determined by limiting dilutions. (C) The same mice were sacrificed at different time points during infection and OT-I CD8⁺ T cells present in the spleen were enumerated by gating on Ly5.2⁺ CD8⁺ cells. Graph represents the average number±se of cells found in the spleen of each individual mouse during the first 41 days of infection. (D) Congenic mice were infected with rVV-SIINFEKL (10⁶ PFU) i.v. the day after receiving 10⁴ OT-I CD8⁺ T cells. Graph represents the average number±se of Ly5.2⁺ CD8⁺ cells found in the spleen of each individual mouse. All graphs show results representative of 2–3 independent experiments; for all experiments, n = 3.

OT-I CD8⁺ T cells were identified by gating on CD8⁺ Ly5.2⁺ cells. Modulation of cell surface markers in PINK (A) and rVV-SIINFEKL (B) infected mice. (A, B) The percentage of gated cells that expressed low/intermediate levels of CD62L is shown in the left panel; the middle panel indicates the percentage of OT-I CD8⁺ T cells that had downregulated CD127; the right panel represents the percentage of OT-I CD8⁺ T cells positive for CD122. (C) Modulation of CD69 expression in mice infected with PINK (left panel) and rVV-SIINFEKL (right panel). Data represent mean percentages±se and is representative of 2 independent experiments, n = 3.

Splenocytes from PINK (left panels) and rVV-SIINFEKL (right panels) infected mice were restimulated in vitro for 4 h with SIINFEKL peptide and IFNγ, TNFα, IL-2 and Granzyme B were assessed by ICS. Graphs represent the percentage of OT-I CD8⁺ T cells producing IFNγ (A), TNFα (B), IL-2 (C), and granzyme B (D). OT-I CD8⁺ T cells were identified by gating on Ly5.2⁺ cells for the triple ICS staining (IFNγ, TNFα, and IL-2) and on CD8⁺ Ly5.2⁺ for the granzyme B staining. Data represent mean percentages±se, representative of 2 independent experiments, n = 3.

On various days after infection, MACS enriched CD11c^hi DC were coincubated with naïve OT-I CD8⁺ T cells labelled with PKH26. Proliferation of OT-I cells was assessed after 72 h. (A) Representative histograms for several time points of infection are shown. (B) Graph represents the average percentage of proliferation during the course of infection and is representative of 2 independent experiments. For each time point, the percentage background proliferation was calculated after incubating OT-I CD8⁺ T cells with naïve DC and was subsequently subtracted from each value.

(A) B7-H1 (left panels) and CD80 (right panels) expression on CD11c^hi splenic DC was assessed on various days after infection. Cells were gated on MHCII^hi CD11c^hi. Dotted lines represent isotyope controls. (B) OT-I CD8⁺ T cells in the spleen of PINK infected mice were identified by gating on Ly5.2⁺ CD8⁺ cells and PD-I expression was assessed at indicated times p.i.. Graph represents mean percentages±se of PD-1 positive cells and show results of 1 of 3 independent experiments, n = 3.

(A, C) Congenic mice received 10⁴ OT-I CD8⁺ T cells prior to infection with 2×10⁷ PINK amastigotes. From day 15 pi on mice were treated biweekly with anti-B7-H1 antibodies. Animals were sacrificed at indicated times pi. (A) OT-I CD8⁺ T cells were identified by gating CD8⁺ Ly5.2⁺ cells. Graph represents the average numbers of cells per spleen at different time points of infection. (C) Graph represents the splenic parasite burden expressed as Leishman Donovan Units (LDU). All data represent mean±se of one of 2 independent experiments, n = 3. (B) On various days after infection, MACS enriched CD11c^hi DC were coincubated with naïve OT-I CD8⁺ T cells labelled with PKH26 in the presence of either 10 µg/ml anti-B7-H1 or an isotype control antibody. Proliferation of OT-I cells was assessed after 72 h. Graph represents the average percentage of proliferation during the course of infection. For each time point, the percentage background proliferation was calculated after incubating OT-I CD8⁺ T cells with naïve DC and was subsequently subtracted from each value. For this experiment, n = 5. (D) C57BL/6 mice were infected with 2×10⁷ LV9 amastigotes and treated biweekly from day 15 pi on with anti-B7-H1 antibodies. Mice were sacrificed at indicated time after infection. Graph represents the splenic parasite burden expressed as LDU. All data represent mean±se of one experiment, n = 5.

10⁴ OT-I CD8⁺ T cells were adoptively transferred into congenic mice, which were subsequently infected with PINK parasites. At day 32 pi, mice were either untreated or challenged s.c. with VV and/or rVV-SIINFEKL. Mice were sacrificed at indicated times pi. (A) OT-I CD8⁺ T cells present in the spleen were enumerated by gating on Ly5.2⁺ CD8⁺ cells. Graph represents the average number±se of cells found in the spleen of each individual mouse at day 2, 6, and 9 after challenge. (B) The splenic parasite burden expressed as Leishman Donovan Units (LDU) is shown. (C) Splenocytes from the 3 groups of infected mice were restimulated in vitro for 4 h with the SIINFEKL peptide and IFNγ and TNF were assessed by ICS. Graphs represent the percentage of OT-I CD8⁺ T cells producing IFNγ (left panel) and TNFα (right panel). All data represent mean±se, n = 3. (D) Representative plots for the ICS with IFNγ and TNFα at day 6 and 9 after superinfection. All data are representative of 2–3 experiments; n = 3.