Processing and presentation of variant surface glycoprotein molecules to T cells in African trypanosomiasis

Abstract

Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.

Figure 1. Expression of MHC II and costimulatory molecules by DCs and MPs from uninfected and Trypanosoma brucei rhodesiense LouTat 1-infected animals

Peritoneal exudate cells and splenocytes were isolated from B10 or C3H mice at 0, 2, 4 and 7 days post-infection. The data points shown represent MFI values derived from flow cytometry for multiple phenotypic markers expressed by the different tissue-specific cell populations; pooled cells from 5 mice per time point per strain were examined in these studies, and the data set shown is from one of five separate representative experiments. MPs were gated based on viability, SSC/FSC profiles, and finally lineage-specific markers to identify peritoneal MPs (F4/80^high CD11b^high) and splenic MPs (F4/80^highCD11c⁻ ). A low density-enriched fraction of splenocytes was prepared for DC analysis; DCs were gates as above using CD11c^highCD8^+/− for DC identification. Asterisks highlight data showing phenotypic changes in CD8a+ and CD8a− DCs that are significantly different between resistant and susceptible mice on the specific days of infection shown.

Figure 2. CD8α− and CD8α+ DCs are selectively depleted in susceptible C3H but not resistant B10 mice infected with African trypanosomes

CD11c^high CD8α− or CD11c^high CD8α+ DCs in live, low density-enriched splenocytes from B10 or C3H animals (5 mice/group) on days 0–7 of infection; percentage of each subset is listed within the gated cell subpopulations. Results of a representative experiment are shown. The values shown in this figure for different time points of infection are: B10.BR (Day 0) CD11c^highCD8α− = 5%; CD11c^highCD8α+ = 2.8%. (Day 2) CD11c^highCD8α− = 6.2%; CD11c^highCD8α+ = 2.6%. (Day 4) = CD11c^highCD8α− = 3.8%; CD11c^highCD8α+ = 1%. (Day 7) = CD11c^highCD8α− = 6.1%; CD11c^highCD8α+ = 1%. C3H (Day 0) CD11c^highCD8α− = 2.5%; CD11c^highCD8α+ = 0.5%. (Day 2) CD11c^highCD8α− = 4.5%; CD11c^highCD8α+ = 1.1%. (Day 4) = CD11c^highCD8α− = 1.1%; CD11c^highCD8α+ = 0.4%. (Day 7) = CD11c^highCD8α− = 0.7%; CD11c^highCD8α+ = 0.03%.

Figure 3. Splenic APCs produce IL-12 upon infection, whereas NO is produced by peritoneal MPs

Cytokine production by DCs and MPs taken from naïve and infected animals (5 mice/group) and cultured ex vivo; supernatants were collected after 24 hr. IL-12p40 and IL-10 production were assessed by ELISA; NO was determined by Griess reaction. Results are mean +/− SEM of triplicate wells in a representative experiment. *, p < 0.05 as compared to naive control; †, p < 0.05 as compared to same measurement in opposite mouse strain.

Figure 4. IL-12 induces the early generation and polarization of VSG-specific Th1 cells during trypanosome infection

Relatively resistant C57BL/6 mice with the IL-12p35 gene deleted were infected with T. b. rhodesiense LouTat 1. SPC were harvested at the time points shown and tested for the secretion of IFN-γ, IL-2 and IL-4 following stimulation in vitro with purified LouTat 1 VSG. Identical results were obtained with IL-12p40 knockout mice and wt mice treated with neutralizing Ab to IL-12 (data not shown; manuscript in preparation). Results presented as +/− SEM of three separate experiments; *,p < 0.05 knockout mouse responses as compared to wt controls.

Figure 5. Splenocytes from infected B10 and C3H mice display impaired ability to process and present newly encountered antigen

Splenocytes from uninfected and infected animals (days 0–13 post infection; 5 mice/group) were irradiated and combined with the 3A9 T cell hybridoma and varying concentrations of HEL antigen. Supernatants were collected after 24 hr and assayed for IL-2 production by [³H] thymidine incorporation of IL-2-dependent CTLL-2 cells. T cell hybridomas cultured alone or with HEL did not produce IL-2 (data not shown). Results are mean +/− SEM of triplicate wells in a representative experiment.

Figure 6. DCs and MPs from infected animals display impaired processing and presentation of newly encountered antigen

DCs, SMPs, or PMPs isolated from uninfected and infected mice (days 0–10 post infection; 5 mice/group) were combined with the 3A9 T cell hybridoma and varying concentrations of HEL antigen. Supernatants were collected after 24 hr and assayed for IL-2 production by ELISA. Results are mean +/− SEM of triplicate wells in a representative experiment.

Figure 7. Splenic APCs activated during trypanosome infection induce naïve CD4+ T cell proliferation and/or IFN-γ production

DCs, SMPs, or PMPs isolated at different times of infection (days 0–10 post infection; 5 mice/group) were incubated with naïve TG T cells and varying doses of HEL. (A) T cell proliferation was determined by [³H]thymidine incorporation during the last 18 hr of a 64 hr culture period, and results are expressed as Δcpm (cpm of wells with HEL–cpm of wells without HEL). (B) IL-2 and (C) IFN-γ were assessed from 48 hr culture supernatants by ELISA. Results are mean +/− SEM of triplicate wells in a representative experiment. At the highest HEL concentration given, all measurements except those noted were significantly different from the naive control. n/s, no significant difference (p > 0.05) as compared to naive control.

Figure 8. APCs ex vivo from infected mice present VSG peptides to T cells

DCs or MPs isolated from uninfected and infected animals (days 0–10 post infection; 5 mice/group) were combined with VSG-specific T hybridomas without the addition of exogenous VSG (50 ug/ml). IL-2 production was measured in 24 hr supernatants by ELISA. Shown are representative responses from four of thirteen T hybridomas. The APC type and T hybridoma used are shown for each graph (note: unlike splenic DCs and MPs, PMPs from resistant and susceptible animals activated different subsets of T hybridomas). Results are mean +/− SEM from triplicate wells in a representative experiment. *, p < 0.05 as compared to naive control.

Figure 9. VSG availability contributes to the sustained presentation of VSG peptides during trypanosome infection

B10 APCs isolated at day 10 of infection (following clearance of LouTat 1 trypanosomes and at a time when such APCs ex vivo were no longer able to present peptides to the T hybridomas; see Figure 8) were supplemented with additional (exogenous) VSG and cultured with VSG-specific T cell hybridomas. Two representative T hybridoma responses are shown. PMPs were also able to activate these hybridomas when provided with exogenous VSG, though they were unable to do so at any time during infection without additional VSG added to cultures. IL-2 was measured by ELISA; results are displayed as mean +/− SEM of triplicate wells in a representative experiment. *, p < 0.05 as compared to control.

Figure 10. DCs from mice infected with LouTat 1.5 display impaired processing and presentation of newly encountered variant LouTat 1 VSG

B10 animals were infected with LouTat 1.5 and DCs were collected on days 0, 4, 7, and 10 of infection. DCs were combined with different LouTat 1 VSG-specific T cell hybridomas (shown in upper left of each graph) and with or without the addition of LouTat 1 VSG. IL-2 was measured by ELISA; results are displayed as mean +/− SEM of triplicate wells in a representative experiment.