Differential ability of surface and endosomal TLRs to induce CD8 T cell responses in vivo

Abstract

TLR activation on dendritic cells (DCs) induces DC maturation and secretion of proinflammatory cytokines, both of which are important for activation and differentiation of CD4 T cells. The importance of TLR activation on DCs for CD8 T cell responses is less clear. In this study, we tested the ability of different TLRs to regulate CD8 T cell responses to pathogens. We found that although all TLRs are able to induce CD8 T cell activation in vitro, there are profound differences in their ability to activate CD8 T cells in vivo. The nucleic acid recognizing endosomal TLRs, TLR3 and TLR9, had a potent ability to induce CD8 T cell activation. However, the surface TLRs, TLR2 and TLR4, that recognize bacterial ligands were not only incapable of inducing CD8 T cell priming, but they had a dominant effect of inhibiting CD8 T cell expansion induced by activation of endosomal TLRs. We found that TLR2 and TLR4, acting in a MyD88-dependent manner, influenced CD8 T cell priming by altering the composition of DCs in the draining lymph nodes. Our results have important implications for combined bacterial and viral infections and suggest that bacterial infections could constrain the ability of the host to mount effective antiviral CD8 T cell immunity.

Figure 1. Differential regulation of in vivo CD8 T cell responses by endosomal and plasma membrane TLRs

Mice that received GFP-OT-I T cells were immunized as indicated and cells from the draining lymph node were stained using K^b-SIINFEKL tetramer. (A) CD8⁺ T cells that express GFP and stain for the Class I tetramer are shown. (B) Mean ± SEM of quantification of GFP+ OT-I T cells as a percentage of total CD8 T cells from 3 independent mice immunized with OVA and different TLR ligands. (C) Representative plots of OT-I T cell expansion in draining lymph nodes, 7 days after immunization. (D) Mean ± SEM from 3 independent mice. (E) Mice without any OT-I T cell transfer were immunized as indicated and cells from draining lymph nodes were stained on day 7 for CD8 and K^b tetramer to reveal SIINFEKL-specific CD8 T cell expansion. (F) Mean ± SEM of tetramer positive, CD8 T cells from 5 independent mice. The data of all above experiments are representative of at least three independent experiments with 3 mice per group. *, P < 0.05; ***, P < 0.005.

Figure 2. LPS mediated suppression of CD8 T cell responses depends on the TLR4-MyD88 signaling axis in CD11c⁺ cells

(A) WT and TLR2/4 DKO mice received WT OT-I T cells and were immunized as indicated. Representative plots show CD8 T cells positive for GFP and K^b tetramer. (B) Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from 3 independent mice per group. (C) WT OT-I T cells were transferred into WT, TRIF KO and MyD88 KO mice and immunized with OVA or OVA mixed with LPS. Representative plots show CD8 T cells positive for GFP and K^b tetramer. (D) Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from 3 independent mice per group. (E) After OT-I CD8 T cell transfer, WT and CD11c MyD88 Tg mice were immunized as indicated and cells were stained for CD8 and K^b tetramer and representative plots show percentage of CD8 T cells that expressed GFP and stained positive for the tetramer. (F) Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from 3 independent mice per group. All the data above are representative of two independent experiments with 3 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure 3. Anti-viral CD8 responses are compromised by activation of plasma membrane TLRs

(A) Mice that received OT-I CD8 T cells were infected using VSV-OVA or VSV-OVA mixed with different TLR ligands or live/heat killed Salmonella typhimurium (Sal/Sal-HK) and draining lymph nodes and the spleen were harvested on day 7 after infection to measure OT-I CD8 T cell expansion. (A, C, E). Representative plots show CD8 T cells that express GFP and stain positive for K^b-SIINFEKL tetramer. (B, D, F). Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from 5 independent mice per group. The data are representative of three (A, B, C and D) or two independent experiments (E and F). *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure 4. TLR2 and TLR4 activation inhibit CD8 T cell responses against Listeria monocytogenes

After OT-I T cell transfer, mice were infected with LM-OVA or LM-OVA mixed with LPS, BLP or heat-killed Salmonella typhimurium (Sal-HK). (A and C) Representative plots show CD8 T cells from draining lymph nodes and spleen that express GFP and stain positive for K^b tetramer. (B and D) Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from three independent mice per group. The experiments are representative of two independent experiments with three mice per group. **, P < 0.01; ***, P < 0.005; ****, P < 0.001.

Figure 5. Inhibition of CD8 T cell responses by LPS is independent of Tregs and CD4 T cells

After OT-I CD8 T cell transfer, groups of mice either received control Rat-IgG or anti-CD25 (clone PC61) to deplete CD25⁺ Tregs. CD8 T cell priming was measured 7 days following immunization with OVA with or without TLR ligands, as indicated (A, B). Groups of mice were depleted of CD4 T cells as described and immunized as indicated following OT-I T cell transfer (C, D). (A, C) Representative plots show CD8 T cells from draining lymph nodes that express GFP and stain positive for K^b tetramer. (B, D) Mean ± SEM of GFP-OT-I T cells as a percentage of total CD8 T cells from three independent mice per group. Data are representative of three independent experiments. **, P < 0.01; ***, P < 0.005.

Figure 6. TLR4 activation in vivo affects the ability of APCs to present antigen to CD8 T cells and induces differential recruitment of lymphoid and myeloid DCs

Mice were injected with OVA or OVA-AF647 with or without different TLR ligands. (A) Cells that take up OVA can be identified in the draining lymph nodes by flow cytometry. (B, C) OVA positive cells from the draining lymph nodes of different groups of mice were sorted and incubated with OT-I CD8 T cells and proliferation was measured at the end of 72 hours by a ³H thymidine incorporation assay. (D) OVA⁺ APCs from draining lymph nodes of OVA primed group were mixed with the OVA⁺ APCs from OVA+LPS group and incubated with OT-I CD8 T cells and proliferation of CD8 T cells was measured as described above. (E). Mice were immunized with OVA-647 with or without different TLR ligands and 24hrs later cells from draining lymph nodes were analyzed for OVA-647⁺ cells and further analyzed for expression of lymphoid DCs (CD11c⁺, CD8⁺, CD11b⁻) and myeloid DC markers (CD11c⁺, CD11b⁺, CD8⁻). Representative plot shows staining for CD11b and CD8 on CD11c+ cells. (F) Ratio of lymphoid DCs to myeloid DCs from several independent mice following immunization and staining as described in E. *, P < 0.05; **, P < 0.01; ***, P < 0.005.