Robust Anti-viral Immunity Requires Multiple Distinct T Cell-Dendritic Cell Interactions

Abstract

Host defense against viruses and intracellular parasites depends on effector CD8(+) T cells, whose optimal clonal expansion, differentiation, and memory properties require signals from CD4(+) T cells. Here, we addressed the role of dendritic cell (DC) subsets in initial activation of the two T cell types and their co-operation. Surprisingly, initial priming of CD4(+) and CD8(+) T cells was spatially segregated within the lymph node and occurred on different DCs with temporally distinct patterns of antigen presentation via MHCI versus MHCII molecules. DCs that co-present antigen via both MHC molecules were detected at a later stage; these XCR1(+) DCs are the critical platform involved in CD4(+) T cell augmentation of CD8(+) T cell responses. These findings delineate the complex choreography of cellular interactions underlying effective cell-mediated anti-viral responses, with implications for basic DC subset biology, as well as for translational application to the development of vaccines that evoke optimal T cell immunity.

Figure 1. Direct priming of CD8⁺ T cells does not require XCR1⁺ DC

(A) Analysis of splenic DC after i.v. infection with MVA-GFP or MVA-NP-SIINFEKL-GFP (8h p.i.). (B) Analysis of OT-I proliferation after ex vivo coincubation with isolated splenic DC from Kbm1 mice infected with MVA-OVA or MVA-OVA-Kb (8h pi). (C) Immunofluorescent (IF) images of a dLN showing clustering/interaction between transferred OT-I cells and infected (GFP-expressing) DC (MVA-OVA-GFP; f.p.; 8h p.i.). (D) Analysis of OT-I proliferation after coincubation with DC subsets sorted ex vivo (MVA-OVA; i.v.; 8h p.i.). (E/F) Activation marker (CD25/CD69) upregulation on transferred OT-I and OT-II cells in the popliteal LN 12h after f.p. infection (MVA-OVA). Representative plots (E) and analysis (F) are shown comparing DTX-treated WT and XCR1-DTR animals. Data are representative of three (n=3) (A-D) and two (n=4) (E/F) independent experiments. ** = p≤0.01, ns = non-significant, scale bars 100/50 μm. See also Figure S1 and Movie S1.

Figure 2. Priming of CD4⁺ and CD8⁺ T cells occurs on spatially distinct DC

(A) Images from IVM of the popliteal LN 3-4h after MVA-OVA f.p. infection. OT-I, OT-II and control cells (polyclonal CD4⁺ T cells) were transferred 24h prior to infection. White arrows indicate brief interaction between clustered OT-I and OT-II or control cells (see also Movie S2). (B) Analysis of the mean velocity of transferred T cells using the data shown in Movie S2, (red bars indicate mean values). (C) Analysis of CD69 upregulation on transferred OT-I and OT-II cells in the dLN at different time-points after f.p. infection (MVA-OVA/MVA WT). (D/E/F) IF images of a dLN showing the localization of (D) transferred OT-I and OT-II cells (MVA-OVA-GFP), (E) transferred P14 and Smarta cells (MVA-GP-Venus), (F) transferred P14, Smarta and polyclonal CD4⁺ T cells (MVA-GP) 10h p.i. (G) Quantification of cluster abundance from four experiments as in F. Data are representative of at least two independent experiments (A/B; n=10), (C/G; n=4-8; pooled data) (D-F; n=10). *** = p≤0.001, ns = non-significant, scale bars (D/E/F) 100 μm, See also Figure S2 and Movie S2.

Figure 3. CD4⁺ and CD8⁺ T cells co-cluster later during infection

(A) Schematic of the experimental set-up to reveal antigen-bearing cells later in the course of infection (MVA-OVA). (B) IF images of the dLN showing the localisation of OT-I, OT-II and control cells (polyclonal CD4 T cells). (C) Histograms showing cellular localisation 10h (see Figure 2) or 38h p.i. (see experimental set-up shown in 3A). (D) IF image of the dLN showing a mixed OT-I/OT-II cell cluster and activation status (CD69). Data are representative of 10 (B/D; n=20) or three (C; n=3) independent experiments. Scale bars (B) 200 μm/100 μm, (D) 10 μm. See also Figure S3.

Figure 4. Non-infected cross-presenting XCR1⁺ DC are the information-transmission platform for CD4⁺ and CD8⁺ T cells

(A) Proliferation of CFSE labeled OT-I cells after ex vivo coincubation with sorted splenic DC subsets (MVA-OVA; i.v.; 30h p.i.). (B) Proliferation of CFSE labeled OT-I cells after ex vivo coincubation with isolated splenic DC from WT or Kbm1 mice 30h p.i. (MVA-OVA-Kb; i.v.). (C) Images of dLN using the experimental set-up as in Figure 3A. XCR1-DTR-Venus mice were treated with PBS or DTX. (D/E) T cell cluster abundance in the presence or absence of XCR1 DC using a (D) semi-automated or (E) fully automated analysis. (F) Analysis and representative plots of CD69 expression on OT-I/OT-II cells that were transferred 28h post infection (MVA-OVA/MVA WT; f.p.) and analyzed 12h later in the dLN. Data are representative of three independent experiments (n=8). *** = p≤0.001, scale bars 50 μm. See also Figure S4.

Figure 5. XCR1⁺ DC are the information transfer platform for CD4⁺ and CD8⁺ T cells during VV infection

(A) IF image and translated tracks from IVM of the popliteal LN 10-11h after VV-OVA infection (f.p.). See also Movie S3. (B) IF images of a dLN showing the localization and cluster formation of transferred OT-I and OT-II cells (VV-OVA; f.p.; 10h p.i.). (C/D) IF images of the dLN of a XCR-DTR-Venus mice treated with PBS (C) or DTX (D) showing the localization of labeled OT-I and OT-II cells following the experimental set-up shown in Figure 3A using VV-OVA. (E/F) T cell cluster abundance in the presence or absence of XCR1 DC using a (D) semi-automated or (E) fully automated analysis. Data are representative of three independent experiments (n=4). Scale bars (A) 50 μm, (B) 100μm, (C) 200 μm, (D) 200 μm/20 μm. See also Figure S5 and Movie S4.

Figure 6. Localization of endogenous activated CD8⁺ T cells during VV infection

(A) IF Images of a LN showing co-localization of transferred OT-I cells and endogenous (non-OT-I) CD69^hi cells (VV-OVA; f.p.; 10h p.i.). (B/C/D/E) Analysis of IFNγ(YFP⁺) reporter animals (VV-OVA; 36h p.i.). (B) Graphs show the gating strategy, (C) the cellular distribution, (D) the size distribution and (E) the mean fluorescence intensity (MFI) of the YFP signal of IFNγ⁺(YFP⁺) cells (red bars indicates mean values). (F) IF images showing the localization of YFP expressing cells 36h after infection of IFNγ (YFP⁺) reporter animals (VV-OVA; f.p.). OT-I cells were transferred 8h before analysis. (G) Histogram shows the distance between IFNγ⁺(YFP⁺) cells and activated (CD69^hi) OT-I cells. Data are representative of two independent experiments (n=8) (C/E/G) shows pooled data. *** = p≤0.001, scale bar (A) 10 μm, (F) 200 μm/10 μm. See also Figure S6.

Figure 7. VV-specific T cells activated in the absence of XCR1⁺ DC are ‘helpless’

(A-C) Analysis of the total numbers of B8R multimer-specific splenic CD8⁺ T cells 8 days after VV-OVA infection (i.p.). Comparison of (A) isotype vs. CD4 depleted mice, (B) WT vs. XCR1-DTR mice treated with DTX and (C) WT vs. XCR1-DTR mice treated with DTX and anti-CD4 antibodies. (D) Schematic of DC composition in bone-marrow (bm) chimeric animals (MHCII KO × XCR1-DTR → WT). (E) Antiviral CD8⁺ immune response is shown comparing DTX vs. PBS treated bm chimeric animals on d8 (VV-OVA i.p.). (F/G) Analysis of B8R specific immune responses d8 p.i. (VV-OVA i.p.), comparing WT vs. XCR1-DTR animals treated with DTX, showing (F) the phenotype of B8R multimer-specific CD8⁺ T cells and (G) the amount of IL-2 producing CD8⁺ T cells after peptide (B8R) stimulation for 5h. (H-L) Analysis of the immune response 60 p.i. (VV-OVA i.p.), comparing WT vs. XCR1-DTR animals treated with DTX. (H) Phenotype and (I) relative distribution of B8R multimer-specific memory subsets. (J) Absolute numbers of IFNγ-producing CD8⁺ T cells, (K) relative distribution of polyfunctional CD8⁺ T cells (gated on IFNγ⁺) after peptide (B8R) stimulation. (L) Recall response d5 after Lm-B8R challenge. Graph shows total numbers of IFNγ-producing CD8⁺ T cells after peptide (B8R) stimulation. Data are representative of three or two (L) independent experiments (n=4). The graphs show mean +SEM. * = p≤0.05, ** = p≤0.01, *** = p≤0.001, ns = “non significant”. See also Figures S7.