DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo

Abstract

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4⁺ and CD8⁺ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8⁺ T cell responses was largely dependent on CD11c⁺CD8⁺ DCs, whereas CD11c⁺CD8^- DCs were critical for priming CD4⁺ T cell responses.

Figure 1.

Expression and internalization analyses of FcγRs. (a) Histogram overlays show expression of FcγRs (FcγRI: CD64, FcγRIIB: CD32, FcγRIII: CD16, and FcγRIV: 9E9) on splenic single lin⁻MHCII⁺ CD11c⁺CD8⁺ DCs, CD11c⁺CD8⁻ DCs, and pDCs (see gating strategy in Fig. S1 a) from C57BL/6 (black) and FcγRI^−/−, FcγRIIB^−/−, FcγRIII^−/−, and FcγRIV^−/− mice (gray). This experiment was repeated three times with similar results. (b) Internalization of αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova into the indicated splenic cell populations. Splenic single-cell suspensions were incubated in media containing the antibodies labeled with an oligo containing an Atto647N fluorochrome for 10, 30, 60, or 120 min at 37°C or kept on ice (0-min time point). Extracellular fluorescence was quenched by incubation with a complementary oligo containing a specific BBQ650 quencher. Cells were gated on single CD19⁺ B cells, CD11c⁺CD8⁺ DCs, CD11c⁺CD8⁻ DCs, CD11c^lowPDCA-1⁺B220⁺ pDCs, Ly6C^high inflammatory and Ly6C^low resident monocytes, T cells, and NK cells as described in Fig. S1 b. The presented experiments were repeated three times with two samples each. All data points ± SD are shown in the graph. MFI, mean fluorescence intensity.

Figure 2.

In vivo proliferation of CD4⁺ and CD8⁺ T cells by antigen delivery to FcγRs in the steady state. (a and b) MACS-purified, CFSE-labeled, congenic 10⁶ CD8⁺ OT-I T cells (a) or 2 × 10⁶ CD4⁺ OT-II T cells (b) were i.v. transferred into C57BL/6 mice. 16 h later, recipients were i.p. injected with doses of 10, 3, 1, 0.3, 0.1, and 0 µg isotype-Ova, αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova targeting antibodies in PBS. 3 d after targeting antibody injection, in vivo T cell proliferation in dependency of the injected antibody amount was measured by CFSE-dilution analysis via flow cytometry of Vα2⁺CD45.1⁺CD8⁺ (a) and Vα2⁺CD45.1⁺CD4⁺ (b) splenic T cells. All experiments were repeated at least three times with similar results.

Figure 3.

T cell proliferation induced by FcγR targeting is ITAM independent, and long-term proliferative responses require additional co-stimulatory signals in vivo. (a) Injection of antigen-targeting antibodies does not change activation status of DCs in vivo. 10 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova, was i.p. injected into C57BL/6 mice. As positive control, a mixture of each 25 µg poly(I:C) and αCD40 or 10⁹ HKLM was used. PBS was i.p. injected as negative control. 12 h later, splenic single lin⁻ (CD3⁻CD19⁻NKp46⁻) MHCII⁺ CD11c⁺CD8⁺ DCs, CD11c⁺CD8⁻ DCs, and CD11c^lowPDCA1⁺ pDCs were analyzed by flow cytometry for the activation markers CD80, CD86, and CD69. Data were analyzed using DIVA and FlowJo Software. This experiment was repeated more than five times with similar results and was used as quality control in the production of antigen-targeting antibodies. (b–e) MACS-purified, CFSE-labeled, congenic 10⁶ CD8⁺ OT-I T cells (b and d) or 2 × 10⁶ CD4⁺ OT-II T cells (c and e) were i.v. transferred into C57BL/6 mice (b–e) or NOTAM mice (b and c). 16 h later, recipients were i.p. injected with 3 µg of the targeting antibodies in PBS (b and c) or together with 25 µg αCD40 antibody and 12.5 µg poly(I:C) (pIC) or HKLM (d and e). (b and c) 3 d after targeting antibody injection, in vivo T cell proliferation was measured by CFSE-dilution analysis via flow cytometry of Vα2⁺CD45.1⁺CD8⁺ (b) and Vα2⁺CD45.1⁺CD4⁺ (c) splenic T cells. The graph shows the proliferation indices of all mice analyzed (n.s., nonsignificant; *, P < 0.05). (d and e) T cell proliferation was analyzed by cell numbers of gated Vα2⁺CD45.1⁺CD8⁺ (d) or Vα2⁺CD45.1⁺CD4⁺ (e) splenic T cells 3 d and 9 d later, when PBS, αCD40 + poly(I:C), or HKLM was coinjected. Graphs show the relative cell number expansion compared with the isotype control. (a) These experiments were repeated at least three times with similar results. (b–e) The data were generated within three independent experiments, and all data points ± SD are presented (n.s., nonsignificant; *, P < 0.05; Mann–Whitney U test).

Figure 4.

Induction of T cell responses by FcγR targeting is dependent on the presence of FcγRs. (a–h) C57BL/6, FcγRIIB^−/− (a–d), or FcγRIV^−/− (e–h) mice were i.v. injected with congenic10⁶ CD45.1⁺CD8⁺ OT-I (a, b, e, and f) or 2 × 10⁶ CD45.1⁺CD4⁺ OT-II (c, d, g, and h) MACS-purified, CFSE-labeled T cells. 3 µg of the antigen-targeting antibodies αDEC205-Ova, αDCIR2-Ova, and αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected 16 h after T cell transfer. T cell proliferation was analyzed by CFSE dilution in gated Vα2⁺CD45.1⁺CD8⁺ OT-I (a, b, e, and f) or Vα2⁺CD45.1⁺CD4⁺ OT-II (c, d, g, and h) T cells 72 h later. (a, c, e, and g) Shown are exemplary overlay histograms (gray: C57BL/6, black: [a and c] FcγRIIB^−/−, [e and g] FcγRIV^−/−) of at least three independent experiments with similar results. (b, d, f, and h) Scatter plots represent the proliferation indices of all mice analyzed ± SD (gray circles: C57BL/6, black squares: [b and d] FcγRIIB^−/−, [f and h] FcγRIV^−/−). (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001 by Mann–Whitney U test.)

Figure 5.

Antigen targeting to FcγRIIB and FcγRIV needs receptor expression on cDCs. (a–h) Mice were i.v. injected with MACS-purified, CFSE-labeled 10⁶ CD45.1⁺CD8⁺ OT-I (a, c, e, and g) or 2 × 10⁶ CD45.1⁺CD4⁺ OT-II (b, d, f, and h) T cells. 3 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 10 µg αFcγRIIB-Ova or isotype-Ova control antibodies was i.p. injected 16 h after T cell transfer. T cell proliferation was evaluated 72 h later. Shown is the CFSE dilution of Vα2⁺CD45.1⁺CD8⁺ OT-I (a, c, e, and g) or Vα2⁺CD45.1⁺CD4⁺ OT-II (b, d, f, and h) gated congenic T cells. (a and b) C57BL/6 mice and CD11c-DTR mice were treated i.p. with PBS or DT 8 h before T cell transfer. (c and d) T cell transfer into FcγRIV^fl/fl, LysM-Cre × FcγRIV^fl/fl, CD11c-Cre × FcγRIV^fl/fl mice. (e and f) C57BL/6 mice were treated three times with 200 µg αPDCA-1 antibody 64 h, 40 h, and 16 h before T cell transfer. (g and h) T cell transfer into C57BL/6 or µMT^−/− mice. All experiments were repeated at least three times with similar results.

Figure 6.

CD11c-Cre × FcγRIV^fl/fl and LysM-Cre × FcγRIV^fl/fl deplete FcγRIV on different cell types. Freshly isolated splenic single-cell suspensions from C57BL/6, FcγRIV^fl/fl, CD11c-Cre × FcγRIV^fl/fl, and LysM-Cre × FcγRIV^fl/fl were stained with antibodies for cell identification and for FcγRIV similar as shown in Fig. S1. Ly6G was used in an additional channel, and neutrophils were gated as Ly6G^hiSSC^im/hi and excluded before starting to gate the other populations. The experiments were repeated five times with similar results.

Figure 7.

Differential antigen presentation to CD8⁺ and CD4⁺ T cells induced by FcγRIIB and FcγRIV targeting to CD11c⁺CD8⁺ or CD11c⁺CD8⁻ DCs. 10 µg αDEC205-Ova, αDCIR2-Ova, or αFcγRIV-Ova or 30 µg αFcγRIIB-Ova or isotype-Ova was i.p. injected into C57BL/6 mice. 12 h later, splenocytes were sorted into CD11c⁺CD8⁺ DCs, CD11c⁺CD8⁻ DCs, pDCs, B cells, and Ly6C^high and Ly6C^low monocytes. (a and b) Antigen-presenting cells were co-cultured in different numbers with 10⁵ MACS-enriched CD8⁺ OT-I T cells (a) or CD4⁺ OT-II T cells (b). Proliferation was evaluated by addition of ³H-thymidine 16 h (a) or 40 h (b) after start of the co-culture. Incorporation of ³H-thymidine was measured 24 h later. This experiment was repeated at least three times, and all data points ± SD are shown in the graph.

Figure 8.

Induction of effector T cell responses in naive mice mediated by antigen delivery through FcγRs. (a–d) C57BL/6 mice were i.p. injected with 10 µg of the targeting antibodies αDEC205-Ova, αDCIR2-Ova, αFcγRIIB-Ova, αFcγRIV-Ova, αFcγRIIB/III-Ova, or isotype-Ova together with 50 µg αCD40 antibody and 25 µg poly(I:C) after injection of PBS or 200 µg αFcγRIV w/o Ova. 14 d later, splenocytes were in vitro restimulated for 12 h with freshly isolated CD11c-positive MACS-enriched DCs loaded with a peptide pool of Ova. Intracellular IFNγ (a and c) and IL-2 (b and d) production was analyzed by flow cytometry. The graphs represent the number of cytokine-positive TCRβ⁺NKp46⁻CD19⁻CD8⁺CD4⁻ (a and b) and TCRβ⁺NKp46⁻CD19⁻CD8⁻CD4⁺ (c and d) T cells. This experiment was performed twice with at least five mice, and all data points were used for the analysis. Shown is the median ± interquartile range (n.s., nonsignificant; **, P < 0.01; ***, P < 0.001; Mann–Whitney U test). (e and f) C57BL/6 mice were immunized with 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (e) or 0.03, 0.10, 0.3, 1, or 3 µg αDEC205-Ova, αDCIR2-Ova, αFcγRIV-Ova, or αFcγRIIB/III-Ova or 10 µg αFcγRIIB or isotype-Ova in combination with 50 µg αCD40 and 25 µg poly(I:C) (f). 8 d later, mice were challenged i.v. with a cocktail of freshly isolated CD45.1⁺ splenocytes labeled with different concentrations of CFSE and/or cell trace violet and loaded with 2.4, 40, 160, or 625 nM SIINFEKL peptide (unloaded cells were used as injection control and control for specificity of the lysis). This allowed for the simultaneous analysis of target cell lysis loaded with different amounts of SIINFEKL within one mouse. 16 h later, splenocytes were analyzed for the presence of the transferred CD45.1⁺ cells. The data points shown in this graph have been generated within three independent experiments. Each dot represents the degree of lysis observed for splenocytes loaded with a specific amount of peptide (median ± interquartile range in e and one line for each single mouse in f). (e) This experiment was performed three times with two mice per group, and all data points are shown in the graphs. (f) This experiment was performed three times with three to five mice per group, and all data points are shown in the graphs.