Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells

Abstract

To initiate an adaptive immune response, rare antigen-specific naive CD4(+) T cells must interact with equally rare dendritic cells (DCs) bearing cognate peptide-major histocompatibility complex (MHC) complexes. Lymph nodes (LNs) draining the site of antigen entry are populated by lymphoid-resident DCs as well as DCs that have immigrated from tissues, although the requirement for each population in initiating the T cell response remains unclear. Here, we show that antigen processing and presentation by both lymphoid-resident and migratory DCs was required for clonal selection and expansion of CD4(+) T cells after subcutaneous immunization. Early antigen presentation by lymphoid-resident DCs initiated activation and trapping of antigen-specific T cells in the draining LN, without sufficing for clonal expansion. Migratory DCs, however, interacted with the CD4(+) T cells retained in the LN to induce proliferation. Therefore, distinct DC subsets cooperate to alert and trap the appropriate cell and then license its expansion and differentiation.

Figure 1. Schematic of migratory and lymphoid-resident DCs

Skin-draining LNs contain multiple DC subsets: Lymphoid-resident DCs that differentiate from blood-derived precursor cells sensitive to γ-irradiation (white) (Naik et al., 2007; Onai et al., 2007), and migratory DCs entering via the afferent lymphatics. Migratory DCs consist of radioresistant (black) epidermal LCs (Merad et al., 2002) as well as heterogenous dermal DCs that are mainly radiosensitive (Bogunovic et al., 2006).

Figure 2. Radioresistant APCs do not efficiently prime T cells in vivo

(A) CFSE dilution of OT-II T cells in cutaneous LN from BM chimeras immunized with 200ug or 20ug OVA protein in CFA. Draining LN from WT→WT or MHCII KO→WT chimeras were analyzed 7 days post-immunization (n=6). (B) Flow cytometry of MHCII expression on cells from cutaneous LN from MHCII KO→WT chimeras analyzed 7 days post-immunization. Histograms are gated on Langerin^high host (CD45.1⁺) and Langerin^low donor (CD45.2⁺) DCs (solid) or endogenous T cells (dashed). Representative data shown of 5 independent experiments. (C) Sorted radioresistant (CD45.1⁺) MHCII⁺ APCs pooled from MHCIIKO→WT chimeras were incubated with OVA protein (100ug/ml) and LPS (75ng/ml) overnight. Irradiated BM splenocytes were similarly treated. CFSE-labeled OT-II cells (10⁵) were added to decreasing numbers of DC or splenocytes and analyzed by flow cytometry on day 5.

Figure 3. Migratory DCs in CD11c/A_β^b mice function poorly

(A) MHCII expression on lymphoid-resident DCs from WT, CD11c/A_β^b, and MHCII KO spleen gated on CD11c⁺CD8α^pos (top) or CD11c⁺CD8α^neg (bottom) subpopulations. (B) MHCII expression on dermal CD11c⁺ cells (DEC205^highLangerin^high or DEC205^intLangerin^low) from 48hr explant culture (n=6). (C) MHCII expression on epidermal CD11c⁺Langerin⁺ cells from 48hr explant cultures (n=6). (D–E) Sorted CD11c⁺ LN or skin DCs were incubated with various doses of OVA protein and LPS (75ng/ml) overnight and then cultured with 10⁵ CFSE-labeled OT-II cells for 5 days in vitro. Histograms of CFSE dilution gated on congenic OT-II CD4⁺ T cells. (D) Sorted CD11c⁺ cutaneous LN DC (10⁵) from WT (left) or CD11c/A_β^b (right) mice (n=3) (E) Sorted CD11c⁺ DC from dermal or epidermal explant cultures from WT or CD11c/A_β^b mice pulsed with 100ug OVA protein (n=3).

Figure 4. Lymphoid-resident DCs poorly prime naïve CD4⁺ T cells

(A–D) Congenic OT-II T cells analyzed for CFSE dilution in the draining LN on day 4 post-immunization. (A) Untreated or (B) Flt3L-treated WT, CD11c/A_β^b, or MHCII KO mice received 2 × 10⁶ OT-II cells prior to immunization with 20ug OVA protein in CFA (n=3). (C) 2 × 10⁴ CFSE-labeled congenic OT-II T cells were analyzed for proliferation after immunization with 20ug OVA protein in CFA (n=3). (D) WT or CD11c/A_β^b mice were immunized with decreasing doses of OVA protein in CFA (200ug, 100ug, 20ug, 0ug) one day after receiving 2×10⁶ CFSE-labeled OT-II T cells. Histogram overlays represent CFSE dilution of congenic OT-II cells in draining LN of WT (line) or CD11c/A_β^b mice (gray shaded) (n=3).

Figure 5. Migratory radioresistant APCs rescue the T cell proliferative defect observed in CD11c/A_β^b mice

(A–B) OT-II cell proliferation analyzed 4 days post-immunization from the draining LN of (A) WT→WT, CD11c/A_β^b→WT, or CD11c/A_β^b→ CD11c/A_β^b BM chimeras, (B) CD11c/A_β^b→ CD11c/A_β^b, CD11c/A_β^b→CCR7^+/− or CD11c/A_β^b→CCR7 KO BM chimeras (n=3). (C) Flow cytometry analysis comparing MHCII expression on CD11c⁺ cutaneous LN cells from CD11c/A_β^b or CD11c/A_β^b→WT chimeras based upon Langerin and/or CD103 expression. Representative data shown. (E) OT-II cell proliferation analyzed 4 days post-immunization from the draining LN of CD11c/A_β^b→H2-DM^+/−, CD11c/A_β^b→H2-DM KO, or H2-DM KO→H2-DM^+/− BM chimeras.

Figure 6. Migratory DC antigen presentation mediates T cell progression into the cell cycle

(A) Time course of CD69 expression on OT-II cells stimulated in WT (■), CD11c/A_β^b (▲), or MHCII KO mice (×) during the first 4 days post-immunization. Data are representative of 5 independent experiments. Standard deviation (48hrs) represents 3–5 mice per group (*p<0.0001 WT vs CD11c/A_β^b). (B) CD69 expression and CFSE dilution on OT-II T cells 48hrs post-immunization in the draining LN from WT→WT or MHCII KO→WT chimeras. (C) Percentage of OT-II T cells expressing CD25 in the draining LN of WT, CD11c/A_β^b or MHCII KO mice 48hrs post-immunization. Standard deviation represents ≥6 mice per group (*p<0.001 WT vs CD11c/A_β^b) (D) CD69 expression and forward scatter of OT-II T cells in draining LN of WT or CD11c/A_β^b mice 48hrs post-immunization.

Figure 7. Lymphoid-resident DC antigen presentation regulates LN trapping of antigen-specific T cells

(A) CFSE-labeled CD45.1⁺ OT-II T cells and CD90.1⁺ TEa T cells were co-transferred into WT, CD11c/A_β^b, MHCII KO, and MHCII KO→WT BM chimeras. At 42hrs post-immunization, the absolute number of antigen-specific (CD45.1⁺ OT-II cells) and antigen-non-specific (CD90.1⁺ TEa cells) was compared in the single draining inguinal LN and the contralateral non-draining axillary LN from the same animal. The ratio of OT-II/TEa cells in the draining LN is shown (n=2; ≥3 mice/group) (B) Analysis of CFSE dilution in OT-II (CD45.1⁺) or TEa (CD45.1⁻) cells from the mice above (C) Realtime PCR analysis of S1P₁ receptor mRNA expression in OT-II cells sorted 42hrs post-immunization with 20ug OVA protein in CFA from draining LN of WT or CD11c/A_β^b, as well as WT mice immunized with only CFA. Representative experiment is shown. Values represent mean ± S.D. of quadruplicate determinations, normalized to GAPDH values.