The Closely Related CD103⁺ Dendritic Cells (DCs) and Lymphoid-Resident CD8⁺ DCs Differ in Their Inflammatory Functions

<div><p>Migratory CD103⁺ and lymphoid-resident CD8⁺ dendritic cells (DCs) share many attributes, such as dependence on the same transcription factors, cross-presenting ability and expression of certain surface molecules, such that it has been proposed they belong to a common sub-lineage. The functional diversity of the two DC types is nevertheless incompletely understood. Here we reveal that upon skin infection with herpes simplex virus, migratory CD103⁺ DCs from draining lymph nodes were more potent at inducing Th17 cytokine production by CD4⁺ T cells than CD8⁺ DCs. This superior capacity to drive Th17 responses was also evident in CD103⁺ DCs from uninfected mice. Their differential potency to induce Th17 differentiation was reflected by higher production of IL-1β and IL-6 by CD103⁺ DCs compared with CD8⁺ DCs upon stimulation. The two types of DCs from isolated lymph nodes also differ in expression of certain pattern recognition receptors. Furthermore, elevated levels of GM-CSF, typical of those found in inflammation, substantially increased the pool size of CD103⁺ DCs in lymph nodes and skin. We argue that varied levels of GM-CSF may explain the contrasting reports regarding the positive role of GM-CSF in regulating development of CD103⁺ DCs. Together, we find that these two developmentally closely-related DC subsets display functional differences and that GM-CSF has differential effect on the two types of DCs.</p></div

CD103⁺ DCs and CD8⁺ DCs differ in expression of costimulatory molecules, inflammasomes and TLR.

<p>(<b>A</b>) Cells of pooled cutaneous LNs from Langerin-EGFP mice and Langerin-EGFP/<i>CD103−/−</i> mice were analyzed. CD103⁺ DCs were identified as CD326⁻CD205⁺langerin⁺ within migratory DCs (mDC, CD11c^intMHC II^high); CD8⁺ DCs were identified as CD205⁺ CD8⁺ within cDCs (CD11c^highMHC II^int). Histograms show the expression of CD103 and langerin-EGFP by CD8⁺ and CD103⁺ DCs. For CD103 expression, CD8⁺ DCs (grey dot line) and CD205⁺CD11b⁻ migratory DCs (equivalent of CD103⁺ DCs, black dot line) from CD103−/− mice were included. (B) CD8⁺ and CD103⁺ DCs from B6 mice were analyzed for the expression of costimulatory molecules. (C&D) CD8⁺ and CD103⁺ DCs from B6 mice were sorted. RT-qPCR was performed for the indicated transcripts with 3 reference genes as controls. One of three repeated experiments is shown.</p

Elevated GM-CSF increase CD103⁺ DCs.

<p>DC-enriched LN cells were isolated from pooled LN for individual GMtg mice and wild type littermates. DC-enriched LN cells were stained for cell surface markers. (A) Gated CD11c⁺ cells were segregated into migratory DC (MHC class II^hi and CD11c^int) and lymphoid-resident DC (MHC class II^int and CD11c^hi) fractions. Numbers inside dot plots indicate percentage of gated populations. (B) CD103 expression by CD103⁺ DCs and CD8⁺ DCs is shown. (C) Mean numbers of DC subsets from cutaneous LNs of individual mice in each group are shown in bar graphs. Data are representative of >3 experiments. (D) Number of CD103⁺ DCs in ear skin. Each dot represents the data derived from an individual mouse. Horizontal line shows the mean ± SEM. Data are pooled from two experiments (*P<0.05, **P<0.01; Two tailed Student's T test).</p

CD103⁺ DCs and CD8⁺ DCs have differential capacity to induce Th17 upon viral infection.

<p>(A) Proliferation of 5×10⁴ CFSE-labeled HSV-specific CD4⁺ T cells (gDT-II) after 60 h of culture together with serial dilutions of DC subsets isolated from brachial lymph nodes of mice infected withn HSV 5 d earlier. (B) Culture supernatants from (A) were measured for the indicated cytokines. (C) Proliferation and cytokine production with isolated DCs and exogenous source of antigenic peptide. Data (mean ± SEM) are one of two individual experiments. (**P<0.01; Two tailed Student's T test).</p

CD103⁺ DCs and CD8⁺ DCs induce naïve T cells to produce different cytokines.

<p>DC subsets from peripheral LNs of B6 mice were sorted. (A&B) DCs (10⁴/well in triplicate) were cultured with CellTrace violet-labeled OT-II cells (5×10⁴/well) with or without 1 mg/mL OVA for 3 d. Cell proliferation and supernatant cytokine levels were determined. (A) CD4⁺ T cell proliferation; (B) Cytokine production. Histograms show proliferation profile of OT-II, Bar graph shows mean ± SEM (*P<0.05, **P<0.01; Two tail Student's T test). (C) DCs (10⁴/well in triplicate) were cultured with CellTrace violet-labeled OT-I cells (5×10⁴/well) with or without 0.1 mg/mL OVA for 3 d. Cell proliferation and supernatant cytokine levels were determined. Bar graph shows mean ± SEM (**P<0.01; Two tail Student's T test).</p

GM-CSF modulates the capacity of CD103⁺ DCs to induce Th differentiation.

<p>CD103⁺ DCs and CD8⁺ DCs were purified from LNs of GMtg and wild type littermates. DCs were cultured with CellTrace violet-labeled OT-II cells and 1 mg/ml OVA for 3 d. (A) Proliferation was determined by dye dilution. Bar graphs show numbers of proliferating OT-II cells from triplicate cultures. (B) Cytokines in culture supernatants were measured. Bar graph shows mean ± SEM (*P<0.05; Two tail Student's T test). >3 experiments were performed with similar results. (C) DCs (5×10³/well in triplicate) were cultured with CellTrace voilet-labelled OT-II cells (1×10⁴/well) for 3 d. Supernatants were collected for analysis of cytokine production. Bar graph shows mean ± SEM of cell proliferation and selected cytokines (*P<0.05; Two tailed Student's T test).</p

CD103⁺ DCs and CD8⁺ DCs produce different cytokines and chemokines.

<p>DCs were sorted from B6 mice and cultured at 5000 DCs per well in 96-well plates with (CpG or Poly I:C) or without (Nil) stimuli for 24 h. Exogenous GM-CSF (2 ng/mL) was included in cultures. Culture supernatants were assayed for cytokines (A) and chemokines (B). Data (mean ± SEM) are one of three individual experiments (*P<0.05, **P<0.01; Two tailed Student's T test).</p