GM-CSF-induced, bone-marrow-derived dendritic cells can expand natural Tregs and induce adaptive Tregs by different mechanisms

Abstract

In our earlier work, we had shown that GM-CSF treatment of CBA/J mice can suppress ongoing thyroiditis by inducing tolerogenic CD8α(-) DCs, which helped expand and/or induce CD4(+)Foxp3(+) Tregs. To identify the primary cell type that was affected by the GM-CSF treatment and understand the mechanism by which Tregs were induced, we compared the effect of GM-CSF on matured spDCs and BMDC precursors in vitro. Matured spDCs exposed to GM-CSF ex vivo induced only a modest increase in the percentage of Foxp3-expressing T cells in cocultures. In contrast, BM cells, when cultured in the presence of GM-CSF, gave rise to a population of CD11c(+)CD11b(Hi)CD8α(-) DCs (BMDCs), which were able to expand Foxp3(+) Tregs upon coculture with CD4(+) T cells. This contact-dependent expansion occurred in the absence of TCR stimulation and was abrogated by OX40L blockage. Additionally, the BMDCs secreted high levels of TGF-β, which was required and sufficient for adaptive differentiation of T cells to Foxp3(+) Tregs, only upon TCR stimulation. These results strongly suggest that the BMDCs differentiated by GM-CSF can expand nTregs and induce adaptive Tregs through different mechanisms.

Figure 1.. GM-CSF-derived BMDCs can increase the percentage of Foxp3⁺ Tregs in cocultures.

spDCs were isolated and cultured with or without GM-CSF for 48 h. BMDCs were generated in vitro with GM-CSF. C-spDCs, G-spDCs, and BMDCs were (A) cocultured with CD4⁺ T cells from mTg-primed mice in the presence of mTg (upper panel) or naïve mice without antigen (lower panel) and stained with FITC-labeled anti-CD4 and allophycocyanin-labeled anti-Foxp3 for FACS analysis. Each scatter plot is representative of five independent experiments gated over 3500 live CD4⁺ T cells. Each in vitro experiment was conducted with T cells, spDCs, and BMDCs pooled from three mice. (B) C-spDCs, G-spDCs, and BMDCs were cocultured with CD4⁺ T cells from naïve mice without antigen for 5 days and stained for the expression of CD4 and IL-17 by FACS. (C) CFSE-labeled CD4⁺ T cells were cultured in different concentrations of GM-CSF ranging from 0 to 2500 ng/ml in the presence and absence of anti-CD3/APCs for 4 days and analyzed for Foxp3 expression by FACS. The numbers indicate the percentage of double-positive (CD4⁺Foxp3⁺) T cells. The experiment was repeated three times with similar results.

Figure 2.. CD11c⁺ BMDCs increase the percentage Foxp3⁺ Tregs in cocultures primarily through a contact-dependent mechanism.

(A) BM cells were analyzed for the expression of CD11b and CD11c on Days 2, 4, 7, and 11 (upper panel). BM cells obtained from the respective days were cocultured with CD4⁺ T cells, and after 5 days, the cells were analyzed for Foxp3 expression by FACS (lower panel). (B) CD11c and CD8α expression on cells from GM-CSF-derived BMDCs (upper panel). CD11c⁺ and CD11c⁻ cells from GM-CSF cultures were cocultured with CD4⁺ cells from naïve mice and percentage of CD4⁺Foxp3⁺ T cells from the coculture analyzed (lower panel). (C) Cocultures of BMDCs with CD4⁺ T cells, together or separated by transwell, were analyzed for Foxp3 expression without anti-CD3 (upper panel) or with anti-CD3 (lower panel). In transwell cocultures, CD4⁺ T cells were cultured in the bottom wells, and the BMDCs were cultured in the top wells. Numbers indicate the percentage of double-positive CD4⁺Foxp3⁺ T cells. Experiments A–C were repeated at least three times with similar results.

Figure 3.. Contact-independent induction of adaptive Tregs in vitro by BM supernatant is TGF-β-dependent.

(A) RT-PCR analysis of cytokine transcripts from BMDC and spDC cultures. The two bands in each category of 1, 2, and 3 indicate the transcript levels after 31 and 33 PCR cycles. (B) CFSE-labeled CD4⁺CD25⁻ T cell cultures, supplemented with TGF-β or BM culture supernatant (SUP; 1×), were stained with allophycocyanin-labeled anti-Foxp3 in the absence (upper panel) and presence (lower panel) of anti-CD3. (C) CFSE-labeled CD4⁺CD25⁻ T cells were cultured with different concentrations of BM supernatant and anti-CD3. The induction of Foxp3⁺ in T cells in the presence of increasing concentrations of BM supernatant (upper panel) and its inhibition by different concentrations of anti-TGF-β (lower panel) are shown. Experiments B and C were repeated three times with similar results.

Figure 4.. BMDCs can directly and selectively expand nTregs in T cell cocultures.

(A) spDCs and BMDCs were cocultured with CFSE-labeled CD4⁺ T cells from naïve mice and analyzed for proliferation and Foxp3 expression. Small panels on the right show the extent of CFSE dilution of Foxp3⁺ and Foxp3⁻ cells in the original histograms. (B) The extent of CFSE dilutions in different T cell subpopulations is measured by gating on Foxp3^+/− or CD25^+/− T cells. The top panel shows the position of the gate, middle panel shows the gated population, and bottom panel shows the extent of CFSE dilution of the gated population. (C) CFSE-labeled CD4⁺CD25⁻ T cells were cocultured with control (spDCs), with or without BM culture supernatant (BM sup), BM cells [BM (day 0)], or BMDCs, and analyzed for Foxp3 expression and CFSE dilution. (A–C) Each scatter plot is representative of five separate experiments.

Figure 5.. The selective expansion of Tregs in BMDC cocultures is OX40L-dependent.

(A) BMDCs and spDCs, cultured in the presence or absence of GM-CSF, were analyzed for the expression of CD80, CD86, MHCII, PDL1, and PDL2. Each scatter plot represents three independent experiments. (B) Coculture of BMDCs and CD4⁺ T cells, in the presence of various blocking and neutralizing antibodies to anti-inflammatory cytokines or cell surface molecules, was stained and analyzed for Foxp3 expression. (C) RT-PCR analysis of OX40L and PDL1 from BMDC and spDC cultures. The two bands in each category indicate the transcript levels at 31 and 33 PCR cycles. HPRT is shown as control. (D) Analysis of surface expression of OX40L in spDCs and BMDCs. (E) Coculture of BMDCs and CFSE-labeled CD4⁺ T cells in the presence of increasing concentrations of a neutralizing antibody to OX40L was analyzed for CFSE dilution and Foxp3 expression in T cells. (F) Cocultures of BMDCs and CD4⁺ T cells in the presence of anti-OX40L antibody, alone or in combination with OX40 agonist at two different concentrations (lo/low=5 μg/ml and hi/high=10 μg/ml). (D–F) Each scatter plot represents five separate experiments.

Figure 6.. BMDC-mediated Treg expansion is dependent on IL-2 but does not require TCR interaction.

(A) Abrogation of Treg proliferation by anti-IL-2. BMDCs were cocultured with CD4⁺ cells, without or with anti-IL-2. (B) Treg expansion is IL-2-dependent. BMDCs and control splenic APCs were cocultured with total CD4⁺ (upper panel) and sorted CD4⁺CD25⁺ (lower panel) T cells in the presence or absence of IL-2. On Day 4, cultures were analyzed for the CFSE dilution of Foxp3-expressing T cells by FACS. (C) Analysis of surface expression of CD11b, CD80, MHCII, and PDL2 on BMDCs of MHCII^−/− mice. (D) BMDCs from C57/B6 mice can also expand Tregs. BMDCs from WT C57/B6 mice were used in cocultures with CD4⁺ T cells, also derived from WT C57/B6 mice. Splenic APCs were used as a negative control. (E) Treg expansion by BMDCs is TCR-independent. BMDCs from MHCII^−/− mice were generated in vitro with GM-CSF, cocultured with CFSE-labeled CD4⁺ cells (upper panel) or CD4⁺CD25⁺ cells (lower panel) in the presence or absence of IL-2. In some cultures, anti-OX40L antibody was added as indicated. (A–E) Experiments were repeated three times with similar results.

Figure 7.. In vitro-expanded Tregs can suppress Teff proliferation.

(A) CD4⁺Foxp3⁺ T cells from cocultures of BMDC and total CD4⁺ T cells (nTregs, upper panel) or TCR-activated CD4⁺CD25⁻ T cells (iTregs, lower panel), supplemented with BM culture supernatant, were stained for different cell surface markers. (B) Histograms show the proliferation of CFSE-labeled CD4⁺CD25⁻ T cells from OVA-immunized mice in the presence of in vitro-generated CD4⁺CD25⁺ T cells (nTregs) and CD4⁺GARP⁺ T cells (iTregs) isolated from BMDC cocultures or TGF-β-supplemented cultures added in different ratios. The numbers in the gated population of the histograms indicate the percentage of cells proliferated. Results shown are representative of three independent experiments.

Figure 8.. GM-CSF treatment leads to the development of CD11b⁺CD11c⁺

tolerogenic DCs in vivo. (A) Mice were treated with GM-CSF for 5 consecutive days for 2 weeks, and spleen cells were stained with FITC-labeled anti-CD11c, allophycocyanin-labeled anti-CD11b, FITC-labeled anti-CD4, and allophycocyanin-labeled anti-Foxp3 and analyzed by FACS. The middle panels indicate the percentage of double-positive CD11b⁺ CD11c⁺ cells that are CD8α⁻. The right panels indicate the percentage of CD4⁺Foxp3⁺ T cells from the control mice and GM-CSF-treated mice. Each scatter plot represents five different experiments. (B) Bar graph indicates the percentage of Foxp3⁺ Tregs in mice immunized with antigen (mTg+CFA), followed by adoptive transfer of buffer, spDCs, or BMDCs. Each column represents the mean ± sd of an experiment conducted with three animals in each group. ***, Statistically significant value.