Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells

Abstract

Induction of antigen-specific tolerance is critical for autoimmunity prevention and immune tolerance maintenance. In addition to their classical role as sentinels of the immune response, dendritic cells (DCs) play important roles in maintaining peripheral tolerance through the induction/activation of regulatory T (T(reg)) cells. The possibility of generating tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Characterizing endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. We here report that the immunosuppressive neuropeptide vasoactive intestinal peptide (VIP) induces the generation of human tolerogenic DCs with the capacity to generate CD4 and CD8 T(reg) cells from their respective naive subsets. The presence of VIP during the early stages of DC differentiation from blood monocytes generates a population of IL-10-producing DCs unable to fully mature after the effects of inflammatory stimuli. CD4 T(reg) cells generated with VIP-differentiated DCs resemble the previously described Tr1 cells in terms of phenotype and cytokine profile. CD8 T(reg) cells generated with tolerogenic VIP DCs have increased numbers of IL-10-producing CD8(+)CD28(-)-CTLA4(+) T cells. CD4 and CD8 T(reg) cells primarily suppress antigen-specific T(H)1-mediated responses. Therefore, the possibility of generating or expanding ex vivo tolerogenic DC(VIPs) opens new therapeutic perspectives for treating autoimmune diseases and graft-versus-host disease after allogeneic transplantation in humans.

Figure 1.

VIP interferes with the differentiation of human DCs and their subsequent maturation. Human DCs generated in the absence (DC_controls) or presence (DC_VIPs) of VIP were stimulated with LPS to induce DC activation/maturation. (A) Cell surface, maturation, and costimulatory markers were analyzed by flow cytometry. Dashed lines in top histograms represent the staining profile with isotype-matched control antibodies. Numbers represent the mean channel fluorescence intensity (MFI) for each phenotypic marker. Histograms are representative of 4 independent experiments. (B) DCs differentiated in the presence of VIP show augmented antigen uptake, evaluated by endocytosis, of FITC-dextran at different times. Results are expressed as fluorescence intensity and are the mean ± SD of 3 experiments performed in duplicate. (C) Differentiation of DCs with VIP modifies their cytokine production after activation. Results are the mean ± SD of 4 experiments performed in duplicate.

Figure 2.

VIP generates human tolerogenic DCs, which induce IL-10-producing anergic CD4 T cells. Human DCs generated in the absence (DC_controls, ▪) or presence (DC_VIPs, □) of VIP were stimulated with LPS to induce DC activation/maturation. (A) Purified naive CD4 T cells were cultured with graded doses of allogeneic DC_controls (▪) or DC_VIPs (□) in primary cultures (panel i). CD4 T cells from primary cultures were recovered, rested, and restimulated with LPS-matured allogeneic DCs (panel ii). Cultures of DC_controls or DC_VIPs without T cells did not proliferate. Cytokine production was determined in secondary cultures at DC/T cell ratios of 1:10 (panel iii). Each result is the mean ± SD of 3 experiments performed in duplicate. (B) TT-primed T_H1 cells were cocultured with TT-pulsed syngeneic DC_controls (▪) or DC_VIPs (□)in primary cultures (panel i). After 3 days of culture, T_H1 cells were rescued, rested, and restimulated with TT-pulsed, LPS-matured syngeneic DCs, and proliferation (panel ii) and IFNγ production (panel iii) were determined. Unpulsed or ovalbumin-pulsed DC_controls or DC_VIPs did not induce the proliferation of TT-primed T_H1 cells. Each result is the mean ± SD of 3 experiments performed in duplicate.

Figure 3.

VIP-differentiated DCs generate human regulatory T cells in vitro. Purified naive CD4 T cells exposed to allogeneic DC_controls (T_{reg controls}) or DC_VIPs (T_{reg VIPs}) were evaluated for suppressive/regulatory activity. (A) T_{reg VIPs} (□) or T_{reg controls} (▪) were cocultured at different ratios with syngeneic T_H1 cells in the presence of allogeneic mature DCs (mDCs). Proliferation of T cells and IL-2 production was determined. T_{reg VIPs}, alone or with allogeneic mDCs, did not proliferate. (B) T_H1 cells and T_{reg VIPs} were generated by priming of CD4 T cells from donor C with allogeneic DC_controls or DC_VIPs from donor A (T_H1^anti-A/T_{reg VIPs}^anti-A) or donor B (T_H1^anti-B/T_{reg VIPs}^anti-B) and were cocultured in the presence of mDCs from donor A or donor B, or both, as indicated. Proliferation of responder T cells was determined. Each result is the mean ± SD of 3 experiments performed in duplicate. (C) Polarized T_H1 cells were cocultured with T_{reg VIPs} and allogeneic mDCs in the presence or absence of blocking anti-IL-10, anti-TGFβ, anti-CTLA-4, or IL-2 antibody. Additionally, T_H1 + mDCs were separated from T_{reg VIPs}+mDCs in a Transwell system. The proliferative response of T_H1 cells was determined. (D) Phenotype of T_{reg VIPs} and T_{reg controls}. Expression of surface markers (CD25, CD69, CD70, CD154), intracellular IL-10 and CTLA-4, and cytosolic and membrane-bound TGFβ were determined by flow cytometry. Numbers represent the percentage of positive cells in each quadrant. Foxp3 and neuropilin 1 (Nrp1) mRNA expression in sorted CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25^- T_{reg controls} (▪) or T_{reg VIPs} (□) was determined by real-time RT-PCR. Results are representative of 5 independent experiments.

Figure 4.

DC_VIPs induce T cell anergy in alloreactive cytotoxic CD8 T cells. (A) Human CD8 T cells (5 × 10⁶) obtained from the PBMCs primed with allogeneic fibroblasts (donor 1) were cultured with or without syngeneic DC_controls or DC_VIPs (5 × 10⁵) obtained from the same donor pulsed without (unpulsed) or with the necrotic fibroblasts used in the priming culture (pulsed 1) or unrelated donor-derived necrotic fibroblasts (pulsed 2). After 3 days of coculture, CD8⁺ cells (5 × 10⁵) were recovered, rested, and assayed for cytotoxicity against the allogeneic fibroblasts used in the priming culture (donor 1) at different effector-target (E/T) cell ratios. The established allogeneic fibroblast-specific CD8 T cells showed cytotoxicity only against allogeneic fibroblasts used in their generation (donor 1), indicating that their cytotoxicity was antigen specific, because none of the CD8⁺ cells showed cytotoxicity against allogeneic fibroblasts from an unrelated donor (donor 2). Each result is the mean ± SD of 3 experiments performed in duplicate. (B) Human allogeneic fibroblast-specific CD8 T cells (5 × 10⁶) were cocultured without (none) or with unpulsed DC_controls or DC_VIPs (5 × 10³, 5 × 10⁴, or 5 × 10⁵ cells) derived from the indicated donors (allogeneic 1 and syngeneic 2) for 3 days. These CD8 T cells were then rescued and subsequently assayed (5 × 10⁵ cells) for cytotoxicity against the allogeneic fibroblasts (10⁴ cells) used in the priming culture (donor 1). The value of spontaneous release cpm was less than 10% of the total release cpm. Each result is the mean ± SD of 4 experiments performed in duplicate.

Figure 5.

DC_VIP generate human CD8 T_reg. (A) Human naive CD8⁺ T cells (5 × 10⁶) were cultured with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells). After 5 days, CD8 T cells were isolated from the cocultures and assayed for phenotype (left panel) and cytokine profile (right panel) by flow cytometry. Human naive CD8⁺ T cells were used as controls. Numbers represent the percentage of positive cells in each quadrant. Cytokine levels in cultured supernatants (determined by ELISA) were consistent with the intracellular staining (not shown). Results are representative of 4 experiments with similar findings. (B) Total CD8⁺, CD8⁺CD28⁺, or CD8⁺CD28^- T cells were isolated from the coculture of human naive CD8⁺ T cells (5 × 10⁶) with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells) for 5 days and were cultured with human allogeneic mDCs (10⁴). Proliferation of CD8 T cells was determined. CD8 T cells without allogeneic mDCs did not proliferate. Each result is the mean ± SD of 3 experiments performed in duplicate. (C) Total CD8⁺, CD8⁺CD28⁺, or CD8⁺CD28^- were isolated from the coculture of human naive CD8⁺ T cells (5 × 10⁶) with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells) for 5 days and were cocultured at different ratios with human syngeneic T_H1 cells (10⁵) in the presence of allogeneic mDCs (10⁴). (D) Human T_H1 cells were cultured with allogeneic mDCs (10⁴) and CD8⁺ T cells (CD8_VIP, 2 × 10⁴) isolated from cocultures with DC_VIPs, in the presence or absence of blocking anti-IL-10, anti-TGFβ, anti-CTLA-4, or IL-2. Isotype IgG was used as control. Additionally, T_H1 + mDCs were separated from CD8_VIP + mDCs in a Transwell system. The proliferative response of T_H1 cells and the production of IFNγ were determined after 4 days of culture. mDCs alone did not proliferate. Each result is the mean ± SD of 3 experiments performed in duplicate. (E) CTLA-4 expression of naive CD8 T cells or CD8 T cells isolated from cocultures of human naive CD8 T cells and allogeneic DC_controls or DC_VIPs was determined by flow cytometry. Dashed lines correspond to isotype IgG controls. Results are representative of 4 independent experiments.