Novel Insights Into the Immunoregulatory Function and Localization of Dendritic Cells

Abstract

Dendritic cells (DCs) are antigen-presenting cells that normally play a critical role in stimulating T-cell-dependent immune responses. However, tolerogenic DCs (CD11cMHC-IICD80CD86) induce immune tolerance by stimulating regulatory T cells (Tregs: CD4CD25Foxp3). Although tolerogenic DCs are used to treat autoimmune diseases and to prevent transplantation rejection, the mechanisms by which they regulate alloimmunity are poorly understood. Here, we review our previous studies aiming to elucidate the mechanisms involved in immune rejection of corneal allografts using a corneal transplant model. We found that donor-derived tolerogenic DCs significantly prolonged corneal allograft survival by suppressing indirect allosensitization. We also reported the precise distribution of intraepithelial corneal DCs, termed Langerhans cells (LCs: CD11cLangerinMHC-II) in the cornea, which we maintain play a critical role in regulating corneal immunity. By confocal microscopy, we constructed 3-dimensional images of corneal LCs, which demonstrated that their cell bodies are present in the basal cell layer of the corneal epithelium. Furthermore, LC dendrites extend toward the ocular surface, but do not connect to epithelial tight junctions, indicating that they cannot directly interact with ocular surface antigens. We confirm the potential of DC therapy for corneal graft rejection and report the function of intraepithelial DCs (LCs) in the normal cornea.

FIGURE 1

Effect of immature DCs (iDCs), mature DCs (mDCs) and tolerogenic DCs on T cell immunity. The steady state iDCs remain quiescent after capturing and processing exogenous antigen. These quiescent iDCs express low levels of co-stimulatory molecules and therefore cause a deficient activation of naive T cells, and induce T cell apoptosis or anergy and probably the generation and/or expansion of regulatory T cells (Treg). In contrast, after exposure to danger signals and/or activation and maturation stimuli, iDCs increase the surface expression of MHC and co-stimulatory molecules, which enhances their ability to stimulate naive or memory T cells. Tolerogenic DCs present antigen to antigen-specific T cells but fail to deliver adequate co-stimulatory signals for effector T cell activation and proliferation. This may manifest as T-cell death, T cell anergy, or Treg expansion or generation.

FIGURE 2

Donor-derived regulatory dendritic cells (DCregs) prolong corneal graft survival. Immature DCs (iDCs), mature DCs (mDCs), and DCregs were generated from C57BL/6 mouse bone marrow cells as described previously by Sato et al. Donor-derived mDC (1×10⁶), iDC (1×10⁶), or DCreg (0.2×10⁶, 1×10⁶)-infused BALB/c mice or untreated mice were transplanted with C57BL/6 corneas and graft survival was followed microscopically for 8 weeks (n = 6 per group). Kaplan–Meier survival curves indicate that infusion of 1×10⁶ donor-derived DCregs prolonged graft survival (*P = 0.043). Reproduced and modified from Hattori et al with permission from the Journal of Leukocyte Biology.

FIGURE 3

Direct and indirect allorecognition in transplantation. Allorecognition can occur by two distinct, but not mutually exclusive, pathways: the direct and indirect pathways. The direct pathway results from the recognition of intact foreign major histocompatibility molecules (MHC) on the surface of donor cells. Indirect allorecognition occurs when donor histocompatibility molecules are internalized, processed, and presented as peptides by host antigen presenting cells (APC)—this is how the immune system normally encounters antigen.

FIGURE 4

Infusion of donor-derived regulatory dendritic cells (DCregs) impairs T cell differentiation in corneal graft recipients. C57BL/6 corneas were transplanted to BALB/c mice. For the infusion of DCs into recipients, 1×10⁶ immature DCs (iDCs), mature DCs (mDCs), or DCregs, generated from C57BL/6 mouse bone marrow cells were injected intravenously via the lateral tail vein 7 days before transplantation. (A) T cells were isolated from draining lymph nodes 3 weeks post-transplantation (n = 3 per group). Isolated T cells obtained from DCreg/mDC-infused recipients or untreated recipients were stimulated with PMA/ionomycin and IFN-γ⁺ (CD4⁺ and CD4⁻ fractions) and were subsequently measured by flow cytometry. (B) mRNA was isolated from draining lymph nodes 3 weeks post-transplantation (n = 3 per group). Expression of Foxp3 in draining lymph nodes post corneal transplantation was measured by real-time PCR. *P = 0.03. Reproduced and modified from Hattori et al. with permission from the Journal of Leukocyte Biology.

FIGURE 5

Donor-derived regulatory dendritic cells (DCregs) suppress the indirect pathway in corneal transplant recipients. C57BL/6 corneas were transplanted to BALB/c mice. For the infusion of DCregs into recipients, 1×10⁶ DCregs generated from C57BL/6 mouse bone marrow cells were injected intravenously via the lateral tail vein 7 days before transplantation. T cells were isolated from draining lymph nodes of untreated and DCreg-infused recipients (n = 4 per group) 3 weeks post-transplantation, and were measured by IFN-γ ELISPOT assay. Isolated T cells were cocultured with allogeneic antigen-presenting cells to measure the direct pathway or cocultured with syngeneic antigen-presenting cells in the presence of alloantigen to measure the indirect pathway. (A, B) Triplicate enzyme-linked immunospot wells for IFN-γ production responder T cells after 48 h of culture from each group (direct pathway: A, indirect pathway: B). (C) Donor-derived DCregs reduced the frequency of direct pathway-type alloreactivity compared with untreated recipients. *P = 0.007. (D) Donor-derived DCregs also reduced the frequency of indirect pathway-type alloreactivity relative to untreated recipients. **P = 0.0007. Moreover, the reduction (30.2-fold) was much higher than in the direct pathway (1.5-fold). Data are representative of two experiments. Reproduced and modified from Hattori et al. with permission from the Journal of Leukocyte Biology.

FIGURE 6

3D visualization of epithelial tight junctions and Langerhans cells (LCs) in normal mouse cornea. Corneas were harvested from naïve C57BL/6 mice and stained with anti-ZO-1 antibody (red) and anti-MHC-II antibody (green). Images of stained corneas were captured by confocal microscopy and analyzed by an image analyzer. (A) Reconstituted 3D images of epithelial tight junctions and LCs are shown. (B) En face imaging is demonstrated by confocal microscopy. (C) A 90° rotated image of the area in (B) is shown. The white arrow indicates the tight junctions of the corneal epithelium. The gray arrows indicate dendrites of corneal LCs.