The presence and activity of dendritic cells (DC) in retina is controversial, as these cells are difficult to identify in retina due to limited markers and sparse numbers. Transgenic mice that express green fluorescent protein (GFP) on the CD11c promoter to label DC allowed the visualization and quantification of retinal DC. Two retina injury models, the optic nerve crush (ONC) and light injury, were used to study their injury response. Many GFP+ DC were tightly associated with retinal ganglion cell nerve fibers following ONC, while very few microglia (GFP−CD11b+ cells) were found in close contact. The GFP+ cells were greatly elevated in the outer plexiform layer following photic injury. All of the GFP+ DC were CD11b+, suggesting a myeloid origin. In addition, the GFP+ DC upregulated expression of MHC class II after injury, while the GFP−CD11b+ microglia did not. This study shows that DC were found in the retina and that they rapidly responded to neural injuries. We propose that they are a previously overlooked population, distinct from microglia, and may be important in the injury response.

Interest in the identities, properties, functions and origins of local antigen presenting cells (APC) in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina, and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of antigen presenting cell activity of DC from quiescent retina showed that they promoted generation of Foxp3+ T cells, and inhibited activation of naive T cells by splenic DC and antigen. Conversely, in vitro assays of DC purified from injured retina revealed an enhanced ability to activate T cells, and reduced induction of Foxp3+ T cells. These findings were supported by the observation that in situ activation of DC prior to adoptive transfer of β-galactosidase-specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of antigen in vivo showed that quiescent retina promoted development of parenchymal Foxp3+ T cells, but assays of pre-injured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function, and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

Extrathymically derived regulatory T cells (iTregs) protect against autoimmunity to tissue-specific antigens (Ag). However, whether Ag-specific iTreg generation and function is limited to secondary lymphoid tissue or whether it can occur within the tissue-specific, local environment of the cognate Ag remains unresolved. Mice expressing beta-galactosidase (βgal) on a retina specific promoter (βgal mice) in conjunction with mice expressing green fluorescent protein (GFP) and diphtheria toxin (DTx) receptor (DTR) under control of the FoxP3 promoter, and βgal-specific T cell receptor transgenic (BG2) mice were used to examine this question. Local depletion (ocular DTx), but not systemic depletion (intra-peritoneal DTx), of βgal-specific iTregs enhanced experimental autoimmune uveoretinitis induced by activated βgal-specific effector T cells. Injections of small amounts of βgal into the anterior chamber of the eye produced similar numbers of βgal-specific iTregs in the retina whether or not the mouse was depleted of pre-existing, circulating Tregs. Together these results suggest that protection from tissue-specific autoimmunity depends on the function of local Ag-specific iTregs, and that the retina is capable of local, “on-demand” iTreg generation that is independent of circulating Tregs.

Although several observations show local T cell recognition of retinal Ag, there has been no direct demonstration that the APC were retinal derived, rather than recruited. In this study, CD45+ cells isolated from immunologically quiescent murine retina were tested in vitro for functional evidence of Ag presentation to naive and Ag-experienced CD4 T cells specific for β-galactosidase. Because CD45+ cells from brain have been reported to be efficient APC, they were included for comparison. Measures of activation included changes in CD4, CD25, CD44, CD45RB, CD62L, CD69, caspase-3 activation, CFSE dilution, size, number of cells recovered, and cytokine production. Retinal CD45+ cells gave no evidence of Ag-dependent TCR ligation in naive T cells, unlike splenic APC and CD45+ cells from brain, which supported potent responses. Instead, addition of retinal CD45+ cells to cocultures of naive 3E9 T cells plus splenic APC reduced the yield of activated T cells and cytokine production by limiting T cell activation at early time points. Ag-experienced T cells responded weakly to Ag presented by retinal CD45+ cells. Activating the retinal cells with IFN-γ, anti-CD40, or LPS incrementally increased their APC activity. Addition of neutralizing Abs to TGF-β did not reveal suppressed retinal APC activity. Because retina lacks tissue equivalents of meninges and choroid plexus, rich sources of dendritic cells in brain, cells from retina may better represent the APC activity of fresh, adult CNS parenchymal and perivascular cells. The activity of the retinal CD45+ cells appears to be directed to limiting T cell responses.

To study retinal immunity in a defined system, a CD4+ TCR transgenic mouse line (βgalTCR) specific for β-galactosidase (βgal) was created and used with transgenic mice that expressed βgal in retinal photoreceptor cells (arrβgal mice). Adoptive transfer of resting βgalTCR T cells, whether naive or Ag-experienced, into arrβgal mice did not induce retinal autoimmune disease (experimental autoimmune uveoretinitis, EAU) and gave no evidence of Ag recognition. Generation of βgalTCR T cells in arrβgal mice by use of bone marrow grafts, or double-transgenic mice, also gave no retinal disease or signs of Ag recognition. Arrβgal mice were also resistant to EAU induction by adoptive transfer of in vitro-activated βgalTCR T cells, even though the T cells were pathogenic if the βgal was expressed elsewhere. In vitro manipulations to increase T cell pathogenicity before transfer did not result in EAU. The only strategy that induced a high frequency of severe EAU was transfer of naive, CD25-depleted, βgalTCR T cells into lymphopenic arrβgal recipients, implicating regulatory T cells in the T cell inoculum, as well as in the recipients, in the resistance to EAU. Surprisingly, activation of the CD25-depleted βgalTCR T cells before transfer into the lymphopenic recipients reduced EAU. Taken together, the results suggest that endogenous regulatory mechanisms, as well as peripheral induction of regulatory T cells, play a role in the protection from EAU.