Antigen-specific accumulation of naïve, memory and effector CD4 T cells during anterior uveitis monitored by intravital microscopy

Abstract

Uveitis is an immune-mediated ocular disease and a leading cause of blindness. We characterized a novel model of uveitis with intravital microscopy. Transfer of ovalbumin-specific T cells from DO11.10 spleen to BALB/c recipients and subsequent challenge with ovalbumin in the anterior chamber of the eye resulted in anterior uveitis. Antigen-specificity was verified by injection of irrelevant antigen and transfer of T cells with a different specificity. Subsets of CD4 T cells, including naive (DO11.10 RAG^−/−) and in vitro-activated Th2 effector CD4 T cells, infiltrated anterior segment tissues early in the inflammation. Memory-like CD44^high CD4 T cells from unprimed transgenic mice and in vitro-activated Th1 effector CD4 T cells accumulated to larger numbers than naive or Th2 effector cells at 48 and 72 h. Of these, the α₂-integrin + CD4 unprimed T cells entered the eye more efficiently, and antibody to α₂-integrin markedly inhibited the inflammatory response. Intravital microscopy revealed the early arrival and antigen-specific accumulation of CD4 T cells in inflamed tissue and should be helpful in understanding T cell migration to other organs.

Introduction

The location of T cells in lymphoid and non-lymphoid organs depends, firstly, upon the phenotype of the T cell and, secondly, on the expression of chemokines and adhesion molecules by the target organs. Murine naïve T cells (CD44^low, CD45RB^high) express the high CD62L levels needed to enter lymphoid tissues and do not express the homing receptors for non-lymphoid organs [1], [2], [3]. Effector cells (CD45RB^low, CD44^high) induced by antigen stimulus express the receptors needed for infiltration of inflamed tissues. T cells activated in lymph nodes that drain the gut tend to express α₄β₇ integrin, which preferentially binds to the Mad-CAM-1 found on inflamed mucosal endothelial vasculature [2], [4], whereas T cells extravasating in skin express high levels of cutaneous lymphocyte-associated antigen (CLA) [5]. The differential expression of α and β integrin heterodimers regulates T cell homing to sites of infection as demonstrated by the upregulation of α₂β₁ and α₁β₁ integrins on T cells infiltrating virally infected sites [6]. CD44^high memory cells are a heterogeneous population: effector memory CD4 T cells circulate via the blood and lymph through non-lymphoid and lymphoid organs, whereas central memory CD4 T cells revert to a naïve-like phenotype that expresses high levels of CD62L and is found predominantly in lymph nodes [7].

Uveitis is the term used to describe intraocular inflammation secondary to an immune-mediated process. Uveitis is roughly comparable to diabetes as a frequent cause of visual loss. Uveitis may occur as an isolated medical problem or it may be a part of a systemic illness such as sarcoidosis, spondyloarthritis, juvenile idiopathic arthritis, multiple sclerosis, or inflammatory bowel disease [8]. The transparency of the cornea allows ready observation of the anterior segment: cells infiltrating the eyes of uveitis patients can be observed by slit lamp examination and intravital confocal microscopy has even been performed on cells in the blood vessels of patients’ conjunctiva [9]. We have used intravital microscopy to investigate the movement of antigen-presenting cells and neutrophils in the anterior segments of mouse eyes [10], [11], [12], [13]. The anterior segment, defined as the ocular tissues anterior to the lens, consists of the iris, ciliary body, limbus, trabecular meshwork and cornea (Fig. 1A). Of these tissues, the iris and cornea are most easily observed by intravital microscopy. The adjacent ciliary body and limbus are more difficult to distinguish from each other by this technique. Intravital microscopy studies of lymphocyte movement and location have been performed in a number of tissues and organs, such as Peyer’s patches and gut vasculature [14], scrotal cremaster and lymph node [15], [16], [17], [18], [19]. Ocular studies using intravital microscopy and scanning laser ophthalmoscopy have followed the adhesion and migration of neutrophils [11], [12], T cells and antigen-presenting cells in the anterior segment and retina [10], [20]. Ocular studies have the advantage that surgical exposure, with the resulting trauma to tissues, is avoided and an individual animal can be examined serially for days or weeks. In addition, the mouse iris is a thin plane so that migrating cells are less likely to escape the focus of the microscope.

The role of T cells in ocular diseases has been studied using a number of animal models [21], [22], [23], [24]. The most commonly used models are experimental autoimmune uveoretinitis (EAU), experimental melanin-associated uveitis (EMIU) and experimental autoimmune encephalitis (EAE). In these models, autoimmune responses are induced by challenge with antigens, such as interphotoreceptor retinoid-binding protein, melanin-associated antigen, and myelin basic protein, or by adoptive transfer of antigen-specific CD4 T cells [22], [25], [26]. The normal progression of EAU depends upon the induction of systemic Th1 type responses [22], [27]. In this study, we developed a novel model of anterior uveitis using T cells from transgenic mice so that antigen-specific T cells could be readily tracked. We investigated the presence of various CD4 T cell populations in the anterior segment in response to direct antigen challenge in the anterior chamber. Our results reveal that initially in the course of the uveitis (6–24 h) there is no restriction on CD4 cell types infiltrating the anterior segment tissues as all types of CD4 T cells (naïve, effector and memory) enter the tissues. This initial influx results in an antigen-specific accumulation of transgenic CD4 T cells. Subsequently the numbers of naïve and Th2 effector cells present in the tissues diminish in the continuing inflammation while Th1 effectors and CD44^high memory-like CD4 T cells preferentially accumulate.