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. Author manuscript; available in PMC: 2012 Sep 15.
Published in final edited form as: J Immunol. 2011 Aug 10;187(6):3338–3346. doi: 10.4049/jimmunol.1004019

Autoreactive Memory CD4+ T Lymphocytes that mediate Chronic Uveitis Reside in the Bone Marrow through STAT3-dependent Mechanisms

Hyun-Mee Oh *,†, Cheng-Rong Yu *,†, YongJun Lee *, Chi-Chao Chan ‡, Arvydas Maminishkis §, Charles E Egwuagu *,‖
PMCID: PMC3304102  NIHMSID: NIHMS310722  PMID: 21832158

Abstract

Organ-specific autoimmune diseases are usually characterized by repeated cycles of remission and recurrent inflammation. However, where the autoreactive memory T-cells reside in-between episodes of recurrent inflammation is largely unknown. In this study, we have established a mouse model of chronic uveitis characterized by progressive photoreceptor-cell loss, retinal-degeneration, focal retinitis, retinal vasculitis, multifocal-choroiditis and choroidal neovascularization, providing for the first time a useful model for studying long-term pathological consequences of chronic inflammation of the neuroretina. We show that several months after inception of acute uveitis that autoreactive memory T-cells specific to retinal autoantigen, IRBP, relocated to bone marrow (BM). The IRBP-specific memory T-cells (IL-7RαHiLy6CHiCD4+) resided in BM in resting state but upon re-stimulation converted to IL-17-/IFN-γ-expressing effectors (IL-7RαLowLy6CLowCD4+) that mediated uveitis. We further show that T-cells from STAT3-deficient (CD4-STAT3KO) mice are defective in α4β1 and osteopontin expression; defects that correlated with inability of IRBP-specific memory CD4-STAT3KO T-cells to traffic into BM. We adoptively transferred uveitis to naïve mice using BM cells from WT mice with chronic uveitis but not BM cells from CD4-STAT3KO, providing direct evidence that memory T-cells that mediate uveitis reside in BM and that STAT3-dependent mechanism may be required for migration into and retention of memory T-cells in BM. Identifying BM as survival-niche for T-cells that cause uveitis, suggests that BM stromal cells that provide survival signals to autoreactive memory T-cells and STAT3-dependent mechanisms that mediate their relocation into BM, are attractive therapeutic targets that can be exploited to selectively deplete memory T-cells that drive chronic inflammation.

Introduction

Survival and homeostatic expansion of memory T cell pools are essential aspects of host immunity that allow for maintenance of herd immunity to pathogens such as those that cause smallpox, poliomyelitis and yellow fever (1). In contrast to memory T cells that confer protective immunity, autoreactive memory T cells that recognize and attack myelin sheath of neurons or retinal tissues mediate multiple sclerosis (MS) and uveitis, respectively (2, 3). These relapsing-remitting CNS autoimmune diseases are characterized by unpredictable, recurrent, inflammatory attacks that can subside spontaneously with no evidence of overt inflammation in-between attacks (3–5). An unresolved issue pertinent to development of effective treatment for uveitis, MS and other CNS autoimmune diseases is where autoreactive pathogenic memory T cells reside in-between episodes of acute inflammation and how to deprive them of factors that promote their survival. For a long time it was assumed that memory T cells require contact with residual priming Ag for survival and the blood, spleen and lymph nodes (LN) were suspected as survival niches for memory T cells (6). However, recent evidence suggest that memory CD4+ T cells preferentially reside in bone marrow (BM) and require IL-7 (to a lesser extent IL-15) for survival and basal homeostatic proliferation (7, 8). Direct in vivo assessment of whether memory T cells that cause uveitis or MS reside in BM is however challenging due to their low numbers in CNS tissues, particularly several weeks or months after episodes of active disease. In this study, we tracked and have defined the location where Ag-specific memory CD4+ T cells that mediate chronic uveitis reside in the body.

Non-infectious uveitis is a potentially blinding intraocular inflammatory disease thought to be mediated by autoreactive T-cells with specificity for retinal proteins (3). Human uveitis commonly begins as an acute intraocular inflammation that often progresses to a chronic inflammatory stage accompanied by vascular, fibrotic and neurodegenerative changes (9). Experimental autoimmune uveitis (EAU) shares essential features with human uveitis and current understanding of the pathophysiology of uveitis derives largely from study of EAU (10, 11). However, EAU in the mouse is generally considered a self-limiting retinal/uveal inflammatory disease and because the insidious and clinically important manifestations of chronic uveitis are not well addressed in the mouse, many clinicians have questioned the value of EAU as model of human uveitis.

In this study, we have developed a mouse model of chronic uveitis that exhibits all clinical features of progressive uveitis observed in humans. We have utilized this model to track over a period of more than six months residual autoantigen-specific memory T cells derived from the initial acute inflammatory responses to the ocular autoantigen. We show that these cells reside mainly in BM and that they are able to initiate uveitis upon re-stimulation with cognate autoantigen. We also provide suggestive evidence that localization and retention of the autoreactive memory T cells in BM is facilitated by STAT3-dependent mechanisms.

Materials and Methods

Mice

C57BL/6 and B10.A mice (6–8 weeks old) were from Jackson Laboratory (Bar Harbor, ME). Mice with conditional deletion of STAT3 in CD4 T cell compartment (CD4-STAT3KO) have previously been described (12). Animal care and use was in compliance with NIH guidelines.

Induction of EAU and Histology

We induced EAU by active immunization with bovine interphotoreceptor retinoid-binding protein (IRBP, 150 µg for C57BL/6 mice, 50 µg for B10.A mice) and human IRBP peptide (amino acid residues 1–20; 300µg for C57BL/6 mice), in a 0.2 ml emulsion (1:1 v/v with complete Freund’s adjuvant (CFA) containing mycobacterium tuberculosis strain H37RA (2.5 mg/ml). Mice also received Bordetella pertussis toxin (0.2µg/mouse) concurrent with immunization. All experiments comprised of an EAU group (immunized with IRBP in CFA) and control group (received CFA alone). For each study 12 mice were used per group and they were matched by age and sex. Clinical disease was established and scored by fundoscopy and histology as described previously (13);(14). Eyes for histological EAU evaluation were harvested 0, 21, 75, and 92 days post-immunization, fixed in 10% buffered formalin and serially sectioned in the vertical pupillary-optic nerve plane. All sections were stained with hematoxylin and eosin.

Imaging mouse fundus

Funduscopic examinations were performed every 3 weeks for six months after EAU induction using a modified Karl Storz veterinary otoendoscope coupled with a Nikon D90 digital camera, as previously described (15). Briefly, following systemic administration of systemic anesthesia [intraperitoneal injection of ketamine (1.4 mg/mouse) and xylazine (0.12 mg/mouse)], the pupil was dilated by topical administration of 1% tropicamide ophthalmic solution (Alcon Inc, Fort Worth, Texas). To avoid a subjective bias, evaluation of the fundus photographs was conducted without knowledge of the mouse identity by a masked observer. At least six images (2 posterior central retinal view, 4 peripheral retinal views) were taken from each eye by positioning the endoscope and viewing from superior, inferior, lateral and medial fields and each individual lesion was identified, mapped and recorded. The clinical grading system for retinal inflammation was as previously established (14).

Imaging mouse retina by Spectral-domain Optical Coherence Tomography (SD-OCT)

Optical coherence tomography (OCT) is a noninvasive procedure that allows visualization of internal microstructure of various eye structures in living animals. An SD-OCT system with 820 nm center wavelength broadband light source (Bioptigen, NC) was used for in vivo non-contact imaging of eyes from control or Day-92 EAU mice as described. Before OCT imaging was performed, each animal was anesthetized and the pupils dilated. The anesthetized mouse was immobilized using adjustable holder that could be rotated easily allowing for horizontal or vertical scan scanning. Each scan was performed at least twice, with realignment each time. The dimension of the scan (in depth and transverse extent) was adjusted until the optimal signal intensity and contrast was achieved. Retinal thickness was measured from the central retinal area of all images obtained from both horizontal and vertical scans from the same eye, using the system software, and averaged. The method used to determine the retinal thicknesses in the system software was as described (16).

Electroretinogram (ERG) Recordings

Before the ERG recordings, mice were dark-adapted over night, and experiments were performed under dim red illumination. Mice were anesthetized with a single intraperitoneal injection of ketamine (1.4 mg/mouse) and xylazine (0.12 mg/mouse) and pupils were dilated with Midrin P containing of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Santen Pharmaceutical Co., Osaka, Japan). ERGs were recorded using an electroretinography console (Espion E2; Diagnosys LLC, Lowell, MA, USA) that generated and controlled the light stimulus. Dark-adapted ERG was recorded with single-flash delivered in a Ganzfeld dome with intensity of −4 to 1 log cd·s/m2 delivered in 6 steps. Light-adapted ERG was obtained with a 20 cd/m2 background, and light stimuli started at 0.3 to 30 cd·s/m2 in 5 steps. Gonioscopic prism solution (Alcon Labs, Fort Worth, TX, USA) was used to provide good electrical contact and to maintain corneal moisture. A reference electrode (gold wire) was placed in the mouth, and a ground electrode (subcutaneous stainless steel needle) was positioned at the base of the tail. Signals were differentially amplified and digitized at a rate of 1 kHz. Amplitudes of the major ERG components (a- and b-wave) were measured (Espion software; Diagnosys LLC) using automated and manual methods. Immediately after ERG recording, imaging of the fundus was performed as previously described.

Adoptive transfer

EAU was induced in WT C57BL/6 by immunization with IRBP in CFA and mice exhibiting clinical features of chronic uveitis 92 days post-immunization were identified by fundoscopic examination as described above. Donor mice were sacrificed and cells isolated from BM were stimulated for 3 days with IRBP (20µg/ml) and cells were then transferred i.v. into naïve WT or CD4-STAT3KO recipient mice at 1×107 cells/mouse. Ten days after cell transfer, disease was assessed by fundoscopy and histopathological examination.

Detection of IRBP-Specific memory CD4+ T Cells

We detected antigen-specific CD4+ T cells according to CD154 expression assay (17) using CD154 Detection Cocktail (Miltenyi Biotec). Cells from spleen, LN, blood, retina and BM were isolated as previously described (12, 13, 18). For surface detection of CD154, the cells were stimulated in medium with 20 µg/ml IRBP and CD154 Detection Cocktail for 6 or 12 h, washed twice and stained with antibodies against CD4, IL-7Rα or Ly6C for 30 min on ice. For intracellular detection of CD154, Brefeldin A (5 µg/ml) was added for the last 2 hr of stimulation. After staining with antibodies against CD4, IL-7Rα or Ly6C and fixation with 2% buffered paraformaldehyde, the cells were permeabilized and detection of intracellular IFN-γ, IL-17 and CD154 was as described (19) on a Becton-Dickinson FACSCalibur (BD Pharmingen, San Diego, CA) using Ag-specific mAbs and corresponding isotype control Abs (PharMingen). Determination of the percentage of CD4+ T cells was based on FACS analysis of 100,000 live cells from each tissue. To calculate the absolute number of CD4+ T cells in each tissue, we first determined the total number of cells in each tissue by use of the Vi-CELL™ Cell Viability Analyzer (Beckman Coulter, Inc). Based on the % CD4+ T cells, the absolute number of CD4+ or IL-7RαHigh T cells in each tissue was determined by extrapolation from the total number of live cells, as determined by Vi-CELL™ Cell Viability Analyzer. Similarly, The numbers of IRBP-specific CD4+ T cells were extrapolated from the total numbers of IL-7RαHigh in each tissue.

Reverse transcription (RT) PCR and quantitative RT-PCR analysis

All RNA samples were DNA free. cDNA was generated and RT-PCR analyses were performed as described (19). Each gene-specific primer pair used for RT-PCR analysis spans at least an intron and primers and probes used for qPCR were purchased from Applied Biosystems. The mRNA expression levels were normalized to the levels of ACTB (encoding -Actin) and GAPDH housekeeping genes. Primers for RT-PCR were: For α4 integrin (5'-AACCGGGCACTCCTACAACCTGGAC-3' & 5'-ACCCCCAGCCACTGGTTAT CCCTCT-3'); β1 integrin (5'-GAGACATGTCAGACCTGCCTTGGCG-3' & 5'-GGGATGAT GTCGGGACCAGTAGGAC-3'); osteopontin (5'-ATTTGCTTTTGCCTGTTTGG-3' & 5'-CCTTTCCGTTGTTGTCCTGA-3'); β-actin (5'-GTGGGCCGCTCTAGGCACCAA-3' & 5'-TCTTTG CCAATAGTGATGACTTGGC-3').

Results

Establishment of a mouse model of chronic uveitis

EAU is a predominantly T cell-mediated intraocular inflammatory disease induced in susceptible species by active immunization with ocular-specific proteins (or peptides derived from them) and is transferable to naive syngeneic animals by injection of in vitro-activated CD4+, MHC class 11-restricted T cell lines specific to retinal Ag (5–7). We induced EAU in C57B/6 or B10.A mouse strain by immunization with interphotoreceptor-retinoid-binding protein (IRBP), a 140-kDa retinal glycoprotein produced by photoreceptor cells (20) and monitored pathologic manifestations of EAU over a 6 months period by fundoscopy, Optical Computerized Tomography (OCT), electroretinography (ERG) and histology. Initial signs of intraocular inflammation were observed by day 14 days post-immunization (p.i.) and full-blown occurring between days 18–22 (p.i.), with disease incidence of 100% (19). The acute EAU was characterized by mononuclear and polymorphonuclear cell infiltration into retina, vitritis, choroiditis, and varying degrees of photoreceptor cell damage is observed. Fundus image of retina 21 days after EAU induction (Day-21 EAU) showed severe inflammation with blurred optic disc margins and enlarged juxtapapillary area, retinal vasculitis with moderate cuffing and yellow-whitish retinal and choroidal infiltrates (white arrows). Histological analysis revealed many inflammatory cells in the vitreous, photoreceptor cell damage granuloma, choroiditis and retinal edema (Fig. 1a). However by day 30 post-immunization, the disease appeared to be resolved as indicated by marked diminution of inflammatory cells in the retina (19).

Figure 1.

Figure 1

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Chronic intraocular inflammation induces retinal degeneration and neovascularization. (A) Fundus images were taken from normal or EAU mice using an otoendoscopic imaging system and assessment of severity of the inflammatory disease was based on changes at the optic nerve disc and retinal vessels or tissues (top panels). Left panels: show control retina with well-circumscribed optic disc and normal retinal vessels and histology revealed intact retinal architecture (bottom panels). Middle panels: Fundus image of retina 21 days after EAU induction (d-21 EAU) showing severe inflammation with blurred optic disc margins and enlarged juxtapapillary area (black arrowhead), retinal vasculitis with moderate cuffing (black arrows) and yellow-whitish retinal and choroidal infiltrates (white arrows). Histological analysis reveals substantial numbers of inflammatory cells in the vitreous, photoreceptor cell damage (white asterisk), granuloma (white arrowhead), choroiditis (black arrowhead), and retinal edema (the thickened retina) (bottom panels). Right panels: 92 days after EAU induction (d-92 EAU) fundus image reveal severe vasculitis with cuffing (black arrows) with part of the vessel segment not visible. Retinal structural damage was observed, including evidence of atrophic retina (thinning) and sclerotic vessel (red arrow) with multiple whitish infiltrates (white arrow) and brownish chorioretinal scars (blue arrows). Histology revealed photoreceptor cell loss (red asterisk), retinal vasculitis (black arrows), retinal sclerotic vessel (white arrow), choroiditis (black arrowhead), and retinal degeneration (bottom panels). OpN, optic nerve; V, vitreous; R, retina; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelial layer; CH, choroid. (B) Layered structure of the retina was visualized by Spectral Domain Optical Coherence Tomography (SD-OCT) and used to follow the evolution of pathology induced by chronic uveitis. Change in the thickness of retina (white bar) or RPE-Choroid (RPE-CH) layer (black bar) was used to quantify severity of the retinal degeneration. Oval circle highlights hypo-dense area indicative of retinal edema while speckles in the vitreous represent clusters of residual inflammatory cells. (C) Quantitative analysis of inflammatory cells in the day-21- or day-92-mouse retina. Numbers in circle indicate percent of CD4+ T cells and result is representative of 3 independent experiments.

The endpoint of most EAU studies is generally day 28–30 (p.i.) as it is tacitly assumed that the disease is completely resolved by this time. However, in this study we continued to examine eyes of immunized mice for an additional 197 days and this was made possible by the recent developments in the use of a modified Karl Storz veterinary otoendoscope equiped with a Nikon D90 digital camera to image rodent retinas (15). To our surprise we found that the rapid decline of the severe acute retinal inflammation at day 30 p.i gave rise to a previously unrecognized chronic EAU (Fig. 1). The marked swelling of the optic nerve observed during acute uveitis (Fig. 1), subsided by day 35 p.i (Supplementary Figure 1). Although we observed reduced optic nerve head swelling, reduced retinal vascular cuffing and a decrease in chorioretinal lesions between 8 to 10 weeks post-immunization, the retina did not regain its normal appearance (Supplementary Figure 1): retinal flecks persisted, becoming larger and brownish and accompanied by vitreous haze and exudative retinal detachments. The chronic phase retinal inflammation persisted and did not completely resolve during the 225 days of the experimental period (Fig. 2). Features in the chronic phase include severe retinal vasculitis with cuffing and/or sclerotic vessels, retinal infiltrates, scaring and/or atrophy and multifocal choroiditis (Fig. 1). These pathological changes were observed by fundoscopic examination and histology beginning around day-50 post-immunization (Fig. 1) and persisted 225 d after EAU-induction (Fig. 2). For the first time Optical Computerized Tomography (OCT) was adapted to visualize the layered retinal architecture of the rodent during chronic EAU (Fig. 1b). This technique provided unprecedented clarity that allowed us to follow evolution of the disease and visualize that retina edema and neuroretinal degeneration represent long-term sequelae of prolonged chronic inflammation of the neuroretina (Fig. 1b). We further show that the initial severe inflammation (day 14~30 p.i.) was followed by precipitous decline of CD4+ T cells in retina during chronic EAU (Fig. 1c). We next examined whether prolonged inflammation of the neuroretina adversely affected visual acuity in mouse, as is the case in human. Electroretinogram (ERG) (Fig. 2a) or fundoscopy (Fig. 2b) performed 185 and 225 days after EAU induction revealed progressive retina degeneration, culminating in severe vision loss and blindness.

Figure 2.

Figure 2

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Prolonged intraocular inflammation in neuroretina caused vision loss and blindness. B10.A mice were immunized with CFA alone or IRBP/CFA emulsion and electroretinogram (ERG) recordings were obtained and analyzed 185 or 225 days post-immunization. ERGs of control unimmunized mice or control mice immunized with CFA alone, reveal the characteristic normal a-, b- and cone waves (A). In contrast, light-induced response was barely elicited from retina of IRBP-induced EAU mice (A). Immediately after ERG recordings, imaging of the fundus was performed as previously described (B).

Autoreactive CD4+ T-cells that mediate uveitis reside in the Bone marrow

Because IRBP expression is restricted to retina and thymus (21), the EAU model induced by IRBP is an excellent system to monitor the migration and fate of autoreactive T cells that mediate chronic inflammation. Direct in vivo tracking of Ag-specific autoreactive memory T-cells that mediate autoimmune disease is challenging due to their low numbers in host tissues. In this study, we utilized the very sensitive and specific antigen-induced CD154 expression assay (17, 22, 23) to trace the location of autoreactive T cells in various tissues over six-month period. Mice exhibiting clinical features of chronic uveitis on post-immunization day-92 (d-92 EAU), day-185 (d-185 EAU) or day-225 (d-225 EAU) were identified by fundoscopy and OCT (Fig. 1, 2). Cells were then isolated from the BM, spleen, blood, LN or retina, re-stimulated in vitro with IRBP and the presence of IRBP-responsive T cells, as indicated by induction of CD154 expression, was assessed in each of these tissues. We detected the highest amount of CD4+ T cells in the LN, followed by the blood and then the spleen while the lowest levels were detected in the BM (Fig. 3a, 3b). It is however notable that control and EAU mice contained similar levels of CD4+ T cells in their LN, spleen, blood and BM. Consistent with the immune-privileged status of the retina, CD4+ T cells were not detectable in control mouse retina. In contrast to the massive numbers of CD4 T cells that infiltrate the retina during acute EAU (see Figure 1c), only trace amounts of residual cells were detected in the retina of mice with chronic uveitis (Fig. 3). Despite the relatively low amounts of CD4+ T cells in the BM (Fig. 3b), more than 15% CD4+ T cells in BM of EAU mice were IRBP-specific compared to less than 3.5% in their spleen, blood, LN or retina (Fig. 4a, 4b), suggesting that long-lived IRBP-specific memory T cells may referentially reside in BM. It has recently been suggested that CD4+ memory T cells rely on IL-7 for survival and that each memory T cell docks with one IL-7-secreting stromal cell that serves as its dedicated survival niche in the BM (7). Analysis of T cells in BM of d-92 EAU mice revealed that 45.13% of the CD4+ T cells expressed IL-7Rα while IL-7Rα-expressing T-cells comprised less than 3.37% of CD4+ T cells in spleen or blood (Fig. 4c). Although substantial numbers of IL-7Rα-expressing T-cells were detected in the spleen, the numbers of IRBP-specific (CD154+) memory IL-7Rα) T cells in BM were significantly high compared to the spleen (Fig. 4d, 4e). Interestingly, restimulation with IRBP induced the IRBP-responsive BM memory T cells (IL-7RαHiCD4+) to rapidly down-regulate IL-7Rα expression, with ~96% conversion to cells exhibiting effector T cell phenotype (CD154+IL-7RαLowCD4+) (Fig. 4c). To further verify these results we sorted the BM cells into IL-7Rαlow or IL-7rαhi populations. Following re-stimulated with IRBP we again observed the conversion of CD154+IL-7RαhiCD4+ memory T cells to CD154+IL-7RαLowCD4+ effectors T cells (Fig. 4f). Similar analysis using B10A mouse strain showed that IRBP-specific T cells are not detectable in BM during acute EAU (day 14~30 p.i), but steadily increased in time-dependent manner, with substantial numbers of IRBP-specific memory T-cells in BM of d-75 EAU mice expressing the memory and BM-homing marker, Ly6C (Fig. 4g, 4h) (7). Similar to C57BL/6 strain, re-stimulation with IRBP induced rapid conversion to IL-7RαLow effector CD4+ T cells (Fig. 4h). These observations suggest that autoreactive T cells that initiate acute uveitis do persist in BM for a long time and can convert to potentially pathogenic effector uveitogenic T cells upon encounter with IRBP autoantigen.

Figure 3.

Figure 3

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Analysis of CD4+ T cells in the BM, spleen, lymph nodes, blood and retina of control and d-92 EAU mice. EAU was induced in C57BL6 mice. (a) Cells from the BM, spleen, blood, LN or retina were isolated 92 days post-immunization with IRBP/CFA (EAU) or CFA alone (control), re-stimulated in vitro with IRBP and then subjected to FACS analysis. Numbers in quadrants indicate percent of CD4+ T cells in the various tissues. (b) Graphical representation of the absolute numbers of CD4+ T cells in the various tissues. Results are representative of 3 independent experiments.

Figure 4.

Figure 4

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Autoreactive CD4+ T-cells that mediate uveitis are maintained in resting non-proliferative state in the bone marrow. EAU was induced in mice and primary BM, spleen, blood, LN or retina cells from EAU day-92 (a–f) or d-21, d-50 and d-75 (g, h) mice were re-stimulated in vitro with IRBP (20µg/ml). (a) Cells were gated on CD4 and the percentages of cells responding to IRBP stimulation were detected by FACS analysis of CD154 cell-surface expression. (b) Graphical representation of the percentage of the IRBP-responsive CD4+ T cells. (c) FACS analysis of the relative percentage of IRBP-specific T cells expressing the memory T cell marker, IL-7Rα. Number in quadrants present the percentage of CD4+ T cells. Graphical representation of the absolute numbers of IL-7Rα+ cells (d) or IRBP-responsive CD4+ memory T cells (e). Cells from BM of day-92 EAU mice were sorted into IL-7Rαlow or IL-7rαhigh populations on a cell sorter and were then re-stimulated in vitro with IRBP (f). Conversion of IRBP-specific, IL-7RαHigh memory T cells to IL-7RαLow effectors in response to re-stimulation with IRBP was assessed by FACS analysis of IL-7Rα and CD154 expression. (g) Analysis of absolute numbers of CD4+ T cells responding to IRBP-stimulation in the BM of mice at various time points post-immunization. (h) BM cells from day-75 EAU mice were analyzed for expression of the memory T-cell markers, IL-7Rα and Ly6C. Numbers in quadrants indicate percent of T-cells expressing CD154, IL-7Ra, Ly6C or CD4. Results are representative of 3 independent experiments.

BM cells from mice with chronic uveitis transferred EAU to naïve syngeneic mice

EAU is mediated by Th1 and Th17 cells. We next performed the proof-of-concept experiment to examine whether BM cells from mice with chronic uveitis contain IFN-γ- and/or IL-17-expressing CD4+ T cells and if BM cells derived from mice several months after resolution of acute uveitis could transfer EAU to naïve syngeneic mice. Although significant percentage of the CD4+ T cells detected in the BM did not respond to IRBP stimulation (>91%), 7.71% of the CD4+ T cells of d-92 EAU BM were responsive to IRBP (Fig. 5a; middle panel). Intracellular cytokine analysis of the IRBP-stimulated BM cells from d-92 EAU mice revealed that potentially pathogenic IRBP-responsive Th1, Th17 and IL-17/IFN-γ double positive T cells were indeed present in mouse BM several months after inception of uveitis (Fig.5a; right panel). It is of note that these three T cell populations characterize the T cell repertoire implicated in pathogenic mechanisms of EAU (12, 19, 24). We also isolated BM cells from control and d-92 EAU mice, stimulated the cells in vitro with IRBP and transferred the activated BM cells (1×107) into naïve syngeneic mice. We show here by both fundoscopic and histological analyses that the BM cells from the d-92 EAU mice were able to induce uveitis 10 d after adoptive transfer while BM cells of mice immunized with CFA alone could not (Fig. 5b, left panel). These findings are consistent with the concept that IRBP-specific memory T cells in BM are capable of mediating uveitis upon subsequent encounter with IRBP. Although Th1 cells have been shown to induce EAU and EAE (25), CD4-STAT3KO mice do not develop EAU or EAE (12). Resistance of CD4-STATKO mice to development of these CNS autoimmune diseases derives in part from defects in Th17 differentiation and in mechanisms that mediate extravasation of T cells into the retina, spinal cord or brain (12). We therefore examined whether loss of STAT3 in CD4+ T cells would also prevent recruitment of IRBP-specific memory T cells into BM. We immunized age and sex matched WT and CD4-STAT3KO mice with IRBP. Fundus images acquired day-92 (p.i) revealed features characteristic of chronic uveitis in WT but not CD4-STAT3KO retina (Fig. 5c). Although similar levels of CD4+ T cells were detected in BM of WT and CD4-STAT3KO mice, 14.8% of the CD4+ T cells in WT BM were IRBP-specific compared to 3.63% in CD4-STAT3KO BM (Fig. 5d, 5e). We also found substantial disparity in abundance of memory T cells in BM, as 42.63% of CD4+ T cells in WT BM express IL-7Rα compared to 10.99% in CD4-STAT3KO BM (Fig. 5e), suggesting that recruitment of long-lived CD4+ memory T cells into BM may require STAT3. However, spleen of both mouse strains contained similar levels of IL-7Rα-expressing T-cells, albeit to a much lesser amount than BM, indicating that STAT3 mainly facilitated trafficking of memory T-cells into BM (Fig. 5e). Consistent with paucity of memory T cells in CD4-STAT3KO BM, re-stimulation of the BM cells with IRBP induced marked reduction of IL-7RαH T cells in WT (42.63 to 8.56%) but not CD4-STAT3KO (10.99 to 10.5%) mice (Fig. 5e). Furthermore, 14.81% of T-cells in WT BM were IRBP-specific compared to 3.63% in CD4-STAT3KO, suggesting that BM cells from CD4-STAT3KO mice did not contain sufficient amounts of IRBP-specific T-cells to transfer EAU.

Figure 5.

Figure 5

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BM cells from d-92 EAU mice express IL-17 and IFN-γ and transferred EAU to naïve mice through STAT3-dependent mechanisms. (A) BM cells from control or d-92 EAU mice were stimulated with IRBP for 12 h and then assayed for intracellular cytokine expression. Numbers in quadrants indicate percent CD154-, IFN-γ̃ or IL-17-expressing CD4+ T-cells. (B) IRBP-stimulated BM cells from control or d-92 EAU mice were transferred (1×107 cells/mouse) into naïve syngeneic C57BL/6 recipient mice. Ten days after adoptive cell transfer, fundoscopy and histology reveal the development of retinal vasculitis (black arrows) and inflammatory infiltrates (white arrows). (C) WT or CD4-STAT3KO mice were immunized with IRBP in CFA and fundus images were taken on day-92 post-immunization. (D) BM cells were re-stimulated with IRBP and percentage of IRBP-specific CD4+ T-cells (CD154+) was quantified by Flow cytometry. (E) BM cells from day-92 p.i. mice were re-stimulated in vitro with IRBP and IL-7Rα- and CD154-expressing CD4+ T-cells were detected by FACS analysis.

Recruitment and maintenance of IRBP-specific memory T cells in BM require STAT3

To directly examine whether BM cells from IRBP-immunized CD4-STAT3KO mice could transfer EAU, WT and CD4-STAT3KO mice were immunized with IRBP and 92 days later cells were harvested from BM or spleen and stimulated in vitro with IRBP. Consistent with results presented above, d-92 EAU BM cells contained substantial amounts of Th1, Th17 and IL-17/IFN-γ-expressing T cells (Fig. 6a). In contrast, d-92 immunized CD4-STAT3KO BM cells did not contain appreciable amounts of Th1 or Th17 cells in the BM (Fig. 6a), suggesting that STAT3 mediated pathways may be required for recruitment of the IRBP-specific memory T cells into the BM. In adoptive transfer experiments using day-92 (p.i) IRBP-stimulated BM cells (1×107), the WT cells transferred EAU to naïve WT or CD4-STAT3KO mice while BM cells from CD4-STAT3KO mice did not (Fig. 6b). Because recent works have implicated α4β1 integrin (VLA4) and osteopontin in lymphocyte extravasation, development of EAU (26) and initiating relapses of multiple sclerosis (5, 27) and osteopontin in rheumatoid arthritis (28, 29), we examined whether defects in the expression of these proteins may underlie inability of IRBP-specific T cells to traffic into BM. RT-PCR analysis of peripheral T cells from day-7 p.i. mice revealed reduction of expression of these proteins in STAT3-deficient CD4+ T cells (Fig. 6c). Possible involvement of integrins in migration into and retention of memory T cells in BM is further suggested by increases in integrin expression and activation in BM cells from mice with EAU (Fig. 6d, 6e). It is notable that overwhelming numbers of IL-7RαHi cells in the BM express activated β1 integrin compared to cells in the spleen (Fig. 6d). Comparative analysis of IL-7RαHI CD4+ T cells underscore defect in β1 integrin expression in STAT3-deficient memory T cells (Fig. 6c). The latter observation is consistent with results of a recent study showing that β1 integrin-deficient memory T cells have decreased localization to the BM (30) and suggests that recruitment and maintenance of IRBP-specific memory T cells in BM may require STAT3.

Figure 6.

Figure 6

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Recruitment and maintenance of IRBP-specific memory T cells in BM require STAT3. (A) BM cells from control or d-92 EAU mice were stimulated with IRBP for 12 h and then assayed for intracellular cytokine expression. Numbers in quadrants indicate percent IL-2, IFN-γ̃ or IL-17-expressing CD4+ T-cells. (B) IRBP-stimulated BM cells from IRBP/CFA-immunized WT or CD4-STAT3KO mice were transferred (1×107 cells/mouse) into naïve WT or CD4-STAT3KO mice recipient mice. Ten days after adoptive cell transfer, disease was assessed by fundoscopy. (C) WT and CD4-STAT3KO mice were immunized with IRBP and after 7 days T cells were isolated from peripheral blood and expression of integrins and osteopontin was analyzed by RT-PCR. (D, E) (d, e) EAU was induced in B10A mice (D) or C57BL6 (E) and BM or spleen cells were isolated from control or d-200 p.i. mice. IL-7Rα-, β1 integrin or α4 integrin integrin-expressing CD4+ T-cells were detected by FACS analysis. Results are representative of 3 independent experiments.

Discussion

Several organ-specific autoimmune diseases such as uveitis and multiple sclerosis are mediated by autoreactive CD4+ T cells and are characterized by unpredictable, repetitive, explosive inflammatory attacks, which can subside spontaneously (without treatment). Between attacks, there can be little or no evidence of inflammation in the eyes or brain (2, 4, 5). In this study, we addressed the age-old question of where the autoreactive memory T cells that mediate the repeated cycles of remission and recurrent autoimmune disease reside during remissions of inflammation.

Data presented show that large numbers of autoreactive T cells that initiate uveitis traffic from the LN into the neuroretina where they mediate ocular pathology. However, following systemic clearance of the autoantigen the acute inflammation subsides coincident with diminution of the number inflammatory cells and by day-30 post-immunization very few cells is detectable in the retina of mice with EAU. We performed longitudinal studies on more than 60 mice over a time period spanning 225 days and tracked the autoreactive IRBP-specific T cells in the spleen, blood, LN, BM and retina. These studies revealed that residual memory CD4+ T cells that persist in mice that have recovered from acute uveitis preferentially relocate into the BM. It is of note that CD4+ memory T-cells primarily rely on IL-7 for their survival and each memory T-cell is thought to dock with one IL-7-secreting stromal cell that serves as its dedicated survival niche in the BM (7, 31, 32). We show here that the IRBP-specific CD4+ T cells remain in the BM as resting IL-7RαHiLy6CHiCD4+ memory T-cells where IL-7-secreting BM stromal cells presumably support their survival and basal homeostatic proliferation as was previously suggested (7, 8, 33). It is however interesting that upon restimulation with the ocular autoantigen, IRBP, the BM memory T cells rapidly converted into T cells exhibiting effector T cell phenotype (IL-7RαLowLy6CLowCD4+). Taken together these observations suggest that autoreactive T-cells that initiate acute uveitis may persist in BM for a long time and can convert to potentially pathogenic effector phenotype upon encounter with IRBP/APC. Additional studies are however required to establish where or how the autoreactive memory T cells encounter and respond to cognate recall autoantigen; whether conventional APCs migrate to BM and present their cognate Ag or if there is continuous egress of some memory T cells to secondary lymphoid organs for subsequent activation and conversion into effector T cells. We directly address whether autoreactive memory T cells that may mediate chronic autoimmune uveitis reside in the BM, we harvested BM cells from mice 3 months after recovery from acute uveitis, re-stimulated them with IRBP and performed adoptive transfer experiments. These adoptive transfer studies demonstrate that BM cells from d-92 EAU mice efficiently induced uveitis in naïve recipients and supports the notion that the BM may harbor autoreactive T cells capable of mediating repeated cycles of remission and recurrent ocular inflammation that characterize human uveitis.

Uveitis is comprised of a heterogeneous group of potentially sight-threatening inflammatory diseases that includes, sympathetic ophthalmia, birdshot retinochoroidopathy, Behçet’s disease, Vogt-Koyanagi-Harada, ocular sarcoidosis and about 10% of severe visual handicap in the United States are attributed to this group of disorders (3). Current therapeutic strategies for treating uveitis primarily focus on immunosuppressive agents (e.g. cyclosporine, FK-506, Daclizumab, rapamycin, infliximab) that target T-helper cells, or Abs that neutralize pro-inflammatory cytokines and these drugs are fairly effective in ameliorating disease symptoms (34, 35). However, they are of limited value as long-term therapy because autoreactive T-cells that mediate uveitis are constantly primed by autoantigens that are continuously released from damaged retinal tissue and renal toxicity or other adverse effects preclude prolonged usage (34, 35). Furthermore, memory CD4+ T-cells that are the source of autoreactive T-cells that perpetuate cycles of recurrent autoimmune inflammation are relatively resistant to conventional therapy, including immunosuppression (36). Data presented in this study showing that autoreactive T cells that initiate uveitis relocate to BM after the acute disease subsides, suggest that new therapeutic strategies targeting IL-7RαHiLy6CHiCD4+ memory T-cells or IL-7-secreting BM stromal cells that support their survival may be viable alternative to conventional therapy for uveitis. However, additional studies will be required to establish selective targeting strategies that would spare bystander memory T cells.

Recent works have revealed the role of α4β1 integrin (VLA4) and osteopontin in initiating relapses of multiple sclerosis (5, 27) while others have implicated STAT3 and osteopontin in rheumatoid arthritis (28, 29). Moreover, resistance of CD4-STAT3KO mice to EAU or EAE derives in part from an inability of T cells to enter CNS tissues (12). In contrast to the efficient transfer of EAU to naïve WT or CD4-STAT3KO mice by BM cells from WT mice immunized with IRBP, we could not transfer the disease with CD4-STAT3KO BM cells. We have shown that the STAT3-deficient cells are defective in α4β1 activation and this correlated with the inability of memory T-cells from IRBP-immunized CD4-STAT3 knockout mice to extravasate into the BM. In addition, the defect in recruitment of Stat3-deficient IRBP-specific T cells into BM also correlated with marked reduction in osteopontin expression (Fig. 6C). Together, results from studies on multiple sclerosis, rheumatoid arthritis and uveitis thus establish a nexus between, STAT3, α4β1 integrin, osteopontin, and autoimmune diseases and suggest that STAT3-dependent mechanisms that mediate trafficking of IRBP-specific autoreactive T-cells into BM and CNS tissues may also be potential therapeutic targets.

Supplementary Material

1

Acknowledgments

Authors thank Drs. Ahjoku Amadi-Obi and Xiaoguang Cao (National Eye Institute, NIH) for technical assistance. Authors also thank Dr. Haohua Qian for assistance with ERG recordings and analysis (Visual Function Core Facility, National Eye Institute, NIH) and Rafael Villasmil for cell sorting (Flow Cytometry Core Facility, National Eye Institute, NIH)

Footnotes

The National Eye Institute (NEI) and National Institutes of Health (NIH) Intramural Research Programs funded this research.

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