November 2005
Volume 46, Issue 11
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Anatomy and Pathology/Oncology  |   November 2005
Autologous T-Lymphocytes Stimulate Proliferation of Orbital Fibroblasts Derived from Patients with Graves’ Ophthalmopathy
Author Affiliations
  • Stephen E. Feldon
    From the Departments of Ophthalmology,
  • D. J. John Park
    Keck School of Medicine, University of Southern California, Los Angeles, California.
    Present affiliations: The Wilmer Eye Institute, Baltimore, Maryland; and
  • Charles W. O’Loughlin
    From the Departments of Ophthalmology,
    Environmental Medicine, and
    Lung Biology and Disease Program, University of Rochester, Rochester, New York; and the
  • Vu T. Nguyen
    Keck School of Medicine, University of Southern California, Los Angeles, California.
  • Shira Landskroner-Eiger
    Environmental Medicine, and
    Lung Biology and Disease Program, University of Rochester, Rochester, New York; and the
    The Albert Einstein College of Medicine, Bronx, New York.
  • Donald Chang
    Keck School of Medicine, University of Southern California, Los Angeles, California.
  • Thomas H. Thatcher
    Environmental Medicine, and
    Medicine and the
    Lung Biology and Disease Program, University of Rochester, Rochester, New York; and the
  • Richard P. Phipps
    Environmental Medicine, and
    Lung Biology and Disease Program, University of Rochester, Rochester, New York; and the
Investigative Ophthalmology & Visual Science November 2005, Vol.46, 3913-3921. doi:10.1167/iovs.05-0605
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      Stephen E. Feldon, D. J. John Park, Charles W. O’Loughlin, Vu T. Nguyen, Shira Landskroner-Eiger, Donald Chang, Thomas H. Thatcher, Richard P. Phipps; Autologous T-Lymphocytes Stimulate Proliferation of Orbital Fibroblasts Derived from Patients with Graves’ Ophthalmopathy. Invest. Ophthalmol. Vis. Sci. 2005;46(11):3913-3921. doi: 10.1167/iovs.05-0605.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Graves’ ophthalmopathy (GO) affects 50% to 60% of patients with Graves’ hyperthyroidism, resulting in exophthalmos, periorbital edema, pain, double vision, optic neuropathy, and loss of vision. Fibroblasts are a key autoimmune target in GO and have effector functions that contribute to GO-associated pathologic conditions, including proliferation, production of excess glycosaminoglycans, and fat deposition. GO is also characterized by autoimmune inflammation of orbital connective tissue with mononuclear cell infiltration, including T cells.

methods. To determine whether autologous T cells can drive proliferation of orbital fibroblasts and thus contribute to GO, a novel reverse autologous mixed-cell reaction (rAMCR) was performed. Fibroblasts cultured from orbital tissue of patients with GO that was removed during orbital decompression surgery were mixed with autologous T cells, and fibroblast proliferation was determined.

results. Autologous T cells stimulated proliferation of orbital fibroblasts. Fibroblasts derived from blepharoplasty fat of two different patients did not proliferate, demonstrating that the effect is specific to cells derived from deep orbital fat. Proliferation was dependent on cell contact and on major histocompatibility complex (MHC) class II and CD40–CD154 (CD40 ligand) signaling.

conclusions. The results suggest that T cells and orbital fibroblasts participate in an antigen-dependent positive feedback loop in which presentation of autoantigens by fibroblasts via MHC class II and CD40–CD40L signaling results in T-cell activation. These activated T cells stimulate fibroblast proliferation, leading to fibroblast-associated diseases in GO. Thus, therapies that interfere with CD40–CD40L signaling, antigen expression by fibroblasts, or T-cell function may be effective in preventing progression of GO symptoms.

Graves’ disease is a common autoimmune disease involving stimulatory autoantibodies that bind to the thyroid-stimulating hormone receptor (THSR), leading to hyperthyroidism. 1 Clinically apparent Graves’ ophthalmopathy (GO) affects 50% to 60% of patients with Graves’ hyperthyroidism. 2 Clinical features of GO include exophthalmos, periorbital edema, eyelid retraction, extraocular muscle dysfunction, pain, double vision, optic neuropathy, and loss of vision. 3 4 5 6 7 8 These symptoms are largely the result of pathologic processes within the orbit of the eye that increase the volume of retroocular tissue, which, confined within the bony orbit, results in forward protrusion of the eye, accompanied by nerve and muscle damage. 9 10 Although Graves’ disease has a probable autoimmune origin, the mechanism of induction and perpetuation of the unique orbital inflammation remains unknown. 
A consensus has emerged that orbital fibroblasts are the principal target of autoimmune attack and are key to the pathophysiology of GO. 11 12 13 14 Fibroblasts from patients with GO produce excess matrix glycosaminoglycans including hyaluronan, are unusually proliferative, and can differentiate into fatlike adipocytes. 15 16 Proliferation of fibroblasts and adipocytes and accumulation of excess glycosaminoglycans leads to increased tissue volume and pressure in the orbit, causing exophthalmos, whereas infiltration of muscle fibers by fibroblasts leads to fibrosis of the extraocular muscles. 17 18 Several differences have been identified that distinguish normal orbital fibroblasts and fibroblasts of nonorbital anatomic sites from orbital fibroblasts harvested from patients with GO, resulting in a unique phenotypic signature that contributes to the pathogenesis of GO. 14 19 20 Orbital fibroblasts synthesize high levels of hyaluronan, and hormonal regulation of glycosaminoglycan synthesis differs between orbital and nonorbital fibroblasts. Orbital fibroblasts also express high levels of prostaglandin H synthase (PGHS)-2 (cyclooxygenase (Cox)-2) and PGE2 after stimulation by proinflammatory cytokines. 21 22 Thus, understanding the interaction of orbital fibroblasts and lymphocytes is critical to understanding the inflammatory tissue remodeling in GO. 
Orbital tissue from patients with GO is infiltrated by T and B lymphocytes and macrophages. 23 24 25 Pappa et al. 26 found an increased number of macrophages and CD4+ T cells in muscle biopsy specimens, as well as CD45RO+ (memory) T cells, suggesting that GO involves a T-mediated autoimmune response. Of special interest is the discovery that the CD40–CD40 ligand pathway is active in orbital fibroblasts. 27 28 CD40 is a cell surface receptor found on antigen-presenting cells and B and T cells, whereas CD40 ligand (CD40L, CD154) is expressed on T cells. Ligation of CD40L on T cells by CD40 on B cells or other antigen-presenting cells is necessary for efficient activation of T-cell effector functions, 29 30 whereas ligation of CD40 on B cells promotes B-cell survival and differentiation. 31 32 Recently, it has been shown that orbital fibroblasts express high levels of CD40, and that activation by CD40L induces hyaluronan synthesis, the proinflammatory cytokines IL-6 and IL-8, and Cox-2 and PGE2. 15 28 33 Thus, the CD40–CD40L bridge is one potential pathway by which T cells could influence fibroblast activation and proliferation in Graves’ disease. 
Retrobulbar fibroblasts derived from GO tissues can drive proliferation of autologous T cells. 34 However, T cells from GO have not been shown to drive proliferation of autologous orbital fibroblasts. In this study, we established a new in vitro model for studying T cell-fibroblast interactions in GO, a reverse autologous mixed-cell reaction (rAMCR). In the rAMCR, cultures of orbital fibroblasts and peripheral blood T cells were established from patients with GO who had undergone surgical orbital decompression. The T cells were then irradiated and added to the fibroblasts, and fibroblast proliferation was assessed. We demonstrate that autologous T cells stimulated proliferation of orbital fibroblasts and that stimulation was dependent on major histocompatibility complex (MHC) class II, CD40–CD40L interactions, and direct cell–cell contact. Proliferation was specific to orbital fibroblasts, as fibroblasts isolated from the anterior fat of patients with GO who had undergone blepharoplasty did not proliferate when cultured with autologous T cells, even though fibroblasts isolated from deep orbital fat derived from transantral orbital decompression in these same patients demonstrated such proliferation. The nature of the peripheral blood-derived cells necessary for the rAMCR response was investigated. 
Methods
Tissue Procurement
Biopsy specimens of deep orbital fat were obtained from patients who underwent orbital decompression surgery for GO. Separate specimens of fat from blepharoplasty were obtained from two of these patients. Whole blood (treated with heparin) was obtained during or after surgery. Tissues were harvested at the University of Southern California Keck School of Medicine, the University of Rochester, Allegheny General Hospital, and Columbia Presbyterian Hospital under the supervision of each institution’s institutional review board. The research protocol adhered to the tenets of the Declaration of Helsinki, and informed written consent was obtained from all subjects. All tissue samples were placed in RPMI medium at 4°C, and blood and tissues were shipped to the University of Rochester under refrigeration for culturing. 
Establishment of Fibroblast Cultures
Fibroblasts were cultured from surgical tissue explants in RPMI containing 2-mercaptoethanol (50 μM), glutamine (2 mM), HEPES buffer (10 mM), nonessential amino acids (0.1 mM), pyruvate (1 mM), and gentamicin (0.05 mg/mL; Invitrogen, Carlsbad, CA). The explants were initially cultured in 40% fetal bovine serum (Hyclone, Logan, UT), which was gradually reduced to 10% over 7 days. Cells were examined for fibroblast markers using immunostaining and flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). The adherent cells stained positively for vimentin and negatively for CD45, cytokeratin, and factor VIII, consistent with a fibroblast phenotype. Fibroblast strains were stored in liquid nitrogen until needed and were used between passages 4 and 9. 
T-Cell Purification and Expansion
Peripheral blood mononuclear cells (PBMCs) were isolated from 60 mL of whole blood obtained during or after orbital decompression surgery (Ficoll-Paque-Plus; GE Healthcare, Piscataway, NJ) and frozen until needed. T cells were enriched from thawed PBMCs with nylon wool fiber columns (Polysciences, Warrington, PA), according to the manufacturer’s instructions. The T-cell-depleted fraction was eluted from the column by plunging. The enriched T cells were expanded by coculturing for 8 days with the autologous T-cell-depleted fraction that had been irradiated (15 Gy), in RPMI containing 10% heat-inactivated human AB serum (Gemini Bioproducts, Woodland, CA) and supplemented with recombinant human IL-2 (25 U/mL; Sigma-Aldrich, St. Louis, MO) on days 0 and 4 and phytohemagglutinin (PHA, 5 μg/mL; Sigma-Aldrich) on day 0. The expanded T cells were isolated (Ficoll-Paque-Plus; GE Healthcare) and readied for downstream application. All enriched T-cell preparations were stained with CD3-PE marker (SouthernBiotech, Birmingham, AL, or BD Biosciences) and analyzed on the flow cytometer to determine purity. After expansion, the enriched T-cell populations were >90% pure. 
For some experiments the expanded enriched T-cell population was sorted into CD4+ and CD4 fractions, by using a labeled CD4 antibody (BD Biosciences) and the flow cytometer. The CD4+ and CD4 populations were reanalyzed by flow cytometry and found to be >98% and >97% pure, respectively. 
For other experiments, the expanded enriched T-cell population was further enriched for CD4+ T cells or depleted of CD4+, CD1a+, CD83+, or CD14+ cells by magnetic bead separation (Dynal Biotech, Brown Deer, WI) according to the manufacturer’s recommended protocol. Briefly, enriched T cells were incubated with specific antibodies coupled to magnetic beads (10:1 bead-to-target cell ratio) for 30 minutes at 4°C. The cells were washed four times with cold PBS and 2% human AB serum and resuspended in RPMI with 1% human AB serum. The supernatant of the first wash containing antibody-depleted cells was saved. CD4+ cells were released from the beads and washed extensively. Both the antibody-enriched and -depleted fractions were resuspended in RPMI containing 10% human AB serum for further use. Samples were analyzed by flow cytometry to determine purity. CD4+ cell preparations were routinely >95% pure. Antibody-depleted cell fractions routinely contained <0.1% of the depleted cell population. 
T-Cell–Fibroblast Coculture
Proliferation of orbital fibroblasts was measured by [3H]thymidine incorporation. 35 Proliferation was verified in many experiments by counting the number of fibroblast cells with a hemacytometer. Orbital fibroblasts were plated in 96-well plates at 5,000 to 10,000 cells/well in RPMI containing 10% FBS. The next day, the medium was changed to RPMI with 10% human AB serum, and irradiated (15 Gy) autologous T cells (20:1 ratio of T cells to fibroblasts) were added. In some experiments, orbital fibroblasts and enriched T cells were cultured separately in cell migration assay culture dishes (0.4-μm pore size, Transwell; Corning Costar, Corning NY), or were incubated with antibodies to CD40, CD40 ligand (CD154), or MHC class II (HLA-DR, DP, DQ) at 0.2 and 2 μg/mL (BD Biosciences). After 24 hours, 1 μCi [methyl-3H]thymidine (Dupont NEN Products, Boston, MA) was added, and 3H incorporation was determined after a further 24 hours, with a microplate scintillation counter (TopCount; PerkinElmer, Meriden, CT). Negative controls included fibroblasts without added T cells and irradiated T cells alone. All assays were performed on triplicate cultures, and the results are reported as mean counts per million ± SD. Statistical significance was determined with the Students t-test. 
Immunofluorescence Microscopy
Orbital fibroblasts and autologous enriched T cells (20:1 T cell-fibroblast ratio) were cocultured in eight-well glass chamber slides (Falcon; BD Biosciences) for 3 days in RPMI medium, as described earlier. Some wells were cultured with 500 U/mL IFN-γ as a positive control. Slides were washed in PBS, blocked with 1% BSA, and stained with 5 μg/mL FITC-labeled mouse anti-human HLA-DR, DP, DQ (BD Biosciences) or a FITC-labeled isotype control antibody, in 1% BSA for 2 hours at 4°C. After they were washed in PBS, the slides were mounted (Fluoromount-G; Southern Biotech) and photographed on a fluorescence-equipped microscope. 
Results
Effect of Autologous T Cells on Proliferation of Orbital Fibroblasts from GO
Primary fibroblast cultures were established from explants of orbital tissue obtained during orbital decompression surgery for GO. A T-cell-enriched fraction of PBMCs obtained from the same patient was expanded in vitro by culturing with PHA and IL-2. To determine whether proliferation of the orbital fibroblasts could be stimulated by autologous T cells, the expanded T-cell population was irradiated and added to fibroblast cultures in an rAMCR. Irradiated T cells alone did not demonstrate significant proliferation (Fig. 1A) . Primary human orbital fibroblasts from patients with GO proliferated well when cultured with fetal bovine serum (for strain propagation), but proliferated poorly in human AB serum. At low T-cell-fibroblast ratios, addition of autologous T cells stimulated a small increase in fibroblast proliferation that was not statistically significant. Higher ratios stimulated a significant increase in fibroblast proliferation, with an optimal T-cell-fibroblast ratio of 20:1 (Fig. 1A)
To verify that this is a common property of orbital fibroblasts from patients with GO, orbital fibroblast and T-cell cultures were established from five additional patients, and orbital fibroblast proliferation was measured by rAMCR. In all cases, autologous T cells stimulated a significant increase in proliferation compared with orbital fibroblasts alone (Fig. 1B) . It should be noted that although the absolute levels of thymidine incorporation and the relative degree of stimulation varied from patient to patient, the ability of autologous irradiated T cells to stimulate fibroblast proliferation was a consistent finding among all patients tested. 
Effect of Autologous T Cells on Proliferation of Fibroblasts from a Second Periocular Site
The observation that autologous T cells can drive proliferation of Graves’ orbital fibroblasts strongly implies T-cell–fibroblast interactions unique to the pathogenesis of GO. However, it is also possible that the T cells are capable of stimulating any fibroblast to proliferate. To rule out this possibility, we obtained tissue from the fat discarded during blepharoplasty in two patients who had previously undergone orbital decompression. Fibroblasts were cultured from orbital and blepharoplasty fat and then cocultured with autologous irradiated enriched T cells in an rAMCR. Figure 2shows that irradiated autologous T cells stimulated a significant increase in proliferation of deep orbital fibroblasts but not of fibroblasts cultured from the blepharoplasty site. 
Contact-Dependence of Stimulation of Orbital Fibroblast Proliferation
The ability of T cells to stimulate proliferation of autologous orbital fibroblasts could be dependent on cell-surface signaling that requires cell–cell contact, such as T-cell receptor (TCR)-MHC and CD40–CD40L interactions, or it could involve diffusible factors such as cytokines. To determine the cell contact dependency of orbital fibroblast proliferation, T cells and orbital fibroblasts were separated by a semi-permeable membrane (Transwell; Corning Costar). Data from two representative patients are shown (Fig. 3) . Autologous irradiated T cells stimulated significant proliferation of orbital fibroblasts when cultured together; however, when separated by a permeable membrane, T cell-dependent proliferation was reduced by 65%. 
MHC Class II and CD40–CD40L Interaction in Orbital Fibroblast Proliferation
Orbital fibroblasts can express CD40 and MHC class II, 11 36 37 whereas activated T cells express CD40L and the TCR complex. It is well established that TCR-MHC and CD40–CD40L interactions are necessary for stimulation of T-cell effector functions. To determine whether TCR-MHC or CD40–CD40L interactions are necessary for stimulation of orbital fibroblast proliferation, orbital fibroblasts were cultured with autologous enriched T cells in the presence of blocking antibodies. As shown in Figure 4 , the ability of autologous T cells to stimulate proliferation of orbital fibroblasts was completely inhibited by antibodies to MHC class II, CD40, and CD40L. To investigate further the role of MHC class II in fibroblast proliferation, fluorescence immunocytochemistry was performed on orbital fibroblasts cultured with or without autologous T cells. Orbital fibroblasts cultured alone expressed little or no MHC class II (Fig. 5B) , whereas coculture with autologous T cells stimulated significant expression of MHC class II (Fig. 5C)
Cell Types Involved in Stimulation of Orbital Fibroblast Proliferation
Results in studies have shown that the T cells infiltrating orbital tissues are CD4+ T-helper cells expressing a Th1 cytokine profile, although there is also evidence of the involvement of CD8+ T cells. 23 26 38 39 To investigate the nature of T cells needed for stimulation of proliferation of orbital fibroblasts, the autologous enriched T-cell population was separated into highly purified CD4+ and CD4 fractions by magnetic bead separation with a CD4 antibody. Whereas the unfractionated T cells supported a significant increase in fibroblast proliferation, neither the rigorously purified CD4+ nor the CD4 fraction stimulated any increased proliferation over fibroblasts alone (Fig. 6A) . These results were confirmed in cells derived from multiple patients (data not shown). We also considered that the result might be an artifact of the magnetic bead separation method. To investigate this possibility, we used fluorescence activated cell sorting (FACS) to separate the enriched T-cell fraction into CD4+ and CD4 fractions. The resultant preparations were >99% and >97% pure, respectively (data not shown). Neither the CD4+ nor the CD4 fraction stimulated proliferation of autologous orbital fibroblasts (Fig. 6B)
One explanation for our results is that stimulation of proliferation requires both CD4+ T cells and a CD4 accessory cell that is present in the enriched T-cell population. Such an accessory cell may be necessary for efficient costimulation of the T cells to express the soluble factors and surface proteins necessary for efficient stimulation of orbital fibroblast proliferation. Dendritic cells (DCs) are potent professional antigen-presenting cells (APCs) that can stimulate multiple effector functions in T cells. Flow cytometric analysis with two DC markers, CD1a and CD83, 40 41 demonstrated that the expanded enriched autologous T-cell population contains 3% to 5% DCs (data not shown). If these DCs are necessary for stimulation of orbital fibroblast proliferation, their removal should result in decreased proliferation. DCs were removed from the enriched T cells using magnetic beads coupled to specific CD1a or CD83 antibodies and the resulting T cell fractions were incubated with autologous orbital fibroblasts. Flow cytometric analysis confirmed that the depleted cell fractions contained <0.1% of antibody-positive cells (data not shown). Although the unfractionated T cells supported a significant increase in proliferation, there was no significant decrease in proliferation of fibroblasts cultured with CD1a depleted (Fig. 7A)or CD83-depleted (Fig. 7B)T cells. 
Because the enriched T cells are prepared from PBMCs, they would be expected to contain some monocyte-macrophages. Macrophages can express T-cell costimulatory molecules and can release soluble cytokines that are essential in T-cell-mediated immune functions. Flow staining with a CD14 antibody confirmed the presence of CD14+ monocytes in the enriched T cell population (data not shown). However, CD14-depleted T cells had the same stimulatory effect on the proliferation of autologous orbital fibroblasts as the unfractionated population (Fig. 7C)
Discussion
GO develops in approximately 50% to 60% of patients with Graves’ disease. 9 42 Graves’ disease is thought to be an autoimmune process involving autoantigens common to the thyroid and the orbit, so that autoimmune attack on the thyroid leads to hyperthyroidism and associated hypermetabolic consequences, and autoimmune attack on the cells of the orbit, particularly fibroblasts, leads to GO. 1 13 Differentiation of fibroblasts to adipocytes leads to accumulation of fatty tissue in the orbit, whereas proliferation of orbital fibroblasts causes accumulation of matrix components and connective tissue, and fibrosis of the ocular muscles. It has been shown that orbital tissue from patients with GO is infiltrated with B and T cells, and it has been reported that orbital fibroblasts from Graves’ patients can stimulate proliferation of autologous T cells. 23 24 25 26 34 Here, for the first time, we demonstrate that T cells from patients with GO can stimulate proliferation of autologous orbital fibroblasts, thus providing a mechanism by which infiltration of orbital tissue by autoimmune lymphocytes can drive the pathogenic features of GO. 
We established an rAMCR assay in which irradiated T cells (enriched from patient PBMCs) are mixed with orbital fibroblasts cultured from the same patient. In all patient samples examined, autologous T cells drove a significant increase in fibroblast proliferation over orbital fibroblasts cultured alone (Figs. 1A 1B) . It is worth noting that this phenomenon was not seen in fibroblasts derived from a nearby anatomic site in patients who were undergoing blepharoplasty and had undergone orbital decompression surgery (Fig. 2) . This result agrees with earlier studies showing that deep orbital fibroblasts express a unique phenotype 14 19 20 and suggests that this phenotype makes them susceptible to autoimmune attack in Graves’ disease. 
To determine whether direct cell–cell contact is necessary for stimulation of orbital fibroblast proliferation, T cells and fibroblasts were cultured separated by a semi-permeable membrane. Separating the orbital fibroblasts from direct T-cell contact resulted in a 65% decrease in proliferation (Fig. 3) , indicating that whereas cell–cell contact is essential for maximum stimulation of proliferation, one or more T cell-derived diffusible factors contribute to the net increase in proliferation. To identify which ligand–receptor interactions are necessary, we performed rAMCR assays with blocking antibodies to MHC class II, CD40, and CD40L. Each antibody completely inhibited T-cell stimulation of increased orbital fibroblast proliferation (Fig. 4) . This indicates that in the absence of signals from the fibroblasts, the T cells do not express proproliferative effector functions, and that the fibroblasts signal via the MHC–TCR and CD40–CD40L pathways. This suggests that the proproliferative signals are dependent on recognition of fibroblast autoantigens by the T cells. However, the antibody results also present an apparent paradox, in that blocking MHC class II and CD40–CD40L interactions completely inhibited fibroblast proliferation, whereas blocking cell contact via the cell-migration plate membrane only partially inhibited fibroblast proliferation. It should be noted that the enriched T cells were expanded in vitro for 8 days with PHA and IL-2 and were probably in a state of partial activation. We hypothesize that the IL-2-activated T cells produce some soluble factors (probably cytokines and chemokines) that stimulate fibroblast proliferation suboptimally, while also inducing expression on the fibroblasts of MHC class II (Fig. 5) . With increased expression of class II, the fibroblasts can fully activate the T cells via an antigen-dependent mechanism requiring MHC–TCR (signal 1) and CD40–CD40L (signal 2) interactions. 30 43 The activated T cells then produce increased cytokines, driving fibroblast proliferation. In the cell-migration membrane experiments, the IL-2-activated T cells produced low levels of stimulatory factors but could not be further activated in the absence of cell contact. However, in the antibody blocking experiments, blockade of class II MHC resulted in signal 2 without signal 1, whereas blockade of CD40 or CD40L costimulation resulted in signal 1 without signal 2, both of which conditions result in T cell anergy. 29 44 Thus, the T cells no longer produce even low levels of proproliferative factors. 
Previous reports have shown that orbital muscle and fat tissue of Graves’ disease patients is infiltrated with CD4+ T cells. 23 25 26 To investigate whether the stimulation of orbital fibroblast proliferation is dependent on CD4+ T cells, we separated the enriched expanded T-cell population into CD4+ and CD4 fractions, by using magnetic bead separation (Fig. 6A) . Neither fraction by itself stimulated orbital fibroblast proliferation. The enriched T-cell fractions (prepared from PBMCs on nylon wool columns followed by activation in vitro for 8 days) were typically >90% pure, as determined by flow cytometry. This raised the possibility that efficient stimulation of fibroblast proliferation requires both CD4+ T cells and a CD4 accessory cell, so that fractionating the enriched T cells on the basis of CD4 expression separated the accessory cells from the CD4+ T cells. An alternative possibility is that the magnetic bead separation technique alters the T cells so that they no longer efficiently stimulate proliferation. To address this possibility, T cells were sorted into CD4+ and CD4 fractions by using a different technology, fluorescence activated cell sorting (FACS; Fig. 6B ). Again, neither fraction stimulated the orbital fibroblasts to proliferate. Attempts to restore proliferation by combining the CD4+ and CD4 fractions were unsuccessful (data not shown), suggesting that this result could be an artifact caused by ligating CD4 with a specific antibody, a step common to both the magnetic bead method and FACS. Alternatively, a population of accessory cells may have been lost during purification, or the cells were not recombined in the optimal ratios. 
To explore further the possibility that efficient stimulation of fibroblast proliferation requires an accessory cell, we used magnetic bead separation to remove potential accessory cells without affecting the CD4+ T cells. We reasoned that the most likely accessory cells would be professional APCs such as DCs or monocyte-macrophages, which could present autoantigens to CD4+ T cells resulting in T-cell activation. Magnetic beads coupled to antibodies for CD1a (DCs and DC precursors in peripheral blood), CD83 (DCs) and CD14 (monocyte-macrophages) were used to deplete the enriched T cells of antigen-expressing cells (<0.1%, data not shown) before rAMCR. None of these antibodies resulted in a decrease in orbital fibroblast proliferation (Fig. 7) . This further supports the hypothesis that orbital fibroblast proliferation is dependent on CD4+ T cells, but that current methods of purifying CD4+ T cells alter their stimulatory capacity in vitro. However, it remains possible that one or more accessory cell types are involved in the rAMCR. It should be noted that the phenotypes and sorting characteristics of human APCs are much less well understood than in the mouse 45 and it is difficult to obtain sufficient blood cells for complex analysis from our surgical patients. Further analysis is necessary to determine whether CD4+ T cells are sufficient for stimulation of orbital fibroblast proliferation or whether an accessory cell is required, and if so, what it is. 
It should be noted that in the present study, fibroblasts were isolated from deep orbital fat obtained during decompression surgery, whereas the T cells were derived from circulating blood mononuclear cells. The ocular muscles are also an important site of disease, and have been shown to be infiltrated by T cells by immunohistochemistry. 23 26 However, biopsy of the extraocular muscles for research cannot be performed on ethical grounds, due to the risk of muscle impairment and increased patient symptoms. The number of T cells available from the fat biopsy tissue is too low to permit conventional analysis; however, it would be interesting to compare the response of fibroblasts to circulating and tissue infiltrating T cells by using more sensitive methods. 
Previously, we and others have reported that Graves’-derived fibroblasts express MHC class II and CD40, and that ligation of CD40 on fibroblasts results in increased production of inflammatory cytokines. 19 22 28 In the present study, we extended these results by demonstrating that T cells from patients with Graves’ disease interacted with autologous orbital fibroblasts via the class II and the CD40–CD40L pathways to promote increased fibroblast proliferation. Although glycosaminoglycan synthesis was not measured in the present study, we have reported that CD40L stimulates orbital fibroblasts to produce hyaluronan, 15 and this study demonstrated that the interaction between orbital fibroblasts and autologous T cells was CD40–CD40L dependent. Thus, stimulation by T cells is responsible for many of the pathogenic properties of orbital fibroblasts in GO. This T-cell dependence supports previous indications that the pathogenesis of GO is antigen dependent. The exact antigens involved in GO have not been identified, although it has been hypothesized that they will be shared by thyroid and orbital tissue. Several candidates have been proposed, including TSHR, 46 47 48 49 and serum immunoglobulins from Graves’ patients can stimulate orbital fibroblasts to produce T-cell chemoattractants. 50 51  
In addition to the role of T cells in stimulating autologous orbital fibroblasts reported herein, orbital fibroblasts can stimulate autologous T cells. 34 Taken together, these results describe a positive feedback loop in which thyroid-stimulating autoantibodies promote migration of T cells to the orbit, which stimulate fibroblasts in orbital tissue to upregulate expression of MHC class II and the presentation of autoantigens. This in turn further activates the T cells to produce surface and/or diffusible factors that drive activation and proliferation of orbital fibroblasts, leading to expression of fibroblast-based diseases including proliferation of fibroblasts and excess connective tissue, deposition of matrix glycosaminoglycans, intramuscular fibrosis, and differentiation and proliferation of adipocytes. New therapies that interrupt these feedback processes hold the potential to halt the progression of GO and may even allow for nonsurgical reduction of GO. 
 
Figure 1.
 
Autologous T cells stimulated proliferation of orbital fibroblasts from patients with Graves’ disease. (A) Orbital fibroblasts (OF) from a patient were cultured with autologous irradiated (irr) enriched T cells, and proliferation was measured by [3H]thymidine incorporation. irrT, 1 × 105 T cells alone, OF, 5 × 103 orbital fibroblasts alone; OF+irrT, 5 × 103 orbital fibroblasts plus the indicated ratio of irradiated T cells (5 × 103, 1 × 104, 1 × 105). The mean ± SD of triplicate cultures is shown. *P < 0.05, t-test. (B) Orbital fibroblasts and enriched, irradiated T cells were cultured from five additional patients, and proliferation was measured as described. irrT, irradiated fibroblasts alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). The mean ± SD of results from triplicate cultures is shown. *P < 0.05, t-test.
Figure 1.
 
Autologous T cells stimulated proliferation of orbital fibroblasts from patients with Graves’ disease. (A) Orbital fibroblasts (OF) from a patient were cultured with autologous irradiated (irr) enriched T cells, and proliferation was measured by [3H]thymidine incorporation. irrT, 1 × 105 T cells alone, OF, 5 × 103 orbital fibroblasts alone; OF+irrT, 5 × 103 orbital fibroblasts plus the indicated ratio of irradiated T cells (5 × 103, 1 × 104, 1 × 105). The mean ± SD of triplicate cultures is shown. *P < 0.05, t-test. (B) Orbital fibroblasts and enriched, irradiated T cells were cultured from five additional patients, and proliferation was measured as described. irrT, irradiated fibroblasts alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). The mean ± SD of results from triplicate cultures is shown. *P < 0.05, t-test.
Figure 2.
 
Autologous T cells did not stimulate proliferation of fibroblasts from a noninvolved site. Fibroblasts were cultured from tissue obtained from the eyelid (blepharoplasty) or orbit (orbital decompression) of two patients (A, B). The fibroblasts were cultured with autologous irradiated T cells (100,000 T cells and 5,000 fibroblasts; 20:1 ratio), and proliferation was measured as described. irrT, irradiated T cells alone. The mean ± SD of results from triplicate cultures is shown. P < 0.05, t-test.
Figure 2.
 
Autologous T cells did not stimulate proliferation of fibroblasts from a noninvolved site. Fibroblasts were cultured from tissue obtained from the eyelid (blepharoplasty) or orbit (orbital decompression) of two patients (A, B). The fibroblasts were cultured with autologous irradiated T cells (100,000 T cells and 5,000 fibroblasts; 20:1 ratio), and proliferation was measured as described. irrT, irradiated T cells alone. The mean ± SD of results from triplicate cultures is shown. P < 0.05, t-test.
Figure 3.
 
Stimulation of proliferation of orbital fibroblasts was partly dependent on cell–cell contact. Orbital fibroblasts were cultured with autologous irradiated T cells, either together or separated by a permeable membrane. After 24 hours, proliferation was measured as described. irrT, irradiated T cells alone; OF, alone, OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). (A) and (B) are two representative patients. The mean ± SD of results from triplicate cultures is shown. The increase in proliferation with autologous T cells is significant (*P < 0.05). Proliferation in the membrane assay was significantly increased from OFs alone and also significantly decreased from coculture (**P ≤ 0.01).
Figure 3.
 
Stimulation of proliferation of orbital fibroblasts was partly dependent on cell–cell contact. Orbital fibroblasts were cultured with autologous irradiated T cells, either together or separated by a permeable membrane. After 24 hours, proliferation was measured as described. irrT, irradiated T cells alone; OF, alone, OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). (A) and (B) are two representative patients. The mean ± SD of results from triplicate cultures is shown. The increase in proliferation with autologous T cells is significant (*P < 0.05). Proliferation in the membrane assay was significantly increased from OFs alone and also significantly decreased from coculture (**P ≤ 0.01).
Figure 4.
 
Stimulation of proliferation of orbital fibroblasts by autologous T cells was inhibited by antibodies to MHC class II, CD40, and CD40L. Orbital fibroblasts were cultured with irradiated enriched autologous T cells, as described, with the addition of the indicated concentrations of antibody, and proliferation was measured by [3H]thymidine incorporation. (A) and (B) are two representative patients. irrT, irradiated enriched T cells; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated T cells. The mean ± SD of results from triplicate wells are shown. Fibroblast proliferation was significantly stimulated by autologous T cells (*P < 0.05), whereas each antibody completely inhibited the T-cell-dependent increase in proliferation (no significant difference from OF alone). Irrelevant isotype control antibodies had no significant effect on fibroblast proliferation (data not shown).
Figure 4.
 
Stimulation of proliferation of orbital fibroblasts by autologous T cells was inhibited by antibodies to MHC class II, CD40, and CD40L. Orbital fibroblasts were cultured with irradiated enriched autologous T cells, as described, with the addition of the indicated concentrations of antibody, and proliferation was measured by [3H]thymidine incorporation. (A) and (B) are two representative patients. irrT, irradiated enriched T cells; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated T cells. The mean ± SD of results from triplicate wells are shown. Fibroblast proliferation was significantly stimulated by autologous T cells (*P < 0.05), whereas each antibody completely inhibited the T-cell-dependent increase in proliferation (no significant difference from OF alone). Irrelevant isotype control antibodies had no significant effect on fibroblast proliferation (data not shown).
Figure 5.
 
Orbital fibroblasts expressed MHC class II when cocultured with autologous T cells. Orbital fibroblasts were cultured for 3 days in glass chamber slides, with or without autologous enriched T cells and then were stained with an FITC-labeled MHC class II antibody (HLA-DR, DP, DQ) and photographed. (A) Fibroblasts incubated with IFN-γ expressed MHC class II (positive control). Orbital fibroblasts expressed MHC class II when cultured with autologous T cells (C) but not when cultured alone (B).
Figure 5.
 
Orbital fibroblasts expressed MHC class II when cocultured with autologous T cells. Orbital fibroblasts were cultured for 3 days in glass chamber slides, with or without autologous enriched T cells and then were stained with an FITC-labeled MHC class II antibody (HLA-DR, DP, DQ) and photographed. (A) Fibroblasts incubated with IFN-γ expressed MHC class II (positive control). Orbital fibroblasts expressed MHC class II when cultured with autologous T cells (C) but not when cultured alone (B).
Figure 6.
 
CD4+ and CD4-depleted T-cell fractions did not stimulate proliferation of orbital fibroblasts. (A) CD4+ T cells were purified from the expanded enriched T-cell population by using magnetic beads. Orbital fibroblasts were cocultured with an equal number of cells of the whole autologous enriched T-cell population, the CD4+ fraction (>98% pure), and the CD4-depleted fraction (<2% CD4+), and proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown for a representative experiment. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05). (B) The expanded enriched T-cell population was separated into CD4+ (>99% pure) and CD4 (>97% pure) fractions by FACS. Equal cell numbers of total T cells, CD4+ cells, and CD4 cells were cultured with autologous orbital fibroblasts, as described. Proliferation was measured as described. The mean ± SD of triplicate wells is shown. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05).
Figure 6.
 
CD4+ and CD4-depleted T-cell fractions did not stimulate proliferation of orbital fibroblasts. (A) CD4+ T cells were purified from the expanded enriched T-cell population by using magnetic beads. Orbital fibroblasts were cocultured with an equal number of cells of the whole autologous enriched T-cell population, the CD4+ fraction (>98% pure), and the CD4-depleted fraction (<2% CD4+), and proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown for a representative experiment. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05). (B) The expanded enriched T-cell population was separated into CD4+ (>99% pure) and CD4 (>97% pure) fractions by FACS. Equal cell numbers of total T cells, CD4+ cells, and CD4 cells were cultured with autologous orbital fibroblasts, as described. Proliferation was measured as described. The mean ± SD of triplicate wells is shown. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05).
Figure 7.
 
Depletion of the enriched T cells of dendritic cells or monocytes-macrophages had no effect on stimulation of fibroblast proliferation. Orbital fibroblasts were cocultured with the unfractionated enriched T cells or T cells depleted of CD1a+ (A), CD83+ (B), or CD14+ (C) cells by magnetic bead separation (<0.1% remaining antibody-positive cells). Fibroblast proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown. The increase in proliferation of OF+irrT over OF alone is significant (*P < 0.05); the differences between OF+irrT and OF+antibody-depleted T cells are not.
Figure 7.
 
Depletion of the enriched T cells of dendritic cells or monocytes-macrophages had no effect on stimulation of fibroblast proliferation. Orbital fibroblasts were cocultured with the unfractionated enriched T cells or T cells depleted of CD1a+ (A), CD83+ (B), or CD14+ (C) cells by magnetic bead separation (<0.1% remaining antibody-positive cells). Fibroblast proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown. The increase in proliferation of OF+irrT over OF alone is significant (*P < 0.05); the differences between OF+irrT and OF+antibody-depleted T cells are not.
The authors thank Mike Kasim, MD, and Kimberly Cockerham, MD, for providing surgical specimens. 
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Figure 1.
 
Autologous T cells stimulated proliferation of orbital fibroblasts from patients with Graves’ disease. (A) Orbital fibroblasts (OF) from a patient were cultured with autologous irradiated (irr) enriched T cells, and proliferation was measured by [3H]thymidine incorporation. irrT, 1 × 105 T cells alone, OF, 5 × 103 orbital fibroblasts alone; OF+irrT, 5 × 103 orbital fibroblasts plus the indicated ratio of irradiated T cells (5 × 103, 1 × 104, 1 × 105). The mean ± SD of triplicate cultures is shown. *P < 0.05, t-test. (B) Orbital fibroblasts and enriched, irradiated T cells were cultured from five additional patients, and proliferation was measured as described. irrT, irradiated fibroblasts alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). The mean ± SD of results from triplicate cultures is shown. *P < 0.05, t-test.
Figure 1.
 
Autologous T cells stimulated proliferation of orbital fibroblasts from patients with Graves’ disease. (A) Orbital fibroblasts (OF) from a patient were cultured with autologous irradiated (irr) enriched T cells, and proliferation was measured by [3H]thymidine incorporation. irrT, 1 × 105 T cells alone, OF, 5 × 103 orbital fibroblasts alone; OF+irrT, 5 × 103 orbital fibroblasts plus the indicated ratio of irradiated T cells (5 × 103, 1 × 104, 1 × 105). The mean ± SD of triplicate cultures is shown. *P < 0.05, t-test. (B) Orbital fibroblasts and enriched, irradiated T cells were cultured from five additional patients, and proliferation was measured as described. irrT, irradiated fibroblasts alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). The mean ± SD of results from triplicate cultures is shown. *P < 0.05, t-test.
Figure 2.
 
Autologous T cells did not stimulate proliferation of fibroblasts from a noninvolved site. Fibroblasts were cultured from tissue obtained from the eyelid (blepharoplasty) or orbit (orbital decompression) of two patients (A, B). The fibroblasts were cultured with autologous irradiated T cells (100,000 T cells and 5,000 fibroblasts; 20:1 ratio), and proliferation was measured as described. irrT, irradiated T cells alone. The mean ± SD of results from triplicate cultures is shown. P < 0.05, t-test.
Figure 2.
 
Autologous T cells did not stimulate proliferation of fibroblasts from a noninvolved site. Fibroblasts were cultured from tissue obtained from the eyelid (blepharoplasty) or orbit (orbital decompression) of two patients (A, B). The fibroblasts were cultured with autologous irradiated T cells (100,000 T cells and 5,000 fibroblasts; 20:1 ratio), and proliferation was measured as described. irrT, irradiated T cells alone. The mean ± SD of results from triplicate cultures is shown. P < 0.05, t-test.
Figure 3.
 
Stimulation of proliferation of orbital fibroblasts was partly dependent on cell–cell contact. Orbital fibroblasts were cultured with autologous irradiated T cells, either together or separated by a permeable membrane. After 24 hours, proliferation was measured as described. irrT, irradiated T cells alone; OF, alone, OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). (A) and (B) are two representative patients. The mean ± SD of results from triplicate cultures is shown. The increase in proliferation with autologous T cells is significant (*P < 0.05). Proliferation in the membrane assay was significantly increased from OFs alone and also significantly decreased from coculture (**P ≤ 0.01).
Figure 3.
 
Stimulation of proliferation of orbital fibroblasts was partly dependent on cell–cell contact. Orbital fibroblasts were cultured with autologous irradiated T cells, either together or separated by a permeable membrane. After 24 hours, proliferation was measured as described. irrT, irradiated T cells alone; OF, alone, OF+irrT, orbital fibroblasts plus irradiated autologous T cells (1:20 ratio). (A) and (B) are two representative patients. The mean ± SD of results from triplicate cultures is shown. The increase in proliferation with autologous T cells is significant (*P < 0.05). Proliferation in the membrane assay was significantly increased from OFs alone and also significantly decreased from coculture (**P ≤ 0.01).
Figure 4.
 
Stimulation of proliferation of orbital fibroblasts by autologous T cells was inhibited by antibodies to MHC class II, CD40, and CD40L. Orbital fibroblasts were cultured with irradiated enriched autologous T cells, as described, with the addition of the indicated concentrations of antibody, and proliferation was measured by [3H]thymidine incorporation. (A) and (B) are two representative patients. irrT, irradiated enriched T cells; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated T cells. The mean ± SD of results from triplicate wells are shown. Fibroblast proliferation was significantly stimulated by autologous T cells (*P < 0.05), whereas each antibody completely inhibited the T-cell-dependent increase in proliferation (no significant difference from OF alone). Irrelevant isotype control antibodies had no significant effect on fibroblast proliferation (data not shown).
Figure 4.
 
Stimulation of proliferation of orbital fibroblasts by autologous T cells was inhibited by antibodies to MHC class II, CD40, and CD40L. Orbital fibroblasts were cultured with irradiated enriched autologous T cells, as described, with the addition of the indicated concentrations of antibody, and proliferation was measured by [3H]thymidine incorporation. (A) and (B) are two representative patients. irrT, irradiated enriched T cells; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus irradiated T cells. The mean ± SD of results from triplicate wells are shown. Fibroblast proliferation was significantly stimulated by autologous T cells (*P < 0.05), whereas each antibody completely inhibited the T-cell-dependent increase in proliferation (no significant difference from OF alone). Irrelevant isotype control antibodies had no significant effect on fibroblast proliferation (data not shown).
Figure 5.
 
Orbital fibroblasts expressed MHC class II when cocultured with autologous T cells. Orbital fibroblasts were cultured for 3 days in glass chamber slides, with or without autologous enriched T cells and then were stained with an FITC-labeled MHC class II antibody (HLA-DR, DP, DQ) and photographed. (A) Fibroblasts incubated with IFN-γ expressed MHC class II (positive control). Orbital fibroblasts expressed MHC class II when cultured with autologous T cells (C) but not when cultured alone (B).
Figure 5.
 
Orbital fibroblasts expressed MHC class II when cocultured with autologous T cells. Orbital fibroblasts were cultured for 3 days in glass chamber slides, with or without autologous enriched T cells and then were stained with an FITC-labeled MHC class II antibody (HLA-DR, DP, DQ) and photographed. (A) Fibroblasts incubated with IFN-γ expressed MHC class II (positive control). Orbital fibroblasts expressed MHC class II when cultured with autologous T cells (C) but not when cultured alone (B).
Figure 6.
 
CD4+ and CD4-depleted T-cell fractions did not stimulate proliferation of orbital fibroblasts. (A) CD4+ T cells were purified from the expanded enriched T-cell population by using magnetic beads. Orbital fibroblasts were cocultured with an equal number of cells of the whole autologous enriched T-cell population, the CD4+ fraction (>98% pure), and the CD4-depleted fraction (<2% CD4+), and proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown for a representative experiment. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05). (B) The expanded enriched T-cell population was separated into CD4+ (>99% pure) and CD4 (>97% pure) fractions by FACS. Equal cell numbers of total T cells, CD4+ cells, and CD4 cells were cultured with autologous orbital fibroblasts, as described. Proliferation was measured as described. The mean ± SD of triplicate wells is shown. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05).
Figure 6.
 
CD4+ and CD4-depleted T-cell fractions did not stimulate proliferation of orbital fibroblasts. (A) CD4+ T cells were purified from the expanded enriched T-cell population by using magnetic beads. Orbital fibroblasts were cocultured with an equal number of cells of the whole autologous enriched T-cell population, the CD4+ fraction (>98% pure), and the CD4-depleted fraction (<2% CD4+), and proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown for a representative experiment. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05). (B) The expanded enriched T-cell population was separated into CD4+ (>99% pure) and CD4 (>97% pure) fractions by FACS. Equal cell numbers of total T cells, CD4+ cells, and CD4 cells were cultured with autologous orbital fibroblasts, as described. Proliferation was measured as described. The mean ± SD of triplicate wells is shown. Only the unfractionated T cells stimulated a significant increase in orbital fibroblast proliferation (*P < 0.05).
Figure 7.
 
Depletion of the enriched T cells of dendritic cells or monocytes-macrophages had no effect on stimulation of fibroblast proliferation. Orbital fibroblasts were cocultured with the unfractionated enriched T cells or T cells depleted of CD1a+ (A), CD83+ (B), or CD14+ (C) cells by magnetic bead separation (<0.1% remaining antibody-positive cells). Fibroblast proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown. The increase in proliferation of OF+irrT over OF alone is significant (*P < 0.05); the differences between OF+irrT and OF+antibody-depleted T cells are not.
Figure 7.
 
Depletion of the enriched T cells of dendritic cells or monocytes-macrophages had no effect on stimulation of fibroblast proliferation. Orbital fibroblasts were cocultured with the unfractionated enriched T cells or T cells depleted of CD1a+ (A), CD83+ (B), or CD14+ (C) cells by magnetic bead separation (<0.1% remaining antibody-positive cells). Fibroblast proliferation was measured by [3H]thymidine incorporation. irrT, irradiated T cells alone; OF, orbital fibroblasts alone; OF+irrT, orbital fibroblasts plus unfractionated enriched T cells. The mean ± SD of results from triplicate wells is shown. The increase in proliferation of OF+irrT over OF alone is significant (*P < 0.05); the differences between OF+irrT and OF+antibody-depleted T cells are not.
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