In Vitro Expanded CD4⁺CD25⁺Foxp3⁺Regulatory T Cells Maintain a Normal Phenotype and Suppress Immune-Mediated Ocular Surface Inflammation

Abstract

PURPOSE.To determine whether in vitro expanded CD4 ϩ CD25 ϩ Foxp3 ϩ regulatory T cells can suppress immunemediated ocular surface inflammation in a mouse model of dry eye. METHODS. C57BL/6 or BALB/c mice were exposed to a dry, desiccating environment produced by maintaining low humidity (Ͻ40%), injections of scopolamine, and air flow produced by a fan. CD4 ϩ CD25 ϩ regulatory T cells were isolated and expanded in vitro in the presence of rmIL-2 and beads coated with anti-CD28 and anti-CD3. In vitro expanded regulatory T cells were phenotypically compared with freshly isolated regulatory T cells by flow cytometry and immunofluorescence. T-cell-deficient nude mice were reconstituted with CD4 ϩ Teffector cells from donor mice exposed to a desiccating environment for 5 days, in combination with or without freshly isolated or in vitro expanded regulatory T cells. Tear cytokine levels were determined by a multiplex bead-based immunoassay. RESULTS. In vitro regulatory T cells maintained normal levels of CD4 ϩ , CD25 ϩ , and intracellular Foxp3 ϩ , as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4 ϩ pathogenic T cells (CD4 ϩPath T cells) in reconstitution experiments and most efficiently ablated tear cytokine levels and conjunctival cellular infiltration at a ratio of 1:1 (T Regs: CD4 ϩPath ). CONCLUSIONS. Regulatory T cells expressed CD4 ϩ , CD25 ϩ , and intracellular Foxp3 ϩ at normal levels and retained their inhibitory function after in vitro expansion, providing a useful tool to determine the mechanism regulatory T cells use to sustain a homeostatic environment on the ocular surface. (Invest Ophthalmol Vis Sci. 2008;49:5434 -5440)

Spontaneous Autoimmune Dacryoadenitis in Aged CD25KO Mice

“…30,49 The importance of Tregs in suppressing ocular surface inflammation has been demonstrated by reconstituting Tcell-deficient nude mice with either pathogenic CD4 ϩ T cells from donors exposed to dessicating stress alone, or in combination with in vitro expanded CD4ϩCD25ϩ Foxp3ϩ regulatory T cells. 50 Nude mice reconstituted with both pathogenic CD4 ϩ T cells and in vitro Tregs had significantly lower conjunctival cellular infiltration and tear levels of pro-inflammatory cytokines compared with the mice receiving only pathogenic effector T cells. 50 Other than dry eye, defective Treg function has been linked to the pathogenesis of several autoimmune diseases including multiple sclerosis and rheumatoid arthritis.…”

“…50 Nude mice reconstituted with both pathogenic CD4 ϩ T cells and in vitro Tregs had significantly lower conjunctival cellular infiltration and tear levels of pro-inflammatory cytokines compared with the mice receiving only pathogenic effector T cells. 50 Other than dry eye, defective Treg function has been linked to the pathogenesis of several autoimmune diseases including multiple sclerosis and rheumatoid arthritis.…”

Ocular Surface APCs Are Necessary for Autoreactive T Cell-Mediated Experimental Autoimmune Lacrimal Keratoconjunctivitis

“…injection into nude recipient mice as previously described (4,21). Mice were sacrificed 3 d posttransfer, and tears and ocular surface tissues were collected for analysis.…”

“…Cytokine and chemokine levels in tears during experimental ALKC Relative levels of select cytokines and chemokines present in the tears were evaluated over the course of experimental ALKC using Luminex analysis (Luminex, Austin, TX) as previously described (21). In brief, tears were collected at various time points (e.g., 0 or 5 d post-DS); 1.5 ml Beadlyte assay buffer (Millipore, Bellerica, MA) was placed on each eye, and 1 ml/eye was collected and combined with 8 ml Beadlyte buffer.…”

Levels of Interleukin-35 and Its Relationship with Regulatory T-Cells in Chronic Hepatitis B Patients

“…Interleukin-35 (IL-35) was discovered and designated by the end of 2007, and identified as a novel immunosuppressive/ anti-inflammatory cytokine of the IL-12 family, which includes IL-12, IL-23, and IL-27 (15). IL-35 is a heterodimeric protein with two subunits, Epstein-Barr virus induced gene 3 (EBI3) and IL-12p35, which shares EBI3 with IL-27.…”