In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation

Karyn F. Siemasko, Jianping Gao, Virginia L. Calder, Rebecca Hanna, Margarita Calonge, Stephen C. Pflugfelder, Jerry Y. Niederkorn, Michael E. Stern

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To determine whether in vitro expanded CD4+CD25 +Foxp3+ regulatory T cells can suppress immune-mediated 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 (<40%), injections of scopolamine, and air flow produced by a fan. CD4+CD25+ 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 + T-effector 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+, CD25+, and intracellular Foxp3+, as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4+ pathogenic T cells (CD4+Path 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 +Path). CONCLUSIONS. Regulatory T cells expressed CD4+, CD25+, and intracellular Foxp3+ 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.

Original languageEnglish (US)
Pages (from-to)5434-5440
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume49
Issue number12
DOIs
StatePublished - 2008

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Regulatory T-Lymphocytes
Inflammation
Phenotype
Tears
T-Lymphocytes
Flow Cytometry
Cytokines
In Vitro Techniques
Scopolamine Hydrobromide
Humidity
Immunoassay
Nude Mice
Fluorescent Antibody Technique
Immunohistochemistry
Air
Injections

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

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In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation. / Siemasko, Karyn F.; Gao, Jianping; Calder, Virginia L.; Hanna, Rebecca; Calonge, Margarita; Pflugfelder, Stephen C.; Niederkorn, Jerry Y.; Stern, Michael E.

In: Investigative Ophthalmology and Visual Science, Vol. 49, No. 12, 2008, p. 5434-5440.

Research output: Contribution to journalArticle

Siemasko, Karyn F. ; Gao, Jianping ; Calder, Virginia L. ; Hanna, Rebecca ; Calonge, Margarita ; Pflugfelder, Stephen C. ; Niederkorn, Jerry Y. ; Stern, Michael E. / In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation. In: Investigative Ophthalmology and Visual Science. 2008 ; Vol. 49, No. 12. pp. 5434-5440.
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abstract = "PURPOSE. To determine whether in vitro expanded CD4+CD25 +Foxp3+ regulatory T cells can suppress immune-mediated 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 (<40{\%}), injections of scopolamine, and air flow produced by a fan. CD4+CD25+ 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 + T-effector 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+, CD25+, and intracellular Foxp3+, as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4+ pathogenic T cells (CD4+Path 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 +Path). CONCLUSIONS. Regulatory T cells expressed CD4+, CD25+, and intracellular Foxp3+ 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.",
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T1 - In vitro expanded CD4+CD25+Foxp3+ regulatory T cells maintain a normal phenotype and suppress immune-mediated ocular surface inflammation

AU - Siemasko, Karyn F.

AU - Gao, Jianping

AU - Calder, Virginia L.

AU - Hanna, Rebecca

AU - Calonge, Margarita

AU - Pflugfelder, Stephen C.

AU - Niederkorn, Jerry Y.

AU - Stern, Michael E.

PY - 2008

Y1 - 2008

N2 - PURPOSE. To determine whether in vitro expanded CD4+CD25 +Foxp3+ regulatory T cells can suppress immune-mediated 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 (<40%), injections of scopolamine, and air flow produced by a fan. CD4+CD25+ 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 + T-effector 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+, CD25+, and intracellular Foxp3+, as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4+ pathogenic T cells (CD4+Path 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 +Path). CONCLUSIONS. Regulatory T cells expressed CD4+, CD25+, and intracellular Foxp3+ 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.

AB - PURPOSE. To determine whether in vitro expanded CD4+CD25 +Foxp3+ regulatory T cells can suppress immune-mediated 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 (<40%), injections of scopolamine, and air flow produced by a fan. CD4+CD25+ 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 + T-effector 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+, CD25+, and intracellular Foxp3+, as determined by flow cytometry and immunohistochemistry. Freshly isolated and in vitro regulatory T cells were titrated in the presence of CD4+ pathogenic T cells (CD4+Path 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 +Path). CONCLUSIONS. Regulatory T cells expressed CD4+, CD25+, and intracellular Foxp3+ 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.

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