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Subconjunctivally applied naïve Tregs support corneal graft survival in baby rats.

Hildebrand A, Jarsch C, Kern Y, Böhringer D, Reinhard T, Schwartzkopff J - Mol. Vis. (2014)

Bottom Line: Corneal transplantation is the most frequent and successful form of tissue transplantation in adults (<10% rejection).No significant delay in rejection was observed when Tregs were applied systemically before keratoplasty.Our data suggest that local application of unprimed regulatory T cells may be a therapeutic strategy for preventing corneal graft rejection in young recipients.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Killianstrasse 5, 79106 Freiburg, Germany ; Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany.

ABSTRACT

Purpose: Corneal transplantation is the most frequent and successful form of tissue transplantation in adults (<10% rejection). In young children, any corneal opacity should be corrected as early as possible to prevent lifelong visual impairment. However, the corneal graft rejection rate is dramatically increased in infants younger than 12 months of age (up to 85% rejection), and immunosuppressive therapy is particularly challenging in this age group. Regulatory T cells (Tregs) are a well-characterized T cell subpopulation with the potential to prevent autoimmune disorders or transplant rejection. Antigen-specific Tregs were shown to inhibit graft rejection in adult stem cell transplantation. Less is known about the role of naïve Tregs.The purpose of the present study was to elucidate the relevance of naïve Tregs in juvenile corneal transplantation in a baby rat keratoplasty model that reproduces the accelerated rejection in young patients.

Methods: Counts and inhibitory potential of Tregs were studied in spleens of 3- and 10-week-old rats. Unprimed Tregs (CD4+CD25+) were isolated from the spleens of 10-week-old Lewis rats and systemically or subconjunctivally administered in vivo in allogenic keratoplasty in 3- and 10-week-old Lewis recipient rats. In subconjunctival tissue, transcription was analyzed for induction of transforming-growth-factor beta (TGF-β).

Results: In 3-week-old rats, CD4 T cell counts, but not FoxP3 T cell counts were lower than in 10-week-old rats. The Tregs of both age groups had the potential to inhibit T cell activation in vitro. No significant delay in rejection was observed when Tregs were applied systemically before keratoplasty. However, subconjunctival application of Tregs abrogated rejection in 66.7% and 33.3% of the 3- and 10-week-old recipients, respectively. Analysis of the conjunctival tissue revealed a transplantation-induced increase in TGF-β transcription in the 3-week-old rats.

Conclusions: Our data suggest that local application of unprimed regulatory T cells may be a therapeutic strategy for preventing corneal graft rejection in young recipients.

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Related in: MedlinePlus

Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p<0.01). Between the cohorts, no significant difference was observed (p=0.141).
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f2: Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p<0.01). Between the cohorts, no significant difference was observed (p=0.141).

Mentions: To address the question whether the Tregs of 3-week-old and 10-week-old rats act inhibitory, we studied the Tregs of both cohorts in a T cell stimulation assay in vitro. The splenic lymphocytes of the 10-week-old rats were stimulated with anti-CD3 and anti-CD28 antibodies and cocultured with or without the addition of Tregs from 3- or 10-week-old rats. The Tregs were sorted with flow cytometry using the surface markers CD4 and CD25 and were added in a 1:1 ratio to a total of 105 cells per well. Cell proliferation was assessed as a loss of CFSE, and the ratio of divided to undivided cells was calculated (Figure 2). Adding Tregs clearly inhibited lymphocyte proliferation (p<0.01). Control T cell stimulation showed a ratio of 2.4 (n=4), while in the T cell stimulation with added Tregs, the ratio was reduced to 0.40±0.17 and 0.74±0.34, respectively. The Treg-mediated inhibition was not age-dependent (n=3 resp. n=9, p=0.141).


Subconjunctivally applied naïve Tregs support corneal graft survival in baby rats.

Hildebrand A, Jarsch C, Kern Y, Böhringer D, Reinhard T, Schwartzkopff J - Mol. Vis. (2014)

Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p<0.01). Between the cohorts, no significant difference was observed (p=0.141).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4279769&req=5

f2: Inhibitory potential of Tregs in a T cell stimulation assay. T cells were stimulated with CD3 and CD28 antibodies and Tregs of 3- and 10-week old rats were added in a co-culture experiment. Division of cells was traced with CFSE and ratio of divided to undivided cells was determined. Treg of both cohorts showed inhibitory potential (*p<0.01). Between the cohorts, no significant difference was observed (p=0.141).
Mentions: To address the question whether the Tregs of 3-week-old and 10-week-old rats act inhibitory, we studied the Tregs of both cohorts in a T cell stimulation assay in vitro. The splenic lymphocytes of the 10-week-old rats were stimulated with anti-CD3 and anti-CD28 antibodies and cocultured with or without the addition of Tregs from 3- or 10-week-old rats. The Tregs were sorted with flow cytometry using the surface markers CD4 and CD25 and were added in a 1:1 ratio to a total of 105 cells per well. Cell proliferation was assessed as a loss of CFSE, and the ratio of divided to undivided cells was calculated (Figure 2). Adding Tregs clearly inhibited lymphocyte proliferation (p<0.01). Control T cell stimulation showed a ratio of 2.4 (n=4), while in the T cell stimulation with added Tregs, the ratio was reduced to 0.40±0.17 and 0.74±0.34, respectively. The Treg-mediated inhibition was not age-dependent (n=3 resp. n=9, p=0.141).

Bottom Line: Corneal transplantation is the most frequent and successful form of tissue transplantation in adults (<10% rejection).No significant delay in rejection was observed when Tregs were applied systemically before keratoplasty.Our data suggest that local application of unprimed regulatory T cells may be a therapeutic strategy for preventing corneal graft rejection in young recipients.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Killianstrasse 5, 79106 Freiburg, Germany ; Faculty of Biology, Albert-Ludwigs-University Freiburg, Germany.

ABSTRACT

Purpose: Corneal transplantation is the most frequent and successful form of tissue transplantation in adults (<10% rejection). In young children, any corneal opacity should be corrected as early as possible to prevent lifelong visual impairment. However, the corneal graft rejection rate is dramatically increased in infants younger than 12 months of age (up to 85% rejection), and immunosuppressive therapy is particularly challenging in this age group. Regulatory T cells (Tregs) are a well-characterized T cell subpopulation with the potential to prevent autoimmune disorders or transplant rejection. Antigen-specific Tregs were shown to inhibit graft rejection in adult stem cell transplantation. Less is known about the role of naïve Tregs.The purpose of the present study was to elucidate the relevance of naïve Tregs in juvenile corneal transplantation in a baby rat keratoplasty model that reproduces the accelerated rejection in young patients.

Methods: Counts and inhibitory potential of Tregs were studied in spleens of 3- and 10-week-old rats. Unprimed Tregs (CD4+CD25+) were isolated from the spleens of 10-week-old Lewis rats and systemically or subconjunctivally administered in vivo in allogenic keratoplasty in 3- and 10-week-old Lewis recipient rats. In subconjunctival tissue, transcription was analyzed for induction of transforming-growth-factor beta (TGF-β).

Results: In 3-week-old rats, CD4 T cell counts, but not FoxP3 T cell counts were lower than in 10-week-old rats. The Tregs of both age groups had the potential to inhibit T cell activation in vitro. No significant delay in rejection was observed when Tregs were applied systemically before keratoplasty. However, subconjunctival application of Tregs abrogated rejection in 66.7% and 33.3% of the 3- and 10-week-old recipients, respectively. Analysis of the conjunctival tissue revealed a transplantation-induced increase in TGF-β transcription in the 3-week-old rats.

Conclusions: Our data suggest that local application of unprimed regulatory T cells may be a therapeutic strategy for preventing corneal graft rejection in young recipients.

Show MeSH
Related in: MedlinePlus