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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

Graft survival after keratoplasty and s.c. Tregs. A: Graft survival after keratoplasty in 3-week-old recipients with subconjunctival Treg application. Application of Tregs led to 66.7% graft survival in 3-week-old recipients (n=6; *p<0.01), application of CD4+CD25- cells induced a graft survival of 25% and a median of POD11 (n=4), while without cells, grafts were rejected at POD9. Recipients of syngenic grafts did not reject until the end of the experiment on POD29 (n=8; *p<0.01). B: Graft survival after keratoplasty in 10-week-old recipients with subconjunctival application. In the control group (CD4+CD25-) the median of rejection was POD 12, in Treg group (CD4+CD25+) median was POD 16 and 37.5% of grafts survived POD35 (both n=6; *p<0.01) while in allogenic transplantations without addition of cells, rejection occurred on POD15 (n=4). In syngenic transplantations no rejection occurred until the end of the experiment on POD 35 (n=4).
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f4: Graft survival after keratoplasty and s.c. Tregs. A: Graft survival after keratoplasty in 3-week-old recipients with subconjunctival Treg application. Application of Tregs led to 66.7% graft survival in 3-week-old recipients (n=6; *p<0.01), application of CD4+CD25- cells induced a graft survival of 25% and a median of POD11 (n=4), while without cells, grafts were rejected at POD9. Recipients of syngenic grafts did not reject until the end of the experiment on POD29 (n=8; *p<0.01). B: Graft survival after keratoplasty in 10-week-old recipients with subconjunctival application. In the control group (CD4+CD25-) the median of rejection was POD 12, in Treg group (CD4+CD25+) median was POD 16 and 37.5% of grafts survived POD35 (both n=6; *p<0.01) while in allogenic transplantations without addition of cells, rejection occurred on POD15 (n=4). In syngenic transplantations no rejection occurred until the end of the experiment on POD 35 (n=4).

Mentions: About 105 Tregs in 30 µl PBS from 10-week-old naïve rats were applied to the conjunctiva at the end of surgery. CD4+CD25- cells were administered in controls. Recipients were clinically monitored until day 29 after keratoplasty. All baby rats with an allogenic transplant and without application of Tregs showed graft rejection “until day 11 after keratoplasty (n=15), while the syngenic transplants were not rejected (n=8). In the group of six baby rats with s.c. application of Tregs, four did not reject the graft. In the control group, three out of four animals rejected the graft (Figure 4A). In the adult age group, two of the six 10-week-old recipients of an allogenic transplant and Tregs s.c. did not reject the grafts while all the controls (n=6) rejected the grafts until day 15 (Figure 4B). Adult recipients of an allogenic transplant without addition of cells reject the graft until day 15 (n=4), while syngenic recipients without addition of cells do not reject the graft (n=4). Results are summarized in Table 1.


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)

Graft survival after keratoplasty and s.c. Tregs. A: Graft survival after keratoplasty in 3-week-old recipients with subconjunctival Treg application. Application of Tregs led to 66.7% graft survival in 3-week-old recipients (n=6; *p<0.01), application of CD4+CD25- cells induced a graft survival of 25% and a median of POD11 (n=4), while without cells, grafts were rejected at POD9. Recipients of syngenic grafts did not reject until the end of the experiment on POD29 (n=8; *p<0.01). B: Graft survival after keratoplasty in 10-week-old recipients with subconjunctival application. In the control group (CD4+CD25-) the median of rejection was POD 12, in Treg group (CD4+CD25+) median was POD 16 and 37.5% of grafts survived POD35 (both n=6; *p<0.01) while in allogenic transplantations without addition of cells, rejection occurred on POD15 (n=4). In syngenic transplantations no rejection occurred until the end of the experiment on POD 35 (n=4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Graft survival after keratoplasty and s.c. Tregs. A: Graft survival after keratoplasty in 3-week-old recipients with subconjunctival Treg application. Application of Tregs led to 66.7% graft survival in 3-week-old recipients (n=6; *p<0.01), application of CD4+CD25- cells induced a graft survival of 25% and a median of POD11 (n=4), while without cells, grafts were rejected at POD9. Recipients of syngenic grafts did not reject until the end of the experiment on POD29 (n=8; *p<0.01). B: Graft survival after keratoplasty in 10-week-old recipients with subconjunctival application. In the control group (CD4+CD25-) the median of rejection was POD 12, in Treg group (CD4+CD25+) median was POD 16 and 37.5% of grafts survived POD35 (both n=6; *p<0.01) while in allogenic transplantations without addition of cells, rejection occurred on POD15 (n=4). In syngenic transplantations no rejection occurred until the end of the experiment on POD 35 (n=4).
Mentions: About 105 Tregs in 30 µl PBS from 10-week-old naïve rats were applied to the conjunctiva at the end of surgery. CD4+CD25- cells were administered in controls. Recipients were clinically monitored until day 29 after keratoplasty. All baby rats with an allogenic transplant and without application of Tregs showed graft rejection “until day 11 after keratoplasty (n=15), while the syngenic transplants were not rejected (n=8). In the group of six baby rats with s.c. application of Tregs, four did not reject the graft. In the control group, three out of four animals rejected the graft (Figure 4A). In the adult age group, two of the six 10-week-old recipients of an allogenic transplant and Tregs s.c. did not reject the grafts while all the controls (n=6) rejected the grafts until day 15 (Figure 4B). Adult recipients of an allogenic transplant without addition of cells reject the graft until day 15 (n=4), while syngenic recipients without addition of cells do not reject the graft (n=4). Results are summarized in Table 1.

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