Host-derived endothelial regeneration of corneal transplants in a rat keratoplasty model.

BACKGROUND: It has been observed that formerly rejected opaque corneal transplants can regain clarity in the rat. We hypothesized that graft endothelium is regenerated by the host. Therefore, we used green fluorescent protein (GFP) transgenic rats to assess the origin of cells following keratoplasty. METHODS: Allogeneic corneal transplantations were carried out between Fischer strain rats as graft donors and GFP-transgenic Lewis rats as recipients. In a second group, syngeneic transplantations were performed between GFP-negative Lewis donors and GFP-positive Lewis recipients, where endothelial-cell-free grafts after mechanical endothelial debridement were used. All grafts were followed up clinically for signs of opacity and rejection. After 6 weeks, corneal flatmounts counterstained with DAPI were analyzed by confocal microscopy. RESULTS: Syngeneic transplantation of endothelial-cell-free grafts led to medium opacity levels without rejection and to subsequent clearing. All grafts showed a population of GFP-positive, host-derived endothelial cells on the graft after 6 weeks. In the allogeneic transplantation group, all grafts but one were rejected after a median of 17 days. While the graft that was not rejected maintained the GFP-negative transplant endothelium, all formerly rejected grafts showed GFP-positive endothelium on the transplant after 6 weeks, accompanied by clinical clearing of the graft. CONCLUSION: GPF positivity shows that in both a syngeneic and an allogeneic setting, the host-derived corneal endothelium can compensate for the endothelial cell loss of the graft. Following rejection, the grafts are repopulated by host-derived endothelial cells in the rat. This finding demonstrates a high regenerative capacity of the peripheral corneal endothelium in the rat, which should be considered whenever interpreting rat keratoplasty results.

Regeneration of corneal endothelial cells following keratoplasty in rats with bullous keratopathy.

PURPOSE: Little is known about the behavior of the endothelial cell (EC) layer following keratoplasty. In vitro experiments suggested that the peripheral endothelium might have a higher regenerative capacity than the central endothelium, and some authors hypothesized that endothelial progenitor cells are present in the limbal area. Thus, we analyzed the corneal endothelial regenerative capacity in vivo in a rat model of bullous keratopathy using either bullous central grafts or bullous peripheral recipient corneas to analyze differences in EC regeneration depending on central versus peripheral cell origin. METHODS: Bullous keratopathy was induced in Lewis rats with an intracameral injection of benzalkonium chloride (0.05%; BAK). Three days later, the central area of the bullous cornea was excised and used as a bullous graft, transplanted to a healthy, green fluorescent protein (GFP)-transgeneic Lewis receipient rat (group 'bullous graft'). In return, the mentioned rat eye with the bullous keratopathy received a healthy GFP-transgeneic corneal transplant (group 'bullous host'). A subgroup of these animals received a healthy, excentrically trephined including parts of the limbus (group 'bullous host, limbo-keratoplasty'). The grafts were monitored clinically for 7 weeks. Subsequently, the mean EC density was calculated on corneal whole mounts with Alizarin Red S staining. GFP was analyzed with confocal microscopy to determine EC origin. Untreated fellow eyes served as controls. RESULTS: BAK injection led to a significant decrease in the mean EC density with subsequent bullous keratopathy. In the control eyes, the mean EC density was 3,744 cells/mm² in the center and 2,811 cells/mm² in the periphery. In eyes with bullous keratopathy, only 233 cells/mm² in the center and 622 cells/mm² in the periphery were observed three days after BAK-injection. Bullous transplants in the group 'bullous graft' cleared, and GFP-positive cells were detected on the transplant. In contrast, no GFP-positive ECs were detected on the host cornea in the groups 'bullous host'. CONCLUSIONS: ECs from the peripheral cornea have the ability to cross the graft border and compensate for the pathologically altered/absent endothelium on the graft. In contrast, ECs derived from the central cornea remain central on the graft and do not replace or regenerate peripheral ECs in our model of bullous keratopathy.

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

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-ß). 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-ß 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.

Short-term azithromycin treatment promotes cornea allograft survival in the rat.

BACKGROUND: Any inflammatory response following corneal transplantation may induce rejection and irreversible graft failure. The purpose of this study is to analyze the anti-inflammatory effect of azithromycin (AZM) following experimental keratoplasty in rats. METHODS: Corneal transplants were performed between Fisher-donor and Lewis-recipient rats. Recipients were postoperatively treated three times daily with AZM, miglyol, ofloxacin or dexamethasone eye drops. As an additional control, AZM was applied following syngeneic keratoplasty. Furthermore, short-term treatments with AZM for seven days perioperatively or with AZM only three days prior to the transplantation were compared to appropriate controls. All transplants were monitored clinically for opacity, edema, and vascularization. Infiltrating CD45(+), CD4(+), CD8(+), CD25(+), CD161(+) and CD163(+) cells were quantified via immunohistochemistry. RESULTS: AZM significantly promoted corneal graft survival compared with miglyol or ofloxacin treatment. This effect was comparable to topical dexamethasone. No adverse AZM effect was observed. Histology confirmed a significant reduction of infiltrating leukocytes. The short-term application of AZM for three days prior to transplantation or for seven days perioperatively reduced corneal graft rejection significantly compared with the controls. CONCLUSIONS: Along with antibiotic properties, topical AZM has a strong anti-inflammatory effect. Following keratoplasty, this effect is comparable to topical dexamethasone without the risk of steroid-induced adverse effects. Short-term treatment with AZM three days prior to the transplantation was sufficient to promote graft survival in the rat keratoplasty model. We therefore suggest further assessing the anti-inflammatory function of topical AZM following keratoplasty in humans.

Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease.

PURPOSE: To assess the expression of human leucocyte antigen (HLA)-DR in epithelial cells and cluster of differentiation (CD8)-positive lymphocytes as possible markers of chronic ocular graft versus host disease (cGvHD) after hematological stem cell transplantation (HSCT). METHODS: Twenty-seven consecutive patients with dry-eye symptoms following HSCT (24 [89%] with peripheral blood stem cell transplantation and 3 [11%] with bone marrow transplants; 17 [63%] familiar allogenic grafts) and 19 age-matched controls were included. Conjunctival impression cytology specimens were stained for HLA-DR, cytokeratin 19, and CD8. Oxford grading scale, blinking frequency, Schirmer test, tear film break-up time (TBUT), and Ocular Surface Disease Index (OSDI) were also recorded. Wilcoxon nonparametric testing was used to compare controls and HSCT recipients and to assess HSCT recipient subgroups with and without clinical cGVHD. RESULTS: Eighteen patients showed clinical signs of ocular cGVHD. TBUT and Schirmer test scores were significantly lower in patients, while Oxford grades and OSDI were significantly higher than in controls. Epithelial HLA-DR expression was generally higher in HSCT recipients than in controls, but it did not correlate with ocular cGVHD status. CD8-positive lymphocytes were identified in five patients with ocular cGvHD and one control. CONCLUSIONS: A strong HLA-DR expression as detected by impression cytology appears to indicate a general HSCT response and fails to predict ocular cGVHD. However, the detection of CD8-positive lymphocytes using impression cytology was frequently associated with ocular cGvHD. Our data warrant further evaluation of CD8 expression in impression cytology, along with comparison to conjunctival biopsies and brush cytology, as impression cytology may offer a less invasive strategy for assessing cGVHD status.

Enhanced TKTL1 expression in malignant tumors of the ocular adnexa predicts clinical outcome.

PURPOSE: Malignant tumors metabolize glucose to lactate even in the presence of oxygen via the pentose-phosphate pathway. The metabolic switch from oxidative glycolysis to nonoxidative fermentation in tumors has been associated with overexpression of the transketolase-like-1-gene (TKTL1), which encodes an essential and rate-limiting enzyme in the nonoxidative part of the pentose-phosphate pathway. This study investigates the role of TKTL1 in ocular adnexal tumors and analyzes how its expression correlates with the clinical outcomes against the background of tumor thickness and mitotic rate. DESIGN: Comparative case studies. PARTICIPANTS: We included 89 subjects with malignant tumors of the ocular adnexa (44 squamous cell carcinomas, 26 lymphomas, 19 malignant melanomas) who had been treated at the University Eye Hospital Freiburg from 1994 to 2008. Sixteen subjects with conjunctival nevi, 19 with conjunctival papilloma, and 2 with conjunctival-reactive lymphoid hyperplasia were included as controls. METHODS: TKTL1 expression was assessed by reverse transcriptase-polymerase chain reaction and immunohistochemistry and semiquantitatively analyzed using an established immunoreactive score (IRS). The tumor recurrence rate, metastasis occurrence, and survival time of each patient were assessed retrospectively and correlated with the TKTL IRS using Kaplan-Meier and Cox regression analyses. MAIN OUTCOME MEASURES: TKTL1 expression, mitotic rate within the tumor mass, and tumor thickness and its association with clinical outcome. RESULTS: We identified increased TKTL1 protein levels in malignant conjunctival tumors compared with control samples and detected an average IRS of 1.78 (standard deviation [SD], ± 0.46) for melanomas, 1.3 for lymphomas (SD, ± 0.79), and 1.22 for squamous cell carcinomas (SD, ± 0. 97) compared with 0.86 for conjunctival nevi (SD, ± 0.57) and 0.5 for conjunctival papilloma (SD, ± 0.83). Multifactorial survival analysis showed that TKTL1 overexpression correlated with the patient outcomes in malignant tumors (P = 0.045). In the squamous cell carcinomas, tumor thickness and mitotic rate correlated more strongly with prognosis compared with TKTL1 overexpression (P = 0.0061, P = 0.015, and P = 0.061, respectively). CONCLUSIONS: TKTL1 is dysregulated in malignant tumors of the ocular adnexa, and enhanced expression seems to predict clinical outcome, especially the tumor recurrence rate.

Inhibition of corneal inflammation following keratoplasty by birch leaf extract.

The objective of this study was to determine the effect of birch leaf (Betula pendula) extract (BPE) on corneal inflammation following keratoplasty in the rat model. T cells were stimulated in vitro in the presence of BPE. Proliferation, activation phenotype and the number of apoptotic/necrotic cells in cell culture were analyzed by flow cytometry. Corneal transplantation was performed between Fisher and Lewis rats. Recipient rats were either treated with cyclosporine A at a low dosage (Low-dose CsA=LDCsA) or received LDCsA in combination with BPE (2×1ml/day). Clinical signs for corneal inflammation and rejection time points were determined. Infiltrating leukocytes were analyzed histologically. BPE specifically inhibited T cell proliferation in vitro by inducing apoptosis. The phenotype was not affected. In vivo, BPE significantly delayed the onset of corneal opacification (p<0.05). The amount of infiltrating CD45(+) leukocytes and CD4(+) T cells (p<0.001) was significantly reduced by BPE, whereas infiltration of CD163(+) macrophages was not significantly different between the two groups. BPE selectively induces apoptosis of activated T cells. Accordingly, BPE treatment significantly reduces infiltrating T cells and subsequent corneal opacification following keratoplasty. Our findings suggest BPE as a promising anti-inflammatory drug to treat corneal inflammation.

Allogenic limbo-keratoplasty with conjunctivoplasty, mitomycin C, and amniotic membrane for bilateral limbal stem cell deficiency.

OBJECTIVE: To present the technique and report the results of up to 36 months after allogenic central penetrating limbo-keratoplasty in conjunction with conjunctivoplasty, mitomycin C (MMC), and amniotic membrane (AM) transplantation in patients with bilateral limbal stem cell deficiency (LSCD). DESIGN: Retrospective, consecutive subject cohort study. PARTICIPANTS: Case records of 20 eyes from 20 patients who presented with bilateral LSCD due to aniridia, chemical/thermal burn, cicatrizing pemphigoid, and chronic ocular surface inflammation and who were treated at the University Eye Hospital, Freiburg. METHODS: All eyes were treated with central limbo-keratoplasty in conjunction with conjunctivoplasty, MMC, and AM. There were 20 human leukocyte antigen-typed allolimbal transplants from cadaveric donors. All patients received systemic immunosuppression with mycophenolate mofetil or cyclosporine A. MAIN OUTCOME MEASURES: Surgical success was measured by the duration for which a healthy corneal epithelium was maintained. Visual success was measured by an improvement in visual acuity (VA) in the eye during follow-up and directly correlated with central clear graft survival. RESULTS: The follow-up period was up to 34 months (mean, 20 months; median, 22.4 months). Mean VA, measured in decimal fractions, increased from 0.029 (∼20/400; median, 0.005; first quartile 0.005; third quartile 0.005) before surgery to 0.281 (20/70; median, 0.2; first quartile 0.04; third quartile 0.55) after surgery. Healthy corneal epithelium showing survival of limbal stem cells was observed in 14 eyes (70%) during complete follow-up. CONCLUSIONS: Penetrating limbo-keratoplasty with conjunctivoplasty, MMC, and AM transplantation is a promising new surgical technique for improving vision and conjunctivalization in patients with severe bilateral LSCD necessitating allogenic transplants.

The intrastromal corneal ring in penetrating keratoplasty-long-term results of a prospective randomized study.

PURPOSE: Postoperative astigmatism after penetrating keratoplasty is a major problem in corneal transplantation. The purpose of this prospective randomized study was to evaluate the efficacy and safety of an intrastromal corneal ring after penetrating keratoplasty. METHODS: Twenty patients were included, 10 of whom received an intracorneal ring (group 1) and 10 who did not (group 2, control group). Astigmatism in Orbscan corneal topography, occurrence of immune reactions, and occurrence of side effects were this study's main outcome criteria. RESULTS: Mean follow-up time was 27.6 ± 5.3 months. Mean astigmatism (Orbscan) was 4.4 diopters in group 1 and 4.4 diopters in group 2 (P = 0.695). Spontaneous suture rupture occurred in 5 patients with corneal ring but in none of those in the control group. We observed 3 immune reactions in 3 patients with corneal ring, whereas group 2 experienced no rejection (P < 0.05). Endothelial cell loss was 15.1% in the group with the ring and 8.7% in the control group. That difference was not statistically significant (P = 0.146). CONCLUSIONS: The use of the intrastromal corneal ring after penetrating keratoplasty caused no reduction in postoperative astigmatism. However, its use was statistically significantly associated with adverse events.

Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits.

PURPOSE: To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits. METHODS: Total limbal stem cell deficiency was produced in 21 right eyes of 21 New Zealand rabbits; 6 eyes served as controls (group 1, G1). After removal of the conjunctival overgrowth, five eyes received amniotic membrane transplantation (AMT; G2). In four eyes, autologous limbal stem cell transplantation from the healthy eye was performed with AMT (G3). In another six eyes, enriched autologous MSC were injected under the amniotic membrane (AM) (G4). Within 280 days, corneoscleral discs were analysed for goblet cells, cytokeratin (CK) 3/12, connexin 43, ß(1) -integrin, CK 19, α-enolase, p63 and ATP-binding cassette transporter subtype G-2 (ABCG-2) distribution patterns. RESULTS: Cultivated MSC were positive for CK 3/12 and α-enolase, but negative for ABCG-2, p63 and connexin 43. On rabbit corneas, CK 3/12 was expressed in all corneal regions in all groups, but with significantly different intensities. Among all other parameters, expression levels of ABCG-2, ß(1) -integrin and connexin 43 were significantly different between the transplanted groups and the control group. After a mean follow-up time of 172 (47-280) days, goblet cells were rarely present in the central cornea (G1-4). CONCLUSION: CK 3/12 is not highly specific for differentiated corneal epithelium. Further, goblet cells are not a reliable marker for conjunctivalization in rabbits. Expression of ABCG-2, ß(1)-integrin and connexin 43 after mesenchymal stem cell transplantation may indicate their ability to maintain their stem cell character or to transdifferentiate to epithelial progenitor cells.

Operational post-keratopasty graft tolerance due to differential HLAMatchmaker matching.

PURPOSE: Penetrating keratoplasty can restore vision in corneal blindness. However, immunologic rejection threatens graft survival. Matching donors at swine leukocyte antigen (SLA)-class II convey allo-specific tolerance in a large animal kidney-transplantation model despite mismatches at SLA-class I. The same matching pattern seems to account for the blood transfusion effect in kidney transplantation. Relying on the molecular basis of HLAMatchmaker eplets, we assessed whether this finding would also apply to keratoplasty, and if it would enhance the benefit from matching human leukocyte antigen (HLA)-class I alone. METHODS: We retrospectively selected two independent cohorts comprising 586 and 975 penetrating keratoplasties. Our computations revealed a quantitative tolerogenicity factor analogous to the animal model. The number of mismatched HLA-class I eplets functioned as a factor for conventional histocompatibility. In the first cohort, we empirically determined the thresholds with the highest predictive power on graft rejection for both factors, and confirmed those thresholds in the second cohort. We applied Cox proportional hazards regression for these analyses. RESULTS: The thresholds with highest predictive power revealed 220 eplets(2) for the tolerance factor and 10 eplets for HLA-class I histocompatibility. The respective hazards ratios were 2.22 (p=0.04) versus 3.63 (p<0.01) in the first cohort and 2.09 (p<0.01) versus 1.51 (p=0.02) in the second, confirmatory cohort. The threshold factors proved to be additive in predicting immune reactions in both cohorts, (hazard ratios 2.66 in cohort 1 versus 1.70; p<0.01 in cohort 2). CONCLUSIONS: Operational tolerance may be inducible by balanced matching of HLA-class I and II HLAMatchmaker eplets. Furthermore, such tolerance is additive to histocompatibility at HLA-class I.

NK cell depletion delays corneal allograft rejection in baby rats.

PURPOSE: Penetrating keratoplasty has a very poor outcome compared with adults if performed in the first years of life. Rejection in these young patients occurs even in the absence of known immunological risk factors. Recently, a baby rat model was introduced and an essential contribution of natural killer (NK) cells during allograft rejection was suggested. To analyze this, NK cells were depleted in baby rats before keratoplasty. METHODS: Allogeneic keratoplasty was performed between Lewis and Fisher rats. The recipient's ages were 10 and 3 weeks, respectively. NK cells were depleted by an intraperitoneal injection of a monoclonal antibody. All experiments were controlled by the injection of isotypic control antibodies and syngeneically. Survival rates were calculated and cellular infiltrates were analyzed histologically. RESULTS: NK cell depletion did delay median graft survival times in a statistically significantly way compared with the control animals (p<0.01). At median rejection time points, macrophages, CD4(+) T cells and CD25(+) leukocytes infiltrated to a greater extent in the depleted recipients. No significant changes in the cell numbers of infiltrating CD8(+) T cells were observed. CONCLUSIONS: We conclude that NK cells play a role during allograft rejection in baby rats, but their effect is replaceable. A greater infiltration of macrophages and CD4(+) T cells suggests that they might compensate for the missing NK cells' response in this experimental setting. Our results represent another step toward understanding the complex mechanisms of an accelerated corneal graft rejection in infant recipients.

Long-term graft survival in penetrating keratoplasty: the biexponential model of chronic endothelial cell loss revisited.

AIM: To present a novel interpretation of the biexponential nature of chronic endothelial cell loss after penetrating keratoplasty (PK). We hypothesize that the fast component of endothelial cell loss reflects the endothelial cells of graft origin. The slow component might just reflect cell loss of the recipient endothelium. We investigate herein whether this hypothesis is in line with long-term survival in bullous keratopathy (BK: almost no endothelium in the recipient bed) and keratoconus (KK: recipient bed with plenty of endothelium). METHODS: We reviewed endothelial graft failures in PK for BK (n = 88) and KK (n = 87). Patients with immune reactions or a history of glaucoma were excluded. We built a statistical model to predict graft failures from biexponential endothelial cell loss and compared this data to the actual outcomes. RESULTS: After 15 years, the incidence of late endothelial failures was 8% in KK and 33% in BK. The 95% confidence intervals of the simulated outcomes corresponded completely to the actual outcomes during follow-up. CONCLUSIONS: Our novel interpretation of the biexponential model is in line with long-term data of PK for BK and KK. Our findings highlight the importance of the recipient bed endothelial reservoir on the long-term prognosis in PK.

Accelerated corneal graft rejection in baby rats.

BACKGROUND: Penetrating keratoplasty in infants has a very poor outcome compared to adults. It is of intrinsic interest to gain insight into the still unknown immunological mechanisms of graft failure because any form of uncorrected corneal opacity leads to amblyopia. METHODS: Allogeneic keratoplasty was performed between Lewis and Fisher rats. The recipients' ages were 10 and 3 weeks, respectively. All experiments were controlled syngeneically. Survival rates were calculated and cellular infiltrates analysed histologically. RESULTS: Median graft survival times were 15 days in old recipients and 9 days in young recipients (p<0.01). There were fewer infiltrating cells in the younger rats than in the older ones on the day of rejection. Despite the fact that T cells dominated there were significantly more NK cells in young recipients at all time points after transplantation when compared to old recipients. CONCLUSIONS: An animal model has been established that shows similar rejection kinetics as in children, that is corneal graft failure occurs sooner in young rats. Already little infiltration was sufficient to reject a corneal allograft. The dominance of infiltrating NK cells and the vigorous rejection process suggest an involvement of the innate immune system in this process.

The role of ACAID and CD4+CD25+FOXP3+ regulatory T cells on CTL function against MHC alloantigens.

PURPOSE: Anterior chamber associated immune deviation (ACAID) is an antigen-specific form of peripheral immune tolerance that is induced to exogenous antigens placed in the ocular anterior chamber, which leads to a suppression in delayed-type hypersensitivity (DTH). Considerable work has been done on ACAID induction to major histocompatibility (MHC) alloantigens. However, its role on cytotoxic T lymphocyte (CTL) activity is currently unknown. METHODS: C57BL/6 (H-2(b)) mice received an intracameral (IC) inoculation with BALB/c (H-2(d)) splenocytes. Splenic CD4(+) and CD8(+) T cell populations were characterized by flow cytometry and proliferation assays during induction and expression phases of ACAID. Percentages of CD4(+)CD25(+)FoxP3(+) T regulatory cells (Treg) were also followed. Lastly, CTL function was measured at various time points during ACAID expression, and Treg were added to identify potential alterations in CTL function. RESULTS: CD4(+) and CD8(+) T cell percentages and proliferation increased in the spleen during ACAID induction but then sharply decreased in response to an allospecific immunization. Expression of ACAID also exhibited a significant drop in CTL function. However, while Treg expansion was observed, these cells did not directly mediate the CTL inhibition. CONCLUSIONS: ACAID mediates an inhibition of CTL function against MHC alloantigens. Furthermore, we found that ACAID induction leads to the expansion and proliferation of CD4(+) and CD8(+) T cells while ACAID expression is associated with a diminishment in T cell percentages due to proliferation impairment. Lastly, Treg also expand during ACAID induction. However, our data suggest that Treg do not directly inhibit CTL activity.

Topical pimecrolimus does not prolong clear graft survival in a rat keratoplasty model.

BACKGROUND: Long-term application of topical steroids following penetrating keratoplasty is disadvantageous due to side effects (steroid response, cataract, surface disorders). In this study we investigated the efficacy of topical pimecrolimus regarding clear graft survival following allogeneic orthotopic keratoplasty in rats. METHODS: A total of 46 penetrating keratoplasties were performed using Fisher rats (allogeneic groups) and Lewis rats (syngeneic group) as donors and Lewis rats as recipients: group 1 (n = 11), allogeneic control without therapy; group 2 (n = 12), syngeneic control; group 3 (n = 11), mycophenolate mofetil (MMF) 40 mg/kg body weight; group 4 (n = 12), pimecrolimus 1% ointment twice daily. Four animals of each group were sacrificed for immunohistological evaluation on day 14. Therapy was administered for 18 days. The grafts were evaluated once every 3 days regarding opacity, oedema and vascularisation. Graft rejection was defined as total graft opacity. RESULTS: Mean rejection-free graft survival was 11.4 days in group 1 (allogeneic control), 100 days (total follow-up time) in group 2 (syngeneic control), 24.0 days in group 3 (MMF 40 mg/kg) and 11.6 days in group 4 (topical pimecrolimus). The immunohistological evaluation showed no statistically significant difference in cell infiltration of the grafts comparing groups 1 and 4. CONCLUSIONS: Topical immunosuppression with pimecrolimus does not prolong graft survival in the allogeneic keratoplasty rat model.

CTLA-4+CD8+ T cells that encounter B7-2+ iris pigment epithelial cells express their own B7-2 to achieve global suppression of T cell activation.

Pigment epithelial (PE) cells cultured from the eye possess the novel property of suppressing TCR-dependent activation of T cells in vitro. Iris PE (IPE) cells accomplish this suppression by a direct cell contact mechanism in which B7-2 expressed by the PE cells interacts with CTLA-4 on responding T cells. Because CTLA-4 expression is constitutively expressed on a very small proportion of naive splenic T cells and since exposure of splenic T cells to IPE leads to global T cell suppression, we have inquired into the mechanism by which suppression is achieved. Using splenic T cells and IPE from donor mice with disrupted genes for CD80 (B7-1), CD86 (B7-2), CTLA-4, and/or CD28, we report that B7-2(+) IPE in the presence of anti-CD3 supported selectively the activation of CTLA-4(+) CD8(+) T cells that express their own B7-2 and secrete enhanced amounts of active TGFbeta. By contrast, activation of CTLA-4-negative T cells, especially CD4(+) cells, in these cultures was profoundly suppressed. Because global suppression of T cell activation in these cultures was obtained only when both IPE and T cells possessed B7-2 genes and expressed the costimulators as surface molecules, we propose that T cells activated in the presence of parenchymal cells from the eye (an immune privileged site) express B7-2 in a manner that equips them to suppress bystander T cells. Thus, B7-2 expression on T cells participates in their eventual ability to function as regulators in vitro.

A critical requirement of interferon gamma-mediated angiostasis for tumor rejection by CD8+ T cells.

It is thought that tumor rejection by CD8(+) T-cell effectors is primarily mediated by direct killing. We show that rejection of different tumors (fibrosarcoma, ras-transformed fibroblasts, colon carcinoma, and plasmacytoma) by CD8(+) T cells is always preceded by inhibition of tumor-induced angiogenesis. Angiostasis and tumor rejection were observed in perforin but not in IFN-gamma-deficient mice. Furthermore, adoptive transfer of tumor-specific CD8(+) T cells from IFN-gamma-competent mice inhibited angiogenesis of lung metastases in comparison to those from IFN-gamma gene-deficient mice. Taken together with our previous findings, we conclude that IFN-gamma-dependent antiangiogenesis is a general mechanism involved in tumor rejection by CD4(+) and CD8(+) T-cell effectors.

CD40 ligation in the presence of self-reactive CD8 T cells leads to severe immunopathology.

Previous work has shown that stimulation of APCs via CD40 strongly influences the outcome of a CD8 T cell response. In this study, we examined the effect of CD40 ligation on peripheral tolerance induction of self-reactive CD8 T cells in an adoptive transfer model. Naive CD8 T cells from TCR-transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus were tolerized when transferred into H8-tg mice expressing the gp33 epitope under the control of a MHC class I promoter. However, if the H8 recipient mice were treated with agonistic anti-CD40 Abs, TCR-tg cells vigorously proliferated, and induced destruction of lymphoid organs and hepatitis. Break of peripheral tolerance induction was B cell independent and did not require CD28/B7 interactions. These findings provide further in vivo evidence for the crucial role of the activation state of the APC in peripheral tolerance induction and suggest the need for caution in systemically activating APC via CD40 ligation in the presence of self-reactive T cells.