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Investigative Ophthalmology & Visual Science, Vol. 31, No. 11, November 1990 Copyright © Association for Research in Vision and Ophthalmology The Role of Closs II Antigen-Expressing Cells in Corneol Allogroft Immunity Bryan M. Gebhardr The goal of this study was to investigate the relationship between the presence of Class II antigen-expressing cells (Class II+) cells in the cornea and the generation of allograft immunity. Wistar/Furth (W/F) rat Class II+ cells were injected in various numbers (0.01, 0.1,1.0, 5.0, 10.0, and 20.0 X 106) into the corneal stroma of Fischer 344 (F344) rats. For comparison, the same numbers of W/F Class II+ cells were injected directly into the peritoneal cavity of F344 rats. Also, W/F cells were injected into the corneas of F344 rats and the corneas were "grafted" intraperitoneally in F344 hosts. The results showed that up to 20 X 106 class II+ cells injected in situ into the corneal stroma did not elicit a serum cytotoxic antibody response or a splenic or blood cytotoxic T-cell response against donor Class II antigens. In contrast, systemic immune responses were elicited by both direct intraperitoneal injection of large numbers (10 or 20 X 106) of allogeneic Class II+ cells and by intraperitoneal grafting of syngeneic corneas carrying similar numbers of allogeneic Class II+ cells. F344 recipients of a syngeneic cornea containing 10 or 20 X 106 W/F Class II+ cells or a suspension of W/F cells exhibited an accelerated rejection of W/F skin allografts (13.3 versus 9.0 days, first- versus second-set rejection). These results indicate that the number of Class II+ cells required to elicit a systemic immune response is larger than the number of Class II+ cells present in the normal cornea. Invest Ophthalmol Vis Sci 31:2254-2260, 1990 corneal allografts.2'5'6"101213 Although it has been shown that both Class I and II histocompatibility antigens expressed on corneal cells may stimulate an allograft reaction,67 it has been suggested that cells expressing Class II antigens are more efficient in this capacity.7'9'10 Cells in various tissues undergoing immunologic attack are induced to express Class II antigens that they normally do not express.14"22 Young and McMillan23 showed that corneal stromal keratocytes in culture can be induced to express Class II histocompatibility antigens when exposed to the lymphokine, gamma interferon. These investigators speculated that the induced expression of Class II histocompatibility antigens could be an important early event in the initiation of the corneal allograft immune reaction.23 Our purpose was to determine the relationship between the presence of Class II+ cells in the cornea and the stimulation of a host antigraft reaction. These results demonstrate that the presence of large numbers of allogeneic Class II+ cells in the cornea does not elicit a systemic immune reaction; this suggests that the immunity engendered by Class II+ allogeneic cells in the cornea is a local rather than a systemic phenomenon. The role of donor Class II+ cells in eliciting immune rejection of corneal grafts in the human eye requires further study. The cells of the normal cornea consist almost exclusively of epithelial cells, stromal keratocytes, and endothelial cells, with relatively few immune accessory cells expressing Class II antigens.1"9 The Class II antigen-expressing (Class II+) Langerhans cells present in the normal cornea are distributed in a gradient from the peripheral cornea and conjunctiva, where the largest numbers are found, to the central cornea, where there are few or none.3'4'6"8 Both resident and infiltrating Class II+ cells are present in larger numbers in corneas subjected to various stresses;1"10 their distribution changes markedly in various corneal disease states, including corneal inflammatory reactions,7"9 corneal infections,611 and the corneal allograft immune response.6'710 Several investigators proposed that Class II+ cells of donor origin contribute to the immunogenicity of From the Lions Eye Research Laboratories, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans, Louisiana. Supported in part by Public Health Service grants EY03150 and EY02377 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, May 5, 1989, Sarasota, Florida. Reprint requests: Bryan M. Gebhardt, PhD, LSU Eye Center 2020 Gravier Street, Suite B, New Orleans, LA 70112. 2254 Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 CLASS II ANTIGEN-EXPRESSING CELLS / Gebhordr No. 11 Materials and Methods Animals Female rats of the inbred strains, Wistar/Furth (W/F, Rtl u ) and Fischer 344 (F344, Rtlvl), weighing approximately 120 g, were used (Harlan Sprague Dawley, Indianapolis, IN). All animals were maintained and handled according to the ARVO Resolution on the Use of Animals in Research. Cell Suspensions The W/F and F344 rats were killed by sodium pentobarbital euthanasia. The spleens were removed using sterile technique, prepared as single cell suspensions, and freed of erythrocytes and dead cells on Lympho-paque gradients (Accurate Chemical and Scientific, Westbury, NY). Total cell number and cell viability were determined by hemocytometer count using the vital dye, trypan blue. Cell Fractionation Two micrograms of mouse monoclonal, rat antiClass II antibody were incubated with 1 mg of magnetic immunobeads (Dynabeads M-450; Robbins, Mountainview, CA) for 4 hr at room temperature with gentle rocking. After incubation the labeled beads were suspended in 10 ml of RPMI-1640 (Grand Island Biological, Grand Island, NY), containing 0.3% bovine serum albumin (Sigma, St. Louis, MO) (RPMI/BSA), and concentrated using a Dynal Magnetic Particle Concentrator (Robbins). The supernatant was discarded, and the labeled beads were resuspended in an additional 10 ml of RPMI/ BSA and again collected with the particle concentrator. Washing and concentration of the beads were repeated two additional times. Ten microliters of the suspension containing approximately 4 X 106 antibody-coated beads was adequate for the isolation of 5 X 106 Class II+ cells. In practice, 0.5 ml of antibody-coated beads was incubated with 1 X 108 rat spleen cells (SC) for 30 min at 4°C on a rocking platform. The RMPI/BSA was added to a final volume of 10 ml, the beads with attached cells were collected in the magnetic particle concentrator, and the supernatant removed. The washing and concentration procedure was repeated three additional times. The bead-cell complexes were incubated at 37°C for 8 hr, during which time the cells separated from the beads. At the end of the incubation, the detached beads were collected on the magnetic particle concentrator and the cell suspension collected in the supernatant. Cell number and viability were determined by hemocytometer count. The viability of cells Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 2255 purified by this method was uniformly greater than 97%. The cell suspensions were held at 4°C for use in the experimental protocol. The presence of Class II histocompatibility antigens on the cells was confirmed by immunoperoxidase staining as previously reported.24 At least 95% of the immunobead-purified cells were Class II+. Cell-Mediated Cytotoxicity Peripheral blood lymphocytes (PBLs), peritoneal exudate lymphocytes (PELs), and SCs were used as the sources of cytotoxic effector cells. Animals that had been injected with allogeneic Class II+ cells were killed at the times indicated in the experimental design. To obtain PBLs, 5-7 ml of whole blood was collected in heparinized syringes. For PELs, the peritoneal cavity was lavaged with 10 ml of RPMI/BSA. For SCs, the spleen was removed with sterile dissecting instruments and reduced to a cell suspension. The cell suspensions obtained from blood, peritoneal exudates, and spleens were fractionated over Lympho-paque gradients. Next, each cell suspension was separately enriched for T-cells by nylon-wool fractionation. Approximately 0.5 g of scrubbed nylon-wool (type 200L; Dupont, Wilmington, DE) was packed in a 10-ml syringe and equilibrated with RPMI/BSA at 37°C for 1 hr. The mononuclear cells obtained from the peritoneal washout, peripheral blood gradient, or approximately one third of a total spleen cell suspension (30 X 106 cells) were added to 2 ml of RPMI/BSA and pipetted onto each column. After 1 hr at 37°C the nonadherent T-lymphocytes were eluted from the nylon column with 50 ml of RPMI/BSA. The T-cells were washed, counted, and resuspended in complete tissue culture medium consisting of RMPI-1640 supplemented with 10% fetal bovine serum (Grand Island Biological). Class II+ cells used as 51Cr-labeled target cells were obtained from the spleens of W/F strain rats and putor. Washing and concentration of the beads were suspended in culture medium containing 100 nC\ of Na251CrO4 (ICN Radiochemicals, Irvine, CA) at a concentration of 1 X 107 cells/ml and incubated at 37°C for 60 min. The cells were washed three times in culture medium, incubated for an additional 30 min at 37°C, and washed once more. The effector and target cells were mixed together in round-bottom microcytotoxicity plates (Falcon Labware; Becton Dickinson, Lincoln Park, NJ) in ratios of 1:1, 10:1, 20:1, 40:1, 80:1, and 160:1. Groups of eight replicate wells were established for each mixture of cells. The plates were incubated for 5 hr at 37°C, then centrifuged at 250 X g for 5 min. Fifty-microliter aliquots of the supernatant from each 2256 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1990 well were collected individually in 12 X 75-mm plastic test tubes (Falcon; Becton Dickinson) and the amount of radioactivity determined in a gamma counter. Control wells for determining the amount of spontaneous 5lCr release contained target cells alone. The total releasable 5lCr was determined by adding 50 n\ of a 1% solution of Triton X-100 to wells containing 51Cr-labeled cells. The percentage of specific cytotoxicity was calculated according to the formula: Percent Specific Cytotoxicity Experimental CPM - Background Release CPM •X 100 Release CPM - Background Release CPM Serum Alloantibody Assay Endogenous complement in sera obtained from F344 strain recipients was inactivated by heating at 56°C for 30 min. The anti-Class I antibody was removed from the sera by repeated absorption with W/F strain erythrocytes. Removal of the anti-Class I antibody was assured by testing the absorbed serum in hemagglutination assay as previously reported.25 Spleen cells from a W/F donor were gradient purified on Lympho-paque and the Class II+ cells enriched using the magnetic immunobead technique. The purified cells were suspended at a concentration of 5 X 106 cells/ml in RPMI/BSA and dispensed in 25-/il volumes into the wells of round-bottom microtiter plates containing serial twofold dilutions of serum samples obtained from F344 donors. Next, 25 fA of a 1:10 dilution of Low Tox M rabbit complement (Accurate Chemical and Scientific) was added to all wells, and the plates were incubated for 45 min at 37°C. The percentage of cells killed was determined by hemocytometer chamber counts using trypan blue as the indicator. Control sera obtained from nonimmune F344 donors and control wells containing only the complement were done with each assay. Skin Grafting The technique of skin grafting rats originally reported by Krapienis26 and used in previous studies25 was employed. Rats of the W/F and F344 strains were anesthetized with an intramuscular injection of Ketaset 60 mg/kg (ketamine hydrochloride; Bristol, Syracuse, NY) and Rompun 0.6 mg/kg (xylazine; Butler, Columbus, OH). The W/F skin was obtained from the dorsal basal region of the tail and grafted onto the dorsal cervical region of the F344 recipients. The grafts were inspected daily after removal of the gauze covering. Complete rejection of the skin graft was Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 Vol. 31 recorded as the day on which the graft was dry to the touch, discolored, and thought to be inviable.25'26 Experimental Design To test the capacity of Class II+ cells to elicit humoral and cellular immune responses after introduction into allogeneic recipients, several experimental groups were studied. The W/F Class II+ cells were injected into the corneas or peritoneal cavities of F344 rats. For corneal injection, six groups of 12 F344 rats each were anesthetized and given injections of 0.01, 0.1, 1.0, 5.0, 10.0, or 20.0 X 106 purified W/F strain Class II+ cells into the corneal stroma using a previously described technique.24 Two other groups of F344 rats received equivalent injections of W/F Class II+ cells as a suspension or as corneal implants into which W/F cells had first been injected. This latter group was included to determine the effect of the corneal milieu on the immunogenicity of cells. The technique of implanting corneas into the peritoneal cavity was done as previously described.25 Negative-control F344 rats received 1.0-ml volumes of RPMI-1640. The time course of the development of humoral and cellular immunity was determined by bleeding and killing two F344 recipients from each experimental and control group at 1,2, 3, 4, and 5 weeks after grafting or injection. To determine whether the introduction of large numbers of Class II+ cells is capable of generating second-set graft rejection, four groups of F344 rats were grafted with W/F skin. One group consisted of eight F344 rats that had been injected with 20 X 106 W/F Class II+ cells into the corneal stroma 5 weeks earlier. A second group involved eight F344 rats that had received a syngeneic cornea containing 20 X 106 W/F Class II+ cells implanted intraperitoneally 5 weeks earlier. A third group included eight F344 rats that had received an intraperitoneal injection of 20 X 106 W/F Class II+ cells 5 weeks earlier. The control group consisted of 12 normal F344 rats; this group was used to determine the median survival time (MST) of W/F skin on the allogeneic F344 recipients. Data Analysis Each experiment was repeated at least three times. The data in the 51Cr release assays and the serum alloantibody assays were reduced to means and standard deviations of the means, and results of the different experimental and control groups were compared for statistical significance by analysis of variance. CLASS II ANTIGEN-EXPRESSING CELLS / Gebhordr No. 11 2257 Results Serum Alloantibody Response None of the F344 rats that received W/F Class II+ cells injected into the corneal stroma developed a serum alloantibody response over the 5-week period of the study (Fig. 1A). In contrast, the animals that received an intraperitoneal injection of either 10 X 106 or 20 X 106 W/F strain Class II+ cells showed a serum alloantibody response measurable at both 1 and 2 weeks after injection (Fig. 1 A). Lower cell numbers did not elicit an antibody response. The F344 corneas injected with W/F Class II+ cells and implanted intraperitoneally in F344 rats also elicited an antibody response. The corneas containing the largest number of cells (20 X 106) generated the greatest response, which became evident 14 days after transplantation and reached a peak at approximately 21 days (Fig. IB). Corneas carrying 10 X 106 Class II+ cells produced a small, but measurable response; lower numbers of cells did not elicit a response above control levels (Fig. 1B). Cell-Mediated Immune Response The W/F Class II+ cells injected into the corneal stroma did not elicit a measurable cellular cytotoxic response in the blood, spleen, or peritoneal cavity of the F344 recipients over the 5 weeks of the study. The Fig. 1. Serum cytotoxic antibody assay results. F344 rats were injected intrastromally, intraperitoneally, or were "grafted" intraperitoneally with 0.01, 0.1, 1.0,5.0, 10.0, or 20.0 X 106 W/F Class II + cells and their sera tested for cytotoxic antibody at five weekly intervals. Positive cytotoxic responses were obtained in the recipients of 10 (circles) and 20 (squares) X 106 Class II + cells given either as a suspension (A) or as a syngeneic cornea (B) containing the allogenic cells placed intraperitoneally. The response to lower cell numbers fell within the 0-10% range (shaded area). Similarly, the recipients of intrastromal injections failed to produce serum alloantibody; all the serum titers of these animals fell within the 0-10% range (shaded area). By analysis of variance (ANOVA) the differences between the cytotoxicity elicited by 10 and 20 X 106 cells and the lower cell numbers tested were statistically significant (P < 0.0005). Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 Fig. 2. Cell-mediated cytotoxic responses of F344 rats immunized with W/F Class II + cells. F344 rats were injected intrastromally or intraperitoneally with 0.01, 0.1, 1.0, 5.0, 10.0, or 20.0 X 106 Class II + cells and the presence of cytotoxic T cells in the blood, spleen, and peritoneal cavity was assessed. No cytotoxic cells were found in the blood. Ten (circles) and 20 (squares) X 106 allogeneic Class II + cells injected intraperitoneally elicited positive peritoneal (A) or splenic (B) cytotoxic responses, whereas the response to lower cell numbers fell within the range (shaded area, 0-10%) of cytotoxicity obtained with peritoneal and spleen cells from nonimmune animals. Intrastromal in situ injections of allogenic cells did not elicit a cellular cytotoxic response, and the percent specific 5 l Cr release fell within the range of the control values (shaded areas). The results obtained with 10 and 20 X 106 cells 7 and 14 days after immunization were statistically significantly different from one another (P < 0.0001). Similarly, the results obtained with these cell concentrations at 7 and 14 days were statistically significantly different from the results with the lower cell numbers tested (P < 0.0001). F344 rats that received 10 X 106 or 20 X 106 Class II+ cells injected into the peritoneum had a peritoneal and splenic cellular cytotoxic response by 7 days (Fig. 2). Fewer than 10 X 106 allogeneic Class II+ cells did not elicit a significant response (Fig. 2). The peritoneal and splenic cellular responses differed from each other in two respects: (1) the peritoneal response peaked earlier than the splenic response and (2) the peritoneal cytotoxic response was greater than the splenic response (Figs. 2A-B). The F344 corneas injected with either 10 or 20 X 106 W/F Class II+ cells and implanted intraperitoneally elicited both peritoneal and splenic cellular cytotoxic responses measurable at 21 and 28 days (Figs. 3A-B). The cellular cytotoxic response given by the peritoneal cells was of greater magnitude than that given by spleen cells, but the peak response occurred at approximately 21 days in both cases. Skin Allograft Rejection The MST of W/F skin grafts on naive F344 recipients was 13.3 ± 1.6 days. The W/F skin grafts subjected to a second-set reaction became vascularized by day 5, underwent rapid discoloration, shrinkage, 2258 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1990 Fig. 3. Cell-mediated cytotoxic responses of F344 rats "grafted" intraperitoneally with syngeneic corneas carrying 0.01, 0.1, 1.0, 5.0, 10.0, or 20.0 X 106 W/F Class II+ cells. The presence of cytotoxic T cells in the blood, spleen, and peritoneal cavity was assessed. No cytotoxic cells were found in the blood. Ten (circles) and 20 (squares) X 106 cells elicited positive peritoneal (A) or splenic (B) cytotoxic responses, whereas the response to lower cell numbers fell within the range (shaded area, 0-10%) of cytotoxicity obtained with peritoneal or spleen cells from nonimmune animals. The results obtained with 10 and 20 X 106 cells at 21 and 28 days after immunization were statistically significantly different from one another (P < 0.005). The results obtained with these cell concentrations at 21 and 28 days were statistically significantly different from the lower cell numbers tested. and became dry scabs within 3-5 days thereafter. The W/F grafts which were rejected in 10 days or less were considered to have been subjected to a second-set response. None of the F344 rats that received allogeneic Class II+ cells injected into the corneal stroma had a second-set skin allograft rejection (Table 1). In contrast, F344 recipients of intraperitoneally transplanted syngeneic corneas carrying either 10 X 106 or 20 X 106 allogeneic Class II+ cells rejected W/F strain skin allografts in an accelerated fashion (Table 1). Similarly, F344 animals injected with suspensions of 10 X 106 or 20 X 106 allogeneic Class H+ cells also mounted second-set skin allograft reactions (Table 1). Discussion The cellular constituents of the normal cornea primarily express Class I histocompatibility anti1-7,10.11,27,28 Class II histocompatibility antigens gens. have restricted expression and occur primarily on the plasma membranes of B lymphocytes, macrophages, various dendritic cells, and Langerhans cells of the skin and conjunctiva.29'30 Under ordinary conditions, the corneal epithelial, stromal, and endothelial cells do not express Class II antigens. Also, there are very few migratory Class II+ cells in the normal cornea. The normal human cornea and the corneas of some animal species may contain a few Class II+ Langerhans cells in the periphery.1"13 The number of Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 Vol. 31 such cells, however, is vanishingly small; in an extensive analysis, not reported here, it was found that young adult rats of the strains used in this study have, on the average, 500 or fewer Class II+ cells in their corneas (Gebhardt, unpublished). Rubsamen et al31 and Williams et al7>9 speculate that cells expressing Class II antigens in the cornea could be expected to be potent immunogens and that the larger the number of these cells present in the cornea, the more likely it is that an immune graft reaction will occur. The results of this investigation indicate that while Class II+ cells may elicit local immune reactions in the cornea they do not generate systemic immunity in the recipient. It is known that certain anatomic locations in the body are immunologically privileged and that allografts in these sites are less likely to be subjected to immunologic attack.32"37 The cornea is one such site.38 In contrast, there are sites in the body, such as the peritoneal cavity, in which immune recognition of alloantigens is facilitated.25'33 In agreement with this concept, the current study showed that large numbers of allogeneic Class II+ cells injected directly Table 1. Survival times of W/F skin allografts on F344 hosts* Graft Number of cells (XI 0s) recipient Normal F344 12 NA Mean survival time (days) 13.3 ± 1.6 F344 recipient/intrastromal injection of W/F Class 11+ cells 8 0.01 12.1 ± 1.9 8 0.1 13.4 ±0.8 8 1.0 11.9 ±1.5 8 5.0 13.9 ±2.0 8 10.0 12.6 ±1.7 8 20.0 12.7 ±1.6 F344 recipient/intraperitoneal graft of F344 cornea W/F Class 11+ cells 8 0.01 8 0.1 8 1.0 8 5.0 8 10.0 8 20.0 containing 12.0 ±1.9 13.7 ±2.1 14.5 ±1.4 11.8 ±1.3 10.1 ±1.1* 8.9 ± 0.8J F344 recipient/intraperitoneal injection of W/F Class 11+ cells 8 0.01 14.5 ± 1.5 8 0.1 14.3 ±1.5 8 1.0 13.2 ±2.1 8 5.0 12.1 ±1.8 8.7±0.7J 8 10.0 8 20.0 8.4 ± 0.9* * F344 strain rats were grafted with W/F skin. The grafts were observed daily after the dressing was removed. Graft rejection was recorded as the day the graft was judged in viable. t N, the number of skin allograft recipients in each group. X Second set graft reactions were characterized by an earlier onset of graft discoloration, hemostasis, and scab formation. The differences in mean survival time for these grafts, compared to the other groups, were statistically significant, P < 0.005. No. 11 2259 CLASS II ANTIGEN-EXPRESSING CELLS / Gebhordr into the peritoneum or implanted in a syngeneic carrier cornea produce a measurable systemic cellular and humoral immune response. In situ intrastromal injection of allogeneic Class II+ cells resulted in no measurable systemic response, regardless of the number of cells used. The failure of allogeneic Class II+ cells in situ to stimulate an immune response is not the result of the death of the cells injected into the cornea. Previous studies established that such cells must be viable in order to mediate immunologic reactions.24 Furthermore, it has been shown that inviable cells are very poorly antigenic.39 Mononuclear cells, including lymphocytes, migrate into the cornea under various circumstances, and they emigrate with equal facility.7'91024 Thus, the difference between the immunogenicity of corneas bearing allogeneic Class II+ cells placed intraperitoneally or in situ would not appear to be due to the failure of the cells to emigrate from the cornea in situ and gain exposure to host immunocompetent cells. Resident cornea cells may be induced to express Class II antigens as a consequence of an alteration in the homeostasis of the tissue. In particular, lymphokines such as gamma interferon may induce Class II antigen expression in cornea cells.23 The syngeneic F344 corneas used as carriers for the allogeneic Class II+ cells in these experiments contained very few resident Class II+ cells. Immunohistochemical staining of corneas retrieved from the peritoneal cavities of syngeneic recipients revealed that the expression of Class II antigens is restricted to the injected passenger cells and that the carrier corneal cells remain Class II antigen negative (Gebhardt, unpublished). In the model used in this study spleen cells "present" their endogenous Class II antigens directly. This model mimics the classic T-cell mediated recognition of alloantigens in which foreign major histocompatibility complex molecules act as foreign antigens and elicit an allograft reaction.40 Direct evidence for the theory that Class II+ cells are a potent source of antigen in the generation of corneal allograft immune reactions is lacking at the present time. The results of this study suggest that Class II+ cells are not an important source of the antigens that generate an allograft reaction. It may be that coexpression of Class I and II antigens is necessary to initiate such reactions. It is also possible that the corneal allograft immune response that is initiated and carried out in the local ocular environment does not engender systemic cellular and humoral antibody responses; previous work supports this idea.25 Corneal allograft rejection continues to be a major clinical problem in patients whose graft beds are highly vascularized before transplantation.27 In such Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 grafts, host blood vessels provide a route by which immunologically competent cells can enter the graft, recognize graft alloantigens, and mount an immune attack. These graft reactions may take place regardless of the Class II antigen content of the allograft. The results of the present investigation indicate that factors in addition to Class II antigen-expressing cells may be operative in the initiation of corneal allograft immune rejection. Key words: allograft, Class II antigens, cornea, immunity, inbred rats References 1. Mayer DJ, Daar AS, Casey TA, and Fabre JW: Localization of HLA-A,B,C and HLA-DR antigens in the human cornea: Practical significance for grafting technique and HLA typing. Transplant Proc 15:126, 1983. 2. Treseler PA, Foulks GN, and Sanfilippo F: The expression of HLA antigens by cells in the human cornea. Am J Ophthalmol 98:763, 1984. 3. Whitsett CF and Stulting RD: The distribution of HLA antigens on human corneal tissue. Invest Ophthalmol Vis Sci 25:519, 1984. 4. Vantrappen L, Geboes K, Missotten L, Maudgal PC, and Desmet V: Lymphocytes and Langerhans cells in the normal human cornea. Invest Ophthalmol Vis Sci 26:220, 1985. 5. Pels E and van der Gaag R: HLA-A,B,C, and HLA-DR antigens and dendritic cells in fresh and organ cultured preserved corneas. Cornea 3:231, 1984. 6. Pepose JS, Gardner KM, Nestor MS, Foos RY, and Pettit TH: Detection of HLA class I and II antigens in rejected human corneal allografts. Ophthalmology 92:1480, 1985. 7. Williams KA, Ash JK, and Coster DJ: Histocompatibility antigen and passenger cell content of normal and diseased human cornea. Transplantation 39:265, 1985. 8. Treseler PA and Sanfilippo F: The expression of major histocompatibility complex and leukocyte antigens by cells in the rat cornea. Transplantation 41:248, 1986. 9. Williams KA, Mann TS, Lewis M, and Coster DJ: The role of resident accessory cells in corneal allograft rejection in the rabbit. Transplantation 42:667, 1986. 10. Williams KA, White MA, Ash JK, and Coster DJ: Leukocytes in the graft bed associated with corneal graft failure. Ophthalmology 96:38, 1989. 11. McBride BW, McGill JI, and Smith JL: MHC class I and class II antigen expression in normal human corneas and in corneas from cases of herpetic keratitis. Immunology 65:583, 1988. 12. Chandler JW, Ray-Kiel L, and Gillette TE: Experimental corneal allograft rejection: Description of murine model and a new hypothesis of immunopathogenesis. Curr Eye Res 2:387, 1983. 13. Ray-Kiel L and Chandler JW: Rejection of murine heterotopic corneal transplants. Transplantation 39:473, 1985. 14. Steiniger B, Klempnauer J, and Wonigeit K: Expression of class I and class II major histocompatibility complex antigens during heart allograft rejection in the rat. Transplant Proc 17:1907, 1985. 15. Hickey WF, Osborn JP, and Kirby WM: Expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat. Cell Immunol 91:528, 1985. 16. Traugott U, Scheinberg LC, and Raine CS: On the presence of la-positive endothelial cells and astrocytes in multiple sclerosis 2260 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1990 lesions and its relevance to antigen presentation. J Neuroimmunol 8:1, 1985. Demetris AJ, Lasky S, Van Thiel DH, Starzl TE, and Whiteside T: Induction of DR/Ia antigens in human liver allografts: An immunocytochemical and clinicopathologic analysis of twenty failed grafts. Transplantation 40:504, 1985. Bottazzo GF, Dean BM, McNally JM, MacKay EH, Swift PGF, and Gamble DR: In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 313:353, 1985. Matsunaga M, Eguchi K, Fukuda T. Kurata A, Tezuka H, Shimomura C, Otsubo T, Ishikawa N, Ito K, and Nagataki S: Class II major histocompatibility complex antigen expression and cellular interactions in thyroid glands of Graves' disease. J Clin Endocrinol Metab 62:723, 1986. Chan C-C, Hooks JJ, Nussenblatt RB, and Detrick B: Expression of la antigen on retinal pigment epithelium in experimental autoimmune uveoretinitis. Curr Eye Res 5:325, 1986. 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