Role of CD4+ and CD8+ T Cells in Allorecognition: Lessons from

This information is current as Florence Boisgérault, Ying Liu, Natalie Anosova, Elana of September 27, 2021. Ehrlich, M. Reza Dana and Gilles Benichou J Immunol 2001; 167:1891-1899; ; doi: 10.4049/jimmunol.167.4.1891 http://www.jimmunol.org/content/167/4/1891 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Role of CD4؉ and CD8؉ T Cells in Allorecognition: Lessons from Corneal Transplantation1

Florence Boisge´rault, Ying Liu, Natalie Anosova, Elana Ehrlich, M. Reza Dana, and Gilles Benichou2

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II؉ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite survival. In rejecting mice the T cell response was mediated by Downloaded from (two T cell subsets: 1) CD4؉ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2 IFN-␥-producing CD8؉ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8؉ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4؉ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8؉ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal http://www.jimmunol.org/ provided by professional APCs in the periphery. The Journal of Immunology, 2001, 167: 1891–1899.

ecognition by recipient T lymphocytes of alloantigens geneic MHC molecules displayed on donor cells. This response is expressed by donor tissues is known to initiate immune dictated by the migration of donor passenger leukocytes out of the destruction of allogeneic transplants (1–3). Although the graft to the host’s lymphoid tissues (11–13). Additionally, it has R ϩ ϩ role of CD4 and CD8 T cells in allorecognition is firmly es- been demonstrated that donor Ags are presented in peptide form tablished, the relative contribution of each of these T cell subsets during acute graft rejection (14–16). The T cell recognition of

to the rejection process remains controversial. Initial studies using processed alloantigens in association with self-MHC molecules at by guest on September 27, 2021 reconstituted nude mice have supported the view that collaboration the surface of recipient APCs has been called indirect allorecog- between helper and effector functional T cell subsets is required to nition. While the direct response is polyclonal, the indirect re- ϩ ϩ ensure rejection. Either CD4 or CD8 T cells could mediate sponse is oligoclonal and usually restricted to the recognition of a ϩ these functions (4). In several models CD4 T cells have been few immunodominant donor-derived peptides (17). Based upon shown to be necessary and sufficient for the initiation of allograft this and despite the presence of both types of alloresponses at early rejection (5–7). Conversely, some evidence has been provided sug- ϩ stages after transplantation, a direct alloresponse is often viewed as gesting that CD8 T cells alone can reject MHC class I-disparate the only driving force in acute rejection. In turn, presentation of allografts (8–10). These studies underscore that the contributions ϩ ϩ ϩ alloantigens by recipient MHC class II APCs is thought to play of CD4 and CD8 T cell subsets in graft rejection are primarily a critical role in late acute and chronic graft rejection processes dictated by intrinsic features of the transplanted tissue. (18–20). Ag presentation by both recipient- and donor-derived APCs Under certain circumstances, the contribution of the direct re- contributes to T cell alloresponse and initiation of graft rejection. sponse to acute rejection has been shown to be minimal. Abroga- Indeed, alloantigen recognition occurs via two distinct mecha- tion of the direct CD4ϩ T response has been achieved by depletion nisms, referred to as the direct and indirect pathways. The direct ϩ of donor MHC class II cells from grafted tissues or by treatment alloresponse results from the stimulation of T cells by intact allo- of recipient with immunosuppressive drugs. In these situations, graft rejection is presumably initiated by the indirect response. In support of this view, Auchincloss et al. (21) have reported that the Cellular and Molecular Immunology Laboratory, Schepens Eye Research Institute, ϩ Department of Ophthalmology, Harvard Medical School, Boston, MA 02114 CD4 T cell-mediated indirect response alone could trigger the Received for publication October 24, 2000. Accepted for publication May 25, 2001. rejection of grafts from MHC class II-deficient mice. It is The costs of publication of this article were defrayed in part by the payment of page noteworthy that in this model, anti-donor cytolytic activity medi- charges. This article must therefore be hereby marked advertisement in accordance ated by CD8ϩ T cells was thought to be the effector mechanism with 18 U.S.C. Section 1734 solely to indicate this fact. (22, 23). Other lines of evidence have been provided suggesting 1 This work was supported by grants from the Massachusetts Lions Eye Foundation that in the absence of a direct pathway, an indirect alloresponse is (to G.B.), National Institutes of Health (Grant RO1EY12963), and Research to Pre- vent Blindness Foundation (to M.R.D.). F.B. was supported by a fellowship from the sufficient to ensure rejection of allografts (5, 24). Valujskikh et al. Fondation pour la Recherche Me´dicale. (24) have reported that reconstitution of SCID mice with a CD4ϩ 2 Address correspondence and reprint requests to Dr. Gilles Benichou, Cellular and T cell line specific for a single donor MHC class II peptide is Molecular Immunology Laboratory, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. sufficient to ensure the rejection of skin allografts. Furthermore, E-mail address: [email protected] nude mice bearing an MHC class I-disparate skin graft have been

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1892 DICHOTOMY IN THE T CELL RECOGNITION OF CORNEAL ALLOGRAFTS shown to reject their graft after adoptive transfer of CD4ϩ T cells Preparation of responder cells (5). However, in many instances, efficient destruction of MHC ϩ Spleen cells from naive, cornea-grafted, as well as skin-grafted BALB/c class I-disparate allografts requires the presence of a CD8 T cell- mice were used as a source of responder cells to measure the total allore- mediated direct recognition of the alloantigens (25). sponse and the direct and indirect responses. RBC were lysed for 2 min in Recent studies using genetically engineered mice have shed Tris-NH4Cl solution. Spleen cells were washed twice in AIM-V (Life some light on the roles of direct and indirect allorecognition in Technologies, Grand Island, NY) medium containing 0.5% FCS and re- suspended at 107 cells/ml with 0.5% FCS in AIM-V for further use. graft rejection. However, the exact contribution of each of these pathways to the alloresponse and the rejection process in normal T cells and T cell subsets isolation individuals remains unclear. Corneal transplantation represents a T cells as well as CD4ϩ or CD8ϩ T cell subsets were isolated from mouse useful model to address this issue. Corneal allografts enjoy high spleen cells by negative selection using commercially available T cell pu- rates of spontaneous acceptance compared with other types of rification columns according to the manufacturer’s instructions (Accurate transplantation (40–50%) (26). This feature is particularly helpful Chemical and Scientific, Westbury, NY; R&D Systems, Minneapolis, MN). Purified T cells were washed in HBSS and used at 5 ϫ 105 cells/well to analyze the mechanisms underlying the graft rejection process. in ELISPOT assays. Convergent studies have shown that CD4ϩ T cells play an essen- tial role in the rejection of orthotopic corneal allografts (7, 27, 28). Preparation of APC In this regard, corneal transplantation does not differ from other Mitomycin C (MMC)3-treated splenocytes from donor and recipient naive types of tissue transplantation (6). In turn, a number of character- mice were used as allogeneic stimulator cells or syngeneic APCs, respec- istics clearly distinguish corneal transplantation from other models tively. Single-cell suspensions of splenocytes devoid of RBC were pre-

␮ Downloaded from (29): 1) normal corneas are devoid of MHC class IIϩ APCs (30); pared in AIM-V containing 0.5% FCS and treated with MMC (50 g/ml) for 30 min at 37°C. The cells were washed once in HBSS, incubated for 10 2) the cornea expresses low amounts of MHC class I molecules min at 37°C, and washed once again and finally resuspended in AIM-V/ (31); and, 3) minor histocompatibility Ags are more potent than 0.5% FCS at 1–3 ϫ 107 cells/ml. MHC Ags in mediating corneal allograft rejection (32). Based Preparation of sonicates upon these observations, it has been proposed that the indirect alloresponse represents the driving force in corneal graft rejection, Stimulator spleen cells were suspended at 3 ϫ 107 cells/ml in AIM-V while the direct response does not occur (33). However, the precise containing 0.5% FCS and sonicated with 10 pulses of 1 s each. The re- http://www.jimmunol.org/ sulting suspension was frozen in a dry ice/ethanol bath, thawed at room nature of the T cell alloresponse and the actual contributions of temperature, and centrifuged at 1200 rpm for 10 min to remove intact cells. direct and indirect pathways in corneal transplantation remain to be determined. ELISPOT assays Here, we have used the sensitive ELISPOT technique to char- Ninety-six-well ELISPOT plates (Polyfiltronics, Rockland, MA) were acterize the T cell alloresponse in a murine model of fully alloge- coated with a capture mAb in sterile PBS overnight. Anti-IL-2, -IFN-␥, neic corneal transplantation. We demonstrate that in the absence of -IL-4, and -IL-5 capture mAb were used at 3, 4, 2, and 5 ␮g/ml, respec- MHC class IIϩ cells within the graft, allorecognition is mediated tively (PharMingen, San Diego, CA). On the day of the experiment, the plates were washed twice with sterile PBS, blocked for 1.5 h with PBS by two distinct T cell subsets that differ by their Ag specificities,

containing 1% BSA, then washed three times with sterile PBS. Responder by guest on September 27, 2021 cytokine profiles, and pathways of Ag recognition (direct vs indi- cells or purified T cells were added to wells previously filled with intact rect pathways). The implication of this finding in understanding donor cells (direct response) or syngeneic APCs together with donor soni- the mechanisms underlying T cell allorecognition in vivo and its cates (indirect response) as previously described (36). Cells were incubated for different periods of time depending on the cytokine measurement: 20 h relationship to alloresponse and allograft rejection are discussed. for IL-2, 42 h for IFN-␥ and IL-4, and 48 h for IL-5. The plates were washed three times with PBS, then four times with PBS containing 0.025% Materials and Methods Tween. Biotinylated anti-lymphokine detection mAbs were added at 2 Mice ␮g/ml (PharMingen) and incubated either for5hatroom temperature or overnight at 4°C. After washing three times with PBS containing 0.025% d d Six- to 8-wk-old female BALB/c (H-2 ), B10.D2/nSnJ (H-2 ), C57BL/10 Tween, avidin-horseradish peroxidase (1/2000) was added to each well for b b b b (H-2 ), C57BL/6 (H-2 ), and BALB.B (C.B10-H2 ; H-2 ) mice were pur- 1.5 h. Four washes with PBS were performed before the spots were re- chased from The Jackson Laboratory (Bar Harbor, ME). They were main- vealed by the addition of the developing solution composed of 800 ␮lof tained in our pathogen-free facility at the Schepens Eye Research Institute 3-amino-9-ethylcarbazole (Sigma; 10 mg dissolved in 1 ml dimethylfor- and treated according to the Association for Research in Vision and Oph- ␮ mamide) in 24 ml 0.1 M sodium acetate, pH 5.0, catalyzed by 12 lH2O2. thalmology resolution. The resulting spots were counted and analyzed on a computer-assisted Orthotopic corneal transplantation and scoring ELISPOT image analyzer (C.T.L., Cleveland, OH). Donor central corneas were marked with a 2-mm diameter microcurette, In vitro treatment with anti-CD4 and anti-CD8 mAb excised by vannas scissors, and placed in PBS. Recipients were anesthe- Commercially available rat anti-mouse CD4 (GK1.5) and CD8 (53-6.72) tized by i.p. injection of ketamine and xylazine, and the right eye was mAbs were used in in vitro blockade experiments at 10 ␮g/ml excised from a 1.5-mm diameter piece in the central cornea to prepare the (PharMingen). graft bed. The donor cornea was placed in the recipient bed and secured with interrupted 11–0 nylon sutures (Sharpoint, Vanguard, Houston, TX). After application of antibiotic ointment, the eye lids were closed for 3 days. Results Seven days later, the sutures were removed. The degree of opacity as well Rejection of fully allogeneic corneal grafts is associated with an as the degree of neovascularization were assessed using a slitlamp biomi- alloresponse dominated by type 1 cytokine-producing cells croscopy as previously described (34). Briefly, the opacification of the graft was quantified using a scoring opacity scale from 0 to 5ϩ. The cornea was Although an allospecific delayed-type hypersensitivity (DTH) re- considered in a rejecting phase for a score equal to or greater than 2ϩ. sponse has been reported after allogeneic corneal transplantation (7, 33, 34, 37), the precise nature of the T cells activated in this Skin grafts model remains unclear. Here, we first investigated the pattern of Tail to back allografts were performed according to the technique previ- cytokines produced by T cells after allogeneic corneal transplan- ously described by Billingham and Medawar (35). Tail was harvested from tation in mice. The total T cell response toward donor alloantigens euthanized donor mice and placed in a graft bed prepared on the left side of a recipient mouse previously anesthetized with a cocktail of ketamine and xylazine. The graft was secured using Vaseline gauze and a bandage, 3 Abbreviations used in this paper: MMC, mitomycin C; DTH, delayed-type hyper- which were removed 9 days later. sensitivity; wt, wild type. The Journal of Immunology 1893 was analyzed in rejecting and nonrejecting BALB/c mice grafted cell response to corneal allografts is mediated by two phenotypi- with fully allogeneic C57BL/6 (B6) corneas. In this combination, cally and functionally distinct T cell subsets: CD4ϩ T cells secret- 60% of the corneal allografts were rejected 4 wk after transplan- ing IL-2 and CD8ϩ T cells producing IFN-␥. tation. The rejection was estimated on the basis of the graft opacity ϩ from 2 to 5 wk post-transplantation. The frequency of IL-2-, IFN- Indirect recognition of alloantigens is mediated by CD4 T ␥-, IL-4-, and IL-5-producing splenocytes activated in response to cells allogeneic stimulation was measured in all cornea-transplanted Traditionally, the immune response to allogeneic transplants is ini- mice. To test this, recipient splenocytes (BALB/c) were cultured in tiated by the activation of MHC class II-restricted T cells. In cor- vitro with MMC-treated donor splenocytes (B6), a situation in neal transplantation, since MHC class II molecules have not been which T cells can be activated via both direct and indirect al- detected in corneal tissues, it has been proposed that the CD4ϩ T lorecognition pathways. After 20–48 h (depending on the cytokine cell alloresponse is mediated exclusively toward donor peptides tested) the frequency of type 1 and 2 cytokine-producing cells was presented by MHC class IIϩ recipient APCs, i.e., through the in- determined using the ELISPOT technique as previously described direct pathway. However, no direct evidence has been provided in (36). As expected, a primary MLR, mainly characterized by the support of this hypothesis. To address this question, we measured activation of IL-2 and IL-4 producers, was detected when lympho- the frequency of cytokine-producing T cells activated through the cytes from naive mice stimulated in vitro with allogeneic cells indirect pathway in cornea-grafted mice using the ELISA spot (Fig. 1). The number of IFN-␥- and IL-2-producing cells was sig- technique. This technique has been applied recently to measure the nificantly increased in mice undergoing corneal graft rejection indirect response in skin-grafted mice (36). To detect indirect re- compared with nontransplanted animals. No IL-5 and a few IL-4- sponse in cornea-transplanted mice, T cells from rejector animals Downloaded from producing cells were detected in the same conditions. Strikingly, in were cultured with syngeneic APCs and donor sonicates. As nonrejecting mice, the frequencies of cytokine producers were shown in Fig. 3A, a potent indirect response was triggered in cor- equivalent to those found in naive animals (data not shown). These nea-grafted mice. This response was mediated predominantly by results demonstrate that corneal transplantation elicits a vigorous IL-2-producing cells, while a significant number of IL-4-producers type 1 response in recipients that is associated with the rejection. were also observed. We surmise that these IL-4-producing CD4ϩ

T cells may play an essential role in the induction of DTH response http://www.jimmunol.org/ ␥ IL-2 and IFN- cytokines are produced by two distinct T cell after corneal transplantation, a possibility supported by a recent subsets study from Heeger’s laboratory (38). The frequency of T cells Next, we investigated the contribution of CD4ϩ and CD8ϩ T cells producing IL-2 through the indirect pathway of allorecognition Ϫ to the cytokine production in mice undergoing corneal graft rejec- averaged 8 ϫ 10 5 (Fig. 3B). In contrast, the number of IFN-␥ tion. We performed in vitro blockade experiments with mAbs producers was not significantly different in grafted vs nongrafted ϩ against CD4 and CD8 molecules (Fig. 2). Activation of IL-2-pro- mice (Fig. 3A), a result suggesting that only CD4 T cells were ducing T cells during primary MLR was abrogated by anti-CD4 activated via indirect allorecognition. mAb. In addition, IL-2 release by activated alloreactive T cells To determine the phenotype of IL-2 producers activated through by guest on September 27, 2021 harvested from mice undergoing corneal was the indirect pathway, we performed in vitro blockade experiments significantly inhibited by in vitro treatment with anti-CD4, but not with anti-CD4 and anti-CD8 mAbs. As shown in Fig. 4, the acti- anti-CD8 mAb. Conversely, the frequency of alloreactive IFN-␥- vation of IL-2-producing T cells was blocked by anti-CD4 mAb, producing cells found in these mice was markedly decreased after while treatment with anti-CD8 mAb had marginal effect (Fig. 4A). ϩ addition of anti-CD8 mAb. In turn, anti-CD4 mAb treatment had To further demonstrate that CD4 T cells were activated through ϩ negligible influence on IFN-␥ production. We conclude that the T the indirect pathway, the CD4 T cell population was purified from graft-rejecting mice and restimulated in vitro with recipient APCs and donor sonicates. As shown in Fig. 4B, purified CD4ϩ T cells harvested from cornea-grafted mice elicited a potent indirect response. Together, these data show that CD4ϩ T cells producing IL-2 cytokine mediate indirect recognition of alloantigens in cor- neal transplantation. In cornea-transplanted mice, the indirect alloresponse is not directed toward MHC-derived peptides Both donor MHC and minor histocompatibility protein Ags are potential sources of peptides in indirect alloresponses. Some evi- dence suggests that in skin and models, the majority of T cells activated via indirect allorecognition recognize processed allo-MHC proteins. Here, since minor Ag-mismatched corneas are more readily rejected than their MHC-disparate coun- terparts, it was crucial to determine whether the same rules of FIGURE 1. Frequency of type 1 and type 2 alloreactive cells in mice immunodominance apply to cornea-grafted mice. To test this, we undergoing fully allogeneic corneal graft rejection. BALB/c mice grafted compared indirect T cell responses in MHC-disparate and fully with C57BL/6 corneas were selected 2–5 wk post-transplantation at the allogeneic corneal transplant models. BALB/c mice were grafted time of rejection. Spleen cells harvested from cornea-grafted, naive (neg- either with C57BL/10 corneas (MHC- and minor histocompatibil- ative control), and skin-grafted BALB/c mice (positive control) were mixed with MMC-treated C57BL/6 APCs (ratio 1:1), and tested by ELIS- ity-mismatched) or BALB.B corneas (MHC-mismatched). Mice POT for IL-2, IFN-␥, IL-4, and IL-5 production. The relative frequency of undergoing rejection were selected (60 and 20% rejection rates, cytokine-producing cells in the spleen is expressed as the number of spots respectively). Recipient splenocytes were restimulated with syn- per million splenocytes. Data are the mean Ϯ SEM of three to four inde- geneic APCs and BALB.B sonicates, and the frequency of IL-2- pendent experiments including at least five mice tested individually. producing cells activated against donor MHC H-2b-derived Ags 1894 DICHOTOMY IN THE T CELL RECOGNITION OF CORNEAL ALLOGRAFTS

FIGURE 2. Phenotype of type 1 alloreactive T cells activated in BALB/c mice rejecting C57BL/6 corneas. In vitro blockade experiments of type 1 cytokine pro- duction were performed using anti-CD4 and anti-CD8 mAbs. Spleen cells collected from cornea-grafted and naive BALB/c mice were incubated with MMC-treated B6 APCs (ratio 1:2) alone (Ⅺ) or together with blocking mAbs (10 ␮g/ml): anti-CD4 (o), anti-CD8 ( ), or anti- CD4 plus anti-CD8 mAbs ( ). The frequency of IL-2 (A) and IFN-␥ (B) producers is expressed as the number of spots per million splenocytes. Data are the mean Ϯ SEM obtained from four or five mice tested individually in two independent experiments. was measured. As shown in Fig. 5, we did not detect any indirect corneal grafts are devoid of professional APCs. Based upon this, it response to allogeneic MHC molecules in mice grafted with fully has been postulated that these grafts cannot activate T cells disparate or MHC-mismatched corneas. In contrast, vigorous through the direct pathway. Here, we investigated whether a direct indirect responses to allo-MHC were found in the same response occurs after fully allogeneic corneal transplantation. To donor-recipient mouse combinations after . We con- address this question, the T cell response to intact donor cells was Downloaded from clude that the indirect recognition of MHC Ags by CD4ϩ T cells measured. Recipient T cells were purified using negative selection does not contribute to corneal graft specific alloimmunity. It is columns and were mixed with MMC-treated donor splenocytes. As noteworthy that in mice rejecting MHC-mismatched corneas, the expected, some IL-2 and IFN-␥ producers were activated via pri- frequency of IL-2 producers was comparable to that recorded in mary MLR. Strikingly, we detected very high numbers of IFN-␥- naive mice, while some alloreactive IFN-␥-producing cells were producing activated cells in mice experiencing corneal graft rejec- detected in some recipient mice only. Therefore, we cannot ex- tion (Fig. 6). Indeed, while some IL-2-producing cells were http://www.jimmunol.org/ clude that some T cell-independent mechanisms may be involved detected in the assay shown in Fig. 6, this response was extremely in the rejection process. low compared with that observed in skin-grafted mice. In addition,

ϩ we found that these few alloreactive IL-2-producing cells were CD8 T cells are activated through the direct pathway of resistant to in vitro treatment with anti-CD4 mAb in cornea-grafted allorecognition mice (Fig. 2; while in vitro treatment with anti-CD4 mAb com- Within days after transplantation, graft bone marrow-derived pletely abolished the primary MLR observed in naive mice). These APCs (B cells, dendritic cells, and macrophages) migrate to the observations suggest that a few T cells other than CD4ϩ T cells ␥

host’s lymphoid organs. Presentation of intact allogeneic MHC might produce some IL-2. The frequency of alloreactive IFN- - by guest on September 27, 2021 molecules on these so-called passenger leukocytes is known to producing T cells averaged 10Ϫ3 in cornea-grafted mice vs 10Ϫ4 trigger a powerful direct T cell response that plays a critical role in in naive animals. To determine the phenotype of these the rejection of all forms of tissue allografts. Corneal transplanta- IFN-␥-producing cells, CD4ϩ and CD8ϩ T cells were purified and tion may not follow this rule, since at the time of transplantation tested for their ability to produce IFN-␥ after stimulation with intact

FIGURE 3. The indirect alloresponse in corneal graft rejectors. The number of IL-2, IFN-␥, and IL-4 cytokine producers (A) as well as the frequency of IL-2-producing T cells (B) activated through the indirect pathway was evaluated using ELISPOT assay. A, Spleen cells collected from cornea-grafted and naive BALB/c mice were incubated with MMC-treated BALB/c APCs (ratio 2:1) and C57BL/6 sonicates. The relative frequencies of IL-2, IL-4, and IFN-␥ producers per million splenocytes were determined. The results shown are representative of one of three independent experiments. B, Spleen T cells harvested from cornea-grafted, naive (negative control), and skin-grafted BALB/c mice (positive control) were mixed with MMC-treated syngeneic BALB/c APCs (ratio 1:3) and C57BL/6 donor sonicates. The frequency of IL-2-producing T cells activated through the indirect pathway was then determined. Symbols represent data obtained from individually tested mice (duplicate). The dotted line represents the averaged number of spots found in naive mice stimulated indirectly. The black symbol represents the average frequency of IL-2-producing T cells activated through the indirect pathway in cornea-grafted mice. The Journal of Immunology 1895

FIGURE 4. The indirect recognition of alloantigens is mediated by CD4ϩ T cells in cornea-transplanted mice. A, Phenotype of T cells activated indirectly. In vitro blockade experiments of IL-2 production were performed with anti-CD4 and anti-CD8 mAbs. Spleen cells collected from cornea-grafted, naive, and skin-grafted BALB/c mice were incubated either exclusively with MMC-treated BALB/c APCs (ratio 2:1) plus C57BL/6 sonicate (Ⅺ) or together with anti-CD4 (u) or anti-CD8 (f) blocking mAbs (10 ␮g/ml). B, Frequency of CD4ϩ T cells activated indirectly. CD4ϩ T cells were first purified from splenocytes collected in cornea-grafted, naive (negative control), and skin-grafted BALB/c mice (positive control) and then mixed with MMC-treated BALB/c APCs (ratio 1:5) and C57BL/6 sonicate. The frequency of IL-2-producing CD4ϩ T cells activated through the indirect pathway was determined. Downloaded from Data are the mean Ϯ SEM from four mice. donor cells. As shown in Fig. 7, CD8ϩ but not CD4ϩ T cells secreted tivation of CD8ϩ T cells was required for corneal graft rejection. IFN-␥ through the direct pathway of allorecognition. To confirm that For this purpose, BALB/c mice were grafted either with wild-type ϩ ϩ CD4 T cells were not activated directly, purified CD4 T cells were (wt) B6 corneas or B6 corneas lacking MHC class I expression http://www.jimmunol.org/ ␤ cultured with intact donor cells, and the frequency of IL-2-secreting (due to a disruption of 2-microglobulin gene). In the last combi- cells was measured. We found that the frequency of IL-2-secreting nation, no CD8ϩ T cell-mediated direct recognition could occur. cells was identical in cornea-grafted mice and naive animals (Fig. 8). The survival of corneal grafts was monitored after assessing the ϩ This confirmed that CD4 T cells are not activated through the direct degree of opacity of the graft as previously described (34). As pathway of allorecognition. Taken together these results show that shown in Fig. 9, BALB/c mice rejected B6 MHC I-deficient and wt corneal transplantation elicits a vigorous direct response mediated ex- B6 corneas in similar fashions. This demonstrates that the CD8ϩ T ϩ clusively by CD8 T cells secreting IFN-␥ cytokine. cell-mediated direct response found in cornea-transplanted mice is

not required for graft rejection. by guest on September 27, 2021 Direct recognition of allogeneic MHC class I molecules is not required for orthotopic corneal graft rejection Cytolysis of donor cells, resulting from the direct recognition of Peripheral sensitization of T cells toward allogeneic MHC allogeneic MHC class I molecules by CD8ϩ T cells, is thought to molecules triggers corneal graft rejection be the effector mechanism of rejection in different models of trans- Apparently, CD8ϩ T cells activated directly following corneal plantation (4, 9, 10). Here, we determined whether the direct ac- transplantation do not influence the fate of the allograft. Two main factors may be responsible for the apparent inability of activated CD8ϩ T cells to mediate corneal graft rejection: 1) an improper stimulation of CD8ϩ T cells by the corneal allograft, and 2) a lack of recognition of MHC class I-positive target cells in the donor cornea. To discriminate between these possibilities, we determined whether corneal graft rejection could be induced following a po- tent peripheral activation of T cells toward allogeneic MHC mol- ecules. For this purpose, mice that had permanently accepted al- logeneic corneas were grafted with allogeneic skin derived from the same donor. BALB/c mice were engrafted with MHC-mis- matched BALB.B corneas. We selected recipient mice exhibiting no signs of rejection at 11 wk post-transplantation (80% of the mice in this combination never reject their grafts). These mice FIGURE 5. The indirect recognition is not directed toward MHC Ags in were then transplanted with BALB.B skin. Strikingly, 15 days af- cornea-grafted mice. Fully allogeneic (C57BL/10, H-2b) and MHC-mis- ter skin transplantation all mice underwent corneal graft rejection matched (BALB. B, H-2b) corneas were transplanted onto BALB/c recip- (Fig. 10B). No rejection of corneal graft was observed in mice that ient mice. At the time of rejection, splenocytes from cornea-grafted mice had not received a skin graft (Fig. 10A). We conclude that allo- were tested for IL-2 production in an ELISPOT assay after being stimu- geneic MHC molecules expressed on the skin graft had activated Ⅺ lated with syngeneic MMC-treated BALB/c APCs (ratio 2:1; ), or MMC- alloreactive T cells that caused rejection of corneal allografts. treated BALB/c APCs plus MHC-mismatched BALB.B sonicate (u). Therefore, in acceptor mice, the failure to reject was not due to the Splenocytes obtained from naive and skin-grafted BALB/c mice were used as negative and positive controls, respectively. The frequency of spleno- lack of target recognition. Alternatively, our results support the cytes activated through the indirect pathway toward H-2b-derived deter- view that in the majority of mice with MHC-mismatched corneal minants in cornea- and skin-grafted mice is represented as number of spots allografts, improper/suboptimal activation of alloreactive T cells per million splenocytes. accounted for the lack of rejection. 1896 DICHOTOMY IN THE T CELL RECOGNITION OF CORNEAL ALLOGRAFTS

FIGURE 6. The direct response in corneal graft rejectors. The frequency of IL-2-producing (A) and IFN-␥-producing (B) T cells responding to intact do- nor cells was evaluated using ELISPOT assay. Spleen T cells harvested from cornea-grafted, naive (negative control), and skin-grafted BALB/c mice (positive control) were mixed with MMC-treated C57BL/6 APCs (ratio 1:3). Open and dark symbols represent data obtained from individually tested na- ive and grafted mice, respectively. The dotted line represents the averaged number of spots found in naive mice stimulated directly.

Discussion planted mice (36). These observations suggest that donor APCs are Downloaded from ϩ In this article, we reported that mice rejecting corneal allografts not critical for the differentiation of naive CD8 T cells into IFN- ϩ mount a potent type 1 T cell response associated with the activa- ␥-producing alloreactive CD8 T cells. Previous studies have ϩ tion of IL-2-producing CD4ϩ and IFN-␥-producing CD8ϩ allo- pointed out that CD8 T cell activation requires the presence of reactive T cells. Thus, IL-2 and IFN-␥ were produced by two dis- very few MHC class I molecules on APCs (42). It is also believed tinct T cell subsets in these mice. Indeed, recent studies by others that proper costimulation delivered by professional APCs is nec- ϩ also measuring cytokine production at a single-cell level have essary for lymphokine production and CTL activity by CD8 T http://www.jimmunol.org/ demonstrated that the vast majority of individual activated T cells cells (41, 43). Several groups, however, have recently reported that ϩ secrete only one lymphokine (39, 40). In addition, it has been under defined circumstances naive CD8 T cells can be activated reported that alloreactive CD4ϩ Th1 cells from skin-grafted mice in the absence of professional APCs. For instance, bypass of co- produce IL-2, but no IFN-␥ (38). Here, we provide the first dem- stimulation can be achieved when the density of TCR ligands is onstration that in corneal transplantation, alloreactive CD8ϩ T high enough on presenting cells (44, 45). Lanzavecchia et al. (46) cells produce IFN-␥ exclusively (Fig. 2). It is noteworthy that have demonstrated that the number of triggered TCRs required to while alloreactive CD8ϩ T cells are known to be the main pro- reach the T cell activation threshold is much higher in the absence viders of IFN-␥, they also normally secrete some IL-2. In turn, the of costimulation than under normal conditions. In turn, we surmise by guest on September 27, 2021 absence of IL-2 production by CD8ϩ T cells is thought to be as- that under physiological circumstances, costimulation is likely to sociated with split anergy (41). be critical to achieve full T cell activation. In support of this, there Our study demonstrates that allogeneic MHC class I molecules is a markedly reduced Ag-primed population in people suffering expressed on corneal allografts, devoid of MHC class IIϩ APCs, from mutations in the gene encoding for CD40 ligand (47). In elicit a vigorous CD8ϩ T cell-mediated direct response. Auchin- corneal transplantation, some evidence has been provided suggest- closs et al. (22, 23) have previously reported that the expression of ing that the cytotoxic activity of CD8ϩ T cells is impaired (48). allogeneic MHC class II molecules on skin allografts was not re- We conclude that while CD8ϩ T cells from cornea-grafted mice quired to initiate a CD8 direct response. In this situation, Langer- secrete IFN-␥, they apparently fail to mature into cytotoxic T cells, hans cells were shown to carry MHC class I molecules to lym- a phenomenon also previously observed with HIV-specific CD8ϩ phoid organs and activate alloreactive CD8ϩ T cells. Here, we T cells (49). found that MHC class IIϩ passenger leukocytes (i.e., dendritic The precise role of CD8ϩ T cells in allograft rejection is still cells and macrophages) are not required to prime alloreactive controversial. Activation of a CD8ϩ T cell response is usually CD8ϩ T. Strikingly, the frequency of CD8ϩ T cells stimulated sufficient, but not always necessary, to ensure rejection. This is through the direct pathway was similar in cornea- and skin-trans- underscored by a number of observations made in BALB/c mice

FIGURE 7. CD8ϩ T cells mediate the direct re- sponse to corneal allografts. CD4ϩ T cells (A) and CD8ϩ T cells (B) were purified from splenocytes collected in cornea-grafted, naive (negative control), and skin-grafted BALB/c mice (positive control) and then mixed with MMC-treated C57BL/6 APCs (ratio 1:3). The frequency of IFN-␥-producing CD4ϩ (A) and CD8ϩ T(B) cells activated through the direct pathway was determined using ELISPOT assay. The data are representative of one of two ex- periments obtained with at least six mice. The Journal of Immunology 1897

FIGURE 8. CD4ϩ T cells are not activated directly after corneal trans- plantation. CD4ϩ T cells were purified from splenocytes collected in cor- FIGURE 9. MHC class I-deficient and wt allogeneic corneal allografts nea-grafted, naive (negative control), and skin-grafted BALB/c mice (pos- are rejected in similar fashions. BALB/c mice were grafted either with wt ϭ ␤ ϭ itive control) and then mixed with MMC-treated C57BL/6 APCs (ratio B6 corneas (n 15) or with 2-microglobulin knockout B6 corneas (n 1:3). The frequency of IL-2-producing CD4ϩ T cells after direct stimula- 10). The graft survival was assessed at different time points post-transplan- Downloaded from tion was determined using ELISPOT assay. The dotted line represents the tation as described in Materials and Methods. number of spots obtained with T cells from nontransplanted mice. tion of functional alloreactive CD8ϩ T cells. This finding may have interesting implications in corneal transplantation. First, it engrafted with B6 devoid of MHC class II expression (5). In ϩ the absence of direct CD4 alloresponse, CD8ϩ T cells recognizing shows that in mice with long term graft survival, CD8 T cells donor MHC class I in direct fashion mediated acute rejection. In against allogeneic MHC were still available. Second, our findings http://www.jimmunol.org/ turn, after depletion of these CD8ϩ T cells, the remaining CD4ϩ demonstrate that these T cells have retained the capacity to reject T cell indirect response alone was sufficient to ensure the rejection the graft when provided with proper stimulation. Therefore, T cell process. A direct CD8ϩ T cell response is not required to ensure the rejection of orthotopic corneal allografts. Indeed, the rejection of fully allogeneic corneas grafted in either CD8 knockout recip- ients or CD8ϩ T cell-depleted recipients is not impaired (7, 27). It is important to note that many CD8ϩ T cells are regularly found in the graft tissue at the time of rejection (50). This observation rules out the possibility that alloreactive T cells cannot reach their tar- by guest on September 27, 2021 gets. The inability of CD8ϩ T cells to mediate corneal rejection could be related to the environment of the graft itself and/or to the incomplete maturation of these cells into effector T lymphocytes. While orthotopic corneal allografts induce a DTH response, the emergence of CTL activity has been inconsistently reported in mice (34, 48). Conversely, it is clear that corneal allografts placed heterotopically (under the skin) stimulate vigorous allospecific CTL activity (51). Therefore, corneal allograft has the intrinsic ability to stimulate CTL, but this process might not be fully achieved when the graft is placed in the eye. This presumably explains why MHC-disparate orthotopic corneas are poorly rejected. In support of this, we showed that sensitization of T cells to allogeneic MHC molecules by placing an MHC-disparate skin graft resulted in the rejection of initially accepted MHC-mismatched corneas. While an indirect recognition of allo- geneic MHC molecules was observed in skin-grafted mice (Fig. 5), there was no indirect response to MHC-derived peptides in cornea- grafted mice. It is therefore unlikely that this response was neces- sary and sufficient to induce the rejection of corneal grafts. After skin grafting, a vigorous CTL response to donor MHC proteins is normally induced (52). In our experimental model it is likely that the induction of this response accounted for the destruction of the corneal graft. It has been reported that mice that did not reject corneal allo- FIGURE 10. Morphology of BALB.B (H-2b) corneal allografts placed grafts 8 wk after transplantation were tolerant to the graft due to an onto BALB/c (H-2d) recipient mice. Aspect of the corneal graft under active suppression mechanism (53). In this article, we have de- slitlamp bimicroscopy before peripheral sensitization with BALB. B skin scribed a mechanism by which tolerance can be broken and rejec- allografts (A) and 15 days after (B). It is noteworthy that the formerly tion induced in recipients that totally accepted their grafts. Pre- transparent BALB.B corneal allograft (nonrejected allograft; A) became ϩ sumably, both induction of direct and indirect CD4 T cell highly vascularized and opaque after peripheral sensitization (rejected al- responses to donor MHC accounted for the activation/differentia- lograft; B). 1898 DICHOTOMY IN THE T CELL RECOGNITION OF CORNEAL ALLOGRAFTS

Table I. T cells activated after allogeneic corneal transplantation: a 7. Yamada, J., I. Kurimoto, and J. W. Streilein. 1999. Role of CD4ϩ T cells in clear dichotomy immunobiology of orthotopic corneal transplants in mice. Invest. Ophthalmol. Vis. Sci. 40:2614. 8. Rosenberg, A. S., T. Mizuochi, and A. Singer. 1988. Evidence for involvement ϩ ϩ CD4 T cells CD8 T cells of dual-function T cells in rejection of MHC class I disparate skin grafts: assess- ment of MHC class I alloantigens as in vivo helper determinants. J. Exp. Med. Cytokines produced IL-2 Ͼ IL-4 IFN-␥ 168:33. Allorecognition pathways Indirect Direct 9. Lowry, R. P., R. D. Forbes, J. H. Blackburn, and D. M. Marghesco. 1985. Im- Antigenic specificity Non-MHC MHC molecules mune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic- Frequency among T cells 8.10Ϫ5 10Ϫ3 suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat. Transplantation 40:545. 10. Sprent, J., M. Schaefer, D. Lo, and R. Korngold. 1986. Properties of purified T cell subsets. II. In vivo responses to class I vs. class II H-2 differences. J. Exp. Med. 163:998. 11. Larsen, C. P., J. M. Austyn, and P. J. Morris. 1990. The role of graft-derived tolerance to corneal allografts is not absolute and can be broken dendritic leukocytes in the rejection of vascularized organ allografts: recent find- ings on the migration and function of dendritic leukocytes after transplantation. when T cells are challenged with appropriate Ag and costimulation Ann. Surg. 212:308. in the peripheral host’s lymphoid organs. 12. Larsen, C. P., P. J. Morris, and J. M. Austyn. 1990. Migration of dendritic leu- Convergent studies have shown that the CD4ϩ T cell response kocytes from cardiac allografts into host spleens: a novel pathway for initiation of rejection. J. Exp. Med. 171:307. is required for initiating corneal graft rejection (7, 27, 28). Corneal 13. Lechler, R. I., and J. R. Batchelor. 1982. Restoration of immunogenicity to pas- transplantation represents an interesting model, in that the graft is senger cell-depleted kidney allografts by the addition of donor strain dendritic naturally devoid of MHC class IIϩ passenger leukocytes and can- cells. J. Exp. Med. 155:31.

ϩ 14. Benichou, G., P. A. Takizawa, C. A. Olson, M. McMillan, and E. E. Sercarz. Downloaded from not theoretically elicit CD4 T cell-mediated direct alloresponse 1992. Donor major histocompatibility complex (MHC) peptides are presented by (30). In this paper we have clearly identified a CD4 indirect re- recipient MHC molecules during graft rejection. J. Exp. Med. 175:305. sponse, but could not detect any CD4 direct response after fully 15. Fangmann, J., R. Dalchau, and J. W. Fabre. 1992. Rejection of skin allografts by indirect allorecognition of donor class I major histocompatibility complex pep- mismatched corneal transplantation. Together, these results dem- tides. J. Exp. Med. 175:1521. onstrate that the recognition of alloantigens through the indirect 16. Liu, Z., N. S. Braunstein, and N. Suciu-Foca. 1992. T cell recognition of allopep- pathway is essential to corneal graft rejection. Since a potent DTH tides in context of syngeneic MHC. J. Immunol. 148:35. 17. Benichou, G., E. Fedoseyeva, P. V. Lehmann, C. A. Olson, H. M. Geysen, http://www.jimmunol.org/ toward minor Ags is induced following corneal transplantation, M. McMillan, and E. E. Sercarz. 1994. Limited T cell response to donor MHC ϩ CD4 T cells activated indirectly could also be the provider of peptides during allograft rejection: implications for selective immune therapy in cytokines required for graft destruction. Even though IFN-␥ is transplantation. J. Immunol. 153:938. 18. Vella, J. P., L. Vos, C. B. Carpenter, and M. H. Sayegh. 1997. Role of indirect thought to be essential to mediate the rejection in this situation, it allorecognition in experimental late acute rejection. Transplantation 64:1823. ϩ has been shown recently that CD4 T cells deficient in IFN-␥ 19. Fluck, N., O. Witzke, P. J. Morris, and K. J. Wood. 1999. Indirect allorecognition production are still able to initiate the destruction of an allogeneic is involved in both acute and chronic allograft rejection. Transplant. Proc. 31: 842. skin graft via an indirect allorecognition pathway (54). 20. Suciu-Foca, N., P. E. Harris, and R. Cortesini. 1998. Intramolecular and inter- In conclusion, we have shown that an allograft naturally devoid molecular spreading during the course of organ allograft rejection. Immunol. Rev. of MHC class IIϩ APCs at the time of transplantation is able to 164:241. 21. Auchincloss, H., Jr., R. Lee, S. Shea, J. S. Markowitz, M. J. Grusby, and by guest on September 27, 2021 activate T cells through both indirect and direct allorecognition L. H. Glimcher. 1993. The role of “indirect” recognition in initiating rejection of pathways. Each alloresponse is mediated by different T cell sub- skin grafts from major histocompatibility complex class II-deficient mice. Proc. Natl. Acad. Sci. USA 90:3373. sets, displaying different phenotypes and alloantigen specificities 22. Lee, R. S., M. J. Grusby, L. H. Glimcher, H. J. Winn, and H. Auchincloss, Jr. (Table I). Even if the direct CD8 response can induce graft de- 1994. Indirect recognition by helper cells can induce donor-specific cytotoxic T struction when proper costimulation is provided, the CD4 indirect lymphocytes in vivo. J. Exp. Med. 179:865. 23. Lee, R. S., M. J. Grusby, T. M. Laufer, R. Colvin, L. H. Glimcher, and response is essential in the rejection observed under normal cir- H. Auchincloss, Jr. 1997. CD8ϩ effector cells responding to residual class I cumstances. This supports the idea that in the absence of MHC antigens, with help from CD4ϩ cells stimulated indirectly, cause rejection of class II on the graft and presumably in the case of MHC-matched “major histocompatibility complex-deficient” skin grafts. Transplantation 63:1123. transplants, indirect alloresponse represents the main driving force 24. Valujskikh, A., D. Matesic, A. Gilliam, D. Anthony, T. M. Haqqi, and in the rejection process. This implies that in these types of trans- P. S. Heeger. 1998. T cells reactive to a single immunodominant self-restricted plantation, strategies designed to block the indirect alloresponse, allopeptide induce skin graft rejection in mice. J. Clin. Invest. 101:1398. 25. Wecker, H., M. J. Grusby, and H. Auchincloss, Jr. 1995. Effector cells must such as donor-peptide tolerization, may be effective at preventing/ recognize antigens expressed in the graft to cause efficient skin graft rejection in delaying allograft rejection. SCID mice. Transplantation 59:1223. 26. Niederkorn, J. Y. 1999. The immune privilege of corneal allografts. Transplan- tation 67:1503. Acknowledgments 27. He, Y. G., J. Ross, and J. Y. Niederkorn. 1991. Promotion of murine orthotopic We thank Drs. R. Tam and I. Rawe and H. Rolls for critical reading of the corneal allograft survival by systemic administration of anti-CD4 monoclonal manuscript. We also thank M. Ortega for technical assistance. antibody. Invest. Ophthalmol. Vis. Sci. 32:2723. 28. Haskova, Z., N. Usiu, J. S. 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