Tolerance to cardiac allografts via local and systemic mechanisms after adenovirus-mediated CTLA4Ig expression

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 +/- 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-gamma, inducible NO synthase, and TGF-beta; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.     

IDO and regulatory T cell support are critical for cytotoxic T lymphocyte-associated Ag-4 Ig-mediated long-term solid organ allograft survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.     

CTLA4Ig-IRES-OX40Ig重组腺病毒的构建及其生物学特性

利用基因重组技术,拼接目的基因CTLA4Ig-IRES-OX40Ig,并亚克隆入腺病毒载体pTrack-CMV中,构建pT/CTLA4Ig-IRES-OX40Ig载体,与pAdeasy-1共同转染BJ5183以进行同源重组,所获得的重组子用LipofectAMINE2000转染293A细胞包装病毒,获得了可同时分别表达CTLA4Ig和OX40Ig的重组腺病毒AdCTLA4Ig-IRES-OX40Ig,并进行扩增和滴度测定,用Western印迹鉴定293A细胞培养上清中目的基因的表达,用混合淋巴细胞反应研究其免疫抑制活性.利用上述方法成功构建了穿梭质粒pT/CTLA4Ig-IRES-OX40Ig;并且获得了重组腺病毒AdCTLA4Ig-IRES-OX40Ig,在感染的293A细胞的培养上清中可以同时检测到CTLA4Ig和OX40Ig分子;AdCTLA4Ig-IRES-OX40Ig、AdCTLA4Ig和AdOX40Ig感染的Lewis大鼠脾细胞(刺激细胞)对Balb/c小鼠的脾细胞(反应细胞)的增殖均具有抑制作用,但AdCTLA4Ig-IRES-OX40Ig感染组的抑制效率明显强于AdCTLA4Ig和AdOX40Ig感染组,体外实验证实该两种分子可协同作用,强烈抑制混合淋巴细胞反应.     

Induction of xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7 and OX40-OX40L co-stimulatory pathways

It has been demonstrated that prolonged graft survival can be achieved through inhibiting the activation of T cells, and addition of soluble CTLA4Ig and OX40Ig proteins to mixed lymphocyte reactions can effectively inhibit T cell proliferation. To explore the potential of this type of treatment in xenotransplantation, we infected streptozotocin-induced diabetic BalB/c mice (H-2d) (200 mg/kg, IV) with 5×10⁸ pfu AdCTLA4Ig-IRES-OX40Ig on day 1 before islets transplantation through the tail vein. The results showed that this treatment prolonged the islet xenografts survival significantly. The reaction to exogenous glucose stimulation was normal and the cytokine secretion of the type Th1 cells was inhibited. The AdCTLA4Ig-IRES-OX40Ig-mediated treatment effectively induced the T cells into anergy and the Th1/Th2 cells into deviation. These results strongly supported the therapeutic potential of blockade of costimulation by Ad-CTLA4Ig-IRES-OX40Ig genes transfer in inducing the organ transplantation tolerance.     

Induction of xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7 and OX40-OX40L co-stimulatory pathways

Type 1 diabetes mellitus is an autoimmune disease caused by T cell-mediated destruction of pancreatic beta islets. With its incidence continuous rising in the pediatric age group in recent years, it is becoming a serious threat to the human health. Up to now, there has been no effective therapy for diabetes mellitus, and islet transplantation still remains a promising ap-proach to the treatment of Type 1 diabetes. However, two serious problems hinder the successful islets transplantation, tha…     

Long-Term Acceptance of Major Histocompatibility Complex-Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2^b) neonatal hearts were transplanted to CBA/J (H-2^k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.     

Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappa B oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig

Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-kappaB-dependent mechanisms, which can be blocked by double-stranded "decoy" oligodeoxyribonucleotides (ODNs) containing binding sites for NF-kappaB. Herein, we describe the combined use of NF-kappaB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-kappaB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.     

CTLA4IgG gene delivery prevents autoantibody production and lupus nephritis in MRL/lpr mice

MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop an autoimmune syndrome closely resembling systemic lupus erythematosus (SLE) in humans, characterized by hypergammaglobulinemia, various autoantibody production, and the development of fatal glomerulonephritis. We have previously demonstrated that systemic administration of soluble form of CTLA4IgG prevented autoantibody-related diseases in MRL/lpr mice. To test the potential protective effects of CTLA4IgG gene delivery on the development of lupus nephritis, we injected MRL/lpr mice with a recombinant adenovirus vector containing CTLA4IgG gene, Adex1CACTLA4IgG (AdCTLA4IgG). It was demonstrated that a single administration of intravenous injection of AdCTLA4IgG into MRL/lpr mice resulted in almost complete amelioration of lupus nephritis.     

Cutting edge: atopy promotes Th2 responses to alloantigens and increases the incidence and tempo of corneal allograft rejection

A large body of evidence suggests that corneal allograft rejection is mediated by a type 1 Th cell response and that deviation toward type 2 immunity favors graft survival. However, clinical observations indicate that patients with severe ocular allergies have increased risk of corneal allograft rejection. We used a mouse model of atopic conjunctivitis to evaluate the effects of Th2 immune deviation on corneal allograft survival and possible mechanisms of graft rejection. Our results reveal the following novel findings: 1) atopic conjunctivitis promotes systemic Th2 immune responses to corneal graft donor alloantigens; 2) corneal allografts in atopic host eyes have an increased incidence and swifter tempo of rejection; 3) increased rejection is associated with alterations in systemic T cell-mediated responses to donor alloantigens; and 4) corneal allograft rejection in atopic hosts does not require the direct involvement of infiltrating eosinophils.     

Cutting edge: blockade of the CD28/B7 costimulatory pathway inhibits intestinal allograft rejection mediated by CD4+ but not CD8+ T cells.

The effect of blocking the CD28/B7 costimulatory pathway on intestinal allograft rejection was examined in mice. Murine CTLA4Ig failed to prevent the rejection of allografts transplanted into wild-type or CD4 knockout (KO) mice but did inhibit allograft rejection by CD8 KO recipients. This effect was associated with decreased intragraft mRNA for IFN-gamma and TNF-alpha and increased mRNA for IL-4 and IL-5. This altered pattern of cytokine production was not observed in allografts from murine CTLA4Ig-treated CD4 KO mice. These data demonstrate that blockade of the CD28/B7 pathway has different effects on intestinal allograft rejection mediated by CD4+ and CD8+ T cells and suggest that these T cell subsets have different costimulatory requirements in vivo. The results also suggest that the inhibition of CD4+ T cell-mediated allograft rejection by CTLA4Ig may be related to down-regulation of Th1 cytokines and/or up-regulation of Th2 cytokines.     

CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment

Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.     

Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection

Corneal transplantation is the most common solid organ transplantation. The immunologically privileged nature of the cornea results in high success rates. However, T cell-mediated rejection is the most common cause of corneal graft failure. Using antiangiogenesis treatment to prevent corneal neovascularization, which revokes immune privilege, prevents corneal allograft rejection. Endostatin is an antiangiogenic factor that maintains corneal avascularity. In this study, we directly test the role of antiangiogenic and immunological signals in corneal allograft survival, specifically the potential correlation of endostatin production and T cell recruitment. We report that 75% of the corneal allografts of BALB/c mice rejected after postoperative day (POD) 20, whereas all syngeneic grafts survived through POD60. This correlates with endogenous endostatin, which increased and remained high in syngeneic grafts but decreased after POD10 in allografts. Immunostaining demonstrated that early recruitment of allospecific T cells into allografts around POD10 correlated with decreased endostatin production. In Rag(-/-) mice, both allogeneic and syngeneic corneal grafts survived; endostatin remained high throughout. However, after T cell transfer, the allografts eventually rejected, and endostatin decreased. Furthermore, exogenous endostatin treatment delayed allograft rejection and promoted survival secondary to angiogenesis inhibition. Our results suggest that endostatin plays an important role in corneal allograft survival by inhibiting neovascularization and that early recruitment of allospecific T cells into the grafts promotes destruction of endostatin-producing cells, resulting in corneal neovascularization, massive infiltration of effector T cells, and ultimately graft rejection. Therefore, combined antiangiogenesis and immune suppression will be more effective in maintaining corneal allograft survival.     

Hydrodynamics-based delivery of plasmid DNA encoding CTLA4-Ig prolonged cardiac allograft survival in rats

BACKGROUND: Although gene therapy using plasmid vectors is thought to be safer compared with viral vectors, poor efficacy of gene transfer is the obstacle preventing wide application of plasmid vectors. However, high levels of foreign gene expression have been achieved by rapid tail vein injection of a large volume of a plasmid DNA solution into rats. Using this technique, we examined the effect of rat CTLA4-Ig gene transfer on prevention of cardiac allograft rejection in this animal model. METHODS: Recipient Lewis rats were injected with either plasmid pCAGGS-CTLA4-Ig-Glu-tag as a treatment vector or plasmid pCAGGS-signal peptide (SP)-Ig as a control vector by hydrodynamics-based delivery technique on the day before heart transplantation. Hearts from Brown Norway donors were transplanted into the neck of Lewis recipients and graft survival was assessed. RESULTS: The plasma level of CTLA4-Ig reached a peak of nearly 5 microg/mL 1 day after injection, and then slowly decreased but still remained above 0.9 microg/mL until 100 days after injection. The recipient rats treated with the control vector and untreated rats rejected cardiac allografts within 7 days. On the other hand, the median survival time of the grafts treated with pCAGGS-CTLA4Ig-Glu-tag was more than 100 days. Histological examination revealed that long-term survival allografts contained fewer infiltrating lymphocytes. The serum from recipients with long-term survival allograft suppressed allogenic mixed lymphocyte reaction. CONCLUSIONS: CTLA4-Ig gene transfer by means of tail vein injection of plasmid DNA into a recipient rat resulted in remarkable prolongation of cardiac allograft survival with persistent plasma level of CTLA4-Ig protein.     

CTLA4Ig-induced linked regulation of allogeneic T cell responses

The mechanisms by which CTLA4Ig exerts its powerful immunomodulatory effects are not clear. We show here that CTLA4Ig can induce linked regulation of allogeneic porcine T cell responses in vitro. Naive miniature swine SLA(dd) T cells were rendered hyporesponsive to specific allogeneic Ag after coculturing with MHC-mismatched SLA(cc) stimulators in the presence of CTLA4Ig. These Ag-specific hyporesponsive T cells were subsequently able to actively inhibit the allogeneic responses of naive syngeneic T cells in an MHC-linked fashion, as the responses of naive SLA(dd) responders against specific SLA(cc) and (SLA(ac))F(1) stimulators were inhibited, but allogeneic responses against a 1:1 mixture of SLA(aa) (I(a), II(a)) and SLA(cc) (I(c), II(c)) were maintained. This inhibition could be generated against either class I or class II Ags, was radiosensitive, and required cell-cell contact. Furthermore, the mechanism of inhibition was not dependent upon a deletional, apoptotic pathway, but it was reversed by anti-IL-10 mAb. These data suggest that CTLA4Ig-induced inhibition of naive allogeneic T cell responses can be mediated through the generation of regulatory T cells via an IL-10-dependent mechanism.     

Cutting edge: reversal of murine lupus nephritis with CTLA4Ig and cyclophosphamide

Cyclophosphamide (CTX) prevents progression of nephritis and prolongs survival in (NZB x NZW)F(1) (B/W) mice and is used to treat humans with lupus nephritis. To compare the efficacy of CTLA4Ig with CTX and determine whether there is an incremental benefit to combining CTLA4Ig with CTX, we treated B/W mice with CTX, CTLA4Ig, or both agents. In mice with mild renal disease, treatment delayed the onset of proteinuria and prolonged survival in all groups. In mice with advanced renal disease, treatment with both agents reduced proteinuria in 71% of mice, whereas mice treated with either agent alone had no such improvement. Survival was also markedly improved among mice treated with both agents. Thus, combination treatment with CTX and CTLA4Ig is more effective than either agent alone in reducing renal disease and prolonging survival of B/W mice with advanced nephritis. This striking reversal of proteinuria is unprecedented in animal models of SLE.     

Adipose-Derived Mesenchymal Stem Cell Administration Does Not Improve Corneal Graft Survival Outcome

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.     

Prolongation of sheep corneal allograft survival by transfer of the gene encoding ovine IL-12-p40 but not IL-4 to donor corneal endothelium

Immunological rejection is the major cause of human corneal allograft failure. We hypothesized that local production of IL-4 or the p40 subunit of IL-12 (p40 IL-12) by the grafted cornea might prolong allograft survival. Replication-deficient adenoviral vectors encoding ovine IL-4 or p40 IL-12 and GFP were generated and used to infect ovine corneas ex vivo. mRNA for each cytokine was detected in infected corneas, and the presence of secreted protein in corneal supernatants was confirmed by bioassay (for IL-4) or immunoprecipitation (for p40 IL-12). Sheep received uninfected or gene-modified orthotopic corneal allografts. Postoperatively, untreated corneas (n = 13) and corneas expressing GFP (n = 6) were rejected at a median of 21 and 20 days, respectively. Corneas expressing IL-4 (n = 6) underwent rejection at 18.5 days (p > 0.05 compared with controls) and histology demonstrated the presence of eosinophils. In contrast, corneas expressing p40 IL-12 (n = 9) showed prolonged allograft survival (median day to rejection = 45 days, p = 0.003). Local intraocular production of p40 IL-12 thus prolonged corneal graft survival significantly, but local production of the prototypic immunomodulatory cytokine IL-4 induced eosinophilia, inflammation, and rejection. These findings have important implications for the development of novel strategies to improve human corneal graft survival.     

Gamma delta T cells are needed for ocular immune privilege and corneal graft survival

It has been recognized for over a century that the anterior chamber of the eye is endowed with a remarkable immune privilege. One contributing component is the Ag-specific down-regulation of systemic delayed-type hypersensitivity (DTH) that is induced when Ags are introduced into the anterior chamber. This phenomenon, termed anterior chamber-associated immune deviation (ACAID), culminates in the generation of regulatory cells that inhibit the induction (afferent suppression) and expression (efferent suppression) of DTH. Since gamma delta T cells play a major role in other forms of immune regulation, we suspected they might contribute to the induction and expression of ACAID. Mice treated with anti-gamma delta Ab failed to develop ACAID following anterior chamber injection of either soluble Ag (OVA) or alloantigens (spleen cells). Additional experiments with knockout mice confirmed that mice lacking functional gamma delta T cells also fail to develop ACAID. Using a local adoptive transfer of DTH assay, we found that gamma delta T cells were required for the generation of regulatory T cells, but did not function as the efferent regulatory cells of ACAID. The importance of gamma delta T cells in corneal allograft survival was confirmed by blocking gamma delta T cells with GL3 Ab before corneal transplantation. While in vivo treatment with normal hamster serum had no effect on corneal graft survival, infusion of anti-gamma delta Ab resulted in a profound increase in corneal allograft rejection. Thus, gamma delta T cells are needed for sustaining at least one aspect of ocular immune privilege and for promoting corneal allograft survival.     

Antigen presentation by Langerhans cells in vivo: donor-derived Ia+ Langerhans cells are required for induction of delayed-type hypersensitivity but not for cytotoxic T lymphocyte responses to alloantigens

T cell activation in response to allogeneic stimulation and hapten-specific delayed-contact hypersensitivity responses in vivo can be initiated by Ia-bearing epidermal Langerhans cells (LC). By using a murine heterotopic corneal allograft model, we have investigated the requirement for allogeneic LC as antigen-presenting cells (APC) in the in vivo induction of delayed-type hypersensitivity (DTH) and cytolytic T lymphocyte (CTL) responses to alloantigens in fully allogeneic and H-2 I region-disparate strain combinations. LC-deficient, avascular central corneal allografts from BALB/c donors failed to induce DTH responsiveness when grafted to a subdermal bed on C57BL/6 recipients (p greater than 0.05), yet antigen-specific primary CTL reactivity developed within 7 days after grafting. LC-containing corneal-limbus allografts or central corneal allografts containing a latex bead-induced infiltrate of LC resulted in intense DTH as well as CTL responsiveness when grafted in this same strain combination. Similarly, LC-containing but not LC-deficient corneal allografts from A.TL donors induced DTH responsiveness in I region-disparate A.TH hosts despite the fact that these grafts survived for prolonged duration (less than 28 days). By contrast, CTL induction in I region-disparate hosts was independent of the presence of allogeneic LC. Corneal epithelial cells of grafts removed from I region-disparate hosts 7 days posttransplantation were shown by immunohistology to express the Iak antigens of donor origin. The possibility that bone marrow-derived allogeneic LC were a sufficient requirement for DTH induction was confirmed in experiments performed with CB6F1----B6 bone marrow chimeras used as corneal allograft donors. Corneal-limbus grafts obtained from mice 90 days after chimerization were shown by immunohistology to contain Iad-bearing CB6F1 LC as a sole source of class II alloantigens. When grafted to C57BL/6 recipients, LC-containing chimeric corneas induced DTH responsiveness that was similar in magnitude to that observed in C57BL/6 mice grafted with chimeric skin, yet no DTH response to LC-deficient chimeric central corneal grafts was observed. Moreover, in all cases, the chimeric corneal and skin allografts survived for prolonged duration (greater than 28 days). These results demonstrate that donor-derived LC act as APC in the induction of DTH responsiveness to allogeneic tissue; however, there was no apparent requirement for allogeneic LC in the induction of CTL responses to class I or class II MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)