Enhanced survival of skin grafts depleted of Langerhans' cells by treatment with dimethylbenzanthracene

Langerhans' cells (LC) are the only subpopulation of epidermal cells to express the class II major histocompatibility (MHC) glycoproteins (H-2 Ia in the mouse) under normal conditions. Since these antigens are important in the initiation of allograft rejection, the effect of LC depletion on mouse skin graft survival was studied. Treatment with the chemical carcinogen 9, 10-dimethyl-1,2-benzanthracene (DMBA) was observed to deplete LC from the epidermis on ultrastructural examination. DMBA-treated C57BL dorsal trunk or tail skin grafted onto BALB/c recipients had a prolonged survival compared to solvent-treated donor skin. This was observed 1 week after a single DMBA treatment; successive once-weekly treatments or three treatments within 1 week failed to enhance allograft survival further. Tail-skin grafts had consistently longer survival times in comparison to dorsal trunk-skin grafts, for both control and treated skin. Treatment of dorsal trunk or tail skin with DMBA probably enhanced skin graft survival on allogeneic recipients by causing a loss of LC, and therefore of the class II MHC antigens from the graft. To confirm this, congenic mouse strains were used: B10.A(2R) x B10.A differing only at H-2D, and B10.A(2R) x B10.A(4R) differing only at H-2 I-E. Treatment of B10.A(2R) tail skin with DMBA for 1 week did not affect its survival when grafted onto H-2D-disparate B10.A mice, whereas when grafted onto H-2I-E-disparate B10.A(4R) hosts the DMBA-treated grafts were not only accepted permanently, but induced specific unresponsiveness. This confirms that the DMBA-induced enhancement of graft survival was the result of a loss of H-2 Ia antigens, and therefore of LC from the treated graft.     

Effects of gliotoxin on Langerhans' cell function: contact hypersensitivity responses and skin graft survival.

Dendritic Langerhans' cells (LC), which are essential for the induction of cutaneous immunity, express high concentrations of class II major histocompatibility (MHC) glycoproteins (Ia in the mouse) on their plasma membrane. Application of gliotoxin, a member of the epipolythiodoxopiperazine (ETP) group of fungal metabolites, reduces epidermal LC density and alters their morphology from highly dendritic to a more rounded form. Here we demonstrate that gliotoxin also alters LC function, reducing contact hypersensitivity (CHS) responses due to the development of suppressor cells, and enhancing C57BL tail skin graft survival on BALB/c recipients. The reduction in LC density following gliotoxin application was shown to enhance skin graft survival, by reducing the concentration of Ia antigens within the graft, by using congenic mouse strains: B10.A(2R) x B10.A, differing only at H-2D, and B10.A(2R) x B10.A(4R), differing only at H-2 I-E. Treatment of B10.A(2R) tail skin with gliotoxin for 1 week did not affect its survival when grafted onto H-2D-disparate B10.A mice, whereas, when grafted onto H-2 I-E-disparate B10.A(4R) hosts, the grafts were not only accepted permanently, but induced specific unresponsiveness. It is concluded that gliotoxin has a marked effect on LC function, inhibiting CHS responses by the induction of suppressor cells and prolonging graft survival between H-2-disparate and congenic mouse strains.     

Impact of donor MHC class I or class II antigen deficiency on first- and second-set rejection of mouse heart or liver allografts

The influence of donor major histocompatibility complex (MHC) class I- or class II-deficiency on the initiation of first- and second-set rejection of mouse heart and liver allografts was examined. C3H (H-2^k) mice received heterotopic cardiac or orthotopic liver grafts from unmodified B10 (H-2^b), B6 (H-2^b), b2m (H-2^b; class I deficient) or AB⁰ (H-2^b; class II deficient) donors. Organ survival was also investigated in C3H recipients that had been presensitized by a normal B10 skin graft 2–3 weeks before heart or liver transplantation. The absence of cell surface MHC class I or class II resulted in significant prolongation of primary cardiac allograft survival. Three of seven (43%) MHC class I-deficient, and two of five (40%) class II-deficient heart grafts were accepted indefinitely (survival time >100 days). Thus both MHC class I and class II molecules appear to be important for the elicitation of first-set rejection in the heart allograft model. All liver allografts survived >100 days in normal recipients. In C3H recipients that had been presensitized by a B10 skin graft, however, both heart and liver grafts from AB⁰ (class II deficient) donors underwent accelerated rejection (median survival time [MST] 3 and 4 days, respectively). In contrast, liver grafts from class I-deficient mice (b2m) were still accepted indefinitely by B10 skin-presensitized C3H recipients, whereas class I-deficient hearts survived significantly longer than those from class II-deficient or normal donors. These data demonstrate that the expression of donor MHC class I, and not class II is crucial in initiating second-set organ allograft rejection. In vitro monitoring revealed that at the time of organ transplant, both splenocytes and serum of the skin-presensitized animals displayed high cytotoxicity against AB⁰ (class II-deficient) but not against b2m (class I-deficient) targets.     

Temporal pattern of host responses against intrastriatal grafts of syngeneic,allogeneic or xenogeneic embryonic neuronal tissue in rats

The host response to immunologically incompatible intrastriatal neural grafts was studied using immunohistochemical techniques. Dissociated ventral mesencephalic tissue from embryonic donors of either syngeneic, allogeneic or xenogeneic (mouse) origin was stereotaxically implanted into adult rats. The brains were analysed 4 days, 2 weeks or 6 weeks after grafting with antibodies against the following antigenic structures: major histocompatibility complex (MHC) class I antigens; MHC class II antigens; complement receptor (CR) 3 (marker for microglia and macrophages); helper T-lymphocyte antigen-cluster of differentiation (CD) 4; cytotoxic T-lymphocyte antigen-CD8; tyrosine hydroxylase (TH) (marker for transplanted dopaminergic neurons). The number of surviving TH-positive cells was not different at the various time points in either the syngeneic or allogeneic groups, whereas the xenogeneic cells were all rejected by 6 weeks.The host reactions were similar in character in the syngeneic and allogeneic groups. At 4 days after implantation, there were increased levels of expression of MHC class I and II antigens. In and around the grafts, there were cellular infiltrates consisting of activated microglia, macrophages, CD4- and CD8-positive lymphocytes. At 6 weeks, MHC expression was reduced and the cellular infiltrates had subsided with only low numbers of activated microglia cells and CD8-positive lymphocytes remaining. In the xenogeneic group, at 4 days, some grafts contained cavities, possibly reflecting acute rejection. At later stages, the xenografts were heavily infiltrated by macrophages, activated microglial cells and T-lymphocytes, and at 6 weeks all the xenografts were rejected.Taken together, the results suggest that there is an inflammation caused by the implantation process which leads to an accumulation of host defence cells. This, in turn, leads to increased MHC expression in and around the grafts. In syngeneic grafts, these reactions are short lasting and weak; for allografts slightly more pronounced and longer lasting than syngeneic grafts, but not sufficient to cause rejection. For xenografts, the reactions are more intense and lead to transplant rejection. Thus, a strong sustained inflammatory response may be an important determinator for the failure of histoincompatible neural grafts. It can be speculated that a short-term anti-inflammatory treatment of graft recipients may be a sufficient immunosuppressive regimen to allow long-term graft survival.     

Allograft rejection in athymic nude rats by transferred T-cell subsets. I. The response of naive CD4+ and CD8+ thoracic duct lymphocytes to complete allogeneic incompatibilities

PVG.rnu/rnu nude rats were pre-grafted with two allogeneic skin grafts, AO(RTlu) and BN(RTln), 6-14 days in advance of cell transfer. Cellular requirements for rejection were established by transferring graded numbers of B cell-depleted (Ig-) thoracic duct lymphocytes (TDL) or purified W3/25+ (CD4+) or OX8+ (CD8+) TDL subsets. Allografts were rejected by 10(5) to 5 x 10(6) Ig- TDL in a dose-dependent fashion. A similar dose-response relationship was found by transferring 5 x 10(5) to 5 x 10(6) Ig- OX8- TDL (purified by depletion of B cells and OX8+ cells). Larger numbers of Ig- OX8- TDL (10-30 x 10(6)) did not significantly accelerate rejection. W3/25+ TDL alone (10(5)), highly purified by fluorescence-activated cell sorting (FACS), were sufficient to induce allograft rejection in this athymic nude rat model. In contrast, 10 times more FACS purified OX8+ TDL (10(6)) were unable to initiate skin graft rejection despite the complete class I and class II MHC incompatibilities. Furthermore, the addition of 10(6) OX8+ cells did not accelerate or retard the rejection induced by 10(5) W3/25+ cells alone. Pre-grafted nude recipients, irradiated (500 R) 2 hr before W3/25+ TDL injection, in order to eliminate putative nude T cells, rejected allografts on the same day as unirradiated controls. We conclude that when confronted with complete MHC disparities, CD4+ T cells are necessary and sufficient to induce skin allograft rejection whereas CD8+ T cells do not appear to contribute.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

CD4 T cells can reject major histocompatibility complex class I-incompatible skin grafts

We have re-investigated the roles of CD4 and CD8 T cell subsets in skin graft rejection across a single class I MHC disparity. Recipient mice were transplanted with skin from donors transgenic for the class I MHC molecule K^b. As expected, CD8 T cells were sufficient for rapid injection; but surprisingly, CD4 T cells were also competent to do the same. Rejection was dependent on one or the other subset, since elimination of both resulted in indefinite graft survival. The possibility that alloantibody was the downstream effector of CD4 mediated rejection was excluded because CD8-depleted mice rendered B cell deficient still rejected rapidly, but T cell-depleted recipients with pre-existing high titers of alloantibody were unable to do so. In addition, if CD4 cells act to reject by recruiting and/or activating macrophages then this was not dependent on CR3, IFN-γ or TNF-α. Transplantation of skin grafts where the MHC class I disparity was at the level of passenger leukocytes only, demonstrated that transient bystander damage could occur, but that this was insufficient to result in full rejection. We surmise that for CD4 T cells to reject an MHC class I-incompatible graft it is necessary that an appropriate allogeneic peptide is processed and presented in the context of recipient MHC class II. CD4 T cells from B6 mice may fail to reject skin from MHC class I mutants because of the lack of such MHC class II-restricted presentation.     

The mechanism of specific prolongation of class I-mismatched skin grafts induced by retroviral gene therapy

In the present study, we examine the mechanism of specific hyporesponsiveness to major histocompatibility complex (MHC) class I-mismatched skin allografts induced by retrovirus-mediated gene transfer of an allogeneic class I gene into syngeneic bone marrow (BM). Using appropriate congenic recombinant mouse strains, we have mapped MHC determinants that are capable of restoring rapid rejection of K^b-bearing skin grafts. Our results indicate that either a single class I or a single class II alloantigen expressed on skin in association with K^b is able to restore the rapid rejection of K^b -mismatched skin grafts. These data suggest that third-party alloantigens expressed on skin in association with K^b abrogate hyporesponsiveness by providing T cell help. Consistent with this interpretation, spleen cells from mice reconstituted with K^b-transduced BM were unable to elicit a significant anti-K^b cytotoxic T lymphocyte response in vitro unless interleukin-2 was added to the culture medium. Skin graft survival was also analyzed on B10.AKM mice thymectomized 3–4 weeks post-reconstitution with K^b-transduced BM. Thymectomy did not result in significantly prolonged survival of B10.MBR skin grafts compared to euthymic controls, suggesting that even early after reconstitution, intrathymic deletion of K^b-reactive T cells must have been incomplete. Taken together, these data suggest that prolongation of skin allograft survival in this model is controlled at the level of T cell help.     

Immunogenicity of the Mutated H-2Kbm1 Antigen(s). Test of Thyroid Graft Rejection between B6.C-H-2bm1 and C57BL/6 Mice Following Reciprocal Immunization with Normal Versus Malignant Cells

The immunogenic properties of one (or few) selected antigen(s) encoded by the mouse major histocompatibility complex was studied using the C57BL/6(B6) mouse strain and its descendant B6.C-H-2 6m1(bm1) mutant. These strains differ in a point mutation in the H-2K region. We compared the immunogenic and antigenic expression of the mutated antigen on different bm1 tissues by testing the vulnerability of these tissues to graft rejection response in B6 recipients. Previous results demonstrated that B6 and bm1 mice do not reject reciprocal thyroid transplants, despite the acute rejection of reciprocal skin grafts. Thyroid grafts were rejected, however, after presensitizing the recipients with skin graft syngeneic with the thyroid, but not after sensitization with spleen cells. In the present work we induced tumors in bm1 mice by treating them with a chemical carcinogen (3-methylcholanthrene). We found that two out of four tumors demonstrated strict strain specificity and were rejected by all mouse strains (including the B6 recipients) except by their strain of origin. All tumors were found to be sensitive to in vitro lysis by B6 anti-bm1 effector cells. HZ1-A and HZ1-B tumor cells were rejected by B6 recipient mice but could not immunize B6 mice against a subsequent bm1 thyroid graft. When testing the immunogenicity of B6 originated EL4 leukemia cells (which are fatal to B6 mice), we found that the tumor cells were rejected by bm1 recipients, but, unlike B6 skin grafts, were incapable of inducing the rejection of a subsequent B6 thyroid transplant. The results demonstrated that an H-2K molecule may exhibit different immunological properties when expressed on cells of different tissues. The different expression of the mutated antigen on different cell types, its ability to trigger T cells but not B cells responses and the potential involvement of the tissue specific differentiation molecules in the graft rejection response are discussed.     

Rat nigral xenografts survive in the brain of MHC class II-, but not class I-deficient mice

We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients. Dissociated embryonic ventral mesencephalic tissue from Sprague-Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls. All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting. In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts. The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups. Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group. No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group. However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups. These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts.     

MD-1 expression regulates direct and indirect allorecognition

Expression of the molecule MD-1 was previously described to regulate allogeneic and xenogeneic skin graft survival, as documented by the decrease in rejection seen following functional blockade of MD-1 expression in vivo, using antisense oligodeoxynucleotides (ODNs) or anti-MD-1 antibodies. It was unclear from these data whether blockade of expression of MD-1 on donor or recipient cells was crucial. We have investigated the effect on allorecognition of treating skin graft donors, and/or recipients, of either fully major histocompatibility complex (MHC)-mismatched allogeneic skin grafts (C3H with C57BL/6 grafts and vice versa) or grafts differing at only multiple minor alloantigens (C3H with B10.BR grafts; C57BL/6 with C3H.SW), with antisense ODNs to MD-1, or in some cases, following transplantation of class II-deficient cells into class I-deficient mice. Graft-specific cytotoxic T lymphocytes (CTLs) were measured in spleen cells recovered at sacrifice of recipients and following donor-specific restimulation in vitro. In the latter case, we also measured cell proliferation and (by enzyme-linked immunosorbent assay) production of interleukin-2 (IL-2)/interferon-gamma (IFN-gamma) or IL-4/IL-10 in vitro (nominal type-1 vs type-2 cytokines). CTL responses to minor-incompatible grafts were diminished, only if graft recipients were treated with ODNs. However, treatment of graft donor and/or recipient of MHC-incompatible grafts produced inhibition of CTL production. Optimal inhibition came from treating both. Specific suppression of CTL production coincided with inhibition of proliferation and preferential production of IL-4 and IL-10 at the expense of IL-2 and IFN-gamma. Our data are consistent with the hypothesis that MD-1 expression regulates both the direct and indirect pathways of allorecognition and that regulation of MD-1 expression may thus help regulate clinical graft rejection.     

Prolongation of allogeneic skin graft survival by injection of anti-Ly49A monoclonal antibody YE1/48

Ly49A receptors expressed on NK, NKT, and T cells play inhibitory roles in regulating the immune responses in vivo and in vitro. Whether or not injection of anti-Ly49A monoclonal antibody (mAb) YE1/48 can block allograft rejection has not been evaluated. Balb/c mouse recipients received intraperitoneal injections of YE1/48 mAb (0.5 mg) or control mAb or phosphate-buffered saline on days -1 and 10. On day 0, fully MHC-mismatched allogeneic C57BL/6 (B6) skin grafts were implanted. The skin graft survival and anti-donor humoral responses were observed. Whereas allogeneic B6 skin grafts survived 14 days in isotopy control antibody-treated or nontreated Balb/c mice, injection of YE1/48 mAb significantly prolonged B6 skin graft survival to 19 days (P < 0.0005). Injection of YE1/48 mAb into presensitized Balb/c recipients did not significantly delay B6 skin graft rejection. On the other hand, after depleting recipient NK, NKT, and some cytotoxic T cells by injection of anti-asialo GM1, YE1/48 failed to prolong B6 skin graft survival in Balb/c recipients. The present studies indicate that injection of YE1/48 mAb significantly delays allogeneic skin graft rejection in nonsensitized recipients but not in sensitized recipients. The presence of NK, NKT cells, and some cytotoxic T cells may be essential for YE1/48-mediated immunosuppression in vivo.     

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: A role for alloantibody

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8⁺ T cells and the mechanism whereby CD4⁺ T cells are able to independently mediate rejection. In this study of rejection of RT1A^a class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1^u recipients, we show that elimination of CD8⁺ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4⁺ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1^u athymic nude rats reconstituted with CD8⁺ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4⁺ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4⁺ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1^u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4⁺ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4⁺ T cell-dependent alloantibody plays a decisive role in effecting rejection.     

Fetal and neonatal murine skin harbors Langerhans cell precursors

Resident epidermal Langerhans cells (LC) in adult mice express ADPase, major histocompatibility complex (MHC) class II, and CD205 and CD207 molecules, while the first dendritic leukocytes that colonize the fetal and newborn epidermis are only ADPase(+). In this study, we tested whether dendritic epidermal leukocytes (DEL) are end-stage cells or represent LC precursors. In epidermal sheets of fetal and neonatal mice, we found no apoptotic leukocytes, suggesting that these cells do not die in situ. To address whether DEL can give rise to LC, sorted DEL from murine newborn skin were cultured with cytokines used to generate LC from human CD34(+) precursors. After 7-14 days, DEL proliferated and acquired the morphology and phenotype of cells reminiscent of LC. In concordance with this finding, we show that neonatal epidermis harbors 10-20 times the number of cycling MHC class II(+) leukocytes as adult tissue. To test whether LC can differentiate from skin precursors in vivo, we developed a transplantation model. As it was impossible to transplant fetal epidermis, whole fetal skin was grafted onto adult severe combined immunodeficient mice. As opposed to the uniform absence of donor LC at the time of transplantation, examination of the epidermis from the grafts after 2-4 weeks revealed MHC class II(+) donor cells, which had acquired CD205 and CD207, thus qualifying them as LC. Finally, we present evidence that endogenous LC persist in skin grafts for the observation period of 45 days. These studies show that hematopoietic precursors seed the skin during embryonic life and can give rise to LC.     

Cultured keratinocyte grafts are recognized, but not rejected by CD8+ T cells in vivo

It has been shown in the mouse that cultured keratinocyte allografts [fully major histocompatibility complex (MHC) mismatched] survive for at least 100 days without evidence of rejection. In an attempt to analyze the immune mechanisms underlying this phenomenon we have investigated the induction of tolerance to such grafts. Primary cultures of BALB/c keratinocytes were prepared using irradiated 3T3 feeder cells, and the cultured cell sheets were grafted, using a silicone transplantation chamber, onto CBA recipients. After the cultured grafts had been in place for 4-6 weeks full-thickness tail skin from BALB/c donors was grafted onto the dorsal flank opposite the cultured graft. The median graft survival time of these full-thickness allografts was 15.5 days compared to 13 days in the control group. These data show that in the absence of Langerhans cells and MHC class II expression, keratinocytes expressing class I and minor histocompatibility antigens induce a prolonged survival of full-thickness skin allografts. The results from experiments in which T cell subsets were depleted in vivo suggest that CD8+ cells (a) recognize the class I alloantigens of the cultured graft, (b) do not reject the cultured graft and (c) do not progress further in their maturation pathway. We propose that these CD8+ T cells might have been partially primed by receiving only the first signal of activation and that they may be equivalent to "poised" cytotoxic T cells. These CD8+ cells can reject non cultured full-thickness skin grafts, and do so more effectively after removing CD4+ cells.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

Dendritic cells internalise and re-present conformationally intact soluble MHC class I alloantigen for generation of alloantibody

Following organ transplantation soluble MHC class I is released from the graft and may contribute to alloimmunity. We determined in a well-established rat model whether DC are able to internalise soluble MHC class I alloantigen and then re-present intact alloantigen to B cells and T cells for generation of an alloantibody or CD8 T cell response. PVG.RT1(u) BM-derived DC internalised (via an active process) and retained intact a recombinant soluble form of RT1-A(a) (sRT1-A(a)). When PVG.RT1(u) rats were immunised with sRT1-A(a)-pulsed syngeneic DC, they developed a strong anti-sRT1-A(a) alloantibody response and showed accelerated rejection of RT1-A(a)-disparate PVG.R8 heart grafts. Alloantibody production and accelerated heart graft rejection were both dependent on immunisation with viable sRT1-A(a)-pulsed DC. The alloantibody response to sRT1-A(a)-pulsed DC was directed exclusively against conformational epitopes expressed by sRT1-A(a) and not epitopes expressed, for example, by non-conformational sRT1-A(a) heavy chain. Immunisation with sRT1-A(a)-pulsed syngeneic DC did not stimulate a CD8 T cell response. Our findings suggest a novel alloantigen recognition pathway whereby soluble MHC class I alloantigen released from an allograft may be taken up by recipient DC and presented in an intact unprocessed form to B cells for the generation of an alloantibody response.     

Identification of immunodominant donor MHC peptides following rejection and donor strain transfusion-induced tolerance of heart allografts in adult rats

Pre-graft priming of heart allograft recipients with donor strain blood induces tolerance in 100% of adult rats in the congenic LEW.1W to LEW.1A combination. This tolerant state is specific for donor MHC antigens as third-party blood transfusions fail to induce tolerance, and third-party skin grafts are promptly rejected by tolerant graft recipients. In this study we have characterized the immunodominant donor (RT1u) class I and II allogenic peptides which elicit an in vitro proliferative response to splenocytes from recipients (RT1a) undergoing acute rejection or tolerant to a LEW.1A cardiac allograft. Paradoxically, splenocytes from tolerant animals responded more vigorously to a broader set of donor peptides than splenocytes from rejecting animals. In addition, several of these peptides were observed to be stimulatory only for tolerant splenocytes. These findings suggest that regulatory cells may be involved in tolerance induction or maintenance and are selected by specific motifs, which could be utilized for manipulating the immune system of graft recipients.     

Autoimmune-mediated vasculopathy.

The use of the immunosuppressive drug cyclosporine A (CsA) in solid organ transplantation can be associated with the development of vasculopathy as part of the complex immune response involved in chronic rejection, including autoimmune recognition. Although CsA can directly affect endothelial cells, this drug alters the T cell repertoire promoting autoimmune recognition. The present studies evaluated the ability of CsA-induced autoreactive T cells to mediate vascular lesions in syngeneic heart grafts. Graft vasculopathy developed in syngeneic heart grafts following either the primary induction of autoimmunity with CsA or the adoptive transfer of CsA-induced autoreactive T cells. Initially, an inflammatory response occurred in the medial wall of the small arterial vessels, accompanied by a perivascular lymphocytic infiltrate (including a lymphocytic infiltrate into the myocardium), followed by progression of vascular disease with endothelial cell proliferation. The development and progression of vascular disease correlated with the cytokine profile of the infiltrating lymphocytes with type 1 cytokines detected early and type 2 cytokines detected as the disease progressed. Initiation of this response correlated with upregulation of the target antigen recognized by the CsA-induced autoreactive T cells, the MHC class II-invariant chain peptide complex. This antigen complex, when upregulated on endothelial cells by interferon, allowed effective targeting by the autoreactive T lymphocytes. Strategies to inhibit the upregulation of MHC class II antigens by treatment of the recipients with chloroquine truncated the disease process. The results of these studies suggest that CsA-induced autoreactive mechanisms can contribute to the development of graft vasculopathy.     

Clinical evaluation of the endothelial tie-2 crossmatch in ABO compatible and ABO incompatible renal transplants

The necessity of detection of other than the classical major histocompatibility complex (MHC) and MHC class I-related chain A (MICA) directed antibodies prior to organ transplantation has already been repeatedly reported. A commercial flow cytometric endothelial crossmatch (CM) using isolated peripheral blood tie-2 positive cells provides a tool to detect non-MHC antibodies in addition to antibodies directed to MHC class I and II. The vast majority of circulating tie-2 positive cells expresses HLA–DR but not the A, B blood group antigens. Tie-2 cells are circulating surrogate endothelial cells. In this retrospective study we evaluated the endothelial CM in 51 renal transplantations, 30 with ABO compatible grafts and 21 with ABO incompatible grafts. Fifteen of the ABO compatible recipients (group A) developed unexplained rejection episodes (RE) while the remaining 15 had no RE (group B). Five cases of group A and none of group B had a positive tie-2 CM before transplantation (p = 0.042). A positive tie-2 CM was also correlated with graft failure in ABO compatible transplants (p = 0.02). No significant correlation was found between a positive pre-transplant tie-2 CM and RE in the ABO incompatible group. This study strongly suggest that a positive tie-2 CM may predict post-transplantation complications in ABO compatible grafts while negative reactions are not predictive. The test is not significantly correlated with RE in ABO incompatible grafts possibly due to applied desensitization.