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.     

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.     

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.     

Analysis of intraepithelial lymphocytes from major histocompatibility complex (MHC)-deficient mice: No evidence for a role of MHC class II antigens in the positive selection of Vδ4+γδ T cells

Three-color flow cytometric analysis was carried out with intraepithelial lymphocytes from mice deficient in expression of major histocompatibility complex (MHC) antigens. These experiments were done to address the possible role of MHC class II molecules in the positive selection of Vδ4⁺ γδ T cells. By analyzing mice deficient MHC class II antigens alone or in combination with MHC class I antigens, no evidence was found for positive selection of Vδ4⁺ cells among CD8a⁺ or CD4^?CD8^? subpopulations of γδ T cell receptor-positive cells. Because V54⁺, CD8a⁺ cells were reported to be positively selected on I-E^k and hybrid I-E^k/b molecules, class II-deficient animals were crossed with I-E^k transgenic mice and progeny examined for Vδ4 expression. Again, no evidence for positive selection was found. Interestingly, in MHC class I-deficient animals, the total number of γδ T cells was about twofold higher than in control and MHC class II-deficient mice and the proportion of V8δ-expressing cells was correspondingly decreased. Taken together, these results cast doubt on a major role for conventional MHC antigens in shaping the γδ T cell repertoire of intraepithelial lymphocytes.     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

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.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Instillation of allogeneic lung antigen-presenting cells deficient in expression of major histocompatibility complex class I or II antigens have differential effects on local cellular and humoral immunity and on pathology in recipient murine lungs

Recognition of allogeneic major histocompatibility complex (MHC) molecules expressed on donor lung antigen-presenting cells (APCs) by host T lymphocytes is believed to stimulate lung allograft rejection. However, the specific roles of donor MHC molecules in the rejection response is unknown. We report a murine model in which instilling allogeneic lung APCs into recipient lungs induces pathology analogous to acute rejection, and the production of interferon (IFN)-gamma, immunoglobulin (Ig) G2a, and alloantibodies in recipient lungs. Using allogeneic lung APCs (C57BL/6, I-a(b), H-2(b)) deficient in MHC class I, II, or both for instillation into lungs of BALB/c mice (I-a(d), H-2(d)), the purpose of the current study was to determine the specific roles of donor MHC molecules in stimulating local alloimmune responses. The data show that MHC class I or II on donor APCs induced IFN-gamma and IgG2a synthesis locally, though less than that induced by wild-type cells. Both MHC class I and II were required to induce alloantibody production. Instillation of wild-type or class I- or class II-deficient APCs induced comparable pathologic lesions in recipient lungs, and more severe than that induced by MHC-deficient cells. These data show that donor MHC class I and II molecules have differential effects in the stimulation of local alloimmune responses.     

Evidence that soluble class I antigen in donor serum induces the suppression of heart allograft rejection in rats

The effect on heart allograft rejection in the rat of continuous slow infusion of donor MHC type serum is described. DA (RT1a) serum delayed significantly the rejection of PVG.RT1a heart grafts in PVG recipients (p less than 0.01), but did not affect survival of third-party WAG grafts. Grafts at both early and late stages of rejection were prolonged by serum infusion. Removal of soluble class I MHC antigen from DA serum by affinity chromatography on a monoclonal anti-class I antibody column completely abolished the immunosuppressive effect. The results may indicate that soluble class I antigen can act as a specific immunosuppressive agent in allograft rejection.     

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.     

Allogeneic recognition of class I molecules: Anti-H-2Ld repertoire of H-2b mice includes T cells recognizing mutant class II H-2b (Abm12) molecules

Two major histocompatibility complex (MHC) class I-reactive T cell clones derived from H-2^b mice, generated against the allogeneic L^d molecule, were found to recognize the H-2^b class II mutant A^bm12 molecule as well. In addition, these clones also recognize the class II A^s molecule, and display a class II-dependent reactivity to staphylococcal enterotoxin B. Neither the class I nor the class II alloreactivities of the clones were found to be dependent on other MHC molecules. Both clones express CD4+CD8^? phenotypes. The CD4 molecule appears to be involved in their class II reactivity, while little or no role for CD4 could be detected in the class I reactivity. This is the first report of a class I/class II cross-reactivity being mediated by CD4⁺ T cells. The structural basis for this cross-reactivity is discussed.     

Human class I major histocompatibility complex alleles determine central nervous system injury versus repair

Background

We investigated the role of human HLA class I molecules in persistent central nervous system (CNS) injury versus repair following virus infection of the CNS.

Methods

Human class I A11⁺ and B27⁺ transgenic human beta-2 microglobulin positive (Hβ2m⁺) mice of the H-2 ^b background were generated on a combined class I-deficient (mouse beta-2 microglobulin deficient, β2m⁰) and class II-deficient (mouse Aβ⁰) phenotype. Intracranial infection with Theiler’s murine encephalomyelitis virus (TMEV) in susceptible SJL mice results in acute encephalitis with prominent injury in the hippocampus, striatum, and cortex.

Results

Following infection with TMEV, a picornavirus, the Aβ⁰.β2m⁰ mice lacking active immune responses died within 18 to 21 days post-infection. These mice showed severe encephalomyelitis due to rapid replication of the viral genome. In contrast, transgenic Hβ2m mice with insertion of a single human class I MHC gene in the absence of human or mouse class II survived the acute infection. Both A11⁺ and B27⁺ mice significantly controlled virus RNA expression by 45 days and did not develop late-onset spinal cord demyelination. By 45 days post-infection (DPI), B27⁺ transgenic mice showed almost complete repair of the virus-induced brain injury, but A11⁺ mice conversely showed persistent severe hippocampal and cortical injury.

Conclusions

The findings support the hypothesis that the expression of a single human class I MHC molecule, independent of persistent virus infection, influences the extent of sub frequent chronic neuronal injury or repair in the absence of a class II MHC immune response.

     

Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab−/−) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab−/− mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab−/− mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.     

Tracing interactions of thymocytes with individual stromal cell partners

Cellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation, most - if not all - stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substituion of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro-environments is rate limiting for positive selection, and that in positive selection, each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing sytem for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma-derived MHC class II determinants. When MHC class II-deficient precursors are placed in H-2^{b × k} F1 environments (where all stromal cells co-express H-2^b and H-2^k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H-2^b or H-2^k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others.     

Monokine Induced by Interferon-γ (MIG/CXCL9) Is Derived from Both Donor and Recipient Sources during Rejection of Class II Major Histocompatibility Complex Disparate Skin Allografts

Chemokines, including monokine induced by interferon-γ (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig^−/− allografts had a 10-day prolonged survival in B6.H-2^bm12 recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2^bm12 skin allografts had a 5-day prolonged survival in B6.Mig^−/− versus wild-type recipients. Transplantation of B6.Mig^−/− skin grafts onto B6.H-2^bm12.Mig^−/− recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.Acute allograft rejection is mediated by the coordinated infiltration of alloantigen-primed T cells into the graft and the expression of effector functions that destroy the vascular endothelium and the parenchymal tissue.^1,2 Adhesion molecules and chemoattractant cytokines, chemokines, play major roles in directing primed T-cell recruitment and infiltration into allografts.^3,4,5 The role of adhesion molecules in graft rejection is indicated by the ability of specific antibodies or the use of adhesion molecule-deficient graft recipients or donors to delay or inhibit acute allograft rejection in many animal models.^6,7,8 Similarly, many studies have demonstrated the ability to delay or inhibit allograft rejection through administration of antibodies to specific chemokines or chemokine receptors.⁹ In addition, the use of graft recipients with targeted deletions in CXCR3 and CCR5 has supported a role for these receptors in promoting T-cell trafficking to mediate acute rejection.^10,11 Although these studies indicate an important function of specific chemokines in directing T-cell infiltration into allografts, the induction and source of these chemokines during the rejection process remains poorly understood.The CXCR3 ligands, Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11, are potent chemoattractants for antigen-activated T cells.^12,13 These chemokines are induced by interferon (IFN)-γ and are produced during many T-cell-mediated inflammatory responses including allograft rejection. Mig/CXCL9 is produced at low levels in skin and heart allografts early after transplantation in mouse models but this production increases with alloantigen-primed T-cell infiltration and activity in the allograft.^14,15 Consistent with animal models, the expression of Mig in biopsies from clinical renal and heart allografts is indicative of an ongoing acute rejection episode.^16,17 In rodent models, treatment with Mig-specific antibodies delays T-cell infiltration and prolongs the survival of complete major histocompatibility complex (MHC)-mismatched skin allografts 3 to 5 days implicating a role for Mig in optimal T-cell recruitment into grafts.¹⁸ This is supported by the ability of chronic treatment of C57BL/6 recipients with Mig-specific antibodies to promote the survival of ∼75% of single class II MHC-disparate B6.H-2^bm12 full thickness trunk skin allografts until the treatment is stopped.¹⁹Mig is produced by endothelial cells and macrophages during many inflammatory processes.²⁰ The production of Mig by donor- and recipient-derived sources during allograft rejection remains unclear and the relative contribution of each source in allograft rejection is untested. In the current study, we have used mice with a targeted deletion in the Mig gene as allograft donors and recipients to test these aspects of the skin allograft rejection process. The results indicate the production of Mig by both graft- and recipient-derived sources but the production of each source appears in different tissue locations and affects the time of T-cell graft infiltration during the acute rejection process.     

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.     

Dual effects of the alloresponse by Th1 and Th2 cells on acute and chronic rejection of allotransplants

The contribution of direct and indirect alloresponses by CD4⁺ Th1 and Th2 cells in acute and chronic rejection of allogeneic transplants remains unclear. In the present study, we addressed this question using a transplant model in a single MHC class I‐disparate donor–recipient mouse combination. BALB/c‐dm2 (dm2) mutant mice do not express MHC class I L^d molecules and reject acutely L^d+ skin grafts from BALB/c mice. In contrast, BALB/c hearts placed in dm2 mice are permanently accepted in the absence of chronic allograft vasculopathy. In this model, CD4⁺ T cells are activated following recognition of a donor MHC class I determinant, L^d 61–80, presented by MHC Class II A^d molecules on donor and recipient APC. Pre‐transplantation of recipients with L^d 61–80 peptide emulsified in complete Freund's adjuvant induced a Th1 response, which accelerated the rejection of skin allografts, but it had no effect on cardiac transplants. In contrast, induction of a Th2 response to the same peptide abrogated the CD8⁺ cytotoxic T cells response and markedly delayed the rejection of skin allografts while it induced de novo chronic rejection of heart transplants. This shows that Th2 cells activated via indirect allorecognition can exert dual effects on acute and chronic rejection of allogeneic transplants.     

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing E^{k / b} or A^k molecules in addition to the A^b molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4⁺ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, E^{k / b} molecules containd a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than A^b did, which, unlike A^b -CLIP complexes, were not SDS-stable. Unlike A^b molecules, both E^{k / b} and A^k efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by A^k molecules in the mutant animals. To our surprise, expression of E^{k / b} overcame the positive selection defect observed in H-2M-deficient mice expressing A^b alone. In contrast, A^k expression did not augment positive selection of CD4⁺ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.