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.     

Allorecognition of isolated, denatured chains of class I and class II major histocompatibility complex molecules. Evidence for an important role for indirect allorecognition in transplantation.

Classical RT1-A class I and RT1-B class II major histocompatibility complex (MHC) molecules were purified from DA (RT1avl) spleens, and the individual chains separated and purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. LEW (RT1l) rats were immunized with the pure class I heavy chain, the RT1-B alpha chain and the RT1-B beta chain with the aim of priming to indirect allorecognition (i.e. after processing and presentation of DA MHC chains on LEW antigen-presenting cells) in the absence of any priming to direct allorecognition (i.e. to whole, undenatured, dimeric DA MHC molecules). LEW rats immunized with each of the three DA MHC chains produced alloantibodies to these chains, suggesting that indirect allorecognition did occur, because of the requirement for cognate recognition of B cells by T helper cells. This also demonstrated polymorphism of all three chains between the DA and LEW strains. The antibodies to the isolated, denatured MHC chains did not react to the whole MHC molecules on DA cells, with the possible exception of very weak reactions in some class I heavy chain-immunized rats. DA skin grafts placed on LEW recipients immunized with each of the DA MHC chains were rejected in an accelerated fashion. Following DA skin grafting, there was an accelerated production of antibodies to whole, undenatured class I MHC molecules, even in the LEW rats preimmunized with RT1-B alpha and RT1-B beta chains. These data suggest that indirect allorecognition can play an important role in the effector mechanisms of allograft rejection, and demonstrate T helper priming as one possible mechanism whereby this might be effective.     

Differences in the rate of donor leucocyte migration between natural and drug-assisted tolerance following rat liver transplantation

The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1^a) livers into PVG (RT1^c) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1¹) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.     

Mechanisms in passive enhancement of cardiac and renal allografts by serum from liver-grafted rats.

The ability of serum from PVG (haplotype RT1c) rats carrying long-term surviving orthotopic DA (RT1a) liver grafts (OLT serum) to enhance cardiac allografts has been confirmed and extended to renal allografts. One millilitre of OLT serum given at the time of allografting was sufficient to cause permanent acceptance of PVG.RT1a heart or kidney grafts in PVG recipients ('enhanced recipients'); the PVG.RT1a being congenic with respect to PVG, and sharing the RT1a haplotype with DA. Adoptive transfer of thoracic duct lymphocytes (TDL) from rats carrying enhanced liver grafts into irradiated recipients indicated that specific alloreactive clones had been functionally inactivated or deleted; this was accompanied by active suppression in which specific alloreactivity of normal TDL was partially inhibited. In vitro, splenic T cells from rats with enhanced grafts mediated allospecific suppression in mixed lymphocyte reaction (MLR). The serum of rats carrying enhanced grafts was able to specifically suppress MLR of the same donor/recipient combination. Thus enhancement by orthotopic liver transplantation (OLT) serum leads to cellular and serological changes in the recipient associated with maintenance of unresponsiveness. Such changes are similar to those seen in liver graft recipients themselves.     

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.     

Hepatocyte expression of soluble donor MHC class I antigen via gene transfer inhibits multiple aspects of the antidonor immune response in fully sensitized rat transplant recipients

Sensitized organ transplant recipients face an increased risk of hyperacute rejection (HAR) due to donor major histocompatibility complex (MHC) class I antigen (Ag) preexposure. We recently reported a novel donor-specific strategy to address this problem, whereby soluble donor MHC class I Ag gene therapy prevented HAR of heart allografts in passively sensitized rats. Here, we tested this same approach in presensitized rat recipients with a fully preactivated humoral and cellular immune response. Our gene therapy method involved liposomal transfection of cultured recipient (Lewis-RT1.A(1)) hepatocytes with DNA encoding secretable donor MHC class I Ag, RT1.A(a). Control-transfected or RT1.A(a)-transfected hepatocytes were implanted intrasplenically into Lewis rats presensitized with three skin transplants. Subsequently, antidonor antibody, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) assays were performed. Additionally, the effectiveness of our gene therapy on the prevention of ACI (RT1(a)) heart HAR was evaluated. Results indicated that soluble MHC not only decreased cytotoxic antibody levels, but also suppressed antidonor CTL and HTL responses; furthermore, HAR of heart allografts was prevented in all recipients. Therefore, soluble donor MHC class I gene therapy can inhibit multiple aspects of the primed antidonor immune response in actively presensitized rats. Development of this strategy in presensitized humans could improve organ transplant outcome.     

Analysis of the expression and immunostimulatory capacity of class I major histocompatibility antigens on rat trophoblast cell lines.

In rat strains expressing the a and other major histocompatibility complex (MHC) haplotypes, subpopulations of placental trophoblast cells synthesize the nonclassical class I Pa antigen in preference to the classical RT1.Aa antigen. In this study, a rat trophoblast cell line, R8RP.3, which was derived from midgestation placentas of PVG.R8 (RT1.Aa) rats, was shown to express class I antigens similarly to those of trophoblast cells in situ. Both unstimulated and IFN-gamma-exposed metabolically labeled R8RP.3 cells synthesized more Pa than RT1.Aa antigen. The reverse was true for labeled spleen cells from PVG.R8 rats. The R8RP.3 cells failed to stimulate allogeneic lymphocyte proliferation even when high levels of both classical and nonclassical class I MHC antigens were expressed on their membranes after incubation with IFN-gamma. These experiments thus supply the first evidence that the inductive phase of the immune response is not promoted by trophoblast cell class I MHC antigens, which could explain the failure of mothers to mount immune responses to class I MHC positive trophoblast cells.     

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.     

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.     

An Enzyme Immunoassay for Rat Soluble MHC Class I Molecules (RT1) and the Release of Soluble Class I From Mitogenically Stimulated Mononuclear Cells

Soluble MHC class I antigens can be detected in the serum of humans and various animals and appear in the circulation shortly after liver transplantation. The precise role of these antigens is currently uncertain, but soluble MHC class I may be involved in immunomodulation. We have developed an enzyme linked immunosorbent assay for soluble rat MHC class I (RT1^a) molecules and monitored the kinetics of antigen release following in vitro stimulation of splenic mononuclear cells. A 4 day DA splenocyte Con A supernatant provided a source of soluble class I antigens and was arbitrarily assigned a concentration of 1000 units/ml. Ninety six well plates were coated with a rat RT1^a-specific mAb (MN4-91-6) and soluble class I binding was detected using a biotinylated mAb reactive with a monomorphic region of the rat MHC class I molecule (OX18) followed by a streptavidinalkaline phosphatase conjugate and substrate. The intra- and interassay variations were typically less than 5% and 10% respectively, to give a working range for the assay of between 62.5 and 1000 units/ml. Mitogenic stimulation led to a progressive increase in soluble class I levels in culture supernatants. This assay will be valuable in differentiating recipient and graft responses following experimental organ transplantation.     

Immunosuppressive activity of serum from liver-grafted rats: in vitro specific inhibition of mixed lymphocyte reactivity by antibodies against class II RT1 alloantigens

The immunosuppressive activity of serum from PVG rats following orthotopic transplantation of DA liver has been examined in vitro. Liver grafts in this combination are never rejected, but induce a state of specific transplantation tolerance in the recipient. Serum from such tolerant animals was able to inhibit proliferation of normal PVG lymph node cells in response to DA stimulators in the mixed lymphocyte reaction (MLR); inhibition was specific for donor (DA) antigens. Interleukin-2 (IL-2) production during the MLR was also reduced. The production of anti-DA cytotoxic T cells developing in the MLR was not affected, but the total yield of such cells was reduced. Evidence was obtained that part of the inhibitory serum activity was due to IgG antibody against class II RT1a alloantigens. Thus, a purified IgG fraction retained much of the inhibitory activity which could be removed by an anti-IgG absorbent. Studies of MLR inhibition in different rat strains indicated the anti-class II specificity of the inhibitory IgG. Lymph node cells from DA-liver-grafted PVG rats responded normally against DA stimulators in vitro, and this MLR was also blocked by the inhibitory IgG. Our results suggest that anti-class II allo-antibody may play a role in immunosuppression and long-term graft survival following liver transplantation in this combination.     

MHC antigen expression in sequential biopsies from cardiac transplant patients--correlation with rejection

Class I induction on the myocardium of transplanted heart was investigated with regard to its temporal relationship to rejection episodes, how it is affected by anti-rejection therapy and whether it is dependent upon the presence of a T cell infiltrate in the biopsy. Sequential cardiac biopsies (total 114) from 11 patients from the time of transplant to 1 year after transplant were studied using immunocytochemical techniques. The effect of different immunosuppressive regimens on MHC antigen expression was also studied. All the biopsies diagnosed as showing rejection for the first time showed induction of Class 1 on the myocardium with 79% during subsequent rejection episodes. Class I induction was associated with a leucocyte infiltrate, not always containing T cells, and disappeared in 47% of biopsies taken 3-4 weeks after treatment with steroids and/or ATG. Increased expression of Class II, in particular DQ antigens on interstitial structures, paralleled Class 1 induction. MHC antigen expression returned to normal in 8/9 patients, at 1 year after transplant. Different immunosuppressive regimens affected the number of biopsies showing Class 1 induction on the myocardium. Our results suggest that in clinical heart transplantation class I induction is related to the rejection process.     

Transfer of specific immunosuppression of graft rejection using lymph from tolerant liver-grafted rats.

The effect on graft rejection of lymph from rats rendered tolerant of donor antigens by liver transplantation has been studied. Transfer, by daily intravenous injection, of lymph from PVG rats grafted with DA livers prolonged the survival of DA skin, kidney and heart grafts in normal PVG recipients. The effect was specific for the antigens of the liver donor. Suppression was short-term only; thus, after lymph injections were stopped, rejection occurred with a time course approximating a normal first-set reaction. The result suggests a reversible interference by materials in the tolerant lymph with early stages of sensitization of the recipients.     

Prolongation of small bowel allograft survival with a sequential therapy consisting of a synthetic MHC class II peptide and temporarily low-dose cyclosporine A

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 +/- 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 +/- 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 +/- 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.     

Partial NK cell tolerance induced by radioresistant host cells in rats transplanted with MHC-mismatched bone marrow

We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B → PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.     

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.     

Susceptibility of DA Rats to Arthritis Induced With Adjuvant Oil or Rat Collagen is Determined by Genes Both Within and Outside the Major Histocompatibility Complex

Inbred DA rats are remarkably susceptible to arthritis induced both by non-immunogenic mineral oil only (OIA) and by rat collagen II together with mineral oil (rCIA). This fact enables interesting studies concerning which DA genes are associated with the arthritogenicity of adjuvant oil and collagen, respectively. In this paper the authors have investigated the role of genes within and outside the major histocompatibility complex (MHC), in this respect by comparative susceptibility studies in inbred rat strains (DA, LEW) and MHC-congenic strains (DA.1I, DA.1O, DA.1M, DA.1H, LEW.1AV1, PVG.1AV1). The authors confirm earlier reports on the arthritis-promoting nature of the DA MHC haplotype (RT1^av1), but demonstrate that neither OIA nor rCIA is restricted to this haplotype or its class II alleles (B^a,D^a). A decisive role of non-MHC genes in determining susceptibility is also clear since the PVG.1AV1 strain, with the permissive RT1^av1 haplotype, is resistant to both arthritis models. In conclusion, the authors demonstrate that the susceptibility of DA rats to OIA and rCIA is determined similarly, by permissive genes both within and outside the MHC. Second, and more interesting from a general perspective, the investigation demonstrates a notable influence of genes within and outside the MHC on the pathogenic capacity of a non-immunogenic adjuvant oil. This adjuvant is in turn a critical component in provocations leading not only to rCIA but also to many other experimental autoimmune diseases where the influence of 'adjuvant response genes' is rarely considered.     

Long-Term Donor Chimerism after MHC (RT1) Mismatched Bone Marrow Transplantation in the Rat: the Role of Host Alloreactive NK Cells

We have investigated the role of major histocompatibility complex (MHC) (RT1) disparities in the engraftment of bone marrow (BM) cells after whole body irradiation of rats. Mononuclear BM cells from PVG.RT7.2 (RT1c) rats were injected i.v. into sublethally (10Gy) whole body irradiated PVG (RT1c) rats and RT1 congenic and recombinant PVG rats. Repopulation of the BM, spleen, and blood with donor cells was assessed by FACS analysis of cells labelled with the fluorescein isothiocyanate (FITC)-labelled HIS41 monoclonal antibody (MoAb) against the RT7.2 marker. In RT1 matched (PVG.RT7.2 --> PVG) and RT1-mismatched combinations (PVG.RT7.2 --> PVG.1AV1), where radioresistant host natural killer (NK) cells could not recognize the BM inoculum as foreign, a donor chimerism close to 100% was observed after 6-8 weeks. However, in rat strain combinations where host NK cells could recognize an RT1 mismatch, almost no donor cells survived, and the rats were repopulated with leukocytes of host origin. In intra-MHC recombinant rat strains the element determining rejection or acceptance of the allograft mapped to the RT1-B/D-C/E/M region in PVG.R8 and PVG.R23 rats, in accordance with the patterns of NK alloreactivity in these strain combinations. NK cells may therefore be a primary obstacle to successful allogeneic BM engraftment in this model.     

Genes in two major histocompatibility complex class I regions control selection, phenotype, and function of a rat Ly-49 natural killer cell subset.

We have generated a monoclonal antibody (STOK2) which reacts with an inhibitory MHC receptor on a subset of alloreactive NK cells in the rat. This receptor, termed the STOK2 antigen (Ag), belongs to the Ly-49 family of lectin-like molecules and displays specificity for the classical MHC class I molecule RT1-A1c of PVG rats. Here, we have investigated the influence of the MHC on the selection, phenotype and function of the STOK2+ NK subset in a panel of MHC congenic and intra-MHC recombinant strains. STOK2 receptor density was influenced by the presence of its classical MHC I ligand RT1-A1c, as evidenced by a reduction of STOK2 Ag on the surface of NK cells from RT1-A1c+, as compared with RT1-A1c-, strains. In addition, a role for nonclassical MHC I RT1-C/E/M alleles in the selection of the STOK2 Ag was demonstrated. The relative number of STOK2+ NK cells was fivefold higher in rats expressing the RT1-C/E/M(av1) as compared with those expressing the RT1-C/E/M(u) class Ib haplotype. The STOK2 ligand RT1-A1c inhibited cytotoxicity of STOK2+ NK cells regardless of effector cell MHC haplotype. Allospecificity of STOK2+ NK cells varied markedly with effector cell MHC, however, and suggested that inhibitory MHC I receptors apart from STOK2 were variably co-expressed by these cells. These data provide evidence for the MHC-dependent regulation of the allospecific repertoire within a subset of potentially autoreactive Ly-49+ rat NK cells.