Recombinant Rat Strains Research Articles

Disparate MHC class II haplotypes in myelin oligodendrocyte glycoprotein- and myelin basic protein-induced experimental autoimmune encephalomyelitis

The major histocompatibility complex (MHC) regulates multiple sclerosis (MS) and its model experimental autoimmune encephalomyelitis (EAE). We created four new intra-MHC recombinant rat strains, between the MHC haplotypes RT1 n (BN) and RT1 l (LEW) on the LEW background, to define disease regulation and localization within the MHC. Immunization with recombinant myelin oligodendrocyte glycoprotein (a.a.1-125; MOG)/IFA induced EAE in strains expressing the MHC class II allele RT1.B n, whereas strains expressing the RT1.B l were resistant. In myelin basic protein peptide (MBP GP63-88)/CFA-induced EAE, RT1.B l expressing strains were susceptible whereas strains expressing the RT1.B n were resistant. High levels of antigen-specific IFN-γ secreting lymphoid cells and antigen-specific serum IgG antibodies were only recorded in rats with an MHC class II allele that permitted MOG- or MBP-EAE, respectively. Genetically, we localized the MHC regulation of the investigated EAE models to the central part of the MHC, containing the MHC class II (RT1.B/D) and the centromeric parts of the MHC class III. No influences were evident from the classical MHC class I (RT1.A), the telomeric parts of the MHC class III or the non-classical MHC class I (RT1.C/E/M) in contrast to previous reports. The MHC class II haplotype-specific regulation of EAE induced with two different CNS antigens demonstrates a strikingly specific MHC-association even within the same target organ.

Time frame of pancreas allograft rejection: an immunogenetic analysis in MHC-disparate, presensitized inbred rat strains

Aim The pattern of rejection of vascularized pancreas allografts in presensitized recipients was studied among congeneic and recombinant rat strains in order to establish the time frame of rejection. Donors and recipients differed either in their entire MHC or in class I or class II MHC antigens. Material and methods Streptozotocin diabetic (55 mg/kg bone weight) recipients underwent an intra-abdominal heterotopic pancreas whole organ transplantation. The exocrine secretion of the pancreas was suppressed by ligation of the duct. Rejection was defined as recurrence of diabetic hyperglycemia exceeding 14 mmol/L, which was subsequently confirmed by histologic examination. The pancreas recipients were presensitized by repetitive donor-specific skin transplants performed 12, 8, and 4 weeks prior to pancreas grafting. Results The results shown accelerated rejection in all instances. Animals with incompatibility for the entire MHC showed a median rejection time in naive recipients of 12.5 days and in presensitized recipients of 8 days. Class I MHC incompatible native recipients rejected the allografts after 16.5 days (median) and presensitized recipients after 9 days (median). Concerning class II MHC incompatibility rejection appeared in naive recipients after 16 days (median) and in presensitized recipients after 7 days (median). Hyperacute rejection, however, was not observed. Discussion These results show an accelerated pattern of rejection in all instances caused by donor MHC antigens. Hyperacute rejection, however, was not observed. The effect is slightly pronounced in class II MHC incompatibility. Acceleration of rejection may be caused by stimulation of the humoral as well as the cellular pathway of the immune response. The underlying mechanisms concerning the different MHC incompatibilities, however, remains speculative.

MHC gene related effects on microglia and macrophages in experimental autoimmune encephalomyelitis determine the extent of axonal injury.

Myelin-oligodendrocyte-glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in rats is a chronic inflammatory demyelinating disease of the central nervous system (CNS) strongly mimicking multiple sclerosis (MS). We determined the involvement of macrophages and microglia in the lesions of MOG-EAE in relation to different major histocompatibility complex (MHC, RT1 in rat) haplotypes. We used intra-RT1 recombinant rat strains with recombinations between the RT1a and RT1u haplotypes on the disease permissive LEW non-MHC genome. Activated microglia and macrophages were identified morphologically and by expression of ED1 and allograft inhibitory factor-1 (AIF-1), and differentiated by their morphological phenotype. White matter lesions contained more macrophages and less microglia compared to grey matter lesions. Similarly active lesions were mainly infiltrated by macrophages, while microglia were abundant in inactive demyelinated plaques. In addition, we found a highly significant genetic association between a macrophage or microglia dominated lesional phenotype, which was independent from location and activity of the lesions. This was not only the case in demyelinating plaques of chronic EAE, but also in purely inflammatory lesions of acute passive transfer EAE. Rat strains with an u-haplotype in both the Class II and the telomeric non-classical Class I region revealed inflammatory and demyelinating lesions, which were dominated by activated microglia. The a-haplotype in any of these regions was associated with macrophage dominated lesions. A comparison of lesions, exactly matched for stages of demyelinating activity in these different rat strains, showed that in spite of a similar extent of demyelination, axonal injury was significantly less in microglia compared to macrophage dominated lesions. Thus, our studies document a genetic influence of the MHC-region on the relative contribution of macrophages versus microglia in the pathogenesis of EAE.

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.

Rats made congenic for Oia3 on chromosome 10 become susceptible to squalene-induced arthritis.

Several quantitative trait loci (QTLs) regulating the risk of experimental arthritis have been identified by genome-wide linkage analyses, but only the MHC has thus far been reported to transfer arthritis susceptibility in congenic animals. We have produced a congenic strain for Oia3, a genetic factor originally identified as an oil-induced arthritis (OIA) QTL in arthritis-prone DA rats. A 46 cM telomeric region of chromosome 10 encompassing Oia3 was transferred from DA rats to MHC-identical but minutely arthritis-susceptible LEW.1AV1 rats by selective breeding. Arthritis development was provoked in Oia3-congenic rats by intradermal injection of different adjuvant oils. One successful arthritis trigger was squalene, which is approved for vaccinations in humans and has been implicated in Gulf War syndrome. The endogenous cholesterol precursor squalene induced T cell infiltration into joints and macroscopic arthritis in Oia3-congenic rats and DA rats, whereas LEW.1AV1 rats were almost resistant. Arthritis onset, approximately 14 days post-injection, coincided with arrested body-weight gain and increased plasma levels of the inflammation markers fibrinogen and alpha 1-acid glycoprotein. Congenic rats displayed intermediate phenotypes compared with the two parental strains, and similar to rheumatoid arthritis in humans, female preponderance was observed in Oia3-congenic rats. Finally, recombinant rat strains were constructed and were used to map a susceptibility gene(s) in females to a telomeric 4--19 cM Oia3 subregion. The experimental system described allows transformation of multifactorial arthritis susceptibility into dichotomous phenotypes.

Role of classical (RT1.A) and nonclassical (RT1.C) MHC class I regions in natural killer cell-mediated bone marrow allograft rejection in rats.

We have studied the role of the different MHC (RT1) subregions in acute natural killer (NK) cell-mediated bone marrow allograft rejection in lethally irradiated, bone marrow cell (BMC) reconstituted rats. We employed a series of MHC congenic and intra-MHC recombinant rat strains so that effects of mismatches in defined RT1 subregions could be studied systematically. BMC allograft survival was measured as 125IUdR uptake in the spleen between day 5 and day 7 after irradiation and BMC reconstitution. We found that in certain RT1 haplotype combinations, nonclassical RT1.C disparities by themselves could determine graft rejection (i.e., in the u/av1 recombinant haplotypes), whereas in another combination (between the av1 and c haplotypes) a mismatch for an isolated classical RT1.A region was decisive for engraftment. Thus, PVG.R1 BMC failed to proliferate in PVG rats, differing in the RT1.A region only, whereas in PVG.1U rats rejection could be determined by isolated differences in the RT1.C region (LEW.1WR1). Also, RT1 homozygous rats (RT1.U) rejected semi-allogeneic F1 hybrid BMC. The acute rejection of BMC was mediated by NK cells, as athymic nude rats, lacking alloreactive T cells but with normal alloreactive NK cells, showed the same patterns of rejection as did normal rats. Nude rats also rejected allogeneic lymphocytes, a previously documented NK-mediated phenomenon, with identical requirements of MHC disparity. This investigation shows that rat effector NK cells are radioresistant, independent of the thymus, and capable of recognizing and rejecting MHC mismatched transplanted BMC on the basis of mismatches in both classical and nonclassical class I regions in vivo. The studies underline the importance also of NK cells in determining BMC allograft survival.

Genetic control of host-vs-graft reactivity after small bowel transplantation with heterotopic and orthotopic placement

It is well established that small bowel transplants can induce graft-vs-host (GVH) reactions which are potentially lethal for the graft recipients. Using a model of MHC congenic and recombinant rat strains, we have characterized the genetic control of GVH reactions after parental to F1 hybrid small bowel transplantation. Lethal graft-vs-host disease (GVHD) in the rat is primarily mediated by class II MHC incompatibility, while class I MHC and non-MHC antigens do not induce a symptomatic GVH reactivity. Following small bowel transplantation, not only may GVH reactions occur, but the transplant is also a potential victim of rejection. In our rat model, we have now analyzed the genetic control of host-vs-graft (HVG) reactions after F1 hybrid to parental small bowel transplantation and compared the results after heterotopic and orthotopic placement.

The rat MHC haplotype RT1c expresses two classical class I molecules.

Cloning and characterization of classical MHC class I coding sequences of the laboratory rat Rattus norvegicus has been reported so far for only four haplotypes, RT1a, RT1(1), RT1n, and RT1u. In all four cases, only one RT1.A classical class I molecule was found. Here we report that, in contrast, the RT1c haplotype expresses two different classical class I molecules. Using recombinant rat strains, we find that allotypic serologic determinants carried by the two molecules map to the RT1.A region, and so we have named them RT1.A1c and RT1.A2c. Multiple clones of functional cDNAs for each of these two molecules were isolated using a recently developed PCR-based expression-cloning method. Using a panel of 20 RT1.Ac-reactive mAb, we find that six recognize RT1.A1c, seven recognize RT1.A2c, and seven recognize both. We also show that both molecules are recognized and distinguished by primary alloreactive cytotoxic T lymphocytes, and that they correspond to identifiable and distinct molecular species in cells that express RT1c naturally. These data all concur to demonstrate that the RT1.Ac region carries two different loci, each of which encodes a functional classical class I molecule.

Protective influences on experimental autoimmune encephalomyelitis by MHC class I and class II alleles.

Experimental autoimmune encephalomyelitis (EAE) is influenced by polymorphism of the MHC. We have previously found that Lewis rats with certain MHC haplotypes are susceptible to disease induced with the myelin basic protein (MBP) peptide 63-88, whereas Lewis rats with other MHC haplotypes are resistant. Interestingly, rats with the MHC u haplotype develop an immune response to the MBP 63-88, but do not get EAE. In this study we have used intra-MHC recombinant rat strains to compare the influences of the MHC u with the a haplotype. We discovered the following: 1) The class II region of the MHC a haplotype permits EAE and a Th1 type of immune response as measured by IFN-gamma production after in vitro challenge of in vivo-primed T cells with MBP 63-88. 2) The class II region of the u haplotype is associated with a disease-protective immune response characterized by production of not only IFN-gamma, but also of IL-4 mRNA expression by the MBP 63-88-activated cells. 3) The class I region upstream of the class II region of the u haplotype is associated with a disease-protective effect and the expression of mRNA for TGF-beta after MBP 63-88-induced activation. Thus, such a TGF-beta response occurs in all strains expressing the class I Au allele. Treatment with Abs to CD8+ cells abrogates peptide-induced TGF-beta mRNA expression, and aggravates disease in strains with the class I Au allele.

The role of histocompatibility antigens in transplantation of isolated islets of Langerhans in the rat.

In a model of congenic and intra-MHC recombinant rat strains, the differential role of various histocompatibility antigens in renal subcapsular transplantation of purified islets of Langerhans is evaluated. Class I MHC antigens of the RT1.A region, expressed on the endocrine cells of the islets themselves, do not induce graft rejection on their own. MHC class I antigens as encoded by the RT1.C region do not induce rejection either. MHC class II antigens as encoded by the RT1.B/D region are not expressed on the endocrine pancreas, not even during rejection. Although interstitial dendritic cells situated within the islets express these antigens, an isolated RT1.B/D incompatibility of islets is associated with prolonged survival in contrast to rapid rejection of fully MHC-mismatched grafts. Unlike other organs, islets matched for all MHC antigens, but incompatible at minor histocompatibility antigens, undergo rejection early after transplantation.

Genetic Control of the Immune Response to Collagen by the Major Histocompatibility Complex in the Rat: I. Humoral Responses to Bovine and Chick Type II Collagen

A number of congenic and recombinant rat strains with different RT1 haplotypes on a PVG/c background were immunised with native bovine or chick type II collagen. Only strains with the “u” haplotype were good responders to bovine type II collagen whilst all strains responded to chick type II collagen. However, the “u” haplotype was again associated with the production of the highest amounts of antibody. All the strains responded to a non-collagenous antigen, keyhole limpet haemocyanin, showing that this effect was not a general one relating to all antigens.When antibodies were produced to the immunising collagen, binding of antibodies to native rat type II collagen was also seen. This association, of high amounts of antibody with the “u” haplotype, was inherited in a dominant fashion when F1 and F2 hybrids between low responder (RT1a)and high reponder (RT1u) were examined.The recombinant r1 (class I av1, class II c) and r8 (class I av1 and class II u) strains showed that “u” haplotype at class II rather than at class I was associated with the strong response to type II collagen.The absence of arthritis in the “u” haplotype rats, suggests that the “u” haplotype alone is insufficient for arthritis induction after immunisation with type II collagen. It suggests that background genes outside the RT1 complex influence arthritis production as Wistar rats with the RT1u haplotype (but different minor genes outside RT1 complex) give an arthritis incidence of 50% under identical immunisation protocols.

A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule.

The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post-translational modification of RT1.Aa.

Genetic control of rat heart allograft rejection: effect of different MHC and non-MHC incompatibilities.

We investigated the genetic control of heterotopic heart allograft rejection using a family of standard inbred, major histocompatibility complex (MHC)-congenic, and intra-MHC recombinant rat strains. Gene products of the various regions within the rat MHC differed markedly in their capacity to induce rejection. Isolated incompatibility at class I antigens encoded by the RTl.A and RTl.C regions failed to induce rejection within the observation period of 100 days, whereas class II antigens encoded by the RTl.B/D region provoked rapid rejection within 10 days. By comparison of the rejection times of isolated and combined incompatibilities a number of functional interactions could be demonstrated between individual MHC regions which either prolonged or shortened allograft survival. In contrast to rapid rejection of MHC-mismatched heart allografts, differences at non-MHC histocompatibility antigens were associated with graft survival beyond 100 days, although chronic rejection of variable severity was detected histologically. Disparity at non-MHC plus class I antigens, however, provoked acute heart allograft rejection.

Recognition of Rat Dendritic Cells by a Monoclonal Antibody

A mouse monoclonal IgM antibody reactive with dendritic cells (DC) from the Brown Norway (BN) rat was prepared. This antibody (1F119) binds to a membrane-bound antigen present on DC from thoracic duct lymph, spleen, thymus, and lymph node. The antigen is present only in low density on 5% of splenic macrophages (M phi) and absent from peritoneal M luminal diameter. In situ, the antibody exhibits a strong reactivity towards DC in the thymic medulla, whereas no reaction is observed with cortical cells. Furthermore, cells positive for 1F119 can be identified in T-cell areas of spleen, lymph node, and Peyers' patches. 1F119 was genetically restricted in that a strong reactivity was found with DC from rats of the RT1n and RT1u haplotypes, an intermediate reactivity with the RT1c haplotype, only a weak reactivity with the RT1l and RT1b haplotypes, and no reactivity with the RT1a and RT1k haplotypes. The relatively weak reactivity of 1F119 with respect to the RT1l haplotype also appeared from a weak binding of 1F119 to DC from Lewis rats, as was assessed by FACS analysis. This result was comparable to the binding of OX3 (RT1.Bl and RT1.Bu) to DC from BN rats. Studies performed on thymus sections of recombinant rat strains indicate that 1F119, despite its apparent specificity for DC, reacts with a polymorphic RT1.B product.

Polymorphism and mapping of the class II genes in the rat: RT1.B, RT1.D, and RT1.H, a new DP-like region.

The major histocompatibility complex of the rat (RT1) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the alpha- and beta-chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has alpha- and beta-chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1.B. The RT1.H alpha and RT1.H beta genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H beta and D beta genes have high levels of polymorphism; B beta, B alpha, and H alpha have intermediate levels of polymorphism, and D alpha has a low level of polymorphism.

Genetics of the rat CT system: its apparent complexity is a consequence of cross-reactivity between the distinct MHC class I antigens RT1.C and RT1.A.

The rat CT antigens are a system of medial histocompatibility antigens linked to RT1, the rat major histocompatibility complex (MHC). They have aroused interest firstly because, despite their extreme serological weakness, they are targets for 'unrestricted' cytotoxic T lymphocytes (CTL); and secondly because they have appeared to represent a complex genetic system in terms both of the number of genetic loci involved and the number of distinguishable antigenic specificities expressed. The CT system was originally defined by the reactions of LEW anti-F344 (RT1l anti-RT1lv1) secondary in vitro CTL. These CTL reacted strongly on DA(RT1av1) targets, but much more weakly on AUG or PVG (RT1c) targets. We have used the recently derived RT1 recombinant rat strains PVG.R19 (RT1.Aav1Iav1Cc) and PVG.R20 (RT1.AcIcCav1) to investigate the genetic control of this system. Contrary to previous interpretations, the results are consistent with a model in which CT is a single locus, which maps to the RT1.C region. In addition, our results demonstrate that there is cross-reactivity of anti-RT1C CTLs on RT1A products, and we suggest that the earlier placement of a CT locus in the RT1.A region was probably incorrect and a consequence of this cross-reactivity.

Allospecific monoclonal antibodies recognizing rat class I and class II histocompatibility antigens.

Rat-mouse hybridomas have been developed producing monoclonal antibodies which recognize rat major histocompatibility complex specificities. Splenic lymphocytes from a LEW (RT1l) rat alloimmunized against ACI (Rt1a) splenocytes were used for hybridoma production. The six clones presented demonstrate two patterns of alloreactivity against ACI tissues: clone 36.3 produces an antibody which agglutinates ACI RBCs and binds to ACI splenic lymphocytes and RBCs. Five clones, 65.6, 79.7,1, 79.7.5, 155.3.1, and 189.3.5, from four independent wells produce monoclonal antibodies which bind to ACI splenic lymphocytes but not RBCs. Screening of these six monoclonal antibodies against a reference panel of RBCs and lymphocytes from 15 standard, congenic, and recombinant rat strains indicated that the monoclonal antibody 36.3 detects a RTI.Aa product ( a rat major histocompatibility complex (MHC) Class I antigen) and that 65.6, 79.7.1, 79.7.5, 155.3.1, and 189.3.5 are monoclonal antibodies which recognize a RT1.Ba private specificity (Class II or Ia antigens). These reagents are unique as they are rat monoclonal alloantibodies recognizing private Ia specificities of the rat MHC.