SummaryRat major histocompatibility complex (MHC) class II molecules RT1.Bl (DQ‐like) and RT1.Dl (DR‐like) were cloned from the LEW strain using reverse transcription–polymerase chain reaction and expressed in mouse L929 cells. The transduced lines bound MHC class II‐specific monoclonal antibodies in an MHC‐isotype‐specific manner and presented peptide antigens and superantigens to T‐cell hybridomas. The T‐cell‐hybridomas responded well to all superantigens presented by human MHC class II, whereas the response varied considerably with rat MHC class II‐transduced lines as presenters. The T‐cell hybridomas responded to the pyrogenic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and SEC3 only at high concentrations with RT1.Bl‐transduced and RT1.Dl‐transduced cells as presenters. The same was true for streptococcal pyrogenic exotoxin A (SPEA), but this was presented only by RT1.Bl and not by RT1.Dl. SPEC was recognized only if presented by human MHC class II. Presentation of Yersinia pseudotuberculosis superantigen (YPM) showed no MHC isotype preference, while Mycoplasma arthritidis superantigen (MAS or MAM) was presented by RT1.Dl but not by RT1.Bl. Interestingly, and in contrast to RT1.Bl, the RT1.Dl completely failed to present SEA and toxic shock syndrome toxin 1 even after transduction of invariant chain (CD74) or expression in other cell types such as the surface MHC class II‐negative mouse B‐cell lymphoma (M12.4.1.C3). We discuss the idea that a lack of SEA presentation may not be a general feature of RT1.D molecules but could be a consequence of RT1.Dlβ‐chain allele‐specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid) in the extremely conserved region flanking the Zn2+‐binding histidine 81, which is crucial for high‐affinity SEA‐binding.

The M region at the telomeric end of the murine major histocompatibility complex (MHC) contains class I genes that are highly conserved in rat and mouse. We have sequenced a cosmid clone of the LEW rat strain ( RT1 l haplotype) containing three class I genes, RT1.M6-1 , RT1.M4 , and RT1.M5 . The sequences of allelic genes of the BN strain ( RT1 n haplotype) were obtained either from cDNAs or genomic clones. For the coding parts of the genes few differences were found between the two RT1 haplotypes. In LEW, however, only RT1.M5 and RT1.M6 have open reading frames; whereas in BN all three genes were intact. In line with the findings in BN, transcription was found for all three rat genes in several tissues from strain Sprague Dawley. Protein expression in transfectants could be demonstrated for RT1.M6-1 using the monoclonal antibody OX18. By sequencing of transcripts obtained by RT-PCR, a second, transcribed M6 gene, RT1.M6-2 , was discovered, which maps next to RT1.M6-1 outside of the region covered by the cosmid. In addition, alternatively spliced forms for RT1.M5 and RT1.M6 were detected. Of the orthologous mouse genes, H2-M4 , H2-M5 , and H2-M6 , only H2-M5 has an open reading frame. Other important differences between the corresponding parts of the M region of the two species are insertion of long LINE repeats, duplication of RT1.M6 , and the inversion of RT1.M5 in the rat. This demonstrates substantial evolutionary dynamics in this region despite conservation of the class I gene sequences themselves.

Background: Microinjection of foreign DNA into pronuclei of zygotes has been the method of choice for the production of transgenic domestic animals. Following microinjection the transgene is randomly integrated into the host genome which can be associated with insertional mutagenesis and unwanted pathological side effects.Methods: Here, we evaluated the health status of pigs transgenic for the human regulator of complement activation (RCA) CD59 and conducted a complete pathomorphological examination on 19 RCA transgenic pigs at 1 to 32 months of age from nine transgenic lines. Nine wild-type animals served as controls. Expression levels of human complement regulator CD59 (hCD59) mRNA were measured by RT-PCR and distribution of hCD59 protein was determined by immunohistochemistry.Results: Albeit variable transgene expression levels, no specific pathomorphologic phenotype associated with the presence of the transgene in all analyzed pig lines could be detected.Conclusion: Transgenic expression of this human RCA gene construct is not correlated with a specific pathological phenotype in pigs. This is crucial for the application of the technology and the use of transgenic pigs for biomedical and agricultural applications.

In recent years, it has become clear that the polarization of T cells depends on the genetic background. However, due to the complexity of the genetic background of each animal, a direct comparison of the phenotype is difficult. In this study, a new rat strain LEW.BN-4-10 carrying the chromosomal regions on chromosomes 4 and 10, which harbor IL-6 and IL-4 gene clusters of BN, has been bred on the genetic background of LEW. It was asked whether these two gene clusters influence the polarization of T cell responses. As a model, the Mycoplasma arthritidis mitogen (MAM)-induced inflammation was used focusing on the microenvironment of the draining lymph node (LN). The effect of differences in these regions was tested by comparing LEW.BN-4-10 and LEW rats under steady-state conditions and upon injection of MAM into the forepaw. Under steady-state conditions, the two strains showed differences in the dendritic cell (DC) subset composition. When MAM was injected, the number of T cells in LEW.BN-4-10 rats producing T(h)2 cytokines such as IL-4 and IL-13 was significantly increased compared with LEW. The data suggest that these differences in the microenvironments in LN of LEW and LEW.BN-4-10 rats resulted in different susceptibility to the disease (increase of cells in LN and paw swelling). In addition, deviations in the distribution and function of injected effector T cells were found in the LN of LEW and LEW.BN-4-10 rats after MAM treatment. The data indicate that the IL-6 and IL-4 gene clusters are involved in polarizing T cell responses in vivo.

The applicability of tightly regulated transgenesis in domesticated animals is severely hampered by the present lack of knowledge of regulatory mechanisms and the long generation intervals. To capitalize on the tightly controlled expression of mammalian genes made possible by using prokaryotic control elements, we have used a single-step transduction to introduce an autoregulative tetracycline-responsive bicistronic expression cassette (NTA) into transgenic pigs. Transgenic pigs carrying one NTA cassette showed a mosaic transgene expression restricted to single muscle fibers. In contrast, crossbred animals carrying two NTA cassettes with different transgenes, revealed a broad tissue-independent and tightly regulated expression of one cassette, but not of the other one. The expression pattern correlated inversely with the methylation status of the NTA transcription start sites indicating epigenetic silencing of one NTA cassette. This first approach on tetracycline regulated transgene expression in farm animals will be valuable for developing precisely controlled expression systems for transgenes in large animals relevant for biomedical and agricultural biotechnology.

RT1.L class I antigens have originally been identified in LEW rats by LEW.1LV3-anti-LEW.1LM1 antisera and have been classified as nonclassical. We report now that LEW.1LV3-anti-LEW.1LM1 antisera react with three different antigens, termed RT1.L1, RT1.L2, and RT1.L3. This was found by serological analysis of a panel of transfectants expressing different class I genes of strain LEW with a LEW.1LV3-anti-LEW.1LM1 antiserum and two monoclonal antibodies (mAbs HT20 and HT21) generated in the same strain combination. The antiserum reacted with all three antigens: the two mAbs with RT1.L1 and RT1.L2, respectively. Sequence analysis showed that the genes encoding RT1.L1, RT1.L2, and RT1.L3 cluster together in a phylogenetic analysis of rat and mouse alpha(1)-alpha(2) sequences and that they share an unusual MHC class I promoter in which Enhancer A and B, as well as the interferon response element (IRE), are missing. Exchange of the promoter in RT1.L2 against the classical RT1.A promoter resulted in high surface expression in appropriate transfectants, indicating that the deviant promoter is responsible for the weak surface expression of the RT1.L2 gene. The very similar promoter structures of RT1.L1 and RT1.L3 are likely to contribute also to the weak expression of these genes. As RT1.L3 maps closely to the deletion in the mutant haplotype lm1, the RT1.L family can be located in the class I region extending from Bat1 to Pou5f1. Different from other allogeneic mAbs detecting known class I molecules encoded by genes of the RT1.C/E region, HT20 and HT21 react with a wide panel of strains carrying different RT1 haplotypes. This suggests that nonclassical class I genes of the RT1.L family are present in most RT1 haplotypes.

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7(a) mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7(a) mAb treatment was performed in LEW.1W (RT1(u) RT7(a)) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)-congenic BM transplantation making use of a diallelic polymorphism (RT7(a)/RT7(b)) of rat CD45 was applied. Following injection of anti-RT7(a) mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7(a) mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1(u) RT7(b)) and transplantation of BM from LEW.1W rats pretreated with anti-RT7(a) mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7(b)/RT7(a)), very few RT7(a)-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7(a) mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7(a) mAb efficiently depletes early rat hematopoietic stem cells.

Transgenic expression of apoptosis-inducing molecules could be a strategy to protect cells and tissues from destruction by apoptosis-susceptible effector T cells. Some evidence for the potency of this approach has been obtained in mouse and rat transplantation models. However, limited data are available on the capacity of apoptosis-inducing molecules to modulate human alloimmune responses. In this study we analyzed the in vitro consequences of an interaction of human T cells with allogeneic 293 cells and 293 transfectants stably expressing high levels of the apoptosis-inducing CD95 ligand (CD95L). Both, CD95L and CD95L(+) 293 cells were able to activate allogeneic T cells as demonstrated by comparable CD25 expression at day 2 of culture. The analysis of viable T cells at day 7, however, revealed anti-293 cytotoxic activity only in cultures that had been stimulated with CD95L(-) 293 cells. Alloactivated effector T cells lysed CD95L- and CD95L(+) 293 targets with similar efficiency when tested in a 4-h Cr-51-release assay. Prolongation of the effector phase to 20 h resulted in a further increase in the destruction of CD95L- target cells, whereas lysis of CD95L(+) targets remained low. These data suggest that genetically engineered expression of CD95L on cells or tissues could be an approach to control human T cell reactivity towards allografts. During the induction of an alloimmune response depletion of cytotoxic precursor cells may be obtained by overexpressing CD95L on stimulatory cells; CD95L expression on graft tissue might limit T cell-mediated destruction of the transplant during the effector phase of the response.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is widely accepted that a dysregulated immune response against brain resident antigens is central to its yet unknown pathogenesis 1 , 2 , 3 , 4 . Although there is evidence that the development of MS has a genetic component, specific genetic factors are largely unknown 5 , 6 , 7 . Here we investigated the role of a point mutation in the gene ( PTPRC ) encoding protein-tyrosine phosphatase, receptor-type C (also known as CD45) in the heterozygous state in the development of MS. The nucleotide transition in exon 4 of the gene locus interferes with mRNA splicing and results in altered expression of CD45 isoforms on immune cells. In three of four independent case-control studies, we demonstrated an association of the mutation with MS. We found the PTPRC mutation to be linked to and associated with the disease in three MS nuclear families. In one additional family, we found the same variant CD45 phenotype, with an as-yet-unknown origin, among the members affected with MS. Our findings suggest an association of the mutation in PTPRC with the development of MS in some families.

With an organ transplant, hematopoietic donor cells are transferred to the recipient. To study the relevance of the resulting microchimerism for allograft acceptance, we analyzed a rat model of cyclosporine-induced tolerance for strongly incompatible heart allografts. Using a monoclonal antibody that detects a donor-specific CD45 allotype (RT7(a)), we selectively depleted donor leukocytes at different times after transplantation (days 0 or 18). Depletion was similarly effective at both times. However, only depletion on day 0 prevented tolerance induction and was associated with severe acute or chronic graft rejection. This indicates that passenger leukocytes have an essential immunomodulatory effect on the induction phase of allograft acceptance.

152 Purpose. Common leukocyte antigen is expressed on all hematopoietic cells, and is known to have costimulatory function for T cell activation. We have generated a new monoclonal antibody (mAb) against the rat common leukocyte antigen RT7. This mAb specifically binds an allomorphic region of the RT7 molecule. The purpose of this study was to explore the effects of this mAb ex vivo and in vivo. Methods. Ex vivo effects of this anti-RT7 mAb on non-specific and allospecific lymphocyte proliferation were evaluated. Lymph node cells of LEW 1W (RT1u) rats were stimulated by anti-CD3 mAb or Con-A or by irradiated allogeneic lymph node cells of LEW (RT1l) rats. Anti-RT7 mAb was added to the culture medium at final concentrations of 0, 1, 10, and 100 µg/ml. [3H]TdR uptake was measured on days 3 and 5. In vivo immunosuppressive effect of the mAb was evaluated in an allogeneic rat heart transplant model. Heart grafts of LEW donors were transplanted heterotopically to LEW 1W recipients. Recipients were treated with a single dose of anti-RT7 mAb i.v. on day -1 (n=9). Leukocyte numbers and subpopulations in peripheral blood were serially analyzed, and graft survival was compared to recipients without mAb treatment (n=6). Results. Ex vivo lymphocyte proliferation, both for non-specific (anti-CD3, Con-A) and allospecific stimulation, was significantly enhanced by anti-RT7 mAb in dose-dependent fashion. In the heart transplant model, single injection of anti-RT7 mAb induced indefinite graft acceptance (>200 days × 3, >100 days × 6), while all grafts in non-treated recipients were rejected within 8 days. After injection of anti-RT7 mAb recipients showed transient leukopenia most strikingly of CD3 positive cells that recovered to the normal levels over 4-6 weeks. No other side effects were observed in the long-term course. Conclusions. Our newly generated anti-RT7 mAb induced long term allograft acceptance in a rat heart transplant model in vivo, while enhancing non-specific and allospecific lymphocyte proliferation ex vivo. Although the mechanism of action still has to be elucidated, anti-common leukocyte antigen antibody treatment may be a potent strategy for tolerance induction.

In this retrospective study, we have investigated the early intragraft inflammatory events of 12 liver allografts leading to chronic rejection. The cytological findings and clinical follow-up were analyzed in detail. Nine patients underwent at least one typical lymphoid activation of acute rejection, and three of them were treated more than once. Diagnosis of rejection was based on biopsy histology, cytology and liver dysfunction. In addition to the acute rejections, cytological analysis demonstrated in 11 of 12 grafts an unidentified lymphoid episode that differed from that of rejection. These lymphoid responses were associated with viral infections; cytomegalovirus (CMV) infection in 10 of 12 patients, hepatitis C virus (HCV) infection in 2 of 12 patients, 1 combined with CMV, and hepatitis B virus (HBV) infection in 1 patient. Graft dysfunction was still seen at the end of the follow-up. Thus, intragraft inflammation caused either by acute rejection or by viral infections may be involved in the induction of chronic rejection.

Rare cases of graft-versus-host disease after liver transplantation indicate that donor lymphocytes may be transferred to the recipient by human liver grafts. In this study, we have analyzed the number and subpopulations of donor lymphocytes transferred by liver grafts in order to evaluate the potential relevance of these cells after transplantation. Therefore, mononuclear cells were isolated from the tissue of perfused human donor livers and from the associated lymph nodes. The number of lymphocytes, their location, and surface marker expression were determined by immunostaining. The majority of lymphocytes transferred by the grafts were found within the liver tissue (5.3 +/- 2.9 x 10(9) cells). These lymphocytes are mainly T and NK cells, predominantly CD8+, are partially activated (28% HLA-DR+), and show strong adhesion molecule expression (88% LFA-1(3+)). In addition, 20-500 x 10(6) of resting lymphocytes, predominantly T and B cells, are transmitted by lymph nodes. These findings demonstrate that considerable numbers of donor lymphocytes of distinct phenotype are regularly transmitted to the recipient by human liver grafts and may be of functional relevance after transplantation.

Evaluation of graft morphology is regarded as a cornerstone for diagnosis of acute liver graft rejection. Here we have studied the clinical relevance of biopsy findings obtained either by aspiration cytology or by histology in the first month after human liver transplantation, and have assessed the influence of immunosuppressive induction treatment on the incidence of morphological and clinical rejection. Results of 865 aspiration biopsies (TAC) and 155 core biopsies in 141 patients were correlated with the retrospective clinical diagnosis concerning the presence or absence of acute rejection. This analysis demonstrated that there are almost no false negative findings either in cytology or in histology (less than 0.1% of negative biopsies). In contrast, with both methods a large number of positive biopsy results were obtained that were without clinical correlate ("false positive" biopsies; 46% and 41% of positive cytologies and histologies, respectively). The rates of clinical and morphological acute rejections were differently influenced by the type of immunosuppressive induction protocol used. The incidence of clinical rejection was particularly low with a quadruple drug regimen when cyclosporine therapy was started immediately after transplantation (29% vs. 62% when introduction of cyclosporine was delayed for 2-5 days). Morphological rejections were similarly frequent with immediate and delayed introduction of cyclosporine at 2 mg/kg during quadruple therapy (65-75%) and were only reduced with initial high dose cyclosporine treatment (5 mg/kg) (35%). Antirejection treatment was not required in patients with morphological evidence of rejection but without clinical symptoms. The study demonstrates that cytology and histology are similarly reliable for exclusion and similarly unreliable for diagnosis of clinical acute rejection. The clinical relevance of positive biopsy findings is strongly influenced by the basic immunosuppressive treatment. Certain types of induction treatment can obviously alter the alloresponse in a way that no graft damage occurs despite the presence of marked intragraft immune activation. "False-positive" biopsy findings, therefore, seem to represent a qualitatively modified and self-limited type of intragraft alloresponse that is without clinical consequences ("incomplete" or "subclinical" rejection).