Imaging Tolerance Induction in the Classic Medawar Neonatal Mouse Model: Active Roles of Multiple F1-Donor Cell Types.

The immature immune system is uniquely susceptible to tolerance induction and thus an attractive target for immunomodulation strategies for organ transplantation. Newborn mice injected with adult semi-allogeneic lymphohematopoietic cells accept transplants without immunosuppressive drugs. Early in vivo/in situ events leading to neonatal tolerance remain poorly understood. Here, we show by whole body/organ imaging that injected cells home to lymphoid organs and liver where various F1-donor cell types selectively alter neonatal immunity. In host thymus, F1-donor dendritic cells (DC) interact with developing thymocytes and regulatory T cells suggesting a role in negative selection. In spleen and lymph nodes, F1-donor regulatory T/B cells associate with host alloreactive cells and by themselves prolong cardiac allograft survival. In liver, F1-donor cells give rise to albumin-containing hepatocyte-like cells. The neonatal immune system is lymphopenic, Th-2 immunodeviated and contains immature DC, suggesting susceptibility to regulation by adult F1-donor cells. CD8a T cell inactivation greatly enhances chimerism, suggesting that variable emerging neonatal alloreactivity becomes a barrier to tolerance induction. This comprehensive qualitative imaging study systematically shows contribution of multiple in vivo processes leading simultaneously to robust tolerance. These insights into robust tolerance induction have important implications for development of strategies for clinical application.

Extrathymic deletion of CD8+ alloreactive T cells in a transgenic T cell receptor model of neonatal tolerance.

BACKGROUND: Immunological tolerance to foreign antigen is most easily achieved during the neonatal period. Although deletion of T cells has been demonstrated in neonatal tolerance models in which donor and recipient express different MHC class II molecules, the requirement for deletion in MHC class I-disparate models is less clear. To address this issue, we used as recipient the T cell receptor (TCR) transgenic mouse (TgM) strain 2C in which the majority of CD8+ T cells express a single alpha/beta TCR alloreactive to H-2Ld, thus facilitating direct monitoring of the class I alloreactive population. METHODS: Newborn (less than 24 hr of age) 2C TgM received injections i.v.with syngeneic C57BL/6J (H-2b) (B6) or semiallogeneic (B6xDBA)F1 (H-2bd; H-2Ld+) splenocytes. Adults were subsequently analyzed in terms of tolerance, deletion of 2C+ T cells, and chimerism. RESULTS: The results showed that semiallogeneic-, but not syngeneic-, injected neonates were unresponsive as adults to H-2Ld-expressing target cells in vitro and the majority of these mice accepted H-2Ld+ skin grafts. Delaying the injection to 72 hr after birth or reducing the number of cells injected essentially abolished in vivo unresponsiveness in 2C recipients. Thus, the 2C TCR Tg model demonstrates the characteristics typical of neonatal tolerance. Injection of 2C neonates within 24 hr of birth with semiallogeneic versus syngeneic cells led to more than a 12-fold reduction of CD8+ 2C+ T cells in adult spleen and LNCs. In contrast, deletion of CD8+ 2C+ cells in adult thymus was not consistently observed. Based on MHC class II expression to distinguish donor (I-E+) and recipient (I-E-) cells, semiallogeneic-injected mice were chimeric in spleens and lymph nodes (LNs). CONCLUSIONS: These results demonstrate that neonatal MHC class I tolerance in the adult is associated with low level hematopoietic chimerism and extrathymic deletion of alloreactive CD8+ T cells.

Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea.

We have found that the somatic mutation rate at the Dlb-1 locus increases exponentially during low daily exposure to ethylnitrosourea over 4 months. This effect, enhanced mutagenesis, was not observed at a lacI transgene in the same tissue, although the two loci respond very similarly to acute doses. Since both mutations are neutral, the mutant frequency was expected to increase linearly with time in response to a constant mutagenic exposure, as it did for lacI. Enhanced mutagenesis does not result from an overall sensitization of the animals, since mice that had first been treated with a low daily dose for 90 days and then challenged with a large acute dose were not sensitized to the acute dose. Nor was the increased mutant frequency due to selection, since animals that were treated for 90 days and then left untreated for up to 60 days showed little change from the 90-day frequency. The effect is substantial: about 8 times as many Dlb-1 mutants were induced between 90 and 120 days as in the first 30 days. This resulted in a reverse dose rate effect such that 90 mg/kg induced more mutants when delivered at 1 mg/kg per day than at 3 mg/kg per day. We postulate that enhanced mutagenesis arises from increased stem cell proliferation and the preferential repair of transcribed genes. Enhanced mutagenesis may be important for risk evaluation, as the results show that chronic exposures can be more mutagenic than acute ones and raise the possibility of synergism between chemicals at low doses.

Factors affecting somatic mutation frequencies in vivo.

The factors that influence the spontaneous mutant frequencies in mammalian tissues have been ranked on the basis of data from our laboratory together with published data. Some of the data come from the endogenous hprt and Dlb-1 loci, but most come from transgenic mice carrying the bacterial lacI and lacZ genes in recoverable lambda phage vectors. Since there is evidence that these bacterial loci are selectively neutral, the mutant frequency observed is the integral of the mutation rates from the formation of the zygote. The factors that affect the inferred mutation rate, in decreasing order of importance are: site of integration of the transgene, age, tissue, and strain. Insufficient data exist to determine the influence of gender (probably small) and inter-laboratory variables (probably at least as important as age). The two most surprising results are (1) that about half of all mutations arise during development (and half of these in utero) and (2) that most somatic tissues, whether queiscent or actively proliferating, have similar mutant frequencies and similar increases during adult life.

The accumulation and persistence of somatic mutations in vivo.

Mutant frequencies at the native Dlb-1 and the lacI transgene reach a plateau within 1 week of mutagenesis in the small intestine of mice. This indicates that these mutations are selectively neutral and should accumulate additively. In a test with three mutagens, one potent (ethylnitrosourea) and two weak (1,2-dimethylhydrazine and methyl methanesulphonate), ten weekly treatments induced ten times the frequency of Dlb-1 mutations induced by a single treatment in each case, a result that is consistent with perfect additivity. These observations indicate that the mutation frequency at neutral loci will be an integrated measure of the effects of aging, endogenous mutagens, environmental mutagens and antimutagens. They suggest a simple protocol for transgenic mutation assays which would increase sensitivity, require fewer animals, and conform to standard toxicological practice.

Comparison of somatic mutation in a transgenic versus host locus.

Somatic mutations can now be quantified in almost any cell type in mice carrying bacterial genes in a lambda phage shuttle vector. Mutations induced in vivo are detectable ex vivo, after packaging host-cell DNA into phage that are grown on suitable bacteria. However, the transgenic DNA differs from many host loci in several ways: it (i) is prokaryotic DNA, (ii) is present in multiple tandem copies, and (iii) is heavily methylated and probably not expressed. Thus, mutation of a transgene may not be a suitable model of the host loci, which are eukaryotic, unique, and expressed. To test the relevance of the transgene mutation model, the frequencies of the bacterial lacI+ to lacI- mutations induced in half of the small intestine were compared with the frequencies of the host Dlb-1b to Dlb-1a mutations induced in the other half. The loci responded similarly to ethyl nitrosourea (ENU) with respect to the animal's age and sex, sex of the parent transmitting the transgene, and expression time. ENU dose-response curves were similar. Furthermore, no difference was found at the Dlb-1 locus between transgenic and nontransgenic siblings. In contrast, x-rays induced few lacI mutations but many Dlb-1 mutations. Probably few large deletions are detectable at lacI, but many are detectable at Dlb-1. If so, an important class of mutation is not readily detected in these transgenic mice. With this exception, the transgene and host gene responded similarly in this somewhat limited trial, as is necessary if the transgenic mice are to be a useful model.

Mutagenicity of methyl methanesulfonate (MMS) in vivo at the Dlb-1 native locus and a lacI transgene.

Methyl methanesulfonate (MMS) is an extraordinarily poor mutagen compared to ethylnitrosourea (ENU) or even X-rays. In lung fibroblasts in vivo, MMS has been shown to induce many micronuclei but few, if any, mutations at the hpt locus. We wondered if the lack of mutations might be due to the lack of division and DNA synthesis in fibroblasts in vivo, which would permit substantial time for differential repair of DNA lesions. This idea was tested in the small intestine, a tissue in which the cells are actively dividing. Two loci were examined: a native locus (Dlb-1) which determines the presence or absence of a lectin binding site on the surface of the epithelial cells, and a lacl transgene which controls beta-galactosidase synthesis. Locl mutations were detected after in vitro packaging of DNA isolated from the intestinal epithelium into lambda phage and expression in suitable bacteria. Although the epithelial cells are proliferating, acute treatments produced no significant increase in mutations at either locus. Subacute treatments produced low but significant increases in mutation frequency at both loci. The results confirm that MMS is a far more potent clastogen than it is a mutagen and should be regarded as a super-clastogen in the same manner as ENU is a super-mutagen. The carcinogenicity of MMS is probably the result of its potent clastogenicity rather than its weak activity as a point mutagen.