Modulation of F1 cytotoxic potentials by GvHR: role and mode of action of non-MHC genes that determine the hybrid resistance to GvHR-associated suppression of F1 cytotoxic potential

The resistance of unirradiated F1 mice against graft-vs-host reaction (GvHR) induced by lymphocytes from certain parental strains is apparently a violation of the basic law in classical transplantation immunity. To explore genetic mechanisms of this peculiar phenomenon, GvHR-associated immunosuppression was examined on various kinds of F1 mice undergoing GvHR induced by parental lymphocytes. In F1 mice raised by crossing DBA/2 mice with various H-2-congeneic B10-series strains, parental lymphocytes having non-H-2 genetic background of DBA (DBA/2 and DBA/1) invariably could not induce GvHR-associated immunosuppression, irrespective of the H-2 haplotype incompatibility involved, whereas lymphocytes of the partner parental strain induced the immunosuppression. The number of the relevant loci in the DBA non-H-2 was assessed to be three recessive loci by examination of the capability to induce the GvHR-associated immunosuppression on lymphocytes from individual (B 10.D2 X DBA/2)F1 X DBA/2 backcross mice. On the other hand, in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congeneic B10-series strains, parental lymphocytes of H-2k haplotype, irrespective of their non-H-2 haplotype, invariably could not induce the GvHR-associated immunosuppression. Furthermore, it was revealed that non-H-2 genes of parental C3H or AKR incorporated in the F1 mice determine the resistance of the F1 mice against the H-2k-induced GvHR. The results of examination of the resistance on individual (B10 X [B10.BR X C3H/He]F1) and (B10 X [B10.BR X AKR/J]F1) mice suggested that three non-H-2 loci of C3H/He or two non-2 loci of AKR/J incorporated in F1 hybrids could determine the resistance of the respective F1 mice.     

Relationship between genetic differentiation of the thymus in mice of different strains and malignant growth. IV. Genetic analysis of the thymic index in mice]

Relative thymus weight was estimated in C3H/He, C57BL/6 mice, their F1 and backcross hybrids, as well as in the progeny of complete diallel crosses between the BALB/c, C3H/He, C57BL/6 and AKR/J strains. On the basis of the analysis of these measurements, a conclusion is drawn that this character is inherited with incomplete dominance of smaller relative weight. The genes determining greater thymus weight are concentrated in the genetic pool of AKR/J and C57BL/6 strains. These genes are characterized by some degree of recessivity with respect to the genes determining smaller thymus weight which are concentrated in the genetic pool of C3H/He and BALB/c strains. The highest concentration of the "plus" and "minus" genes is found in the genetic pools of AKR/J and C3H/He strains respectively.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila.

Peritoneal macrophages obtained from lipopolysaccharide (LPS)-low responder C3H/HeJ mice (J) permitted the intracellular growth of the bacterium in macrophages of (J x N) F1 progeny was between the parent strains, showing that the traits were co-dominantly expressed. Correlation between intracellular bacterial growth in macrophages and LPS response of spleen cells was examined. Negative correlation was found between the two factors in F2, (J x F1) backcross and (N x F1) backcross progeny. This result implies that Lps gene controls the innate resistance of murine macrophages against the bacteria. Although macrophages of A/J strain also permit intracellular growth of L. pneumophila, gene complementation analysis of A/J and C3H/HeJ mice made clear that the gene control in C3H/HeJ differs from that of A/J strain. Macrophages of C57BL/10ScN, which is LPS-low responder line obtained from C57BL/10, were also defective in controlling the bacterial growth when compared to C57BL/10 mice. We suggest that the Lps gene also controls the natural resistance of murine macrophages against L. pneumophila.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

Chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in DBA/2 mice.

Dystrophic cardiac calcinosis (DCC) occurs in certain inbred strains of mice, including DBA/2 and C3H/He, and is generally found as an incidental lesion in adult animals at necropsy. Preliminary genetic studies into the cause of DCC have been performed in DBA/2 mice and suggest that DCC is inherited as an autosomal recessive trait involving three or four unlinked genes. To investigate the genetics of DCC further, we produced myocardial cell death by freeze-thaw injury to induce DCC. Experiments were conducted with three F1 hybrids made using three inbred strains of mice (DBA/2J and C3H/HeJ, DCC-susceptible strains; C57BL/6J, DCC-resistant strain) to compare the genetic factors in the development of DCC. We found that DBA/2 and C3H/He mice share the same gene pattern(s) that is responsible for DCC. We determined by backcross linkage analysis in DBA/2 and C57BL/6 mice that at least one recessive locus is responsible for DCC. A haplotype analysis of the backcross data demonstrated that the recessive locus, designated dyscalc1, is located on Chromosome 7, 20.5 cM distal to the centromere. The likely candidate genes for dyscalc1 are discussed. Further understanding of the structure and function of these mutant genes will be beneficial in explaining the molecular pathogenesis of DCC.     

Genetics of dystrophic epicardial mineralization in DBA/2 mice

The genetics of dystrophic epicardial mineralization in mice was studied using 6 to 8-week-old hybrids and recombinant inbred strains derived from DBA/2J (high prevalence) and C57B1/6J (low prevalence) mice. DBA/2J mice of both sexes were uniformly affected. No cases were seen among 32 F1 mice and 82 F2 mice. Six out of 31 backcross progeny obtained from F1 females backcrossed to DBA/2J males were affected. Two out of 25 recombinant inbred strains were affected. These results suggest that dystrophic epicardial mineralization is determined by three or four unlinked autosomal recessive alleles.     

AKR/J gene(s) unlinked to H-2 determines dominant inheritance of lymphocyte hyporesponsiveness to acetylcholine receptor

Mice with the H-2b major histocompatibility complex haplotype are high immune responders to nicotinic acetylcholine receptors (AChR), whereas mice with the H-2k haplotype are generally low responders. F1 progeny of C57BL/6 (H-2b) mice crossed with mice of most H-2k strains are high responders to AChR in standard conditions of testing helper T cell proliferation in vitro (4 X 10(5) lymph node cells/microwell, 1 wk after primary challenge in vivo). In contrast, the F1 progeny of AKR/J (H-2k) crossed with high responder (H-2b) strains (B6, A.BY, or C3H.SW) were all hyporesponsive to AChR when lymphocytes were tested at 4 X 10(5) cells/well. However, at a density of 1 X 10(6) or greater/well, a high level of antigen-specific responsiveness was demonstrable in the F1 hybrid lymphocytes. A shift from low to high responsiveness to AChR at high cell densities was observed also in the H-2b strain AKR.B6. Other strains previously demonstrated to be low responders to AChR did not become responsive to AChR when lymphocyte numbers were increased to 1.4 X 10(6)/well. The N2 generation yielded by backcrossing (AKR X B6)F1 mice to AKR/J were all low responders, whereas N2 progeny derived by backcrossing F1 to B6 were high or low responders in a ratio of approximately 1:1 (independent of their H-2 phenotype). Results consistent with this observation were obtained in (AKR X B6) F2 mice. These data suggest that at least one AKR/J gene outside of the H-2 complex exerts a hyporesponsive influence on the I-A-dependent helper T cell response to AChR in H-2b mice.     

Maternally transmitted resistance to lymphoma development in mice of reciprocal crosses of the RF/J and AKR/J strains

Females but not males of the low-lymphoma RF/J strain transmit a non-Mendelian factor which suppresses the development of lymphoma in F1 crosses with mice of the high-lymphoma AKR/J strain. Suppression of lymphoma was also evident in the first backcross generation to the parental AKR strain, but only when (RF female x AKR male)F1 mice had been the female parent. This "maternal resistance factor" was transmitted independently of the dominant, lymphoma-suppressing Fv-1n allele transmitted by both males and females of the RF strain, but the suppressive capacities of the two factors appeared to be additive. In this cross, F1 progeny of RF females also showed marked suppression of ecotropic murine leukemia virus expression by comparison with mice of the reciprocal F1 cross, but this suppression of virus expression was not detected in the lymphoma-suppressed AKR backcross population. The observation of lymphoma suppression in the absence of ectropic virus suppression in mice of the (RF X AKR)F1 female x AKR male backross generation indicates a qualitative or quantitative difference in the determination of these two effects.     

Genetic analysis of intestinal cholesterol absorption in inbred mice.

A genetic mapping strategy was employed to identify chromosomal regions harboring genes that influence the absorption of intestinal cholesterol in the mouse. Analysis of seven inbred strains of male mice (129P3, AKR, BALB/c, C3H/He, C57BL/6, DBA/2, and SJL, all from Jackson Laboratories) revealed substantial differences in their abilities to absorb a bolus of cholesterol delivered by gavage. Crosses between high (AKR, 129) and low (DBA/2, SJL) absorbing strains revealed evidence for the presence of dominant genes that increase and decrease cholesterol absorption. Backcrosses between F1 offspring and parental strains (DBA/2xAKD2F1 and 129xSJL129F1) followed by linkage analyses revealed four quantitative trait loci that influenced cholesterol absorption. Analyses of recombinant inbred strains identified an additional three loci affecting this phenotype. These seven quantitative trait loci, which map to different chromosomes and are termed Cholesterol absorption 1-7 (Chab1-7) loci, together influence the absorption of intestinal cholesterol in mice and are likely to be involved in different steps of this complex pathway.     

Mitogenic,immunoadjuvancy, and genetic studies on fatty acyl maltose

Summary Three synthetic glycolipids, maltose tetrapalmitate (MTP), maltose hexastearate (MHS), and maltose hexalinoleate (MHL) prepared as nontoxic lipid A analogs, and Escherichia coli lipopolysaccharide (LPS) were assayed for their mitogenic activity using spleen lymphocytes in nine inbred mouse strains and three F1 hybrids. The MTP and LPS were also assayed for their ability to enhance plaque-forming cell (PFC) responses using sheep red blood cells as the antigen in th same inbred mouse strains and F1 hybrids, The mitogenic activity of synthetic glycolipids was several fold lower than that of LPS and MHL was inferior to MTP and MHS. DBA/2J was the most responsive strain for MTP and DBA/1J and C3H/HeJ the least. The mitogenic activity of MTP was generally in agreement with the PFC response stimulation by it. Lowdose cyclophosphamide treatment of mice synergized MTP for PFC response augmentation. Genetic studies on MTP mitogenicity revealed that 90% of responder DBA/2J X nonresponder C3H/HeJ F1 hybrids had intermediate mitogenic activity. Among F2, 73% had intermediate-high activity and 27% were nonmitogenic. Among F1 X C3H/HeJ backcrosses 11% had high, 56% intermediate, and 33% had no mitogenic activity, whereas, for the F1 X DBA/2J backcross, 14% had high, 36% intermediate, and 50% low or negligible activity. The data favored a single gene for MTP activation of immune cells.This work was supported, in part, by a grant from the National Cancer Institute of Canada, and by grant from the Cancer Research Society Inc.     

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y^MRL), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y^MRL and MRL-Y^B6. Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y^B6 mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y^MRL. Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8?C17?cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y^MRL.     

Two genes controlling the expression of extended globoglycolipids in mouse kidney are closely linked to each other on chromosome 19

The gene (Gsl-5) controlling the expression of GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) in mouse kidney was suggested to be located near Ea-4 on mouse chromosome 19 by the results of glycolipid analysis of BXD/Ty recombinant inbred strains (Sekine et al. [1987] J. Biochem. 101, 563-568). In this study, Gsl-5 was mapped on mouse chromosome 19. Among 133 backcross progeny produced on mating between DBA/2 mice and (WHT/Ht x DBA/2)F1 mice, 10 recombinants between Lyt-1 and Gsl-5 were detected, indicating that Gsl-5 is located at 7.5 +/- 2.3 centimorgans (cM) from Lyt-1. While among 154 backcross progeny produced on mating between DBA/2 and (DBA/2 x Mus musculus castaneus)F1 mice, 39 recombinants between Got-1 and Gsl-5 were obtained, indicating that the distance between Got-1 and Gsl-5 is 25.3 +/- 3.5 cM and that Gsl-5 is telomeric to Lyt-1. In the latter mating experiment, we detected 3 recombinants between Gsl-5 and the gene (Gsl-6) controlling the expression of the Z1 ganglioside (NeuGc alpha 2-3Gal beta 1-3Gb4Cer) among the 154 backcross mice. These results indicate that these two genes, Gsl-5 and Gsl-6, are closely linked to each other, being 1.9 +/- 1.1 cM apart. This is the report of evidence that two genes controlling the expression of carbohydrates in glycoconjugates are closely linked and the first to suggest that some genes controlling the expression of carbohydrates may be clustered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y^MRL), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y^MRL and MRL-Y^B6. Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y^B6 mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y^MRL. Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8–17 cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y^MRL.

     

Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice

An increased airway response to various bronchoconstricting agents is one of the hallmarks of asthma. An interdependence of heredity and environment appears to determine this nonspecific hyperreactivity of the airways. The present study describes the patterns of inheritance of the airway response to a direct mediator of smooth muscle contraction (acetylcholine) in A/J and C3H/HeJ inbred mice and their offspring. The mean airway response to acetylcholine was greater than sixfold higher in A/J mice as compared with C3H/HeJ mice. Two phenotypes were easily distinguished on the basis of airway responses to acetylcholine in the progeny of A/J and C3H/HeJ mice. These two phenotypes were termed HYPERREACTIVE (after the A/J strain) and HYPOREACTIVE (after the C3H/HeJ strain). The observed frequencies of HYPERREACTIVE and HYPOREACTIVE phenotypes in the (A/J x C3H/HeJ) F1; (C3H/HeJ x A/J) F1 x C3H/HeJ (C3H/HeJ backcross); and the [(A/J x C3H/HeJ) F1 x (C3H/HeJ x A/J) F1] F2 are consistent with a single autosomal recessive gene primarily controlling acetylcholine-mediated airway responses. This single gene difference in airway response is completely inhibited by atropine and therefore mediated entirely by the muscarinic acetylcholine receptor.     

A locus on distal chromosome 11 (ahl8) and its interaction with Cdh23 ahl underlie the early onset, age-related hearing loss of DBA/2J mice

The DBA/2J inbred strain of mice is used extensively in hearing research, yet little is known about the genetic basis for its early onset, progressive hearing loss. To map underlying genetic factors we analyzed recombinant inbred strains and linkage backcrosses. Analysis of 213 mice from 31 BXD recombinant inbred strains detected linkage of auditory brain-stem response thresholds with a locus on distal chromosome 11, which we designate ahl8. Analysis of 225 N2 mice from a backcross of (C57BL/6JxDBA/2J) F1 hybrids to DBA/2J mice confirmed this linkage (LOD>50) and refined the ahl8 candidate gene interval. Analysis of 214 mice from a backcross of (B6.CAST-Cdh23 Ahl+ xDBA/2J) F1 hybrids to DBA/2J mice demonstrated a genetic interaction of Cdh23 with ahl8. We conclude that ahl8 is a major contributor to the hearing loss of DBA/2J mice and that its effects are dependent on the predisposing Cdh23 ahl genotype of this strain.     

Identification of Hepatocarcinogen-Resistance Genes in Dba/2 Mice

Male DBA/2J mice are ~20-fold more susceptible than male C57BL/6J mice to hepatocarcinogenesis induced by perinatal treatment with N,N-diethylnitrosamine (DEN). In order to elucidate the genetic control of hepatocarcinogenesis in DBA/2J mice, male BXD recombinant inbred, D2B6F(1) X B6 backcross, and D2B6F(2) intercross mice were treated at 12 days of age with DEN and liver tumors were enumerated at 32 weeks. Interestingly, the distribution of mean tumor multiplicities among BXD recombinant inbred strains indicated that hepatocarcinogen-sensitive DBA/2 mice carry multiple genes with opposing effects on the susceptibility to liver tumor induction. By analyzing D2B6F(1) X B6 backcross and D2B6F(2) intercross mice for their liver tumor multiplicity phenotypes and for their genotypes at simple sequence repeat marker loci, we mapped two resistance genes carried by DBA/2J mice, designated Hcr1 and -2, to chromosomes 4 and 10, respectively. Hcr1 and Hcr2 resolved the genetic variance in the backcross population well, indicating that these resistance loci are the major determinants of the variance in the backcross population. Although our collection of 100 simple sequence repeat markers allowed linkage analysis for ~95% of the genome, we failed to map any sensitivity alleles for DBA/2J mice. Thus, it is likely that the susceptibility of DBA/2J mice is the consequence of the combined effects of multiple sensitivity loci.     

Genetic Factors Controlling the Proliferative Activity of Mouse Epidermal Melanocytes during the Healing of Skin Wounds

A cut was made on the middorsal skin of newborn mice of strains C57BL/10J, C57BL/10J-A/A, and C3H/He using fine iridectomy scissors. In the epidermis within 1 mm of the wound edge in C57BL/10J and C57BL/10J-A/A, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically until the 3rd day, then gradually decreased. In contrast, the melanocyte population of C3H/He did not increase after wounding, despite that the melanoblast-melanocyte population increased. Pigment-producing melanocytes in mitosis were frequently found in C57BL/10J and C57BL/10J-A/A, but not in C3H/He. The F1, F2, and backcross matings were performed to get some information about the genetic basis of the difference between C57BL/10J and C3H/He. In the F1 generation the offspring from reciprocal crosses exhibited intermediate values in both populations on the 3rd day after wounding. The F2 generation included the C3H/He type, F1 type, and C57BL/10J type in a ratio of 1:2:1 in both populations. Moreover, both reciprocal backcrosses gave 1:1 ratios of parent type to F1 type in both populations. These results indicate that the proliferative activity of mouse epidermal melanocytes during the healing of skin wounds are controlled by semidominant genes.     

Presphenoidal synchondrosis fusion in DBA/2J mice

Cranial base growth plates are important centers of longitudinal growth in the skull and are responsible for the proper anterior placement of the face and the stimulation of normal cranial vault development. We report that the presphenoidal synchondrosis (PSS), a midline growth plate of the cranial base, closes in the DBA/2J mouse strain but not in other common inbred strains. We investigated the genetics of PSS closure in DBA/2J mice by evaluating F1, F1 backcross, and/or F1 intercross offspring from matings with C57BL/6J and DBA/1J mice, whose PSS remain open. We observed that PSS closure is genetically determined, but not inherited as a simple Mendelian trait. Employing a genome-wide SNP array, we identified a region on chromosome 11 in the C57BL/6J strain that affected the frequency of PSS closure in F1 backcross and F1 intercross offspring. The equivalent region in the DBA/1J strain did not affect PSS closure in F1 intercross offspring. We conclude that PSS closure in the DBA/2J strain is complex and modified by different loci when outcrossed with C57BL/6J and DBA/1J mice.     

Genetic and biochemical studies of the adrenal lipid depletion phenotype in mice

Study of F₁ and backcross progenies from crosses between DBA/2 and strains carrying the adrenal lipid depletion (ald) gene indicated that the adrenal lipid depletion character is due to the operation of two or more interacting gene loci. In C57BL/10, DBA/2, and their F₁ hybrids, variation in adrenocortical sudanophilia was highly correlated with both cholesterol ester content and the capacity for the production of corticosterone in vitro. In F₁×DBA/2 backcross progeny, however, the degree of adrenocortical sudanophilia remained correlated only with cholesterol ester content and not with the capacity for corticosterone production. Thus, in these strains, adrenal cholesterol ester concentration is not necessarily the determining factor of corticosterone production in vitro; the two phenotypic characters are controlled by independent genetic mechanisms.This work was supported by MH 10976 and HD 00801 (U.S.P.H.S.). J.G.M.S. is a recipient of a Wellcome Foundation Travel Grant. R.B.C. is a recipient of U.S.P.H.S. Research Scientist Award K5-47, 413 of the National Institutes of Mental Health.     

Importance of suppressor T cells in cyclophosphamide-induced tolerance to the non-H-2-encoded alloantigens. Is mixed chimerism really required in maintaining a skin allograft tolerance?

Mechanisms of cyclophosphamide (CP)-induced tolerance were studied. When AKR/J Sea (AKR: H-2k) mice were primed i.v. with 5 x 10(7) spleen cells plus 1 x 10(7) bone marrow cells from [C57BL/6 Slc (B6; H-2b) x C3H/He Slc (C3H; H-2k)]F1 (B6C3F1) mice and treated i.p. with 200 mg/kg CP 2 days later, the survival of C3H skin was moderately prolonged, but the survival of either B6 or B6C3F1 skin was not prolonged. By this treatment, however, mixed chimerism of B6C3F1 cells in the AKR mice was not established. When C3H cells were used as the tolerogen, a minimal degree of mixed chimerism associated with profound tolerance to C3H skin was established. Similar results were observed in various donor-recipient combinations. When C3H skin was grafted in the AKR mice 12 wk after the treatment with C3H cells and CP, or B6C3F1 cells and CP, survival of the grafted C3H skin was prolonged remarkably or moderately, respectively, although mixed chimerism was not detectable at the timing of grafting in either of the groups. In this late stage of tolerance, a strong level of tolerogen-specific suppressor cell activity was observed in those tolerant AKR mice. The suppressor activity was mainly attributable to T cells. These results suggest that the role of Ts cells in order to maintain skin tolerance is important in our CP-induced tolerance system, especially in the late stage of tolerance. Moreover, the generation of the Ts cells does not necessarily require the establishment of a long term mixed chimeric state.