Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes

We have begun to dissect the cellular requirements for generation of immunity against enteric infection by Listeria monocytogenes using a novel T(-) B(-) NK(-) mouse strain (mice double deficient for the common cytokine receptor gamma-chain (gamma(c)) and the recombinase-activating gene-2 (RAG2/gamma(c) mice). Initial experiments showed that C57BL/6 mice and alymphoid RAG2/gamma(c) mice had similar kinetics of bacterial accumulation in the spleen, liver, and brain early after intragastric L. monocytogenes infection (up to day 3), calling into question the physiologic role of gut-associated lymphoid cells during the passage of this enterobacterium into the host. However, in contrast to C57BL/6 mice, RAG2/gamma(c) mice rapidly succumbed to disseminated infection by day 7. Polyclonal lymph node CD4(+) and CD8(+) alphabeta T cells were able to confer RAG2/gamma(c) mice with long-lasting protection against enteric L. monocytogenes infection in the absence of gammadelta T, NK, and NK-T cells. Moreover, these alphabeta T-reconstituted RAG2/gamma(c) mice produced IFN-gamma at levels comparable to C57BL/6 mice in response to L. monocytogenes both in vitro and in vivo. Protection was IFN-gamma dependent, as RAG2/gamma(c) mice reconstituted with IFN-gamma-deficient alphabeta T cells were unable to control enteric L. monocytogenes infection. Furthermore, alphabeta T cell-reconstituted RAG2/gamma(c) mice were able to mount memory responses when challenged with lethal doses of L. monocytogenes. These data suggest that NK, NK-T, gammadelta T, and B cells are functionally redundant in the immunity against oral L. monocytogenes infection, and that in their absence alphabeta T cells are able to mediate the early IFN-gamma production required for both innate and adaptive immunity.     

An X-encoded alloantigenicity between BALB/c and C57BL/6 strains of mice

To detect minor barriers to histocompatibility that might be encoded on the X chromosome in mice, we grafted reciprocal sets of (C57BL/6xBALB/c)F1, (C57BL/6xDBA/2)F1, and (BALB/cxDBA/2)F1 mice with tail skin from the respective paternal inbred strain. Our histogenic analysis suggests that, compared with the C57BL/6 mouse strain, the BALB/c strain generates X-linked antigen loss. In contrast, we detected no X-linked histogenic differences between strains C57BL/6 and DBA/2, or DBA/2 and BALB/c. To localize this X-linked barrier to histocompatibility, we produced a panel of 25 [(BALB/cxC57BL/6)F1xC57BL/6]N2 males that were grafted with C57BL/6 skin to determine which carried the BALB/c-derived component(s) necessary for graft rejection. DNA marker analysis showed one region of overlapping BALB/c-derived X-chromosomal segments among the graft rejecters, suggesting that this antigen-loss haplotype ( H-hix(c), for histoincompatibility on the X chromosome, c haplotype) may be restricted within the DXMit55 to the Xq telomere interval (which excludes only the centromeric tip of the X). Further backcrossing of H-hix(c) to C57BL/6 resulted in fewer rejecter mice than expected by the N4 generation, suggesting that a second, unlinked locus is also involved in this X-linked alloantigenicity. The vigorous rejection of male (C57BL/6xBALB)F1 and female (B6.C- H2(d)xC57BL/6)F1 skin by (BALB/cxC57BL/6)F1 males, as well as the assessment of markers on Chromosome 17 among N2 and N4 graft-recipient males, suggests that this second locus is H2, and that H-hix(b)-encoded alloantigens require both H2(b) and H2(d)-encoded presentation molecules for efficient graft rejection.     

Background matters: the effects of estrogen receptor alpha gene disruption on male sexual behavior are modified by background strain

One approach to study interactions between behavior and genetics is to use inbred mice with different genetic backgrounds. To examine the effect of background on a specific gene, we conducted a series of experiments with a well-characterized knockout (KO) mouse, the estrogen receptor alpha KO (ERalphaKO). The ERalphaKO mouse has so far been examined in one inbred line, C57BL/6J. Here, we examined the behavior of ERalphaKO mice within three different backgrounds mixed with C57BL/6J; DBA/2J, BALB/c, and A/J. First, we assessed masculine sexual behavior in both intact male and testosterone-treated female offspring. More ERalphaKO males in the DBA/2J (5/12) and BALB/c (5/13) backcrosses displayed intromissions and many ejaculated as compared with males in a C57BL/6J and A/J mixed background. Many fewer ERalphaKO females than males displayed masculine sexual behavior in any of the three hybrid crosses. We assessed fertility in males from the C57BL/6J by DBA/2J cross and found that one of 12 ERalphaKO males sired a litter. Several other characteristics of sexual behavior and physiology were unaffected by genetic background in ERalphaKO mice. Our data suggest that genetic background has dramatic effects on male sexual behavior and its dependence on the ERalpha gene.     

Inheritance of hydronephrosis in the inbred mouse strain DDD

The mode of inheritance of hydronephrosis was investigated by crossing inbred DDD mice having a high incidence of hydronephrosis and C57BL/6 mice having normal kidneys. In the males, incidences of hydronephrosis in F1 animals were intermediate between the two parental strains at a rate of 32.6% in (DDD x C57BL/6)F1 and 23.4% in reciprocal F1. The same tendency was observed in F2 male animals. In BCF1 males, the number of affected mice was higher in (C57BL/6 x DDD) F1 x DDD (72.4%) than in (DDD x C57BL/6)F1 x C57BL/6 (11.1%). A few affected mice were found among the females of hybrids F1, F2 and BCF1. These results suggested that hydronephrosis in the DDD strain of mice was controlled by polygenes, and that male hormones may have some effect on the occurrence of hydronephrosis.     

Further evidence that BALB/c and C57BL/6 gamma 2a genes originate from two distinct isotypes.

Gene conversion by the corresponding gamma 2b gene has been proposed to explain the multiple differences between the nucleic acid sequences of BALB/c (Igh-1a) and C57BL/6 (Igh-1b) gamma 2a immunoglobulin allelic genes. However, genetic analysis indicates that duplicated forms of gamma 2a genes are not only present in Eastern Asia, but also in European wild mouse populations which suggests a widespread phenomenon. In order to verify whether the gamma 2a-related isotypic genes, namely gamma 2c and gamma 2a, could correspond to those present as alleles in domestic mice (Igh-1b and Igh-1a), a genomic library from Mus m.musculus strain (MAI) was constructed. Extensive mapping of the recombinant phages and Southern blot analysis with several restriction enzymes gave the complete organization of these loci: gamma 2b (18 kb) gamma 2c (17 kb) gamma 2a (14 kb) epsilon. The homology in flanking, coding and intervening region sequences indicates that MAI gamma 2c and gamma 2a related genes correspond to C57BL/6 and BALB/c Igh-1 alleles respectively. Also, Southern blot analysis using several probes derived from exonic and intronic regions between gamma 2b and gamma 2a genes shows a 2.0- to 3.0-kb difference in the distance between gamma 2b and gamma 2a genes of BALB/c strain as compared to C57BL/6. Taken together, these results indicate that BALB/c and C57BL/6 gamma 2a genes could originate from different isotypes.     

Genetic factors influence cataract formation in alpha 3 connexin knockout mice

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.     

Immunologic Control of Mus musculus Papillomavirus Type 1

Persistent papillomas developed in ~10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection.     

Dominantly inherited expression of BID, an invariant undiversified T cell receptor delta chain

In BALB/c lung and lymph node gamma delta T cells, a large fraction of the expressed V delta 5 genes consist of an invariant sequence, BID (for BALB/c invariant delta). BID results from a direct joining of the V delta 5, D delta 2, and J delta 1 segments, which conserve their complete germline coding sequences. In C57BL/6 (H-2b) mice, where identical and functional segments are present in the germline, BID is absent. It appears that BID+ gamma delta T cells are positively selected by factors encoded outside of the classical MHC region, as indicated by their dominance in F1(C57BL/6 x BALB/c) and in BALB.B (H-2b) mice. Additional observations, including the expression of BID in BALB/c nu/nu but not in C57BL/6 nu/nu mice, suggest that the expansion of BID+ T cells essentially occurs extrathymically.     

The reproductive performance of XX/XY male chimeric mice

The reproductive performance of male aggregation chimeric mice was examined. C57BL/6 in equilibrium BALB/c male chimeras and control animals, C57BL/6, BALB/c, and their reciprocal F1 crosses, were mated with ICR females. Of 45 overt chimeras, 13 produced mixed-genotype progenies and were revealed to be XY/XY chimeras. By karyotype analysis 16 of 32 single-genotype progeny chimeras were determined to be XX/XY chimeras, but the remaining single-genotype progeny chimeras showed only XY metaphase plates, so that their chromosomal sex could not be determined. The mean litter size of C57BL/6 was significantly higher than that of BALB/c. In contrast, the birth rate of C57BL/6 was lower than that of BALB/c. XY/XY chimeras showed almost the same performance as C57BL/6 for litter size and as BALB/c for birth rate. There were no significant differences for both traits between the reciprocal F1 crosses and XY/XY chimeras. The mean litter size of XX/XY chimeras was lower than that of XY/XY chimeras and the differences was statistically significant. Some XX/XY chimeras had very small testes, while XY/XY chimeras had normal testes. Such results indicate that the reproductive performance of XX/XY male chimeras is inferior to that of XY/XY males.     

Langerhans cells that have matured in vivo in the absence of T cells are fully capable of inducing a helper CD4 as well as a cytotoxic CD8 response

Langerhans cells (LCs) are immature dendritic cells (DCs) present in the skin epithelium. Upon Ag exposure, they migrate to the draining lymph nodes where they mature into potent stimulators of naive T cells. The aim of this study was to investigate the influence of T cells on LC migration and maturation. Therefore, the in vivo migration and maturation of LCs after sensitization with the hapten FITC was compared between C57BL/6 or BALB/c mice used as positive controls, and recombination activating gene (RAG) 1 knockout (-/-) mice or SCID mice used as T cell-deficient mice. Phenotypically, there was no difference between migrated LCs from RAG1-/- or SCID mice vs normal C57BL/6 or BALB/c mice: both populations of FITC+ cells had a dendritic morphology and a mature phenotype as they expressed high levels of MHC class II molecules and costimulatory molecules CD80, CD86, and CD54. Sorted migrated LCs of RAG1-/- or SCID mice were efficient stimulators of allogeneic T cells and Ag-specific CD4+ T cells. The same results were found if migrated LCs were fixed instead of irradiated, excluding the possibility that LCs derived from RAG1-/- or SCID mice would mature in the presence of T cells during the stimulation tests. Importantly, fixed migrated LCs of RAG1-/- mice were also efficient stimulators of cytotoxic CD8+ T cells. These data suggest that T cells are not required for full maturation of LCs.     

Strain-dependent differences in responses to exercise training in inbred and hybrid mice

The aim of this study was to characterize the response to exercise training in several mouse strains and estimate the genetic contribution to phenotypic variation in the responses to exercise training. Male mice from three inbred strains [C57Bl/6J (BL6), FVB/NJ (FVB), and Balb/cJ (Balb/c)] and three hybrid F(1) strains [CB6F1/J (CB6 = female Balb/c x male BL6), B6F F(1) (female BL6 x male FVB), and FB6 F(1) (female FVB x male BL6)] completed an exercise performance test before and after a 4-wk treadmill running program. Distance was used as the primary estimate of endurance exercise performance. FVB mice showed the greatest response to training, with five- to sevenfold greater increases in distance run compared with BL6 and Balb/c strains. Specifically, BL6, FVB, and Balb/c strains increased distance by 33, 172, and 23%, respectively. A similar pattern of changes across strains was observed for run time (17, 87, and 11%) and work (99, 287, and 57%). As a group, F(1) hybrid mice derived from BL6 and FVB strains showed an intermediate response to training (61%). However, further analysis indicated that training responses in FB6 F(1) mice (80%) were approximately 2.5-fold greater than responses in B6F F(1) mice (33%, P = 0.08). A similar pattern of changes between FB6 and B6F F(1) mice was observed for run time (44.5 and 17%) and work (141 and 59%). These data demonstrate that there are large strain-dependent differences in training responses among inbred mouse strains, suggesting that genetic background contributes significantly to adaptation to exercise. Furthermore, the contrasting responses in B6F and FB6 F(1) strains show that a maternal component strongly influences strain-dependent differences in training responses.     

Strain variation in interferon gamma production of BCG-sensitized mice challenged with PPD II. Importance of one major autosomal locus and additional sexual influences

Interferon-gamma (IFN-gamma) production in BCG-sensitized mice challenged with PPD was examined in the sera from BALB/c and C57BL/6 mice. C57BL/6 mice produce about ten times more IFN-gamma than BALB/c mice. Studies on F1, F2, and backcross generations indicate that one partially dominant autosomal locus is involved. Furthermore, females consistently produce more IFN-gamma than males in all of these crosses, though the X chromosome cannot be held responsible for this.     

Absence of CD43 fails to alter T cell development and responsiveness

Genetic elimination of CD43 has been associated with increased T cell adhesiveness and T cell hyperresponsiveness to mitogens and alloantigens. Therefore, we investigated whether T cell development was perturbed in CD43-deficient mice by breeding CD43(null) mice with male Ag (Hy)-specific TCR-transgenic mice. Neither positive nor negative thymic selection of male Ag-specific T cells were affected by CD43 status. Furthermore, we did not observe a substantial or consistent hyperresponsive pattern in HY-CD43(null) lymph node cells compared with littermate HY-CD43(+/-) lymph node cells upon analysis of in vitro T cell stimulation with male Ag or mitogen. These observations challenged original conclusions associating absence of CD43 with T cell hyperresponsiveness and led us to re-examine this association. Reported phenotypes of CD43(null) mice have been based on mice with a mixed 129xC57BL/6 genetic background. To exclude a possible influence of genetic background differences among individual mice we analyzed CD43(null) littermates that had been back-bred onto the C57BL/6 background for seven to eight generations. We found that CD43(+) and CD43(null) littermates with the C57BL/6 background exhibited no differences in response to mitogen or alloantigen, thereby establishing that T cell hyperresponsiveness is not a general correlate of CD43 absence.     

Genotype-dependent mice behavior in cognitive tasks. Effect of noopept

The interstrain differences in performance of C57BL/6J, BALB/c and DBA/2J male mice in two cognitive tasks were found. Mice C57BL/6J showed good learning ability and preservation of memory traces tested 10 days after performance in a simplified version of Morris water maze. Mice BALB/c learned the task but, virtually, no long-term memory traces were revealed, whereas DBA/2J demonstrated poor learning. The effect of nootropic drug Noopept (GVS-111, N-phenil-acetyl-L-prolylglycin ethyl ether) was shown to be genotype-dependent. Its administration (0.5 mg/kg i.p., 15 min before learning) improved the long-term memory in Morris test in BALB/c mice but failed to produce any improvement in C57BL/6J. The ability of mice for extrapolation of the direction of stimulus movement differently changed after Noopept injections: the proportion of correct task solutions increased in C57BL/6J and BALB/c mice, whereas the performance of DBA/2J did not change.     

Strain differences of ether susceptibility in mice

The susceptibility to ether in the following six strains of mice was tested: C57BL/6, DBA/2, BALB/c, C3H/He, ICR and ddY. Mice of 4 weeks old were exposed to a flow of air containing various concentrations of ether for 90 min and the mortalities were assessed. The C57BL/6 strain was the most resistant and the C3H/He strain was the most sensitive to the lethal effect of ether. The susceptibilities of the closed colony mice, ICR and ddY, were intermediate between those of C57BL/6 and C3H/He mice. The DBA/2 and BALB/c strains were more sensitive than these closed colony mice and made up a sensitive group with the C3H/He strain. The LD50 values for ether in male mice of C57BL/6 and C3H/He were 6.0 and 3.1% atm, and in female mice of these strains were 6.6 and 3.2% atm, respectively. The ED50 value of ether which was accompanied by loss of righting reflex after exposure for 10 min was also higher in male C57BL/6 mice than in male C3H/He mice.     

Different patterns of food consumption and locomotor activity among Taiwanese native rodents, Formosan wood mice (Apodemus semotus), and common laboratory mice, C57BL/6 (Mus musculus)

The comparisons of food consumption and locomotor activity among Taiwan native rodents, Formosan wood mice (Apodemus semotus), and laboratory mice, C57BL/6, were examined in this study. The food consumption exhibited the circadian rhythmicity, e.g. higher in the lights-off period and lower in the lights-on period, in either Formosan wood mice (WM) or C57BL/6 mice. We also found that Formosan WM ate more food than C57BL/6 mice in the lights-off period and the whole day in males, but not in females. Similarly, the male Formosan WMs had more locomotor activities than the male C57BL/6 mice in the lights-off period, but this phenomenon did not appear in female mice. These results indicated that even though the Formosan WMs have been successfully inbred in the laboratory, they still keep more native paradigm than the laboratory C57BL/6 mice do. This study is the first report to provide basic physiological comparisons on native and common laboratory mice.     

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.     

Correlation between the avidity maturation of anti-HSP70 IgG autoantibody and recombination activating gene expressions in peripheral lymphoid tissues of Toxoplasma gondii-infected mice

The avidity maturation of anti-TgHSP70 IgG antibody produced by B-2 cells of BALB/c mice (a resistant strain) and that of anti-mHSP70 IgG autoantibody produced by B-1 cells of C57BL/6 mice (B6; a susceptible strain) was observed after Toxoplasma gondii infection. Recombination-activating genes (RAGs) were predominantly expressed in B-1 cells from peritoneal exudate cells (PECs) of T. gondii-infected B6 mice, while RAGs were expressed in B-2 cells from PECs of BALB/c mice. These results suggest that the involvement of RAG gene activations in the peripheral lymphoid tissues in the avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in T. gondii-infected mice.     

Dimethylnitrosamine metabolism: II. In vitro activation of dimethylnitrosamine by hepatic and renal tissues from crosses among BALB/c, DBA and C57BL mice

Crosses among BALB/c, C57BL and DBA mice were performed to investigate the genetic mechanisms involved in metabolism of DMN by renal and hepatic tissues. Liver S-9 fractions from parental strain DBA had the greatest potential to activate DMN and liver fractions from parental strain BALB/c had the lowest. No age or sex-related differences were observed within strain. Crossing of either C57BL or DBA to BALB/c mice resulted in F1 hybrids with liver microsomal enzymes that gave results similar to the BALB/c parental strain. There were no sex or age differences within crossbred strains in the potential of liver to activate DMN. In contrast male DBA and C57BL parental mice renal S-9 fractions did not differ significantly from each other but did differ significantly from male BALB/c renal fractions and from female and immature animals of all strains. Crossing of either DBA or C57BL mice with BALB/c mice resulted in male F1 hybrids whose renal S-9 fractions did not differ significantly from males of the parental BALB/c strain. In all instances, male renal S-9 fractions had a significantly greater potential to activate DMN than female or immature animals. F1 DBA X C57BL hybrids had renal S-9 fractions that did not differ significantly from the parental strains. These data suggest that the gene(s) for low DMN metabolism of BALB/c mice are apparently dominant over the genes from both DBA and C57BL. The exact genetic or physiological mechanism needs further elucidation.     

Synergism of iron and hexachlorobenzene inhibits hepatic uroporphyrinogen decarboxylase in inbred mice.

The combination of a single subcutaneous dose of iron (12.5 mg/mouse) and subsequent treatment with hexachlorobenzene (0.02% of the diet) caused a progressive inhibition of hepatic uroporphyrinogen decarboxylase in male C57BL/10 mice leading to the accumulation of uroporphyrin in 4-6 weeks. There was only a slight inhibition of the enzyme in the absence of iron and none without hexachlorobenzene. Females were less sensitive than males. In addition, comparisons between the C57BL/10, BALB/c, AKR and DBA/2 strains indicated that the susceptibilities of mice to induction of porphyria did not completely correlate with their classification as Ah-responsive or Ah-non-responsive.