Cutaneous Delayed Type Hypersensitivity in SCID Mice Adoptively Transferred with Lymphocytes is B Cell Independent and H-2 Restricted

Severe combined immunodeficiency (SCID) mice are largely devoid of functional T and B lymphocytes but have normal antigen presenting cell (APC) capacity. The authors have examined the requirements for cutaneous delayed type hypersensitivity (DTH) in SCID recipients by i.p. transfer of freshly isolated naive mature T cells or non-fractionated spleen cells of H-2 compatible or incompatible origin. Recipient SCID mice were epicutaneously sensitized with oxazolone (OXA) within 24 h after cell transfer. SCID mice injected with as few as 10⁵ H-2 compatible BALB/c (H-2^d) spleen cells were able to mount DTH ear swelling reaction upon sensitization and challenge with OXA. Non-fractionated spleen cells from H-2 incompatible B6 or NZW mice were also able to restore DTH capacity in SCID recipients. However, when thymocytes were transferred, only donor mice expressing H-2^d, but not H-2^b and H-2^z, haplotype restored DTH reactivity. Serum levels of IgM and IgM anti-OXA antibodies in SCID mice 27 days post-transfer with 10⁷ BALB/c spleen cells were similar to that of intact donor mice. In contrast, specific antibodies of IgG isotype were approximately only one-tenth of that found in BALB/c controls. The results of this study show that for the development of cutaneous DTH, in SCID mice transferred with T cells, H-2 restricted APC-T cell interaction is required, whereas B cells are not mandatory. Also, SCID mice transferred with splenocytes show signs of defect immunoglobulin switch function. The authors believe that this model of DTH will be useful in delineating the effects of immunomodulatory substances in vivo on distinct host versus donor cell populations.     

Impact of MHC Class I Gene on Resistance to Murine AIDS

Development of murine AIDS in mice following infection with LP-BM5 murine leukaemia virus (MuLV) is highly strain dependent, with strain differences determind by genes within and outside H-2. Among H-2 genes, the D^d gene is the most closely associated with resistance to LP-BM5 MuLV infection. However, the D^d-mediated resistance is highly influenced by outside H-2 genes, i. e. A lineage strains are more resistant than mice strains of B6/B10 lineage. In this study, the mice having BALB background were analysed and, similarly to A lineage mice, only D^d gene products were found to be required to provide resistance to LP-BM5 MuLV infection. Furthermore, BALB/c Kh mice bearing both D^d and L^d genes clearly showed obviously higher resistance than BALB/c-H-2^dm2 mice solely having the D^d gene. In addition, in the long-term observation of the effect of the D^d gene on B6/B10 background mice, D8 mice having the D^d gene as a transgene and expressing a high level D^d gene product showed higher resistance than naturally recombinant B10. A(18R) mice. These results suggest that the MAIDS resistance associated with the D end loci is dependent on the level of expression of an MHC class I gene.     

Expression of the H-2 Antigenic Complex on Murine Haematopoietic Stem Cells

We used hybridoma-derived monoclonal antibody to H-2K^k antigens and xenoantibodies to β₂-microglobulin (β₂) to study the expression of the H-2 antigenic molecular complex on murine hematopoietic stem cells and the effect of long-term culture in vitro on the expression of these antigens. Monoclonal anti-H-2K^k antibody produced potent complement-dependent inhibition of pluripotent (CFU-S), myeloid (CFU-C), and erythroid (CFU-E) stem cells from the bone marrow of C3H and AKR (H-2^k) mice and was without effect on stem cells from Balb/C (H-2^d) mice. Anti-β₂m xenoantibodies inhibited stem cells from all three strains. Stem cells could not be 'rescued' from the inhibitory effects of the antibodies by the addition of thymocytes to marrow cells after antibody treatment. Both the anti-H-2K^k monoclonal antibody and the anti-β₂m xenoantibodies produced potent inhibition of AKR (H-2^k) CFU-C that had been maintained in culture for up to 6 weeks. These results indicate that murine CFU-S, CFU-C, and CFU-E express H-2 antigens and that the expression of these antigens by CFU-C is not altered during long-term culture.     

Comparison between the Specificity of Primary and Secondary Killer Cells against Alloantigens and Hapten-modified Syngeneic Lymphoid Cells

Cytotoxic responses of lymphoid cells from different mouse strains against syngeneic cells modified with the haptens fluorescein isothiocyanate and trinitrophenyl were investigated. Mice of the H-2^k strain demonstrated strong primary in vitro hapten-specific cytotoxicity reactions, which were H-2 restricted and involved the K^k specificity. However, cells from H-2^d and H-2^b mice developed hapten-specific cytotoxic reactions that showed H-2 cross-reactivity. This cross-reactivity, with regard to the restriction element, was particularly evident with cells from mice that had been immunized in vivo. No cross-reaction was observed between the two haptens, however. Genetic mapping experiments demonstrated that cross-reactions existed between D^b and D^d target cell antigens. Similar cross-reactions were demonstrated in in vitro experiments in which secondary in vitro responses were induced by stimulation with cross-reacting H-2 antigens. This finding was also investigated in allogeneic cytotoxicity. In vitro induced responses resulting in relatively weak specific cell-mediated lympholysis reactions were H-2 specific, whereas secondary in vitro responses demonstrated cross-reactivity between D^d and D^b antigens. In these test systems, cross-stimulation was also demonstrated in secondary in vitro responses. These results are discussed in terms of similarities of T cell recognition of hapten-modified self antigens and of alloantigens.     

Acute Graft-versus-Host Reaction in SCID Mice Leads to an Abnormal Expansion of CD8+ Vβ14+ and a Broad Inactivation of Donor T Cells Followed by a Host-Restricted Tolerance and a Normalization of the TCR Vβ Repertoire in the Chronic Phase

The persistence and selection of allogeneic CBA/J T lymphocytes were studied during graft-versus-host (GvH) reaction in immunodeficient C. B-17 SCID (SCID) mice. After neonatal injection the donor cells primarily migrated to the spleen plus lymph nodes (SL) and the thymus of the recipients. Thirteen days post engraftment, CD8⁺ cells in SL had increased five times in cell number with an 18-fold increase of CD8⁺ Vβ14⁺ cells, paralleled by clinical signs of GvH disease (GvHD). Donor lymphocytes from these mice were proliferative unresponsive to allogeneic Balb/c or C57B1/6 SL cells, whereas 8 weeks post injection the tolerance was confined to H-2^d specific donor cells. Here, spleens had a total cell content similar to untreated SCID mice but the average percentage of donor cells had reached 25%. Moreover, the CD4/CD8 cell ratio in the donor population in SL and thymus had changed to normal and the TCR Vβ repertoire was similar to that of the originally injected cells. Following secondary transfer into syngeneic CBA/Ca nu/nu recipients donor cells regained a significant but reduced response to H-2^d stimulators indicating that the antigen specific tolerance of allogeneic donor cells in the SCID mice was due, at least in part, to a reversible state of anergy.     

Enhancement of experimental autoimmune encephalomyelitis severity by ultrasound emulsification of antigen/adjuvant in distinct strains of mice

Susceptibility to experimental autoimmune encephalomyelitis (EAE) is associated with the major histocompatibility complex (MHC) haplotype. In this study EAE could be induced in six out of ten mice of the resistant DBA/2 (H-2^d) strain by ultrasound emulsified antigen/adjuvant, whereas none of the mice immunized with the conventional adjuvant developed the disease. Similar results were previously obtained for the MHC identical BALB/c mice. Further, while only few T cells were present in the central nervous systems (CNS) of the diseased DBA/2 mice, macrophages formed the majority of the infiltrates. In congenic BALB.B (H-2^b) and BALB.K (H-2 ^k) mice, EAE could be induced with both sonicated and extruded antigen/adjuvant emulsion. The results indicate that the EAE resistance in mice carrying the H-2^d MHC haplotype is dependent on the physical structure of the immunogen.     

Altered Allogeneic Response in Neonatally Anti-MHC Class I-Treated Mice

Neonatal treatment of C3H mice (H-2^k) with anti-K^k monoclonal antibodies results in altered cytotoxic responses against allogeneic targets. After 2–3 weeks of antibody treatment, no difference in the number of CD4⁺8⁻ or CD4⁻ 8⁺ T cells was observed between the antibody- and saline-treated mice. However, antibody-treated mice had a significantly reduced cytotoxic response against various allogeneic major histocompatibility complex (MHC) class I-expressing targets. The strongest reduction was observed in very young mice (up to 2 weeks of age). As the mice got older, the allo MHC-specific responses reached control levels. No significant changes in T-cell receptor (TCR)-V-region usage was observed even in young antibody-treated mice. The results suggest that the reduction in the number of positively selecting elements reduces alloreactivity and most likely also the diversity of TCR-repertoire. However, the reduced alloresponsiveness was not restricted to either allogeneic K- or D-encoded molecules, suggesting that self MHC-D-region encoded molecules can mediate positive selection of T cells able to react against both K and D region-encoded allogeneic MHC class I molecules.     

A Study on Graft-versus-Host Reaction (GVHR) by Simonsen's Splenomegaly Assay Cells and Antigen Systems Involved in Induction of GVHR

Cells and histocompatibility antigen systems involved in graft-versus-host reactions (GVHR) were analyzed using Simonsen's splenomegaly assay employing various combinations of donor and F₁ hybrid recipients mice. Most of the cells proliferating in spleens of mice undergoing GVHR were J11d⁺, and had histological features of cells of the hematopoietic lineage. The proportions of CD3⁺ T cells were decreased in the spleens. Disparity at minor histocompatibility determinants of AKR, I-E and H-2D regions between B10.A(4R) donors and (4R × AKR) F₁ recipients evoked only negligible GVHR. On the contrary, disparity at H-2K and/or I-A regions appeared to be sufficient to permit induction of full GVHR. When surface markers of donor spleen cells were analyzed, it was shown that Thy-1⁺ and/or MEL-14⁺ cells caused a strong effect on GVHR. Further, either CD4⁺ or CD8⁺ T cell subset could induce significant GVHR. However, synergistic influences of these two T cell subsets on one another in GVHR were observed. The present results raise the possibility of using Simonsen's assay along with a number of reagents to identify the contribution of subsets of T lymphocytes and in analyzing precise contributions of cellular components from both donor and recipient, and also of the target antigen systems of the recipient that contribute to early events involved in GVHR.     

DNA Repair, H-2, and Aging in NZB and CBA Mice

Current evidence suggests that a correlation exists between the capacity to perform excision repair of UV-induced DNA damage and maximum lifespan in different species. Preliminary evidence has also indicated differences of DNA repair capacities in lymphocytes of several strains of mice congenic at the H-2 locus. It is known that the H-2 system influences maximum lifespan potential in mice. In the present studies excision repair of UV-induced DNA damage, but not gamma-induced damage, was found to correlate with mean survival in the adult inbred mouse strains NZB and CBA, using PHA stimulated splenic lymphocytes. Furthermore, in (NZB × CBA)F₂ hybrid adult progeny the level of DNA repair of UV-induced damage corresponded to the H-2 allele (H-2^d/2^d from NZB or H-2^k/2^k from CBA) inherited from the parental strain. These studies suggest the possibility of a tricornered relationship between the main histocompatibility complex, one form of DNA repair, and lifespan within the species.     

Non-Diabetogenic Insulitis in NOD↔BIO. GD Allophenic Mice in Spite of Permissive Class I MHC Antigens

Allophenic mice (embryo aggregation mouse chimeras) enable us to dissect the process of spontaneous autoimmunity under physiological conditions. Our previous experiments showed that the autoimmune process in allophenic mice of the NOD↔C57Bl/6 strain combination does not progress from insulitis to diabetes. One possible explanation for this protection is that H-2 K^d-restricted CD8⁺ T cells kill only NOD ß cells (K^d, D^b) in the chimeric islets, while the B6ß cells (K^b, D^b) are spared from destruction. To test this hypothesis we analysed 22 NOD↔B10. GD chimeras in which the class I MHC are shared by both parental strains. Therefore all the ß cells in these chimeras express H-2 K^d molecules. Ten allophenic mice were killed at 7 weeks and studied for early pathology. No evidence for intra-islet infiltration was obtained at this age, suggesting that the autoimmune process in NOD↔B10. GD chimeras is slower than in NOD mice. Twelve chimeras were followed up for 1 year for disease development and all failed to progress to full-blown diabetes, despite the occurrence of intra-insulitis in six out of 12 mice. The lack of disease in NOD↔B10. GD chimeras demonstrates that class I MHC chimerism does not account for diabetes resistance in NOD-allophenic mice.     

Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.     

TNF microsatellite a2, b3 and d2 alleles are associated with systemic lupus erythematosus

We have investigated TNF microsatellite polymorphisms in SLE and their association with both HLA class II alleles and disease expression. A total of 91 Caucasoid SLE and 109 matched Caucasoid controls were recruited for this study. TNF microsatellites a, b and d were typed using fluorescent based semi-automated gene scanning. TNF a², b³ and d² allele frequencies were significantly increased in the SLE group compared to controls. These alleles were found to be part of an extended HLA-DRB 1*0301 haplotype and have previously been associated with high TNF-α production. When the SLE group was analyzed according to presentation of certain clinical features, photosensitivity and Raynaus phenomenon, the frequency of these alleles (TNF a², b³ and d²) were also significantly increased. No significant increase in the allele frequencies of TNF a², b³ and d² was demonstrated in the group of patients with renal involvement. These data suggest that TNF microsatellite alleles are not independent of HLA associations in SLE and may be important in the expression of certain clinical features in SLE.     

Oligoclonality of TCRint Cells with a Low Diversity of TCR Complementarity-Determining Region 3 in Mice with Graft- Versus-Host Disease

Conventional T cells (i.e. TCR^high) are generated by the main stream of T-cell differentiation in the thymus. However, primordial T cells (i.e. TCR^int) are generated by extrathymic pathways and an alternative intrathymic pathway. Since TCR^int cells contain self-reactive clones, the diversity of the T-cell antigen receptor (TCR) complementarity-determining region (CDR) 3 was examined. The predominant Vβ8.2⁺ clones among TCR^int cells were selected for DNA sequencing. Thymectomized, irradiated mice subjected to bone-marrow transplantation (BMT) were used; graft-versus-host disease (GVHD), B6→(B6 × C3H/He) F ₁ and syngeneic BMT, B6→B6. In these combinations, only TCR^int cells were generated. Vβ8.2⁺ cells with a low diversity of CDR3 of V-gene expanded in GVHD mice. Vβ8.2⁺ cells of TCR^int and TCR^high cells in normal mice were polyclonal, showing that the former has a lower diversity of CDR3 than the latter. The clonality of activated TCR^high cells was examined, in which CD3^high cells (bm12 mice) were injected into 1 Gy-irradiated B6 nude mice. Some Vβ8.2⁺ clones among TCR^high cells were expanding but the diversity of CDR3 was greater than that of CD3^int cells, despite the fact that the recognition site of the H-2 difference was smaller. Taken together with invariant usage of Vα14, these results suggest that TCR^int cells have a low diversity of CDR3 of Vβ genes.     

Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice

L-type calcium currents ( i _Ca) were recorded using the two-microelectrode voltage-clamp technique in single short toe muscle fibres of three different mouse strains: (i) C57/SV129 wild-type mice (wt); (ii) mdx mice (an animal model for Duchenne muscular dystrophy; and (iii) transgenically engineered mini-dystrophin (MinD)-expressing mdx mice. The activation and inactivation properties of i _Ca were examined in 2- to 18-month-old animals. Ca²⁺ current densities at 0 mV in mdx fibres increased with age, but were always significantly smaller compared to age-matched wild-type fibres. Time-to-peak (TTP) of i _Ca was prolonged in mdx fibres compared to wt fibres. MinD fibres always showed similar TTP and current amplitudes compared to age-matched wt fibres. In all three genotypes, the voltage-dependent inactivation and deactivation of i _Ca were similar. Intracellular resting calcium concentration ([Ca²⁺]_i) and the distribution of dihydropyridine binding sites were also not different in young animals of all three genotypes, whereas i _Ca was markedly reduced in mdx fibres. We conclude, that dystrophin influences L-type Ca²⁺ channels via a direct or indirect linkage which may be disrupted in mdx mice and may be crucial for proper excitation–contraction coupling initiating Ca²⁺ release from the sarcoplasmic reticulum. This linkage seems to be fully restored in the presence of mini-dystrophin.     

Residual Minor Histocompatibility Genes Contaminate the B10. AM Congenic Line: No Evidence of C-Region-Controlled Histoincompatibility

After being primed in vivo and restimulated in vitro, cytolytic T lymphocytes (CTL) were produced in the strain combinations B10.AM ami-B10.A(1R) and B10.A(1R) anti-B10.AM. Although the two strains differ in the chromosomal interval between the E_α and the D loci, the CTL are not directed against antigens controlled by loci in this interval. Instead, the CTL detect minor histocompatibility (H) antigens controlled by loci that are notlinked to H-2 . The recognition of the antigens detected by the B10. AM anti-B10.A(lR) CTL is restricted by the K^k and D^b molecules, but the CTL also cross-react with the D^d molecule (or a molecule controlled by a locus closely linked to D^d). The recognition of the antigens detected by these two CTL behave as if controlled by alleles at the same minor H locus or loci. This locus is distinct from H-2 , and the B 10. AM congenic line apparently retained a C3H-derived allele at this locus.     

Differential invasion of Trypanosoma brucei brucei and lymphocytes into the brain of C57BL/6 and 129Sv/Ev mice

Trypanosoma brucei subspecies invade the brain parenchyma at late stages of human and experimental rodent infections. In this study, we compared the outcome of infection with T. b. brucei in MHC-matched (H-2^b) C57BL/6 (B6) and 129Sv/Ev (Sv-129). Sv-129 showed higher parasitaemia and lower specific IgM (but not IgG) antibody levels than B6 mice. The number of trypanosomes, CD4⁺ and CD8⁺ T cells in the brain parenchyma was higher in B6 mice. B6 mice lost weight and showed higher cumulative mortality when compared with Sv-129 mice. Higher levels of IL-1β, IL-6, IL-10, TNF-α, IFN-γ, ICAM-1 and E-selectin, but low levels of TGF-β mRNA were present in brains of B6 when compared with Sv-129-infected mice. Thus, host genetics differentially determine the invasion of T. b. brucei into the brain parenchyma, which is paralleled by the severity of inflammation in the brain and course of the disease, but not by parasitaemia nor by antibody titres.     

Staphylococcal Protein A and Human IgG Subclasses and Allotypes

Staphylocoecal protein A binds molecules belonging to the IgGl, IgG2, and IgG4 sub-classes. IgG3 proteins generally do not bind, except for those coded by the two γ3 alleles, which are G3m(u⁻): G3m(b⁰,b³,b⁵,s,v) and G3m(b⁰,b³,b⁵,s,t,v). G3m(u) is located in the C_H2 domain. The difference between G3m(u⁻) and G3m(u⁺) IgG3 proteins correlates with the sequence at position 339 in the C_H2 domain—Ala and Thr respectively. There is another structural difference in the CH3 domain which correlates with protein A binding and non-binding: all IgG proteins that bind protein A have His at position 435, whereas those that do not, have Arg at that position.     

The kinin B1 receptor contributes to the cardioprotective effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in mice

Recent studies have shown that inhibition of angiotensin-converting enzyme (ACE) or angiotensin II receptors causes upregulation of the B₁ receptor (B₁R). Here we tested the hypothesis that activation of the B₁R partly contributes to the cardiac beneficial effect of ACE inhibitor (ACEi) and angiotensin II receptor blockers (ARB). B₁R knockout mice ( B₁R^−/− ) and C57Bl/6J (wild-type control animals, WT) were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. Three weeks after MI, each strain of mice was treated with vehicle, ACEi (ramipril, 2.5 mg kg⁻¹ day⁻¹ in drinking water) or ARB (valsartan, 40 mg kg⁻¹ day⁻¹ in drinking water) for 5 weeks. We found that: (1) compared with WT mice, B₁R^−/− mice that underwent sham surgery had slightly but significantly increased left ventricular (LV) diastolic dimension, LV mass and myocyte size, whereas systolic blood pressure, cardiac function and collagen deposition did not differ between strains; (2) MI leads to LV hypertrophy, chamber dilatation and dysfunction similarly in both WT and B₁R^−/− mice; and (3) ACEi and ARB improved cardiac function and remodelling in both strains; however, these benefits were significantly diminished in B₁R^−/− mice. Our data suggest that kinins, acting via the B₁R, participate in the cardioprotective effects of ACEi and ARB.     

Cytokine Gene Expression During Infeetion of Miee Lacking CD4 and/or CD8 with Trypanosoma cruzi

The expression of lymphokine genes during infection of virulent (Tulahuén) or mild ( CA-I ) strains of T. cruzi was studied in mice lacking CD4 and/or CD8 molecules. The increased susceptibility of CD4⁻ and CD4⁻ CD8⁻ mice to infection with CA-I or Tuiahuen was parallelled by diminished IFN-γ mRNA levels. Nitric oxide release and inducible nitric oxide synthase mRNA accumulation by cells from Tulahuen infected CD4⁻ mice was also diminished. CD8⁻ (but not CD4⁻ CD8⁻ mice) showed an increased IL-4 and IL-10 mRNA accumulation upon infection with both strains of T. cruzi. A T_h2-like' response (higher IL-4 and IL-10 mRNA to IFN-γ mRNA ratio), was also observed when cells from non-infected CD8 - mice were stimulated with T cell mitogens.