Mapping the extracellular topography of the α-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain

The extracellular surface of theα-chain ofTorpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR. was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptidesα1–16,α89–104,α158–174,α262–276, andα388–408. Lower, but significant, levels of reactivity were obtained with antibodies against peptidesα67–82,α78–93,α100–115, andα111–126. On the other hand, free AChR bound high levels of antibodies against peptidesα34–49,α78–93,α134–150,α170–186, andα194–210. It also bound moderate levels of antibodies against peptidesα262–276 andα388–408. Low, yet significant, levels of binding were exhibited by antibodies against peptidesα45–60,α111–126, andα122–138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).     

H-2 antigens as genetic markers: A two-dimensional gel electrophoretic study

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of theH-2K andH-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five differentH-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity amongK- andD-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.Abbrevations used in this paper NP-40 nonidet P-40 - 2D two-dimensional - SDS sodium dodecyl sulfate - IEF isoelectric focusing     

Eo: a history of a mutation

Eighteen mouse t haplotype-carrying strains were found not to express cell-surface E molecules controlled by class II genes of the H-2 complex (= Eo strains). Northern and Southern blot analysis of these and other, non-t strains that also fail to express the E molecule, has revealed two kinds of defect. Three strains (CRO437, tw2, and presumably to) were found to transcribe the E alpha gene, but they were not able to convert the message into a functional protein. All other Eo strains fail to transcribe the E alpha gene because of a deletion encompassing the promoter region, the RNA initiation site, and the first exon. The length of the deletion is approximately 650 +/- 50 bp. These two defects closely resemble those found previously in standard inbred strains carrying the H-2f, H-2q (failure of E mRNA to be expressed functionally), H-2b, and H-2s (deletion of a part of the E alpha gene) haplotypes. In particular, the location and length of the E alpha deletion appear to be the same in the strains carrying this mutation. The E alpha deletion is in linkage disequilibrium with certain alleles at other H-2 loci in some of the strains. These observations, combined with the growing evidence that H-2 haplotypes associated with t chromosomes derive from a single ancestral haplotype, suggest that the E alpha deletion is an old mutation and that it has been disseminated in mouse populations by the t chromosomes.     

Specificity of the T cell immune response to acetylcholine receptor in experimental autoimmune myasthenia gravis. Response to subunits and synthetic peptides

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent diseases mediated by antibodies against acetylcholine receptor (AChR) on skeletal muscle. Most of the antibodies are directed toward conformation-dependent epitopes on the AChR, whereas T cells recognize denatured AChR. In search of T cell epitopes in EAMG, we tested 24 synthetic peptides covering 62% of the alpha-subunit sequence of Torpedo californica electric organ AChR in the T cell proliferation assay with lymph node cells from rats immunized with AChR. In Lewis rats, 2 of these peptides, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90, strongly stimulated T cells and, of these, [Tyr 100]alpha 100-116 was much more potent; 4 other peptides were weakly mitogenic and 18 were ineffective. None of the 24 synthetic peptides alone stimulated anti-AChR production and, when added to cultures along with AChR, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90 suppressed antibody production. Of twelve cloned T cell lines specific to AChR, 4 responded to [Tyr 100]alpha 100-116, indicating the importance of the epitope in alpha 101-116 in Lewis rats. In three other strains of rats whose responses to AChR and its subunits were similar to those in the Lewis rat, neither [Tyr 100]alpha 100-116 nor [Gly 89, Tyr 90]alpha 73-90 was stimulatory. Instead, completely different sets of peptides stimulated their T cells. When peptides were used as immunogens, each strain (except Lewis rats) responded only to the peptides that stimulated AChR-immune T cells from the same strain. Genetically restricted T cell recognition of AChR peptides in rats suggests that T cells from MG patients with different major histocompatibility haplotypes may recognize different AChR peptides.     

Regulation of the in vitro presentation of minor lymphocyte stimulating determinants by major histocompatibility complex-encoded immune response genes

Activation of murine B lymphocytes in a splenocyte stimulator population with affinity-purified goat anti-mouse IgD (G alpha M delta) antibody was previously shown by this laboratory to enhance the presentation of strongly stimulatory major histocompatibility complex (MHC) and minor lymphocyte-stimulating (Mlsa,d) determinants in a primary mixed lymphocyte reaction. In the present study, the G alpha M delta treatment of murine splenocytes was employed to enhance the detection of the weakly stimulatory non-MHC Mlsc determinant in order to study the role the MHC might play as a restricting element for the recognition of these minor antigens in a primary mixed lymphocyte reaction. Indeed, enhanced T cell proliferation to Mlsc determinants presented on G alpha M delta-treated splenocytes was observed when the responder and activated H-2-compatible stimulator cell shared certain MHC haplotypes. High responsiveness was associated with the H-2a,k,j,p haplotypes, intermediate responsiveness was associated with the H-2f,g haplotypes and low responsiveness was associated with the H-2b,s haplotypes. (Low X high responder)F1 T cells preferentially responded to the Mlsc determinants presented on G alpha M delta-treated stimulator cells of the F1 or parental high responder H-2 haplotype. When mitomycin C instead of irradiation was used to inactivate normal (non-IgD-treated) splenocytes, a similar preferential response of T cells to Mlsc determinants presented on stimulator cells of a high responder H-2 haplotype was also observed. The inability of G alpha M delta-treated splenocytes of the low responder haplotype to elicit substantial levels of T cell proliferation across an Mlsc difference could not be attributed to the failure of these stimulator cells to become activated by the anti-Ig antibody. In addition, co-culture experiments could not identify the poor T cell response to Mlsc determinants presented on certain MHC haplotypes as being caused by the induction of nonspecific suppressor cells. Presentation of Mlsc determinants caused by transgene product complementation was detectable in F1 mice derived by crossing one parent that had the Mlsc non-MHC genes and a poorly permissive H-2 haplotype with a parent that expressed a permissive H-2 haplotype but lacked the Mlsc non-MHC genes.(ABSTRACT TRUNCATED AT 400 WORDS)     

H-2 linked Ir gene control of antibody responses to porcine insulin. I. Development of insulin-specific antibodies in some but not all nonresponder strains injected with proinsulin

Cell-mediated and humoral immune responses to heterologous insulins in mice are controlled by H-2 linked, dominant, immune response (Ir) genes. For example, mice bearing the H-2d haplotype develop T cell proliferative responses and produce antibody after injection with porcine insulin, whereas mice bearing other H-2 haplotypes do not. Data presented in this communication demonstrate that homozygous and heterozygous H-2d mice produce insulin-binding antibodies when immunized with porcine insulin or proinsulin. Some (H-2b,k,s) insulin-nonresponder mice produce insulin-binding antibodies after injection of proinsulin, whereas other insulin-nonresponder strains (H-2q) do not. All strains, except homozygous H-2q mice, produce antibodies specific for proinsulin, suggesting that the response to porcine proinsulin is also controlled by H-2-linked Ir genes. More importantly, F1 hybrids between insulin-nonresponder C57BL/10 (H-2b) and DBA/1 (H-2q) produce no insulin-binding antibodies when injected with proinsulin, despite the fact that proinsulin-binding antibodies are produced by these mice.     

Genetic control of the immune response to the H-2 associated Slp alloantigen in the mouse.

A survey of sixteen standard inbred and congenic resistant strains of mice reveals that the ability to mount an immune response to the Slp allotype is associated with the H-2 type of the recipient. Strains carrying the H-2f, H-2k, and H-2q haplotypes are able to produce specific antibody whereas strains of the H-2b haplotype are non-responders. Analysis of F1-hybrids and four informative intra-H-2-recombinants demonstrates that the ability to respond to the Slp allotype is controlled by a dominant gene associated with the K end of the H-2 complex.     

Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).     

Genetic control of immune response to sperm whale myoglobin in mice. I. T lymphocyte proliferative response under H-2-linked Ir gene control.

Studies on the genetic control of immune response to sperm whale myoglobin were initiated. As demonstrated in this paper, the T lymphocyte proliferative response to whale myoglobin is under H-2-linked Ir gene control. Mice of H-2d, H-2f, and H-2s haplotypes were high responders to the myoglobin, whereas haplotypes H-2b, H-2k, H-2p, H-2q, and H-2r were low responders. The Ir gene(s) was localized between H-2K and H2D regions, since the recombinant strain A.TL (KsIkSkDd) was a low responder and A.TH (KsIsSsDd) was a high responder. Further studies with recombinant strains revealed that the expression of the high-responder I-Ad or Ias alleles was sufficient to give a good response, since strains D2.GD (d d b b b b b b) and B10.HTT (s s s s k k k d) were high responders. The expression of the I-Cd allele in strains B10.A (k k k k k d d d) and B10.A(5R) (b b b k k d d d) also gave high response, and thus suggested a second Ir gene, derived from the H-2d haplotype. The finding that expression of the I-Cs allele in B10.S(8R) (k k ? ? s s s s) did not result in high response suggests the lack of the second Ir gene in the high-responder H-2s haplotype.     

Evidence for CD8-independent T cell maturation in transgenic mice

Double-negative (CD4-/CD8-) T cells expressing the alpha/beta transgenic TCR from the 2C cell line (anti-H-2Ld) were examined in the periphery of animals whose MHC type produces positive, negative, or no selection for differentiation of the TCR on single positive (CD8+) cells. Regardless of the selection haplotype the CD4-/CD8- cells are capable of activation by anticlonotypic mAb indicating that negative selection does not inactivate the "forbidden" TCR. Rather, the lack of response to H-2Ld in the negative haplotype is likely to absence of CD8 required to produce a functional response to H-2Ld. The similarity of surface phenotype and functional activity of these CD4-/CD8- cells maturing in different haplotypes suggests they may arise by an alternative lineage which, unlike the dominant TCR alpha/beta pathway, does not require coexpression of CD4/CD8.     

Localization of the functional sites on the alpha chain of acetylcholine receptor

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.     

Regulation of antibody production by helper T cell clones in experimental autoimmune myasthenia gravis

Ten acetylcholine receptor (AChR)-specific T cell clones from Lewis rats were studied. These clones had various AChR subunit and peptide specificities, and proliferated in response to antigen on appropriate APC. All the T cell clones were CD4+CD8- and OX22-, helped anti-AChR antibody production by AChR-primed lymph node B cells, and could secrete IL-2. However, several lines of evidence suggested that IL-2 was not the lymphokine that mediated T cell help. B cells primed with native AChR and then exposed in culture to very low concentrations of native AChR effectively presented the Ag to the T cell lines, presumably due to uptake via Ag receptors, but primed B cells were no more effective than were non-specific APC at presenting a synthetic AChR peptide which is recognized by AChR-specific T cells but not by AChR-specific B cells. Increasing AChR doses produced an antibody production response that was bell shaped and low doses stimulated, whereas higher AChR concentrations suppressed the antibody production response. Evidence suggested that AChR exerted its inhibitory effect through the T cells, but not via IL-2.     

Nematospiroides dubius: two H-2-linked genes influence levels of resistance to infection in mice

Strains of mice sharing common H-2 haplotypes but different genetic backgrounds, and H-2 congenic strains of mice differing only at H-2 genes were studied to assess the role of H-2 and non-H-2 genes in immunity to challenge infections with the nematode parasite Nematospiroides dubius. Strains of mice sharing the H-2k haplotype were uniformly more susceptible to challenge than strains expressing H-2q alleles, regardless of genetic background. However, in some cases strains of mice sharing the k or q haplotypes differed significantly in levels of resistance. Therefore, non-H-2 genes must influence the response observed. H-2 cogenic strains of mice differed markedly in their ability to resist challenge infections. Mice sharing the C57BL/10 background but expressing k alleles were very susceptible to challenge, while the H-2q, H-2f, and H-2s, haplotypes were associated with resistance. Studies of H-2 congenic recombinant strains of mice suggested that two H-2 genes influence the antiparasite response. One of these genes maps to the left of E alpha and the other to the D-end of the H-2 complex. It is concluded also that the unique configuration of H-2 genes in F1 hybrids contributes to increased resistance to challange.     

Contribution of synthetic polymers of amino acids to knowledge of immune response

The synthetic random polymers poly(Glu,Lys,Phe), poly(Glu,Phe) and poly(Glu,Lys,Tyr), have been used to study some parameters associated with the genetic control of the immune response (Ir) of mice. Mice of haplotypes d and q respond well to GLPhe. Mice of haplotypes k and b were previously shown to be nonresponders, whereas the F₁ (k × b) responded via a phenomenon involving “complementation” between 2 Ir genes, i.e., one gene product from IA, and another from IE form the requisite two-chain Ia “receptor” macromolecules (EE). When it was determined that mice of haplotypes q and k respond to GPhe, and the controlling gene maps to IA, (A_α A_β), we tested the theory that mice having q and k alleles in IA might respond to GLPhe via recognition of GPhe determinants in the terpolymer. Employing the in vitro proliferative response to T-cells from mice immunized with GLPhe and stimulated with GPhe and GLT (cross-reaction), it was determined that different determinant selection patterns exist in the recognition of GLPhe. Mice having q and k alleles in IA can respond to GLPhe via one mechanism, and other mice having d and f alleles respond via other mechanisms. The F₁ of the appropriate nonresponder strains forming the Ia molecule (EE) still exhibit the “complementation” phenomenon. Rabbit antibody against anti-GPhe (ID) from SWR mice (H-2^q)(anti-ID) was prepared. This anti-ID strongly inhibited the binding of ¹²⁵I-GPhe by anti-GPhe antisera produced only in mice of H-2^q haplotype and had no effect on the binding of GPhe by anti-GPhe antisera produced in mice of other haplotypes. The anti-ID also inhibited the binding of ¹²⁵I-GLPhe and ¹²⁵I-GPhe by anti-GLPhe antisera produced only in mice of H-2^q haplotype. These specificities were also confirmed by the inhibition of the plaque-forming cells. It was concluded that the antibodies produced in mice of H-2^q haplotype against GPhe and GLPhe share common idiotypic determinants that are recognized by the anti-idiotypic antiserum.     

Protective immunity in mice vaccinated with the Schistosoma mansoni P-28-1 antigen

The P28-1 Ag induces a strong protective immunity toward Schistosoma mansoni infection in various experimental models. T lymphocytes of mice immunized with the recombinant P28-1 Ag were stimulated in vitro by schistosome Ag of different development stages and by three P28-1 Ag-derived synthetic peptides. The most significant stimulation was achieved with the 24-43 peptide. The use of two fragments of this peptide showed that the P28-1 T lymphocyte specificity concerned essentially the NH2 terminal sequence of the 24-43 peptide. Moreover, T lymphocytes specific for the 24-43 peptide were stimulated by both schistosome Ag and the recombinant P28-1 protein. The passive transfer of (Th + Ts) lymphocytes recovered from P28-1 Ag-immunized mice increased the IgG response to P28-1 and its peptides during infection but did not protect against a challenge infection, such as the passive transfer of anti-P28-1 sera. In contrast, P28-1 specific Th cell lines maintained in culture for 2 mo, passively transferred a strong protection (50%) to infected mice. Supernatants of P28-1-specific T cells obtained after stimulation with the corresponding Ag, were able to confer cytotoxic properties to platelets and macrophages. The presence of IFN-gamma for the cytotoxicity mediated by platelets and macrophage activating factor for the cytotoxicity mediated by macrophages in these supernatants is in a large part responsible for the parasite killing observed. Finally, a preliminary immunogenetic approach with H-2 congenic mice on BALB background showed that the P28-1 Ag T cell response was under the control of the MHC and that the H-2b haplotype determined a low response to P28-1 Ag and its peptides while H-2d and k haplotypes determined high responders.     

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.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

H-2 antigens as genetic markers: a two-dimensional gel electrophoretic study

Direct immunoprecipitation and two-dimensional (2D) gel electrophoresis have been used to identify and characterize genetic variation of the H-2K and H-2D regions. Using inbred strains of mice and alloantisera, haplotype-specific polypeptides were defined for five different H-2 haplotypes. Specific immunoprecipitates prepared from strains of different haplotypes were applied to 2D gels in pairwise combinations to determine whether peptides specific to one haplotype can be distinguished from peptides specific to another. Those haplotype-specific peptides that migrate to unique positions on 2D gels with respect to the positions occupied by haplotype-specific peptides of another haplotype are useful as biochemical genetic markers. Cross-reactivity among K- and D-region antigens of different haplotypes was identified on 2D gels and found to correlate well with existing data based on serological cross-reactivity. An anti-mouse beta 2-microglobulin serum was found to be a useful general reagent for immunoprecipitating haplotype-specific H-2 antigens to permit their visualization on 2D gels.     

Leishmania donovani infection in heterozygous and recombinant H-2 haplotype mice

On a B10 (Lshs) genetic background the development of acquired T-cell-mediated immunity to Leishmania donovani infection in mice is under H-2-linked genetic control. Three phenotypic patterns of recovery were previously observed: "early cure" (H-2s, H-2r), "cure" (H-2b) and "noncure" (H-2d, H-2q, H-2f), with cure behaving as a recessive trait in H-2b/H-2d mice. In this study the long-term response to L. donovani is followed over 130 days of infection in eight recombinant haplotype strains and in six further heterozygous haplotype combinations. Noncure in B10.HTG mice, which carry d alleles for loci at the K end and b alleles for loci at the D end of H-2, confirms that H-2-linked genetic control of the acquired response to L. donovani infection is located in the K end. The complex pattern of dominance relationships observed in the additional heterozygous haplotypes studied, the variable phenotypic response of H-2k mice and of recombinant haplotype strains carrying IEk in common, and the differential early curing activity observed in heterozygotes involving the s but not the r early cure haplotype and in recombinant haplotype mice carrying s alleles to the left of IE suggest, however, that more than one subregion (IE and presumably IA) are involved. Results are interpreted in the light of immunoregulatory T-cell populations previously demonstrated in noncure, cure, and early cure strains.     

Gamma interferon is critical for neuronal viral clearance and protection in a susceptible mouse strain following early intracranial Theiler's murine encephalomyelitis virus infection

We evaluated the role of gamma interferon (IFN-gamma) in protecting neurons from virus-induced injury following central nervous system infection. IFN-gamma(-/-) and IFN-gamma(+/+) mice of the resistant major histocompatibility complex (MHC) H-2(b) haplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-gamma(+/+) H-2(b) mice also cleared virus from the spinal cord white matter, whereas IFN-gamma(-/-) H-2(b) mice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-gamma(-/-) mice of the susceptible H-2(q) haplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-gamma(-/-) H-2(q) mice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-gamma(-/-) H-2(q) mice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-gamma(-/-) H-2(q) mice. IFN-gamma deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-gamma(-/-) H-2(q) mice was lower than those in IFN-gamma(+/+) H-2(q) mice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-gamma exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-gamma plays a critical role in protecting spinal cord neurons from persistent infection and death.