Somatic cell variants express altered H-2Kb allodeterminants recognized by cytolytic T cell clones

The present studies have made use of in vitro derived H-2Kb mutants to analyze the fine specificity of alloreactive cytotoxic T lymphocytes (CTL). The variants were derived by negatively selecting mutagenized tumor cells with a monoclonal anti-H-2Kb antibody and positively selecting for residual cells expressing serologically altered H-2Kb molecules. Details of this procedure are described in the companion paper. Selected populations of bulk alloreactive and cloned CTL were examined for recognition of the variants. In contrast to the serologic findings presented in the companion paper, there does not appear to be a correlation between the monoclonal antibody used to select the R8 variant and the CTL specificities recognized. In several instances, CTL clones could discriminate between variants having identical serologic profiles. Therefore, it would appear that the CTL have a large repertoire of allorecognition, even when generated across a mutant anti-Kb combination reflecting only a few amino acid differences. In addition, a diverse set of epitopes can be recognized on the Kb molecule. Finally, in some instances a change in what would appear to be a single amino acid resulted in a profound alteration of CTL recognition even though the Kb mutant molecule expressed limited serologic changes. These results support the idea that small changes in the H-2Kb molecule can have dramatic effects on CTL even though there are relatively little effects on serologic recognition of the target molecule.     

Single point mutation on the gene encoding dysbindin results in recognition deficits

The dystrobrevin‐binding protein 1 (DTNBP1) gene is a candidate risk factor for schizophrenia and has been associated with cognitive ability in both patient populations and healthy controls. DTNBP1 encodes dysbindin protein, which is localized to synaptic sites and is reduced in the prefrontal cortex and hippocampus of patients with schizophrenia, indicating a potential role in schizophrenia etiology. Most studies of dysbindin function have focused on the sandy (sdy) mice that lack dysbindin protein and have a wide range of abnormalities. In this study, we examined dysbindin salt and pepper (spp) mice that possess a single point mutation on the Dtnbp1 gene predicted to reduce, but not eliminate, dysbindin expression. By western blot analysis, we found that spp homozygous (spp ?/?) mutants had reduced dysbindin and synaptosomal‐associated protein 25 (SNAP‐25) in the prefrontal cortex, but unaltered levels in hippocampus. Behaviorally, spp mutants performed comparably to controls on a wide range of tasks assessing locomotion, anxiety, spatial recognition and working memory. However, spp ?/? mice had selective deficits in tasks measuring novel object recognition and social novelty recognition. Our results indicate that reduced dysbindin and SNAP‐25 protein in the prefrontal cortex of spp ?/? is associated with selective impairments in recognition processing. These spp mice may prove useful as a novel mouse model to study cognitive deficits linked to dysbindin alterations. Our findings also suggest that aspects of recognition memory may be specifically influenced by DTNBP1 single nucleotide polymorphisms or risk haplotypes in humans and this connection should be further investigated.     

Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior

The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential

A human CSF-1 receptor containing an "activating" mutation in its extracellular domain (serine for leucine 301) induced morphologic transformation, anchorage-independent growth, and tumorigenicity in mouse NIH 3T3 cells. A second regulatory mutation within the receptor's intracytoplasmic carboxy-terminal tail (phenylalanine for tyrosine 969) augmented transforming efficiency but was itself insufficient to induce transformation. Like the v-fms oncogene product, receptors bearing the activating mutation retained high-affinity binding sites for CSF-1 but were retarded in transport to the cell surface and were phosphorylated on tyrosine in the absence of ligand. Although the activating mutation does not affect the CSF-1 binding site in the receptor extracellular domain, it must induce a conformational change that mimics the effect of ligand binding, resulting in CSF-1-independent signals for cell growth.     

The role of H-2 in T cell recognition of Mls

The role of H-2 was evaluated in T cell recognition of Mls-encoded antigens during primary mixed lymphocyte responses (MLR). Mlsc was used as a stimulating determinant in MLR and its recognition by T cells was assessed by linear regression analysis under culture conditions in which (A x B)F1 responder cell number was the factor limiting total response. Results of such experiments indicated the presence of distinct (A x B)F1 responder T cell subpopulations capable of differentially recognizing the foreign Mls antigen in association with one or the other parental H-2 haplotype. These findings demonstrate that T cells do not recognize Mlsc products in isolation, but rather are restricted to recognition of Mlsc in the context of "self" H-2 determinants.     

A new point mutation affecting the fourth transmembrane domain of PMP22 results in severe,de novo Charcot-Marie-Tooth disease

A novel TG mutation in exon 4 of the PMP22 gene was identified heterozygously in a girl with severe, de novo CMTIA disease. Duplication of the chromosomal 17p11–12 region, encompassing the PMP22 gene, was ruled out. This is the only known mutation that specifically affects the human fourth transmembrane (TM) domain of PMP22. It results in a substitution of a non-polar amino acid by a polar one (Leu¹⁴⁷4Arg), similar to the nearby Gly¹⁵⁰Asp substitution, underlying the severe Trembler phenotype in the mouse. These mutations suggest that the fourth TM domain plays a crucial role in the normal function of PMP22. The new mutation also augments previous observations that diseases caused by mutations in PMP22 are more severe than those caused by the duplication of 17p11–12.     

A point mutation in the cytoplasmic domain of the transferrin receptor inhibits endocytosis.

The rate of receptor-mediated endocytosis of diferric 125I-transferrin by Chinese-hamster ovary cells expressing human transferrin receptors was compared with the rate measured for cells expressing hamster transferrin receptors. It was observed that the rate of endocytosis of the human transferrin receptor was significantly higher than that for the hamster receptor. In order to examine the molecular basis for the difference between the observed rates of endocytosis, a cDNA clone corresponding to the cytoplasmic domain of the hamster receptor was isolated. The predicted primary sequence of the cytoplasmic domain of the hamster transferrin receptor is identical with that of the human receptor, except at position 20, where a tyrosine residue in the human sequence is replaced with a cysteine residue. To test the hypothesis that this structural change in the receptor is related to the difference in the rate of internalization, we used site-directed mutagenesis to examine the effect of the replacement of tyrosine-20 with a cysteine residue in the human transferrin receptor. It was observed that the substitution of tyrosine-20 with cysteine caused a 60% inhibition of the rate of iron accumulation by cells incubated with [59Fe]diferric transferrin. No significant difference between the rate of internalization of the mutant (cysteine-20) human receptor and the hamster receptor was observed. Thus the substitution of tyrosine-20 with a cysteine residue can account for the difference between the rate of endocytosis of the human and hamster transferrin receptors.     

Immunogenetic analysis of H-2 mutations. III. Genetic mapping and involvement in immune reactions of the H-2ka mutation.

Mutation M523 (H-2ka) occurred spontaneously in strain CBA/CaLacSto and was discovered during routine skin graft testing for genetic homogeneity. By linkage and complementation tests, the mutation was previously mapped in the K end of the H-2 complex. We demonstrate that the mutation occurred in the K region, without affecting the I region in the K end of the complex. The mutant antigens cause rejection of skin grafts, stimulate cells in mixed lymphocyte culture, and function as stimulators as well as targets in cell-mediated lymphocytotoxicity. Yet, they are serologically indistinguishable from the antigens of the original strain and do not induce formation of humoral antibodies upon immunization of the CBA strain. Together with the results obtained on testing of other H-2 mutants, the data strongly support the notion that classical H-2 antigens (i.e., products of the H-2K and H-2D loci) can function as lymphocyte-stimulating determinants, and that I-region differences are not required for the induction of strong cell-mediated lymphocytotoxicity.     

A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression.

The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP-L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity.     

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.