Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

T cells with variable region Vδ1 γδ T cell receptors (TCRs) are distributed throughout the human intestinal epithelium and may function as sentinels that respond to self antigens. The expression of a major histocompatibility complex (MHC) class I–related molecule, MICA, matches this localization. MICA and the closely related MICB were recognized by intestinal epithelial T cells expressing diverse Vδ1 γδ TCRs. These interactions involved the α1α2 domains of MICA and MICB but were independent of antigen processing. With intestinal epithelial cell lines, the expression and recognition of MICA and MICB could be stress-induced. Thus, these molecules may broadly regulate protective responses by the Vδ1 γδ T cells in the epithelium of the intestinal tract.

https://science.sciencemag.org/content/sci/279/5357/1737.full.pdf

Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial γδ T Cells

(First of Two Parts) COLLAGEN is the major macromolecule of most connective tissues, and it is widely believed that many human diseases, particularly chronic diseases, will be found to involve this protein either directly or indirectly. Since collagen biosynthesis was last reviewed in the Journal in 1972,1 much new information has been gained about the relatively complex pathway by which it is synthesized. We should like to consider this new information and some of its consequences in terms of the following questions: What is the structural information in the collagen molecule, and how does it determine the structure of the . . .

Biosynthesis of collagen and its disorders

Major histocompatibility class I proteins display viral and self antigens to potentially responsive cells and are important for the maturation of T cells; beta 2-microglobulin (beta 2M) is required for their normal expression. Mouse chimeras derived from embryonic stem cells with a disrupted beta 2M gene transmitted the inactivated gene to their progeny. Animals homozygous for the mutated beta 2M gene were obtained at expected frequencies after further breeding. The homozygotes appeared normal, although no class I antigens could be detected on their cells and the animals are grossly deficient in CD4- CD8+ T cells, which normally mediate cytotoxic T cell function.

https://science.sciencemag.org/content/sci/248/4960/1227.full.pdf

Normal development of mice deficient in beta 2M, MHC class I proteins, and CD8+ T cells

Maternal immunoglobulin G transmitted to the fetus or newborn provides humoral immunity for the first weeks of mammalian life. Fc receptors on intestinal epithelial cells of the neonatal rat (FcRn) mediate the uptake of IgG from milk. Affinity-purified FcRn is resolved by SDS-PAGE into components of relative molecular masses 45,000-53,000 (p51) and about 14,000 (p14). We report the identification of the smaller component as beta 2-microglobulin. Association of beta 2-microglobulin with p51 was confirmed by crosslinking in intestinal epithelial cell brush borders. We have cloned a cDNA encoding the presumptive Fc-binding subunit, p51, and its predicted primary structure has three extracellular domains and a transmembrane region which are all homologous to the corresponding domains of class I major histocompatibility complex (MHC) antigens. This is the first time a function has been assigned to an MHC antigen-related molecule.

An Fc receptor structurally related to MHC class I antigens.

The lymphoblastoid cell line Daudi lacks both HLA-A and B antigens and beta2 microglobulin. Somatic cell hybrids derived from a fusion between this line and D98/AH--2 were shown to express four HLA antigens not detectable on either parent cell, A1, A10(Aw26), Bw16(Bw38, Bw17. The initial definition by direct cytotoxicity assay was confirmed by absorption of reactions against target T lymphocytes, thus avoiding problems due to contaminating Ia antibodies, and by blocking the reactions by pretreatment with a chicken anti-human beta2 microglobulin serum. That the new specificities were due to the Daudi HLA region was confirmed by the finding that interspecific hybrids between Daudi and A9L, containing a single human chromosome 6, expressed A10 and Bw17. This also defined the haplotypes of Daudi as A10(Aw26), Bw17 and A1, Bw16(Bw38). The re-expression of the Daudi HLA-A and B antigens in two independent sets of hybrids indicates that it does not carry a mutation in the HLA region. It has previously been reported that somatic cell hybrids with Daudi, which contain chromosome 15, do not express human beta2 microglobulin. These results suggest that the reason for the lack of HLA-A and B antigens on Daudi is a secondary effect due to the mutation(s) in the beta2 microglobulin gene.

The genetic control of HLA-A and B antigens in somatic cell hybrids: requirement for beta2 microglobulin.

Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase (PKM2) mannose phosphate isomerase (MPI), N-acetyl hexosaminidase A (HEXA) and β2-microglubulin (β2-m) all segregated in concordance with the X/15 translocation chromosome. The latter markers have been assigned to chromosome 15. Selection against the X/15 chromosome was done using antihuman β2-m serum. Electrophoretic and immunochemical analyses of the N-acetyl hexosaminidases A and B in these hybrids were performed.

Human gene mapping using an X/autosome translocation.

Hybrid clones (MOGs) were made between the mouse line RAG and a primary fibroblast line from an individual of the rare alphaGLU 2 phenotype. Fifteen independent primary clones and 32 subclones were tested for the presence of human alphaGLU after separation of the human and rodent enzymes by starch gel electrophoresis. Twenty-three other human-mouse hybrids from six different crosses were analysed for the presence of human alphaGLU by exploiting a difference in the thermostability of the human and mouse enzymes. The hybrids were also analysed for up to 25 other enzymes which were used as markers for different human chromosomes. Two of the MOG hybrids were karyotyped and karyotype data were already available for a number of the other hybrids. The combined results demonstrate that alphaGLU is located on chromosome 17, and probably on 17q.

Assignment of the human acid alpha-glucosidase gene (alphaGLU) to chromosome 17 using somatic cell hybrids.

COLLAGEN is the major extracellular protein of the body, and forms the fibres which impart tensile strength to all of its structural elements, including skin, bone, tendon and cartilage. It occurs as a family of molecules with closely related structures but distinctive tissue distributions1. Four types have been described with varying degrees of precision; the most abundant and best studied (type I) is a heteropolymer of three polypeptides, two identical (α1(I)), and a third (α2(I)) which differs slightly in primary structure. Types II-IV are homopolymers of the general formula (α1(X))3, where the designation α1 reflects the close sequence homologies shared with α1(I), although this relationship is only tentative in the case of α1(IV). These collagens are probably the products of at least five non-allelic structural genes, and knowledge of their linkage would be useful in view of their possible origin by duplication and divergence from an ancestral collagen gene. Quantitative variations in the expression of these genes are important in normal tissue development, and defective expression has been implied in two inherited disorders, Ehlers-Danlos IV2 and osteogenesis imperfecta3. The chromosomal assignment of these structural genes is a first step towards discovering their linkage relationship and perhaps towards understanding more about their differential expression. Because collagen is readily synthesised by cells in vitro, the problem of chromosome assignments may be investigated by the techniques of somatic cell genetics. When mouse and human cells are fused, random loss of human chromosomes occur, while the full set of mouse chromosomes is usually retained. Assignment of a gene to the chromosome carrying it can be achieved by examining sets of hybrids for expression of the human gene product and ascertaining the segregation of this phenotype with a particular human chromosome. We have examined a series of human–mouse hybrid clones for the production of collagen and report here the consistent segregation of human collagen with human chromosome 7.

Assignment of a type I collagen structural gene to human chromosome 7

The monoclonal antibody OKT-9 has been shown to recognize the human transferrin receptor. We have exploited the species specificity of OKT-9 to map a gene controlling human transferrin receptor expression to chromosome 3, using human-mouse somatic cell hybrids. The gene for the human transferrin receptor and the gene controlling transferrin expression may be linked in humans.

E. Solomon

Papers

The genetic control of HLA-A and B antigens in somatic cell hybrids: requirement for beta2 microglobulin.

Human gene mapping using an X/autosome translocation.

Assignment of the human acid alpha-glucosidase gene (alphaGLU) to chromosome 17 using somatic cell hybrids.

Assignment of a type I collagen structural gene to human chromosome 7

Expression of human transferrin receptor is controlled by a gene on chromosome 3: assignment using species specificity of a monoclonal antibody.