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R N Germain

Publications and source records attributed to R N Germain.

At least 145 records · Page 8Linked to original sources
PubMed1986

Functional expression of the murine Thy-1.2 gene in transfected human T cells.

The interaction of certain mAbs with the Thy-1 molecules of murine T lymphocytes leads to cell activation and proliferation. To examine the signal transduction mechanism underlying this process and to determine what, if any, relationship exists between Thy-1-dependent triggering and T cell activation mediated through the T3-antigen receptor (T3-Ti) complex, a genomic clone of murine Thy-1.2 was isolated and transfected into the human T cell tumor, Jurkat. The transfected gene was actively transcribed in these human cells and high levels of Thy-1.2 glycoprotein were found on the cell membrane. Although certain mAbs to Thy-1.2 failed to bind to the Thy-1 transfected Jurkat cells, several known mitogenic anti-Thy-1 mAbs did react, and in the presence of phorbol ester, induced IL-2 secretion. One Thy-1+ transfectant out of five failed to produce IL-2 in response to anti-T3/Ti antibodies even though it retained the ability to increase intracytoplasmic calcium concentration [( Ca2+]i) in response to these ligands. A Thy-1 negative revertant of this cell regained anti-T3/Ti reactivity, suggesting a regulatory defect in signal transmission via T3/Ti in the original transfectant. These data confirm the ability of Thy-1 to act as an activation receptor for T cells. They reveal a potential role for changes in [Ca2+]i in this process, in common with other pathways of T cell activation, but also indicate a more complex series of events is involved.

Animals↗
PubMed1986

T-cell recognition of Ia molecules selectively altered by a single amino acid substitution.

T lymphocytes recognize foreign antigen together with allele-specific determinants on membrane-bound class I and class II (Ia) gene products of the major histocompatibility complex. To identify amino acids of class II molecules critical to this recognition process, the genes encoding the beta chains of the I-Ak molecule were cloned from a wild-type B-cell hybridoma and from an immunoselected variant subline showing distinct serological and T-cell stimulatory properties. Nucleotide sequencing and DNA-mediated gene transfer established that a single base transition (G----A) encoding a change from glutamic acid to lysine at position 67 in the I-Ak beta molecule accounted for all the observed phenotypic changes of the variant cells. These results confirm the importance of residues 62 to 78 in the amino terminal domain of I-A beta for class II-restricted T-cell recognition of antigen and demonstrate the ability of a single substitution in this region to alter this recognition event.

Amino Acid Sequence↗
PubMed1986

The mouse E beta 2 gene: a class II MHC beta gene with limited intraspecies polymorphism and an unusual pattern of transcription.

Analysis of genomic clones containing the E beta 2 region from the mouse major histocompatibility complex (MHC) reveals a gene that is similar to conventional class II beta genes in its overall organization and sequence but unusual in that it shows limited intraspecies allelic polymorphism. Like the conventional class II beta genes, the E beta 2 gene is transcriptionally active in a variety of B lymphoid cells. However, its transcripts consist of multiple polyadenylated species from 1.8 to 3.6 kb in size which are present at only approximately 1/20 the level of E beta 1 mRNA in the same cells. In addition, unlike other class II genes, E beta 2 transcription is not induced by gamma-interferon treatment of macrophage/monocyte tumor lines. These distinctive features suggest that the E beta 2 gene product may have a unique functional role, different from that of previously studied class II proteins.

Amino Acid Sequence↗
PubMed1985

Influence of allelic polymorphism on the assembly and surface expression of class II MHC (Ia) molecules.

Cell surface Ia expression was examined following transfection of murine alpha and beta class II major histocompatibility complex (MHC) genes into L cells. Although haplotype-matched (e.g., Ak beta Ak alpha) gene pairs yielded high expression in primary transfectants, haplotype-mismatched pairs (e.g., Ak beta Ad alpha) gave unexpectedly low expression. RNA analysis revealed a requirement for greater A beta, and particularly A alpha transcript levels in haplotype-mismatched vs. haplotype-matched transfectants with similar levels of membrane Ia. A beta allelic control of this assembly/expression process was mapped to the NH2-terminal (beta 1) domain, the locus of high intraspecies polymorphism. These data on the effects of allelic variation on Ia levels provide a possible explanation for the strong linkage disequilibrium of A alpha and A beta genes and may account for the current molecular organization of the I region of the MHC.

Alleles↗
PubMed1985

Cell surface expression of an in vitro recombinant class II/class I major histocompatibility complex gene product.

Chimeric histocompatibility genes encoding the amino-terminal (beta 1) domain of the class II Ak beta polypeptide and the carboxy-terminal (C2, transmembrane, and intracytoplasmic) domains of either the class I H-2Ld or H-2Dd molecules were stably introduced into mouse L cells. Although both were transcribed, only 5' Ak beta/3' H-2Dd transformants had significant cell membrane expression of a 30-40 kd, heterogeneous glycoprotein containing Ak beta 1 and H-2Dd (C2) serological epitopes. These transformants had a unique pattern of reactivity with monoclonal antibodies previously identified as requiring the Ak beta 1 domain for recognition of complete I-A molecules. These results allow new insight into the structural requirements for cell surface expression of proteins and provide unique cellular reagents for the dissection of humoral and cell-mediated recognition of MHC molecules.

Animals↗
PubMed1985

Rearrangement and transcription of a T-cell receptor beta-chain gene in different T-cell subsets.

The functionally defined sets of T lymphocytes--helper T cells, cytotoxic T cells, and suppressor T cells--were examined for the possible involvement of a recently identified T-cell receptor beta gene locus in receptor formation. Since gene rearrangements are required for functional gene expression, cloned T-cell lines from each of the groups were surveyed for the expression of unique gene rearrangements. In addition, cell lines that showed gene rearrangements were further tested for the expression of the mature 1.2- to 1.3-kilobase mRNA transcribed from a productive gene rearrangement. The results of such experiments show that helper and cytotoxic T cells may use a common beta chain of the receptor, whereas suppressor cells do so rarely, if at all.

Animals↗
PubMed1985

"Exon-shuffling" maps control of antibody- and T-cell-recognition sites to the NH2-terminal domain of the class II major histocompatibility polypeptide A beta.

To investigate the role of the highly polymorphic amino-terminal (beta 1) domain of the class II major histocompatibility polypeptide A beta during recognition by T cells and antibodies, "exon-shuffling" was carried out between genomic recombinant DNA clones of Ak beta and Ad beta to generate a hybrid gene containing Ak beta exons for the amino-terminal domain followed by the Ad beta exons for the remainder of the molecule. L-cell gene transfectants expressing this hybrid A beta gene in combination with Ak alpha were compared to L cells expressing wild-type Ak beta Ak alpha dimers in tests of antigen-presentation to T-cell clones and hybridomas and for staining by a panel of anti-I-Ak-specific monoclonal antibodies. These antibodies were also tested for their reactivity with a B-lymphoma transfectant expressing Ak beta in the absence of Ak alpha. The results showed no qualitative differences in either T-cell or antibody-mediated recognition of I-Ak molecules containing either the exon-shuffled or wildtype Ak beta. Together with the data involving the B cell transfectant expressing only Ak beta, these results map control of the A beta contribution to the immunologically relevant determinants of I-Ak to the highly polymorphic amino-terminal domain and indicate little, if any, contribution to allele-specific recognition by amino acid sequence variations in the remaining portions of the A beta polypeptide.

Alleles↗
PubMed1985

Comparison of MHC genes among distantly related members of the genus Mus.

The genomic content of class I and class II MHC DNA sequences in a variety of wild mice has been analyzed. The panel of mice includes members of three subgenera of the genus Mus. By genomic hybridization with the use of a variety of DNA probes, both class I and class II DNA sequences appear to be conserved in all of the species examined. However, the number of class I DNA sequences differs among the species. Furthermore, this variation appears to result from differential increases within subsets of class I genes. These data suggest that the class I multigene family is dynamic and changing over short periods of evolutionary time. In contrast, none of the class II genes appears to vary in copy number. More extensive polymorphism was noted amongst the class II beta genes than the alpha genes. Interestingly, the genomic sequence corresponding to E beta 2 is highly conserved, leading to the prediction that it is a genetically functional sequence.

Animals↗
PubMed1985

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells.

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.

Animals↗
PubMed1985

Nucleotide sequence and expression of a mouse interleukin 2 receptor cDNA.

We have isolated and sequenced a 1.3 kilobase (kb) cDNA that contains the entire 804 base pair coding region of the murine interleukin 2 (IL 2) receptor. A comparison with the human IL 2 receptor sequence revealed regions of high conservation, including the short (11 amino acid) intracytoplasmic domain, as well as an internally repeated sequence that is consistent with the receptor containing at least two distinct extracytoplasmic domains. When inserted into the expression vector pcEXV-3, this cDNA directs functional membrane expression of the IL 2 receptor on transfected COS 7 cells. An analysis of cytoplasmic RNA from IL 2 receptor-bearing T cells reveals five distinct polyadenylated transcripts ranging in size from 1.7 to 4.7 kb. All five transcripts are evident in both concanavalin A-stimulated splenic T cells and in the IL 2-dependent line CTLL. Some of this size heterogeneity is due to different sites of polyadenylation, resulting in mRNAs that differ in the 3' untranslated region. We cannot exclude the possibility of alternative splicing, however, which may result in distinct forms of the IL 2 receptor on the cell surface.

Animals↗
PubMed1985

Use of molecular techniques for analysis of Ia structure-function relationships.

An overview of a strategy for the molecular analysis of class II major histocompatibility complex (Ia) gene product structure-function relationships is presented, and results obtained to date by using this approach are summarized. The A beta, A alpha, E alpha, and E beta genes have been cloned and sequenced to yield information on gene organization and primary protein sequence. Comparison of sequences from allelic forms of these genes show the NH2-terminal domain to be the locus of most intraspecies polymorphism. Transfection of I-A alpha and A beta genes into B lymphoma cells or L cells has generated cells expressing the transfected gene products on their membrane. Such Ia+ transfectants present antigen to various T cells, which use the expressed I-A as a restriction element. Exon shuffling has shown the beta 1 domain of A beta to play a predominant role in such restricted antigen recognition. Preliminary data refining this analysis to sites within beta 1, as well as data on control of alpha: beta chain association, are reviewed, and future prospects for use of this approach in resolving questions of immunological interest are discussed.

Animals↗
PubMed1985

Influence of accessory cell and T cell surface antigens on mitogen-induced IL 2 receptor expression.

We have assessed the inhibitory effects of various monoclonal antibodies on the expression of the IL 2 receptor. Anti-LFA-1, but not anti-Ly-2, markedly inhibited the induction of the IL 2 receptor on the Ly-2+ subset. T-depleted spleen cells, L cells, and B lymphoma cells all functioned as potent accessory cells (AC) for the induction of the IL 2 receptor on L3T4+ T cells. Anti-LFA-1 inhibited the induction of the IL 2 receptor irrespective of the type of AC used. Anti-L3T4 only inhibited the induction of IL 2 receptor expression when L cells were the source of AC. The inhibitory capacity of anti-L3T4 was not related to the expression of Ia on the AC population, because the magnitude of inhibition was comparable in cultures containing either Ia+ or Ia- L cells, whereas no inhibition was seen with either Ia+ or Ia-B lymphoma cells. We conclude from these studies that LFA-1 plays a critical role in mitogen-induced activation of both T cell subsets by promoting both T-AC and T-T interactions. Although anti-L3T4 can inhibit T cell activation in the absence of the recognition of Ia, the mechanism of inhibition and the proposed target molecule for L3T4 on the AC or the T cell have not been determined in our studies. A number of different models for the function of this cell surface antigen are discussed.

Animals↗
PubMed1984

Membrane Ia expression and antigen-presenting accessory cell function of L cells transfected with class II major histocompatibility complex genes.

To study the relationship between the structure and function of Ia antigens, as well as the physiologic requirements for antigen presentation to major histocompatibility complex-restricted T cells, class II A alpha and A beta genes from the k and d haplotypes were transfected into Ltk- fibroblasts using the calcium phosphate coprecipitation technique. Individually transfected genes were actively transcribed in the L cells without covalent linkage to, or cotransformation with, viral enhancer sequences. However, cell surface expression of detectable I-A required the presence of transfected A alpha dA beta d or A alpha kA beta k pairs in a single cell. The level of I-A expression under these conditions was 1/5-1/10 that of Ia+ B lymphoma cells, or B lymphoma cells expressing transfected class II genes. These I-A-expressing transfectants were tested for accessory cell function and shown to present polypeptide and complex protein antigens to T cell clones and hybridomas in the context of the transfected gene products. One T cell clone, restricted to I-Ak plus GAT (L-glutamic acid60-L-alanine30-L-tyrosine10), had a profound cytotoxic effect on I-Ak- but not I-Ad-expressing transfectants in the presence of specific antigen. Assays of unprimed T cells showed that both Ia+ and Ia- L cells could serve as accessory cells for concanavalin A-induced proliferative responses. These data indicate that L cells can transcribe, translate, and express transfected class II genes and that such I-A-bearing L cells possess the necessary metabolic mechanisms for presenting these antigens to T lymphocytes in the context of their I-A molecules.

Animals↗
PubMed1983

Murine I-A beta chain polymorphism: nucleotide sequences of three allelic I-A beta genes.

The polymorphism of immune response genes plays a critical role in determining the immune capabilities of a particular individual. The molecular nature of this polymorphism was studied by examining the structure of the coding portions of three alleles of the I-A beta chain gene, an immune response gene whose protein product constitutes a subunit of the I-A molecule. Comparison of the I-A beta chains encoded by these alleles revealed an amino acid sequence divergence of 5 to 8 percent. The differences were found to be a series of short alterations clustered in the amino terminal half of the polypeptide.

Alleles↗
PubMed1983

Polymorphism and complexity of the human DC and murine I-A alpha chain genes.

A cDNA clone encoding the human B cell alloantigen DC alpha chain (pDCH1) has been used to analyse the structure of the human and murine major histocompatibility complexes by the DNA filter hybridization technique. The pDCH1 probe hybridizes to a single DNA sequence present on chromosome 17 in the mouse genome. A restriction enzyme polymorphism enables us to map this sequence to the I-A subregion. Extensive restriction enzyme polymorphism detected in HLA-DR homozygous typing cells is reminiscent of the DR/MT linkage disequilibrium groups, suggesting that the pDCH1 probe could be useful for haplotype typing in the human population. The HLA-DR region appears more complex than the I region since a second DC-like hybridizing sequence is detected in the human genome in these experiments.

Animals↗