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Biomedical subjects

P F Gallagher

Publications and source records attributed to P F Gallagher.

9 recordsLinked to original sources
PubMed1989

Efficient radioiodination and immunopurification of surface proteins from small numbers of cells.

This report describes a method for the cell surface radioiodination and immunoprecipitation of the T cell receptor from as few as 7.5 x 10(4) lymphoid cells. The membranes of cells were labelled using the lactoperoxidase/H2O2 method. In order to minimize the loss of labelled cells after iodination, T cell receptor-negative 'filler' cells were added to the washes and the washing volume was kept small. Before immunoprecipitation, the solid-phase Staphylococcus aureus was precoated with the specific monoclonal antibody to ensure optimal recovery of antibody-antigen complexes. These simple modifications greatly increased the efficiency of the iodination and immunoprecipitation procedures, thus enabling direct analysis of membrane proteins from small numbers of cells.

Antibodies, Monoclonal↗
PubMed1989

CD4 and CD8 molecules can physically associate with the same T-cell receptor.

The expression of the cell-surface glycoproteins CD4 and CD8 on functionally mature T cells is usually mutually exclusive and correlates with class II and class I major histocompatibility complex (MHC) restriction, respectively. CD4 and CD8 function by binding to class II and class I MHC molecules on the antigen-presenting cell (APC), thereby increasing the adhesion between the T cell and the APC. From antibody-blocking studies and from cocapping and comodulation experiments, CD4 and CD8 come into close physical contact with their appropriately restricted T-cell receptor (TCR) at the time of antigen recognition. By the use of affinity chromatography followed by two-dimensional diagonal gel electrophoresis, we have identified a Mr 43,000 disulfide-bonded heterodimer copurifying with CD4. This protein was identified as the TCR by its removal after preclearing with the anti-TCR antibody F23.1 and by its generation after protease digestion of the same peptides as the TCR from this clone. When CD4 and CD8 were similarly isolated from an unusual CD4+ CD8+ class II-restricted T-cell clone, the TCR was identified as associating with either accessory molecule in the absence of activation. Therefore, CD4 and CD8 do not distinguish between class I- and class II-restricted TCRs in their ability to form membrane complexes, indicating a need for both the TCR and its associated accessory molecule to recognize the same individual MHC molecule on the APC to optimize TCR triggering.

Antibodies, Monoclonal↗
PubMed1988

Immunoglobulin gene expression is a normal differentiation event in embryonic thymocytes.

By in situ hybridization to frozen sections of mouse embryos, we have localized cells transcribing the Ig C mu gene during ontogeny. Transcripts were detected from before day 14 of gestation in individual pre-B cells in the liver and, surprisingly, in a large proportion of thymocytes between days 15 and 18. The level of mu RNA sequences in the thymus at day 17 was much higher than has been observed for adult thymocytes; from grain counts, the amount of mu RNA was similar to that observed for Ti gamma RNA. These findings suggest that Ig and Ti genes are under similar transcriptional controls during Ti gene recombination and that elevated mu RNA production is a normal event early in the intrathymic differentiation of T cells.

Animals↗
PubMed1986

Stable expression of Lyt-2 homodimers on L3T4+ T cell clones.

The murine T lymphocyte antigen Lyt-2 is considered to act as an accessory molecule to the class I-restricted T cell receptor during antigen recognition. We have previously described two unusual Lyt-2+L3T4+ class II-restricted T cell clones whose activation by antigen is inhibited by antibodies to L3T4 but not to Lyt-2 (B. Fazekas de St. Groth et al., Proc. Natl. Acad. Sci. USA 1986. 83: 2594). The Lyt-2 immunoprecipitated from one of these clones was indistinguishable from the molecule found on splenic T cells, as analyzed under reducing conditions on polyacrylamide gels, in two-dimensional charge/size separations and in peptide mapping. The molecule from the second clone showed slightly more extensive glycosylation but was within the range described for functional Lyt-2 on cytotoxic T cell lines. Lyt-2 mRNA from both clones showed no abnormalities on Northern analysis. Lyt-2 is normally expressed on thymocytes and peripheral T cells as a heterodimer disulfide bonded to the Lyt-3 glycopeptide, yet Lyt-3 could not be detected on the cell membranes of our clones; Lyt-2 existed as stable homodimers without Lyt-3. Thus Lyt-3 is not required structurally for the spontaneous expression of Lyt-2 on lymphoid cells.

Animals↗
PubMed1986

Involvement of Lyt-2 and L3T4 in activation of hapten-specific Lyt-2+ L3T4+ T-cell clones.

The murine T-cell surface molecules Lyt-2 and L3T4 play a role in the activation of antigen-specific T cells. The currently accepted model for the function of these molecules proposes that Lyt-2 and L3T4 increase the overall avidity of the interaction between the T-cell antigen receptor and antigen in association with the major histocompatibility complex (MHC) molecules on the antigen-presenting cell. We have used two unusual Lyt-2+ L3T4+ class II MHC-restricted T-cell clones to test whether Lyt-2 can substitute for L3T4 when the T-cell antigen receptor is class II MHC-restricted. Monoclonal antibodies against L3T4 profoundly inhibited antigen-induced lymphokine production by both T-cell clones. Anti-Lyt-2 monoclonal antibody had no effect. These results strongly suggest that L3T4 and the class II-restricted T-cell antigen receptors are physically close during antigen recognition, probably as part of a multimolecular complex from which Lyt-2 is excluded. The ability of L3T4 but not Lyt-2 to participate in such a complex with class II-restricted T-cell antigen receptors may explain the striking correlation between class II restriction and L3T4 expression in the peripheral T-cell pool.

Animals↗
PubMed1986

H-2 restriction and H-2 phenotypes of spleen cells from thymus-grafted radiation chimeras: evidence consistent with extrathymic suppression.

As an approach to analyzing the factors that contribute to determining H-2 restriction specificities of cytotoxic T cells, thymectomized semiallogeneic radiation chimeras were given transplants of fetal thymus from parental strain or F1 hybrid donors. A pronounced preference for lysis of infected targets of the same H-2 haplotype as the thymus was observed, the bias ranging from an undetectable preference through to absolute restriction to the thymic H-2. Virus-immune Tc cells could also be restricted to H-2 antigenic determinants of haplotypes expressed only by donated stem cell progeny lymphomyeloid cells. T cells from chimeras that had received thymuses of both parental strains were less effective than normal syngeneic F1 hybrid T cells in lysing allogeneic cells and both infected and uninfected parental targets. Variation in H-2 restriction bias from one chimera to another was not reflected in modulations to the class I H-2 phenotypes of the chimeric spleen cells.

Animals↗
PubMed1986

Self-tolerance and H-2 antigen expression in semi-allogeneic radiation chimeras are controlled by the extrathymic host environment.

Experiments with thymus-grafted radiation chimeras have implicated the H-2 type of the radio-resistant portion of the thymus as determining self for H-2 restricted T-cell responses. (P1 X P2)F1----P1 chimeras, shown to be fully reconstituted with donor haemopoietic cells, have displayed intolerance to P2 H-2 antigens and expressed aberrant levels of P2 H-2 on their spleen cells. We used semi-allogeneic radiation chimeras grafted with fetal thymuses from parental strain donors to compare the relative contributions of the thymus and the extrathymic periphery in determining self-tolerance and in modulating the H-2 phenotype of heterozygous chimeric spleen cells. As analysed by the rosetting titres of anti-H-2 antisera on chimeric spleen cells, by parental skin graft rejection, and by spleen cell anti-parent cytotoxicity after culture with parental stimulators, the decreased expression of non-host parental H-2 antigens was associated with anti-P2 T-cell reactivity, regardless of the H-2 type of the thymus. The donor thymus did not affect tolerance to parental H-2 or cause aberrant expression of H-2 antigens on spleen cells of syngeneic F1----F1 thymus-grafted chimeras.

Animals↗
PubMed1984

Variation in H-2 antigen expression on lymphomyeloid cells in semi-allogeneic irradiation chimeras.

Spleen cells from 30 individual murine irradiation chimeras of the type (P1 X P2)F1----P1 were compared in a rosetting assay for H-2K and H-2D cell surface antigen expression with normal (P1 X P2)F1 hybrid controls. Eleven out of the 30 chimeras were in the normal range, but the other 19 differed from F1 controls by 4- to 100-fold in endpoint titre for at least one H-2K or H-2D antigen. Every possible class of variation was found, i.e. up or down variation of H-2K or H-2D antigens of P1 or P2 type. This evidence, together with data from T6 chromosome marker experiments which also showed full reconstitution of lethally irradiated P1 recipients by (P1 X P2)F1 donor lymphomyeloid stem cells, suggested that incomplete reconstitution was not the cause of H-2 antigenic variation. Low expression of P2 H-2 antigens on spleen cells derived from (P1 X P2)F1----P1 chimeras was investigated further. Fifteen lethally irradiated (P1 X P2)F1 recipients of bone marrow cells from two such chimeras were all of normal F1 H-2 phenotype when tested 10-12 weeks after reconstitution, thus excluding stable, low P2 H-2-expressing variant F1 stem cells as a cause of the phenomenon. If P1 recipients were hyperimmunized against P2 cells before lethal irradiation and reconstitution with (P1 X P2)F1 stem cells, there were significantly fewer Till- McCulloch colonies in their spleens 10 days after reconstitution than in spleens of unimmunized controls. Also greater than 90% of immunized recipients died by 6 weeks after stem cell injection but two survivors both showed very low levels of P2 H-2K and H-2D antigens. These results together with previously published evidence of anti-P2 Tc cell activity and P2 skin graft rejection in (P1 X P2)F1----P1 chimeras suggested that residual anti-P2 immunological capability in lethally irradiated P1 recipients may be associated with low P2 H-2 expression on their F1-derived spleen cells, although the mechanism does not involve selection of stable, variant F1 stem cells. The mechanism(s) of other classes of variation in H-2 expression in (P1 X P2)F1----P1 chimeras were not investigated.

Animals↗