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Biomedical subjects

C G Fathman

Publications and source records attributed to C G Fathman.

At least 145 records · Page 8Linked to original sources
PubMed1985

The effects of autacoids on cloned murine lymphoid cells: modulation of IL 2 secretion and the activity of natural suppressor cells.

The pharmacologic effects of histamine and isoproterenol (autacoids) were studied on clones of murine T helper (TH) and natural suppressor (NS) cells. The data are consistent with receptors for the autacoids being nonrandomly distributed on phenotypically and functionally distinct clones. The effects of histamine on IL 2 secretion by TH cells could be either inhibitory or stimulatory, depending on the conditions of incubation with the autacoid. When TH cells were pretreated with histamine, their secretion of IL 2 was augmented; conversely, if histamine was added to the TH cells in the presence of antigen, IL 2 secretion was inhibited. The suppressor function of NS cells was enhanced by preincubation with histamine (10(-4) M) for 4 hr before the washed NS cells were added to responder and stimulator cells in mixed lymphocyte cultures. Two H1 receptor antagonists, mepyramine (10(-6) M) and pyrobutamine (10(-7) M), each competitively blocked the histamine activation of the NS cells, but an H2 receptor antagonist cimetidine (10(-5) M) did not alter the suppressor-enhancing function of histamine. Activation of NS cells did not occur if histamine was added with responder, stimulator, and co-cultured cells in MLR. The effects of each autacoid were additive in cytolytic T cells alone. The adenylate cyclase pools that can be stimulated by isoproterenol and histamine in cytolytic T cells may be independent of each other, but further work will be needed to prove this point.

Animals↗
PubMed1984

Gene conversion between murine class II major histocompatibility complex loci. Functional and molecular evidence from the bm 12 mutant.

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.

Animals↗
PubMed1984

Multiple functional sites on a single Ia molecule defined using T cell clones and antibodies with chain-determined specificity.

Monoclonal antibodies (mAb) were used to inhibit the proliferation of antigen-reactive (C57BL6/J X A/J)F1 restricted T cell clones. We have been able to subdivide these F1 restricted T cell clones into two groups: one of which recognizes the A alpha k A beta b molecule and the other group which recognizes the A alpha b A beta k molecule. Using clones with defined reactivities, we could assign the reactivities of monoclonals to the A alpha or A beta chains. By immunoprecipitation and two-dimensional analysis of Ia molecules from F1 spleen cells, we could independently map the reactivities of the mAb as being determined by the A alpha or A beta chain. To date, these two methods of chain localization of the antibody reactivity have agreed. Further, the differential blocking of the A alpha k A beta b restricted T cell clones suggests that there exists more than one restriction site per Ia molecule. Increasing the number of possible functional Ia restriction sites, either through combinatorial association of alpha and beta chains or by using more than one site per molecule, should increase the number of ways Ia molecules can function in antigen presentation.

Animals↗
PubMed1984

Clonotypic antibodies which stimulate T cell clone proliferation.

We have prepared a variety of antisera which recognize the T cell receptors on both alloreactive and antigen-specific functional clones. The antisera specifically stimulate the immunizing T cell clones to proliferate without cross-stimulating a variety of other clones and are thus clonotypic. The antisera specifically precipitate an 82k molecular weight complex unique to the immunizing clone and an independent common component present on all the helper T cell clones tested. The 82k molecular weight complex consists of a heterodimer, which can be resolved into an acidic (a) chain and a more basic (beta) chain. The beta subunit differs in its isoelectric focusing pattern when isolated from two A derived clones which recognize and respond to different alloantigens. Thus, the functional specificity of the antibodies is reflected in identifiable changes in molecules at the biochemical level.

Animals↗
PubMed1983

The I-Ab mutant B6.C-H-2bm12 allows definition of multiple T cell epitopes on I-A molecules.

The experiments presented in this study demonstrate that there exist at least two functional epitopes on an I-A molecule that can be recognized by T cell clones. By comparing the abilities of spleen cells from C57BL/6 mice and the congenic I-A mutant line B6.C-H-2bm12 to stimulate alloreactive T cell clones specific for the I-Ab molecule, we have discriminated two sets of clones, those recognizing the I-Ab and I-Abm12 molecule equally well and those able to recognize only the I-Ab molecule. These results imply that the two sets of clones have different receptors for I-A and that they therefore recognize separate epitopes on the I-A molecule. We have similarly been able to separate T cell clones, both alloreactive and L-glutamic acid60-L-alanine30-L-tyrosine10-reactive, specific for the Ab alpha Ak beta hybrid molecule into two groups based on their ability to recognize bm 12 spleen cells. Although the recognition of bm 12 spleen cells by these clones was unexpected since none of them responds to B6 spleen cells, these data again allow us to conclude that these groups of clones have different receptors for the same I-A molecule and therefore that they recognize distinct epitopes on the molecule. Additional studies, in which monoclonal anti-I-A antibodies were used to block the stimulation of T cells by stimulator or antigen-presenting cells, have demonstrated that this blockade can be a steric effect and therefore is not necessarily indicative of direct competition between the antibody and the T cell for the same site on an I-A molecule. Although this study does not reveal the physical nature of an I region-controlled "antigen-restriction site," we can suggest that increasing the number of possible functional Ia restriction sites either through combinatorial association of alpha and beta chains or by using more than one site per molecule will increase the number of configurations the ternary complex of Ia, antigen and T cell receptor(s) can form.

Animals↗
PubMed1983

Arsonate-specific murine T cell clones. I. Genetic control and antigen specificity.

The antigen-induced proliferative response of lymph node cells (LNC) from mice sensitized to the monofunctional antigen L-tyrosine-p-azobenzenearsonate (ABA-Tyr) was used to monitor genetic control. All strains tested mounted significant responses, but those that were H-2(b) at both the I-A and I-E loci [B10., B6., B10.A(18R), A.BY, and C3H.SW] gave consistently weaker responses than other haplotypes. The F(1) progeny of matings between high and low responder phenotype parents (DBA/2 and B6, respectively) were high responders, establishing the dominance of the responder trait. Proliferative responses of LNC to ABA-Tyr were blocked by the appropriate anti-Ia monoclonal reagents. For example, B10.A(4R) LNCI (I-A(k), I-E(b)) were blocked by anti-I-A(k), whereas B10.A(3R) LNC (I-A(b), I-E(k)) were blocked by anti-I-E(k). Long-term cultures of T cell lines specifically reactive to ABA-Tyr were established from LNC of A/J mice immunized with ABA-Tyr and were cloned by limiting dilution. The proliferative responses to ABA-Tyr of 14 out of 15 clones tested were I-A restricted on the basis of activation by antigen-presenting cells from appropriate recombinant strains and the blocking activity of the monoclonal anti-Ia antibodies. The response of the other clone was I-E restricted. The fine antigen specificity of the clones was studied using structural analogs of the homologous antigen to induce proliferation. The clones could be divided into three types with respect to responsiveness to ABA-histidine (ABA-His). One group responded about equally well to ABA-His and ABA-Tyr. A second set responded less strongly to ABA-His than to ABA-Tyr, while the third showed no response above background to ABA- His. In all instances, the ABA-His-responding clones discriminated exquisitely between the 2-azo and 4-azo histidine isomers, responding only to the 4-azo compound. These T cell clones provide extremely useful tools for studies of T cell specificity, antigen recognition and lymphoid cell interaction systems.

Animals↗
PubMed1983

T-lymphocyte subsets during pregnancy and the menstrual cycle.

Many observations suggest that cellular immunity is depressed during gestation. To define alterations in subsets of circulating lymphocytes during pregnancy, we compared the percentages of total T cells, T helper cells, and T suppressor cells in the peripheral blood of 31 pregnant women at various stages of gestation with those in that of nonpregnant controls. Control studies consisted of five women ingesting oral contraceptives and five women menstruating spontaneously. Isolated peripheral blood mononuclear cells were reacted with fluoresceinated monoclonal antibodies that were specific for Leu-2 cells, Leu-3 cells, and Leu-4 cells and that were subsequently analyzed on a fluorescence-activated cell sorter. No variation in the percentage of Leu-2, Leu-3, or Leu-4 cells was observed during the menstrual cycle or oral contraceptive cycle. No differences were seen when T-cell subsets at various stages of gestation were compared with the corresponding T-cell subset in nonpregnant controls. The data suggest that pregnancy does not alter the percentage of T-cell subsets in the peripheral blood.

Adolescent↗
PubMed1983

T-lymphocyte clones.

To date, the most successful uses of T-cell clones have been in the demonstration that a single type of cell can perform multiple functions. However, their potential usefulness is enormous, and the study of cell interactions using clonal populations has just begun. The development and study of more cloned populations will surely lead to a clearer analysis of cellular interactions in the immune system. The use of T-cell clones and hybridomas to analyze T-cell receptors and/or factors is well under way, and will continue to be an area of intense investigation. Molecular biologists will undoubtedly make more extensive use of T-cell clones in the future, both as a source of cloning material and as transfection recipients. The most exciting area for development, from a medical point of view, is the potential for use of these cell lines or their products in immunotherapy and in providing a mechanism for specifically modulating the immune response.

Animals↗
PubMed1982

Immunochemical properties of human low density lipoproteins as explored by monoclonal antibodies. Binding characteristics distinct from those of conventional serum antibodies.

Spleen cells obtained from mice immunized with human plasma low-density lipoproteins (LDL) were fused with mouse myeloma cells. The resulting hybridoma cells secreting immunoglobulin specific for LDL were screened and scored by radioimmunoassay and cloned by multiple limiting dilutions. Immunochemical properties of the monoclonal antibodies were compared with convential mouse serum antibodies. It was found that conventional antibodies precipitated LDL and bound more than 95% of 125I-labeled LDL and the maximal binding was independent of temperature. The monoclonal antibodies were incapable of precipitating LDL and bound a maximum of only 20% of the total 125I-labeled LDL. The maximal binding between monoclonal antibodies and LDL was extremely temperature-dependent. An optimal degree of binding was observed at 4 degrees C, whereas binding at 37 degrees C was only 30% of that achieved at 4 degrees C. Although the binding at 37 degrees C was low, the maximal binding could be re-established following a subsequent incubation at 4 degrees C, suggesting that the antigenic structure of LDL is reversibly modulated at temperatures between 4 and 37 degrees C. Since the orientation of apolipoprotein B in LDL is known to be dynamic at different temperatures, this result suggests that monoclonal antibodies, but not conventional antibodies, are capable of detecting subtle conformational changes in LDL. In addition, we have determined the binding affinity of LDL to monoclonal antibodies and to conventional antibodies. Only monoclonal antibodies showed a linear Scatchard plot, suggesting that the binding was to a single site with a single affinity. The monoclonal antibodies also possessed high specificity and failed to react with porcine LDL, while serum antibodies could recognize both human and porcine LDL.

Animals↗
PubMed1982

T cell clones specific for hybrid I-A molecules. Discrimination with monoclonal anti-I-Ak antibodies.

Alloreactive and soluble antigen-reactive, I-A-restricted T cell clones were examined for their ability to recognize hybrid I-A antigens. Several clones that recognized hybrid I-A(b)/I-A(k) molecules on (C57BL/6 x A/J)F(1) [(B6A)F(1)] spleen cells were studied. We were able to distinguish clones that recognized hybrid I-A molecules of the A(b)(a)A(k)(beta) type from those that recognized A(k)(a)A(b)(beta) molecules. We reached this conclusion by considering data from three independent types of experiments. (a) Monoclonal antibodies were used to inhibit T cell stimulation. Antibodies 10.2.16 and H116.32 distinguished two mutually exclusive "families" of T cell clones. One group of clones was inhibited by 10-2.16 and not H116.32, the other group exhibited reciprocal inhibition. (b) T cell proliferation was assayed using antigen-presenting cells from B6.C-H-2(bml2) (bml2) and [bml2 x B10.A(4R)]F(1) mice. Because the bml2 strain has a mutation that results in an altered A(b)(beta) polypeptide chain (A(bm12)(beta)), we reasoned that clones that could recognize the [bm12 x B 10.A(4R)]F(1) cells were recognizing A(b)(a)A(k)(beta) molecules. Alternatively, clones not recognizing [bml2 x B10.A(4R)]F(1) cells had specificity for A(k)(a)A(b)(beta) molecules. (c) I-A molecules immunoprecipitated from radiolabeled (B6A)F(1) splenocyte extracts were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments confirmed an earlier report that antibody 10.2.16 recognized determinants on the A(k)(beta) chain (12). Antibody H116.32 immunoprecipitated products consistent with recognition of A(k)(a) determinants. Taken together, these three types of results offer conclusive evidence that T cell clones recognizing "hybrid" I-A molecules use either A(b(k)A(k)(beta) or A(k)(a)A(b)(beta) molecules as recognition or restriction sites. Clones whose proliferation was supported by [bm 12 x B10.A(4R)]F(1) cells and blocked by anti-I-A(k) antibody 10-2.16 recognized A(b)(a)A(k)(beta) B molecules. Clones that were blocked by antibody H116.32 and did not recognize [bml2 X B10.A(4R)]F(1) cells use a recognition site(s) on A(b)(a)A(k)(beta) molecules. Thus, we can demonstrate both functionally and biochemically that hybrid F(1) I-A molecules of the structure A(k)(a)A(b)(beta) and A(b)(a)A(k)(beta) both exist on (B6A)F(1) splenocytes and that both configurations are used in immune recognition phenomena.

Animals↗
PubMed1982

Role of the major histocompatibility complex in T cell activation of B cell subpopulations. A single monoclonal T helper cell population activates different B cell subpopulations by distinct pathways.

It has recently been demonstrated that the Lyb-5+ and Lyb-5- B cell subpopulations differ in their requirements for major histocompatibility complex (MHC)-restricted activation by T helper (TH) cells. To determine whether these MHC-restricted and -unrestricted pathways of B cell activation result from differences in the participating TH cell populations or reflect differences exclusively in the responding B cell subpopulations, experiments were carried out using cloned TH cells for in vitro antibody responses to trinitrophenyl-keyhole limpet hemocyanin. The same cloned T helper cells were able to activate both CBA/N (Lyb-5-) B cells and CBA/CaHN (Lyb-5+ + Lyb-5-) B cells under different experimental conditions. The activation of Lyb-5-B cells by cloned T helper cells required both MHC-restricted TH cell-B cell interaction and carrier-hapten linkage. In contrast, the activation of Lyb-5+ B cells required only MHC-restricted T helper cell interaction with accessory cells, while T-B interaction was MHC unrestricted and did not require carrier-hapten linkage. Thus, the differences in activation requirements observed for the Lyb-5- and Lyb-5+ B cell subsets do not result from differences in the TH cell populations activating these B cells, but rather reflect differences in the ability of these B cells to respond to signals from the same TH cells.

Animals↗
PubMed1982

Definition of T cell idiotypes using anti-idiotypic antisera produced by immunization with T cell clones.

Alloreactive T cell clones with distinct specificities were used to raise anti-idiotypic antisera via an F1 anti-(parent anti-F1) protocol. Antisera were raised that could stimulate the proliferation of the appropriate T cell clone, but not other clones. The active fraction of the antisera for T cell proliferation was immunoglobulin. In addition to proliferation, an anti-idiotypic antiserum could induce the appropriate T cell clone to secrete substantial amounts of interleukin 2 (IL-2). Production of IL-2 appeared independent of the involvement of accessory cells. These accessory cells may be unnecessary for IL-2 production in our assay, or their effect may be produced by anti-idiotype. Thus, anti-idiotype may provide two or more specific T cell signals.

Animals↗
PubMed1982

Functional similarities of AeE alpha Ia molecules as determined by analysis with T-cell clones.

Recognition of AeE alpha Ia antigens at the functional level was investigated using T-cell clones. The reactivities of an alloreactive and an antigen-reactive clone, both of which recognized AeE alpha Ia molecules, were compared on a panel of stimulator/antigen-presenting cells of various genotypes. The two clones recognized all tested AebE alpha x Ia molecules, where x is a haplotype capable of expressing an Ia.7-bearing E alpha polypeptide. Ia antigen recognition by either clone could be inhibited by the monoclonal antibody Y-17, which recognizes a combinatorial serologic determinant on certain AeE alpha molecules. There were no differences in the recognition of Ia by the alloreactive versus the antigen-reactive clone, suggesting that Ia antigens are recognized by the two clones in a fundamentally similar way. The recognition of these various Ia molecules by the two cloned T-cell lines provides evidence that the E alpha polypeptides from H-2 haplotypes k, d, r, and u are functionally indistinguishable.

Animals↗
PubMed1982

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway.

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.

Animals↗