PubMed Health⌕ Search

Biomedical subjects

R W Dutton

Publications and source records attributed to R W Dutton.

At least 37 records · Page 2Linked to original sources
PubMed1994

Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor alpha/beta (TCR-alpha/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta 3/V alpha 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly interferon gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only 3-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Monoclonal antibody that distinguishes between a phosphorylated, beta 2-microglobulin-associated, and a free, nonphosphorylated, chain of MHC class I.

We have shown that a mAb, 7.2.14, recognizes a conserved sequence in exon 7 of a number of murine MHC class I molecules. 7.2.14 binding is abolished when the molecule is phosphorylated, presumably at a serine residue in exon 7, whereas treatment of material in cell lysates with alkaline phosphatase increases the intensity of the binding. A genomic construct containing Dd was transfected into human fibroblasts and a clonal cell line expressing high levels of surface MHC was selected. Cell lysates were prepared from surface-iodinated cells and analyzed by using a panel of antibodies. An apparent size heterogeneity was detected in the MHC class I gene product precipitated by different anti-class I MHC antibodies, suggesting that more than one conformational species of Dd was present. This was further investigated regarding the molecules precipitated by antibodies 34-2-12, M1/42, and 7.2.14. After preclearing of surface-iodinated cell lysates by using one antibody, challenge with the others still precipitated a Dd molecule, confirming that there were three independent conformations of the Dd gene product. A similar complexity could be observed in the lysates of surface-labeled spleen cells from C57BL/6 mice. A major polypeptide at approximately 48 to 50 kDa, representing the MHC H chain, was seen, and one or two as yet unidentified but strongly associated polypeptides at 41 kDa and 56 kDa were also visible. Sequential clearing of surface-iodinated material with one antibody followed by precipitation with the other confirmed that the 7.2.14-reactive material was distinct from that which reacted with M1/42. We propose that the 7.2.14-reactive 50-kDa band is the nonphosphorylated form of class I MHC, which exists in a conformation different from that of the conventional 48-kDa, phosphorylated, beta 2-microglobulin-associated entity.

Amino Acid Sequence↗
PubMed1992

Restricted IgH V gene usage in the response to the ese epitope on "Hi" sheep red blood cells.

We had previously shown that the in vitro antibody response to a single epitope (ese; extra sheep E Ag) present on some sheep E but absent from others could be monitored by assay of the plaque-forming cell response on both Lo3 and Hi SRBC. We had shown also that the response was seen only in certain strains of mice and that the gene(s) controlling the response mapped to the IgH V region of the IgH chain complex. An additional feature of the response is that it is only seen in vitro and is absent and, we hypothesize, is suppressed in vivo. The strain distribution of the response to the ese determinant suggested that the response may only use one V gene (or a small set of closely related V genes) that would be present in the responder strains and absent from the nonresponder strains. To test this hypothesis, we made hybridomas with specificity for the ese determinant and for the shared determinants. cDNA from these hybridomas were sequenced. All four anti-ese hybridomas were almost identical in V region sequence, but varied considerably in D and J segment usage, thus confirming the hypothesis that the ese response would be limited at the V segment. The four anti-ese hybridomas used two Vh J558 genes that differed only by one, or possibly two, nucleotide(s). Importantly, these genes are quite different from most other published J558 sequences. The sequence is very similar to an unexpressed sequence from a C57Bl/6 perinatal mouse and slightly less similar to two other Vhb sequences. It was quite similar to two sequences from autoantibodies, one an anti-DNA hybridoma antibody, BXW-14, isolated from an NZB x NZWF1 mouse, and the other, an NZB hybridoma, G8, with specificity for a mouse E Ag. We speculate that the Ig encoded by the V ese gene react with an autoantigen, that the B cells persist in the animal, but that the secretion of Ig is somehow suppressed.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of a trophoblast-endothelial-activated lymphocyte surface protein: cDNA sequence and genomic structure.

We have previously described the distribution of a surface glycoprotein recognized by monoclonal antibody B721. We now report the molecular characterization of this molecule at the protein, cDNA, and genomic level. A 75-kDa glycoprotein can be immunoprecipitated from B721+ tissues. We have isolated a near full-length cDNA containing the complete coding region and a full-length genomic clone. We present evidence that this protein has similarity to several classes of nuclear transcription factors, particularly the myc family of proteins. The 721P protein was found to have a leucine zipper-like structure, a possible basic region immediately upstream from the leucine zipper, and a protein kinase A phosphorylation site. 721P protein is encoded by a gene distinct from any deposited in existing data bases, and displays several features associated with proteins involved in signal transduction and gene regulation.

Amino Acid Sequence↗
PubMed1991

Helper T-cell subsets: phenotype, function and the role of lymphokines in regulating their development.

We have concentrated here on the lymphokines which might serve to regulate the different pathways of precursor development. We suggest that, as a result of antigenic stimulation, specific precursor cells both proliferate and become committed to develop into either an effector cell, a memory cell or an anergized cell. Anergy has not been dealt with in this review, but it is likely to be one of the options available. The development of an effector population takes 4-7 d (quite analogous to the time it takes for CTLp to become CTL and for resting B to become Ab-forming cells). The effector populations are large, generally IL-2R-positive cells. These cells have upregulated many adhesion molecule systems [e.g., Pgp-1, LFA-1 and ICAM-1 (Swain unpublished)], but downregulated the Mel-14 homing receptor. Effectors are ready to respond to APC such as specific B cells with a rapid synthesis and secretion of lymphokines. The effector population is then quickly downregulated, both by the turn off of lymphokine synthesis/secretion and possibly by its own suicide. This kind of pattern makes teleological sense since the cells making such high titers of lymphokines could have many potent pleitropic effects. It also seems to be the strategy employed in the generation of other terminally differentiated effectors (such as CTL and plasma cells). The requirement for restimulation and the requirement for direct and perhaps prolonged contact between the helper effector and the APC-B cell can be expected to help ensure that these lymphokines are localized (reviewed in Swain & Dutton 1987, Swain & Croft 1990) and effectively delivered to specific responding cells. We postulate that at the same time, or perhaps subsequent to this, another set of signals drives precursors to generate prememory cells. Our studies suggest these emerging memory cells may be phenotypically unique and we postulate that they are specialized to become a "long-lived" population of memory cells that will persist indefinitely as a protective population of increased frequency for the antigen encountered and which is also able to respond more rapidly and effectively. The greater effectiveness of the memory response would thus be due to dramatically increased frequency, to characteristic and stable changes in adhesion molecule expression and to the fact that, in addition to IL-2, resting memory cells also secrete at least low titers of IL-3, IL-4, IFN-gamma and other lymphokines upon initial restimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.

Animals↗
PubMed1991

Is autoimmunity good for you? The Molecular Basis of Recognition in the Immune System sponsored by the European Federation of Immunological Societies, Edinburgh, UK, September 10-12, 1990.

In summary, the EFIS meeting highlighted key advances in the development of B and T cells and in the regulation of that development by other components of the immune system. Several of these recent advances were made possible by the use of transgenic mice expressing either a single antigen-specific receptor or an engineered form of that receptor. We can expect further advances as such mice are used to probe the development of immune responses to antigen. The large number of responding cells should allow heretofore difficult analyses of the cell interactions, immune cell localizations, and patterns of response during the generation of the primary immune response and should also facilitate the study of immune memory.

Animals↗
PubMed1989

A role for class I MHC antigens in responses of activated B lymphocytes.

Expression of the H-2 D protein was increased over 10-fold in B cells activated by IL-4 IL-5, IFN-gamma or LPS. Differences in the regulation of H-2 D expression and in growth rates were detected during lymphokine-triggered responses of subpopulations of splenic B cells. The enhancement of H-2 D expression on activated B cells was regulated at the transcriptional level and required synthesis of an intermediate cellular protein(s). B cell growth and differentiation were inhibited by antibodies to the H-2 D Ag suggesting a signaling function for the class I molecules on activated B lymphocytes. In T cells, a similar elevation of H-2 D expression occurred after stimulation with IL-5 or Con A.

Animals↗
PubMed1989

Induction of the H-2 D antigen during B cell activation.

Mitogenic activation causes increased expression of class I Ag of the MHC in mouse B cells. The increased expression was seen in flow cytometry analysis for both K and D in k as well as d haplotypes. A more detailed molecular analysis was carried out for H-2Dd. Increased expression (10- to 20-fold) of the H-2 Dd gene was detected at both protein and messenger RNA levels, and the time course for the accumulation of H-2 Dd protein on the cell surface parallels the increase in the steady-state messenger RNA levels. The increase in H-2 Dd expression in small B cells stimulated with LPS is detectable after 10 h of culture. The present data provide molecular and serologic evidence about alterations in the expression of the H-2 Dd Ag, previously identified as a B cell activation antigen B7.2. Our results indicate a new significance for the function and regulation of the MHC during immune responses, and suggest that the class I molecules may serve some role in the B cell activation process.

Amino Acid Sequence↗
PubMed1988

Role of antigen in the B cell response. Specific antigen and the lymphokine IL-5 synergize to drive B cell lymphoma proliferation and differentiation to Ig secretion.

CH12 tumor B cells specific for SRBC require SRBC as Ag and the lymphokine IL-5 (formerly known as BCGFII) for optimal proliferation and differentiation to Ig-secreting cells. Lysed SRBC and IL-5 purified to homogeneity synergize markedly, especially at low B cell densities. A sizable proportion of CH12 cells differentiate into Ig-secreting plaque-forming cells when low numbers of the B lymphoma cells (100 to 3000) are cultured with Ag and IL-5. IL-2 or IL-4 have no effects. Intact SRBC or lysed SRBC are equally effective as sources of Ag. Even in the presence of the mitogens LPS and dextran sulfate, there is a striking requirement for Ag for both proliferation and differentiation at low B cell density. Because of the low cell numbers used, the results strongly suggest that the effects of Ag and lymphokine are directly on the B cell. The cell surface phenotype of the CH12 lymphoma and the kinetics of their response suggest that CH12 B cells have the characteristics of activated B cells. Thus, it appears that Ag binding to surface Ig gives a direct signal to at least some B cells that is critical in the later phases of the B cell response after initial activation during which proliferation and differentiation to Ig secretion occur and that the lymphokine IL-5 costimulates with Ag to mediate this phase of the response.

Adjuvants, Immunologic↗
PubMed1987

B cell stimulatory factor 1 (interleukin 4) is sufficient for the proliferation and differentiation of lectin-stimulated cytolytic T lymphocyte precursors.

In this report, we demonstrate that IL-4 is sufficient to stimulate both the proliferation and differentiation of Lyt-2+, Ia- splenic CTL precursors stimulated with the mitogenic lectin Con A. The response to IL-4 and Con A was not dependent on a putative endogenous production of IL-2 within the cultures, as demonstrated by an absence of an inhibitory effect by an anti-IL-2-R blocking mAb. Our results indicate that IL-2 and IL-4 can support an equivalent proliferative response by lectin-stimulated Lyt-2+ T lymphocytes, while IL-4 is more efficacious in stimulating their differentiation into mature cytolytically active cells.

Animals↗
PubMed1987

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5).

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.

Amino Acid Sequence↗
PubMed1987

B cell surface glycoprotein induced during growth response: molecular structure and expression pattern.

We present the molecular characterization of a cell surface antigen, B 7.2, that is expressed on activated B lymphocytes. The BCL1 and CH12 B lymphoma cells express the B 7.2 antigen constitutively. In small resting B cells from spleen, the B 7.2 expression is induced during polyclonal growth in response to mitogenic stimulation. B 7.2 expression also occurs with a significant frequency in cells from fresh lymphoid tissues. The endogenous expression of the B 7.2 antigen is high in spleen and lymph nodes, and is undetectable in the thymus. The B 7.2 antigen is a microheterogeneous 45,000 to 50,000 dalton glycoprotein with a single polypeptide chain, intramolecular S--S bonds, and N-linked glycan moieties. The folded structure of the B 7.2 antigen appears to contain a domain with hydrophilic properties exposed on the cell surface and a hydrophobic segment that may comprise a transmembrane portion. Considering the observed expression pattern and the molecular structure, we speculate that the B 7.2 antigen has a specific function in regulation of B cell activation, perhaps as a receptor for a regulatory ligand or as a ligand recognized by other B or T cells.

Animals↗
PubMed1987

Expression of an endothelial surface antigen on activated human lymphocytes: lymphocyte subset distribution and kinetics of antigen activation.

In this study we have utilized a monoclonal antibody, B721, to demonstrate the expression of an endothelial surface antigen on activated human lymphocytes. Using one- and two-color flow cytometry we have demonstrated that this antigen appears in vitro on cultured lymphocytes stimulated by mitogen or by MLC. The appearance and expression of the antigen are similar regardless of the stimulus. The antigen first appears on Day 2 of culture and expression continues through Day 6 of culture. At the time of its maximum expression, the antigen is present on a majority of B lymphoblasts and CD8 T lymphoblasts, but is present on only a subpopulation of CD4 T lymphoblasts. This antigen appears distinct from other lymphocyte activation antigens, endothelial antigens, and trophoblast antigens. It may play a role in lymphocyte activation and immune responses.

Antibodies, Monoclonal↗