T cell costimulation through the CD28 receptor.
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Animals↗
Biomedical subjects
C B Thompson
Publications and source records attributed to C B Thompson.
The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.
The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins.
Amino Acid Sequence↗
Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system.
Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ (beta-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S. Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2. Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2, Bcl-X-L, or Mcl-1 but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.
Bacterial Proteins↗
Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2.
Cyclosporin A, FK-506, and rapamycin are immunosuppressants often used as pharmacological probes to study lymphocyte activation and physiological cell death (PCD). Because cyclosporin A and FK-506 are known to prevent PCD in T-cell hybridomas and thymocytes, we used these reagents, as well as rapamycin, to determine whether they alter the pathway leading to apoptosis in murine WEHI-231 cells following surface IgM cross-linking. We observed that the immunosuppressants themselves induced PCD in WEHI-231 cells, but only in sublines susceptible to anti-IgM-mediated apoptosis. PCD was preceded by growth arrest and characterized by the DNA fragmentation pattern typical of apoptosis. In B-cell lines resistant to anti-immunoglobulin- and immunosuppressant-induced PCD, cyclosporin A, FK-506, and rapamycin caused growth arrest. PCD was also induced by inhibitors of protein synthesis in WEHI-231 cells but not in the mature B-cell line BAL-17. Immunosuppressant-induced and protein synthesis inhibitor-induced PCD, but not growth arrest, could be prevented by the overexpression of bcl-xL, while transfection with bcl-2 did not affect PCD or cell cycle arrest. These results suggest that bcl-2 and bcl-xL may control partially independent systems to inhibit PCD in lymphoid cells and that PCD in B and T cells may be differentially regulated.
Animals↗
The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities.
Although recent data have demonstrated that the chimeric EWS-FLI-1 cDNA isolated from cases of Ewing's sarcoma can transform NIH 3T3 cells, little is known about the basis for this transformation. Since FLI-1 and EWS-FLI-1 contain an Ets domain, both proteins may act as sequence-specific transcription factors. Here the DNA binding properties of FLI-1 and EWS-FLI-1 have been examined. An epitope-tagging strategy was developed to determine the optimum DNA-binding sequence of FLI-1. The alignment of cloned binding sequences showed a consensus DNA-binding site of ACCGGAAG/aT/c. This consensus sequence shows greater specificity for sequence 5' of the GGAA core site than those of other Ets proteins. Using several truncated forms of FLI-1, we show that the Ets domain is necessary and sufficient for the DNA binding specificity of FLI-1. The EWS-FLI-1 protein displayed the same DNA binding specificity and affinity as FLI-1 did. Despite their DNA binding similarities, the EWS-FLI-1 translocation product is likely to have a distinct pattern of expression from that of FLI-1 since the translocation results in the replacement of the 5' regulatory region of Fli-1 with that of EWS. Consistent with this we found that Fli-1 mRNA expression in lymphocytes was high in quiescent cells and disappeared upon activation while EWS mRNA expression was low in resting cells and increased in activated T cells. In summary, our data suggest that EWS-FLI-1 might act through the same target genes normally regulated by FLI-1, and EWS-FLI-1-induced transformation may result from dysregulation of FLI-1 target genes during cell proliferation and differentiation.
3T3 Cells↗
Keratinocyte-derived T cell costimulation induces preferential production of IL-2 and IL-4 but not IFN-gamma.
By using superantigens, we have found previously that keratinocytes activated by IFN-gamma could serve as accessory cells, providing costimulatory signals needed to induce T cell proliferation. Here, we compared the profile of cytokines produced by T cells stimulated in the presence of activated keratinocytes with the response seen using professional APCs. When keratinocytes are used as accessory cells there is a specific defect in T cell IFN-gamma production, whereas IL-2 and IL-4 are induced at levels comparable with those seen when professional APCs are used as accessory cells. Because keratinocytes express BB-1, a CD28-ligand distinct from B7-1 or B7-2 (which are found on professional APCs), we examined the possibility that the defect in IFN-gamma production might be a result of nonproductive CD28 engagement. However, even when the CD28 pathway is directly activated by a stimulatory mAb, there is no induction of IFN-gamma production in keratinocyte-supported cultures. In these same cultures IL-2 production is increased 10-fold, thus demonstrating a specific deficiency in the induction of IFN-gamma rather than a failure to respond to CD28 stimulation. Analysis by reverse transcriptase-PCR and ELISA for the inducible p40 chain of IL-12 reveals that keratinocytes produce little if any messenger RNA and no protein for IL-12 p40 compared with professional APCs. Addition of rIL-12 to keratinocyte-supported cultures restores IFN-gamma levels to those seen when professional APCs are present. Finally, when T cells are restimulated and analyzed at later time points (10 to 14 days) we find a refinement in cytokine profiles: T cells stimulated in the presence of professional APCs produced the Th1 cytokines IL-2 and IFN-gamma, whereas T cells stimulated in the presence of activated keratinocytes produced only the Th2 cytokine IL-4. The specific ability of keratinocytes to induce a Th2 response seems most closely linked to their absence of IL-12 production, and may be important in the maintenance of peripheral tolerance to self-Ags or in the immune response to exogenous Ags, pathogens, or haptens encountered in skin.
Antigen-Presenting Cells↗
Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression.
Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.
Base Sequence↗
AU-A, an RNA-binding activity distinct from hnRNP A1, is selective for AUUUA repeats and shuttles between the nucleus and the cytoplasm.
The 3'-untranslated regions of many labile transcripts contain AU-rich sequences that serve as cis determinants of mRNA stability and translational efficiency. Using a photocrosslinking technique, our laboratory has previously defined three cytoplasmic RNA-binding activities specific for the AUUUA multimers found in the 3'-untranslated regions of lymphokine mRNAs. One of these activities, AU-A, has an apparent molecular mass of 34 kDa, is constitutively expressed in both primary T cells and the Jurkat T cell leukemia line, and binds to a variety of U-rich RNA sequences. Previous studies had shown that AU-A is more prevalent in the nucleus than the cytoplasm, raising the possibility that AU-A is really a nuclear RNA-binding activity that is found in cytoplasmic extracts because of nuclear leakage during cell fractionation. We now show that AU-A shuttles between the cytoplasm and the nucleus. Our results indicate that AU-A is a candidate protein component of ribonucleoprotein complexes that participate in nucleocytoplasmic transport of mRNA and cytoplasmic mRNA metabolism. The properties of AU-A activity are similar to those of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). However, using monoclonal antibodies to hnRNP A1 and protease digestion patterns, we show that AU-A activity and hnRNP A1 protein are distinct. These studies have also allowed us to define a fourth RNA-binding activity of apparent molecular mass 41 kDa with specificity for AUUUA multimers. This activity is restricted to the nucleus and contains the hnRNP C protein.
Base Sequence↗
T cell receptor stimulation, but not CD28 costimulation, is dependent on LFA-1-mediated events.
Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. In this manuscript we specifically examine the role of two accessory molecules, CD28 and LFA-1, in modulating the T cell proliferative response to a variety of stimuli. We demonstrate that the proliferation induced by staphylococcal enterotoxins A and B in combination with CD28 costimulation is dependent on LFA-1-mediated events. This requirement for LFA-1 is independent of T cell-accessory cell adhesion. Similarly, an allogeneic mixed lymphocyte reaction, which has previously been shown to be a CD28-dependent response, can be inhibited by blockade of LFA-1. This suggests LFA-1 plays an essential role in these responses, either by enhancing intercellular adhesion or by an independent signal transduction event. In contrast, when the primary activating stimulus is delivered by immobilized anti-CD3 antibody or by PMA, and the secondary stimulus by either alpha-CD28 or cell-bound CD28 ligand B7, there is no requirement for LFA-1. In addition, we demonstrate that cross-linking of LFA-1 with immobilized monoclonal antibody, or engagement of LFA-1 with ICAM-1 expressed on the surface of a CHO cell, provide an insufficient costimulus for T cell proliferation initiated by enterotoxin, immobilized alpha-CD3 or phorbol ester. Our data suggests that LFA-1, in contrast to CD28, functions not as a costimulatory molecule, but serves primarily to modulate the signal delivered through the T cell receptor.
Animals↗
Modulation of T cell proliferative response by accessory cell interactions.
Antigen-specific activation of the T cell is accomplished by engagement of the T cell receptor (TCR) by an antigen (Ag)/MHC complex presented on the surface of an antigen- presenting cell (APC). However, it has been demonstrated that engagement of the TCR by Ag/HC complexes alone is normally insufficient to lead to a proliferative response and the development of effector function. Thus it has been proposed that the APC also provides additional signals which serve to modulate the T cell's response. These second or costimulatory signals are thought to be critical in the generation of a T cell-driven immune response. Several receptors have been proposed to be capable of serving as costimulatory receptors. Candidate molecules include CD28 and LFA-1 as well as other receptors. In this review the studies that we have performed to clarify the role of both LFA-1 and CD28 in providing costimulatory activity for T cell activation are discussed. In addition, we present evidence that under certain conditions, TCR signalling alone can be sufficient to lead to T cell proliferation.
Animals↗
The B7 and CD28 receptor families.
Current evidence suggests that T-cell receptor (TCR) recognition of antigen bound to the major histocompatibility complex (Ag-MHC) is insufficient to lead to T-cell proliferation or effector function. For a helper T cell to produce sufficient interleukin 2 (IL-2) to allow autocrine-driven clonal expansion, there is a requirement for so-called 'co-stimulatory' or 'accessory' signals in addition to TCR ligation by Ag-MHC. The interaction of the CD28 receptor on T cells with B7 on antigen-presenting cells (APCs) supplies one such co-stimulatory signal. However, the recent discovery that CD28 and B7 are each members of larger gene families suggests that the regulation of co-stimulation is more complex than previously imagined. Here, Carl June and colleagues highlight recent advances in the understanding of the CD28 and B7 receptor families.
Abatacept↗
Transcriptional regulation of T-cell genes during T-cell development.
The expression of many T cell specific genes has been shown to be regulated at the transcriptional level. Recent studies of T-cell specific promoters and enhancers have allowed the identification of a number of transcription factors that appear to play distinct but complementary roles in regulating gene expression during T-cell development and activation.
Animals↗
CTLA-4 can function as a negative regulator of T cell activation.
CD28 and CTLA-4 are related glycoproteins found on T cells. Ligation of CD28 following antigen receptor engagement provides a costimulatory signal required for T cell activation. Anti-CTLA-4 antibodies were generated to examine the role of the CTLA-4 receptor on murine T cells. Expression of CTLA-4 as a homodimer is up-regulated 2-3 days following T cell activation. Anti-CTLA-4 antibodies and Fab fragments augmented T cell proliferation in an allogeneic MLR. However, when optimal costimulation and Fc cross-linking were present, anti-CTLA-4 Mabs inhibited T cell proliferation. Together, these results suggest that the MAb may obstruct the interaction of CTLA-4 with its natural ligand and block a negative signal, or directly signal T cells to down-regulate immune function.
Abatacept↗
Absence of B7-dependent responses in CD28-deficient mice.
Costimulation of T cell proliferation can occur through the CD28 signal transduction pathway. In addition, other cell surface receptors, including the CD28 homolog CTLA-4, have been proposed to be capable of providing costimulatory signals. We have examined the response of CD28-deficient T cells to activation by a variety of agonists. We demonstrate that proliferation of CD28-deficient T cells in the presence of antigen-presenting cells or B7-1 transfectants is markedly reduced. Although CTLA-4 can be expressed on CD28-deficient T cells, we observed no B7-dependent costimulation in the absence of CD28. This data demonstrates that CD28 is the major B7-binding costimulatory ligand on T cells. Furthermore, our data suggest that CD28 is the primary, and perhaps exclusive, costimulatory receptor used by traditional antigen-presenting cells to augment the proliferation of antigen-activated T cells.
Abatacept↗
Solution structure of the ets domain of Fli-1 when bound to DNA.
Members of the ets family of transcription factors share a conserved DNA-binding domain, the ets domain. By using multidimensional NMR, we have determined the structure of the ets domain of human Fli-1 in the DNA-bound form. It consists of three alpha-helices and a four-stranded beta-sheet, similar to structures of the class of helix-turn-helix DNA binding proteins first found in the catabolite activator protein of Escherichia coli. NMR and mutagenesis experiments suggest that in comparison to structurally related proteins, the ets domain uses a new variation of the helix-turn-helix motif for binding to DNA.
Amino Acid Sequence↗
A molecular genetic intervention for AIDS--effects of a transdominant negative form of Rev.
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Acquired Immunodeficiency Syndrome↗
Regulation of RAG-2 protein expression in avian thymocytes.
The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells.
Animals↗
Activation of the granulocyte-macrophage colony-stimulating factor promoter in T cells requires cooperative binding of Elf-1 and AP-1 transcription factors.
The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene has been studied extensively as a model system of transcriptional induction during T-lymphocyte activation. The GM-CSF gene is not expressed in resting peripheral blood T cells but is rapidly induced at the transcriptional level following activation through the cell surface T-cell receptor. A highly conserved 19-bp element located immediately 5' of the human GM-CSF TATA box (bp -34 to -52), herein called purine box 1 (PB1), has been shown to bind a T-cell nuclear protein complex and to be required for transcriptional induction of the GM-CSF gene following T-cell activation. The PB1 sequence motif is highly conserved in both human and murine GM-CSF genes. In this report, we demonstrate that the PB1 element alone confers inducibility on a heterologous promoter following transfection into human Jurkat T cells. In addition, we identify a major PB1 nuclear protein-binding complex that is not present in resting peripheral blood T cells but is rapidly induced following T-cell activation. Sequence analysis revealed that PB1 is composed of adjacent binding sites for Ets and AP-1 transcription factors. In vitro mutagenesis experiments demonstrated that both the Ets and AP-1 sites are required for binding of the inducible PB1 nuclear protein complex and for the transcriptional activity of this element and the GM-CSF promoter in activated T cells. Using antibodies specific for different Ets and AP-1 family members, we demonstrate that the major inducible PB1-binding activity present in activated T-cell nuclear extracts is composed of the Elf-1, c-Fos, and JunB transcription factors. Taken together, these results suggest that cooperative interactions between specific Ets and AP-1 family members are important in regulating inducible gene expression following T-cell activation.
Adult↗