Structure-function relationship of immunosuppressive drugs: a cautionary tale.
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Animals↗
Biomedical subjects
C B Thompson
Publications and source records attributed to C B Thompson.
Guanine ribonucleotide depletion inhibits T cell activation.
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Cell Cycle↗
Avian T cell ontogeny.
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Amino Acid Sequence↗
An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA.
Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.
Base Sequence↗
Avian B-cell development: generation of an immunoglobulin repertoire by gene conversion.
The vertebrate B-cell repertoire is capable of generating up to 10(9) different antibody molecules using relatively few germline immunoglobulin (Ig) gene segments. To generate diversity, humans and mice depend on combinatorial and junctional variations that occur during the gene rearrangement events that produce complete heavy and light chain Ig genes. This gene rearrangement process goes on continuously in the bone marrow, where each developing B cell assembles a unique heavy and light chain Ig gene from families of functional V, D, and J gene segments. In contrast, chickens have only single functional V and J segments for the heavy and light chain loci, and chicken Ig gene rearrangement occurs only during a brief period of embryonic development. A specialized organ involved in avian B-cell development, the bursa of Fabricius, provides the microenvironment necessary for the amplification of B cells that have undergone productive Ig gene rearrangements. Within the bursa, B cells also acquire somatic diversity within the rearranged V gene segments of the heavy and light chain Ig loci. Somatic diversification of chicken V gene segments occurs by intrachromosomal gene conversion, a DNA recombination process which involves unidirectional transfer of nucleotide sequence blocks from families of V region pseudogenes into the functional rearranged VH and VL genes.
Animals↗
Guanine ribonucleotide depletion inhibits T cell activation. Mechanism of action of the immunosuppressive drug mizoribine.
The immunosuppressive drug, mizoribine, has been used to prevent rejection of organ allografts in humans and in animal models. Based on studies in cell lines, mizoribine has been postulated to be an inhibitor of inosine monophosphate (IMP) dehydrogenase (EC1.2.1.14), a pivotal enzyme in the formation of guanine ribonucleotides from IMP. To further characterize the mechanism of action of this drug, we studied the effect of mizoribine on human peripheral blood T cells stimulated with alloantigen, anti-CD3 MAb, or pharmacologic mitogens. Mizoribine (1-50 micrograms/ml) was able to inhibit T cell proliferation by 10-100% in a dose-dependent fashion to all stimuli tested. Measurements of purine ribonucleotide pools by HPLC showed that mizoribine led to a decrease in intracellular GTP levels, and that repletion of GTP reversed its antiproliferative effects. We also examined sequential events occurring after T cell stimulation. Early events in T cell activation, as assessed by steady-state mRNA levels of c-myc, IL-2, c-myb, histone, and cdc2 kinase, as well as surface IL-2 receptor expression, were unaffected. However, cell cycle analysis revealed decreased numbers of cells in S, G2, and M phases, and showed that the G1/S block was reversed with GTP repletion. These data indicate that mizoribine has an effect on T cell proliferation by a mechanism distinct from that of cyclosporine or corticosteroids, and therefore may be useful in combination immunosuppressive regimens.
Adenosine Triphosphate↗
Sequence-specific binding of human Ets-1 to the T cell receptor alpha gene enhancer.
Expression of the human T cell receptor (TCR) alpha gene is regulated by a T cell-specific transcriptional enhancer that is located 4.5 kilobases (kb) 3' to the C alpha gene segment. The core enhancer contains two nuclear protein binding sites, T alpha 1 and T alpha 2, which are essential for full enhancer activity. T alpha 1 contains a consensus cyclic adenosine monophosphate (cAMP) response element (CRE) and binds a set of ubiquitously expressed CRE binding proteins. In contrast, the transcription factors that interact with the T alpha 2 site have not been defined. In this report, a lambda gt11 expression protocol was used to isolate a complementary DNA (cDNA) that programs the expression of a T alpha 2 binding protein. DNA sequence analysis demonstrated that this clone encodes the human ets-1 proto-oncogene. Lysogen extracts produced with this cDNA clone contained a beta-galactosidase-Ets-1 fusion protein that bound specifically to a synthetic T alpha 2 oligonucleotide. The Ets-1 binding site was localized to a 17-base pair (bp) region from the 3' end of T alpha 2. Mutation of five nucleotides within this sequence abolished both Ets-1 binding and the activity of the TCR alpha enhancer in T cells. These results demonstrate that Ets-1 binds in a sequence-specific fashion to the human TCR alpha enhancer and suggest that this developmentally regulated proto-oncogene functions in regulating TCR alpha gene expression.
Base Sequence↗
The genomic organization of the CD28 gene. Implications for the regulation of CD28 mRNA expression and heterogeneity.
CD28 is a 90-kDa homodimeric glycoprotein present on the surface of a large subset of T cells that appears to play an important role in the modulation of T cell activation. Although a number of physiologic effects associated with CD28 stimulation have been defined, relatively less is known about the structure and expression of the CD28 gene itself. We now show that CD28 is expressed in both Th cells and plasma cells as a series of four distinct CD28 mRNA species: 1.3-, 1.5-, 3.5-, and 3.7-kb transcripts. The steady state expression of all four transcripts in CD28+ T cells was stimulated by PMA, suggesting that they might share a common phorbol-sensitive promoter. Consistent with this hypothesis, CD28 was found to be encoded by a single copy gene organized into four exons, each exon defining a functional domain of the predicted protein. All CD28 transcripts appear to initiate within a 61-bp palindrome. Generation of the four CD28 mRNA species from the CD28 gene involves two distinct posttranscriptional events. The longer pair of transcripts (3.5/3.7 kb) is generated by the use of an alternate nonconsensus polyadenylation signal. This results in the addition of 2167 bp beyond the first polyadenylation site utilized by the shorter (1.3/1.5 kb) pair of transcripts. The size difference between the 3.7- and 3.5-kb messages and between the 1.5- and 1.3-kb messages is generated by an internal splicing event that deletes 252 bp within exon 2, which encodes the extracellular domain. This deletion would result in the loss of 84 amino acids, including 4 of 5 extracellular cysteine residues. Although this deletion would result in significant disruption of CD28 secondary structure, it would not be expected to interfere with the ability of the resultant protein to be expressed on the cell surface. These findings suggest that variant isotypes of CD28 may be expressed on the cell surface with potentially different physiologic roles.
Amino Acid Sequence↗
CD28 ligation in T-cell activation: evidence for two signal transduction pathways.
The CD28 homodimer is thought to function as a signal transducing receptor during activation of T cells. Evidence is presented that the degree of aggregation of CD28 on the cell surface regulates two distinct CD28-associated signals. Binding of bivalent CD28 monoclonal antibody (MoAb) 9.3 upregulates lymphokine production by messenger RNA (mRNA) stabilization, without direct initiation of lymphokine mRNA transcription. This signal was not dependent on inositol phospholipid production or activation of a protein tyrosine kinase (PTK). In contrast, further crosslinking of CD28 on the cell surface rapidly induced formation of large amounts of inositol trisphosphate (InsP3) and increased cytoplasmic calcium concentration [( Ca2+]i), but did not stimulate PTK. CD28 crosslinking directly activated a subset of resting T cells, since CD25 (interleukin [IL]-2 receptor alpha chain) mRNA was rapidly induced in purified T cells, and proliferation, even without addition of exogenous IL-2, was sometimes observed. CD25 expression was detected on the cell surface of approximately 20% of CD4+ T cells. The degree of CD28 aggregation required for activation was investigated by preparing soluble 9.3 x 9.3 conjugates ranging in size from approximately 300 Kd to greater than 1,000 Kd, and comparing their function in T-cell proliferation assays with phorbol-12-myristate-13-acetate (PMA), anti-CD3, or IL-2. There was a correlation between conjugate size and proliferation with IL-2, whereas costimulation with PMA or CD3 was optimized at a lower degree of CD28 aggregation. The inositol phospholipid (InsP) generation and increase in [Ca2+]i after CD28 receptor aggregation appeared to proceed through a pathway different from the CD3/T-cell receptor (TCR) pathway since it was enhanced by pretreatment with PMA, while the InsP and [Ca2+]i signal from crosslinking CD3 was suppressed by PMA. Furthermore, the proliferation response to CD28 aggregation was resistant to inhibition by CD3 modulation. Thus, CD28 aggregation appears to trigger a phospholipase C activation pathway that differs from the CD3/TCR-linked pathway.
Animals↗
CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes.
The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.
Antibodies, Monoclonal↗
Role of the CD28 receptor in T-cell activation.
Antigen-specific T-cell activation is initiated through the T-cell receptor. Recent evidence has shown that a number of additional T-cell surface receptors serve to regulate the responses of antigen-activated T cells. One such molecule, CD28, is a member of a heterophilic cell adhesion complex, and is the receptor for the B-cell-restricted B7/BB-1 antigen. As Carl June, Jeffrey Ledbetter, Peter Linsley and Craig Thompson review here, CD28 serves as the surface component of a novel signal transduction pathway that modulates T-cell lymphokine production and increases the resistance of T-cell responses to various immunosuppressive agents.
Animals↗
Somatic diversification of the chicken immunoglobulin light-chain gene.
The bursa of Fabricius provides a unique organ for the study of lineage-specific development in a multicellular organism. Unlike mammalian B cells, B cells in the chicken develop in a single wave of differentiation, beginning with the commitment of progenitor cells to the B cell lineage between days 10 and 15 of embryogenesis. By day 18 of embryogenesis, all lymphoid progenitor cells capable of differentiation along the B cell lineage have migrated to the bursa of Fabricius. Following migration to the bursa, these lymphoid progenitors enter exponential growth and begin to populate each of the 10(4) bursal follicles. Between day 18 of embryogenesis and 2-4 weeks of age, B cells undergo a stage of bursal-dependent differentiation. By the end of this period, chickens are able to mount primary immune responses against virtually all antigens. In addition, by this time sufficient numbers of B cells have migrated from the bursa to peripheral lymphoid organs so that the B cell immune system can be maintained even if the bird is bursectomized. Bursectomy of chicks after 4 weeks of age has no long-term effects on the development and maintenance of the B cell immune system in adult birds. Because of the central nature of the surface Ig molecule to B cell development in mammals, the chicken IgL gene locus has been intensively studied during avian B cell development. The chicken IgL locus is a particular interest because it has only one V region capable of rearrangement. Rearrangement of the IgL gene is not dependent on the bursal environment. B cell progenitors rearrange their IgL gene between days 10-15 of embryogenesis, prior to migration to the bursa. IgL gene rearrangement occurs by a deletional mechanism in which a precise joining of the IgL recombination signal sequences leads to a circular episomal element. During this deletion it appears that single nonrandom bases are added to both the V and J coding segments. Subsequent V-J joining occurs at random. Most progenitor B cells appear to rearrange only a single IgL allele. The high frequency of in-frame alleles observed in avian B cell lines appears to result from the selective amplification of cells with productive IgL rearrangements during bursal development between days 12 and 18 of embryogenesis. To create an immunological repertoire, chickens must diversify the coding sequence of this single functional V gene segment during development.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Reciprocal expression of human ETS1 and ETS2 genes during T-cell activation: regulatory role for the protooncogene ETS1.
The expression of the protooncogenes ETS1 and ETS2 has been studied in purified human T cells activated either by cross-linking of the T-cell receptor-CD3 complex on their cell surface or by direct stimulation with phorbol esters and ionomycin. Our results show that resting T cells express high levels of ETS1 mRNA and protein, while expression of ETS2 is undetectable. Upon T-cell activation, ETS2 mRNA and proteins are induced, while ETS1 gene expression decreases to very low levels. Late after stimulation, ETS1 mRNA is reinduced and maintained at a high level, while ETS2 gene expression decreases to undetectable levels. Therefore, it appears that in human T cells, ETS2 gene products are associated with cellular activation and proliferation, while ETS1 gene products are preferentially expressed in a quiescent state.
Calcium↗
Evolutionary conservation of antigen recognition: the chicken T-cell receptor beta chain.
T cells play important regulatory roles in the immune responses of vertebrates. Antigen-specific T-cell activation involves T-cell receptor (TCR) recognition of a peptide antigen presented by a major histocompatibility complex molecule, and much has been learned about this antigen-recognition process through structural and genetic studies of mammalian TCRs. Although previous studies have demonstrated that avian T cells express cell-surface molecules analogous to the mammalian TCR heterodimers, TCR genes have not been identified in nonmammalian species. We now report the cloning of a cDNA that encodes the beta chain of the chicken TCR. Southern blot analysis using this TCR beta cDNA probe demonstrated that the chicken TCR beta locus was clonally rear-ranged in chicken T-cell lines. TCR beta mRNA was expressed in cells isolated from the thymus but not in cells from the bursa of Fabricius where B cells are generated. Sequence analysis of six additional TCR beta cDNAs suggested the existence of at least two variable (V) region families, three joining (J) elements, and single diversity (D) and constant (C) elements. As in mammals, considerable nucleotide diversity was observed at the junctions of the variable, diversity, and joining elements in chicken TCR beta cDNAs. Genomic V beta and J beta elements were also cloned and sequenced. Both elements are flanked by classical heptamer/nonamer recombination signal sequences. Although the chicken and mammalian TCR beta chains displayed only 31% overall amino acid sequence identity, a number of conserved structural features were observed. These data indicate that (i) the chicken TCR beta repertoire is generated by combinatorial and junctional diversity and (ii) despite divergent evolution at the level of nucleotide sequence, important structural features of the TCR beta polypeptide are conserved between avian and mammalian species.
Amino Acid Sequence↗
Characterization of chicken octamer-binding proteins demonstrates that POU domain-containing homeobox transcription factors have been highly conserved during vertebrate evolution.
The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, we identified two homologous octamer-binding proteins in chicken B cells. When the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, we sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, we were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. Low-stringency screening of a chicken embryonic cDNA library allowed us to clone one of these genes. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. The predicted protein sequence of the chicken Oct-1 gene demonstrated that the gene for Oct-1 has been highly conserved during vertebrate evolution with an overall 96% amino acid sequence identity between the chicken and human proteins. The previously described POU domain (termed POU for its presence in the Pit-1, Oct-1/Oct-2, and Unc-86 genes) and homeobox domain are 100% conserved between the two protein products. Together, our data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.
Amino Acid Sequence↗
Estimates of exposure rates and fallout arrival times near the Nevada Test Site.
One of the tasks of the Off-Site Radiation Exposure Review Project (ORERP) was to estimate doses to individuals resulting from exposure to fallout from nuclear testing at the Nevada Test Site (NTS). Dose estimates are based on estimates of the exposure rate 12 h post-detonation (H + 12) and the time of fallout arrival from events producing discernible fallout at locations off the NTS. These estimates are derived from both published fallout patterns and survey meter readings taken by monitors in the field. Each fallout pattern is digitized, and kriging is used to interpolate estimates of exposure rate and arrival time onto a 10-km grid covering the contours of the pattern. These grid values are then used to calculate estimates at any location of interest within the pattern. Exposure rate is also estimated from the survey meter readings for a particular nuclear event by decay-correcting the measurements near a selected location to H + 12 and then aggregating them into one estimate. Estimates of discernible exposure rate and time of fallout arrival from 74 nuclear events at the NTS are contained in the Town Data Base. Estimates are given for 352 locations in Arizona, California, Nevada, and Utah. Each record in the data base contains estimates for a specified event and location. The distribution of exposure rate is represented by a geometric mean and standard deviation; the distribution of fallout arrival time is represented by an arithmetic mean and standard deviation. A more extensive description and a listing of the Town Data Base are included in a separate report.
Arizona↗
Expertise: the basis for expert system development.
Expert systems in nursing are developed with traditional knowledge engineering techniques. These techniques focus on the behavior and logic of the expert, not qualities of expertise. Expertise has been described but not explained. This article proposes a theoretical framework for the study of expertise that can be used to facilitate the development of expert systems.
Clinical Competence↗
Templated insertions in the rearranged chicken IgL V gene segment arise by intrachromosomal gene conversion.
Chickens create a repertoire for their immunoglobulin light-chain gene by a novel process of sequence substitution within a unique rearranged V gene segment (VL1) during B-cell development in the bursa of Fabricius. Sequence analysis has shown that these nucleotide substitutions are not random. Potential donors for observed sequence substitutions are present within the 25 psi VL segments located 5' of the VL1 gene. In this report, we demonstrate that VL1 sequence substitutions: (1) are derived from the psi VL donor segment templates in cis, (2) do not result in reciprocal transfer of VL1 gene sequences to the psi VL segments, and (3) lead to the rapid disappearance of cells with nondiversified rearranged VL1 genes during B-cell development in the bursa of Fabricius. Together, these data provide evidence that VL1 sequence diversity arises as a result of intrachromosomal gene conversion.
Animals↗