Transcription factors in lymphocyte development--T and B cells get together.
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B-Lymphocytes↗
Biomedical subjects
L H Glimcher
Publications and source records attributed to L H Glimcher.
Using thymus anatomy to dissect T cell repertoire selection.
The thymic development of CD4(+) T cells incorporates the opposing processes of positive and negative selection to produce mature lymphocytes which respond to foreign peptides in the context of self-major histocompatibility complex class II molecules. Here, we present a model in which these events occur in two temporally and anatomically distinct steps. We propose that an initial positive selection step is mediated exclusively by thymic cortical epithelium. Subsequently, all those thymocytes which have been positively selected will interact with medullary epithelium and bone marrow-derived cells. Those thymocytes reacting with excess affinity or avidity to these antigen presenting cells will be negatively selected. Although acknowledging the importance of differential signalling to the developing thymocyte, we will emphasize the centrality of the phenotype of the tissues which comprise the thymus.
Animals↗
The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines.
IL-4 promotes the differentiation of naive CD4+ T cells into IL-4-producing T helper 2 (Th2) cells. Previous work provided suggestive but not conclusive evidence that the transcription factor c-Maf directed the tissue-specific expression of IL-4. It was not known whether c-Maf controlled the transcription of other Th2 cytokine genes. To elucidate the role of c-Maf in vivo, we examined cytokine production in mice lacking c-Maf (c-maf(-/-)). CD4+ T cells and NK T cells from c-maf(-/-) mice were markedly deficient in IL-4 production. However, the mice produced normal levels of IL-13 and IgE, and, when differentiated in the presence of exogenous IL-4, c-maf(-/-) T cells produced approximately normal levels of other Th2 cytokines. We conclude that c-Maf has a critical and selective function in IL-4 gene transcription in vivo.
Animals↗
Marking IL-4-producing cells by knock-in of the IL-4 gene.
IL-4 is a cytokine which can be expressed by a number of cell types including Th2 cells, mast cells and a population of CD4+ NK1.1+ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-secreting Th2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells.
Animals↗
A minimal level of MHC class II expression is sufficient to abrogate autoreactivity.
The establishment of CD4(+) T cell tolerance requires that self-reactive thymocytes are negatively selected during thymic development. A threshold of antigen concentration appears to exist for both MHC class I- and class II-mediated negative selection, below which clonal deletion of a self-reactive transgenic TCR does not occur. Similarly, both the specificity and thymic concentration of MHC molecules affect the efficiency with which autoreactive thymocytes are deleted. However, this threshold for MHC class II concentration has not been well established. Here, we show that this threshold must be extraordinarily low. We have used the human lysozyme promoter to re-express an A(beta)(b) cDNA on macrophages and other phagocytic myelomonocytic cells of class II-deficient A(beta)(b) -/- mice. Surface expression of I-A(b) could be detected on mature peritoneal macrophages and, minimally, on thymic dendritic cells; however, this level of expression was not sufficient for antigen-specific T cell activation. Nevertheless, when backcrossed onto an autoreactive K14 background, this minimal level of class II was sufficient to induce negative selection of a polyclonal self-reactive population. We conclude that provision of extremely low levels of class II to thymic dendritic cells confers on them the ability to mediate clonal deletion of autoreactive T cells.
Animals↗
Tissue-specific regulation of cytokine gene expression.
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Animals↗
Identification of c-myc promoter-binding protein and X-box binding protein 1 as interleukin-6 target genes in human multiple myeloma cells.
Interleukin-6 (IL-6) is implicated in the in vivo proliferation of malignant plasma cells in multiple myeloma. To define the molecular basis of the IL-6-induced mitogenic response in myeloma cells, we applied STAR (subtractive transcriptional amplification of mRNA), a new differential expression analysis technology, to isolate mRNAs preferentially expressed in IL-6-treated versus untreated cultures of the factor-responsive myeloma cell line U266. From the resulting collection of STAR clones, sequence information was obtained for a total of 72 distinct transcripts. Of these, 29 were found to correspond to known genes, 22 matched expressed sequence tags in public databases and 21 showed no sequence similarity to any existing entries. Among the known genes uncovered in the screen were those encoding proteins that function in cell division, cell signalling and gene/protein expression. Northern blot analysis documented that two transcription factor genes chosen for further study, c-myc promoter-binding protein (MBP-1) and X-box binding protein 1 (XBP-1), were up-regulated in U266 cells about 3-fold relative to the cell cycle-dependent beta-actin gene 12 h after IL-6 treatment. Both genes were also similarly up-regulated by IL-6 in factor-dependent ANBL-6 myeloma cells. These results indicate that MBP-1 and XBP-1 are IL-6 genes in myeloma cells; as such, they may play a role in IL-6-mediated growth control in multiple myeloma.
Base Sequence↗
A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis.
NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.
3T3 Cells↗
c-maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms.
The c-maf protooncogene is a T helper cell type 2 (Th2)-specific transcription factor that activates the interleukin (IL)-4 promoter in vitro. Although it has been postulated that c-maf directs the Th2-specific expression of the IL-4 gene in vivo, direct evidence that c-maf functions during the differentiation of normal, primary T cells is lacking. We now demonstrate that overexpression of c-maf in vivo skews the Th immune response along a Th2 pathway, as evidenced by increased production of Th2 cytokines and the IL-4-dependent immunoglobulins, IgG1 and IgE. The overproduction of IgGl and IgE in the CD4 promoter/c-maf transgenic mice was IL-4 dependent since this was not observed in c-maf transgenic mice bred onto an IL-4-deficient background. Ectopic expression of c-maf in mature Th1 cells did not confer on them the ability to produce IL-4, but did decrease the production of IFN-gamma. The attenuation of Th1 differentiation by c-maf overexpression occurred by a mechanism that was independent of IL-4 and other Th2 cytokines, and could be overcome by IL-12. These studies demonstrate that c-maf promotes Th2 differentiation by IL-4-dependent mechanisms and attenuates Th1 differentiation by Th2 cytokine-independent mechanisms.
Animals↗
The transcription factor NF-ATc is essential for cardiac valve formation.
Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.
Animals↗
Delayed rejection of soluble tumor necrosis factor receptor-secreting tumor allografts.
BACKGROUND: Exogenous soluble tumor necrosis factor receptor (TNFR) has been shown to be an effective immunosuppressant. It has yet to be tested whether tissues secreting soluble TNFR, when transplanted into a foreign host, could locally generate immunosuppression and therefore manifest prolonged survival. METHODS: A murine tumor line was transfected with the gene encoding a chimeric protein consisting of the extracellular domain of the human 75-kDa TNFR fused to the Fc region of the human IgG1 heavy chain. This tumor line was then injected into allogeneic recipients. RESULTS: Transfected tumor cells were shown to secrete soluble TNFR. When transplanted into minor histocompatibility antigen-disparate allogeneic recipients, these tumor cells grew as a solid tumor and resisted rejection, whereas untransfected tumors and interleukin-4 receptor transfectant controls were rejected within 4 weeks. The resistance to rejection could be reversed by coadministration of an anti-TNFR monoclonal antibody. CONCLUSIONS: Prolongation of graft survival can be achieved by genetically altering transplanted tissue to secrete soluble cytokine receptors.
Animals↗
Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc.
The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.
Alleles↗
The transcription factor NFAT4 is involved in the generation and survival of T cells.
Nuclear factor of activated T cells (NFAT) is a family of four related transcription factors implicated in cytokine and early response gene expression in activated lymphocytes. Here we report that NFAT4, in contrast to NFATp and NFATc, is preferentially expressed in DP thymocytes. Mice lacking NFAT4 have impaired development of CD4 and CD8 SP thymocytes and peripheral T cells as well as hyperactivation of peripheral T cells. The thymic defect is characterized by increased apoptosis of DP thymocytes. The increased apoptosis and hyperactivation may reflect heightened sensitivity to TcR-mediated signaling. Further, mice lacking NFAT4 have impaired production of Bcl-2 mRNA and protein. NFAT4 thus plays an important role in the successful generation and survival of T cells.
Animals↗
Inhibitory function of two NFAT family members in lymphoid homeostasis and Th2 development.
Nuclear factor of activated T cells (NFAT) is a critical regulator of early gene transcription in response to TCR-mediated signals. Here, we show that mice lacking both NFATp and NFAT4 develop a profound lymphoproliferative disorder likely due to a lowered threshold for TCR signaling coupled with increased resistance to apoptosis secondary to defective FasL expression. NFAT mutant mice also have allergic blepharitis, interstitial pneumonitis, and a 10(3) to 10(4) fold increase in serum IgG1 and IgE levels, secondary to a dramatic and selective increase in Th2 cytokines. This phenotype may be ascribed to unopposed occupancy of the IL-4 promoter by NFATc. Our data demonstrate that lymphoid homeostasis and Th2 activation require a critical balance among NFAT family members.
Alveolitis, Extrinsic Allergic↗
Single amino acid mutations in the murine MHC class II A beta cytoplasmic domain abrogate antigen presentation.
Class II MHC molecules are heterodimeric transmembrane glycoproteins that function in the presentation of Ag to CD4+ T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-chains has previously been shown to diminish Ag presentation and abrogate rejection of class II-transfected tumor cells. To examine the contributions of individual amino acid residues of the A beta cytoplasmic domain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the five conserved amino acid residues, Q224, K225, L235, L236, and Q237 (delta5). In addition, cDNA were produced in which alanine was individually substituted for A beta(k) cytoplasmic domain residues 224 through 237 or doubly substituted at residues G226 and P227 or L235 and L236. These mutant cDNAs were individually cotransfected with wild-type A alpha cDNA into the class II-negative M12.C3 B lymphoma and Sal sarcoma cell lines. As was previously reported for transfectants lacking the entire A beta(k) cytoplasmic domain, the delta5 M12.C3 transfectant could not effectively present Ag to an autoreactive Ak-restricted T cell hybrid, and the delta5 Sal transfectant was not rejected when inoculated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrogated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domain in Ag presentation both in vitro and in vivo can be disturbed by mutation of single amino acid residues.
Amino Acid Sequence↗
An isotype-specific activator of major histocompatibility complex (MHC) class II genes that is independent of class II transactivator.
Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.
Blotting, Western↗
CIITA-dependent and -independent class II MHC expression revealed by a dominant negative mutant.
The MHC class II transactivator gene (CIITA) coordinately controls the expression of the three major human class II genes, HLA-DR, HLA-DQ, and HLA-DP. Indeed, patients with one form of MHC class II immunodeficiency disease, due to defective CIITA genes, lack expression of all three isotypes. Nevertheless, there is substantial evidence that human class II genes are not always coordinately regulated, raising the possibility that CIITA-independent, isotype-specific class II regulatory pathways exist. To address this issue, we have generated a dominant negative mutant of CIITA that lacks the acidic transcription-activating N terminus, but retains the proline/serine/threonine-rich domain. Three newly produced anti-CIITA mAbs revealed that this mutant protein lacked N-terminal epitopes. In this study, we report that this CIITA dominant negative mutant repressed the constitutive expression of all three class II isotypes in human EBV-B cell lines, as well as IFN-gamma-induced class II transcription in HeLa cells. However, in a CIITA-deficient, EBV-transformed B cell line, clone 13, the dominant negative mutant did not alter the endogenous expression of the HLA-DQ gene. Taken together, these data demonstrate the existence of both CIITA-dependent and -independent class II regulatory pathways. Furthermore, our data provide evidence that the latter pathways can be isotype specific.
Gene Expression Regulation↗
CD8+ effector cells responding to residual class I antigens, with help from CD4+ cells stimulated indirectly, cause rejection of "major histocompatibility complex-deficient" skin grafts.
BACKGROUND: Skin grafts from mice that are deficient in the expression of both class I and class II major histocompatibility complex (MHC) antigens are rejected rapidly by normal recipients. METHODS: To determine the mechanism of this rejection, MHC-deficient skin grafts were placed on recipients with different degrees of antigenic disparity and on recipients depleted of selected T cell subpopulations. In addition, the recipient's T cells were examined in vitro for their responses before and after graft rejection. RESULTS: The results indicate that (1) CD4+ cells provide help for this rejection by recognizing donor antigens presented by recipient class II antigens, and (2) CD8+ cells can participate as effector cells, recognizing residual class I antigens expressed by the MHC-deficient grafts. CONCLUSIONS: The primary conclusion from these studies is that the supposedly MHC-deficient mice actually do have sufficient class I antigen expression to cause skin graft rejection. This finding prevents the use of these mice to answer definitively the question of whether grafts entirely lacking MHC antigens would be rejected. However, these studies do illustrate two important (although previously recognized) features of allogeneic skin graft rejection: (1) that rejection can be initiated by help provided entirely through the indirect pathway, and (2) that help provided through the indirect pathway is available for effector T cells sensitized directly by donor cells. However, the results from these and other studies suggest that indirect effector mechanisms would probably be able to destroy truly MHC-deficient grafts under some circumstances.
Animals↗