Biomedical subjects

K A Forbush

Publications and source records attributed to K A Forbush.

At least 19 recordsLinked to original sources

PubMed2001

Dynamic tuning of T cell reactivity by self-peptide-major histocompatibility complex ligands.

Intrathymic self-peptide-major histocompatibility complex class II (MHC) molecules shape the T cell repertoire through positive and negative selection of immature CD4(+)CD8(+) thymocytes. By analyzing the development of MHC class II-restricted T cell receptor (TCR) transgenic T cells under conditions in which the endogenous peptide repertoire is altered, we show that self-peptide-MHC complexes are also involved in setting T cell activation thresholds. This occurs through changes in the expression level of molecules on thymocytes that influence the sensitivity of TCR signaling. Our results suggest that the endogenous peptide repertoire modulates T cell responsiveness in the thymus in order to enforce tolerance to self-antigens.

Animals↗

PubMed2001

Activated p56lck directs maturation of both CD4 and CD8 single-positive thymocytes.

p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.

Animals↗

PubMed2001

Positive and negative selection of thymocytes depends on Lck interaction with the CD4 and CD8 coreceptors.

Considerable evidence supports a role for the Src family protein tyrosine kinase Lck in regulating multiple aspects of thymocyte development. In this report, we establish that early events in T lymphopoiesis are restored to Lck-deficient mice by provision of a transgene encoding a version of Lck that cannot interact with the coreceptors CD4 and CD8. In addition, we demonstrate that later events in thymocyte development, specifically, the processes of positive and negative selection, are compromised in mice where the only Lck available cannot associate with either CD4 or CD8. We conclude that not only is Lck activity required for positive and negative selection, but that that activity must be coupled to the CD4 and CD8 coreceptors.

Animals↗

PubMed2001

Identification and characterization of thymus LIM protein: targeted disruption reduces thymus cellularity.

We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.

Actins↗

PubMed2000

Down-regulation of the orphan nuclear receptor ROR gamma t is essential for T lymphocyte maturation.

Thymocyte development is a tightly regulated process. CD4+CD8+ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor ROR gamma t in mature T cells down-regulates their surface TCR expression. The ROR gamma t transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of ROR gamma t on c-Rel transcription. Furthermore, ectopic expression of ROR gamma t inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that ROR gamma t controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of ROR gamma t expression in thymocytes is essential for their maturation.

Animals↗

PubMed2000

Caspase enzyme activity is not essential for apoptosis during thymocyte development.

Caspases, a family of cysteine proteases, are critical mediators of apoptosis. To address the importance of caspases in thymocyte development, we have generated transgenic mice that express the baculovirus protein p35, a viral caspase inhibitor, specifically in the thymus. p35 expression inhibited Fas (CD95)-, CD3-, or peptide-induced caspase activity in vitro and conferred resistance to Fas-induced apoptosis. However, p35 did not block specific peptide-induced negative selection in OT1 and HY TCR transgenic mouse models. Even the potent pharmacological caspase inhibitor zVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone) could not prevent peptide-induced deletion of OT1 thymocytes, although it improved basal thymocyte survival in vitro. Moreover, the developmental block observed in rag1-/- thymocytes, which lack pre-TCR signaling, was also not rescued by p35 expression. These results indicate that caspase-independent signal transduction pathways can mediate thymocyte death during normal T cell development.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes.

Notch proteins regulate many developmental processes. Notch1 is highly expressed on thymocytes, but its role in regulating their development is not known. We show that activation of Notch1 signaling in CD4+CD8+ double positive thymocytes promotes the maturation of both CD4+ and CD8+ single positive thymocytes and that this occurs in the absence of interactions between the T cell receptor and MHC molecules expressed on thymic epithelial cells. We have also identified several genes that are transcriptionally regulated by Notch1 in T cells and show that they are upregulated during maturation into both single positive lineages. These observations suggest that Notch1 signaling plays a role in promoting maturation into both the CD4 and CD8 T cell lineages.

ADAM Proteins↗

PubMed1999

Distinct signals mediate maturation and allelic exclusion in lymphocyte progenitors.

Successful in-frame rearrangement of immunoglobulin heavy chain genes or T cell antigen receptor (TCR) beta chain genes in lymphocyte progenitors results in formation of pre-BCR and pre-TCR complexes. These complexes signal progenitor cells to mature, expand in cell number, and suppress further rearrangements at the immunoglobulin heavy chain or TCRbeta chain loci, thereby ensuring allelic exclusion. We used transgenic expression of a constitutively active form of c-Raf-1 (Raf-CAAX) to demonstrate that activation of the Map kinase pathway can stimulate both maturation and expansion of B and T lymphocytes, even in the absence of pre-TCR or pre-BCR formation. However, the same Raf signal did not mediate allelic exclusion. We conclude that maturation of lymphocyte progenitors and allelic exclusion require distinct signals.

Alkyl and Aryl Transferases↗

PubMed1998

Requirement for Jak3 in mature T cells: its role in regulation of T cell homeostasis.

The tyrosine kinase Jak3 plays a key role in transducing signals from the IL-2, -4, -7, -9, and -15 receptors. Mice lacking Jak3 exhibit a profound, early block in both B and T cell development. To examine the mechanisms whereby Jak3 influences T cell function, we have reconstituted thymic development in Jak3-/- animals by introducing a Jak3 transgene in which expression was driven by the lck proximal promoter. Thymic reconstitution required Jak3 kinase activity, as catalytically inactive Jak3 did not restore early thymic development. Furthermore, the thymus-restricted expression pattern of the transgene allowed us to assess the requirement for Jak3 in peripheral T cells. In these mice, loss of Jak3 expression was associated with a failure to proliferate in response to antigen receptor crosslinking, the accumulation of T cells manifesting an activated cell surface phenotype, and an increased CD4/CD8 ratio among peripheral T cells, all of which are characteristics that were observed in Jak3-/- animals. Finally, we present data which suggest that peripheral T cells proliferate more rapidly in vivo and also undergo apoptosis more rapidly, upon loss of Jak3. Hence Jak3 exerts effects on mature peripheral T lymphocytes, as well as on thymocytes, resulting in the proper maintenance of circulating, quiescent cells.

Animals↗

PubMed1997

Control of B cell development by Ras-mediated activation of Raf.

Cell fate commitment in a variety of lineages requires signals conveyed via p21ras. To examine the role of p21ras in the development of B lymphocytes, we generated transgenic mice expressing a dominant-negative form of Ras in B lymphocyte progenitors, using a novel transcriptional element consisting of the Emu enhancer and the lck proximal promoter. Expression of dominant-negative Ras arrests B cell development at a very early stage, prior to formation of the pre-B cell receptor. Furthermore, an activated form of Raf expressed in the same experimental system could both drive the maturation of normal pro-B cells and rescue development of progenitors expressing dominant-negative Ras. Hence p21ras normally regulates early development of B lymphocytes by a mechanism that involves activation of the serine/threonine kinase Raf.

Animals↗

PubMed1997

Characterization of Lnk. An adaptor protein expressed in lymphocytes.

Stimulation of the T cell antigen receptor (TCR) activates a set of non-receptor protein tyrosine kinases that assist in delivering signals to the cell interior. Among the presumed substrates for these kinases, adaptor proteins, which juxtapose effector enzyme systems with the antigen receptor complex, figure prominently. Previous studies suggested that Lnk, a 38-kDa protein consisting of a single SH2 domain and a region containing potential tyrosine phosphorylation sites, might serve to join Grb2, phospholipase C-gamma1, and phosphatidylinositol 3-kinase to the TCR. To elucidate the physiological roles of Lnk in T cell signal transduction, we isolated the mouse Lnk cDNA, characterized the structure of the mouse Lnk gene, and generated transgenic mice that overproduce Lnk in thymocytes. Here we report that although Lnk becomes phosphorylated during T cell activation, it plays no limiting role in the TCR signaling process. Moreover, we have distinguished p38(Lnk) from the more prominent 36-kDa tyrosine phosphoproteins that appear in activated T cells. Together these studies suggest that Lnk participates in signaling from receptors other than antigen receptors in lymphocytes.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Replacement of pre-T cell receptor signaling functions by the CD4 coreceptor.

An important checkpoint in early thymocyte development ensures that only thymocytes with an in-frame T cell receptor for antigen beta (TCR-beta) gene rearrangement will continue to mature. Proper assembly of the TCR-beta chain into the pre-TCR complex delivers signals through the src-family protein tyrosine kinase p56lck that stimulate thymocyte proliferation and differentiation to the CD4+CD8+ stage. However, the biochemical mechanisms governing p56lck activation remain poorly understood. In more mature thymocytes, p56lck is associated with the cytoplasmic domain of the TCR coreceptors CD4 and CD8, and cross-linking of CD4 leads to p56lck activation. To study the effect of synchronously inducing p56lck activation in immature CD4-CD8- thymocytes, we generated mice expressing a CD4 transgene in Rag2-/- thymocytes. Remarkably, without further experimental manipulation, the CD4 transgene drives maturation of Rag2-/- thymocytes in vivo. We show that this process is dependent upon the ability of the CD4 transgene to bind Lck and on the expression of MHC class II molecules. Together these results indicate that binding of MHC class II molecules to CD4 can deliver a biologically relevant, Lck-dependent activation signal to thymocytes in the absence of the TCR-alpha or -beta chain.

Animals↗

PubMed1997

Restoration of thymopoiesis in pT alpha-/- mice by anti-CD3epsilon antibody treatment or with transgenes encoding activated Lck or tailless pT alpha.

Mice deficient for the pre-TCR alpha (pT alpha) chain cannot form a pre-T cell receptor (TCR) and exhibit a severe defect in early T cell development, characterized by lack of "beta selection" and impaired generation of double-positive (DP) thymocytes. Here, we demonstrate that intraperitoneal injection of CD3epsilon-specific antibodies into pT alpha-/- x RAG-/- mice or introduction of an activated p56(lck) transgene in pT alpha-/- mice fully restores the number of DP thymocytes, and that expression of a transgenic pT alpha chain lacking its cytoplasmic portion can overcome all developmental defects associated with pT alpha deficiency. These results allow a better definition of the role of pT alpha in pre-TCR signal transduction and provide conclusive evidence that the cytoplasmic tail of pT alpha is not essential for pre-TCR signaling.

Animals↗

PubMed1997

Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone.

Antibody reactivity to self-antigens is a normal component of the immune system. To study the mechanism by which self-reactive B cells are generated and maintained, we analyzed B cell development in transgenic mice that express a rearranged VH81X heavy chain from the pre-immune repertoire. In these mice, > 95% of B cells express the transgene in association with a variety of kappa light chains but V kappa 1 C being the dominant light chain. These transgenic B cells with identical V kappa 1C-J kappa 5 joins do not normally secrete IgM in vivo, but antibodies derived from these B cells, through LPS activation in vitro or after hybridoma immortalization, are self-reactive and recognize an ubiquitous epitope(s) on intracytoplasmic proteins from different tissues. They have the phenotype and localization pattern of long-lived marginal zone B cells and their development in vivo is blocked by injection of soluble VH81X-V kappa 1CJ kappa 5 IgM antibody. The observations in this transgenic mouse provide evidence for positive selection of a population of self-reactive B cells. These B cells enter the peripheral pool of B cells where they localize in the marginal zone of the spleen and, in contrast to other transgene-expressing B cells, do not secrete IgM antibody.

Animals↗

PubMed1995

Functional dissection of the murine lck distal promoter.

The lymphocyte-specific proto-oncogene lck is transcribed from two developmentally regulated, independently functioning promoters. The proximal promoter is used in thymocytes, but not in peripheral T lymphocytes. The distal promoter operates in all stages of T cell development, but predominates in more mature cells. Both promoters lack a TATAA element and they share little sequence similarity with each other. Using transgenic mice to locate in vivo functional cis-acting regions of the murine distal promoter, we defined a region from -1786 to -2913 that is essential for consistent insertion site-independent expression of a heterologous cDNA reporter. The transgene is lymphoid specific and expressed predominantly in T cells. One of four transgenic mice bearing a shortened distal promoter (-886 to +41) expressed the reporter in the expected developmental pattern, suggesting that important regulatory elements that require favorable flanking sequences for expression are present nearer the transcription start site. The DNA sequence from -4032 to +623 contains few consensus binding sites for previously described T lymphocyte-specific trans-acting factors, and their locations do not correlate well with the functional data. However, the locations of tissue-specific modifications of chromatin structure in the promoter region, manifest as sites of DNase hypersensitivity, correlated with these two functional regions in normal mice. The identification of lck distal promoter regulatory regions provides a useful control element for deliberate expression of transgenes in mature T lymphocytes. In addition, these regulatory regions should assist in defining T cell-specific trans-acting factors.

Animals↗

PubMed1995

Selective requirement for MAP kinase activation in thymocyte differentiation.

ENGAGEMENT of the T-cell receptor (TCR) with cognate ligands provokes different outcomes depending on the developmental stage of the T cell and on the properties of the ligand. In immature thymocytes TCR stimulation may result in maturation (positive selection) or death (negative selection), whereas in mature T cells it may induce proliferation, death or unresponsiveness. To investigate the different signals involved in these processes, we have analysed the role of the MAP kinase (MAPK) cascade, which is required for growth-factor-stimulated replication and for differentiation in other cell types, by expressing a catalytically inactive form of MAPK kinase (MEK-1) in thymocytes, thereby blocking MAPK activation. We find that positive selection of these cells is inhibited but that negative selection and TCR-induced proliferation are unaffected. Our results indicate that the intracellular signals regulating lineage commitment in T cells parallel those in photoreceptor cell specification in Drosophila and vulval cell differentiation in Caenorhabditis elegans, suggesting that general rules for cell-type specification could apply among all metazoans.

Animals↗

PubMed1995

Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.

Animals↗

PubMed1993

CD8 inhibits signal transduction through the T cell receptor in CD4-CD8- thymocytes from T cell receptor transgenic mice reconstituted with a transgenic CD8 alpha molecule.

T cell repertoire selection processes involve intracellular signaling events generated through the TCR. The CD4 and CD8 coreceptor molecules can act as positive regulators of TCR signal transduction during these developmental processes. In this report, we have used TCR transgenic mice to determine whether TCR signaling can be modulated by the CD8 coreceptor molecule. These mice express on the majority of their T cells a TCR specific for the male (H-Y) Ag presented by the H-2Db MHC class I molecule. We show that CD4-CD8-, but not CD4-CD8+, thymocytes expressing the H-Y TCR responded with high intracellular calcium fluxes to TCR/CD3 stimulation without extensive receptor cross-linking. To examine the effects of CD8 expression on intracellular signaling responses in the CD4-CD8- cells, the H-Y TCR transgenic mice were mated with transgenic mice that constitutively expressed the CD8 alpha molecule on all T cells. The expression of the CD8 alpha alpha homodimer in the CD4-CD8-thymocytes led to impaired intracellular calcium responses and less efficient protein tyrosine phosphorylation of substrates after TCR engagement. In male H-2b H-Y transgenic mice, the majority of thymocytes have been deleted with the surviving cells expressing a high density of the transgenic TCR and exhibiting either a CD4-CD8- or CD4-CD8lo phenotype. It has been postulated that these cells escaped deletion by down-regulating the CD8 molecule. In the H-Y TCR/CD8 alpha double transgenic male mice, the CD4-CD8lo cells were completely eliminated as a result of CD8 alpha expression. However, the CD4-CD8- T cells were not deleted despite normal levels of the CD8 alpha transgene expression. These results suggest that the CD4-CD8- thymocytes may not be susceptible to the same deletional mechanisms as other thymocytes expressing TCR-alpha beta.

Animals↗