Biomedical subjects

C Geisler

Publications and source records attributed to C Geisler.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

CD4-Positive T-Lymphocytes↗

PubMed1999

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.

Antibodies, Monoclonal↗

PubMed1999

TCRzeta is transported to and retained in the Golgi apparatus independently of other TCR chains: implications for TCR assembly.

It is generally assumed that TCR assembly occurs in the endoplasmic reticulum (ER), and ER retention/degradation signals have been identified in several of the TCR chains. These signals are probably responsible for retention of incompletely assembled TCR complexes and free TCR chains in the ER. This study focused on the intracellular localization and transport of partially assembled TCR complexes as determined by confocal microscopy analyses. We found that none of the TCR chains except for TCRzeta were allowed to exit the ER in T cell variants in which the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was not formed. Interestingly, TCRzeta was exported from the ER independently of other TCR chains and was predominantly located in a compartment identified as the Golgi apparatus. Furthermore, in the TCRzeta-negative cell line MA5.8, the hexameric CD3gammaepsilonTi alphabetaCD3 deltaepsilon complex was allowed to exit the ER and was also predominantly located in the Golgi apparatus. However, neither hexameric TCR complexes nor TCRzeta chains were efficiently expressed at the cell surface without the other. The observations that TCRzeta and hexameric TCR complexes are transported from the ER to the Golgi apparatus independently of each other and that these partial TCR complexes are unable to be efficiently expressed at the cell surface suggest that final TCR assembly occurs in the Golgi apparatus.

Biological Transport↗

PubMed1999

Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

Animals↗

PubMed1999

Autoimmunity and extranodal lymphocytic infiltrates in lymphoproliferative disorders.

OBJECTIVE: To examine the relationship between autoimmunity and extranodal lymphocytic infiltrates in different lymphoproliferative disorders with immunoglobulin alterations. SUBJECTS AND DESIGN: A clinical review combined with a retrospective cohort study of 380 patients, 28 with monoclonal gammopathy of undetermined significance, three with common variable immunodeficiency, 147 with chronic lymphocytic leukaemia, 57 with Waldenström's macroglobulinaemia and 145 with non-Hodgkin's malignant lymphoma. SETTING: A university hospital and The State Serum Institute in Copenhagen. INTERVENTION: Clinical examination of each patient with special attention to chronic inflammatory and autoimmune manifestations. Biopsies were taken from non-infectious infiltrates, some of which were additionally tested with PCR analysis for gene rearrangements. Serological screening with a test battery for various autoantibodies was used in combination with techniques for the detection of M-components and monoclonal B-cell proliferation. MAIN OUTCOME MEASURES: Clinical and/or serological autoimmune manifestations, M-component and other immunoglobulin alterations, and inflammatory tissue changes were studied in patients with chronic inflammatory, polyclonal or oligoclonal pseudolymphomas and in monoclonal, malignant extranodal lymphomas. RESULTS: In 380 consecutive patients, 49 (12.9%) had extranodal manifestations, of whom 47 also had autoimmune manifestations. Nearly half of the 47 patients had more than one autoimmune manifestation. There was a strong correlation between clinical signs and corresponding autoantibodies such as anti-SSA and -SSB antibodies in Sjögren's syndrome (10 cases), antithyroid peroxidase antibodies in thyroiditis and Graves' disease (10 cases), and parietal cell antibodies in gastric ulcers with maltoma (12 cases). Clinical and serological signs of autoimmunity correlated strongly with female sex (34, 72% women; and 13, 28% men) and with immunoglobulin alterations. CONCLUSIONS: To our knowledge this is the first systematic review of B-lymphoproliferative and autoimmune disorders indicating that pseudolymphoma and malignant lymphomas, including maltomas, may develop in the context of a permanent autoantigenic drive.

Aged↗

PubMed1998

T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif.

The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently of phosphorylation and leads to rapid internalization and sorting of these chimeras to lysosomal degradation. Because the TCRzeta chain rescues incomplete TCR complexes from lysosomal degradation and allows stable surface expression of fully assembled TCR, we addressed the question whether TCRzeta has the potential to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface. Thus, our data demonstrate that TCRzeta allows stable cell surface expression of receptors containing CD3gamma leucine-based motifs by its potential to mask such motifs.

Amino Acid Sequence↗

PubMed1998

TCR alpha beta chains associate with the plasma membrane independently of CD3 and TCR zeta chains in murine primary T cells.

The TCR is a multisubunit complex composed of the clonotypic alpha/beta disulfide-linked heterodimer and noncovalently linked invariant CD3 gamma epsilon and CD3 delta epsilon and TCR zeta chains. Recent studies demonstrate that the surface expression of CD3 components can occur independently of the clonotypic TCR complexes in both thymocytes and splenic T cells. In this study, we report that free noncovalently associated TCR alpha beta heterodimers that exist independently of CD3 and TCR zeta chains are expressed on the cell surface of immature thymocytes and peripheral T cells, but not of T cell lines and T cell hybridomas. This suggests that the regulation of surface expression of TCR alpha beta heterodimers differs between primary T cells and T cell lines or T cell hybridomas. The isolation and biochemical characterization of surface clonotype-independent CD3 complexes and free membrane-associated TCR alpha beta complexes may provide a structural basis for the quantitative difference in amount of T cell proliferation stimulated by anti-CD3 epsilon and anti-TCR beta.

Animals↗

PubMed1998

The phosphorylation state of CD3gamma influences T cell responsiveness and controls T cell receptor cycling.

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3gamma. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3gamma, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56(Lck) and p59(Fyn). Studies of mutated TCR and chimeric CD4-CD3gamma molecules demonstrated that CD3gamma did not contain a recycling signal in itself. In contrast, the only sorting information in CD3gamma was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3gamma molecules. Finally, we found a correlation between the phosphorylation state of CD3gamma and T cell responsiveness. Based on these observations a physiological role of CD3gamma and TCR cycling is proposed.

Amino Acid Sequence↗

PubMed1998

Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane.

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic residue of leucine-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs. Thus, our observations suggest that the position, the exact sequence, and surrounding residues are major determinants of the function of leucine-based receptor sorting motifs.

Amino Acid Sequence↗

PubMed1998

Two distinct pathways exist for down-regulation of the TCR.

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. Down-regulation of the TCR is induced by engagement of the TCR by specific ligands and/or by activation of protein kinase C (PKC). We report here that ligand- and PKC-induced TCR down-regulation is mediated by two distinct, independent mechanisms. Ligand-induced TCR down-regulation is dependent on the protein tyrosine kinases p56(lck) and p59(fyn) but independent of PKC and the CD3gamma leucine-based (L-based) internalization motif. In contrast, PKC-induced TCR down-regulation is dependent on the CD3gamma L-based internalization motif but independent of p56(lck) and p59(fyn). Finally, our data indicate that in the absence of TCR ligation, TCR expression levels can be finely regulated via the CD3gamma L-based motif by the balance between PKC and serine/threonine protein phosphatase activities. Such a TCR ligation-independent regulation of TCR expression levels could probably be important in determining the activation threshold of T cells in their encounter with APC.

Amino Acid Sequence↗

PubMed1998

Interleukin-2 induces beta2-integrin-dependent signal transduction involving the focal adhesion kinase-related protein B (fakB).

beta2 integrin molecules are involved in a multitude of cellular events, including adhesion, migration, and cellular activation. Here, we studied the influence of beta2 integrins on interleukin-2 (IL-2)-mediated signal transduction in human CD4(+) T cell lines obtained from healthy donors and a leukocyte adhesion deficiency (LAD) patient. We show that IL-2 induces tyrosine phosphorylation of a 125-kDa protein and homotypic adhesion in beta2 integrin (CD18)-positive but not in beta2-integrin-negative T cells. EDTA, an inhibitor of integrin adhesion, blocks IL-2-induced tyrosine phosphorylation of the 125-kDa protein but not other proteins in beta2-integrin-positive T cells. Likewise, a beta2 integrin (CD18) antibody selectively inhibits induction of the 125-kDa phosphotyrosine protein, whereas cytokine-mediated tyrosine phosphorylation of other proteins is largely unaffected. Immunoprecipitation experiments indicate that the IL-2-induced 125-kDa phosphotyrosine protein is the focal adhesion kinase-related protein B (fakB). Thus, IL-2 induces strong tyrosine phosphorylation of fakB in beta2-integrin-positive but not in beta2-integrin-negative T cells, and CD18 mAb selectively blocks IL-2-induced fakB-tyrosine phosphorylation in beta2-integrin-positive T cells. In parallel experiments, IL-2 does not induce or augment tyrosine phosphorylation of p125(FAK). In conclusion, our data indicate that IL-2 induces beta2-integrin-dependent signal transduction events involving the tyrosine kinase substrate fakB.

CD18 Antigens↗

PubMed1998

Peptides derived from self-proteins as partial agonists and antagonists of human CD8+ T-cell clones reactive to melanoma/melanocyte epitope MART1(27-35).

The self-peptide MART1(27-35) derives from the melanocyte/melanoma protein Melan A/MART1 and is a target epitope of CD8+ T cells, commonly recovered from tumor-infiltrating lymphocytes of HLA-A2.1+ melanoma patients. Despite their prevalence in such patients, these CTLs generally appear to be ineffective in mediating tumor regression in vivo. We have noted previously that numerous peptides from both endogenous and foreign proteins are similar to MART1(27-35) and, potentially, are capable of productively engaging the T-cell receptors of patient-derived CTLs. This observation raised the question of whether CTLs in vivo might encounter self-peptide analogues of MART1(27-35) that lack full agonist activity, perhaps to the detriment of the antitumor CTL response. This possibility was evaluated using cloned, patient-derived CTLs with a panel of self-derived natural analogues of MART1(27-35) in assays for cytolysis, cytokine release, and phosphorylation of T-cell receptor signaling constituents. Several peptides were identified as partial agonists, capable of eliciting cytolysis and/or release of cytokines tumor necrosis factor-alpha and IFN-gamma but not interleukin 2. Several other peptides showed antagonist behavior, effectively inhibiting cytolysis of MART1(27-35)-pulsed targets, but did not inhibit killing of cells prepulsed with a synthetic, heteroclitic variant of MART1(27-35). Some of these antagonists also had lasting effects on interleukin 2 secretion by CTLs under experimental conditions involving sequential exposure to ligands. Together, these observations suggest that encounters with self-peptide analogues of MART1(27-35) may contribute to the peripheral maintenance of these CTLs, while ultimately impairing the efficacy of this antitumor T-cell response.

Antigens, Neoplasm↗

PubMed1998

The role of caspase 3 and BclxL in the action of interleukin 7 (IL-7): a survival factor in activated human T cells.

The effects of interleukin 7 (IL-7) on apoptosis in interleukin 2 (IL-2)-dependent, activated, primary, human T lymphocytes (hT cells) was examined. IL-7 (like IL-2) rescued cells from apoptosis, as measured by their cellular DNA profile and fragmentation. IL-2 also acted as a mitogen in these T cells. Both cytokines abrogated the dexamethasone-induced stimulation of Caspase 3 and prevented the cleavage of poly (ADP-ribose) polymerase (PARP), a substrate for the Caspase 3. IL-7 upregulated the expression of Bc1xL and counteracted the downregulation of this anti-apoptotic protein by the synthetic glucocorticoid, dexamethasone. Bcl-2 protein expression was uupregulated by IL-7 with or without dexamethasone, but Bc1-2 was expressed at a much lower level than BclxL in these cells. Levels of Bax did not markedly change on either cytokine stimulation or dexamethasone treatment. An unidentified 23-kDa band, which was recognized by the anti-Bc1-2 antibody, was induced by dexamthasone and suppressed by IL-7 and IL-2. This protein was subject to independent regulation as compared to the p26 Bc1-2 protein, suggesting that it may be a novel factor, possibly involved in the regulation of apoptosis. A clear role for IL-7 as a survival factor for cytokine withdrawal and glucocorticoid induced apoptosis in activated primary hT cells is implicated. In addition, regulation of BclxL and downstream inhibition of Caspase 3 activity may mediate this rescue signal.

Apoptosis↗

PubMed1998

Staphylococcus enterotoxin A modulates interleukin 15-induced signaling and mitogenesis in human T cells.

T cells expressing the appropriate T-cell receptor Vbeta chain proliferate in response to Staphylococcus enterotoxin A (SEA) pulsed antigen-presenting cells (APC), whereas other T cells do not (SEA "non-responders"). Activated human T cells express MHC class II molecules that are high affinity receptors for SEA. Here we show that, in the absence of APC, SEA induces a profound inhibition of IL-15-driven proliferation in MHC class II+, human SEA-"responder" T-cell lines. In contrast, proliferation induced by phorbol esther (PMA) was enhanced by SEA. The inhibitory effect on cytokine-mediated mitogenesis correlates with an inhibition of IL-2Rbeta expression and ligand-induced tyrosine phosphorylation of IL-2R. Cyclosporin A (CyA), an inhibitor of the protein phosphatase (PP2B) calcineurin, strongly inhibits the SEA-induced modulations of cytokine receptor expression. Moreover, CyA inhibits both the anti-mitogenic effect of SEA on cytokine-induced proliferation and the pro-mitogenic effect of PMA. In contrast, inhibitors of PP1, PP2A, protein kinase C (PKC), phosphatidyl-inositol-3-kinase (PI-3K) and mammalian target of rapamycin (mTOR) are unable to inhibit the effects of SEA. In a SEA "non-responder" T-cell clone obtained from the affected skin of a patient with psoriasis vulgaris, SEA does not inhibit IL-2Rbeta expression and IL-15-driven proliferation. On the contrary, SEA enhances IL-15- and IL-2-induced proliferation via a CyA-sensitive pathway in this T-cell clone. In conclusion, the present data show that (i) SEA selectively inhibits IL-15- (but not PMA-) mediated proliferation in SEA "responder" T cells, (ii) SEA enhances cytokine-driven growth in psoriasis T cells with a "non-responder" phenotype, and (iii) crosstalk between SEA receptors and the IL-15R (and IL-2R) pathway is mediated via a PP2B-dependent and PP1/PP2A-, PKC-, PI-3 kinase- and mTOR-independent pathway in human T-cell lines.

Cell Division↗

PubMed1998

Staphylococcal enterotoxin-A directly stimulates signal transduction and interferon-gamma production in psoriatic T-cell lines.

Bacterial superantigens such as staphylococcal enterotoxin-A (SEA) have been implicated in the pathogenesis of psoriasis vulgaris. Major histocompatibility complex (MHC) class II molecules are high affinity receptors for SEA, and T cells found in psoriatic skin lesions express high levels of MHC class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen-presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T-cell receptor (TCR), and iii) production of interferon-gamma (IFN-gamma), but not autocrine mitogenesis in CD8-positive T clones obtained from skin lesions of a patient with psoriasis vulgaris. Psoriatic T cells do not respond to SEA molecules if mutations are introduced in the TCRbeta- or in both the two MHC class II alpha- and beta-binding sites of SEA. Mutations in only one of the two MHC class II binding sites of SEA has different effects on T-cell activation. Thus, SEA molecules with a mutation in the MHC class II beta-binding site induce protein tyrosine phosphorylation, but not IFN-gamma production or co-stimulation of cytokine-mediated proliferation. In contrast, SEA with a mutation in the MHC class II alpha-binding site induces IFN-gamma and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co-stimulatory effect on cytokine-mediated proliferation. This suggests that both MHC class II binding sites are involved in the autopresentation of SEA by psoriatic T cells. In conclusion, we provide evidence that SEA directly activates MVHC class H-positive psoriatic T-cell lines to produce IFN-gamma, a key cytokine in the pathogenesis of psoriasis vulgaris.

Antigen-Presenting Cells↗

PubMed1998

MHC class II ligation induces CD58 (LFA-3)-mediated adhesion in human T cells.

MHC class II positive T cells found in areas of inflammation are believed to play an important pathogenetic role in autoimmunity. In experimental models , class II molecules have been shown to regulate adhesion between human T cells. It is, however, not known in detail how class II molecules are functionally linked to adhesion molecules. Some data suggest that beta2 integrin (CD11a/CD18) molecules play a role in class-II-induced homotypic adhesion in B cells, monocytes, and virus-transformed or neoplastic cell lines. We have previously obtained evidence that adhesion molecules other than beta2 integrins might play a role in class-II-mediated adhesion in T cells. To study further class-II-mediated adhesion in T cells, we have taken advantage of (allo)antigen-specific beta2-integrin-negative, CD4-positive T cell lines obtained from a leukocyte adhesion deficiency patient. We show that class II ligation induces homotypic adhesion in both beta2-integrin-positive and negative, CD4-positive T cell lines. Anti-CD18 monoclonal antibody (mAb) weakly inhibited the adhesion response in beta2-integrin-positive T cells and had no effect on beta2-integrin-negative T cells. In contrast, an anti-CD58 (LFA-3) mAb almost completely inhibited the adhesion response in beta2-integrin-negative T cells. Antibodies against the CD58 ligand, CD2, partly inhibited the adhesion response in beta2-integrin-negative T cells whereas antibodies against other adhesion molecules did not. The adhesion response in beta2-integrin-positive T cells was partly inhibited by antibodies against CD58 and CD2. Taken together, these data provide the first evidence that CD58 molecules are involved in class-II-induced homotypic adhesion between T cells.

Antibodies, Monoclonal↗

PubMed1998

Recognition of melanoma-derived antigens by CTL: possible mechanisms involved in down-regulating anti-tumor T-cell reactivity.

Several T cell-recognized epitopes presented by melanoma cells have been identified recently. Despite the large array of epitopes potentially available for clinical use, it is still unclear which of these antigens could be effective in mediating anti-tumor responses when used as a vaccine. Preliminary studies showed that immunization of melanoma patients with epitopes derived from proteins of the MAGE family may result in significant clinical regressions. However, no sign of systemic immunization could be observed in peripheral blood of treated patients. Conversely, significant immunization (detected as increased antigen-specific CTL activity in peripheral blood) was obtained by vaccinating HLA-A2.1+ melanoma patients with the immunodominant epitope (residues 27-35) of the differentiation antigen MART-1, but this immunization was not accompanied by a significant clinical response. To implement immunotherapeuties capable of significantly impacting disease outcome, it is necessary to identify the potential mechanisms responsible for the failure of some antigens to mediate significant anti-tumor responses in vivo. In the case of the MART-1(27-35) epitope, we hypothesize that one of these mechanisms may be related to the existence of natural analogs of this peptide in other human normal proteins.

Antigens, Neoplasm↗

PubMed1997

Regulation and function of the CD3gamma DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro.

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3gamma. Thus, phosphorylation of CD3gamma serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3gamma internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3gamma chimeras; and in vitro by binding CD3gamma peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3gamma D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3gamma molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3gamma S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.

Adaptor Protein Complex alpha Subunits↗