The pro-oxidative drug WF-10 inhibits serial killing by primary human cytotoxic T-cells.

Cytotoxic T-cells (CTLs) play an important role in many immune-mediated inflammatory diseases. Targeting cytotoxicity of CTLs would allow to interfere with immune-mediated tissue destruction. Here we demonstrate that WF-10, a pro-oxidative compound, inhibits CTL-mediated cytotoxicity. WF-10 did not influence early steps of target-cell killing, but impaired the ability of CTLs to detach from the initial target cell and to move to a second target cell. This reduced serial killing was accompanied by stronger enrichment of the adhesion molecule LFA-1 in the cytolytic immune synapse. LFA-1 clustering requires activation of the actin-bundling protein L-plastin and was accordingly diminished in L-plastin knockdown cells. Interestingly, WF-10 likely acts through regulating L-plastin: (I) It induced L-plastin activation through phosphorylation leading to enhanced LFA-1-mediated cell adhesion, and, importantly, (II) WF-10 lost its influence on target-cell killing in L-plastin knockdown cells. Finally, we demonstrate that WF-10 can improve immunosuppression by conventional drugs. Thus, while cyclosporine A alone had no significant effect on cytotoxicity of CTLs, a combination of cyclosporine A and WF-10 blocked target-cell killing synergistically. Together, our findings suggest that WF-10 - either alone or in combination with conventional immunosuppressive drugs - may be efficient to control progression of diseases, in which CTLs are crucially involved.

Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells.

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.

The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Molecular alterations of the Fyn-complex occur as late events of human T cell activation.

Two-dimensional gel electrophoresis of anti-p59fyn immunoprecipitates obtained from non-transformed resting human T lymphocytes resulted in the identification of an oligomeric protein complex which is constitutively formed between Fyn and several additional phosphoproteins (pp43, pp72, pp85, the protein tyrosine kinase Pyk2, as well as the two recently cloned adaptor proteins, SKAP55 and SLAP-130). With the exception of pp85, these proteins seem to preferentially interact with Fyn since they are not detectable in Lck immunoprecipitates prepared under the same experimental conditions. Among the individual members of the Fyn-complex pp85, SKAP55 and pp43 are constitutively phosphorylated on tyrosine residue(s) in vivo and likely interact with Fyn via its src homology 2 (SH2)-domain. In contrast to non-transformed T lymphocytes, continuously proliferating transformed human T cell lines express an altered Fyn-complex. Thus, despite normal expression and tyrosine phosphorylation, SKAP55 does not associate with Fyn in Jurkat cells and in other human T cell lines. Instead two novel proteins interact with Fyn among which one has previously been identified as alpha-tubulin. Importantly, almost identical alterations of the Fyn-complex as observed in Jurkat cells are induced in non-transformed T lymphocytes following mitogenic stimulation. These data suggest that Fyn and its associated proteins could be involved in the control of human T cell proliferation. Moreover, the analogous constitutive alterations in transformed T cell lines could indicate that deregulation of the Fyn-complex might be functionally associated with the malignant phenotype of these cells.

SHP2-interacting transmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulating human T cell activation.

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain-containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain-containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor- and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.

T cell receptor (TCR) interacting molecule (TRIM), a novel disulfide-linked dimer associated with the TCR-CD3-zeta complex, recruits intracellular signaling proteins to the plasma membrane.

The molecular mechanisms regulating recruitment of intracellular signaling proteins like growth factor receptor-bound protein 2 (Grb2), phospholipase Cgamma1, or phosphatidylinositol 3-kinase (PI3-kinase) to the plasma membrane after stimulation of the T cell receptor (TCR)- CD3-zeta complex are not very well understood. We describe here purification, tandem mass spectrometry sequencing, molecular cloning, and biochemical characterization of a novel transmembrane adaptor protein which associates and comodulates with the TCR-CD3-zeta complex in human T lymphocytes and T cell lines. This protein was termed T cell receptor interacting molecule (TRIM). TRIM is a disulfide-linked homodimer which is comprised of a short extracellular domain of 8 amino acids, a 19-amino acid transmembrane region, and a 159-amino acid cytoplasmic tail. In its intracellular domain, TRIM contains several tyrosine-based signaling motifs that could be involved in SH2 domain-mediated protein-protein interactions. Indeed, after T cell activation, TRIM becomes rapidly phosphorylated on tyrosine residues and then associates with the 85-kD regulatory subunit of PI3-kinase via an YxxM motif. Thus, TRIM represents a TCR-associated transmembrane adaptor protein which is likely involved in targeting of intracellular signaling proteins to the plasma membrane after triggering of the TCR.

Biochemical analysis of the CD45-p56(lck) complex in Jurkat T cells lacking expression of lymphocyte phosphatase-associated phosphoprotein.

In human and mouse lymphocytes the protein tyrosine phosphatase CD45, a key molecule involved in T cell activation, non-covalently associates with the tyrosine kinase p56(lck) and lymphocyte phosphatase-associated phosphoprotein (LPAP), a 32 kDa phosphoprotein of unknown function. In order to gain insight into the function of LPAP we have generated an LPAP-deficient Jurkat variant by means of antisense strategies. Analysis of the CD45-p56(lck) molecular complex in this cell line revealed that loss of LPAP does not alter the expression or the enzymatic activity of CD45 or p56(lck). In addition, the association between CD45 and p56(lck) is not affected in LPAP-deficient T cells. These data suggest that LPAP does not regulate the enzymatic activity of CD45 or p56(lck) and is not required for the association between these two proteins. In order to identify polypeptides that preferentially associate with LPAP we established a Jurkat variant expressing a chimeric receptor which was composed of the extracellular portion of the human HLA-A2.1 molecule and the full-length LPAP protein. Comparative two-dimensional analysis of CD45 and HLA-A2 immunoprecipitates obtained from these cells following metabolic labeling resulted in the identification of a 43 kDa protein that preferentially associates with LPAP under mild detergent conditions.

Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes.

In human T-lymphocytes the Src family protein tyrosine kinase p59(fyn) associates with three phosphoproteins of 43, 55, and 85 kDa (pp43, pp55, and pp85). Employing a GST-Fyn-Src homology 2 (SH2) domain fusion protein pp55 was purified from lysates of Jurkat T-cells. Molecular cloning of the pp55 cDNA reveals that the pp55 gene codes for a so far nondescribed polypeptide of 359 amino acids that comprises a pleckstrin homology domain, a C-terminal SH3 domain, as well as several potential tyrosine phosphorylation sites, among which one fulfills the criteria to bind Src-like SH2 domains with high affinity. Consistent with this observation, pp55 selectively binds to isolated SH2 domains of Lck, Lyn, Src, and Fyn but not to the SH2 domains of ZAP70, Syk, Shc, SLP-76, Grb2, phosphatidylinositol 3-kinase, and c-abl in vitro. Based on these properties the protein was termed SKAP55 (src kinase-associated phosphoprotein of 55 kDa). Northern blot analysis shows that SKAP55 mRNA is preferentially expressed in lymphatic tissues. SKAP55 is detected in resting human T-lymphocytes as a constitutively tyrosine phosphorylated protein that selectively interacts with p59(fyn). These data suggest that SKAP55 represents a novel adaptor protein likely involved in Fyn-mediated signaling in human T-lymphocytes.

Sequence, genomic organization, and chromosomal localization of the human LPAP (PTPRCAP) and mouse CD45-AP/LSM-1 genes.

Human LPAP (lymphocyte phosphatase associated phosphoprotein) and its mouse homologue, CD45-AP (CD45 associated protein) or LSM-1, represent 32- and 30-kDa transmembrane phosphoproteins that noncovalently associate with the tyrosine phosphatase CD45, a key molecule involved in T- and B-lymphocyte activation. Here we report the isolation and sequencing of genomic clones of the human and mouse genes. LPAP (HGMW-approved symbol PTPRCAP) maps to human chromosome 11q13, distal to the BCL-1 breakpoint, and mouse CD45-AP/LSM-1 maps to Chromosome 19B. Both genes span 3 kb and consist of two exons that are separated by a 1.2-kb intron. The promoter regions do not contain TATA boxes, but possess consensus transcriptional initiator sequences that have also been described for other TATA-less genes. The genomic sequences also provide a genetic basis for two different cDNAs (termed CD45-AP and LSM-1, respectively) that have been described in the mouse system.

Human T lymphocyte activation induces tyrosine phosphorylation of alpha-tubulin and its association with the SH2 domain of the p59fyn protein tyrosine kinase.

A glutathione-S-transferase-src-homology domain 2 (GST-SH2) fusion protein was employed to identify molecules interacting with the protein tyrosine kinase p59fyn. Among several proteins which bound to the fyn SH2 domain in lysates of human Jurkat T lymphocytes, alpha- and beta-tubulin were identified by N-terminal sequencing. Further analysis established that alpha-tubulin exists as a tyrosine-phosphorylated protein in Jurkat cells, where it interacts with p59fyn, but not with p56lck. By contrast, in untransformed resting human T lymphocytes alpha-tubulin is not detectable as a tyrosine phosphorylated protein. However, following T cell activation, it becomes rapidly phosphorylated on tyrosine residues and subsequently associates with the SH2 domain of fyn. Interestingly, constitutively tyrosine-phosphorylated alpha-tubulin that is able to interact with the fyn-SH2 domain is expressed in peripheral blood T lymphoblasts isolated from leukemic patients in the absence of external stimulation.

LPAP, a novel 32-kDa phosphoprotein that interacts with CD45 in human lymphocytes.

CD45, a leukocyte-specific protein tyrosine phosphatase involved in signal transduction, has previously been shown to associate with a 32-kDa phosphoprotein in human T-lymphocytes and T-lymphoma cell lines. The 32-kDa protein was purified and its coding cDNA cloned. Since expression of the protein was found to be restricted to B- and T-lymphocytes it was termed LPAP (lymphocyte phosphatase-associated phosphoprotein). LPAP exists in two differentially phosphorylated forms in resting human T-lymphocytes c, both of which undergo alterations during T-lymphocyte activation. Analysis of LPAP protein and mRNA expression in CD45-deficient mutant T-cell lines suggests that LPAP protein is subjected to degradation in the absence of its binding partner, CD45. Stable expression of LPAP protein seems to require particular portions of CD45 distinct from the phosphatase domains. In pervanadate-treated human T-lymphocytes LPAP undergoes phosphorylation on tyrosine residues in vivo. Since tyrosine phosphorylation of LPAP is undetectable in T-lymphocytes expressing enzymatically active CD45, these data suggest that LPAP likely represents a novel substrate for CD45.

Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells.

Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR). Since tyrosine phosphorylated pp29/30 selectively interacts with the Src homology type 2 domains (SHZ) of the protein tyrosine kinases p56lck and p59fyn but not ZAP70 the present data suggest that pp29/30 represents a novel signaling receptor associated phosphoprotein likely involved in the activation of human T lymphocytes and NK cells.

Alterations of CD2 association with T cell receptor signaling molecules in "CD2 unresponsive" human T lymphocytes.

CD2-associated molecules were identified by means of in vitro kinase assays of CD2 immunoprecipitates obtained from nontransformed human T lymphocytes lysed in the detergent brij 58. Under these conditions CD2 was found to be associated with the protein tyrosine kinases p56lck and p59fyn as well as two low molecular weight phosphoproteins. The latter were identified as the zeta and epsilon chains, the major signaling components of the CD/7 TcR complex. Importantly, induction of T cell unresponsiveness towards CD2-mediated stimuli by means of CD3/T cell receptor (TcR) modulation results in uncoupling of zeta and epsilon from the CD2 molecule, while its associations with p56lck and p59fyn remain unaffected. Moreover, despite the incapacity of T lymphocytes to undergo DNA synthesis in the CD3/TcR-modulated state, CD2 triggering still results in tyrosine phosphorylation of some unknown protein substrates. Thus, the same zeta and epsilon chains which are components of a functional TcR complex appear to also couple to the CD2 molecular complex. Moreover, dissociation of TcR and CD2 complexes in intact cells seems to block CD2-mediated T cell growth but does not result in complete abolishment of the signal transducing capacity of the CD2 receptor.

Four CD45/P56lck-associated phosphorproteins (pp29-pp32) undergo alterations in human T cell activation.

In human T lymphocytes a functional complex is formed between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and a phosphoprotein, pp32, a possible common substrate. Here we demonstrate that the previously described pp32 protein is composed of two distinct molecules (pp29 and pp32) in both resting human T lymphocytes and continuously proliferating T lymphoma lines. Importantly, T lymphocyte activation employing CD2 monoclonal antibodies (mAb), CD3 mAb or phorbol 12, 13 dibutyrate results in loss of pp29 and pp32 from the CD45/p56lck molecular complex and concomitant association of two distinct phosphoproteins with different molecular weights (pp30 and pp31). These events appear to be unrelated to clonal T cell growth but rather depend on receptor-mediated differentiation signals. Reprecipitation experiments employing an antiserum directed at a consensus sequence of GTP-binding proteins suggest that all four pp29-pp32 molecules might represent proteins with GTP-binding properties. Biochemical analysis of pp29-pp32 employing V8-protease digestion indicates that they differ in low-molecular weight fragments of 8, 5, 4.5, 4 and 3 kDa, respectively.

A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate.

In vitro protein kinase assays of CD45 immunoprecipitates prepared from digitonin lysates of resting human T lymphocytes resulted in exclusive tyrosine phosphorylation of a 32-kDa protein (pp32). Reprecipitation of the in vitro phosphorylated proteins and Western blot analysis of whole CD45 immunoprecipitates employing antisera specifically directed at different protein tyrosine kinases demonstrated that the p56lck protein tyrosine kinase was responsible for in vitro phosphorylation of pp32. Since in vitro kinase assays of p56lck immunoprecipitates also resulted in phosphorylation of pp32, the present data strongly suggest that a functional complex is formed between CD45, p56lck and pp32. Such a notion is supported by the findings that phosphorylation of pp32 by p56lck correlated with expression of the CD45 molecules and that in vitro phosphorylated pp32 was completely dephosphorylated by purified CD45.

Melanoma targeting with a cocktail of monoclonal antibodies to distinct determinants of the human HMW-MAA.

The monoclonal antibodies (MoAbs) 149.53, 225.28, and 763.74 which recognize distinct and spatially distant determinants of the human high molecular weight-melanoma associated antigen (HMW-MAA) do not influence the binding of each other to cultured human melanoma cells. In vitro incubation of melanoma cells with a combination of the three 125I-labeled anti-HMW-MAA MoAbs results in a marked additive binding only when the MoAbs are used at saturating concentrations. Injection of the combination of the three 125I-labeled MoAbs (up to 300 micrograms per mouse) into human melanoma-bearing nude mice does not increase the amount of radioactivity specifically localized in melanoma lesions above the level observed upon injection of corresponding doses of individual MoAbs. These results may reflect the low concentration of MoAbs which reaches tumor lesions in vivo. Therefore, administration of combinations of MoAbs to distinct determinants of HMW-MAA may not increase the sensitivity of immunoscintigraphy to visualize lesions in patients with melanoma.

Antibody targeting to the murine lymphoma ESb-MP: increased accumulation due to reduced internalization into lymphoma cells as compared to normal lymphoid cells.

Using rat monoclonal antibody (MAb) 12-15A against the spontaneously metastasizing mouse lymphoma variant ESb-MP, we elaborated conditions for targeting. In vitro, high binding of labelled antibody to ESb-MP cells and low binding to lymphoid cells (e.g., spleen cells) was noted. In vivo, we observed pronounced accumulation in spleen, lymph nodes and bone marrow, the uptake kinetics indicating high accessibility of the target antigen in these tissues, and rapid clearance of radioactivity from blood and most normal tissues, indicating degradation of the antibody and excretion of the label. Binding to lymphoma tissue was slow but persistent, resulting in high tumor:tissue ratios only after 2-3 days. Biodistribution could be dramatically changed by pre-treatment of animals with excess cold antibody, which reduced trapping of labelled antibody in normal lymphatic tissue, leading to prolonged persistence in the blood and preferential uptake into tumor tissue. Monovalent 12-15A fragments showed less pronounced binding to lymphatic tissue, while being rapidly cleared from the circulation by virtue of their inherent tendency to bind to kidney tissue. Tumor:tissue ratios up to 56:1 were obtained by a combination of pre-treatment with unlabelled 12-15A IgG or Fab fragment, followed by injection of labelled fragment or IgG, respectively. This is interpreted on the basis of differences in the internalization and retention of antigen-antibody complexes. Pre-treatment obviously leads to a temporary blockade or removal of the target antigen, which is much more efficient with normal lymphoid cells than with tumor cells. Thus, it may become possible to target antibodies into the tumor despite concurrent antigen expression on normal tissue.

Iodination of monoclonal IgG antibodies at a sub-stoichiometric level: immunoreactivity changes related to the site of iodine incorporation.

Thirteen monoclonal antibodies (MAbs) were labeled with 125I to a different degree such as to cover the range from 0.5-20 microCi/micrograms. By SDS polyacrylamide gel electrophoresis, the amount of iodine incorporated into heavy (h) and light (l) chains was determined. Comparing different MAbs, h:1 ratios varied from 0.6-34.6, but virtually no variation was observed with individual MAbs labeled at different levels. Immunoreactivity of labeled MAbs was analyzed with antigen-positive tumor cells according to the Lineweaver Burk method. Immunoreactive fractions were found to decrease with increasing iodine incorporation in 9/12 MAbs, while binding affinities decreased in 5/12 MAbs; only 1 MAb was stable in both respects. Immunoreactivity changes were not linked to preferential h or 1 chain labeling, nor to the isotype. This result indicated incorporation of the first iodine atom to take place at individually distinct residues, with a minimum estimate of two or four sites, depending on whether preferential chain labeling or random incorporation took place. In cases where increasing labeling led to a gradual decrease of binding affinity, a shift in the spectrum of acceptor residues has to be assumed.

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part II. Comparison of immunoreactivity and biodistribution of monoclonal antibodies labeled with the 67Ga-chelate or with 131I.

Coupling of the 67Ga-P-EDDHA chelate via carbodiimide to the anti-melanoma monoclonal antibody (Mab) M.2.9.4 resulted in a low degree of oligomerization, but a considerable degree of intra-molecular (inter-chain) cross-linking. However, this did not impair immunoreactivity, nor did the half-life in vivo differ substantially from that of 131I-M.2.9.4. Biodistribution analysis in normal mice showed Ga:I ratios near 1 in the blood and other tissues not involved in degradation and label excretion. In tissues of the reticulo-endothelial system (RES) and the kidneys, Ga:I ratios up to 2.51 were reached within 4 days of administration. In antigen-positive MeWo tumor tissue, retention of 67Ga also excreted that of 131I, so that tumor; organ ratios (except tumor:liver) were superior for the 67Ga-labeled MAb. It is concluded that the method of coupling pre-established 67Ga-P-EDDHA chelate to antibody results in a functionally intact tracer molecule, whose persistence in vivo is not significantly impaired. The major difference to I-labeled MAbs may be a prolonged retention of Ga in tissues (cells) physiologically involved in antibody catabolism.

Immunoreactivity of monoclonal anti-melanoma antibodies in relation to the amount of radioactive iodine substituted to the antibody molecule.

The damage to monoclonal anti-melanoma antibodies caused by iodination was investigated by comparing the results obtained using the chloramine-T method and the 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenyl-glycoluril (IODOGEN) method at different levels of iodine substitution to the molecule. The level of substitution at which losses in immunoreactivity occurred was evaluated in each monoclonal antibody (MAb) studied. This phenomenon was not dependent on the method of substitution, provided that mild conditions of reaction were used. Lineweaver-Burk plots and--in cases of alterations in binding affinity--Scatchard plots were found to provide an adequate description of the binding behaviour of individual MAbs after labelling. Immunoreactivity was shown to be determined not only by the proportion of bona fide reactive MAb molecules, but also by a substitution-dependent decrease in affinity constants. The practical consequences of altered binding parameters were demonstrated by quantitating specific antibody accumulation in melanoma transplants in vivo.