Lck inhibitors as a therapeutic approach to autoimmune disease and transplant rejection.

T-cells play an important role in the pathogenesis of many diseases. These include diseases with large commercial markets and also with significant unmet medical needs, such as rheumatoid arthritis and asthma in addition to those with smaller markets such as organ transplantation, multiple sclerosis, inflammatory bowel diseases, type 1 diabetes, systemic lupus erythematosus and psoriasis. The use of currently available immunomodulatory agents is often limited by the appearance of dose-limiting side effects that result from the actions of these agents on non-lymphoid tissues. LSTRA cell kinase (lck), one of eight known members of the human src family of non-transmembrane protein tyrosine kinases, has a pivotal role in T-cell signaling. Lck expression is restricted to lymphoid cells, so an lck-selective inhibitor would be expected to have a significantly improved safety profile for the treatment of T-cell-driven diseases.

Pyrrolo[2,3-d]pyrimidines containing an extended 5-substituent as potent and selective inhibitors of lck I.

Pyrrolo[2,3-d]pyrimidines containing a 5-(4-phenoxyphenyl) substituent are potent and selective inhibitors of Ick in vitro; some compounds are selective for lck over src. Data are shown for two compounds demonstrating that they are potent and selective inhibitors of IL2 production in 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.

Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

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.

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

The cytoplasmic domain of CD4 is required for stable association with the lymphocyte-specific tyrosine protein kinase p56lck.

The CD4 T cell surface molecule binds MHC class II determinants expressed on antigen-presenting cells. CD4 is thought to enhance T cell activation by serving as an adhesion molecule as well as possibly by transducing an independent intracellular signal during the process of antigen stimulation. The recent observation that CD4 is physically associated with the Src-related tyrosine protein kinase p56lck suggests that tyrosine phosphorylation might be involved in these CD4 "signaling" events. The results presented in this report demonstrate that deletion of the cytoplasmic domain of CD4 significantly diminishes its ability to stably associate with p56lck. This observation provides a biochemical basis for the decreased ability of this mutant CD4 molecule to enhance T cell activation during suboptimal antigen stimulation.

Direct evidence for binding of CD8 to HLA class I antigens.

Adhesion of T lymphocytes is an essential step for antigen recognition and lymphocyte activation. mAbs to T cell surface proteins have been used to define the receptor-ligand proteins that appear to be involved in adhesion. Since most assays measure the effects of mAbs on T lymphocyte function, it is not known whether mAb-mediated blocking is due to a disruption of receptor-ligand interactions or results in inhibition of some aspect of receptor-mediated triggering. It has been suggested that the CD8 molecule augments T cell avidity for the target cells by binding to determinants on target cell MHC class I molecules. In the present report, we demonstrated that purified CD8 molecules incorporated into large lipid vesicles (artificial target cells) mediate the adhesion of these vesicles to cells expressing HLA proteins, while vesicles expressing purified HLA class I antigens bind to CD8+ T cells. Furthermore, vesicles bearing CD8 will form conjugates with vesicles expressing HLA class I proteins. These conjugates were found to be specifically inhibited by mAbs to CD8 or HLA class I molecules. We also demonstrate that CD2-reconstituted vesicles can form conjugates with vesicles bearing LFA-3. These experiments provide direct evidence for an interaction of the CD8 molecule with class I MHC proteins as well as between CD2 proteins and LFA-3 proteins, thus supporting the hypothesis that these molecules can mediate cell-cell adhesion.

Expression and function of CD8 in a murine T cell hybridoma.

In general, the human CD8 molecule is expressed on T cells specific for HLA class I molecules. Studies designed to delineate the function and to define the ligand of the CD8 molecule have been complicated by the fact that the presumptive ligand for CD8 is on the HLA class I molecule, the same molecule encoding the ligand for the antigen-specific T cell receptor. The ability to express genes in cells other than their natural host has produced a new technology with which to approach CD8 functional studies. The insertion of a cDNA clone for CD8 in a defective retroviral vector has allowed the transfer of CD8 by infection with the resulting defective retrovirus. CD8 was then expressed in an HLA class II-specific T cell, thus separating the ligand requirements of the TCR and CD8. By this approach, the human CD8 molecule was expressed in a murine T cell hybridoma specific for human class II antigens. The resulting CD8+ hybridomas demonstrated a 10-fold increase in IL-2 production over the parent cell line when stimulated with JY, a human B lymphoblastoid cell line expressing both class I and II HLA antigens, demonstrating that expression of CD8 increases T cell activation. mAbs directed against the CD8 molecule inhibited the response of CD8+ hybridomas to JY, supporting the conclusion that the CD8 molecule was fractional. The role of CD8 as a receptor for class I MHC antigens was addressed by stimulation with a cell line expressing HLA-DR antigens, but lacking the expression of HLA class I antigens (Daudi). Stimulation of the CD8+ hybridomas by Daudi did not result in increased IL-2 production. The response to Daudi was unaltered by the addition of anti-CD8 mAb, in contrast to the ability of anti-CD8 mAb to block JY stimulation. Furthermore, mAbs directed against the class I antigens present on JY cells were able to block the enhanced response of the CD8+ hybridomas to JY. These data support the hypothesis that HLA class I molecules are the ligands involved in the CD8-dependent enhancement of T cell activation.

Excretory urography in current practice: evidence against overutilization.

Excretory urography could be performed less frequently if some combinations of genitourinary signs and symptoms were found to be predictive of either a specific disease or normality. To explore this possibility, the authors conducted a prospective study involving more than 3,000 patients at three institutions (a teaching hospital, a community hospital, and a health maintenance organization). Predictive algorithms were obtained by application of a polychotomous logistic regression model but did poorly at differentiating normal from abnormal patients or arriving at a specific diagnosis. Selection of patients on the basis of the logistic model would have required testing 90% of all patients in order to detect 95% of those with abnormal urograms. These results suggest that current clinical selection criteria for excretory urography are effective, and that present frequency of utilization is appropriate.

Selection criteria for upper gastrointestinal examinations: attempts at improvement.

An attempt was made to improve upon selection criteria for the performance of upper gastrointestinal (UGI) series in three settings: a teaching hospital, a community hospital, and a health maintenance organization. Two statistical techniques, the polychotomous logistic model (to develop predictive algorithms for the identification of specific diseases) and the maximum attainable discrimination technique, were used to show the relationship between the percentage of patients with any disease detected and the percentage of UGI examinations performed. Results showed that neither technique improved significantly upon selection criteria for identifying patients with abnormal UGI series.

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells.

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant. A soluble factor can be obtained from the helper T cell cultures that can also augment the CTL response. The suppressor T cells generated in culture do not demonstrate the specificity observed with the helper T cells; however, they are antigen-dependent in their induction. Whether helper or suppressor activity is obtained depends upon the length of time cells are cultured in vitro.

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells.

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.

T lymphocyte-mediated suppression of myeloma function in vitro. II. Evidence for regulation of hapten-binding myelomas by syngeneic hapten-specific cytolytic T lymphocytes.

BALB/c splenocytes stimulated in vitro with trinitrophenyl (TNP)-modified syngeneic cells inhibit the secretion of antibody by the TNP-binding BALB/c myeloma MOPC 315 in the presence of soluble TNP-Keyhole limpet hemocyanin (KLH). The effector cells are hapten-specific, H-2-restricted, Thy-1.2-bearing, Ly-2-positive T lymphocytes whose precursors are resistant to pretreatment with cyclophosphamide. These phenotypic properties are typical of hapten-specific cytolytic T lymphocytes (CTL). The TNP-reactive CTL that inhibit MOPC 315 cells fail to suppress H-2d myelomas that do not bear TNP-specific surface receptors, and this is not attributable to differences in total binding of TNP-KLH to the different myeloma cells. Moreover, azobenzene arsonate (ABA)-specific CTL inhibit MOPC 315 cells in the presence of the double conjugate TNP-ABA-KLH, but not in the presence of soluble TNP-KLH or ABA-KLH. These results show that H-2-restricted, hapten-specific lymphocytes regulate the function of myeloma cells that bind the hapten only to specific surface receptors, and provide a model for associative recognition of surface H-2 determinants and receptor-bound antigen. The results are discussed with reference to the mechanisms of T lymphocyte-target cell interactions, and the possible physiologic role of hapten-reactive CTL in specifically regulating anti-hapten antibody responses.

Mouse cytolytic T lymphocytes induced by xenogeneic rat stimulator cells exhibit specificity for H-2 complex alloantigens.

When mouse spleen cells were stimulated with irradiated xenogeneic, allogeneic, or trinitrophenyl-modified syngeneic lymphoid cells, the strongest cytolytic response was induced by alloantigens. Mouse cytolytic T lymphocytes generated to rat lymphoid cells demonstrated specificity for the immunizing rat strain, but extensive lysis of allogeneic target cells from certain mouse strains was also observed. Cold target inhibition studies indicated that separate clones of xenoantigen-induced cytolytic T lymphocytes lysed each of the allogeneic murine targets. [3H]Thymidine suicide of the effector cells generated to the rat stimulators revealed that only some of all potentially reactive mouse cytolytic T lymphocyte precursors with specificity for a given allogeneic target are activated by the stimulation with rat cells. This evidence that xenoantigens induce alloreactive cytolytic T lymphocyte receptor repertoire is directed at variants of autologous major histocompatibility complex antigens.