Karel Drbal

GPI-microdomains: a role in signalling via immunoreceptors

Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids are assembled on the leukocyte surface within membrane microdomains, which also accommodate a set of cytoplasmic signalling molecules (Src family kinases, G-proteins, linker proteins). Recent results suggest that these membrane specializations mediate not only signal transduction via GPI-proteins and glycolipids but also play important roles in initiation of signalling via immunoreceptors.

Genetically Encoded Förster Resonance Energy Transfer Sensors for the Conformation of the Src Family Kinase Lck

The current model for regulation of the Src family kinase member Lck postulates a strict correlation between structural condensation of the kinase backbone and catalytic activity. The key regulatory tyrosine 505, when phosphorylated, interacts with the Src homology 2 domain on the same molecule, effectively suppressing tyrosine kinase activity. Dephosphorylation of Tyr505 upon TCR engagement is supposed to lead to unfolding of the kinase structure and enhanced kinase activity. Studies on the conformation-activity relationship of Lck in living cells have not been possible to date because of the lack of tools providing spatiotemporal resolution of conformational changes. We designed a biochemically active, conformation-sensitive Förster resonance energy transfer biosensor of human Lck using the complete kinase backbone. Live cell imaging in Jurkat cells demonstrated that our biosensor performed according to Src family kinase literature. A Tyr505 to Phe mutation opened the structure of the Lck sensor, while changing the autophosphorylation site Tyr394 to Phe condensed the molecule. The tightly packed structure of a high-affinity YEEI tail mutant showed that under steady-state conditions the bulk of Lck molecules exist in a mean conformational configuration. Although T cell activation commenced normally, we could not detect a change in the conformational status of our Lck biosensor during T cell activation. Together with biochemical data we conclude that during T cell activation, Lck is accessible to very subtle regulatory mechanisms without the need for acute changes in Tyr505 and Tyr394 phosphorylation and conformational alterations.

Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor α-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

A New Type of Membrane Raft-Like Microdomains and Their Possible Involvement in TCR Signaling

Membrane rafts and signaling molecules associated with them are thought to play important roles in immunoreceptor signaling. Rafts differ in their lipid and protein compositions from the rest of the membrane and are relatively resistant to solubilization by Triton X-100 or similar detergents, producing buoyant, detergent-resistant membranes (DRMs) that can be isolated by density gradient ultracentrifugation. One of the key signaling molecules present in T cell DRMs is the transmembrane adaptor protein LAT (linker for activation of T cells). In contrast to previous results, a recent study demonstrated that a LAT construct not present in the buoyant DRMs is fully able to support TCR signaling and development of T cells in vivo. This finding caused doubts about the real physiological role of rafts in TCR signaling. In this study, we demonstrate that these results can be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization “heavy DRMs” containing a number of membrane molecules. At a moderate level of expression, LAT supported TCR signaling more efficiently than constructs targeted to the microdomains producing heavy DRMs or to nonraft membrane. We suggest that different types of membrane microdomains provide environments regulating the functional efficiencies of signaling molecules present therein.

Characterization of the Human Leukocyte GPI-Anchored Glycoprotein CDw108 and its Relation to Other Similar Molecules

The CDw108 glycoprotein is expressed on the surface of some leukemic cell lines, erythrocytes and on activated lymphocytes. Its surface expression is rapidly upregulated following various activating stimuli (PHA, PWM, Con A, PMA, anti-CD3) and subsequently gradually decreases. The molecule is anchored in the membrane via glycosylphosphatidylinositol (GPI) moiety, it has molecular mass of 75-80 kDa and pI of 5.0-5.5. Endoglycosidase F and H reduce its apparent size as determined by SDS PAGE by approx. 15 and 22 kDa, respectively. It is a component of large, detergent-resistant GPI-complexes associated with protein kinases. In addition to the previously described identity of CDw108 with the JMH blood group antigen, we demonstrate here its identity to the previously described glycoprotein recognized by monoclonal antibodies H105 and KS.2, and exclude its identity with another GPI-anchored glycoprotein of similar size, melanotransferrin (gp97).

A Novel Anti-CD 18 mAb Recognizes an Activation-Related Epitope and Induces a High-Affinity Conformation in Leukocyte Integrins

Monoclonal antibody MEM-148 was previously shown to recognize CD18 chains in a free form unassociated within leukocyte integrin heterodimers, but yet it is paradoxically able to induce a high-affinity conformation in the native, cell surface expressed LFA-1 molecules. Our results based on kinetics of binding, immunoprecipitation and cell-aggregation experiments demonstrate that the mAb does bind to and stabilizes a specific conformation of LFA-1 heterodimers apparently distinguished by an increased affinity to its cellular ligand(s). A similar high-affinity conformation of LFA-1, in which the MEM-148 epitope becomes exposed, is induced also by a Mg2+/EDTA or low pH (5.5-6.5) treatments which may mimic physiologically relevant situations in normal or inflamed tissues. Thus, mAb MEM-148 is a novel valuable tool for detection and induction of specific conformations of human leukocyte integrins.

Defining the repeating elements in the cysteine-rich region (CRR) of the CD18 integrin β2 subunit

The cysteine‐rich region (CRR) of the integrin β subunits is organised into four repeating elements. By expression of a panel of truncated β2 subunits, and CRR segments fused to the C‐terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482–Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of ‘xC‐‐‐C‐‐‐C‐‐‐CxCxxCxC‐‐‐Cx’, where ‘x’ represents a single residue, and ‘‐‐‐’ represents a stretch of four to 14 residues.

PRR7 Is a Transmembrane Adaptor Protein Expressed in Activated T Cells Involved in Regulation of T Cell Receptor Signaling and Apoptosis

Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells.

Large-Scale Production and Characterization of Novel CD4+ Cytotoxic T Cells with Broad Tumor Specificity for Immunotherapy

Immune-cell–based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4+ cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer. (Mol Cancer Res 2009;7(3):339–53)

Folate Receptor β Regulates Integrin CD11b/CD18 Adhesion of a Macrophage Subset to Collagen.

Folate, also known as vitamin B9, is necessary for essential cellular functions such as DNA synthesis, repair, and methylation. It is supplied to the cell via several transporters and receptors, including folate receptor (FR) β, a GPI-anchored protein belonging to the folate receptor family. As FRβ shows a restricted expression to cells of myeloid origin and only a subset of activated macrophages and placental cells have been shown to express functional FRβ, it represents a promising target for future therapeutic strategies. In this study, we performed affinity purification and mass spectrometric analysis of the protein microenvironment of FRβ in the plasma membrane of human FRβ+ macrophages and FRβ-transduced monocytic THP-1 cells. In this manner, we identified a novel role of FRβ: that is, we report functional interactions of FRβ with receptors mediating cellular adhesion, in particular the CD11b/CD18 β2 integrin heterodimer complement receptor type 3/Mac-1. This interaction results in impeded adhesion of FRβ+ human primary macrophages and THP-1 cells to collagen in comparison with their FRβ− counterparts. We further show that FRβ is only expressed by human macrophages when differentiated with M-CSF. These findings thus identify FRβ as a novel CD11b/CD18 regulator for trafficking and homing of a subset of macrophages on collagen.