The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. The src family kinase Lck plays a pivotal role in T cell signaling. We show that MAL is required in T cells for efficient expression of Lck at the plasma membrane and activation of IL-2 transcription. To investigate the mechanism by which MAL regulates Lck targeting, we analyzed the dynamics of Lck and found that it travels to the plasma membrane in specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated an association of MAL with Lck. Both carrier formation and partitioning of Lck into detergent-insoluble membranes were ablated in the absence of MAL. Polarization of T cell receptor for antigen (TCR) and microtubule-organizing center to immunological synapse (IS) were also defective. Although partial correction of the latter defects was possible by forced expression of Lck at the plasma membrane, their complete correction, formation of transport vesicles, partitioning of Lck, and restoration of signaling pathways, which are required for IL-2 transcription up-regulation, were achieved by exogenous expression of MAL. We concluded that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signaling and IS assembly.

The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 μg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.

Expression of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) at the plasma membrane is essential for it to fulfill its pivotal role in signal transduction in T lymphocytes. MAL, an integral membrane protein expressed in specific types of lymphoma, has been shown to play an important role in targeting Lck to the plasma membrane. Here we report that MAL interacts with Inverted IntroductionThe activation of the src-family kinase lymphocyte-specific protein tyrosine kinase (Lck) is one of the earliest intracellular events downstream T-cell receptor (TCR) recognition. Activated Lck phosphorylates tyrosine residues of CD3, -chain-associated protein kinase 70, and other substrates, initiating various signaling cascades that result in T-cell activation and proliferation. 1,2 Lck is predominantly associated with the cytosolic side of the plasma membrane, a localization that is consistent with its importance in TCR-mediated early signaling events. 3 Transport of Lck to the plasma membrane relies on the exocytic pathway 4 and requires addition of myristate and palmitate to the glycine and the 2 cysteine residues of its N-terminal Gly-Cys-Val-Cys sequence. 5,6 Palmitoylation/depalmitoylation of Lck can also contribute to exchange Lck between the plasma membrane and the cytosol. 7 Lck acylation is also essential for activation of downstream signaling pathways 6 and partitioning into detergent-resistant membranes 5 that are postulated to contain specialized membrane microdomains. 8 MAL is an integral membrane protein exclusively detected in detergentresistant membranes of epithelial cells and human T lymphocytes. 9,10 Recent work established that MAL, which was originally characterized as a component of the machinery for specialized transport from the Golgi to the apical surface of polarized epithelial cells, 10-12 mediates a specialized route responsible for Lck transport to the plasma membrane of human T cells. 13 The questions remain as to what other machinery collaborates with MAL to transport Lck and how this process is regulated.The important role of MAL in intracellular protein transport implies that alterations in MAL expression or subcellular distribution could be reflected in abnormal functioning of the cells. It has been reported that MAL is overexpressed in cutaneous T-cell lymphoma resistant to ␣-interferon therapy. 14 Although MAL expression is normally absent from normal B lymphocytes, MAL expression has been found in primary mediastinal B-cell lymphoma 15,16 and in a subset of Hodgkin lymphoma with an adverse outcome. 17,18 Therefore, in addition to the interest of the elucidation of the mechanism of MAL-mediated transport of Lck in normal T cells, the understanding of MAL function in normal cells would be of great help to unveil its role in tumor cells.The mechanism and regulation of MAL-mediated Lck transport in T lymphocytes are addressed in the present paper through the identification of human Inverted Formin2 (INF2) as a binding partner of MAL. INF2 domain organization is similar...

The formin INF2 promotes the formation of stabilized, detyrosinated microtubules, which are important for centrosome reorientation to the immunological synapse of T cells.

T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.

Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells.

IL-15 bound to the IL-15 receptor α chain (IL-15Rα) is presented in trans to cells bearing the IL-2 receptor β and γc chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor–ligand interactions. In this study, human NK cells were tested for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Rα. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIR–HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.

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