CD82 controls CpG-dependent TLR9 signaling.

The tetraspanin CD82 is a potent suppressor of tumor metastasis and regulates several processes including signal transduction, cell adhesion, motility, and aggregation. However, the mechanisms by which CD82 participates in innate immunity are unknown. We report that CD82 is a key regulator of TLR9 trafficking and signaling. TLR9 recognizes unmethylated cytosine-phosphate-guanine (CpG) motifs present in viral, bacterial, and fungal DNA. We demonstrate that TLR9 and CD82 associate in macrophages, which occurs in the endoplasmic reticulum (ER) and post-ER. Moreover, CD82 is essential for TLR9-dependent myddosome formation in response to CpG stimulation. Finally, CD82 modulates TLR9-dependent NF-κB nuclear translocation, which is critical for inflammatory cytokine production. To our knowledge, this is the first time a tetraspanin has been implicated as a key regulator of TLR signaling. Collectively, our study demonstrates that CD82 is a specific regulator of TLR9 signaling, which may be critical in cancer immunotherapy approaches and coordinating the innate immune response to pathogens.-Khan, N. S., Lukason, D. P., Feliu, M., Ward, R. A., Lord, A. K., Reedy, J. L., Ramirez-Ortiz, Z. G., Tam, J. M., Kasperkovitz, P. V., Negoro, P. E., Vyas, T. D., Xu, S., Brinkmann, M. M., Acharaya, M., Artavanis-Tsakonas, K., Frickel, E.-M., Becker, C. E., Dagher, Z., Kim, Y.-M., Latz, E., Ploegh, H. L., Mansour, M. K., Miranti, C. K., Levitz, S. M., Vyas, J. M. CD82 controls CpG-dependent TLR9 signaling.

Cancer-malaria: hidden connections.

Cancer and malaria exemplify two maladies historically assigned to separated research spaces. Cancer, on the one hand, ranks among the top priorities in the research agenda of developed countries. Its rise is mostly explained by the ageing of these populations and linked to environment and lifestyle. Malaria, on the other hand, represents a major health burden for developing countries in the Southern Hemisphere. These two diseases also belong to separate fields of medicine: non-communicable diseases for cancer and communicable diseases for malaria.

Determining antigen specificity of a monoclonal antibody using genome-scale CRISPR-Cas9 knockout library.

An essential step in monoclonal antibody (mAb) development is the characterization and final identification of the specific target antigen and its epitope. Antibody validation is rather straightforward when immunization is carried out with peptide or purified protein, but is more difficult when whole cells or other complex antigens are used for the immunization. Determining antigen specificity of a mAb is further complicated, when reactivity of an antibody is not detected in Western blotting and/or immunoprecipitation assay. In addition to protein-based methods used for antibody characterization, a number of gene-based techniques, such as cDNA expression or short-interfering RNA (siRNA) knockdown have been applied for validation of antibodies with restricted reactivities. Earlier we have generated, characterized, but not identified the BF4 mAb that specifically stains viral biofilms on the surface of the Human T-lymphotropic Virus Type I (HTLV-1) infected T cells. In this study, using the recently developed genome-scale CRISPR-Cas9 knockout (GeCKO) library vectors, we have established the CEM T- and the Raji B cell lines with pooled libraries. After immunofluorescent staining of these cells, negative cell sorting, and guide-RNA (gRNA) sequencing, we have identified BF4 as an anti-CD82 mAb. A deep sequence analysis of GeCKO library transferred to the cells shows that the chance to succeed in the selection of antibody-negative cells and, therefore, to identify a mAb depends on the quality of cell library preparation. We believe that the described method is applicable for identification of many other hybridomas and represents a good alternative to the current protein- and gene-based methods used for mAb validation.

Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains.

CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis.

Autoantibodies to tetraspanins (CD9, CD81 and CD82) in demyelinating diseases.

Tetraspanin family proteins, CD9, CD81 and CD82 are expressed in the oligodendrocytes and Schwann cells. We investigated autoantibodies to tetraspanin proteins in patients with demyelinating diseases. Sera were collected from 119 multiple sclerosis patients, 19 neuromyelitis optica, 42 acute inflammatory demyelinating polyneuropathy, 23 chronic inflammatory demyelinating polyneuropathy and 13 acute motor axonal neuropathy as well as 55 healthy controls. Few multiple sclerosis and acute inflammatory demyelinating polyneuropathy patients had autoantibodies that were weakly reactive to CD9 or CD81 but the significance is unclear. It is unlikely that these autoantibodies are pathogenic or serve as potential biomarkers in demyelinating diseases.

Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37.

This study supports a new concept where the opposing functions of the tetraspanins CD37 and CD82 may coordinate changes in migration and Ag presentation during dendritic cell (DC) activation. We have previously published that CD37 is downregulated upon monocyte-derived DC activation, promotes migration of both skin and bone marrow-derived dendritic cells (BMDCs), and restrains Ag presentation in splenic and BMDCs. In this article, we show that CD82, the closest phylogenetic relative to CD37, appears to have opposing functions. CD82 is upregulated upon activation of BMDCs and monocyte-derived DCs, restrains migration of skin and BMDCs, supports MHC class II maturation, and promotes stable interactions between T cells and splenic DCs or BMDCs. The underlying mechanism involves the rearrangement of the cytoskeleton via a differential activation of small GTPases. Both CD37(-/-) and CD82(-/-) BMDCs lack cellular projections, but where CD37(-/-) BMDCs spread poorly on fibronectin, CD82(-/-) BMDCs are large and spread to a greater extent than wild-type BMDCs. At the molecular level, CD82 is a negative regulator of RhoA, whereas CD37 promotes activation of Rac-1; both tetraspanins negatively regulate Cdc42. Thus, this study identifies a key aspect of DC biology: an unactivated BMDC is CD37(hi)CD82(lo), resulting in a highly motile cell with a limited ability to activate naive T cells. By contrast, a late activated BMDC is CD37(lo)CD82(hi), and thus has modified its migratory, cytoskeletal, and Ag presentation machinery to become a cell superbly adapted to activating naive T cells.

Identification of the tetraspanin CD82 as a new barrier to xenotransplantation.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galß1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal-independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal-independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal-independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major "xenogenicity sensor" and open new avenues of intervention to making xenotransplantation a clinical reality.

The tetraspanin CD82 is specifically recruited to fungal and bacterial phagosomes prior to acidification.

CD82 is a member of the tetraspanin superfamily, whose physiological role is best described in the context of cancer metastasis. However, CD82 also associates with components of the class II major histocompatibility complex (MHC) antigen presentation pathway, including class II MHC molecules and the peptide-loading machinery, as well as CD63, another tetraspanin, suggesting a role for CD82 in antigen presentation. Here, we observe the dynamic rearrangement of CD82 after pathogen uptake by imaging CD82-mRFP1 expressed in primary living dendritic cells. CD82 showed rapid and specific recruitment to Cryptococcus neoformans-containing phagosomes compared to polystyrene-containing phagosomes, similar to CD63. CD82 was also actively recruited to phagosomes containing other pathogenic fungi, including Candida albicans and Aspergillus fumigatus. Recruitment of CD82 to fungal phagosomes occurred independently of Toll-like receptor (TLR) signaling. Recruitment was not limited to fungi, as bacterial organisms, including Escherichia coli and Staphylococcus aureus, also induced CD82 recruitment to the phagosome. CD82 intersected the endocytic pathway used by lipopolysaccharide (LPS), implicating CD82 in trafficking of small, pathogen-associated molecules. Despite its partial overlap with lysosomal compartments, CD82 recruitment to C. neoformans-containing phagosomes occurred independently of phagosome acidification. Kinetic analysis of fluorescence imaging revealed that CD82 and class II MHC simultaneously appear in the phagosome, indicating that the two proteins may be associated. Together, these data show that the CD82 tetraspanin is specifically recruited to pathogen-containing phagosomes prior to fusion with lysosomes.

Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.

Previous reports suggested that culture as 3D aggregates or as spheroids can increase the therapeutic potential of the adult stem/progenitor cells referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). Here we used a hanging drop protocol to prepare human MSCs (hMSCs) as spheroids that maximally expressed TNFalpha stimulated gene/protein 6 (TSG-6), the antiinflammatory protein that was expressed at high levels by hMSCs trapped in the lung after i.v. infusion and that largely explained the beneficial effects of hMSCs in mice with myocardial infarcts. The properties of spheroid hMSCs were found to depend critically on the culture conditions. Under optimal conditions for expression of TSG-6, the hMSCs also expressed high levels of stanniocalcin-1, a protein with both antiinflammatory and antiapoptotic properties. In addition, they expressed high levels of three anticancer proteins: IL-24, TNFalpha-related apoptosis inducing ligand, and CD82. The spheroid hMSCs were more effective than hMSCs from adherent monolayer cultures in suppressing inflammatory responses in a coculture system with LPS-activated macrophages and in a mouse model for peritonitis. In addition, the spheroid hMSCs were about one-fourth the volume of hMSCs from adherent cultures. Apparently as a result, larger numbers of the cells trafficked through the lung after i.v. infusion and were recovered in spleen, liver, kidney, and heart. The data suggest that spheroid hMSCs may be more effective than hMSCs from adherent cultures in therapies for diseases characterized by sterile tissue injury and unresolved inflammation and for some cancers that are sensitive to antiinflammatory agents.

Controlling cell surface dynamics and signaling: how CD82/KAI1 suppresses metastasis.

The recent identification of metastasis suppressor genes, uniquely responsible for negatively controlling cancer metastasis, are providing inroads into the molecular machinery involved in metastasis. While the normal function of a few of these genes is known; the molecular events associated with their loss that promotes tumor metastasis is largely not understood. KAI1/CD82, whose loss is associated with a wide variety of metastatic cancers, belongs to the tetraspanin family. Despite intense scrutiny, many aspects of how CD82 specifically functions as a metastasis suppressor and its role in normal biology remain to be determined. This review will focus on the molecular events associated with CD82 loss, the potential impact on signaling pathways that regulate cellular processes associated with metastasis, and its relationship with other metastasis suppressor genes.

Tetraspanins regulate ADAM10-mediated cleavage of TNF-alpha and epidermal growth factor.

Several cytokines and growth factors are released by proteolytic cleavage of a membrane-anchored precursor, through the action of ADAM (a disintegrin and metalloprotease) metalloproteases. The activity of these proteases is regulated through largely unknown mechanisms. In this study we show that Ab engagement of several tetraspanins (CD9, CD81, CD82) increases epidermal growth factor and/or TNF-alpha secretion through a mechanism dependent on ADAM10. The effect of anti-tetraspanin mAb on TNF-alpha release is rapid, not relayed by intercellular signaling, and depends on an intact MEK/Erk1/2 pathway. It is also associated with a concentration of ADAM10 in tetraspanin-containing patches. We also show that a large fraction of ADAM10 associates with several tetraspanins, indicating that ADAM10 is a component of the "tetraspanin web." These data show that tetraspanins regulate the activity of ADAM10 toward several substrates, and illustrate how membrane compartmentalization by tetraspanins can control the function of cell surface proteins such as ectoproteases.

Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration.

It has been proposed that the metastasis suppressor CD82/KAI-1, which is a member of the tetraspanin superfamily, regulates biological activity by associating with cell surface receptors or proteins. We show a novel association between CD82 and the hepatocyte growth factor (HGF) receptor c-Met. Although ectopic expression of CD82 in nonsmall cell lung carcinoma cells did not affect the tyrosine phosphorylation of c-Met, these cells showed significant suppression of HGF-induced lamellipodial protrusion and cell migration. CD82 selectively attenuated c-Met signaling via the Ras-Cdc42/Rac and the phosphatidylinositol 3-kinase/Cdc42/Rac pathways. In contrast, another c-Met signaling pathway that involves phosphatidylinositol 3-kinase/Akt and phosphatidylinositol 3-kinase/mitogen activated protein kinase was not affected by CD82. Signaling adapter proteins for c-Met, such as Grb2 and p85, exhibited reduced association with c-Met in cells that ectopically expressed CD82. These results indicate that the CD82-c-Met complex inhibits HGF-induced cancer cell migration by the inactivation of small GTP-binding proteins of the Rho family via c-Met adapter proteins.

Characterization of an antibody that can detect the Kai1/CD82 murine metastasis suppressor.

BACKGROUND: Kai1, also known as CD82, is a member of the tetraspanin family (TM4SF). The human homolog, KAI1, is an activation antigen of T-cells and is a metastasis suppressor for prostate and other cancers. Little is known about the mouse protein because of the lack of antibody reagents. METHODS: Peptide immunized rabbits were used to generate polyclonal antibody to Kai1. The antibody was analyzed using immunoblotting, flow cytometry, and immunohistochemistry. RESULTS: This antibody specifically recognizes murine Kai1 protein, crossreacts with rat Kai1 but not with human KAI1. The normal tissue distribution of this protein in mice is shown to be similar to that of the human homolog. Interestingly, mouse prostatic epithelium showed differential expression within the lobes. CONCLUSION: This antibody, the first described that can specifically detect murine Kai1/CD82, should be very useful in addressing the mechanism of action of Kai1 in metastatic suppression.