Tetraspanins in cell stemness and cancer initiation: markers or active players?

Tetraspanins mark stem cells and tumor initiating cells. Recent studies in adipose development, intestinal crypt remodeling, and muscle stem cells shed new light on the contribution of tetraspanins and their associated partners in cell fate determination. These studies reveal that these partnerships actively help guide precursor cell fate.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

Targeting the tetraspanin CD81 reduces cancer invasion and metastasis.

Tetraspanins are an evolutionary conserved family of proteins involved in multiple aspects of cell physiology, including proliferation, migration and invasion, protein trafficking, and signal transduction; yet their detailed mechanism of action is unknown. Tetraspanins have no known natural ligands, but their engagement by antibodies has begun to reveal their role in cell biology. Studies of tetraspanin knockout mice and of germline mutations in humans have highlighted their role under normal and pathological conditions. Previously, we have shown that mice deficient in the tetraspanin CD81 developed fewer breast cancer metastases compared to their wild-type (WT) counterparts. Here, we show that a unique anti-human CD81 antibody (5A6) effectively halts invasion of triple-negative breast cancer (TNBC) cell lines. We demonstrate that 5A6 induces CD81 clustering at the cell membrane and we implicate JAM-A protein in the ability of this antibody to inhibit tumor cell invasion and migration. Furthermore, in a series of in vivo studies we demonstrate that this antibody inhibits metastases in xenograft models, as well as in syngeneic mice bearing a mouse tumor into which we knocked in the human CD81 epitope recognized by the 5A6 antibody.

CD81 is a novel immunotherapeutic target for B cell lymphoma.

The tetraspanin CD81 was initially discovered by screening mAbs elicited against a human B cell lymphoma for their direct antiproliferative effects. We now show that 5A6, one of the mAbs that target CD81, has therapeutic potential. This antibody inhibits the growth of B cell lymphoma in a xenograft model as effectively as rituximab, which is a standard treatment for B cell lymphoma. Importantly, unlike rituximab, which depletes normal as well as malignant B cells, 5A6 selectively kills human lymphoma cells from fresh biopsy specimens while sparing the normal lymphoid cells in the tumor microenvironment. The 5A6 antibody showed a good safety profile when administered to a mouse transgenic for human CD81. Taken together, these data provide the rationale for the development of the 5A6 mAb and its humanized derivatives as a novel treatment against B cell lymphoma.

EspH Suppresses Erk by Spatial Segregation from CD81 Tetraspanin Microdomains.

Enteropathogenic Escherichia coli (EPEC) belongs to a group of enteric human pathogens known as attaching-and-effacing (A/E) pathogens, which utilize a type III secretion system (T3SS) to translocate a battery of effector proteins from their own cytoplasm into host intestinal epithelial cells. Here we identified EspH to be an effector that prompts the recruitment of the tetraspanin CD81 to infection sites. EspH was also shown to be an effector that suppresses the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) signaling pathway at longer infection times. The inhibitory effect was abrogated upon deletion of the last 38 amino acids located at the C terminus of the protein. The efficacy of EspH-dependent Erk suppression was higher in CD81-deficient cells, suggesting that CD81 may act as a positive regulator of Erk, counteracting Erk suppression by EspH. EspH was found within CD81 microdomains soon after infection but was largely excluded from these domains at a later time. Based on our results, we propose a mechanism whereby CD81 is initially recruited to infection sites in response to EspH translocation. At a later stage, EspH moves out of the CD81 clusters to facilitate effective Erk inhibition. Moreover, EspH selectively inhibits the tumor necrosis factor alpha (TNF-α)-induced Erk signaling pathway. Since Erk and TNF-α have been implicated in innate immunity and cell survival, our studies suggest a novel mechanism by which EPEC suppresses these processes to promote its own colonization and survival in the infected gut.

Immune Targeting of Tetraspanins Involved in Cell Invasion and Metastasis.

Metastasis is the ultimate consequence of cancer progression and the cause of patients' death across different cancer types. Patients with initial diagnosis of distant disease have a worst 5-year survival compared to patients with localized disease. Therapies that target primary tumors fail to eradicate distant dissemination of cancer. Recently, immunotherapies have improved the survival of patients with metastatic disease, such as melanoma and lung cancer. However, only a fraction of patients responds to immunotherapy modalities that target the host immune system. The need to identify new druggable targets that inhibit or prevent metastasis is, therefore, much needed. Tetraspanins have emerged as key players in regulating cell migration, invasion, and metastasis. By serving as molecular adaptors that cluster adhesion receptors, signaling molecules, and cell surface receptors; tetraspanins are involved in all steps of the metastatic cascade. They regulate cell proliferation, participate in EMT transition, modulate integrin-mediated cell adhesion, and participate in angiogenesis and invasion processes. Tetraspanins have also been shown to modulate metastasis indirectly through exosomes and by regulating cellular interactions in the immune system. Importantly, targeting individual tetraspanin with antibodies has impacted tumor progression. This review will focus on the contribution of tetraspanins to the metastatic process and their potential as therapeutic tumor targets.

Administration of low-dose combination anti-CTLA4, anti-CD137, and anti-OX40 into murine tumor or proximal to the tumor draining lymph node induces systemic tumor regression.

The delivery of immunomodulators directly into the tumor potentially harnesses the existing antigen, tumor-specific infiltrating lymphocytes, and antigen presenting cells. This can confer specificity and generate a potent systemic anti-tumor immune response with lower doses and less toxicity compared to systemic administration, in effect an in situ vaccine. Here, we test this concept using the novel combination of immunomodulators anti-CTLA4, -CD137, and -OX40. The triple combination administered intratumorally at low doses to one tumor of a dual tumor mouse model had dramatic local and systemic anti-tumor efficacy in lymphoma (A20) and solid tumor (MC38) models, consistent with an abscopal effect. The minimal effective dose was 10 µg each. The effect was dependent on CD8 T-cells. Intratumoral administration resulted in superior local and distant tumor control compared to systemic routes, supporting the in situ vaccine concept. In a single tumor A20 model, injection close to the tDLN resulted in similar efficacy as intratumoral and significantly better than targeting a non-tDLN, supporting the role of the tDLN as a viable immunotherapy target in addition to the tumor itself. Distribution studies confirmed expected concentration of antibodies in tumor and tDLN, in keeping with the anti-tumor results. Overall intratumoral or peri-tDLN administration of the novel combination of anti-CTLA4, anti-CD137, and anti-OX40, all agents in the clinic or clinical trials, demonstrates potent systemic anti-tumor effects. This immunotherapeutic combination is promising for future clinical development via both these safe and highly efficacious routes of administration.

CD81 association with SAMHD1 enhances HIV-1 reverse transcription by increasing dNTP levels.

In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.

CD81 as a tumor target.

CD81 participates in a variety of important cellular processes such as membrane organization, protein trafficking, cellular fusion and cell-cell interactions. In the immune system, CD81 regulates immune synapse, receptor clustering and signaling; it also mediates adaptive and innate immune suppression. CD81 is a gateway in hepatocytes for pathogens such as hepatitis C virus and Plasmodium; it also confers susceptibility to Listeria infection. These diverse biological roles are due to the tendency of CD81 to associate with other tetraspanins and with cell-specific partner proteins, which provide the cells with a signaling platform. CD81 has also been shown to regulate cell migration and invasion, and has therefore been implicated in cancer progression. Indeed, we have recently shown that CD81 contributes to tumor growth and metastasis. CD81 is expressed in most types of cancer, including breast, lung, prostate, melanoma, brain cancer and lymphoma, and the overexpression or down-regulation of this molecule has been correlated with either good or bad prognosis. Here, we discuss the role of CD81 in cancer and its potential therapeutic use as a tumor target.

Tetraspanin CD81, a modulator of immune suppression in cancer and metastasis.

Cancer cells can escape the antitumor immune response by recruiting immune suppressor cells. However, although innate myeloid-derived suppressor cells (MDSCs) and T regulatory (Treg) cells accumulate normally in tumor-bearing CD81-deficient mice, both populations are impaired in their ability to suppress the antitumor immune response.

Tetraspanin CD81 promotes tumor growth and metastasis by modulating the functions of T regulatory and myeloid-derived suppressor cells.

Tumor cells counteract innate and adaptive antitumor immune responses by recruiting regulatory T cells (Treg) and innate myeloid-derived suppressor cells (MDSC), which facilitate immune escape and metastatic dissemination. Here we report a role in these recruitment processes for CD81, a member of the tetraspanin family of proteins that have been implicated previously in cancer progression. We found that genetic deficiency in CD81 reduced tumor growth and metastasis in two genetic mouse backgrounds and multiple tumor models. Mechanistic investigations revealed that CD81 was not required for normal development of Treg and MDSC but was essential for immunosuppressive functions. Notably, adoptive transfer of wild-type Treg into CD81-deficient mice was sufficient to promote tumor growth and metastasis. Our findings suggested that CD81 modulates adaptive and innate immune responses, warranting further investigation of CD81 in immunomodulation in cancer and its progression.

Role of an arginine-lysine rich motif in maturation and trafficking of CD19.

Normal expression of CD19 on the surface of B cells requires the presence of the tetraspanin molecule CD81. Previous studies have shown that surface expression of CD19 is highly reduced in CD81-deficient mouse B cells and that it is completely absent in an antibody deficient human patient with a mutation in the CD81 gene. The current study explored the contribution of an arginine-lysine rich motif, present in the membrane-proximal cytoplasmic domain of CD19, for the maturation and trafficking of this molecule. We demonstrate that this motif plays a role in the maturation and recycling of CD19 but in a CD81-independent manner.

A mutation in the human tetraspanin CD81 gene is expressed as a truncated protein but does not enable CD19 maturation and cell surface expression.

A homozygous mutation in a splice site of the CD81 gene was identified previously in a patient, as the cause in a case of common variable immune deficiency (CVID). CD19 expression is reduced in mice that lack CD81; however, B cells in this patient lacked completely CD19 surface expression. The mutation led to an absence of the CD81 protein on the cell surface and it was assumed that the CD81 protein was not produced. Here we demonstrate that a truncated human CD81 mutant (CD81mut) was actually produced, but retained intracellularly. We also demonstrate that the truncated CD81mut protein is in close proximity to the intracellularly sequestered CD19. However, this interaction does not enable normal CD19 maturation and surface expression. In addition, we show that specific domains of CD81 enable retrieval and trafficking of human CD19 to the cell surface. Finally, we demonstrate that surface expression of CD19 requires CD81, even in non-B cells.

Identification of a novel drug lead that inhibits HCV infection and cell-to-cell transmission by targeting the HCV E2 glycoprotein.

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

TSPAN33 is a novel marker of activated and malignant B cells.

We have identified Tspan33 as a gene encoding a transmembrane protein exhibiting a restricted expression pattern including expression in activated B cells. TSPAN33 is a member of the tetraspanin family. TSPAN33 is not expressed in resting B cells, but is strongly induced in primary human B cells following activation. Human 2E2 cells, a Burkitt's lymphoma-derived B cell model of activation and differentiation, also upregulate TSPAN33 upon activation. TSPAN33 is expressed in several lymphomas including Hodgkin's and Diffuse large B cell lymphoma. TSPAN33 is also expressed in some autoimmune diseases where B cells participate in the pathology, including rheumatoid arthritis patients, systemic lupus erythematosus (SLE), and in spleen B cells from MRL/Fas(lpr/lpr) mice (a mouse model of SLE). We conclude that TSPAN33 may be used as a diagnostic biomarker or as a target for therapeutic antibodies for treatment of certain B cell lymphomas or autoimmune diseases.

The CD19/CD81 complex physically interacts with CD38 but is not required to induce proliferation in mouse B lymphocytes.

In B lymphocytes, the cell surface receptor CD38 is involved in apoptosis of immature B cells, proliferation and differentiation of mature B cells. Although CD38 has been establish as a receptor, its signaling has been only partially characterized. As a result of the lack of signaling motifs in the cytoplasmic domain, CD38 must use a co-receptor to induce signaling within the cell. Accordingly, CD38 has been associated with different receptors such as the T-cell receptor/CD3 complex on T cells, CD16 on natural killer cells and MHC class II molecules on monocytes. The CD19/CD81 complex has been proposed as a co-receptor for CD38 in human B lymphocytes, but little or no characterization has been performed in mice. In this study the contribution of the CD19/CD81 complex in murine CD38 signaling was evaluated. Proliferation assays were performed using CD19(-/-) or CD81(-/-) deficient mice; CFSE-labeled B lymphocytes from wild-type mice and CD19(-/-) , CD81(-/-) and CD38(-/-) deficient mice were stimulated with agonistic antibodies against CD38. Immunoprecipitation and immunofluorescence were also performed to detect protein-protein interactions. Our results indicate that the CD19/CD81 complex interacts with CD38 but this interaction is not required to induce proliferation in mouse B lymphocytes, suggesting that other receptors may contribute to the proliferation induced by CD38 in B lymphocytes.

Consequences of two naturally occurring missense mutations in the structure and function of Bruton agammaglobulinemia tyrosine kinase.

Bruton agammaglobulinemia tyrosine kinase (BTK) is a key protein in the B-cell receptor (BCR) signaling pathway and plays an essential role in the differentiation of B lymphocytes. X-linked agammaglobulinemia (XLA) is a primary humoral immunodeficiency caused by mutations in the gene encoding BTK. Previously, we identified two novel variations, L111P and E605G, in BTK; these are localized within the pleckstrin homology and Src homology 1 domains, respectively. In the present study, we evaluated the potential effects of these variations on the structural conformation and the function of BTK. Using in silico methods, we found that the L111P and E650G variations are not located directly in protein-protein interfaces but close to them. They distorted the native structural conformation of the BTK protein, affecting not only its geometry and stability but also its ability for protein recognition and in consequence its functionality. To confirm the results of the in silico assays, WT BTK, L111P, and E650G variants were expressed in the BTK-deficient DT40 cell line. The mutant proteins exhibited an absence of catalytic activity, aberrant redistribution after BCR-crosslinking, and deficient intracellular calcium mobilization. This work demonstrates that L111 and E605 residues are fundamental for the activation and function of BTK.

CD38 through the life of a murine B lymphocyte.

CD38 is a 45 kDa transmembrane receptor expressed in B lymphocytes and other cells from the immune system. It is involved in apoptosis, cell activation, differentiation, and proliferation. CD38 has been used extensively to classify various subpopulations of lymphocytes in both humans and mice. It has also been used as a marker of poor prognosis in some lymphoid pathologies. However, CD38 is not a marker but rather an ectoenzyme and a receptor, where it performs several functions. The CD38 signaling pathway has only been partially studied in various cells of the immune system, where apparently the signaling is different depending on the lineage and differentiation state of the cell, leading to distinct outcomes. In this review, we provide an overview of well-established roles of CD38 signaling B lymphocytes from mice. We also discuss areas that need further clarification to get a broader image of how CD38 performs different functions in B cells and to understand its role in B lymphocyte biology under normal versus pathological conditions.