Chiung-Chi Kuo

CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency.

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2

ABSTRACT Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

CD81-Dependent Binding of Hepatitis C Virus E1E2 Heterodimers

ABSTRACT Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.

Building of the Tetraspanin Web: Distinct Structural Domains of CD81 Function in Different Cellular Compartments

ABSTRACT The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web.

Cell-free production of Gaussia princeps luciferase - antibody fragment bioconjugates for ex vivo detection of tumor cells

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

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.

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.

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

ABSTRACT 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.

Abstract B061: CD81: A new target for therapy of B cell lymphoma

CD81 belongs to an evolutionary conserved family of proteins named tetraspanins and it was originally described as a B cell target. Tetraspanins associate in multi-molecular complexes in tetraspanin-enriched microdomains (TEMS). Indeed, in B cells CD81 associates with CD19 and CD21, and together function as co-receptor to lower the cell activation threshold initiated by the BCR. Recently, we have demonstrated the importance of CD81 in tumor growth and metastasis of solid tumors1. However, the role of CD81 in B cell malignancies has not been explored. Previously, we have shown that anti-CD81 antibodies had anti-proliferative effects on human B cell lymphoma cell lines in vitro2. Here we tested the therapeutic effect of a mouse anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-CD81 antibody had therapeutic effect comparable to rituximab. Moreover, we found ADCC as one of the mechanism of action. Furthermore, to enhance the anti-CD81-mediated ADCC we engineered chimeric antibodies containing human IgG1ADCC-HIGH and human IgG4 ADCC-LOW Fc constant regions. Indeed, the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC compared to Rituximab in vitro. These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC. The Human IgG1 version is being developed as a therapeutic candidate. 1. Vences-Catalan, F., et al. Cancer Res 75, 4517-4526 (2015). 2. Oren, R., Takahashi, S., Doss, C., Levy, R. & Levy, S. Mol Cell Biol 10, 4007-4015 (1990). Citation Format: Felipe Vences-Catalan, Chiung-Chi Kuo, Ranjani Rajapaksa, Ronald Levy, Shoshana Levy. CD81: A new target for therapy of B cell lymphoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B061.