Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

Prospecting for Planarian Pluripotency.

Planarians are renowned for extraordinary regenerative abilities that are driven by stem cells maintained throughout their lives. In this issue of Cell, Zeng et al. report the prospective isolation of planarian pluripotent stem cells. Their work opens new directions for understanding how these remarkable cells are established, maintained, and activated.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

Actin Dynamics Regulates Dendritic Cell-Mediated Transfer of HIV-1 to T Cells.

Whereas human dendritic cells (DCs) are largely resistant to productive infection with HIV-1, they have a unique ability to take up the virus and transmit it efficiently to T lymphocytes through a process of trans-infection or trans-enhancement. To elucidate the molecular and cell biological mechanism for trans-enhancement, we performed an shRNA screen of several hundred genes involved in organelle and membrane trafficking in immature human monocyte-derived dendritic cells (MDDCs). We identified TSPAN7 and DNM2, which control actin nucleation and stabilization, as having important and distinct roles in limiting HIV-1 endocytosis and in maintaining virus particles on dendrites, which is required for efficient transfer to T lymphocytes. Further characterization of this process may provide insights not only into the role of DCs in transmission and dissemination of HIV-1 but also more broadly into mechanisms controlling capture and internalization of pathogens.

Tetraspanin proteins in membrane remodeling processes.

Membrane remodeling is a fundamental cellular process that is crucial for physiological functions such as signaling, membrane fusion and cell migration. Tetraspanins (TSPANs) are transmembrane proteins of central importance to membrane remodeling events. During these events, TSPANs are known to interact with themselves and other proteins and lipids; however, their mechanism of action in controlling membrane dynamics is not fully understood. Since these proteins span the membrane, membrane properties such as rigidity, curvature and tension can influence their behavior. In this Review, we summarize recent studies that explore the roles of TSPANs in membrane remodeling processes and highlight the unique structural features of TSPANs that mediate their interactions and localization. Further, we emphasize the influence of membrane curvature on TSPAN distribution and membrane domain formation and describe how these behaviors affect cellular functions. This Review provides a comprehensive perspective on the multifaceted function of TSPANs in membrane remodeling processes and can help readers to understand the intricate molecular mechanisms that govern cellular membrane dynamics.

Role of a novel uropod-like cell membrane protrusion in the pathogenesis of the parasite Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease worldwide. As an extracellular parasite, adhesion to host cells is essential for the development of infection. During attachment, the parasite changes its tear ovoid shape to a flat ameboid form, expanding the contact surface and migrating through tissues. Here, we have identified a novel structure formed at the posterior pole of adherent parasite strains, resembling the previously described uropod, which appears to play a pivotal role as an anchor during the attachment process. Moreover, our research demonstrates that the overexpression of the tetraspanin T. vaginalis TSP5 protein (TvTSP5), which is localized on the cell surface of the parasite, notably enhances the formation of this posterior anchor structure in adherent strains. Finally, we demonstrate that parasites that overexpress TvTSP5 possess an increased ability to adhere to host cells, enhanced aggregation and reduced migration on agar plates. Overall, these findings unveil novel proteins and structures involved in the intricate mechanisms of T. vaginalis interactions with host cells.

Tetraspanin-enriched microdomains play an important role in pathogenesis in the protozoan parasite Entamoeba histolytica.

Tetraspanins (TSPANs) are a family of highly conserved proteins present in a wide variety of eukaryotes. Although protein-protein interactions of TSPANs have been well established in eukaryotes including parasitic protists, the role they play in parasitism and pathogenesis remains largely unknown. In this study, we characterized three representative members of TSPANs, TSPAN4, TSPAN12, and TSPAN13 from the human intestinal protozoan Entamoeba histolytica. Co-immunoprecipitation assays demonstrated that TSPAN4, TSPAN12 and TSPAN13 are reciprocally pulled down together with several other TSPAN-interacting proteins including TSPAN binding protein of 55kDa (TBP55) and interaptin. Blue native-PAGE analysis showed that these TSPANs form several complexes of 120-250 kDa. Repression of tspan12 and tspan13 gene expression led to decreased secretion of cysteine proteases, while repression of tspan4 led to a four-fold increase in the activity of cysteine proteases in crude extracellular vesicles (EVs) fraction. Meanwhile, strains overexpressing HA-tagged TSPAN12 and TSPAN13 demonstrated reduced adhesion to collagen. Altogether, this study reveals that the TSPANs, especially TSPAN12 and TSPAN13, are engaged with complex protein-protein interactions and are involved in the pathogenicity-related biological functions such as protease secretion and adhesion, offering insights into the potential regulatory mechanisms of tetraspanins in protozoan parasites.

CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

Phase 1 study of CAR-37 T cells in patients with relapsed or refractory CD37+ lymphoid malignancies.

ABSTRACT: We report a first-in-human clinical trial using chimeric antigen receptor (CAR) T cells targeting CD37, an antigen highly expressed in B- and T-cell malignancies. Five patients with relapsed or refractory CD37+ lymphoid malignancies were enrolled and infused with autologous CAR-37 T cells. CAR-37 T cells expanded in the peripheral blood of all patients and, at peak, comprised >94% of the total lymphocytes in 4 of 5 patients. Tumor responses were observed in 4 of 5 patients with 3 complete responses, 1 mixed response, and 1 patient whose disease progressed rapidly and with relative loss of CD37 expression. Three patients experienced prolonged and severe pancytopenia, and in 2 of these patients, efforts to ablate CAR-37 T cells, which were engineered to coexpress truncated epidermal growth factor receptor, with cetuximab were unsuccessful. Hematopoiesis was restored in these 2 patients after allogeneic hematopoietic stem cell transplantation. No other severe, nonhematopoietic toxicities occurred. We investigated the mechanisms of profound pancytopenia and did not observe activation of CAR-37 T cells in response to hematopoietic stem cells in vitro or hematotoxicity in humanized models. Patients with pancytopenia had sustained high levels of interleukin-18 (IL-18) with low levels of IL-18 binding protein in their peripheral blood. IL-18 levels were significantly higher in CAR-37-treated patients than in both cytopenic and noncytopenic cohorts of CAR-19-treated patients. In conclusion, CAR-37 T cells exhibited antitumor activity, with significant CAR expansion and cytokine production. CAR-37 T cells may be an effective therapy in hematologic malignancies as a bridge to hematopoietic stem cell transplant. This trial was registered at www.ClinicalTrials.gov as #NCT04136275.

Cutting Edge: The Tetraspanin CD53 Promotes CXCR4 Signaling and Bone Marrow Homing in B Cells.

B cell trafficking involves the coordinated activity of multiple adhesive and cytokine-receptor interactions, and the players in this process are not fully understood. In this study, we identified the tetraspanin CD53 as a critical regulator of both normal and malignant B cell trafficking. CXCL12 is a key chemokine in B cell homing to the bone marrow and secondary lymphoid organs, and both normal and malignant B cells from Cd53-/- mice have reduced migration toward CXCL12 in vitro, as well as impaired marrow homing in vivo. Using proximity ligation studies, we identified the CXCL12 receptor, CXCR4, as a novel, to our knowledge, CD53 binding partner. This interaction promotes receptor function, because Cd53-/- B cells display reduced signaling and internalization of CXCR4 in response to CXCL12. Together, our data suggest that CD53 interacts with CXCR4 on both normal and malignant B cells to promote CXCL12 signaling, receptor internalization, and marrow homing.

Casein kinase 1 gamma regulates oxidative stress response via interacting with the NADPH dual oxidase complex.

Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.

Rice tetraspanins express in specific domains of diverse tissues and regulate plant architecture and root growth.

Tetraspanins (TETs) are small transmembrane scaffold proteins that distribute proteins into highly organized microdomains, consisting of adaptors and signaling proteins, which play important roles in various biological events. In plants, understanding of tetraspanin is limited to the Arabidopsis TET genes' expression pattern and their function in leaf and root growth. Here, we comprehensively analyzed all rice tetraspanin (OsTET) family members, including their gene expression pattern, protein topology, and subcellular localization. We found that the core domain of OsTETs is conserved and shares a similar topology of four membrane-spanning domains with animal and plant TETs. OsTET genes are partially overlapping expressed in diverse tissue domains in vegetative and reproductive organs. OsTET proteins preferentially targeted the endoplasmic reticulum. Mutation analysis showed that OsTET5, OsTET6, OsTET9, and OsTET10 regulated plant height and tillering, and that OsTET13 controlled root growth in association with the jasmonic acid pathway. In summary, our work provides systematic new insights into the function of OsTETs in rice growth and development, and the data provides valuable resources for future research.

Phospho-eIF4E stimulation regulates coronavirus entry by selective expression of cell membrane-residential factors.

The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3, CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3, CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3, CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3, CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3, CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.

Mutations in TSPAN12 gene causing familial exudative vitreoretinopathy.

BACKGROUND: To report newly found TSPAN12 mutations with a unique form of familial exudative vitreoretinopathy (FEVR) and find out the possible mechanism of a repeated novel intronic variant in TSPAN12 led to FEVR. RESULTS: Nine TSPAN12 mutations with a unique form of FEVR were detected by panel-based NGS. MINI-Gene assay showed two splicing modes of mRNA that process two different bands A and B, and mutant-type shows replacement with the splicing mode of Exon11 hopping. Construction of wild-type and mutant TSPAN12 vector showed the appearance of premature termination codons (PTC). In vitro expression detection showed significant down-regulated expression level of TSPAN12 mRNAs and proteins in cells transfected with mutant vectors compared with in wild-type group. On the contrary, translation inhibitor CHX and small interfering RNA of UPF1 (si-UPF1) significantly increased mRNA or protein expression of TSPAN12 in cells transfected with the mutant vectors. CONCLUSIONS: Nine mutations in TSPAN12 gene are reported in 9 FEVR patients with a unique series of ocular abnormalities. The three novel TSPAN12 mutations trigger NMD would cause the decrease of TSPAN12 proteins that participate in biosynthesis and assembly of microfibers, which might lead to FEVR, and suggest that intronic sequence analysis might be a vital tool for genetic counseling and prenatal diagnoses.

Hemolymph exosomes inhibit Spiroplasma eriocheiris infection by promoting Tetraspanin-mediated hemocyte phagocytosis in crab.

Exosomes released from infected cells are thought to play an important role in the dissemination of pathogens, as well as in host-derived immune molecules during infection. As an intracellular pathogen, Spiroplasma eriocheiris is harmful to multiple crustaceans. However, the immune mechanism of exosomes during Spiroplasma infection has not been investigated. Here, we found exosomes derived from S. eriocheiris-infected crabs could facilitate phagocytosis and apoptosis of hemocytes, resulting in increased crab survival and suppression of Spiroplasma intracellular replication. Proteomic analysis revealed the altered abundance of EsTetraspanin may confer resistance to S. eriocheiris, possibly by mediating hemocyte phagocytosis in Eriocheir sinensis. Specifically, knockdown of EsTetraspanin in E. sinensis increased susceptibility to S. eriocheiris infection and displayed compromised phagocytic ability, whereas overexpression of EsTetraspanin in Drosophila S2 cells inhibited S. eriocheiris infection. Further, it was confirmed that intramuscular injection of recombinant LEL domain of EsTetraspanin reduced the mortality of S. eriocheiris-infected crabs. Blockade with anti-EsTetraspanin serum could exacerbate S. eriocheiris invasion of hemocytes and impair hemocyte phagocytic activity. Taken together, our findings prove for the first time that exosomes modulate phagocytosis to resist pathogenic infection in invertebrates, which is proposed to be mediated by exosomal Tetraspanin, supporting the development of preventative strategies against Spiroplasma infection.

Deciphering a crucial dimeric interface governing Norrin dimerization and the pathogenesis of familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a hereditary eye disease that could cause blindness. It has been established that Norrin forms dimers to activate ß-catenin signaling, yet the core interface for Norrin dimerization and the precise mechanism by which Norrin dimerization contributes to the pathogenesis of FEVR remain elusive. Here, we report an NDP variant, c.265T>C (p.Phe89Leu), that interrupted ß-catenin signaling by disrupting Norrin dimerization. Structural and functional analysis revealed that the Phe-89 of one Norrin monomer interacts with Pro-98, Ser-101, Arg-121, and Ile-123 of another, forming two core symmetrical dimerization interfaces that are pivotal for the formation of a "hand-by-arm" dimer. Intriguingly, we proved that one of the two core symmetrical interfaces is sufficient for dimerization and activation of ß-catenin signaling, with a substantial contribution from the Phe-89/Pro-98 interaction. Further functional analysis revealed that the disruption of both dimeric interfaces eliminates potential binding sites for LRP5, which could be partially restored by over-expression of TSPAN12. In conclusion, our findings unveil a core dimerization interface that regulates Norrin/LRP5 interaction, highlighting the essential role of Norrin dimerization on ß-catenin signaling and providing potential therapeutic avenues for the treatment of FEVR.

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function.

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP-12 and TSP-14, function redundantly to promote both Notch signaling and bone morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP-14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the N-terminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism.

Transmembrane proteins tetraspanin 4 and CD9 sense membrane curvature.

Multiple membrane-shaping and remodeling processes are associated with tetraspanin proteins by yet unknown mechanisms. Tetraspanins constitute a family of proteins with four transmembrane domains present in every cell type. Prominent examples are tetraspanin4 and CD9, which are required for the fundamental cellular processes of migrasome formation and fertilization, respectively. These proteins are enriched in curved membrane structures, such as cellular retraction fibers and oocyte microvilli. The factors driving this enrichment are, however, unknown. Here, we revealed that tetraspanin4 and CD9 are curvature sensors with a preference for positive membrane curvature. To this end, we used a biomimetic system emulating membranes of cell retraction fibers and oocyte microvilli by membrane tubes pulled out of giant plasma membrane vesicles with controllable membrane tension and curvature. We developed a simple thermodynamic model for the partitioning of curvature sensors between flat and tubular membranes, which allowed us to estimate the individual intrinsic curvatures of the two proteins. Overall, our findings illuminate the process of migrasome formation and oocyte microvilli shaping and provide insight into the role of tetraspanin proteins in membrane remodeling processes.

Tetraspanin-5-mediated MHC class I clustering is required for optimal CD8 T cell activation.

MHC molecules are not randomly distributed on the plasma membrane but instead are present in discrete nanoclusters. The mechanisms that control formation of MHC I nanoclusters and the importance of such structures are incompletely understood. Here, we report a molecular association between tetraspanin-5 (Tspan5) and MHC I molecules that started in the endoplasmic reticulum and was maintained on the plasma membrane. This association was observed both in mouse dendritic cells and in human cancer cell lines. Loss of Tspan5 reduced the size of MHC I clusters without affecting MHC I peptide loading, delivery of complexes to the plasma membrane, or overall surface MHC I levels. Functionally, CD8 T cell responses to antigen presented by Tspan5-deficient dendritic cells were impaired but were restored by antibody-induced reclustering of MHC I molecules. In contrast, Tspan5 did not associate with two other plasma membrane proteins, Flotillin1 and CD55, with or the endoplasmic reticulum proteins Tapasin and TAP. Thus, our findings identify a mechanism underlying the clustering of MHC I molecules that is important for optimal T cell responses.