Membrane organization by tetraspanins and galectins shapes lymphocyte function.

Immune receptors are not randomly distributed at the plasma membrane of lymphocytes but are segregated into specialized domains that function as platforms to initiate signalling, as exemplified by the B cell or T cell receptor complex and the immunological synapse. 'Membrane-organizing proteins' and, in particular, tetraspanins and galectins, are crucial for controlling the spatiotemporal organization of immune receptors and other signalling proteins. Deficiencies in specific tetraspanins and galectins result in impaired immune synapse formation, lymphocyte proliferation, antibody production and migration, which can lead to impaired immunity, tumour development and autoimmunity. In contrast to conventional ligand-receptor interactions, membrane organizers interact in cis (on the same cell) and modulate receptor clustering, receptor dynamics and intracellular signalling. New findings have uncovered their complex and dynamic nature, revealing shared binding partners and collaborative activity in determining the composition of membrane domains. Therefore, immune receptors should not be envisaged as independent entities and instead should be studied in the context of their spatial organization in the lymphocyte membrane. We advocate for a novel approach to study lymphocyte function by globally analysing the role of membrane organizers in the assembly of different membrane complexes and discuss opportunities to develop therapeutic approaches that act via the modulation of membrane organization.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma.

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis.

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Reclassification of diffuse large B cell lymphoma to large B cell lymphoma with IRF4 rearrangement in an adult population.

AIMS: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4. METHODS AND RESULTS: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008. CONCLUSIONS: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region.

Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.

Tetraspanin CD53 controls T cell immunity through regulation of CD45RO stability, mobility, and function.

T cells depend on the phosphatase CD45 to initiate T cell receptor signaling. Although the critical role of CD45 in T cells is established, the mechanisms controlling function and localization in the membrane are not well understood. Moreover, the regulation of specific CD45 isoforms in T cell signaling remains unresolved. By using unbiased mass spectrometry, we identify the tetraspanin CD53 as a partner of CD45 and show that CD53 controls CD45 function and T cell activation. CD53-negative T cells (Cd53-/-) exhibit substantial proliferation defects, and Cd53-/- mice show impaired tumor rejection and reduced IFNγ-producing T cells compared with wild-type mice. Investigation into the mechanism reveals that CD53 is required for CD45RO expression and mobility. In addition, CD53 is shown to stabilize CD45 on the membrane and is required for optimal phosphatase activity and subsequent Lck activation. Together, our findings reveal CD53 as a regulator of CD45 activity required for T cell immunity.

IRF8 is a transcriptional activator of CD37 expression in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) represents the most common form of non-Hodgkin lymphoma (NHL) that is still incurable in a large fraction of patients. Tetraspanin CD37 is highly expressed on mature B lymphocytes, and multiple CD37-targeting therapies are under clinical development for NHL. However, CD37 expression is nondetectable in ∼50% of DLBCL patients, which correlates with inferior treatment outcome, but the underlying mechanisms for differential CD37 expression in DLBCL are still unknown. Here, we investigated the regulation of the CD37 gene in human DLBCL at the (epi-)genetic and transcriptional level. No differences were observed in DNA methylation within the CD37 promoter region between CD37-positive and CD37-negative primary DLBCL patient samples. On the contrary, CD37-negative DLBCL cells specifically lacked CD37 promoter activity, suggesting differential regulation of CD37 gene expression. Using an unbiased quantitative proteomic approach, we identified transcription factor IRF8 to be significantly higher expressed in nuclear extracts of CD37-positive as compared with CD37-negative DLBCL. Direct binding of IRF8 to the CD37 promoter region was confirmed by DNA pulldown assay combined with mass spectrometry and targeted chromatin immunoprecipitation (ChIP). Functional analysis indicated that IRF8 overexpression enhanced CD37 protein expression, while CRISPR/Cas9 knockout of IRF8 decreased CD37 levels in DLBCL cell lines. Immunohistochemical analysis in a large cohort of primary DLBCL (n = 206) revealed a significant correlation of IRF8 expression with detectable CD37 levels. Together, this study provides new insight into the molecular mechanisms underlying differential CD37 expression in human DLBCL and reveals IRF8 as a transcriptional regulator of CD37 in B-cell lymphoma.

Site-specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.

Improving Therapeutic CD20 Antibodies Requires Insight into Their Mechanism of Action.

CD20-targeting antibodies are the current standard of care for patients with mature B-cell malignancies. However, many patients relapse or develop therapy resistance, which emphasizes the urgent need for new therapies. Here, we provide an overview of the biology of the CD20 protein and the mechanisms of action of CD20 antibodies currently used in the clinic. In addition, we discuss different mechanisms underlying therapy resistance, and recent advances made in the development of novel antibody-based therapeutics to improve clinical outcome of patients with mature B-cell malignancies.

High frequency of inactivating tetraspanin C D37 mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in Cd37-knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, CD37 mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. CD37 mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site CD37 mutations. Modeling and functional analysis of CD37 missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without CD37 mutations.

C-type lectin-like receptor 2 (CLEC-2)-dependent dendritic cell migration is controlled by tetraspanin CD37.

Cell migration is central to evoking a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2; encoded by the gene Clec1b), expressed by DCs, with podoplanin, expressed by lymph node stromal cells, although the underlying molecular mechanisms remain elusive. Here, we show that CLEC-2-dependent DC migration is controlled by tetraspanin CD37, a membrane-organizing protein. We identified a specific interaction between CLEC-2 and CD37, and myeloid cells lacking CD37 (Cd37-/-) expressed reduced surface CLEC-2. CLEC-2-expressing Cd37-/- DCs showed impaired adhesion, migration velocity and displacement on lymph node stromal cells. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs. Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. Finally, Cd37-/- DCs failed to inhibit actomyosin contractility in lymph node stromal cells, thus phenocopying CLEC-2-deficient DCs. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights into the mechanisms underlying CLEC-2-dependent DC migration.This article has an associated First Person interview with the first author of the paper.

Antitumor Immunity Is Controlled by Tetraspanin Proteins.

Antitumor immunity is shaped by the different types of immune cells that are present in the tumor microenvironment (TME). In particular, environmental signals (for instance, soluble factors or cell-cell contact) transmitted through the plasma membrane determine whether immune cells are activated or inhibited. Tetraspanin proteins are emerging as central building blocks of the plasma membrane by their capacity to cluster immune receptors, enzymes, and signaling molecules into the tetraspanin web. Whereas some tetraspanins (CD81, CD151, CD9) are widely and broadly expressed, others (CD53, CD37, Tssc6) have an expression pattern restricted to hematopoietic cells. Studies using genetic mouse models have identified important immunological functions of these tetraspanins on different leukocyte subsets, and as such, may be involved in the immune response against tumors. While multiple studies have been performed with regards to deciphering the function of tetraspanins on cancer cells, the effect of tetraspanins on immune cells in the antitumor response remains understudied. In this review, we will focus on tetraspanins expressed by immune cells and discuss their potential role in antitumor immunity. New insights in tetraspanin function in the TME and possible prognostic and therapeutic roles of tetraspanins will be discussed.

Interleukin-6 is essential for glomerular immunoglobulin A deposition and the development of renal pathology in Cd37-deficient mice.

Immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is characterized by IgA depositions in the kidney. Deficiency of CD37, a leukocyte-specific tetraspanin, leads to spontaneous development of renal pathology resembling IgAN. However, the underlying molecular mechanism has not been resolved. Here we found that CD37 expression on B cells of patients with IgAN was significantly decreased compared to B cells of healthy donors. Circulating interleukin (IL)-6 levels, but not tumor necrosis factor-α or IL-10, were elevated in Cd37-/- mice compared to wild-type mice after lipopolysaccharide treatment. Cd37-/- mice displayed increased glomerular neutrophil influx, immune complex deposition, and worse renal function. To evaluate the role of IL-6 in the pathogenesis of accelerated renal pathology in Cd37-/-mice, we generated Cd37xIl6 double-knockout mice. These double-knockout and Il6-/- mice displayed no glomerular IgA deposition and were protected from exacerbated renal failure following lipopolysaccharide treatment. Moreover, kidneys of Cd37-/- mice showed more mesangial proliferation, endothelial cell activation, podocyte activation, and segmental podocyte foot process effacement compared to the double-knockout mice, emphasizing that IL-6 mediates renal pathology in Cd37-/- mice. Thus, our study indicates that CD37 may protect against IgA nephropathy by inhibition of the IL-6 pathway.

Differential expression of tetraspanin superfamily members in dendritic cell subsets.

Dendritic cells (DCs), which are essential for initiating immune responses, are comprised of different subsets. Tetraspanins organize dendritic cell membranes by facilitating protein-protein interactions within the so called tetraspanin web. In this study we analyzed expression of the complete tetraspanin superfamily in primary murine (CD4+, CD8+, pDC) and human DC subsets (CD1c+, CD141+, pDC) at the transcriptome and proteome level. Different RNA and protein expression profiles for the tetraspanin genes across human and murine DC subsets were identified. Although RNA expression levels of CD37 and CD82 were not significantly different between human DC subsets, CD9 RNA was highly expressed in pDCs, while CD9 protein expression was lower. This indicates that relative RNA and protein expression levels are not always in agreement. Both murine CD8α+ DCs and its regarded human counterpart, CD141+ DCs, displayed relatively high protein levels of CD81. CD53 protein was highly expressed on human pDCs in contrast to the relatively low protein expression of most other tetraspanins. This study demonstrates that tetraspanins are differentially expressed by human and murine DC subsets which provides a valuable resource that will aid the understanding of tetraspanin function in DC biology.

Novel Insights into Membrane Targeting of B Cell Lymphoma.

Standard therapy of patients with B cell non-Hodgkin lymphoma (B-NHL) predominantly consists of chemotherapy combined with anti-CD20 (e.g., rituximab) immunotherapy. However, relapse of aggressive B-NHL occurs frequently, and this may coincide with therapy resistance. This demonstrates the urgent need for exploring new lymphoma-targeted therapies. We review here recent insights in the pathophysiology of B-NHL and discuss CD20 and three alternative membrane targets (B cell receptor, immune checkpoints PD-1/PD-L1, tetraspanin CD37) that are currently in the spotlight for B-NHL treatment. Furthermore, we present a novel concept in which the plasma membrane organization of the lymphoma B cell determines the efficacy of membrane-targeted therapies, and this has consequences for treatment application and clinical outcome in patients with B cell lymphoma.

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37-) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37-, including TP53 mutation, NF-κBhigh, Mychigh, phosphorylated STAT3high, survivinhigh, p63-, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37- status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37- DLBCL, ICOSLG upregulation in CD37+ GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.

Tetraspanin CD37 protects against the development of B cell lymphoma.

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.

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.

Semaphorin 7A Promotes Chemokine-Driven Dendritic Cell Migration.

Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.

Tetraspanin CD37 Regulates ß2 Integrin-Mediated Adhesion and Migration in Neutrophils.

Deciphering the molecular basis of leukocyte recruitment is critical to the understanding of inflammation. In this study, we investigated the contribution of the tetraspanin CD37 to this key process. CD37-deficient mice showed impaired neutrophil recruitment in a peritonitis model. Intravital microscopic analysis indicated that the absence of CD37 impaired the capacity of leukocytes to follow a CXCL1 chemotactic gradient accurately in the interstitium. Moreover, analysis of CXCL1-induced leukocyte-endothelial cell interactions in postcapillary venules revealed that CXCL1-induced neutrophil adhesion and transmigration were reduced in the absence of CD37, consistent with a reduced capacity to undergo ß2 integrin-dependent adhesion. This result was supported by in vitro flow chamber experiments that demonstrated an impairment in adhesion of CD37-deficient neutrophils to the ß2 integrin ligand, ICAM-1, despite the normal display of high-affinity ß2 integrins. Superresolution microscopic assessment of localization of CD37 and CD18 in ICAM-1-adherent neutrophils demonstrated that these molecules do not significantly cocluster in the cell membrane, arguing against the possibility that CD37 regulates ß2 integrin function via a direct molecular interaction. Moreover, CD37 ablation did not affect ß2 integrin clustering. In contrast, the absence of CD37 in neutrophils impaired actin polymerization, cell spreading and polarization, dysregulated Rac-1 activation, and accelerated ß2 integrin internalization. Together, these data indicate that CD37 promotes neutrophil adhesion and recruitment via the promotion of cytoskeletal function downstream of integrin-mediated adhesion.