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Lymph nodes are uniquely organised to form specialised niches for immune interactions. Fibroblastic reticular cells (FRCs) are an essential stromal component of lymph nodes – forming intricate 3-dimensional networks to facilitate communication between immune cells and depositing and ensheathing extracellular matrix on the conduit network. However, beyond these structural roles, FRCs regulate immune function through the production of growth factors, chemokines and inflammatory cues. Here we sought to determine how the immunoregulatory properties of FRCs are determined. Since PDPN has been implicated in lymph node development, we directly tested how the PDPN/CLEC-2 signalling axis impacted the immunoregulatory properties of FRCsin vitroandin vivo. We find that FRCs use the PDPN/CLEC-2 signalling axis to switch transcriptional states and alter the expression of immune related genes.In vivo, genetic deletion of PDPN from fibroblastic stroma in PDGFRαmGFPΔPDPNmice downregulated key immunoregulatory molecules CCL21, VCAM-1 and ICAM-1 and attenuated the activation, proliferation and differentiation of lymphocyte populations. Further, PDGFRαmGFPΔPDPNmice exhibited severe disruption of the FRC network structure, leading to a failure to separate B and T lymphocytes and misdistribution of myeloid cells through the tissue. We conclude that PDPN expression controls signalling pathways beyond cytoskeletal regulation and cell mechanics and that PDPN expression is required for FRC phenotype and function in lymph nodes.

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian. Instituto de Patología Vegetal Fil: Kent, Brianne A. Simon Fraser University. Department of Psychology; Canadá Fil: Holman, Constance. Universitätsmedizin Berlin. BIH QUEST Center for Responsible Research. Berlin Institute of Health at Charité; Alemania Fil: Amoako, Emmanuella. Cape Coast Teaching Hospital. Department of Paediatrics and Child Health; Ghana Fil: Amoako, Emmanuella. University of Cape Coast. School of Medicine. Department of Paediatrics and Child Health; Ghana Fil: Antonietti, Alberto. Politecnico di Milano. Department of Electronics, Information and Bioengineering; Italia Fil: Azam, James M. Stellenbosch University. DSI-NRF Center of Excellence in Epidemiological Modelling and Analysis. Department of Mathematics; Sudáfrica Fil: Ballhausen, Hanne. Universitätsmedizin Berlin. BIH QUEST Center for Responsible Research. Berlin Institute of Health at Charité; Alemania Fil: Fil: Ballhausen, Hanne. Universitätsmedizin Berlin. Department of Paediatric Endocrinology and Diabetes, Charité; Alemania Fil: Bediako, Yaw . University of Ghana. West African Centre for Cell Biology of Infectious Pathogens; Ghana Fil: Belasen, Anat M. Cornell University. Society for Conservation Biology. Department of Ecology and Evolutionary Biology; Estados Unidos Fil: Carneiro, Clarissa F. D. Federal University of Rio de Janeiro. Institute of Medical Biochemistry Leopoldo de Meis; Brasil Fil: Chung Chen, Yen. New York University. Department of Biology; Estados Unidos Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Debat, Humberto Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Unidad de Fitopatología y Modelización Agrícola (UFyMA); Argentina Fil: Weissgerber, Tracey L. Universitätsmedizin Berlin. BIH QUEST Center for Responsible Research. Berlin Institute of Health at Charité; Alemania

Writing recommendation letters on behalf of students and other early-career researchers is an important mentoring task within academia. An effective recommendation letter describes key candidate qualities such as academic achievements, extracurricular activities, outstanding personality traits, participation in and dedication to a particular discipline, and the mentor’s confidence in the candidate’s abilities. In this Words of Advice, we provide guidance to researchers on composing constructive and supportive recommendation letters, including tips for structuring and providing specific and effective examples, while maintaining a balance in language and avoiding potential biases.

Contains fulltext : 248786.pdf (Publisher’s version ) (Open Access) 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.

Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells that act to bridge innate and adaptive immunity. DCs are critical in mounting effective immune responses to tissue damage, pathogens and cancer. Immature DCs continuously sample tissues and engulf antigens via endocytic pathways such as phagocytosis or macropinocytosis, which result in DC activation. Activated DCs undergo a maturation process by downregulating endocytosis and upregulating surface proteins controlling migration to lymphoid tissues where DC-mediated antigen presentation initiates adaptive immune responses. To traffic to lymphoid tissues, DCs must adapt their motility mechanisms to migrate within a wide variety of tissue types and cross barriers to enter lymphatics. All steps of DC migration involve cell–cell or cell–substrate interactions. This review discusses DC migration mechanisms in immunity and cancer with a focus on the role of cytoskeletal processes and cell surface proteins, including integrins, lectins and tetraspanins. Understanding the adapting molecular mechanisms controlling DC migration in immunity provides the basis for therapeutic interventions to dampen immune activation in autoimmunity, or to improve anti-tumour immune responses.

Summary Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilize amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by Rho GTPase signalling. Migrastatic drugs targeting actin polymerization and contractility are therefore a promising treatment option for metastatic melanoma. To predict amoeboid invasion and metastatic potential, biomarkers functionally linked to contractility pathways are needed. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts and is overexpressed in many cancers. We show that podoplanin enhances amoeboid invasion in melanoma. Podoplanin expression in murine melanoma drives rounded cell morphology, increasing motility, and invasion in vivo. Podoplanin expression is increased in a subset of dedifferentiated human melanoma, and in vitro is sufficient to upregulate melanoma-associated marker Pou3f2/Brn2. Together, our data define podoplanin as a functional biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target., Graphical abstract, Highlights • A dedifferentiated subset of human melanomas expresses high levels of podoplanin • Amoeboid invasion is promoted by expression of the membrane glycoprotein, podoplanin • Podoplanin induces phosphorylation of ERM and myosin to drive blebbing protrusions, Molecular biology; Cell biology; Transcriptomics

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes, an immunologically-relevant, adult mammalian tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive tissue expansion upon immune challenge. We find that following immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that CLEC-2/podoplanin signalling regulates the cell surface mechanics of fibroblasts, permitting cell elongation and interdigitation through expedited access to plasma membrane reservoirs. Increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation, restoring homeostatic cellular ratios and tissue structure through expansion.

In adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC–FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin., Summary: There is a multi-faceted response of lymph node fibroblasts to dendritic cells in an adaptive immune reaction, which requires the coordinated activity of partner proteins podoplanin, CD44 and CD9.

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in the conduct and communication of science. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve science. The event was focused on why ECRs are needed to improve science and the obstacles they face when trying to promote reform. Our discussions also highlighted the additional obstacles that ECRs in countries with limited research funding experience when working to improve the scientific system. We provide the lessons learned from successful ECR-led or ECR-focused initiatives and outline actions that individuals and organizations can take to further support ECRs who are working to improve research culture and practice.

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes, an immunologically-relevant, adult mammalian tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive tissue expansion upon immune challenge. We find that following immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that CLEC-2/podoplanin signalling regulates the cell surface mechanics of fibroblasts, permitting cell elongation and interdigitation through expedited access to plasma membrane reservoirs. Increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation, restoring homeostatic cellular ratios and tissue structure through expansion.

Mentorship is experience and/or knowledge-based guidance. Mentors support, sponsor and advocate for mentees. Having one or more mentors when you seek advice can significantly influence and improve your research endeavours, well-being and career development. Positive mentee-mentor relationships are vital for maintaining work-life balance and success in careers. Early-career researchers (ECRs), in particular, can benefit from mentorship to navigate challenges in academic and nonacademic life and careers. Yet, strategies for selecting mentors and maintaining interactions with them are often underdiscussed within research environments. In this Words of Advice, we provide recommendations for ECRs to seek and manage mentorship interactions. Our article draws from our experiences as ECRs and published work, to provide suggestions for mentees to proactively promote beneficial mentorship interactions. The recommended practices highlight the importance of identifying mentorship needs, planning and selecting multiple and diverse mentors, setting goals, and maintaining constructive, and mutually beneficial working relationships with mentors.

Securing research funding is a challenge faced by most scientists in academic institutions worldwide. Funding success rates for all career stages are low, but the burden falls most heavily on early career researchers (ECRs). These are young investigators in training and new principal investigators who have a shorter track record. ECRs are dependent on funding to establish their academic careers. The low number of career development awards and the lack of sustained research funding result in the loss of ECR talent in academia. Several steps in the current funding process, from grant conditions to review, play significant roles in the distribution of funds. Furthermore, there is an imbalance where certain research disciplines and labs of influential researchers receive more funding. As a group of ECRs with global representation, we examined funding practices, barriers, and facilitators to the current funding systems. We also identified alternatives to the most common funding distribution practices, such as diversifying risk or awarding grants on a partly random basis. Here, we detail recommendations for funding agencies and grant reviewers to improve ECR funding prospects worldwide and promote a fairer and more inclusive funding landscape for ECRs. Instituto de Virología Fil: de Winde, Charlotte M. University College London. MRC Laboratory for Molecular Cell Biology; Reino Unido Fil: de Winde, Charlotte M. Amsterdam University Medical Center. Department of Molecular Cell Biology & Immunology; Países Bajos Fil: Sarabipour, Sarvenaz. Johns Hopkins University. Department of Biomedical Engineering. Institute for Computational Medicine; Estados Unidos Fil: Carignano, Hugo Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina Fil: Davla, Sejal. City University of New York. Advanced Science Research Center; Estados Unidos Fil: Eccles, David. Malaghan Institute of Medical Research; Nueva Zelanda Fil: Hainer, Sarah J. University of Pittsburgh. Department of Biological Sciences; Estados Unidos Fil: Haidar, Mansour. Hasselt University; Bélgica Fil: Ilangovan, Vinodh. Aarhus University; Dinamarca Fil: Jadavji, Nafisa M. Midwestern University. Department of Biomedical Sciences; Estados Unidos Fil: Jadavji, Nafisa M. Carleton University. Department of Neuroscience; Canadá Fil: Kritsiligkou, Paraskevi. German Cancer Research Center; Alemania Fil: Lee, Tai-Ying. University of Oxford; Reino Unido Fil: Ólafsdóttir, H. Freyja. Radboud University. Donders Institute for Brain, Cognition and Behaviour; Países Bajos

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilise amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by the RhoA signalling pathway. Migrastatic drugs targeting actin polymerization and contractility to inhibit invasion and metastasis are therefore a promising treatment option. To predict amoeboid invasion and metastatic potential, there is a need for biomarkers functionally linked to contractility pathways. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts, and is overexpressed in several cancer types. Here, we show that podoplanin enhances amoeboid invasion in melanoma. Expression of podoplanin in murine melanoma models drives rounded cell morphology, increasing motility and invasionin vivo. Podoplanin expression is upregulated in a subset of dedifferentiated human melanoma, andin vitrois sufficient to suppress melanogenesis and upregulate melanoma-associated markersMitfandPou3f2. Together, our data indicates that podoplanin is both a potential biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

The eLife Early-Career Advisory Group (ECAG), an international group of early-career researchers committed to improving research culture, calls for radical changes at eLife and other journals to address racism in the scientific community and to make science more diverse and inclusive. Fil: Mehta, Devang. University of Alberta; Canadá Fil: Bediako, Yaw. University Of Ghana; Ghana Fil: De Winde, Charlotte M.. Colegio Universitario de Londres; Reino Unido Fil: Ebrahimi, Hedyeh. No especifíca; Fil: Fernández, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina Fil: Ilangovan, Vinodh. University Aarhus; Dinamarca Fil: Paz Quezada, Carolina. Universidad Bernardo O'higgins; Chile Fil: Riley, Julia L.. Dalhousie University Halifax; Canadá Fil: Saladi, Shyam M.. California Institute of Technology; Estados Unidos Fil: Tay, Andy. No especifíca; Fil: Weissgerber, Tracey. No especifíca

The need to protect public health during the current COVID-19 pandemic has necessitated conference cancellations on an unprecedented scale. As the scientific community adapts to new working conditions, it is important to recognize that some of our actions may disproportionately affect early-career researchers and scientists from countries with limited research funding. We encourage all conference organizers, funders and institutions who are able to do so to consider how they can mitigate the unintended consequences of conference and travel cancellations and we provide seven recommendations for how this could be achieved. The proposed solutions may also offer long-term benefits for those who normally cannot attend conferences, and thus lead to a more equitable future for generations of researchers.

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilise amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by the RhoA signalling pathway. Migrastatic drugs targeting actin polymerization and contractility to inhibit invasion and metastasis are therefore a promising treatment option. To predict amoeboid invasion and metastatic potential, there is a need for biomarkers functionally linked to contractility pathways. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts, and is overexpressed in several cancer types. Here, we show that podoplanin enhances amoeboid invasion in melanoma. Expression of podoplanin in murine melanoma models drives rounded cell morphology, increasing motility and invasion in vivo. Podoplanin expression is upregulated in a subset of dedifferentiated human melanoma, and in vitro is sufficient to suppress melanogenesis and upregulate melanoma-associated markers Mitf and Pou3f2 . Together, our data indicates that podoplanin is both a potential biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

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.

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

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.

Cell migration is central to evoke 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) expressed by DCs, with podoplanin expressed by lymph node stromal cells (LNSCs). However, the underlying molecular mechanisms by which CLEC-2 influences DC migration remain elusive. Here, we show that CLEC-2-dependent DC migration is tightly controlled by tetraspanin CD37, a membrane-organizing protein. Our findings demonstrate a specific molecular interaction between CLEC-2 and CD37. Myeloid cells lacking CD37 (Cd37-/-) expressed less CLEC-2 on their surface compared to wild-type cells, indicating that CD37 is required to stabilize membrane expression of CLEC-2. In addition, CLEC-2-expressing DCs lacking CD37 showed impaired adhesion, migration velocity and displacement on LNSCs. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs (CD11cΔCLEC-2). Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights underlying CLEC-2-dependent DC migration.

Contains fulltext : 177846.pdf (Author’s version preprint ) (Open Access) 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.

Contains fulltext : 171804.pdf (Publisher’s version ) (Closed access) 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 approximately 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-kappaBhigh, 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.

Contains fulltext : 165795.pdf (Publisher’s version ) (Closed access) 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.

Multispectral imaging is a novel microscopy technique that combines imaging with spectroscopy to obtain both quantitative expression data and tissue distribution of different cellular markers. Tetraspanins CD37 and CD53 are four-transmembrane proteins involved in cellular and humoral immune responses. However, comprehensive immunohistochemical analyses of CD37 and CD53 in human lymphoid organs have not been performed so far. We investigated CD37 and CD53 protein expression on primary human immune cell subsets in blood and in primary and secondary lymphoid organs. Both tetraspanins were prominently expressed on antigen-presenting cells, with highest expression of CD37 on B lymphocytes. Analysis of subcellular distribution showed presence of both tetraspanins on the plasma membrane and on endosomes. In addition, CD53 was also present on lysosomes. Quantitative analysis of expression and localization of CD37 and CD53 on lymphocytes within lymphoid tissues by multispectral imaging revealed high expression of both tetraspanins on CD20+ cells in B cell follicles in human spleen and appendix. CD3+ T cells within splenic T cell zones expressed lower levels of CD37 and CD53 compared to T cells in the red pulp of human spleen. B cells in human bone marrow highly expressed CD37, whereas the expression of CD53 was low. In conclusion, we demonstrate differential expression of CD37 and CD53 on primary human immune cells, their subcellular localization and their quantitative distribution in human lymphoid organs. This study provides a solid basis for better insight into the function of tetraspanins in the human immune response. Electronic supplementary material The online version of this article (doi:10.1007/s00418-015-1326-2) contains supplementary material, which is available to authorized users.

In this chapter, we describe the technique of electroporation as an efficient method to load primary leukemic cells with the double-stranded RNA (dsRNA) analogue, polyriboinosinic polyribocytidylic acid (poly(I:C)), and detail on the delicate freezing and thawing procedure of primary leukemic cells.Electroporation is a non-viral gene transfer method by which short-term pores in the membrane of cells are generated by an electrical pulse, allowing molecules to enter the cell. RNA electroporation, a technique developed in our laboratory, is a widely used and versatile transfection method for efficient introduction of both coding RNA (messenger RNA) and non-coding RNA, e.g., dsRNA and small interfering (siRNA), into mammalian cells. Accurate cell processing and storage of patient material is essential for optimal recovery and quality of the cell product for downstream applications.

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures (“physical entities”) that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.