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The behavior of cells and tissues in vivo is determined by the integration of multiple biochemical and mechanical signals. Of the mechanical signals, stretch has been studied for decades and shown to contribute to pathophysiological processes. Several different stretch devices have been developed for in vitro investigations of cell stretch. In this work, we describe a new 3D-printed uniaxial stretching device for studying cell response to rapid deformation. The device is a bistable compliant mechanism holding two equilibrium states—an unstretched and stretched configuration—without the need of an external actuator. Furthermore, it allows multiple simultaneous measurements of different levels of stretch on a single substrate and is compatible with standard immunofluorescence imaging of fixed cells as well as live-cell imaging. To demonstrate the effectiveness of the device to stretch cells, a test case using aligned myotubes is presented. Leveraging material area changes associated with deformation of the substrate, changes in nuclei density provided evidence of affine deformation between cells and substrate. Furthermore, intranuclear deformations were also assessed and shown to deform non-affinely. As a proof-of-principle of the use of the device for mechanobiological studies, we uniaxially stretched aligned healthy and dystrophic myotubes that displayed different passive mechanical responses, consistent with previous literature in the field. We also identified a new feature in the mechanoresponse of dystrophic myotubes, which is of potential interest for identifying the diseased cells based on a quick mechanical readout. While some applications of the device for elucidating passive mechanical responses are demonstrated, the simplicity of the device allows it to be potentially used for other modes of deformation with little modifications., Biomaterials Advances, 141, ISSN:2772-9508

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC–MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC–MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC–MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of malignancies with an aggressive clinical course. Molecular and immunophenotypic analysis have linked PTCL subgroups to derivation from differentiated T cells, including T follicular helper (Tfh), Th1, and Th2 cells. Effective therapies for these PTCLs are undefined and tumor models are limited. We developed a novel mouse model of PTCL and applied it to evaluate responses to therapy. An in vivo transplantable cell line was isolated by passaging a splenic lymphoma arising from a mouse with B cell-specific Blimp1-deletion (Cγ1-Cre Blimp1fl/fl) into C57bl/6J mice. Lymphomas were profiled by flow cytometry, immunohistochemistry, RNA, and exome sequencing. Mice intravenously inoculated with lymphoma cells developed disseminated disease affecting primarily lymphoid tissues and the liver. Model latency to welfare endpoint was 3-4 weeks. Transplantable cells expressed multiple T-cell lineage markers, were CD8 negative and CD4 positive, but gradually downregulated CD4 with passaging. Lymphoma cells (“mPTCL”) also expressed a single TCRvβ chain, suggesting origin from a T helper cell clone. Immunophenotypically, mPTCL stably expressed Tfh markers ICOS, PD-1, CD40L, and low BCL6, and thus was potentially classifiable as a surrogate PTCL of Tfh phenotype. However, the transcriptome of purified mPTCL or spleens indicated a resemblance to Th2 cells and corresponding PTCL-GATA3 subtype (a PTCL-not otherwise specified). In accord with Tfh and Th2 helper function, B cells in the tumor microenvironment had an activated phenotype but were nonexpansive. Investigation into oncogenic pathways in mPTCL revealed a predicted deleterious mutation in Ctnnb1 (β-catenin) and enrichment for Myc-activated, cell cycle regulation, and DNA damage response genes. In vivo, mPTCL progression was significantly and potently delayed by monotherapy inhibition of ataxia telangiectasia and Rad3-related (ATR), a kinase central to resolving replication stress. Taken together, we established a novel model of PTCL with overlapping Tfh and Th2 features. Our findings in this model suggest the use of a DNA damage response inhibitor as a potential treatment strategy for aggressive PTCL-GATA3 lymphomas. Citation Format: Elizabeth Kuczynski, Giulia Morlino, Alison Peter, Dinis Calado, Charles Sinclair, Larissa Carnevalli. Molecular characterization of a mouse model of peripheral T-cell lymphoma with Tfh and Th2 features [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-20.

Diminished overall survival rate of non-Hodgkin lymphoma (NHL) patients treated with a combination regimen of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) has been recently linked to recurrent somatic mutations activating FOXO1. Despite of the clinical relevance of this finding, the molecular mechanism driving resistance to R-CHOP therapy remains largely unknown. Herein, we investigated the potential role of FOXO1 in the therapeutic efficacy of rituximab, the only targeted therapy included in the R-CHOP regimen. We found CD20 transcription is negatively regulated by FOXO1 in NHL cell lines and in human lymphoma specimens carrying activating mutations of FOXO1. Furthermore, both the expression of exogenous mutants of FOXO1 and the inhibition of AKT led to FOXO1 activation in lymphoma cells, increased binding to MS4A1 promoter and diminished CD20 expression levels. In contrast, a disruption of FOXO1 with CRISPR/Cas9 genome-editing (sgFOXO1) resulted in CD20 upregulation, improved the cytotoxicity induced by rituximab and the survival of mice with sgFOXO1 tumors. Accordingly, pharmacological inhibition of FOXO1 activity in primary samples upregulated surface CD20 levels. Importantly, FOXO1 was required for the downregulation of CD20 levels by the clinically tested inhibitors of BTK, SYK, PI3K and AKT. Taken together, these results indicate for the first time that the AKT-unresponsive mutants of FOXO1 are important determinant of cell response to rituximab-induced cytotoxicity, and suggest that the genetic status of FOXO1 together with its transcriptional activity need further attention while designing anti-CD20 antibodies based regimens for the therapy of pre-selected lymphomas.

© 2015 Elsevier B.V. The tetraspanin CD9 has been shown to interact with different members of the β1 and β3 subfamilies of integrins, regulating through these interactions cell adhesion, migration and signaling. Based on confocal microscopy co-localization and on co-immunoprecipitation results, we report here that CD9 associates with the β2 integrin LFA-1 in different types of leukocytes including T, B and monocytic cells. This association is resistant to stringent solubilization conditions which, together with data from chemical crosslinking, in situ Proximity Ligation Assays and pull-down experiments, suggest a primary/direct type of interaction mediated by the Large Extracellular Loop of the tetraspanin. CD9 exerts inhibitory effects on the adhesive function of LFA-1 and on LFA-1-dependent leukocyte cytotoxic activity. The mechanism responsible for this negative regulation exerted by CD9 on LFA-1 adhesion does not involve changes in the affinity state of this integrin but seems to be related to alterations in its state of aggregation., SAF2012-34561 from the Spanish «Ministerio de Economía y Competitividad-MINECO

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.

During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

Rituximab, a therapeutic anti-CD20 monoclonal antibody, is effective against B-cell malignancies, when combined with chemotherapy, such as cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP therapy). However, in case of 30-40% patients the resistance to rituximab-based therapy appears to be a serious concern. One of the major mechanisms of resistance relies on the reduced level of CD20 antigen on the surface of tumor B-cells. While exploring the molecular mechanisms affecting the level of CD20, we found that transcription factor FOXO1 is a negative regulator of MS4A1 expression, the gene encoding CD20 antigen. Using the CRISPR/Cas9 genome-editing technology, we disrupted the loci of FOXO genes in lymphoma cell lines and discovered that ablation of FOXO1 (but not FOXO3) was sufficient for upregulation of the surface level of CD20 by 3-fold. Consistently, the complement-dependent cytotoxicity (CDC), induced by rituximab, was significantly improved in cell clones with abrogated expression of FOXO1, but not FOXO3. Importantly, the treatment with pharmacological inhibitor of FOXO1 activity, AS1842856, resulted in increased levels of CD20 on the surface of both lymphoma cell lines and patients-derived chronic lymphocytic leukemia (CLL) cells cultured ex vivo. In order to verify our findings in the animal model, we inoculated SCID mice intravenously with Raji cells, what resulted in the development of lymphoma-like tumors. We demonstrated that mice treated systemically with rituximab, administered at a dose of 10 mg/kg, survived longer when inoculated with sgFOXO1-transduced Raji cells as compared with mice inoculated with control Raji cells (median survival 49 days versus 29 days, respectively). These results confirmed that FOXO1 ablation in lymphoma cells resulted in higher efficacy of rituximab treatment in vivo. Taken together, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity and indicate that FOXO1 inhibitors could be exploited for the therapeutic purposes, in combination with anti-CD20 monoclonal antibodies. Novel FOXO1 inhibitors with improved potency and selectivity are however urgently needed for further exploration of our discoveries in the near future. Acknowledgments: NCN, Poland (grant no: 2013/11/B/NZ5/03240) and H2020 (STREAM project, CSA action, grant no: 692180). Citation Format: Beata Pyrzynska, Abdessamad Zerrouqi, Michal Dwojak, Giulia Morlino, Piotr Zapala, Nina Miazek, Agnieszka Zagozdzon, Kamil Bojarczuk, Malgorzata Bobrowicz, Marta Siernicka, Marcin M. Machnicki, Joanna Barankiewicz, Ewa Lech-Maranda, Przemyslaw Juszczynski, Dinis Calado, Jakub Golab, Magdalena Winiarska. FOXO1 is transcriptional regulator of malignant B-cell surface antigen CD20, the target for therapeutic monoclonal antibodies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B17.

Lack of remission or early relapse remains a major clinical concern in diffuse large B-cell lymphoma (DLBCL), with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent targets for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild-type and mutated) on the expression of the rituximab-target, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available Chip-Seq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK-2206 and GDC-0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab-mediated complement-dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild-type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1-AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA-binding defective mutant of FOXO1-H215R, suggesting a FOXO1-mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA, and the analysis of ChIP-Seq data using anti-FOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity effectively suppress CD20 expression in comparison to wild-type FOXO1, through a binding to CD20 promoter. In addition, the disruption of FOXO1 with CRISPR/Cas9 genome editing resulted in CD20 upregulation and improved the cytotoxicity induced by rituximab and survival of mice with sgFOXO1 tumors. Consistently, in primary samples pharmacologic inhibition of FOXO1 activity upregulated surface CD20 levels. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity needs further attention while designing rituximab-based regimens in the therapy of selected lymphomas. Supported by National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwoyak, giulia Morlino, Piotr Zapala, Nina Miazek, Jakub Golab, Magdalena Winiarska. FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B51.

The adaptive immune response depends on the interaction of T cells and antigen-presenting cells at the immune synapse. Formation of the immune synapse and the subsequent T-cell activation are highly dependent on the actin cytoskeleton. In this work, we describe that T cells express drebrin, a neuronal actin-binding protein. Drebrin colocalizes with the chemokine receptor CXCR4 and F-actin at the peripheral supramolecular activation cluster in the immune synapse. Drebrin interacts with the cytoplasmic tail of CXCR4 and both proteins redistribute to the immune synapse with similar kinetics. Drebrin knockdown in T cells impairs the redistribution of CXCR4 and inhibits actin polymerization at the immune synapse as well as IL-2 production. Our data indicate that drebrin exerts an unexpected and relevant functional role in T cells during the generation of the immune response.

In many cell types, nuclear A-type lamins regulate multiple cellular functions, including higher-order genome organization, DNA replication and repair, gene transcription, and signal transduction; however, their role in specialized immune cells remains largely unexplored. We showed that the abundance of A-type lamins was almost negligible in resting naïve T lymphocytes, but was increased upon activation of the T cell receptor (TCR). The increase in lamin-A was an early event that accelerated formation of the immunological synapse between T cells and antigen-presenting cells. Polymerization of F-actin in T cells is a critical step for immunological synapse formation, and lamin-A interacted with the linker of nucleoskeleton and cytoskeleton (LINC) complex to promote F-actin polymerization. We also showed that lamin-A expression accelerated TCR clustering and led to enhanced downstream signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, as well as increased target gene expression. Pharmacological inhibition of the ERK pathway reduced lamin-A-dependent T cell activation. Moreover, mice lacking lamin-A in immune cells exhibited impaired T cell responses in vivo. These findings underscore the importance of A-type lamins for TCR activation and identify lamin-A as a previously unappreciated regulator of the immune response. Spanish Ministerio de Economia y Competitividad (MINECO) [SAF2011-25834, SAF2010-16044]; Comunidad de Madrid [INDISNET-S2011/BMD-2332]; Instituto de Salud Carlos III (ISCIII) [RD12/0042/0028, RD12/0042/0056, CP11/00145]; Progeria Research Foundation [PRF 2012-42]; European Commission [ERC-2011AdG 294340-GENTRIS, 317916-Liphos]; Sara Borrell ISCIII program [CP11/00145]; Miguel Servet ISCIII program [CP11/00145]; Fundacion Mario Losantos del Campo; Fundacion Ferrer para la Investigacion; Obra Social Cajastur; Spanish MINECO; Pro-CNIC Foundation Sí

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution., This study was supported by SAF2011-25834 from the Spanish Ministry of Science and Innovation, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII), and ERC-2011-AdG 294340-GENTRIS. J.M.G.-G. received salary support from the Miguel Servet (CP11/00145) ISCIII program. R.V.-B. was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Economy and Competiveness (JCI-2011-09663).

Neo-angiogenesis is a complex phenomenon modulated by the concerted action of several molecular factors. We have generated a congenic line of knockout mice carrying null mutations of both placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS), two genes that play a pivotal role in the regulation of pathological angiogenesis. In the present study, we describe the phenotype of this new experimental animal model after surgically induced hind-limb ischemia. Plgf–/–, eNos–/–, Plgf–/– eNos–/–, and wild-type C57BL/6J mice were studied. Plgf–/– eNos–/– mice showed the most severe phenotype: self-amputation, and death occurred in up to 47% of the animals studied; in ischemic legs, capillary density was severely reduced; macrophage infiltration and oxidative stress increased as compared to the other groups of animals. These changes were associated with an up-regulation of both inducible NOS (iNOS) expression and vascular endothelial growth factor (VEGF) protein levels in ischemic limbs, and to an increased extent of protein nitration. Our results demonstrate that the deletion of these two genes, Plgf, which acts in synergism with VEGF, and eNos, a downstream mediator of VEGF, determines a significant change in the vascular response to an ischemic stimulus and that oxidative stress within the ischemic tissue represents a crucial factor to maintain tissue homeostasis.—Gigante, B., Morlino, G., Gentile, M. T., Persico, M. G., De Falco, S. Plgf–/–eNos–/– mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia.