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Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L²⁶⁵P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L²⁶⁵P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas. CA extern

The ability of a cell to migrate, adhere and change its morphology is determinant to develop its functions; these capacities reach their maximum relevance in immune cells. For an efficient immune response, immune cells must localize in the right place at the right time; that implies crossing tissue barriers and migrating in the interstitial space of the tissues at high velocities. The dependency on trafficking abilities is even higher for B cells, one of the arms of the adaptive immune system, considering that they must encounter specific antigen for their clonal receptor in the enormous tissue volume of the secondary lymphoid organs (spleen, lymph nodes, Peyer patches). The regulated interplay between cell motililty and cell adhesion allows B cells to reach distinct lymphoid tissues and, within them, to explore the stromal cell networks where antigen might be exposed. In this meeting-invited review, I summarize the current knowledge on the molecular cues and mechanisms that shapes B cell dynamics at the initial phase of the humoral immune response, including homeostatic chemoatractants and innate/inflamatory stimuli. I also revised the B cell behaviour alterations caused by BCR recognition of antigen and the molecular mechanisms involved. The publication of this review was supported by a grant (RTI2018‐101345‐B‐I00) from the MCIU/AEI/FEDER EU to Y.R.C.

41 p.-7 fig., ICAP-1 regulates β1 integrin activation and cell adhesion. Here we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single positive (SP) CD8+ cell generation, thus unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T and B cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B cell numbers., This work was supported by grants SAF2017-85146-R and PID2020-116291RB-I00 from the Ministerio de Ciencia e Innovación (MICINN) to J.T, PID2019-105623RB-I00 from MICINN to M.L.T,BFU2013-48828-P from MICINN to Y.R.C., ERC Synergy Grant (2018) to R.F., RTI2018-095497-B-I00 from MICINN to A.H, and RTI2018-093938-B-I100 from MICINN and (RD16/0011/0002, TERCEL) from Instituto de Salud Carlos III to AGZ.

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-04-2019, Esta tesis tiene embargado el acceso al texto completo hasta el 11-10-2020, Cuando la célula linfoide reconoce antígeno a través de su receptor específico, las Diacilglicerol Kinasas (DGK) limitan la señalización a través del receptor mediante el consumo de su sustrato, el diacilglicerol (DAG). La relevancia de su producto, el ácido fosfatídico (AF), en los procesos de activación y respuesta a antígeno de los linfocitos es aún desconocida. En esta tesis doctoral, mostramos que el AF derivado de la isoforma DGKz regula moléculas importantes para la estructura de la sinapsis inmune de las células B, particularmente la adhesión mediada por la integrina LFA-1 y la polimerización de F-actina. La medición de la fuerza mecánica que ejerce la célula B durante la sinapsis utilizando dos tecnologías diferentes reveló que la deficiencia en DGKz resulta en una menor generación de fuerzas en la sinapsis con respecto a las células B normales. También encontramos que DGKz controla la activación de la proteína motora no-muscular miosina-II y la translocación del MTOC a la sinapsis, ambos eventos necesarios para la adquisición y procesamiento de antígeno. Todos estos defectos correlacionaron con una disminución en la habilidad de las células B para presentar el antígeno a las células T en ensayos in vitro. En experimentos in vivo de inmunización, la respuesta de centro germinal de las células B deficientes para DGKz también estuvo afectada, sugiriendo que estas células presentan una desventaja durante la competición por la ayuda de las células T. En conjunto, nuestros datos indican que el AF derivado de DGKz modula la respuesta de las células B a través del control de la reorganización del citoesqueleto de actina y la adhesión mediada por LFA-1/ICAM-1, que participan en la generación de fuerza, y de eventos relacionados con la polaridad celular en la sinapsis inmune. Para que la célula B pueda progresar y realizar correctamente su función es necesario que todos estos eventos moleculares asociados a la formación de la sinapsis inmune ocurran adecuadamente; ello asegura que la adquisición, internalización y presentación de antígeno se lleve a cabo de forma eficiente y la respuesta de la célula B avance con éxito. Por lo tanto, es necesario el balance entre el DAG y el AF por DGKz para que las células B puedan llevar a cabo su función., When the lymphoid cell recognizes the antigen through its specific receptor, the Diacylglycerol Kinases (DGK) limit receptor signaling through the consumption of their substrate, the diacylglycerol (DAG). The relevance of its product, phosphatidic acid (PA), in lymphocyte activation and response against antigen is still unknown. In this thesis, we show that PA derived from the DGKz isoform regulates important molecules for the B cell immune synapse structure, particularly the adhesion mediated by LFA-1 integrin and the F-actin polymerization. Measurements of the mechanical force exerted by B cells during the synapse using two different technologies revealed that DGKz deficiency results in lower force generation compared to normal B cells. Moreover, we found that DGKz controls non-muscle motor protein myosin-II activation and the MTOC translocation to the synapse, both events required for antigen acquisition and processing. All these defects correlated with a decreased B cell ability for antigen presentation to T cells in in vitro assays. In in vivo immunization experiments, the germinal center response of DGKz-deficient B cells was also affected, suggesting that these cells have a disadvantage during the competition for T cell help. Overall, our data indicate that DGKzderived PA modulates the B cell response through the control of actin cytoskeleton reorganization and LFA-1/ICAM-1 adhesion, which are involved in force generation, and cell polarity events at the immune synapse. For the progress and appropriate response of B cells, it is necessary that all these molecular events associated with synapse formation occur properly; that ensures efficient antigen acquisition, internalization and presentation and the success of B cell response. Therefore, the balance between DAG and PA by DGKz is necessary for the B cell function.

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88 . We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88 L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88 L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma, accounting for the 40% of diagnosed cases worldwide. The heterogeneous nature of this disease is one of its main features and is directly related to the prognosis shown by the patients. There are many indicators linked to the development of the pathology but the most important one is the subtype of DLBCL present in the patient. The most extended classification distinguishes between germinal centre B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL. Both subtypes are defined by the differences detected in their genetic profile, that delineates not only the subtype but also the major mutations associated with each of them. ABC DLBCL associates with a worse prognosis, so specific compounds have been added to the treatment for fighting ABC DLBCL. Ibrutinib is one of those compounds, inhibitor of the kinase activity of Btk. ABC DLBCL neoplastic cells depend for their survival on the activation of the NF- kB pathway via Btk. Ibrutinib promotes neoplastic cell death by inhibiting Btk activity, and therefore NF- kB route. It has been shown to be effective in the treatment of ABC DLBCL, however, its effectiveness is reduced in the presence of the L265P mutation in the adaptor protein MyD88. This mutation allows MyD88 constitutive activation, promoting the assembly of the Btk signalosome and thus modulating Btk activity.The expression of the chemokine receptor CXCR4 is also related to a worse prognosis of the DLBCL. High CXCR4 expression levels at the cell surface are associated with a more aggressive nature of the tumour due to its involvement in cell migration and adhesion events. In addition, there is controversy about the involvement of Btk in the CXCR4-mediated functions.Taking into account the relevance of Btk and MyD88 in the development of ABC DLBCL, and that of CXCR4 in DLBCL, this work will evaluate the relationship between the three proteins in the molecular environment of the pathology and their functional effects on the development of the disease.