Differential Engraftment of Parental A20 PD-L1 WT and PD-L1 KO Leukemia Cells in Semiallogeneic Recipients in the Context of PD-L1/PD-1 Interaction and NK Cell-Mediated Hybrid Resistance

[EN] The contribution of natural killer (NK) cells to tumor rejection in the context of programmed death-ligand 1/programmed death 1 (PD-L1/PD-1) blockade is a matter of intense debate. To elucidate the role of PD-L1 expression on tumor cells and the functional consequences of engaging PD-1 receptor on cytotoxic cells, PD-L1 expression was genetically inactivated and WT or PD-L1-deficient parental tumor cells were adoptively transferred intravenously into F1 recipients. The engraftment of PD-L1-deficient A20 tumor cells in the spleen and liver of F1 recipients was impaired compared with A20 PD-L1 WT tumor counterparts. To elucidate the mechanism responsible for this differential tumor engraftment and determine the relevance of the role of the PD-L1/PD-1 pathway in the interplay of tumor cells/NK cells, a short-term competitive tumor implantation assay in the peritoneal cavity of semiallogeneic F1 recipients was designed. The results presented herein showed that NK cells killed target tumor cells with similar efficiency regardless of PD-L1 expression, whereas PD-L1 expression on A20 tumor cells conferred significant tumor protection against rejection by CD8 T cells confirming the role of the co-inhibitory receptor PD-1 in the modulation of their cytotoxic activity. In summary, PD-L1 expression on A20 leukemia tumor cells modulates CD8 T-cell-mediated responses to tumor-specific antigens but does not contribute to inhibit NK cell-mediated hybrid resistance, which correlates with the inability to detect PD-1 expression on NK cells neither under steady-state conditions nor under inflammatory conditions.S

Manual de disección del ratón para la localización y el aislamiento de los órganos linfoides

[ES] En los órganos linfoides primarios o centrales tiene lugar el origen y diferenciación de diversas células competentes del sistema inmunitario. Forman parte de este grupo la médula ósea y el timo. El timo es un órgano linfoide primario que ocupa un lugar fundamental en la biología de las células T, que está localizado cranealmente al corazón. Aparte de ser la fuente primordial de células T, es el lugar en el que las células T se diferencian y maduran para generar un amplio repertorio de células T, que posteriormente serán exportadas a la periferia como células T naïve maduras capaces de reconocer péptidos en el contexto del complejo mayor de histocompatibilidad (MHC), tras sufrir un proceso de selección positiva (restricción MHC) y negativa (tolerancia a lo propio) que tiene lugar en la cortical y medular, respectivamente. La médula ósea, como órgano linfoide primario, es el sitio donde se produce la he-matopoyesis y en el que se originan y maduran las células B en los mamíferos. Las células B proliferan y se diferencian dentro de la médula ósea y, de manera similar a lo que ocurre en el timo, mediante un proceso de selección se eliminarán aquellas células B que porten receptores de alta afinidad para antígenos propios. En los órganos linfoides secundarios o periféricos (bazo y nódulos linfáticos) es donde se produce el encuentro entre el antígeno y las células del sistema inmunita-rio. Existen varios tipos de organización de tejido linfoide que se localiza a lo largo de los vasos del sistema linfático. En algunos casos, este tejido se dispone de manera difusa en el pulmón y en la lámina propia, mientras que en otros lugares el tejido linfoide está organizado en forma de folículos linfoides, que consisten en agregados tanto de células linfoides, como no linfoides rodeados por una red de capilares de drenaje linfático. El bazo es un órgano linfoide secundario localizado en el hipocondrio izquierdo y ejerce un papel primordial en la respuesta inmunitaria frente a microorganismos procedentes del torrente sanguíneo. Los nódulos linfáticos están distribuidos por todo el organismo y están conectados entre sí a través del sistema linfático y su función se centra en servir de sitio de encuentro entre los antígenos que son transportados por la linfa o capturados por las células fagocíticas en los tejidos para ser presentados a las células efectoras del sistema inmunitario. Tejidos linfoides terciarios asociados a las mucosas. A pesar de que el bazo y los nódulos linfáticos son los órganos linfoides secundarios más organizados, existe otro tejido linfoide, menos organizados, que se denomina tejido linfoide asociado a mucosas (MALT). Las placas de Peyer, por ejemplo, son acúmulos linfoides macroscópicos que están presentes en la submucosa del intestino delgado murino y humano. Estas estructuras constituyen los sitios donde se produce la respuesta inmunitaria frente a antígenos de la dieta, bacterias comensales o microorganismos patógenos y, a diferencia, de otros órganos linfoides periféricos, como son los nódulos linfáticos mesentéricos, que recogen el drenaje linfático de las vellosidades intestinales y de las placas de PeyerLa identificación y obtención de los órganos linfoides primarios (timo y médula ósea), secundarios (bazo y nódulos linfoides) y terciarios (placas de Peyer) en el ratón.Contenido docenteActiv

Critical role of PD-L1 expression on non-tumor cells rather than on tumor cells for efective anti-PD-L1 immunotherapy in a transplantable mouse hematopoietic tumor model

[EN] The expression of PD-L1 on tumor cells or within the tumor microenvironment has been associated with good prognosis and sustained clinical responses in immunotherapeutic regimens based on PD-L1/PD-1/CD80 immune checkpoint blockade. To look into the current controversy in cancer immunotherapy of the relative importance of PD-L1 expression on tumor cells versus non-tumor cells of the tumor microenvironment, a hematological mouse tumor model was chosen. By combining a genetic CRISPR/Cas9 and immunotherapeutic approach and using a syngeneic hematopoietic transplantable tumor model (E.G7-cOVA tumor cells), we demonstrated that dual blockade of PD-L1 interaction with PD-1 and CD80 enhanced anti-tumor immune responses that either delayed tumor growth or led to its complete eradication. PD-L1 expression on non-tumor cells of the tumor microenvironment was required for the promotion of tumor immune escape and its blockade elicited potent anti-tumor responses to PD-L1 WT and to PD-L1-defcient tumor cells. PD-L1+ tumors implanted in PDL1-defcient mice exhibited delayed tumor growth independently of PD-L1 blockade. These fndings emphasize that PD-L1 expression on non-tumor cells plays a major role in this tumor model. These observations should turn our attention to the tumor microenvironment in hematological malignancies because of its unappreciated contribution to create a conditioned niche for the tumor to grow and evade the anti-tumor immune response.SIThis work has been supported by Grant FIS PI# 1300029 (Fondo de Investigaciones Sanitarias, Ministry of Health, Spanish Government, and co-funded by European Union ERDF/ESF, “Investing in your future”), LE093U13 and Unit of Excellence Research UIC #012 (Department of Education of the Regional Government, Junta de Castilla y Leon) and Gerencia Regional de Salud (BIO/01/15) to JIRB. It was also funded by Miguel Servet National Grant (Health National Organization Research) CP12/03063, CPII17/00002 and FIS PI16/00002 (Instituto de Salud Carlos III and co-funded by European Union ERDF/ESF, “Investing in your future”), and Gerencia Regional de Salud GRS963/A/2014, GRS1142/A/2015 and GRS 1505/A/2017 to M.L.R.G. This work has been partially funded by the National Network CIBER-ONC (oncology research) CB16/12/00480

PD-1/PD-L1, PD-1/PD-L2, and other co-inhibitory signaling pathways in transplantation

[EN] Transplantation of cells, tissues and vascularized solid organs is a successful therapeutic intervention for many end-stage chronic diseases. The combination of co-stimulatory blockade with the delivery of negative signals to T cells through co-inhibitory receptors would provide a robust approach to modulating T-cell receptor signaling and improving alloantigen-specific control of transplant rejection. This approach based on fundamental knowledge of APC/T-cell interactions may complement conventional therapies in the near future to reinforce long-term allograft survival, and permit minimal immunosuppression. The focus of this review was primarily on two major co-inhibitory signaling pathways, namely PD-1/PD-L1/PD-L2 and BTLA/CD160/HVEM/LIGHT that have been thoroughly characterized in murine models of transplantation using genetically modified mice, specific monoclonal antibodies and fusion proteinsSIThis work has been supported by grants FIS 01-3026 and FIS PI-050021 of Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Spanish Government, Spain) to JIR

The role of TNFR2 and DR3 in the in vivo expansion of tregs in T cell depleting transplantation regimens

[EN] Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNF

Molecular characterization of Haemophilus parasuis ferric hydroxamate uptake (fhu) genes and constitutive expression of the FhuA receptor

P. 49-59Bacteria have evolved a set of highly specialized proteins to capture iron in irondepleted environments. The acquisition and uptake of iron present in the extracellular milieu of eukaryotic organisms is indispensable for the growth and survival of microbial pathogens in the course of infection. Haemophilus parasuis is the causative agent of Glässer disease, which is responsible for considerable financial losses in pig-rearing worldwide. To gain insight into the mechanisms involved in siderophore-mediated iron uptake in H. parasuis, genes in the H. parasuis ferric hydroxamate uptake (Fhu) region were amplified in the work being reported here. As has been described in A. pleuropneumoniae, an Fhu genomic region was also present in H. parasuis, being composed of four potential consecutive open reading frames (ORF) designated as fhuC, fhuD, fhuB, and fhuA, respectively. By immunoblotting, using a cross-reactive polyclonal antibody raised against Actinobacillus pleuropneumoniae FhuA protein, it was demonstrated that this protein was constitutively expressed in H. parasuis and its level of expression was not modified under conditions of restricted iron availability. This is the first report describing the presence of the fhu genes in H. parasuis. Our results indicate that FhuA protein expression is not affected under ironrestricted conditions, however, it is one of the targets of the humoral immune responseS

LIGHT/HVEM/LTβR Interaction as a Target for the Modulation of the Allogeneic Immune Response in Transplantation

[EN] The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC-cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell-mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR) engagement. LIGHT-deficient mice, or WT mice treated with LIGHT-targeting decoy receptors HVEM-Ig, LTβR-Ig or sDcR3-Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTβR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor-specific tolerance. This review discusses how targeting the interaction of LIGHT with HVEM and/or LTbR using recombinant soluble decoy receptors or monoclonal antibodies may represent an innovative therapeutic intervention for the prevention and treatment of allograft rejection and promotion of donor-specific tolerance. © 2013 The American Society of Transplantation and the American Society of Transplant SurgeonsSIThis work has been supported by grants FIS reference # PI10/01039 from Ministry of Health and Department of Education from Junta of Castilla and Leon reference # LE007A10-2 (to JIRB), and by the Swiss National Science Foundation (to PS

HVEM, a cosignaling molecular switch, and its interactions with BTLA, CD160 and LIGHT

[EN] Temporal and spatial expression of cosignaling receptors and their ligands regulates the early stages of T-cell activation (signal 1, T-cell receptor (TCR) signaling and signal 2, costimulation/ coinhibition), clonal expansion and T-cell survival during their differentiation towards effector T cells. Once the inflammatory stimulus is eliminated, effector T cells return to homeostasis after undergoing a contraction phase by activation-induced cell death and the intervention of ligands for coinhibitory receptors, leaving a population of long-term memory T cells. The expression of ligands for coinhibitory receptors on hematopoietic cells and, more importantly, on non-hematopoietic cells of peripheral tissues is a key process in tuning the functional activity of effector T cells to prevent excess tissue inflammation that may lead to immunopathology and subsequent tissue dysfunctionS

Genetic deletion of HVEM in a leukemia B cell line promotes a preferential increase of PD-1- stem cell-like T cells over PD-1+ T cells curbing tumor progression

[EN] Introduction: A high frequency of mutations affecting the gene encoding Herpes Virus Entry Mediator (HVEM, TNFRSF14) is a common clinical finding in a wide variety of human tumors, including those of hematological origin. Methods: We have addressed how HVEM expression on A20 leukemia cells influences tumor survival and its involvement in the modulation of the antitumor immune responses in a parental into F1 mouse tumor model of hybrid resistance by knocking-out HVEM expression. HVEM WT or HVEM KO leukemia cells were then injected intravenously into semiallogeneic F1 recipients and the extent of tumor dissemination was evaluated. Results: The loss of HVEM expression on A20 leukemia cells led to a significant increase of lymphoid and myeloid tumor cell infiltration curbing tumor progression. NK cells and to a lesser extent NKT cells and monocytes were the predominant innate populations contributing to the global increase of immune infiltrates in HVEM KO tumors compared to that present in HVEM KO tumors. In the overall increase of the adaptive T cell immune infiltrates, the stem cell-like PD-1- T cells progenitors and the effector T cell populations derived from them were more prominently present than terminally differentiated PD-1+ T cells. Conclusions: These results suggest that the PD-1- T cell subpopulation is likely to be a more relevant contributor to tumor rejection than the PD-1+ T cell subpopulation. These findings highlight the role of co-inhibitory signals delivered by HVEM upon engagement of BTLA on T cells and NK cells, placing HVEM/BTLA interaction in the spotlight as a novel immune checkpoint for the reinforcement of the anti-tumor responses in malignancies of hematopoietic originSIDepartment of Education of Castilla and Leon Regional Government (Grant # LE-006P20)Gerencia Regional de Salud (GRS1142/A/2015)Gerencia Regional de Salud (GRS1505/A/2017

Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells

[EN] Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFSFR14) and the lymphotoxin β receptor (LTβR, TNFSFR3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.SIThis work has been supported by grants of the Spanish Ministry of Health (Fondo de Investigaciones Sanitarias, PI13/00029), Department of Education of Castilla and Leon Regional Government (Grant# LE093U13) and Mutua Madrile~na Foundation (Basic research grants 2012) to J.I.R.B; by Miguel Servet National Grant (Health National Organization Research Program) CP12/03063 and by Gerencia Regional de Salud GRS963/A/2014 and GRS1142/A/2015 to M.L.R.G; and by the Swiss National Science Foundation to PS