Jose-Ignacio Rodriguez-Barbosa

CD103− and CD103+ Bronchial Lymph Node Dendritic Cells Are Specialized in Presenting and Cross-Presenting Innocuous Antigen to CD4+ and CD8+ T Cells

Dendritic cells (DC) are able to capture, process, and present exogenous Ag to CD8+ T lymphocytes through MHC class I, a process referred to as cross-presentation. In this study, we demonstrate that CD103+ (CD11chighCD11blow) and CD103− (CD11cintCD11bhigh) DC residing in the lung-draining bronchial lymph node (brLN) have evolved to acquire opposing functions in presenting innocuous inhaled Ag. Thus, under tolerogenic conditions, CD103− DC are specialized in presenting innocuous Ag to CD4+ T cells, whereas CD103+ DC, which do not express CD8α, are specialized in presenting Ag exclusively to CD8+ T cells. In CCR7-deficient but not in plt/plt mice, Ag-carrying CD103+ DC are largely absent in the brLN, although CD103+ DC are present in the lung of CCR7-deficient mice. As a consequence, adoptively transferred CD8+ T cells can be activated under tolerizing conditions in plt/plt but not in CCR7-deficient mice. These data reveal that CD103+ brLN DC are specialized in cross-presenting innocuous inhaled Ag in vivo. Because these cells are largely absent in CCR7−/− mice, our findings strongly suggest that brLN CD103+ DC are lung-derived and that expression of CCR7 is required for their migration from the lung into its draining lymph node.

Development and functional specialization of CD103+ dendritic cells

Summary:  CD103 (αE) integrin expression distinguishes a population of dendritic cells (DCs) that can be found in many if not all lymphoid and non‐lymphoid organs. CD103+ DCs display distinct functional activities. Migratory CD103+ DCs derived from skin, lung, and intestine efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8+ T cells through a mechanism known as cross‐presentation. On the T cells they prime, intestinal CD103+ DCs can drive the induction of the chemokine receptor CCR9 and α4β7 integrin, both known as gut‐homing receptors. CD103+ DCs also contribute to control inflammatory responses and intestinal homeostasis by fostering the conversion of naive T cells into induced Foxp3+ regulatory T cells, a mechanism that relies on transforming growth factor‐β and retinoic acid signaling. This review discusses recent findings that identify murine CD103+ DCs as important regulators of the immune response.

Induction of Tolerance to Innocuous Inhaled Antigen Relies on a CCR7-Dependent Dendritic Cell-Mediated Antigen Transport to the Bronchial Lymph Node

Allergic airway diseases such as asthma are caused by a failure of the immune system to induce tolerance against environmental Ags. The underlying molecular and cellular mechanisms that initiate tolerance are only partly understood. In this study, we demonstrated that a CCR7-dependent migration of both CD103+ and CD103− lung dendritic cells (DC) to the bronchial lymph node (brLN) is indispensable for this process. Although inhaled Ag is amply present in the brLN of CCR7-deficient mice, T cells cannot be tolerized because of the impaired migration of Ag-carrying DC and subsequent transport of Ag from the lung to the draining lymph node. Consequently, the repeated inhalation of Ag protects wild-type but not CCR7-deficient mice from developing allergic airway diseases. Thus, the continuous DC-mediated transport of inhaled Ag to the brLN is critical for the induction of tolerance to innocuous Ags.

HVEM/LIGHT/BTLA/CD160 cosignaling pathways as targets for immune regulation

Immunosuppression is currently the treatment of choice to attenuate the chronic deterioration of tissue function as a result of the effector mechanisms of the immunological response in transplant rejection and autoimmune diseases. However, global immunosuppression greatly increases the risk of acquiring life‐threatening infections and is associated with organ toxicity when used long‐term. Thus, alternative approaches that inhibit only the unwanted immune responses and preserve general immunity are highly desirable. The receptor/ligand pairs involved in the cross‐talk between DC and T cells have been the focus of intense and exciting research during the last decade. The HVEM/LIGHT/BTLA/CD160 costimulatory/coinhibitory pathway has emerged as a potential target for the development of immune therapeutic interventions. Herein, we will summarize and discuss how blockade of the costimulatory HVEM/LIGHT interaction or agonist signaling through the inhibitory BTLA and CD160 receptors could contribute to the control of deleterious immune responses.

Isolation and characterization of immortalized porcine aortic endothelial cell lines

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).

CX3CR1+ c-kit+ bone marrow cells give rise to CD103+ and CD103- dendritic cells with distinct functional properties.

Dendritic cells (DC) represent a rather heterogeneous cell population with regard to morphology, phenotype, and function and, like most cells of the immune system, are subjected to a continuous renewal process. CD103+ (integrin αE) DC have been identified as a major mucosal DC subset involved in the induction of tissue-specific homing molecules on T cells, but little is known about progenitors able to replenish this DC subset. Herein we report that lineage (lin)−CX3CR1+c-kit+ (GFP+c-kit+) bone marrow cells can differentiate to either CD11c+CD103− or CD11c+CD103+ DC in vitro and in vivo. Gene expression as well as functional assays reveal distinct phenotypical and functional properties of both subsets generated in vitro. CD103− DC exhibit enhanced phagocytosis and respond to LPS stimulation by secreting proinflammatory cytokines, whereas CD103+ DC express high levels of costimulatory molecules and efficiently induce allogeneic T cell proliferation. Following adoptive transfer of GFP+c-kit+ bone marrow cells to irradiated recipients undergoing allergic lung inflammation, we identified donor-derived CD103+ DC in lung and the lung-draining bronchial lymph node. Collectively, these data indicate that GFP+c-kit+ cells contribute to the replenishment of CD103+ DC in lymphoid and nonlymphoid organs.

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

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 proteins.

Antibody-mediated signaling through PD-1 costimulates T cells and enhances CD28-dependent proliferation.

Programmed death‐1 (PD‐1, CD279) is a molecule expressed on activated T, B and myeloid cells. The role of the interaction of PD‐1 ligands (PD‐L1 and PD‐L2) with PD‐1 receptor and the type of signals (costimulatory or inhibitory) that are delivered is a subject of intense debate. Our study has characterized two monoclonal antibodies (mAb) against murine PD‐1, termed clone 1H10 and clone 4F10, that recognized different epitopes from that of anti‐PD‐1, clone J43. We showed that neither of them inhibited anti‐CD3‐mediated proliferation, but 1H10 mAb induced direct T cell proliferation in the absence of any other stimulus. Moreover, PD‐1 engagement with 1H10 mAb costimulated anti‐CD3‐mediated proliferation and enhanced anti‐CD3/CD28 proliferation on both CD4+ and CD8+ T cells in the low range of anti‐CD3 concentrations. Anti‐PD‐1‐mediated proliferation induced with 1H10 mAb was also observed in vivo on CD4+ and CD8+ T cells, when CFSE‐labeled splenocytes were adoptively transferred to irradiated syngeneic and allogeneic recipients. Overall, our data indicate that PD‐1 might not only deliver negative signals to T cells upon interaction through one of its ligands, PD‐L1 as reported, but also could costimulate T cells, suggesting a dual potential functional activity of the extracellular domains of this receptor.

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells.

BACKGROUND Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved. MATERIALS AND METHODS Multiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry. RESULTS Peripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. CONCLUSION Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässers disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 x 10(9) CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10(5) CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45(+), CD3(+), CD4(+), CD8alpha(+), CD25(+), CD4(+) naïve, alphaIgM(+) and SWC3(+) cells in single-colour fluorescence, and CD4(+)/CD8alpha(+) and CD8alpha(+)/CD8beta(+) combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P<0.05) in the relative proportions of monocytes and granulocytes (SWC3(+)) and B cells (alphaIgM(+)), as well as a significant reduction in CD3(+) cells (P<0.05). These changes were similar for the five groups compared, except for the significant increase of CD25(+) cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.