Gloria Martínez del Hoyo

Origin and differentiation of dendritic cells

Despite extensive, recent research on the development of dendritic cells (DCs), their origin is a controversial issue in immunology, with important implications regarding their use in cancer immunotherapy. Although, under defined experimental conditions, DCs can be generated from myeloid or lymphoid precursors, the differentiation pathways that generate DCs in vivo remain unknown largely. Indeed, experimental results suggest that the in vivo differentiation of a particular DC subpopulation could be unrelated to its possible experimental generation. Nevertheless, the analysis of DC differentiation by in vivo and in vitro experimental systems could provide important insights into the control of the physiological development of DCs and constitutes the basis of a model of common DC differentiation that we propose.

Characterization of a common precursor population for dendritic cells

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs—including CD8- and CD8+ DCs, as well as the B220+ DC subset—but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.

Langerhans cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation.

Dendritic cell (DC) reconstitution experiments and phenotypic analysis of DC subpopu‐lations have allowed the definition in the mouse of two main DC categories: CD8+ lymphoid DCs and CD8− myeloid DCs. With regard to Langerhans cells (LCs), which represent immature DCs differentiating into mature DCs on migration to the lymph nodes after an antigenic stimulation, although classically considered as myeloid DCs, there is no experimental evidence of their origin. It has been recently shown that mouse LCs, negative for CD8 and LFA‐1, undergo CD8/LFA‐1 up‐regulation on migration, suggesting that LCs belong to the CD8+ lymphoid DC lineage. To further reinforce this hypothesis, we have analyzed the modulation of CD8 expression by LCs on culture with molecules known to induce LC maturation. Our results show that LC acquired a CD8+ lymphoid phenotype on CD40 ligation. J. Leukoc. Biol. 67: 206–209; 2000.

Functional Role of P-Selectin Glycoprotein Ligand 1/P-Selectin Interaction in the Generation of Tolerogenic Dendritic Cells

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-β genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4+CD25+Foxp3+ regulatory T cells, which expressed high levels of TGF-β1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4+CD25− T cells. Accordingly, we found that DCs from PSGL-1−/− mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.

Immunological synapse formation inhibits, via NF-κB and FOXO1, the apoptosis of dendritic cells

The immunological synapse (IS) is a cell–cell junction formed between CD4+ T cells and dendritic cells (DCs). Here we show in vitro and in vivo that IS formation inhibits apoptosis of DCs. Consistent with these results, IS formation induced antiapoptotic signaling events, including activation of the kinase Akt1 and localization of the prosurvival transcription factor NF-κB and the proapoptotic transcription factor FOXO1 to the nucleus and cytoplasm, respectively. Inhibition of phosphatidylinositol 3-OH kinase and Akt1 partially prevented the antiapoptotic effects of IS formation. Direct stimulation of the IS component CD40 on DCs leads to the activation of Akt1, suggesting the involvement of this receptor in the antiapoptotic effects observed upon IS formation.

Functional and phenotypic analysis of thymic B cells: role in the induction of T cell negative selection

The phenotype of mouse thymic B cells and their capacity to induce T cell negative selection in vitro were analyzed. Thymic B cells expressed B cell markers such as IgM, Fcγ receptor, CD44, heat‐stable antigen, LFA‐1 and CD40. In addition, they were positive for the activation molecule CD69 and displayed high levels of B7‐2. Although thymic B cells expressed CD5 on their surface, no CD5‐specific mRNA was detected. Moreover, thymic B cells induced a stronger deletion of TCR‐transgenic (TG) thymocytes than splenic B cells, which had low CD69 and B7‐2 levels. Interestingly, CD40‐activated splenic B cells up‐regulated CD69 and B7‐2 and acquired a capacity to induce T cell deletion comparable to that of thymic B cells. Moreover, thymic B cells from CD40‐deficient mice displayed lower CD69 and B7‐2 levels than control thymic B cells, and lower capacity to induce the deletion of TCR TG thymocytes. These results support the hypothesis that CD40‐mediated activation of thymic B cells determines a high efficiency of antigen presentation, suggesting that within the thymus B cells may play an important role in the elimination of autoreactive thymocytes.

Imaging of plasmacytoid dendritic cell interactions with T cells

Plasmacytoid dendritic cells (pDCs) efficiently produce type I interferon and participate in adaptive immune responses, although the molecular interactions between pDCs and antigen-specific T cells remain unknown. This study examines immune synapse (IS) formation between murine pDCs and CD4(+) T cells. Mature pDCs formed canonical ISs, involving relocation to the contact site of the microtubule-organizing center, F-actin, protein kinase C-, and pVav, and activation of early signaling molecules in T cells. However, immature pDCs were less efficient at forming conjugates with T cells and inducing IS formation, microtubule-organizing center translocation, and T-cell signaling and activation. Time-lapse videomicroscopy and 2-photon in vivo imaging of pDC-T-cell interactions revealed that immature pDCs preferentially mediated transient interactions, whereas mature pDCs promoted more stable contacts. Our data indicate that, under steady-state conditions, pDCs preferentially establish transient contacts with naive T cells and show a very modest immunogenic capability, whereas on maturation, pDCs are able to form long-lived contacts with T cells and significantly enhance their capacity to activate these lymphocytes.

Prion Protein Expression by Mouse Dendritic Cells Is Restricted to the Nonplasmacytoid Subsets and Correlates with the Maturation State

Expression of the physiological cellular prion protein (PrPC) is remarkably regulated during differentiation and activation of cells of the immune system. Among these, dendritic cells (DCs) display particularly high levels of membrane PrPC, which increase upon maturation, in parallel with that of molecules involved in Ag presentation to T cells. Freshly isolated mouse Langerhans cells, dermal DCs, and DCs from thymus, spleen, and mesenteric lymph nodes expressed low to intermediate levels of PrPC. Highest levels of both PrPC and MHC class II molecules were displayed by lymph node CD8αint DCs, which represent fully mature cells having migrated from peripheral tissues. Maturation induced by overnight culture resulted in increased levels of surface PrPC, as did in vivo DC activation by bacterial LPS. Studies on Fms-like tyrosine kinase 3 ligand bone marrow-differentiated B220− DCs confirmed that PrPC expression followed that of MHC class II and costimulatory molecules, and correlated with IL-12 production in response to TLR-9 engagement by CpG. However, at variance with conventional DCs, B220+ plasmacytoid DCs isolated from the spleen, or in vitro differentiated, did not significantly express PrPC, both before and after activation by TLR-9 engagement. PrP knockout mice displayed higher numbers of spleen CD8α+ DCs, but no significant differences in their maturation response to stimulation through TLR-4 and TLR-9 were noticed. Results are discussed in relation to the functional relevance of PrPC expression by DCs in the induction of T cell responses, and to the pathophysiology of prion diseases.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

The Leukocyte Activation Receptor CD69 Controls T Cell Differentiation through Its Interaction with Galectin-1

ABSTRACT CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and function.