Laura Carramolino

CCL25 mediates the localization of recently activated CD8alphabeta(+) lymphocytes to the small-intestinal mucosa.

The recruitment of antigen-specific T lymphocytes to the intestinal mucosa is central to the development of an effective mucosal immune response, yet the mechanism by which this process occurs remains to be fully defined. Here we show that the CC chemokine receptor 9 (CCR9) is selectively and functionally expressed on murine alpha(E)beta(7)(+) naive CD8alphabeta(+) lymphocytes and a subset of recently activated CD69(+) CD8alphabeta(+) lymphocytes. Using a T cell receptor transgenic transfer model, we demonstrate that CCR9 expression is functionally maintained on CD8alphabeta(+) lymphocytes following activation in mesenteric lymph nodes but rapidly downregulated on CD8alphabeta(+) lymphocytes activated in peripheral lymph nodes. These recently activated CCR9(+) CD8alphabeta(+) lymphocytes selectively localized to the small-intestinal mucosa, and in vivo neutralization of the CCR9 ligand, CCL25, reduced the ability of these cells to populate the small-intestinal epithelium. Together these results demonstrate an important role for chemokines in the localization of T lymphocytes to the small-intestinal mucosa and suggest that targeting CCL25 and/or CCR9 may provide a means to selectively modulate small-intestinal immune responses.

CCR6-deficient mice have impaired leukocyte homeostasis and altered contact hypersensitivity and delayed-type hypersensitivity responses

CCR6 expression in dendritic, T, and B cells suggests that this beta-chemokine receptor may regulate the migration and recruitment of antigen-presenting and immunocompetent cells during inflammatory and immunological responses. Here we demonstrate that CCR6-/- mice have underdeveloped Peyers patches, in which the myeloid CD11b+ CD11c+ dendritic-cell subset is not present in the subepithelial dome. CCR6-/- mice also have increased numbers in T-cell subpopulations within the intestinal mucosa. In 2,4-dinitrofluorobenzene-induced contact hypersensitivity (CHS) studies, CCR6-/- mice developed more severe and more persistent inflammation than wild-type (WT) animals. Conversely, in a delayed-type hypersensitivity (DTH) model induced with allogeneic splenocytes, CCR6-/- mice developed no inflammatory response. The altered responses seen in the CHS and DTH assays suggest the existence of a defect in the activation and/or migration of the CD4(+) T-cell subsets that downregulate or elicit the inflammation response, respectively. These findings underscore the role of CCR6 in cutaneous and intestinal immunity and the utility of CCR6-/- mice as a model to study pathologies in these tissues. This article was published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.

Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest

Polycomb group (PcG) proteins are key epigenetic regulators of hematopietic stem cell (HSC) fate. The PcG members Ezh2 and Ezh1 are important determinants of embryonic stem cell identity, and the transcript levels of these histone methyltransferases are inversely correlated during development. However, the role of Ezh1 in somatic stem cells is largely unknown. Here we show that Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response. Epigenomic and gene expression changes induced by Ezh1 deletion in senesced HSCs demonstrated that Ezh1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27. Deletion of Cdkn2a on the Ezh1 null background rescued HSC proliferation and survival. Our results suggest that Ezh1 is an important histone methyltransferase for HSC maintenance.

Platelets Play an Essential Role in Separating the Blood and Lymphatic Vasculatures During Embryonic Angiogenesis

Rationale: Several mutations that impair the development of blood lineages in the mouse also impair the formation of the lymphatic vasculature and its separation from the blood vasculature. However, the basis for these defects has remained unknown because the mutations characterized affect more than one blood lineage. Objective: We tested the hypothesis that megakaryocytes/platelets are required for the formation of the lymphatic vasculature and its separation from the blood vascular system. Methods and Results: We characterized the vascular patterning defects of mice deficient for the homeodomain transcription factor Meis1 (myeloid ecotropic viral integration site 1), which completely lack megakaryocyte/platelets. Meis1 null embryos fail to separate the blood and lymphatic vasculature, showing blood-filled primary lymphatic sacs and superficial lymphatic vessels. To test the involvement of megakaryocytes/platelets in this phenotype, we generated megakaryocyte/platelet-specific deficient mice by targeted lineage ablation, without affecting other blood lineages. This model reproduces the lymphatic/blood vasculature separation defects observed in Meis1 mutants. A similar phenotype was induced by antibody-mediated ablation of circulating platelets in wild type mice. Strong association of platelets with vascular endothelium at regions of contact between lymphatic sacs and veins confirmed a direct role of platelets in the separation of the 2 vasculatures. Conclusions: In addition to their known protective function in the response accidental vascular injury, platelets are also required during embryonic lymphangiogenesis for the separation of the nascent lymphatic vasculature from blood vessels.

Absence of CCR8 Does Not Impair the Response to Ovalbumin-Induced Allergic Airway Disease

Interaction of chemokines with their specific receptors results in tight control of leukocyte migration and positioning. CCR8 is a chemokine receptor expressed mainly in CD4+ single-positive thymocytes and Th2 cells. We generated CCR8-deficient mice (CCR8−/−) to study the in vivo role of this receptor, and describe in this study the CCR8−/− mouse response in OVA-induced allergic airway disease using several models, including an adoptive transfer model and receptor-blocking experiments. All CCR8−/− mice developed a pathological response similar to that of wild-type animals with respect to bronchoalveolar lavage cell composition, peripheral blood and bone marrow eosinophilia, lung infiltrates, and Th2 cytokine levels in lung and serum. The results contrast with a recent report using one of the OVA-induced asthma models studied here. Similar immune responses were also observed in CCR8−/− and wild-type animals in a different model of ragweed allergen-induced peritoneal eosinophilic inflammation, with an equivalent number of eosinophils and analogous increased levels of Th2 cytokines in peritoneum and peripheral blood. Our results show that allergic diseases course without critical CCR8 participation, and suggest that further work is needed to unravel the in vivo role of CCR8 in Th2-mediated pathologies.

Down-regulation of the beta-chemokine receptor CCR6 in dendritic cells mediated by TNF-alpha and IL-4.

Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response. We have generated DC from human umbilical cord blood CD34+ progenitors cultured with granulocyte‐macrophage colony‐stimulating factor, tumor necrosis factor α (TNF‐α), and stem cell factor. Using an anti‐CCR6 monoclonal antibody, we observed that these cells showed maximum expression of this β‐chemokine receptor when they were immature, as determined by their relatively low expression of several DC maturation markers such as CD1a, CD11c, CD14, CD40, CD80, and CD83. Immature DC responded strongly to macrophage inflammatory protein‐3α (MIP‐3α), the CCR6 ligand, in migration and calcium mobilization assays. CCR6 expression decreased in parallel with the DC maturation induced by prolonged TNF‐α treatments. Interleukin‐4 was also able to decrease CCR6 protein levels. Our findings suggest that the MIP‐3α/CCR6 interaction plays an important role in the trafficking of immature DC to chemokine production sites such as injured or inflamed peripheral tissues, where DC undergo maturation on contact with antigens. J. Leukoc. Biol. 66: 837–844; 1999.

Involvement of CCL25 (TECK) in the generation of the murine small-intestinal CD8alpha alpha+CD3+ intraepithelial lymphocyte compartment.

The CC chemokine CCL25 (TECK) is selectively expressed in the thymus and small intestine, indicating a potential role in T lymphocyte development. In the present study we examined the role of CCL25 in the generation of the small‐intestinal CD8α α+CD3+ intraepithelial lymphocyte (IEL) compartment. CCL25 mRNA expression in the murine small intestine increased at three weeks of age and corresponded with the appearance of CD8α α+CD3+ lymphocytes in the small‐intestinal epithelium. Administration of monoclonal neutralizing anti‐CCL25 antibody to two‐week‐old mice led to a ∼50% reduction in the total number of CD8α α+TCRγ δ+ and CD8α α+TCRα β+ IEL at four weeks of age. Freshly isolated murine CD8α α+CD3+ IEL migrated in response to CCL25 and expressed the CCL25 receptor, CCR9. Analysis of CCR9 expression on putative IEL precursor populations demonstrated the presence of both CCR9– and CCR9+ cells and indicated that up‐regulation of this receptor occurred during IEL precursor differentiation. Finally, data from wild‐type and RAG–/– mice suggested that the reduction in CD8α α+CD3+ IEL in anti‐CCL25 antibody treated mice resulted primarily from defective maintenance and/or development of IEL precursors rather than a direct effect on mature CD8α α+CD3+ IEL.

Clonal analysis identifies hemogenic endothelium as the source of the blood-endothelial common lineage in the mouse embryo

The first blood and endothelial cells of amniote embryos appear in close association in the blood islands of the yolk sac (YS). This association and in vitro lineage analyses have suggested a common origin from mesodermal precursors called hemangioblasts, specified in the primitive streak during gastrulation. Fate mapping and chimera studies, however, failed to provide strong evidence for a common origin in the early mouse YS. Additional in vitro studies suggest instead that mesodermal precursors first generate hemogenic endothelium, which then generate blood cells in a linear sequence. We conducted an in vivo clonal analysis to determine the potential of individual cells in the mouse epiblast, primitive streak, and early YS. We found that early YS blood and endothelial lineages mostly derive from independent epiblast populations, specified before gastrulation. Additionally, a subpopulation of the YS endothelium has hemogenic activity and displays characteristics similar to those found later in the embryonic hemogenic endothelium. Our results show that the earliest blood and endothelial cell populations in the mouse embryo are specified independently, and that hemogenic endothelium first appears in the YS and produces blood precursors with markers related to definitive hematopoiesis.

The Transient Expression of C-C Chemokine Receptor 8 in Thymus Identifies a Thymocyte Subset Committed to Become CD4+ Single-Positive T Cells

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a β-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4− CD8− double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4+ CD8+ double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4+ cell differentiation proceeded, reaching a maximum at the CD4+ CD8− single-positive stage. These CD4+ cells expressing CCR8 were also CD69high CD62Llow thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4− CD8+ thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4+ lineage.

Vegetative propagation of the olive tree from in vitro cultured embryos

Abstract A procedure to establish in vitro cultures of the olive tree, starting with isolated embryos, is described. Embryos germinated in a much higher proportion and at a faster rate than whole seeds. Germination of embryos occurs at a higher frequency when fruits of the same season are used as a source. A yield of 40% viable plantlets, depending on the cultivar, was obtained from these embryos. From this material in vitro cultures of the olive tree can be established in approx. 3 months. Shoot and root induction were readily achieved in nodal explants from plantlets derived from aseptically germinated embryos. Induction of shoots was equally efficient with either zeatin, 2iP or benzylaminopurine (BAP). Roots, induced by IBA, required darkness for their subsequent development.