Beatriz Garcillán

Alteration of medial-edge epithelium cell adhesion in two Tgf-β3 null mouse strains

Although palatal shelf adhesion is a crucial event during palate development, little work has been carried out to determine which molecules are responsible for this process. Furthermore, whether altered palatal shelf adhesion causes the cleft palate presented by Tgf-β3 null mutant mice has not yet been clarified. Here, we study the presence/distribution of some extracellular matrix and cell adhesion molecules at the time of the contact of palatal shelves in both wild-type and Tgf-β3 null mutant palates of two strains of mice (C57/BL/6J (C57), and MF1) that develop cleft palates of different severity. We have performed immunohistochemistry with antibodies against collagens IV and IX, laminin, fibronectin, the α5- and β1-integrins, and ICAM-1; in situ hybridization with a Nectin-1 riboprobe; and palatal shelf cultures treated or untreated with TGF-β3 or neutralizing antibodies against fibronectin or the α5-integrin. Our results show the location of these molecules in the wild-type mouse medial edge epithelium (MEE) of both strains at the time of the contact of palatal shelves; the heavier (C57) and milder (MF1) alteration of their presence in the Tgf-β3 null mutants; the importance of TGF-β3 to restore their normal pattern of expression; and the crucial role of fibronectin and the α5-integrin in palatal shelf adhesion. We thus provide insight into the molecular bases of this important process and the cleft palate presented by Tgf-β3 null mutant mice.

Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells.

Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression

BackgroundThe T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/−) or CD3D (δ+/−, δ+/leaky) with that of normal controls.ResultsAlthough the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/− individuals, whereas CD3δ+/− and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.ConclusionsThe results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

Enrichment of the rare CD4+ γδ T-cell subset in patients with atypical CD3δ deficiency

demonstrate the novel finding that human ILC2s highly express the signaling lymphocyte activation molecule family member CD84, although this expression is not selective for ILC2s. Allergen challenge did not increase levels of CD84 expression on ILC2s or CD4 cells. We next assessed the percentage of CRTH21 ILC2s in the peripheral blood of cat-allergic subjects before and 4 hours after nasal cat allergen or diluent challenges (Fig 2). The baseline percentage of CRTH21 cells within the lineage-negative population was 10.7 6 1.9 and 12.0 6 1.3 at the diluent and cat allergen challenge visit, respectively (Fig 2, B). Four hours after diluent challenge, the percentage of CRTH21 cells did not change significantly (9.7 6 1.8) compared with time zero. However, after cat allergen challenge, the percentage of CRTH21 cells nearly doubled to 19.1 6 2.6 compared with baseline (P5 .05) and compared with diluent challenge at 4 hours (P < .05) (Fig 2, B). Thus, nasal cat allergen challenge induced an increased percentage of peripheral blood CRTH21 ILC2s when measured 4 hours after challenge. ILC2s produce large amounts of IL-5 and IL-13 in response to IL-25, IL-33, TSLP, and LTD4 and could initiate and/or propagate allergic airway inflammation. Our studies demonstrate that the percentage of CRTH21 ILC2s in the peripheral blood is rapidly increased (within 4 hours) after allergen challenge. Potential mechanisms for the increase in ILC2s in the peripheral blood may be due to enhanced recruitment of ILC2s from the bone marrow triggered by either humoral (cytokine, chemokine, or mediator production in the nose) and/or cellular mechanisms (cells released from the nasal mucosa trafficking to the bone marrow). The human ILC2 marker CRTH2 is the receptor for prostaglandin D2 (PGD2), a lipid mediator that has a known role in chemotaxis and activation of immune cells. Importantly, a previous study demonstrated that high levels of serum 9a,11b-PGF2, the major PGD2 metabolite, are induced within 5 minutes after airway allergen challenge, suggesting that PGD2 is rapidly available systemically for the recruitment of CRTH21 cells after allergen exposure. We have also recently determined that PGD2 induces chemotaxis of CRTH21 human blood ILC2s in vitro, suggesting that PGD2 may directly regulate the migration of human ILC2s into tissues. The role of increased peripheral blood ILC2 numbers after allergen challenge is unclear. One hypothesis is that greater ILC2 availability in the blood (within 4 hours after challenge) may result in greater numbers of cytokine-producing nasal mucosa ILC2s at later time points, but this would need to be investigated in future studies. Strategies to inhibit the recruitment of ILC2s in allergic individuals may reduce tissue TH2 cytokine levels that contribute to allergic inflammation.

γδ T Lymphocytes in the Diagnosis of Human T Cell Receptor Immunodeficiencies.

Human T cell receptor (TCR) immunodeficiencies (TCRID) are rare autosomal recessive disorders caused by mutations affecting TCR, CD3, or CD247 chains, which share developmental, functional, and TCR expression defects (1). Their rapid diagnosis is fundamental for patient survival and early hematopoietic stem cell transplantation. Here, we propose that studying γδ T cells, which are often neglected, can be helpful for a timely diagnosis. We thus offer a diagnostic flowchart and some lab tricks based on published cases.