Differentiation of T helper lymphocyte subsets is crucial for immune and inflammatory responses. In addition to the two classical T helper cell subsets (Th1 and Th2), a third T-lymphocyte subpopulation, designated Th17, characterized by the synthesis of interleukin 17A (IL-17A), IL-17F, and IL-22, has emerged as an independent differentiation pathway (9). Differentiation toward each Th subset is regulated by a variety of molecules, including cytokines and transcription factors. The key transcription factors that drive the differentiation of the Th1 and Th2 lineages are, respectively, T-bet and GATA-3, while the differentiation of Th17 cells is directed by retinoic acid-related orphan receptor ␥t (ROR␥t) (12). Th17 differentiation, moreover, is regulated by the balance of Stat3/Stat5 activation; Stat3 is necessary for Th17 differentiation, whereas the transcription factor Stat5 negatively regulates the development of these cells (1). The key cytokines involved in Th17 cell differentiation are transforming growth factor ␤ (TGF-␤) and IL-6 (3, 33). Another important cytokine for Th17 biology is IL-23. Although Th17 cells can arise in the absence of IL-23, the cytokine is required for their maintenance and survival (29, 33) and for their pathogenicity (20).Dysregulated activation of T helper subpopulations is associated with immune pathogenesis (21). Th1 cells are clearly involved in autoimmune and inflammatory disorders mediated by the cellular immune response, and Th2 cells are involved in antibody-mediated allergic and inflammatory conditions (24).Recent evidence indicates that Th17 cells also exert a pathogenic effect in several autoimmune and hypersensitivity reactions. Indeed, a number of immune pathologies previously thought to be related to uncontrolled activation of Th1 or Th2 populations now appear to be related, at least in part, to Th17 cell differentiation. For example, involvement of Th17 lymphocytes has been reported in collagen-induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE), experimental autoimmune myocarditis (EAM), contact hypersensitivity (CHS), and airway hyperresponsiveness (AHR) (11,13,18,22,23,30).We previously found that CD69 ϩ T cells are localized at sites of chronic inflammation and that these lymphocytes seem to be able to downregulate the inflammatory process. Although antigen-dependent T-cell activation and proliferation do not appear to be altered in CD69-deficient lymphocytes (16), CD69 knockout (CD69 KO) mice develop an exacerbated form of CIA characterized by diminished local synthesis of TGF-␤ (26). These and other studies (5) suggest that CD69 is a negative regulator of the immune response, in part through modulation of local levels of TGF-␤. Here, we explore the role of CD69 in the differentiation of T helper lineages. Our results show that, while Th1 and Th2 differentiation remain unchanged in CD69-deficient mice, lymphocytes from these animals show an enhanced potential to differentiate toward Th17 cells both in vitro and in vivo. Biochemical and funct...

f 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. C D69, a C-type lectin, is a member of the natural killer (NK) receptor family and is induced early following activation of leukocytes (1). The physiological role of this receptor, which is persistently expressed by infiltrating leukocytes in different chronic inflammatory diseases, has been studied in CD69-deficient mice in multiple different models of chronic inflammation (2-5). Thus, we have previously described that CD69 Ϫ/Ϫ mice develop an exacerbated form of collagen-induced arthritis (CIA) (3), a Th1 and Th17 cell-mediated autoimmune condition. Moreover, in an experimental model of autoimmune myocarditis (EAM), CD69 negatively regulates cardiac inflammation through the regulation of heart-specific Th17 responses (4). In this regard, we have detected that CD69 modulates the in vitro differentiation of T cells toward the Th17 lineage through the activation of the Jak3/Stat5 inhibitory pathway (5). On the other hand, CD69 negatively regulates the chemotactic responses of effector lymphocytes and dendritic cells (DCs) to sphingosine 1 phosphate (S1P); CD69 can associate with S1P1 in the cell membrane and induce a conformation of S1P1 that favors its internalization and degradation (6-8). It is clear that the identification of cellular ligands for CD69 is a critical next step to better understand the physiological role of this receptor.Galectins are characterized by a common structural fold and a conserved carbohydrate recognition domain (CRD) with a high affinity for beta-galactosides (9). Despite being soluble proteins, galectins are also expressed on the cell surface due to their association with membrane glycoproteins. Thus, galectin-1 (Gal-1) is expressed by most activated but not resting T and B cells, and it is significantly upregulated in activated macrophages and T regulatory lymphocytes (10). In addition, tolerogenic DCs show a high expression of Gal-1 (11), which is rapidly downregulated in response to maturation signals. Furthermore, Gal-1-deficient DCs show a greater immunogenic potential and an impaired ability to halt the inflammatory phenomenon in a ...

Background-Experimental autoimmune myocarditis (EAM), a mouse model of post-infectious cardiomyopathy, reflects mechanisms of inflammatory cardiomyopathy in humans. EAM is characterized by an infiltration of inflammatory cells into the myocardium that can be followed by myocyte fibrosis, edema, and necrosis, leading to ventricular wall dysfunction and heart failure. Different data indicate that CD69 exerts an important immunoregulatory effect in vivo. However, the possible role of CD69 in autoimmune myocarditis has not been studied. Methods and Results-We have explored the role of the leukocyte regulatory molecule CD69 in the inflammation that leads to cardiac dysfunction after myocardial injury in EAM. We have found that after induction of EAM, the draining lymph nodes from CD69-deficient mice developed an exacerbated Th17 inflammatory response, resulting in increases in the numbers of infiltrating leukocytes in the myocardium. In the chronic phase of EAM, transthoracic echocardiography revealed a significantly reduced left ventricular fractional shortening and a decreased ejection fraction in CD69-deficient mice, indicative of an impaired cardiac contractility. This condition was accompanied by a greater extent of myocardial fibrosis, an elevated number of sinus pauses on ECG, and an enhanced ratio of heart weight to body weight in CD69 Ϫ/Ϫ mice. Moreover, both bone marrow transplantation and adoptive transfer of Th17 cells isolated from immunized CD69Ϫ/Ϫ mice with EAM into naive wild-type recipients reproduced the severity of the disease, demonstrating that CD69 exerts its function within the lymphocyte compartment. Conclusion-Our findings indicate that CD69 negatively regulates heart-specific Th17 responses, cardiac inflammation, and heart failure progression in EAM. (Circulation. 2010;122:1396-1404.)Key Words: cardiomyopathy Ⅲ echocardiography Ⅲ immune system Ⅲ inflammation Ⅲ myocarditis M yocarditis and subsequent dilated cardiomyopathy (DCM) are major causes of heart failure in young patients. 1 This condition is characterized by infiltration of inflammatory cells into the myocardium with consequent loss of myocytes and development of fibrosis and necrosis. 2 In a significant fraction of patients, the loss of cardiomyocytes leads to ventricular remodeling, permanent ventricular wall dysfunction, DCM, heart failure, and/or arrhythmias. Myocarditis is induced by a variety of agents, including genetic susceptibility, toxins, viruses, bacteria, and parasites. 3,4 In addition, myocardial injury can induce an autoimmune response to heart tissue, which has an important role in the pathogenesis of myocarditis and DCM. 5-7 Experimental autoimmune myocarditis (EAM) is a mouse model of postinfectious myocarditis that can be induced in susceptible mouse strains by immunization with cardiac ␣-myosin heavy chain (MyHC-␣)-derived peptides or by injection of activated MyHC-␣-loaded dendritic cells. 8 -10 In this regard, it has been shown that EAM is a CD4 ϩ T-cell-mediated disease and its development depends critical...

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Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.

Rho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell-derived factor-1 (SDF-1␣). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1␣-activated human lymphocytes, where it transiently interacts with the SDF-1␣ receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1␣-mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4. IntroductionLeukocyte migration in and out of target tissues during homeostasis and inflammation is a finely regulated process mediated by many receptors, which regulate rolling, adhesion and/or detachment, and motility. Chemotactic receptors play an important role in the modulation of cell adhesion as well as in controlling the morphology of migrating leukocytes. 1 In particular, chemokines are chemotactic cytokines that, acting through heterotrimeric G-proteincoupled receptors (GPCRs), regulate cell adhesion through crosstalk with integrin receptors and also modulate the morphology of migrating leukocytes. 1,2 The chemokine stromal cell-derived factor-1 (SDF-1␣) is the most pleiotropic of chemokines, being involved in many processes, from migration of hematopoietic progenitors and most leukocytes to morphogenesis in mammals and lower organisms such as zebrafish. 3,4 SDF-1␣ interacts exclusively with the chemokine receptor CXCR4, the specificity of this pair being underscored by the similar phenotype of mice deficient for SDF-1␣ or CXCR4. [5][6][7] SDF-1␣ has been shown to modulate adhesion through the integrins very late activation antigen-4 (VLA-4, ␣ 4 ␤ 1 ) and leukocyte function associated antigen-1 (LFA-1, ␣ L ␤ 2 ), 8 although the intracellular mechanisms involved in such cross-talk are largely undefined. We and others have shown that SDF-1␣ induces profound morphological changes in adherent leukocytes and the acquisition of a bipolar shape with a front leading edge, in which chemokine receptors are clustered, 9-12 and a trailing edge or uropod that accumulates adhesion molecules such as ICAM-1, -3, or CD44 (Vicente-Manzanares and Sanchez-Madrid 1 ). CXCR4 trigge...

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.

SummaryAccumulating evidence shows that galectins play roles in the initiation and resolution phases of inflammatory responses by promoting anti-or proinflammatory effects. This study investigated the presence of three members of the galectin family (galectin-1, -3 and -9) in induced sputum samples of asthma patients, as well as their possible implication in the immunopathogenesis of human asthma. Levels of interleukin (IL)-5, IL-13, and galectins were determined in leucocytes isolated from induced sputum samples by reverse transcription-polymerase chain reaction (RT-PCR) immunofluorescence and flow cytometry. High levels of IL-5 and IL-13 mRNA were detected in sputum cells from asthma patients. In parallel, immunoregulatory proteins galectin-1 and galectin-9 showed a reduced expression on macrophages from sputum samples compared with cells from healthy donors. In-vitro immunoassays showed that galectin-1 and galectin-9, but not galectin-3, are able to induce the production of IL-10 by peripheral blood mononuclear cells from healthy donors. These findings indicate that macrophages from sputum samples of asthma patients express low levels of galectin-1 and galectin-9, favouring the exacerbated immune response observed in this disease.