Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A 1 adenosine receptors, or A2B adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC 50 for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADAanchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3-to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-␥, TNF-␣, and IL-6. adenosine deaminase ͉ costimulation ͉ immunosynapse A denosine deaminase (ADA; EC 3.5.4.4) an enzyme involved in purine metabolism, catalyzes the hydrolytic deamination of adenosine or 2Ј-deoxyadenosine to inosine or 2Ј-deoxyinosine and ammonia. Congenital defect of ADA causes severe combined immunodeficiency, which is characterized by the absence of functional T and B lymphocytes in affected individuals (1). For many years, ADA was considered to be cytosolic, but it has been found on the cell surface of many cell types; therefore, it can be considered an ectoenzyme. In addition, ecto-ADA has been proposed to have a catalytic-independent function as a costimulatory molecule in lymphocytes (2).So far, two types of surface anchoring proteins for ecto-ADA have been described. The first type, with only one member, is CD26, a multifunctional protein of 110 KDa strongly expressed on epithelial cells (kidney proximal tubules, intestine, and bile duct) and on several types of endothelial cells and fibroblasts and on leukocyte subsets (3-5). The second type of ecto-ADA-binding proteins includes the adenosine receptors (AR) A 1 (A 1 R) (6) and A 2B (A 2B R) (7). The association between ADA and CD26 on the T cell surface has been proposed to have a costimulatory function during T cell antigen receptor-CD3 complex engagement (2). Because CD26 has a short cytoplasmatic tail, it needs partners to transduce the signal. Ishii et al. (8) have described that CD26-mediated signaling occurs through its association with CD45RO. At present, it is not known whether ADA generates a signal when it binds to AR. However, we have previously demonstrated that ADA binding to A 1 R or A 2B R is required for high efficiency affinity binding of the agonist and for efficient agonist-dependent signaling (6, 7).Dendritic cells (DC) are the most potent antigen-presenting cells (APC) specialized in the initiation of immune responses by directing the activation and differentiation of naïve T lymphocytes (9, 10). Immature DC (iDC) reside in most tissues to uptake antigen; they are engaged when exposed to danger ...

Adaptive immune responses begin after productive immunosynaptic contacts formation established in secondary lymphoid organs by dendritic cells (DC) presenting the Ag to T lymphocytes. Despite its resemblance to the neurosynapse, the participation of soluble small nonpeptidic mediators in the intercellular cross-talk taking place during T cell–DC interactions remains poorly studied. In this study, we show that human DC undergoing maturation and in contact with T cells release significant amounts of glutamate, which is the main excitatory neurotransmitter in mammalians. The release of glutamate is nonvesicular and mediated by the DC-expressed Xc− cystine/glutamate antiporter. DC-derived glutamate stimulating the constitutively expressed metabotropic glutamate receptor 5 impairs T cell activation. However, after productive Ag presentation, metabotropic glutamate receptor 1 is expressed in T cells to mediate enhanced T cell proliferation and secretion of Th1 and proinflammatory cytokines. These data suggest that, during T cell–DC interaction, glutamate is a novel and highly effective regulator in the initiation of T cell-mediated immune responses.

Defensins are effector molecules of the innate immunity with a broad antimicrobial spectrum, including HIV. They also link innate and adaptive immunity, displaying chemotactic activity for monocytes, T cells, and dendritic cells (DCs). alpha-Defensins 1-3 are mainly produced by neutrophils, but their production by other leukocyte subsets has also been reported. Herein, we studied whether monocyte-derived DCs (MDDCs), which are regarded as a model for myeloid DCs, produce alpha-defensins 1-3. We found that immature MDDCs (imMDDCs) produce alpha-defensins 1-3 mRNA, but this production is undetectable or barely detectable following 48 h of maturation with the proinflammatory cytokine cocktail (IL-1beta+IL-6+TNF-alpha) or LPS. It is surprising that alpha-defensins 1-3 production was up-regulated when exposed to each one of the proinflammatory cytokines alone, especially IL-1beta. alpha-Defensins 1-3 produced by imMDDCs were mainly secreted peptides. Production and secretion of alpha-defensins 1-3 by imMDDCs can have biological relevance for the antigen processing of pathogens and can contribute to understanding differences in susceptibility to infections, an issue of special interest in the field of HIV infection.

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca 2؉ and/or Mg 2؉ in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4°C, increases at 37°C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg 2؉ is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.

BackgroundDefensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.Methodology/Principal FindingsMonocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated “elite” and “viremic” controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of α-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted α-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher α-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm3, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.Conclusions/SignificanceHigh production of α-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of α-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.

Summary The cell surface association between CD26 and adenosine deaminase (ADA) has a costimulatory function during T‐cell activation. Several studies have revealed correlations among CD4+ CD26+ T‐cell depletion, increased serum levels of ADA, and the evolution of human immunodeficiency virus (HIV) infection, implicating CD26 and ADA in HIV disease progression. In this context, we aimed to determine whether ADA costimulation could be altered during HIV infection. ADA costimulation was investigated in cells from HIV‐infected patients (n = 36) in terms of proliferation and cytokine secretion. An effect of ADA on T‐cell proliferation was found in HIV‐1‐infected patients and correlated positively with the CD4+ percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADA‐induced increase in cytokine production [interferon (IFN)‐γ, interleukin (IL)‐6 and IL‐10] was markedly reduced in T cells from HIV‐1‐infected subjects. To eliminate some of the variables associated with immunological defects in HIV‐1‐infected patients, anti‐CD3 plus ADA assays with T cells from healthy volunteers were performed in the presence of recombinant glycoprotein 120 (gp120). It was found that gp120 was responsible for the impairment of the ADA–CD26 interaction and consequently of the ADA‐induced effect on both costimulation and cytokine production. The gp120‐mediated disruption of the CD26–ADA interaction is a novel mechanism that might explain, at least in part, the altered immunological features observed in HIV‐1‐infected patients and may have significant relevance in AIDS pathogenesis.

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