Pilar Portolés

T-cell receptor.

The T-cell antigen receptor complex (TCR/CD3) is a cell surface structure that defines the T lymphocyte lineage, where it fulfills two basic functions, namely antigen recognition and triggering of signals needed to mount adequate responses to foreign aggression and/or to undergo differentiation. Knowing the precise structure of the complex in terms of its components and their relative arrangement and interactions before and after antigen recognition is essential to understand how ligand binding transforms into functionally relevant T-cell responses. These include not only full responses to foreign peptide antigens by mature T-cells, but also other phenomena like modulation ofT-cell activation with altered peptide ligands, positive and negative selection ofthymocytes, alloreactivity and autoimmune reactions. A wealth of new data has accumulated in recent years on the structure of TCR/antigen complexes and CD3 polypeptides and on the stoichiometry of the TCR/CD3 complex and intersubunit interactions. In this review, we discuss how these data fit into a meaningful model of the TCR/CD3 function.

Crry/p65, a Membrane Complement Regulatory Protein, Has Costimulatory Properties on Mouse T Cells

It is known that certain type I membrane molecules (complement receptors type 1 and 2) belonging to the regulators of complement activation (RCA) family are involved in the regulation of B lymphocyte activation. In contrast, only GPI-anchored RCA molecules (CD55) have been described to be involved in T lymphocyte activation. In this study, we describe a novel function for the mouse RCA type I membrane protein Crry/p65 as a costimulatory molecule in CD4+ T cell activation. This is shown by increased anti-CD3-induced proliferation of CD4+ spleen T lymphocytes in the presence of the Crry/p65-specific mAb P3D2. Furthermore, Ab-induced coligation of Crry/p65 and CD3 favors IL-4 rather than IFN-γ secretion in these cells. Crry/p65 signaling was also observed regardless of additional Ca2+, protein kinase C, or CD28-mediated costimuli. Analysis of intracellular intermediaries shows that Crry/p65-CD3 coligation enhances certain TCR/CD3-mediated signals, producing increased early tyrosine phosphorylation of many substrates and enhanced activation of the mitogen-activated protein kinase, extracellular signal-related kinase. These data fit well with the association of Crry/p65 with the tyrosine kinase Lck found in T cell lysates. The epitope recognized by the mAb P3D2 interferes with the protective role of Crry/p65 on C3 deposition. The relationship between protective function and costimulation by Crry/p65 is discussed. Our results support a multifunctional role for Crry/p65 in T cells and suggest new links between the natural and adaptive immune responses.

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways.

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4+ Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti‐H4/ICOS antibodies strongly enhanced CD3‐mediated tyrosine phosphorylation of ZAP‐70, ζ, or Vav, as well as extracellular signal‐regulated kinase (ERK), Jun N‐terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3‐kinase (PI‐3K) to H4/ICOS was enhanced by H4/ICOS cross‐linking, and PI‐3K inhibitors inhibited ERK and JNK activation andIL‐4/IL‐10 secretion, but not p38 MAP kinase or ZAP‐70 activation. H4/ICOS‐mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP‐70, MAP kinase activation, or IL‐4/IL‐10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP‐70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL‐4, but not IL‐10 secretion. ERK or JNKinhibitors partially inhibited IL‐4 and IL‐10 secretion, while PKC or p38 inhibitors had no significant effects on IL‐4 or IL‐10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.

Complement regulatory protein Crry/p65-mediated signaling in T lymphocytes: role of its cytoplasmic domain and partitioning into lipid rafts

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I‐related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR‐dependent activation signals, including p56lck‐, ζ‐associated protein‐70 (ZAP‐70), Vav‐1, Akt, and extracellular signal‐regulated kinase (ERK) phosphorylation but also costimulation‐dependent mitogen‐activated protein kinases (MAPK), such as the stress‐activated c‐Jun N‐terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent‐insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3ɛ, and p59‐60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol‐3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin‐4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP‐70, Akt, Vav‐1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.

NF‐κB in Th2 cells : delayed and long‐lasting induction through the TCR complex

Nucelar NFκB was analyzed in murine Th2 cells after stimulation via the TCR pathway. Signals delivered through the TCR/CD3 complex induced active NFκB translocation to the nucleus of Th2 cells after a late phase (24 h) of the activation process, which is in contrast to the rapid appearance of nuclear NFκB (3 h) in Th1 cells after the same stimulation. The slow kinetic of NFκB nuclear uptake in Th2 cells was not accelerated by CD28 triggering or under stimulation with antigen plus antigen‐presenting cells. Th1 and Th2 cells were also different in the composition of NFκB complexes induced. Whereas in Th1 cells TCR triggering induced the presence of nuclear p50.p65 heterodimers, in Th2 cells the complexes induced were shown to be composed of p65 plus another NFκB protein distinct from p50. The delayed NFκB induction in Th2 cells was dependent on protein synthesis and the significance of this is discussed.

CD4 Dependence of Activation Threshold and TCR Signalling in Mouse T Lymphocytes

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4− mutants obtained from the SR.D10 clone. This non‐tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4− mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4− clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA‐1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti‐CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen‐plus self MHC may become CD4‐independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low‐affinity TCR reactivities like those directed at self MHC. Moreover, by using anti‐TCR/CD3 antibodies, the authors have confirmed the importance of CD4‐associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co‐precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck‐TCR co‐precipitation.

Enhancement of lymphocyte proliferation, interleukin-2 production and NK activity by inmunoferon (AM-3), a fungal immunomodulator: Variations in normal and immunosuppressed mice

The in vivo effect of Inmunoferon (AM-3), a glycophosphopeptide of fungal origin, has been studied on T and B lymphocyte mitogenesis, Interleukin 2 (IL-2) synthesis and natural killer (NK) activity. Inmunoferon (30 mg/kg/day) was administered to several groups of mice 2, 3 or 7 days/week for 2 weeks, and its effect assessed on day 15 of treatment. Every treatment assayed enhanced IL-2 and NK activity in the spleen. The greater effect was produced by daily administration of the immunomodulator. No enhancement was found in mitogen-induced proliferation of T and B lymphocytes. Similar treatments with Inmunoferon enhanced NK activity in the spleens of mice treated with 250 mg/kg cyclophosphamide. In addition, mitogenic responses of T lymphocytes, but not IL-2 production, were also increased in immunosuppressed mice after treatment with the immunomodulator.

Biased binding of class IA phosphatidyl inositol 3-kinase subunits to inducible costimulator (CD278).

To better understand T lymphocyte costimulation by inducible costimulator (ICOS; H4; CD278), we analyzed proteins binding to ICOS peptides phosphorylated at the Y191MFM motif. Phosphorylated ICOS binds class IA phosphatidyl inositol 3-kinase (PI3-K) p85α, p50-55α and p85β regulatory subunits and p110α, p110δ and p110β catalytic subunits. Intriguingly, T cells expressed high levels of both p110α or p110δ catalytic subunits, yet ICOS peptides, cell surface ICOS or PI3-kinase class IA regulatory subunits preferentially coprecipitated p110α catalytic subunits. Silencing p110α or p110δ partially inhibited Akt/PKB activation induced by anti-CD3 plus anti-ICOS antibodies. However, silencing p110α enhanced and silencing p110δ inhibited Erk activation. Both p110α- and p110δ-specific inhibitors blocked cytokine secretion induced by TCR/CD3 activation with or without ICOS costimulus, but only p110α inhibitors blocked ICOS-induced cell elongation. Thus, p110α and p110δ are essential to optimal T cell activation, but their abundance and activity differentially tune up distinct ICOS signaling pathways.

Complement regulatory protein Crry/p65 costimulation expands natural Treg cells with enhanced suppressive properties in proteoglycan-induced arthritis

OBJECTIVE To investigate the costimulatory role of Crry/p65 (Crry), a membrane complement regulatory protein, on the expansion and function of natural Treg cells and their ability to ameliorate proteoglycan-induced arthritis (PGIA), an animal model of inflammatory arthritis in which the role of natural Treg cells is not well established. METHODS CD4+CD25+ natural Treg cells from BALB/c mice were activated in vitro and costimulated by Crry. The expanded cells were phenotypically characterized, and their suppressive effect on T cell proliferation was assayed in vitro. The potential prophylactic and therapeutic effects of this population versus those of natural Treg cells in PGIA were studied. The clinical score, histology, the antigen-specific isotype antibody pattern, in vitro T cell responses, and the presence of Treg cells in the paws were studied. RESULTS Crry costimulation enhanced the in vitro expansion of natural Treg cells while maintaining their phenotypic and suppressive properties. Crry-expanded Treg cells had stronger suppressive properties in vivo and a longer ameliorating effect in the PGIA model than did natural Treg cells. Crry-expanded Treg cells suppressed T cell- and B cell-dependent responses in PGIA, changing the pathogenic antibody isotype pattern and decreasing antigen-dependent secretion of cytokines, including interferon-γ, interleukin-12 (IL-12), and IL-17. Increased FoxP3 expression was detected in the paws of mice transferred with Crry-expanded Treg cells. CONCLUSION Crry-mediated costimulation facilitates in vitro expansion of natural Treg cells while maintaining their suppressive properties in vitro and in vivo in the PGIA model. These results highlight the potential of the complement regulatory protein Crry to costimulate and expand natural Treg cells capable of suppressing disease in an animal model of chronic inflammatory arthritis.

Variability of invariant mouse CD3ε chains detected by anti-CD3 antibodies

Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the α β (or γ δ) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4+ T lymphocytes and T cell lines are bound with different avidity by anti‐CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3ϵ δ to CD3ϵ γ chain pairing. Rather, they are linked to heterogeneity of the N‐terminal region of CD3ϵ chains, as detected by peptide‐specific antibodies. In turn, these differences among CD3ϵ chains correlate with variations in the strength of TCR / CD3 interaction. N‐terminal CD3ϵ heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription‐PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3ϵ N‐terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.