Galectin-1-induced down-regulation of T lymphocyte activation protects (NZB x NZW) F1 mice from lupus-like disease.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by a hyperactive immune system, including activation of autoreactive T and B cells. These studies demonstrate that administration of recombinant galectin-1, a ß-galactose binding protein, to SLE-prone (NZB × NZW) F1 mice reduced lymphocyte activation, inhibited serum anti-double-stranded DNA(dsDNA) IgG antibody production, decreased the incidence of proteinuria, and increased survival rate. In addition, recombinant galectin-1'-treated mice had a higher frequency of Foxp3 expression, which suggested an increase in the percentage of peripheral regulatory T cells. Consistent with the finding that there were fewer activated T lymphocytes, ex vivo T cells from mice treated with recombinant galectin-1 exhibited less proliferation in response to TCR stimulation. Furthermore, these cells were less efficient at lipid raft clustering in response to TCR/CD28 engagement, consistent with published reports that galectin-1 can reorganize the synaptic contact to interfere with TCR signaling and activation to prevent T cell activation. Aged galectin-1-deficient mice had higher serum levels of antibodies against dsDNA, elucidating a role for endogenous galectin-1 in decreasing susceptibility to autoimmunity. Together, the findings highlight galectin-1 as a novel potential therapeutic immune modulator for treatment of lupus-like disease.

Co-stimulation and counter-stimulation: lipid raft clustering controls TCR signaling and functional outcomes.

T cell receptor (TCR) antigen recognition induces the formation of a specialized 'immunological synapse' at the T cell : antigen presenting cell (APC) junction. This junction is generated by the recruitment and exclusion of particular proteins from the contact area and is required for T cell activation. We and others have hypothesized that lipid raft/non-raft partitioning provides a molecular basis for protein sorting which organizes the TCR, co-stimulators, signal transducers and the actin cytoskeleton at the T cell : APC interface. Here we discuss the emerging paradigm that co-stimulators induce the directional transport and clustering of lipid rafts at the T cell : APC interface, thus generating platform(s) specialized for processive and sustained TCR signal transduction and T cell activation. We also discuss recent data implicating the involvement of 'counter-stimulators' and other negative regulators which prevent optimal raft clustering at the TCR contact site and, thus, facilitate T cell inactivation and tolerance induction.

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events.

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.

Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction.

Galectin-1 is an endogenous lectin with known T cell immunoregulatory activity, though the molecular basis by which galectin-1 influences Ag specific T cell responses has not been elucidated. Here, we characterize the ability of galectin-1 to modulate TCR signals and responses by T cells with well defined hierarchies of threshold requirements for signaling distinct functional responses. We demonstrate that galectin-1 antagonizes TCR responses known to require costimulation and processive protein tyrosine phosphorylation, such as IL-2 production, but is permissive for TCR responses that only require partial TCR signals, such as IFN-gamma production, CD69 up-regulation, and apoptosis. Galectin-1 binding alone or together with Ag stimulation induces partial phosphorylation of TCR-zeta and the generation of inhibitory pp21zeta. Galectin-1 antagonizes Ag induced signals and TCR/costimulator dependent lipid raft clustering at the TCR contact site. We propose that galectin-1 functions as a T cell "counterstimulator" to limit required protein segregation and lipid raft reorganization at the TCR contact site and, thus, processive and sustained TCR signal transduction. These findings support the concept that TCR antagonism can arise from the generation of an inhibitory pp21zeta-based TCR signaling complex. Moreover, they demonstrate that TCR antagonism can result from T cell interactions with a ligand other than peptide/MHC.

Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation.

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.

Engagement of GPI-linked CD48 contributes to TCR signals and cytoskeletal reorganization: a role for lipid rafts in T cell activation.

GPI-linked proteins coassociate with intracellular tyrosine kinases in "lipid rafts" proposed to function as platforms for signal transduction and cytoskeletal reorganization. TCR activation requires both tyrosine kinase signals and cytoskeletal reorganization. How receptor engagement initiates cytoskeletal changes remains poorly understood. We investigated the consequences of recruiting GPI-linked CD48 and associated rafts to the site of T cell:APC contact by stimulating T cells with APCs that express the CD48 ligand CD2. We demonstrate that CD2:CD48 interactions enhance TCR-mediated functions. CD48/TCR coengagement qualitatively and quantitatively enhances lipid raft-dependent zeta association with the actin cytoskeleton and zeta tyrosine phosphorylation. This implicates lipid rafts as sites where receptor-induced signals and cytoskeletal reorganization are integrated and reveals a novel component of accessory molecule function.

The Lck SH2 phosphotyrosine binding site is critical for efficient TCR-induced processive tyrosine phosphorylation of the zeta-chain and IL-2 production.

The lymphocyte-specific tyrosine kinase Lck is essential for TCR-mediated signal transduction. This is in part due to its enzymatic activity as a tyrosine kinase responsible for TCR-induced tyrosine phosphorylation of zeta and CD3 receptor subunits. In addition to its catalytic domain, the Lck protein contains SH3 and SH2 domains capable of associating with other signaling molecules. It has been proposed that phosphotyrosine binding by the Lck SH2 domain may enhance substrate tyrosine phosphorylation by facilitating the processive phosphorylation of multiple sites within the TCR complex. Alternatively or additionally, it may function in adapter activity for facilitating required protein-protein interactions. Previous experiments demonstrate that overexpression of a constitutively activated form of Lck (F505) in the BI-141 T cell hybridoma leads to the Lck kinase activity-dependent enhancement of TCR-mediated signals. Here we demonstrate that mutation of amino acids important for SH2 phosphotyrosine binding significantly compromises the ability of F505 to enhance TCR-mediated protein tyrosine phosphorylation and Ag-induced IL-2 production in BI-141. Examination of the effects of TCR-regulated phosphorylation of the Lck substrate zeta provides in vivo evidence for a role for the Lck SH2 domain in the processive phosphorylation of a multiply phosphorylated substrate.

T cell antigen receptor-induced IL-2 production and apoptosis have different requirements for Lck activities.

The T cell hybridoma BI-141 has been previously used to dissect the roles of Lck in Ag-induced IL-2 production. Here we demonstrate that BI-141 undergoes apoptosis in response to TCR stimulation using Ag or anti-TCR Abs. Using a panel of BI-141 transfectants expressing constitutively activated Lck (F505) or phosphotyrosine-binding (K154F505 and C156F505) or kinase-impaired (R273F505) mutants, we assess the relative requirements for Lck in TCR-mediated IL-2 production and apoptosis. While BI-141 transfectants expressing F505 are dramatically enhanced in their ability to produce IL-2 in response to Ag relative to K154F505-, C156F505-, or R273F505-expressing transfectants, no differences between these transfectants are observed in their ability to undergo TCR-induced apoptosis. TCR-induced Fas ligand (FasL) expression is demonstrated to be dependent on Lck SH2 and kinase activities, although FasL expression cannot be correlated with apoptosis. Low levels of Fas are constitutively expressed at equal levels on transfectants and are not increased in response to TCR ligation. Together, these data indicate that TCR-induced apoptosis in BI-141 is regulated through a mechanism(s) distinct from the Lck-induced expression of Fas, FasL, or IL-2 production. This TCR signal may be independent of Lck kinase and SH2 activities, or may require lower threshold activity. The identification of differential requirements for particular T cell functions is crucial to understanding how TCR engagement affects downstream T cell functions and may aid in the rational design of therapeutics aimed at specifically modulating particular T cell responses.

Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 and has been shown to play a role in B cell proliferation and differentiation. Using the polymerase chain reaction we have isolated and sequenced cDNA clones encoding the serologically defined mouse Lyb-2a, Lyb-2b, and Lyb-2c alleles. We confirmed that our full length cDNA clones encode the Lyb-2a, -2b, and -2c alleles, respectively, by transfecting the isolated Lyb-2/CD72 cDNA clones into L cells and demonstrating that the transfectants bind only the appropriate allele specific anti-Lyb-2/CD72 antibodies. Sequence comparisons demonstrate that the Lyb-2/CD72 allels are highly conserved in their cytoplasmic and transmembrane domains but exhibit a high degree of polymorphism in their extracellular domains. This polymorphism in the extracellular region involves amino acid substitutions at a minimum of 20 residues and is concentrated primarily in the membrane distal region. cDNA sequence comparisons also demonstrate two distinct seven amino acid insertion/deletions among these allelic variants. A form of Lyb-2b cDNA lacking the sequence encoding the transmembrane region was isolated from a C57B1/6 mouse and a CH12.LX subline. The Lyb-2/CD72 PCR products from mRNA of mice expressing Lyb-2a and Lyb-2c contain a DNA fragment that corresponds in size to the transmembraneless form, suggesting that these mouse strains also express this mRNA.

The roles of CD4 and CD8 in T cell activation.

CD4 and CD8 T cell surface molecules play a role in T cell recognition and activation by binding to their respective class II and class I major histocompatibility complex (MHC) ligands on an antigen presenting cell (APC). Though CD4 and CD8 are capable of binding to MHC molecules in the absence of the T cell receptor (TCR), increasing evidence suggests that they may primarily function by complexing with the TCR to form a 'co-receptor' for recognition of antigen-bound MHC. Using gene transfer studies we have demonstrated that CD4 and CD8 can augment antigen-induced IL-2 production through different mechanisms dependent on whether or not they can bind MHC independently of the TCR or complexed with the TCR. Under circumstances where CD4 and CD8 can bind to the same MHC ligand as the TCR, they potentiate antigen-induced IL-2 production maximally by a mechanism in large part dependent on their cytoplasmic tails. Enhancement of antigen-induced IL-2 production can also occur under circumstances where CD4 and CD8 bind on MHC ligand distinct from that recognized by the TCR. In this instance, the magnitude of this enhancement is not as great and appears (at least for CD8) to be independent of the cytoplasmic tail and the associated p56lck. The dependence of co-receptor function on the cytoplasmic tail of CD4 or CD8 may reflect the activity of the associated intracellular tyrosine kinase p56lck.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesion versus coreceptor function of CD4 and CD8: role of the cytoplasmic tail in coreceptor activity.

CD4 and CD8 play an important role in T-cell recognition and activation; however, their mechanisms of action are not well understood. We compare the effects of expressing CD4 and CD8 alpha either individually or together in a class II-restricted T-cell hybridoma. We also compare the effects of expressing truncated forms of CD4 or CD8 alpha that do not have a cytoplasmic tail and thus do not associate with the T-cell-specific tyrosine kinase p56lck, which has been implicated in T-cell activation. We demonstrate that, although CD4 and CD8 alpha can specifically enhance interleukin 2 secretion, maximal potentiation occurs with expression of CD4, which, unlike CD8, can bind to the same major histocompatibility complex protein as the T-cell receptor. Our data further indicate that the cytoplasmic tail and/or the associated p56lck are primarily significant for interleukin 2 secretion by the hybridomas we have examined when CD4 or CD8 can bind to the same major histocompatibility complex ligand as the T-cell receptor.

Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck.

Lymphocyte-specific tyrosine protein kinase p56lck is physically associated with CD4 and CD8 T-cell surface molecules, suggesting that it may transduce CD4/CD8-triggered tyrosine phosphorylation signals during antigen stimulation. Indeed, antibody-mediated aggregation of CD4 (to mimic interaction with its ligand, major histocompatibility complex (MHC) class II molecules), rapidly elevates the kinase activity of p56lck and is associated with marked changes in tyrosine protein phosphorylation. Genetic analyses suggest that the interaction of CD4/CD8 with p56lck results in a positive signal during antigen-induced T-cell activation. To evaluate directly the role of p56lck in T-cell activation, we introduced a constitutively activated form of Lck protein (tyrosine 505 to phenylalanine 505 mutant); in a CD4-negative, MHC-class II restricted mouse T-cell hybridoma. We report here that, as for transfection of CD4, expression of the Lck mutant enhanced T-lymphocyte responsiveness. This finding provides direct evidence that p56lck can positively regulate T-cell functions and that it mediates at least some of the effects of CD4 and CD8 on T-cell activation.

Equivalence of human and mouse CD4 in enhancing antigen responses by a mouse class II-restricted T cell hybridoma.

We have examined the ability of hCD4 to interact functionally with mouse class II MHC molecules using the mouse T cell hybridoma BI-141, specific for beef insulin. We have previously shown that expression of mouse CD4 results in a marked enhancement of IL-2 release by BI-141 cells in response to beef insulin or, in a cross-reactive response, to pork insulin, on the appropriate mouse APCs. We now demonstrate that expression of hCD4 results in an equivalent stimulation of antigen responses by this mouse T cell hybridoma. The specificity of this effect was demonstrated by mAb and gp120 blocking studies. These data provide the first direct evidence for function of hCD4 and in an exclusively mouse system.

[Epidemiologic data on breast feeding in Florence and the province. Survey carried out in 1364 children born between 1985 and 1987]. / Dati epidemiologici sull'allattamento al seno in Firenze e provincia. Indagine condotta su 1364 bambini nati tra il 1985 ed il 1987.

We have studied the breast-feeding frequency in Florentine Area. We investigate a group of 1364 children born between January 1985 and June 1987. We collected following data: 1) Kind of feeding at birth. 2) Duration of breast-feeding related with birth-weight, kind of delivery, mother's age, mother's working activity. 3) Growth in relation to feeding practices from birth to 3rd month. The percentage of infants breast-feed in the hospital was 81.6%, 50.5% at the end of third month, 21.9% at the end of sixth month. We have found that children weighing more than 3000 g at birth had an higher frequency of breast-feeding at birth and last longer than children weighing less than 3000 g (p less than 0.01). Maternal age had a clear effect on both the incidence and the duration of breast-feeding. Mother aged 30 years or more begin to breast-feed in lower percentage but they continue breast-feeding for a longer time. One of the factors that has a negative influence on breast-feeding at birth is caesarean section since it precludes early mother-infant contact and early initiation of breast-feeding. Mother's resume working has a negative influence on breast-feeding duration. With regard to growth in first three months of life related with different kind of feeding we have found no differences between breast-feeding and artificial-fed infants.

T cell receptor beta-chain selection in human allograft rejection.

We have analyzed a series of T cell lines established from renal needle biopsies taken from renal allograft recipients with clinical signs of rejection. These T cells show strong cytotoxicity directed against donor HLA and their proliferative capacity in vitro is highly correlated with irreversible graft rejection. A total of 10 of 12 lines examined by Southern blot analysis using a J beta C beta DNA probe show predominant beta-chain rearrangements. In one instance DNA was isolated from cell lines generated from sequential biopsies taken from the same patient at different times of rejection. These lines show the same predominant beta-chain rearrangements. To determine whether these predominant rearrangements are due to expansion of a single clone or different T cell clones rearranging similar beta-chains, the same blots were analyzed with a J gamma probe. Cell line MH3 shows two predominant beta-chain rearrangements and at least seven of eight possible rearranged gamma-chain bands, implying that multiple clones share similar beta-chains. In contrast, the cell line King shows a single beta-chain and a single gamma-chain rearrangement. Many of the other cell lines fall between these two extremes, indicating that both beta-chain selection and clonal dominance are operating during graft rejection, resulting in the appearance of predominant beta-chain rearrangements.

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. I. Suppressor cell induction, function, and specificity.

The functional characteristics of T suppressor (Ts) cells generated from the peripheral blood lymphocytes (PBL) of a kidney transplant recipient who had excellent graft function for 1 year were examined. Ts cells were induced by co-culture of PBL with an autologous alloreactive cytotoxic T lymphocyte (CTL) line (EE-1) previously grown from a routine renal allograft biopsy of this patient performed 10 days posttransplant. The EE-1 line included CD3+ T cells of CD8+ and CD4+ phenotypes with cytotoxic specificity for disparate class 1 (HLA-B8) and class II (HLA-DR1 and 3) antigens of the kidney donor (JC). The EE-1 induced Ts cell lines (designated TsEE) were found to significantly suppress (50%-95%) autologous fresh responder EE-PBL stimulation by donor EBV-transformed cells (JC-EBV) in mixed lymphocyte reaction (MLR) assay. TsEE cells were CD3+ (98%) and predominantly CD8+ (68-80%), showed no cytotoxic activity, and were suppressive only at the early phase of MLR stimulation. In three-party cell test MLR assays, TsEE-mediated suppression appeared restricted to responder cells sharing HLA-B7 with the suppressor line, and was not abrogated by the addition of exogenous interleukin-2 (IL-2). TsEE cells also showed restricted suppression of CTL generation but not mature CTL activity. The restricted suppressor activity of TsEE lines was dependent upon their induction and restimulation with the autologous EE-1 line.

Human allograft-derived T-cell lines: donor class I- and class II-directed cytotoxicity and repertoire stability in sequential biopsies.

In order to characterize directly the T-cell repertoire utilized in human renal allograft rejection, we analyzed 20 long-term T-cell lines established from lymphocytic infiltrates present in renal tissue obtained by needle biopsy from transplant patients with rejection indications. All cell lines are strongly cytotoxic against one or more of the HLA antigens for which the kidney donor and the recipient were mismatched. Traditionally, cytotoxicity in allograft rejection has been attributed to CD8-positive cytotoxic T lymphocytes (CTLs) directed against class I alloantigens. Cell lines described here are mixtures of distinct CD4-antigen-positive and CD8-antigen-positive subpopulations, and exhibit class I- as well as class II-directed killing. Using a cell line that demonstrates class II-directed cytotoxicity and a set of class II deletion mutants as target cells, we show that class II antigens, in particular HLA-DP, can serve as targets in renal allograft rejection. The role of CD4-positive CTLs was shown by analysis of clonal populations or sorted CD8-positive and CD4-positive subpopulations. In several instances we have obtained cell lines from serial biopsies performed on the same patient at distinct time points during an ongoing rejection. Comparisons of the phenotype, function, and specificities allow for speculation regarding T-cell population dynamics within the rejection response.