Differential induction of IL-17, IL-10, and IL-9 in human T helper cells by B7h and B7.1.

ICOS and CD28 are expressed by T cells and are involved in costimulation of cytokine production in T helper (TH) cells. ICOS binds B7h expressed by several cell types, whereas CD28 binds B7.1 and B7.2 expressed by activated antigen presenting cells. This work investigated the role of B7h and B7.1 in TH17 and TH9 cell differentiation by assessing activity of recombinant B7h-Fc and B7.1-Fc on human naïve TH cells activated in the presence of different combinations of exogenous cytokines. In the presence of TGF-ß1 and IL-1ß (TH17 promoting condition), B7h-Fc was more effective than B7.1-Fc in inducing IL-17A and IL-10 secretion, whereas B7.1-Fc was more effective in inducing IL-17F. Dual costimulation with B7h-Fc and B7.1-Fc displayed an intermediate pattern with predominance of IL-17F over IL-17A, secretion of high levels of IL-10, and secretion of IL-9 levels lower than those induced by B7.1-Fc alone. In the presence of TGF-ß1 and IL-4 (TH9 promoting condition), B7h-Fc induced IL-17A only, whereas B7.1-Fc induced also IL-17F, IL-10, and high levels of IL-9. Experiments on memory TH cells showed that B7h-Fc mainly supported secretion of IL-17A and IL-10, whereas B7.1-Fc supported secretion of IL-17A, IL-17F, IL-10, and IL-9. These data indicate that B7h and B7.1 play different roles in modulation of TH17 and TH9 differentiation. This plasticity might be important in the immune response to pathogens and tumors, and in the development of autoimmune diseases, and should be taken in consideration in designing of immunotherapeutic protocols triggering ICOS or CD28.

SAP-mediated inhibition of diacylglycerol kinase α regulates TCR-induced diacylglycerol signaling.

Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.

B7h triggering inhibits umbilical vascular endothelial cell adhesiveness to tumor cell lines and polymorphonuclear cells.

Vascular endothelial cells (ECs) are key players in leukocyte recruitment into tissues and metastatic dissemination of tumor cells. ECs express B7h, which is the ligand of the ICOS T cell costimulatory molecule. The aim of this work was to assess the effect of B7h triggering by a soluble form of ICOS (ICOS-Fc) on the adhesion of colon carcinoma cell lines to HUVECs. We found that B7h triggering inhibited HUVEC adhesiveness to HT29 and DLD1 cells (by 50 and 35%, respectively) but not to HCT116 cells. The effect was dependent on the ICOS-Fc dose and was detectable as early as 30 min after treatment and was still present after 24 h. It was inhibited by soluble anti-ICOS reagents (mAb and B7h-Fc) and silencing of B7h on HUVECs, and it was not displayed by an F119S mutated form of ICOS-Fc that does not bind B7h. HUVEC treatment with ICOS-Fc did not modulate expression of adhesion molecules and cytokines, but it substantially downmodulated ERK phosphorylation induced by E-selectin triggering or osteopontin, which may influence HUVEC adhesiveness. Moreover, HUVEC treatment with ICOS-Fc also inhibited adhesion of polymorphonuclear cells and several tumor cell lines from different origins. Therefore, the B7h-ICOS interaction may modulate spreading of cancer metastases and recruitment of polymorphonuclear cells in inflammatory sites, which opens a view on the use of ICOS-Fc as an immunomodulatory drug.

Role of tissue inhibitor of metalloproteinases-1 in the development of autoimmune lymphoproliferation.

BACKGROUND: Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome and its variant Dianzani's autoimmune lymphoproliferative disease. Analysis of the lymphocyte transcriptome from a patient with this latter condition detected striking over-expression of osteopontin and tissue inhibitor of metalloproteinases-1. Since previous work on osteopontin had detected increased serum levels in these patients, associated with variations of its gene, the aim of this work was to extend the analysis to tissue inhibitor of metalloproteinases-1. DESIGN AND METHODS: Tissue inhibitor of metalloproteinases-1 levels were evaluated in sera and culture supernatants from patients and controls by enzyme-linked immunosorbent assay. Activation- and Fas-induced cell death were induced, in vitro, using anti-CD3 and anti-Fas antibodies, respectively. RESULTS: Tissue inhibitor of metalloproteinases-1 levels were higher in sera from 32 patients (11 with autoimmune lymphoproliferative syndrome and 21 with Dianzani's autoimmune lymphoproliferative disease) than in 50 healthy controls (P<0.0001), unassociated with variations of the tissue inhibitor of metalloproteinases-1 gene. Both groups of patients also had increased serum levels of osteopontin. In vitro experiments showed that osteopontin increased tissue inhibitor of metalloproteinases-1 secretion by peripheral blood monocytes. Moreover, tissue inhibitor of metalloproteinases-1 significantly inhibited both Fas- and activation-induced cell death of lymphocytes. CONCLUSIONS: These data suggest that high osteopontin levels may support high tissue inhibitor of metalloproteinases-1 levels in autoimmune lymphoproliferative syndrome and Dianzani's autoimmune lymphoproliferative disease, and hence worsen the apoptotic defect in these diseases.

Defective Fas-mediated T-cell apoptosis predicts acute onset CIDP.

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are immune-mediated neuropathies. GBS is characterized by acute onset and subsequent remission of symptoms, whereas CIDP displays slow progression over at least 2 months. However, a small proportion of CIDP patients display acute onset CIDP (a-CIDP) resembling that of GBS. The Fas receptor is involved in shutting off the immune response and its defective function predisposes to auto-immune diseases. In CIDP patients, Fas function is lower than in GBS patients and healthy controls. This study is aimed at assessing whether evaluation of T-cell Fas function helps in early discrimination between GBS and a-CIDP. Fas function was evaluated in patients with acute onset polyneuropathy: 55 retrospective patients analyzed after development of GBS or a-CIDP before year 2005; 50 prospective patients analyzed at onset after year 2005 and followed up for development of GBS or a-CIDP. In both groups, a-CIDP patients displayed defective Fas function, whereas GBS patients displayed normal function. These findings suggest that the evaluation of Fas function in acute onset polyneuropathy helps in early prediction of long-term outcome.

Serum levels of osteopontin are increased in SIRS and sepsis.

OBJECTIVE: In sepsis, dysregulation of the immune response leads to rapid multiorgan failure and death. Accurate and timely diagnosis is lifesaving and should discriminate sepsis from the systemic inflammatory response syndrome (SIRS) caused by non-infectious agents. Osteopontin acts as an extracellular matrix component or a soluble cytokine in inflamed tissues. Its exact role in immune response and sepsis remains to be elucidated. Therefore, we investigated the role of osteopontin in SIRS and sepsis. DESIGN: Prospective, observational study. SETTING: Intensive care unit of a university hospital. PATIENTS AND PARTICIPANTS: Fifty-six patients with SIRS or sepsis and 56 healthy subjects were enrolled. INTERVENTIONS: We analyzed the serum levels of osteopontin and TH1-TH2 cytokines and investigated the role of osteopontin on interleukin 6 secretion by monocytes. MEASUREMENTS AND MAIN RESULTS: Serum osteopontin levels were strikingly higher in patients than in controls and in sepsis than in SIRS, and decreased during the resolution of both the disorders. Receiver operating characteristic curves showed that osteopontin levels have discriminative power between SIRS and sepsis with an area under the curve of 0.796. Osteopontin levels directly correlated with those of interleukin 6 and in vitro, recombinant osteopontin increased interleukin 6 secretion by monocytes in both the absence and presence of high doses of lipopolysaccharide. CONCLUSION: These data suggest that osteopontin might be a mediator involved in the pathogenesis of SIRS and sepsis, possibly by supporting interleukin 6 secretion. DESCRIPTOR: 45. SIRS/Sepsis: clinical studies.

ICOS gene haplotypes correlate with IL10 secretion and multiple sclerosis evolution.

Human ICOS is a T cell costimulatory molecule supporting IL10 secretion. A pilot study investigating variations of the ICOS gene 3'UTR detected 8 polymorphisms forming three haplotypes (A, B, C). Haplotype-A and -C displayed the highest difference. Activated T cells from healthy AA homozygotes expressed significantly less ICOS and secreted more IL10 than AC heterozygotes, whereas AB heterozygotes displayed intermediate levels. Analysis of 441 multiple sclerosis patients and 793 controls showed that frequency of AA homozygosity was significantly lower in MS patients with relapsing-remitting onset (N=416) than in controls (OR=0.70). Moreover, AA patients with relapsing-remitting onset had lower relapse rate and multiple sclerosis severity score than non-AA patients.

Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes.

1. The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 microg ml(-1) phytohemagglutinin plus 2 U ml(-1) interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 microg ml(-1); 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). 2. L-Glutamate (1 x 10(-8)-1 x 10(-4) M) significantly (P < or = 0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 x 10(-8) M; maximum inhibition 54.8+/-6.3% at 1 x 10(-6) M). 3. The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 x 10(-7) M for (1S,3R)-ACPD; 4.5 x 10(-8) M for quisqualate; 1.0 x 10(-6) M for (S)-3,5-DHPG; 2.0 x 10(-5) M for CHPG. 4. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 x 10(-6) M. The IC50 values calculated were: 8.7 x 10(-5), 4.3 x 10(-6) and 6.3 x 10(-7) M for AIDA, LY 367385 and MPEP, respectively. 5. L-Glutamate (1 x 10(-6) M; 18 h) significantly (P < or = 0.05) inhibited FasL expression (40.8+/-11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase-PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. 7. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms.

Osteopontin gene haplotypes correlate with multiple sclerosis development and progression.

Osteopontin (OPN) is an inflammatory cytokine highly expressed in multiple sclerosis (MS) plaques. In a previous work, we showed that four OPN polymorphisms form three haplotypes (A, B, and C) and that homozygotes for haplotype-A display lower OPN levels than non-AA subjects. In this work, we evaluated the distribution of these OPN haplotypes in 425 MS patients and 688 controls. Haplotype-A homozygotes had about 1.5 lower risk of developing MS than non-AA subjects. Clinical analysis of 288 patients showed that AA patients displayed slower switching from a relapsing remitting to a secondary progressive form and milder disease with slower evolution of disability. MS patients displayed increased OPN serum levels, which were partly due to the increased frequency of non-AA subjects. Moreover in AA patients, OPN levels were higher than in AA controls and similar to those found in both non-AA patients and controls, which suggests a role of the activated immune response. These data suggest that OPN genotypes may influence MS development and progression due to their influence on OPN levels.

Human CD38 interferes with HIV-1 fusion through a sequence homologous to the V3 loop of the viral envelope glycoprotein gp120.

CD38 is a progression marker in HIV-1 infection, it displays lateral association with CD4, and down-modulates gp120/CD4 binding. The aim of this study was to elucidate the mechanism behind the interplay between CD4, CD38, and HIV-1. We used mouse cell transfectants expressing human CD4 and either CD38 or other CD4-associated molecules to show that CD38 specifically inhibits gp120/CD4 binding. Human cell transfectants expressing truncated forms of CD38 and bioinformatic analysis were used to map the anti-HIV activity and show that it is concentrated in the membrane-proximal region. This region displayed significant sequence-similarity with the V3 loop of the HIV-1 gp120 glycoprotein. In line with this similarity, synthetic soluble peptides derived from this region reproduced the anti-HIV effects of full-length CD38 and inhibited HIV-1 and HIV-2 primary isolates from different subtypes and with different coreceptor use. A multiple-branched peptide construct presenting part of the sequence of the V3-like region potently and selectively inhibited HIV-1 replication in the nanomolar range. Conversely, a deletion in the V3-like region abrogated the anti-HIV-1 activity of CD38 and its lateral association with CD4. These findings may provide new insights into the early events of HIV-1 fusion and strategies to intervene.

Role of FAS in HIV infection.

Direct cytopathic effects cannot explain the massive CD4+ T cell depletion in acquired immunodeficiency syndrome (AIDS) patients and several indirect mechanisms may be involved. A role has been proposed for apoptosis of uninfected lymphocytes, since lymphocytes from human immunodeficiency virus-1+ (HIV-1) individuals display increased levels of spontaneous apoptosis. This process may be ascribed in part to cell exhaustion by the chronic uncontrolled infection, but can also be directly induced by viral components, such as gp120, tat or nef. A key role is played by the death receptor Fas, but a role can also be played by other death receptors, such as the TNF and TRAIL receptors. By contrast, death of HIV-infected cells seems to be Fas-independent and driven by other viral components such as vpr and HIV proteases. A further role may be played by depletion of CD4+ T cell itself and hence the withdrawal of survival factors such as cytokines. Different ability of HIV strains to induce death of infected and uninfected cells might play a role in the clinical and biological differences displayed by HIV strains. A further variability may be ascribed to the intrinsic resistance of cells to apoptosis, which may depend on the individual genetic background or the use of drugs inhibiting apoptosis. The observation that when progression of HIV infection is slow due to "apoptosis-resistant" genetic backgrounds of the patients, or defective HIV-1 strains, or successful highly active antiretroviral therapy (HAART), generally also T cell apoptosis is low, suggests that HIV-infected subjects may benefit from therapies aimed to inhibit Fas function and/or spontaneous apoptosis.

Serum cytokine profile during Mycobacterium ulcerans infection (Buruli ulcer).

BACKGROUND: Buruli Ulcer (BU) is a severe cutaneous and subcutaneous disease due to Mycobacterium ulcerans infection, mainly distributed in sub-Saharan Africa and tropical areas. The role of T helper (TH) cytokines in the development and clinical course of the disease has been previously studied by investigating the in vitro immune response of lymphocytes from affected patients and immunohistochemical analyses of bioptic samples. METHODS: TH cytokine levels (IFNγ, TNF-α, IL-2, IL-10, IL-4, IL-5, IL-17) were evaluated in serum of 34 Beninese subjects by cytofluorimetric and immunoenzymatic assays: 16 patients affected with active BU, 4 patients who had healed after specific therapy, and 14 matched controls. RESULTS: Levels of IFNγ were higher in patients with late BU (>2 months from onset) and healed patients than in controls, and in ulcerative than in pre-ulcerative patients. Analysis of 4 patients with "late" disease evaluated both at the beginning of antibiotic therapy and 6 months later showed that IFNγ levels were always lower in the second evaluation. By contrast, no differences were found in levels of the other cytokines. CONCLUSIONS: IFNγ production is low in early BU, and increases in late BU and healing, suggesting a role of this cytokine in infection clearance. Moreover, evaluation of IFNγ serum levels may be a useful tool to monitor the immune response during the BU course.

The 423Q polymorphism of the X-linked inhibitor of apoptosis gene influences monocyte function and is associated with periodic fever.

OBJECTIVE: Hereditary periodic fever syndromes (HPFs) develop as a result of uncontrolled activation of the inflammatory response, with a substantial contribution from interleukin-1beta or tumor necrosis factor alpha (TNFalpha). The HPFs include familial Mediterranean fever (FMF), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), TNF receptor-associated syndrome (TRAPS), and cryopyrinopathies, which are attributable to mutations of the MEFV, MVK, TNFRSF1A, and CIAS1 genes, respectively. However, in many patients, the mutated gene has not been determined; therefore, the condition in these patients with an HPF-like clinical picture is referred to as idiopathic periodic fever (IPF). The aim of this study was to assess involvement of X-linked inhibitor of apoptosis (XIAP), which plays a role in caspase inhibition and NF-kappaB signaling, both of which are processes that influence the development of inflammatory cells. METHODS: The XIAP gene (X-linked) was sequenced in 87 patients with IPF, 46 patients with HPF (13 with HIDS, 17 with TRAPS, and 16 with FMF), and 182 healthy control subjects. The expression of different alleles was evaluated by sequencing XIAP-specific complementary DNA mini-libraries and by real-time polymerase chain reaction and Western blot analyses. The functional effect of XIAP on caspase 9 activity was assessed by a fluorimetric assay, and cytokine secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: Sequencing disclosed a 1268A>C variation that caused a Q423P amino acid substitution. The frequency of 423Q-homozygous female patients and 423Q-hemizygous male patients was significantly higher in the IPF group than in the control group (69% versus 51%; odds ratio 2.17, 95% confidence interval 1.23-3.87, P = 0.007), whereas no significant difference was detected in the HPF group (59%) compared with controls. In primary lymphocytes and transfected cell lines, 423Q, as compared with 423P, was associated with higher XIAP protein and messenger RNA expression and lower caspase 9 activation. In lipopolysaccharide-activated monocytes, 423Q was associated with higher secretion of TNFalpha. CONCLUSION: These results suggest that 423Q is a predisposing factor for IPF development, possibly through its influence on monocyte function.

Altered expression of UVB-induced cytokines in human papillomavirus-immortalized epithelial cells.

Keratinocytes can be induced to produce cytokines by exogenous stimuli, such as UVB, and dysregulation of this production has been described in various skin diseases, including cancer. In this study, we compared the effect of UVB on the secretion of several cytokines involved in inflammation by human keratinocytes immortalized or not with human papillomavirus (HPV)16 or HPV38 at the mRNA and protein levels. We show that expression of the HPV E6/E7 oncoproteins influences not only the basal cytokine secretion profile of keratinocytes, but also its modulation upon UVB irradiation. In particular, UVB upregulates interleukin (IL)-6, IL-8 and transforming growth factor (TGF)-beta in HPV-immortalized cells to a higher extent than in control keratinocytes. Moreover, expression of other pro-inflammatory molecules such as S100A8/9 and interferon (IFN)-kappa was downregulated in HPV-immortalized cells. These data support the functional similarity between HPV16 and 38, and suggest an active role of these viruses in modulation of the inflammatory process.

ICOS cooperates with CD28, IL-2, and IFN-gamma and modulates activation of human naïve CD4+ T cells.

Several sets of data indicate that ICOS regulates cytokine production in activated T cells, but is less effective on naïve T cells. This work evaluates ICOS function in human naïve CD4+ T cells through an assessment of the effect of soluble forms of the ICOS and CD28 physiological ligands on activation driven by anti-CD3 mAb. ICOS strikingly potentiated secretion of IL-2, IFN-gamma, IL-10, and TNF-alpha, but not IL-4, promoted by optimal stimulation of CD3+CD28, and it was the key switching-factor of activation when cells received suboptimal stimulation of CD3+CD28 or stimulation of CD3 alone in the presence of exogenous IL-2. In these conditions, blockade of IL-2 and IFN-gamma showed that ICOS builds up a positive feedback loop with IFN-gamma, which required IL-2 and was inhibited by IL-4. By contrast, in the absence of CD28 triggering or exogenous IL-2, ICOS-induced costimulation mainly supported expression of TGF-beta1 and FoxP3 and differentiation of regulatory T cells capable to inhibit proliferation of naïve CD4+ T cells driven by allogeneic cells. These data suggest that ICOS favors differentiation of Th effector cells when cooperates with appropriate activation stimuli such as CD3+CD28 or CD3+IL-2, whereas it supports differentiation of regulatory T cells when costimulatory signals are insufficient.

Fas-mediated T-cell apoptosis is impaired in patients with chronic inflammatory demyelinating polyneuropathy.

The Fas death receptor is expressed by activated lymphocytes and is involved in switching-off the immune response. Its inherited defects cause auto-immune lymphoproliferative syndrome. Impaired Fas function may also play a role in other auto-immune diseases, such as multiple sclerosis and type 1 diabetes mellitus. The aim of this work was to evaluate Fas function in T cells from patients with chronic inflammatory demyelinating polyneuropathy (CIDP). We evaluated Fas-induced apoptosis in T-cell lines from 27 patients with CIDP, 12 patients with acute inflammatory demyelinating polyneuropathy (AIDP), and 110 controls. CIDP patients displayed lower Fas function than both AIDP patients and controls, whereas no statistically significant difference was found between AIDP patients and controls. Moreover, Fas function was lower in CIDP patients with progressive course than in those with relapsing-remitting course and lower in CIDP patients with axonal damage than in those with pure demyelination. These data suggest that defective Fas function favours CIDP development and aggressive evolution.

Glutamate modulation of human lymphocyte growth: in vitro studies.

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-(S,R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-gamma (+44.3+/-8.2%) and IL-10 (+31.6+/-9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-alpha was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.

High levels of osteopontin associated with polymorphisms in its gene are a risk factor for development of autoimmunity/lymphoproliferation.

The autoimmune/lymphoproliferative syndrome (ALPS) displays defective function of Fas, autoimmunities, lymphadenopathy/splenomegaly, and expansion of CD4/CD8 double-negative (DN) T cells. Dianzani autoimmune/lymphoproliferative disease (DALD) is an ALPS variant lacking DN cells. Both forms have been ascribed to inherited mutations hitting the Fas system but other factors may be involved. A pilot cDNA array analysis on a DALD patient detected overexpression of the cytokine osteopontin (OPN). This observation was confirmed by enzyme-linked immunosorbent assay (ELISA) detection of higher OPN serum levels in DALD patients (n = 25) than in controls (n = 50). Analysis of the OPN cDNA identified 4 polymorphisms forming 3 haplotypes (A, B, and C). Their overall distribution and genotypic combinations were different in patients (N = 26) and controls (N = 158) (P <.01). Subjects carrying haplotype B and/or C had an 8-fold higher risk of developing DALD than haplotype A homozygotes. Several data suggest that these haplotypes influence OPN levels: (1) in DALD families, high levels cosegregated with haplotype B or C; (2) in healthy controls, haplotype B or C carriers displayed higher levels than haplotype A homozygotes; and (3) in AB and AC heterozygotes, mRNA for haplotype B or C was more abundant than that for haplotype A. In vitro, exogenous OPN decreased activation-induced T-cell death, which suggests that high OPN levels are involved in the apoptosis defect.