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Summary To better understand the kinetics of the delayed reconstitution of peripheral CD4+ T-cells after depletion with a single administration of alemtuzumab (AL) for renal transplantation, we evaluated in these patients the percentage and absolute number of recent thymic emigrants (RTEs) CD4+ T cells, together with naive and memory subsets, defined by the analysis of CD31, CD45RA and CCR7 expression, and compared with patients treated with a nondepleting protocol based on basiliximab, and with healthy controls. In AL-treated patients, the number of circulating CD4+ T cells was greatly reduced 1 year after the infusion (P

Antiphospholipid antibodies (aPL) are known to be pathogenic in experimental models and are predictive of thrombosis and miscarriages in patients, so it is important to correctly evaluate their presence for identifying patients at risk. Despite many years of work, the standardization of aPL ELISA remains an open problem, so evaluation of newly introduced commercial preparations is mandatory. A total of 80 sera were collected (10 primary antiphospholipid syndromes (APSs), 10 APSs associated with systemic lupus erythematosus, 20 infectious diseases, 20 rheumatoid arthritis and 20 normal blood donors) and tested for IgG/IgM anti-cardiolipin and anti-beta2GPI antibodies on commercial ELISA kits (ETI) by DiaSorin and on home-made ELISA. Both methods displayed good sensitivity and specificity for APS and were found to be concordant, especially in determining antibodies of IgG class.

Aim Immune activation in severe congestive heart failure (CHF) due to idiopathic conditions is a well-known phenomenon. The question remains: is it confined to a specific aetiology or is a disease-dependent derangement? We investigated immune alterations in ischaemic compared to idiopathic patients with CHF and healthy subjects. Methods and results We evaluated alterations of immune activation studying both in vivo and in vitro parameters. In both idiopathic and ischaemic CHF patients: (a) lymphocyte count was reduced (P < 0.01); (b) lymphocyte subsets were altered (CD4/CD8 ratio increased vs. controls: 2.53 ± 0.8 and 3.3 ± 2.1% in idiopathic and ischaemic patients, vs. 1.8 ± 0.6% in controls; P < 0.01); (c) lymphocytes from patients underwent a higher apoptosis (27.1 ± 4.3% in idiopathic CHF and 25.4 ± 3.5% in ischaemic CHFvs. 4.5 ± 1.4%; P < 0.0001); (d) TNF-a production from lymphocytes and monocytes of patients was higher than controls. A positive correlation was observed between TNF-a from monocytes of patients and the relevant serum levels (r = 0.58; P < 0.01); (e) conditioned media of lymphocytes and monocytes from patients significantly increased rate of endothelial apoptosis. Conclusions In severe CHF, irrespectively from aetiology, significant activation of immune system occurs: several pro-inflammatory cytokines and soluble factors are spontaneously released in serum, possibly contributing to disease progression, as they induce apoptosis.

T cell clones are classified as type 0, 1 or 2 depending on the lymphokines they produce. However, it has remained unclear whether single cells of a given type produce one or several cytokine species. Flow cytometric analysis of peripheral blood lymphocytes (PBL) obtained from 20 healthy donors for the production of the type 1 cytokines IFN-gamma and IL-2 revealed very few cells that co-expressed both cytokines independently of the mitogenic stimulus used for PBL activation. Similarly, kinetic studies of cytokine synthesis indicated a low percentage of IFN-gamma/IL-2 double-positive T cells at all time points. Reverse transcription-PCR analysis of sorted IL-2- and IFN-gamma-positive T cells showed the presence of IL-2- or IFN-gamma-specific mRNA only in those cells expressing the corresponding cytokine. This segregation of the two type 1 cytokines was lost in long-term cultured T cells and in T cell clones. A high percentage of cells expressing only IL-2 or IFN-gamma was observed even when the production of these cytokines was evaluated on CD4- and CD8+ subsets. Moreover, in some healthy individuals, IFN-gamma and IL-2 production by CD8+ T cells was related to CD8+ expression levels and cell size, i. e. IL-2-expressing cells were generally smaller with more intense CD8+ staining as compared with IFN-gamma-producing T cells. These data indicate that activated T lymphocytes are strongly committed in vivo to produce IFN-gamma or IL-2 and emphasizes the independent regulation of the two cytokine genes.

Background: Common Variable Immunodeficiency (CVID) is characterized by defective antibody production. This has been variably attributed to intrinsic B-cell defects or to T-cell disfunctions. Recently, it has been reported that the expression of the CD40 Ligand (CD40L), a T-cell surface molecule that plays a critical role in the cell-contact-mediated helper signals provided to B-cells, is defective in a subset of patients with CVID. Methods: To demonstrate that the defective expression is due to intrinsic functional abnormalities of CD4+ lymphocytes, CD4+ cells were purified from eight patients with CVID and eight age-paired controls, stimulated with PMA+Ionomycin. and studied for CD40L expression by flow cytome-try using specific monoclonal antibodies. Results and conclusions: The percentage of CD4+ cells expressing CD40L after optimal stimulation was correlated with age both in patients with CVID (r: 0. 74: p: 0.04) and in healthy controls (r: 0. 73; p: 0.04). The percentage of CD40L+ cells was reduced in patients with CVID compared to that of controls (p: 0.02 when data are paired for age) with a reduced density of expression (p: < 0.01). The defect was variable in different patients and in some cases it was marginal.

The CD40 ligand (CD40L) is a molecule expressed by activated T cells which plays a critical role in the regulation of B-cell responses, including differentiation into Ig-producing cells. Using the specific monoclonal antibody TRAP1 we have evaluated the ontogeny of CD40L expression in 97 normal individuals between birth and 50 years of age. The expression of CD40L is a function of age; it is severely reduced at birth, progressively increases during the first months of life, and reaches a plateau in the second decade. This progressive attainment of the ability to express CD40L is due to a process of maturation of the CD4 + subset, being significantly correlated with the expression of the CD45RO antigen.

Alterations of B cell subpopulations have been described up to date as characterizing advanced stage of HIV-1 infection. However, whether such defects are relevant in subjects with a preserved number of CD4+T cells (>350 cells/μL) is unclear. In a cross-sectional study, we investigated if signs of B cells exhaustion and impaired viral immune surveillance are present in a cohort of 43 asymptomatic HIV-1-infected patients with preserved CD4+T cell counts (>350 cells/μL) and highly active antiretroviral therapy (HAART) untreated. A dramatic expansion of exhausted tissue-like memory B cells (CD10-CD21lowCD27-) was observed. B cells alteration was related to an increase in Torque teno virus (TTV) load, used as surrogate marker of immune function. Successfully HAART-treated patients showed normalization of B cell subpopulations frequency and TTV load. These results provide new insights on B cell in HIV-1 infection and show that development of B cell abnormalities precedes CD4+T cell decline.

To better understand the kinetics of the delayed reconstitution of peripheral CD4+ T-cells after depletion with a single administration of alemtuzumab (AL) for renal transplantation, we evaluated in these patients the percentage and absolute number of recent thymic emigrants (RTEs) CD4+ T cells, together with naive and memory subsets, defined by the analysis of CD31, CD45RA and CCR7 expression, and compared with patients treated with a nondepleting protocol based on basiliximab, and with healthy controls. In AL-treated patients, the number of circulating CD4+ T cells was greatly reduced 1 year after the infusion (P0.01), but the proportions of central memory, effector memory and terminally differentiated effector memory subsets among CD4+ cells were significantly increased. On the contrary, the proportion and the absolute number of naïve CD4+ T cells, although progressively increasing with time, were severely reduced. In particular, the absolute number of RTEs had only very slight increase with time (P = 0.049) and was dramatically low 1 year after the therapy (P0.01 vs. healthy controls; P0.05 vs. basiliximab-treated transplant recipients). These data suggest that a prolonged defective thymic output after AL therapy in renal transplant recipients is one of the main causes of the persistent CD4+ T-cell lymphopenia observed in these patients.

The objectives of the study were to characterize the production, function and survival of T lymphocytes of children with prolonged fetal exposure to dexamethasone for anti-Ro/SS-A antibodies associated congenital complete heart block. The analysis of thymic function, studied by measuring the level of T-cell receptor excision circles, was performed by real time PCR, the composition of T-cell subpopulation was evaluated by flow cytometry and the T-cell diversity was assayed by heteroduplex analysis. T-cell competence was gauged at two functional levels by determining the proliferation and the number of T-cell divisions and by measuring γ-interferon production after mitogenic stimulation. We observed that the thymic output, distribution of T-cell subsets, thymidine incorporation, number of T-cell divisions, and γ-interferon production were comparable to those of age-matched control. On the contrary, heteroduplex analysis demonstrated the presence of both polyclonal and oligoclonal peripheral T-cell repertoires.In conclusion, the analysis of the T-cell compartment in children with prolonged intrauterine exposure to high dose dexamethasone did not disclose any relevant abnormality, except a restriction of T-cell receptor diversity in some patients.

Impairment of purine metabolism due to adenosine deaminase (ADA) deficiency is associated with a severe combined immunodeficiency (SCID). Polyethylene glycol-modified ADA (PEG-ADA) has provided noncurative, life-saving treatment for these patients, but full immune recovery is not achieved with this therapy. Since ADA-SCID is perhaps the most difficult form of SCID to handle clinically, understanding the benefits and limitations of PEG-ADA therapy may be relevant for treatment selection. To this purpose, we analyzed the rate of thymic output, T and B cell repertoires, number of T cell divisions, IFN-gamma and IL-4 production, and the extent of cell death in five ADA-SCID patients following a prolonged period of treatment with PEG-ADA. We found that thymic output was low in these patients. However, their T cell repertoire was heterogeneous, and their T lymphocytes produced cytokines upon activation and responded to mitogen stimulation, although with different kinetics. Furthermore, a high number of peripheral T lymphocytes were committed to apoptosis. Anomalies were also observed in the B cell compartment, with oligoclonal expansions of B cell clonotypes in two patients. Our data indicate that decreased thymic function, B cell oligoclonality, and increased spontaneous apoptosis may be the mechanisms by which the immunodeficiency of ADA-SCID patients persists in spite of treatment with PEG-ADA.

In utero transplantation of hematopoietic stem cells allows immune reconstitution of fetuses with severe combined immunodeficiency. The objective of this work was to study the quality of T-cell reconstitution following this procedure.We evaluated the kinetics and extent of T-cell reconstitution in five infants with severe combined immune deficiency (SCID), three with a B+ and two with a B- phenotype, who received haploidentical stem cell transplantation before birth. To this end, we measured the frequency of T-cell receptor excision circles (TREC) and the diversity of the T-cell repertoire.In utero transplantation led to engraftment of donor-derived T lymphocytes which attained normal numbers in four infants, who are in good health. In the three patients with a B+ phenotype, generation of a heterogeneous T-cell repertoire was associated with development of TREC levels comparable to those of SCID patients treated by post-natal transplantation and of healthy babies. Of the two patients with a B- phenotype, one developed mixed T-cell chimerism and a substantial number of circulating T cells, associated with a variable heterogeneity of the T-cell repertoire; TREC levels were normal soon after birth, but declined thereafter. The remaining B- patient remained lymphopenic with a skewed T-cell repertoire and very low TREC levels. This patient eventually required transplantation from a matched unrelated donor at 5 years of age, but died of EBV-related lymphoproliferative disease.These data indicate that in utero transplantation of fetuses with B+ SCID allows generation of newly diversified T lymphocytes and ensures long-term reconstitution of cell-mediated immunity.

Objective: The SEN virus (SENV) represents a recently described group of DNA viruses, two members of which (SENV-D and SENV-H) are linked with posttransfusion hepatitis. Since patients on hemodialysis have a high risk of being infected by blood-borne viruses, we investigated the prevalence of seven SENV isolates in two distinct units of our hospital. Methods: The presence of SENV was investigated in 171 hemodialysis patients and in 163 controls by using a polymerase chain reaction based methodology, with which the specificity of amplified products was detected by hybridization with probes specific for each variant. Polymerase chain reaction products from 4 patients were sequenced. Results: The overall detection of SENV DNA as well as of SENV-D, SENV-E, and SENV-G was significantly higher in one of the two units, and there was a higher degree of homology in the sequences prepared from patients of the same unit. Furthermore, we demonstrated that mixed infections with multiple SENV were common. No relationship was observed between presence of SENV and sex, age, duration of hemodialysis, previous transfusions or transplantation, hepatitis infection, and routine liver test results. Conclusion: Our results indicate that patients who undergo hemodialysis can be at high risk of SENV transmission and suggest an intraunit transmission of specific SENV variants.

Since it has been demonstrated that concomitant infections could influence the outcome of antiviral treatment, we investigated whether the presence of SENV infection may interfere with highly active antiretroviral therapy (HAART) in HIV+ coinfected patients. In spite of persistent fluctuations in SENV-A positivity we could not find any correlation between SENV-DNA and the immunological and virologic parameters found in the patients, suggesting SENV has no apparent clinical relevance during highly active antiretroviral therapy.

Lamivudine treatment in chronic hepatitis B leads to the reconstitution of virus-specific T cells in the circulation, but it is not clear whether this is the preferential result of T cell efflux from the liver or lymph nodes. To address this question, the frequency and function of liver-, lymph node-, and blood-derived hepatitis B virus (HBV)-specific CD8 T cells were analyzed in patients treated with lamivudine and undergoing liver transplantation. HBV-specific CD8 T cells, identified in portal lymph nodes, were able to expand in vitro after antigen-specific stimulation and displayed a heterogeneous profile of cytokine production. These findings suggest that the peripherally reconstituted HBV-specific CD8 T cells can originate from precursor cells within lymph nodes.

The mechanism causing the increasing number of peripheral T cells after highly active antiretroviral therapy (HAART) is still unclear. The bcl-2 oncogene prevents spontaneous apoptosis (SA) in lymphocytes. Spontaneous apoptosis could be a determinant of HIV immunodeficiency and can be reversed by HAART including protease inhibitors (PI-HAART). The aims of our study were to measure Bcl-2 protein expression in memory (CD45RO+) and naive (CD45RO-) CD4+ and CD8+ T lymphocytes of HIV+ patients and to correlate it with efficacy of PI-HAART. Forty-nine HIV+ patients (cases) and 26 HIV- individuals (controls) were evaluated. Patients receiving PI-HAART, and who had undetectable HIV plasma viral load (VL-, n = 21), had higher levels of Bcl-2 than did VL+ patients (n = 28), both in CD4+ cells (p0.0001) and in CD8+ cells (p0.001). VL+ patients had lower Bcl-2 levels than did controls in CD8+ cells (p = 0.02), but not in CD4+ cells (p0.05). Interestingly, VL- patients had higher Bcl-2 expression than did controls both in CD4+ cells (p0.0001) and in CD8+ cells (p = 0.03). In a subcohort of the same patients, Bcl-2 was significantly higher in VL- patients (n = 10) than in controls (n = 12), both in naive CD4+ cells (p0.0001) and in naive CD8+ cells (p = 0.01). Naive CD4+ cells had higher Bcl-2 expression in VL- than in VL+ patients (p = 0.01). In a subsequent longitudinal study of nine HIV patients, naive CD4+ cells increased after effective PI-HAART (p = 0.03), which paralleled an increase in Bcl-2 expression in the same cells (p = 0.02). In conclusion, upregulation of bcl-2 could be a mechanism of immune reconstitution of naive CD4+ T cells induced by PI-HAART.

A population of Vdelta1(+)Vgamma9(-) gammadelta T cells that represented almost the totality (84%) of circulating lymphocytes in a patient with chronic, non-HIV-related, CD4 lymphocytopenia complicated by a disseminated Mycobacterium intracellulare infection was characterized. These gammadelta(+) T cells expressed a single killer inhibitory receptor (CD158b) and their phenotype (CD8(+)CD57(+)CD27(-)CD28(-)) indicated that, although CD45RA(+), they were not naive. However, the absence of large granular lymphocyte morphology, the impaired proliferative activity, the high susceptibility to apoptosis, and the total lack of cytotoxic ability suggested that these gammadelta cells were in a resting state. A high percentage of the cells did not harbor the CD11b integrin alpha chain and exhibited a decreased capability to bind endothelial cells. This defect might represent the mechanism whereby they remained trapped in the circulation.

In vitro spontaneous apoptosis (SA) of lymphocytes was studied in HIV infection to evaluate possible clinical and prognostic correlations, in a transsectional study of 101 individuals in different clinical categories and in a prospective longitudinal study of 18 asymptomatic individuals (mean follow-up, 17.2 months). The rate of SA was higher in HIV+ patients than in healthy controls (p < 0.001) and was higher in patients with AIDS than in the other HIV+ individuals (p < 0.001). It was inversely correlated with the peripheral blood CD4+ (R -0.61; p < 0.001) and CD8+ (R -0.46; p < 0.001) cell numbers. In a group of long-term survivors (LTS), it was significantly lower than in a control group of asymptomatic HIV+ patients with a similar number of circulating CD4+ lymphocytes but a shorter follow-up (p < 0.02). In the five asymptomatic HIV-infected individuals who showed a clinical progression, peaks of SA rates above the normal range before the clinical event were much more frequent than in those who remained asymptomatic (p < 0.0001), even though they were fairly homogeneous as far as CD4+ cell count and viral load were concerned. The median levels of SA rates were moreover correlated with the rate of total T cell loss (R -0.46; p 0.053). This study suggests that evaluation of the SA levels may provide a predictive factor for clinical and immunological progression of HIV-related immunodeficiencies and strengthen the hypothesis for the role of this phenomenon in the pathogenesis of this progression.

SUMMARY To evaluate whether vertical HIV infection interferes with the expression of CD28 on T lymphocytes, 25 HIV-infected children and 29 seroreverted children born to HIV+ mothers were studied. The percentage of CD28− cells among CD8+ T lymphocytes was higher in HIV-infected children than in controls (P

A major feature of the immature immune system in the newborn is its inability to produce significant levels of immunoglobulins other than IgM in response to antigens. It has recently been demonstrated that interaction of the CD40 molecule on B cells with the CD40 ligand (CD40L) on activated T cells is pivotal for immunoglobulin switching. In view of these findings, we have tested cord blood mononuclear cells (CBMC) for expression of CD40L. Our data clearly demonstrate that freshly isolated CBMC do not express significant levels of CD40L upon in vitro activation; this defect is intrinsic to CD4+ cord blood lymphocytes. In vitro priming of CBMC with phytohemagglutinin and interleukin-2 for several days induced a conversion from the "naive" to the "memory" phenotype (as assessed by expression of CD45 isoforms) and led to substantial CD40L expression upon appropriate restimulation. These data indicate that ineffective CD40L expression might represent a major factor for reduced immunoglobulin production in the neonate, and suggest that antigenic exposure in vivo leads to changes in the genetic program, enabling T cells to express CD40L.