Francesca Mazzetta

Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency

We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.

Hepatitis C Virus Drives the Unconstrained Monoclonal Expansion of VH1–69-Expressing Memory B Cells in Type II Cryoglobulinemia: A Model of Infection-Driven Lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the VH1–69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of VH1–69+ cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of VH1–69+ B cells: naive (small, IgMhighIgDhighCD38+CD27−CD21highCD95−CD5−), “early memory” (medium-sized, IgMhighIgDlowCD38−CD27+CD21lowCD95+CD5+), and “late memory” (large-sized, IgMlowIgDlow-negCD38−CD27lowCD21low-negCD5−CD95−). The B cells expanded in cryoglobulinemia patients have a “memory” phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated VH1–69+ B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal VH1–69+ B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a VH1–69+ natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.

NK cell activity controls human herpesvirus 8 latent infection and is restored upon highly active antiretroviral therapy in AIDS patients with regressing Kaposi's sarcoma.

Kaposis sarcoma (KS) develops upon reactivation of human herpesvirus 8 (HHV8) infection and virus dissemination to blood and tissue cells, including endothelial and KS spindle cells where the virus is mostly present in a latent form. However, this may likely require the presence of compromised host immune responses and/or the evasion of infected cells from the host immune response.In this regard, mechanisms of evasion of productively infected cells from both CTL and NK cell responses, and resistance of latently infected cells from specific CTL, have already been shown. Here we show that cells which are latently infected by HHV8 are indeed efficiently lysed by NK cells from individuals with a normal immune response. Notably, NK cell‐mediated immunity was found to be significantly reduced in AIDS patients with progressing KS as compared to both HIV‐negative patients with indolent classic KS or normal blood donors. However, it was restored after treatment with the highly active antiretroviral therapy (HAART) in AIDS‐KS patients, that showed regression and clearance of HHV8 from PBMC. By contrast, AIDS‐KS patients with a more aggressive disease and no clinicalresponse had persistent HHV8 viremia associated with reduced NK cell cytotoxicity. These results suggest a key role for NK cells in the control of HHV8 latent infection, KS development, and in disease remission upon HAART.

Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by repeated infections and hypogammaglobulinaemia. Additionally, T‐cell abnormalities including lymphopenia, decreased proliferation to mitogens and antigens, and the reduced production and expression of cytokines, have also been observed. In this study we have investigated the expression of naive, memory and activation markers in T‐cell subpopulations in 17 CVID patients in comparison to age‐matched normal controls. The numbers of CD4+ T cells, including CD45RA+CD62L+ and, to a lesser extent, CD45RA–CD62L+/RA+CD62L– were significantly reduced in patients, whereas CD8+ T cells were within normal range. In contrast, HLA‐DR+ cells were increased both in CD4+ and CD8+ T cells. To assess the thymic output, we analysed the presence of T‐cell receptor excision circles (TRECs) in CD4+ and CD8+ T cells by quantitative PCR. TRECs were decreased significantly in patients and the rate of TREC loss was higher with increasing age. TRECs correlated with naive CD4+ T cells, whereas there was an inverse relationship between TRECs and CD8+HLA–DR+ and CD8+CD45RA–CD62L+/RA+CD62L– T cells. Our results suggest the presence of a defect in the naive T cell compartment with origin at the thymic level in CVID, and indicate that TREC may be a useful marker to monitor thymic function in this primary immunodeficiency.

Biased T-cell receptor repertoires in patients with chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome)

Chromosome 22q11.2 deletion (del22q11.2) syndrome (DiGeorge syndrome/velocardiofacial syndrome) is a common syndrome typically consisting of congenital heart disease, hypoparathyroidism, developmental delay and immunodeficiency. Although a broad range of immunologic defects have been described in these patients, limited information is currently available on the diversity of the T‐cell receptor (TCR) variable β (BV) chain repertoire. The TCRBV repertoires of nine patients with del22q11.2 syndrome were determined by flow cytometry, fragment size analysis of the third complementarity determining region (CDR3 spectratyping) and sequencing of V(D)J regions. The rate of thymic output and the phenotype and function of peripheral T cells were also studied. Expanded TCRBV families were detected by flow cytometry in both CD4+ and CD8+ T cells. A decreased diversity of TCR repertoires was also demonstrated by CDR3 spectratyping, showing altered CDR3 profiles in the majority of TCRBV families investigated. The oligoclonal nature of abnormal peaks detected by CDR3 spectratyping was confirmed by the sequence analysis of the V(D)J regions. Thymic output, evaluated by measuring TCR rearrangement excision circles (TRECs), was significantly decreased in comparison with age‐matched controls. Finally, a significant up‐regulation in the percentage, but not in the absolute count, of activated CD4+ T cells (CD95+, CCR5+, HLA‐DR+), IFN‐γ ‐ and IL‐2‐expressing T cells was detected. These findings suggest that the diversity of CD4 and CD8 TCRBV repertoires is decreased in patients with del22q11.2 syndrome, possibly as a result of either impaired thymic function and/or increased T‐cell activation.

CCR5 and CXCR4 chemokine receptor expression and β-chemokine production during early T cell repopulation induced by highly active anti-retroviral therapy

Expression of chemokine receptors and β‐chemokine production by peripheral blood mononuclear cells (PBMC) were determined in HIV‐1‐infected individuals before and after highly active anti‐retroviral therapy (HAART) and their relationship to viral load, T cell phenotype and the expression of immunological activation markers was examined. We found that the expression of CCR5 is up‐regulated in HIV‐1‐infected individuals while CXCR4 appears down‐regulated on both CD4 and CD8 T cells compared with normal controls. These alterations are associated with the high levels of viral load. In addition, a relationship was observed between the degree of immune activation and chemokine receptor expression on T cells. However, after 3 months of combined anti‐retroviral regimen, expression of CXCR4 significantly increased while CCR5 decreased when compared with pretherapy determinations. This was seen in strict association with a dramatic decrease of viral load and an increase of both CD45RA+/CD62L+ (naive) and CD45RA−/CD62L+ or CD45RA+/CD62L− (memory) T cells accompanied by a significant decrease of the expression of immune activation markers such as HLA‐DR and CD38. At enrolment, both spontaneous and lectin‐induced RANTES, macrophage inflammatory protein‐1α (MIP‐1α) and MIP‐1β production by PBMC were higher in HIV‐1‐infected individuals compared with normal controls, although differences for MIP‐1β were not statistically significant. However, RANTES and MIP‐1α production decreased during HAART at levels closer to that determined with normal controls, while MIP‐1β production was less consistently modified. These data indicate that the expression of chemokine receptors CCR5 and CXCR4 and the production of β‐chemokines are altered in HIV‐infected individuals, and suggest that their early modifications during HAART reflect both the peripheral redistribution of naive/memory T cell compartments and the decrease in levels of T cell activation. Such modifications in the expression of host determinants of viral tropism and the production of anti‐viral molecules may play a role in the emergence of virus variants when a failure of HAART occurs.

T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity

The immunological correlates of highly active antiretroviral therapy (HAART)‐induced suppression of human immunodeficiency virus type 1 (HIV‐1) replication have been investigated.

Changes in CCR5 and CXCR4 expression and β-chemokine production in HIV-1-infected patients treated with highly active antiretroviral therapy

&NA; The effect of highly active antiretroviral therapy (HAART) on the expression of CCR5 and CXCR4 HIV coreceptors and the production of the &bgr;‐chemokines regulated upon activation, normal T‐cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)‐1&agr;, and MIP‐1&bgr; has been investigated in 30 HIV‐1‐infected individuals during 12‐36 months of therapy. CCR5 expression was increased in both CD4+ and CD8+ subsets, whereas CXCR4 expression was upregulated only in CD4+ cells. CCR5 levels normalized during 36 months of therapy and positively correlated with the levels of memory, CD95+, and HLA‐DR+ T cells. In contrast, the frequency of CXCR4‐expressing cells was not significantly modified by HAART, although a downregulation was observed early after starting treatment. CXCR4 levels were significantly associated with the frequencies of naive T cells and negatively correlated with plasma viral load, CD95, and HLA‐DR expression. An increased production of both spontaneous and lectin‐induced RANTES, MIP‐1&agr;, and MIP‐1&bgr; was found at baseline in HIV‐infected individuals. The spontaneous &bgr;‐chemokines production was not modified by 12 months of HAART, although a significant reduction was seen during the first months of therapy. A transient decrease of lectinstimulated RANTES production was also observed, whereas the reduction of lectininduced MIP‐1&agr; persisted for up to 12 months of therapy. In contrast, MIP‐1&bgr; secreted by phytohemagglutinin antigen‐stimulated peripheral blood mononuclear cells progressively increased during HAART. In conclusion, our data indicate a normalization of CCR5 but not CXCR4 expression during suppressive therapy and changes in &bgr;‐chemokine production that may play a part in dictating the efficiency of viral infection and consequently the disease course.

Free peritoneal tumor cells detection in gastric and colorectal cancer patients

Free peritoneal tumor cells (FPTC) derive from the detachment of primary cancer and may result in peritoneal carcinomatosis. Since peritoneal lavage cytology has low sensitivity in detecting FPTC, our aim was to estimate the clinical relevance of FPTC detected using an approach based on multiple molecular techniques.

Persistently biased T-cell receptor repertoires in HIV-1-infected combination antiretroviral therapy-treated patients despite sustained suppression of viral replication

In most HIV-1-infected patients, highly active antiretroviral therapy (HAART) reduces plasma viral load to <50 copies/mL and increases CD4+ T-cell number and function. However, it is still unclear whether alterations of T-cell receptor (TCR) beta-chain variable region (BV) repertoire, tightly related to disease progression, can be fully recovered by long-term treatment with HAART. This study analyzed the evolution of both T-cell subset composition and TCRBV perturbations in chronically HIV-1-infected patients with moderate immunodeficiency during 36 months of HAART. Despite persistently suppressed HIV replication, the rate of CD4+ T-cell repopulation, after an initial burst, progressively declined throughout the study period, resulting in a mean CD4+ T-cell count at the end of follow-up that was still significantly lower in HIV patients than in HIV-seronegative controls. This was seen in association with an incomplete restitution of both CD4 and CD8 TCRBV repertoire disruptions and was also demonstrated by the appearance of new TCRBV oligoclonal expansions occurring during HAART. In conclusion, these data indicate that 3 years of fully suppressive HAART may be not adequate to normalize CD4 counts and TCRBV repertoires in patients starting HAART with moderately advanced disease.