Alessandra Sottini

The Different Extent of B and T Cell Immune Reconstitution after Hematopoietic Stem Cell Transplantation and Enzyme Replacement Therapies in SCID Patients with Adenosine Deaminase Deficiency

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle+ lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA–treated patients, it was accompanied by a significant and progressive decrease in circulating CD19+ lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle+ cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.

Dacarbazine Treatment before Peptide Vaccination Enlarges T-Cell Repertoire Diversity of Melan-A―Specific, Tumor-Reactive CTL in Melanoma Patients

Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse.

Detection of skewed T-cell receptor V-β gene usage in the peripheral blood of patients with multiple sclerosis

The ex vivo analysis of the T-cell receptor V-beta (TCRBV) gene usage by circulating T lymphocytes in Multiple Sclerosis (MS) patients may contribute to understanding disease pathogenesis. In the present study, TCRBV gene usage was analyzed in freshly collected unstimulated peripheral blood mononuclear cells (PBMC) isolated from 40 MS patients and 20 healthy controls. Nine patients presented abnormal repertoires, with expansion of one or more TCRBV segments. Among these patients, six presented expansion of TCRBV9 chain expression, three also having an expansion of TCRBV1, TCRBV11 and TCRBV22 segments. The most frequently observed TCRBV chain expansion, TCRBV9, was further analyzed and identified as polyclonal. Evaluation of clinical variables showed that median disease duration was shorter in patients with TCRBV gene expression abnormalities. Longitudinal evaluation of five patients with a skewed repertoire showed regression of expanded TCRBV chains expression to normal values. These data indicate that certain MS patients have abnormal TCRBV gene expression. Such abnormalities are caused by polyclonal expansions of T lymphocyte subpopulations that use the same TCRBV gene families, are unstable and preferentially observed early in the course of the disease.

IFNβ bioavailability in multiple sclerosis patients: MxA versus antibody-detecting assays ☆

Anti-IFNbeta antibodies are related to IFNbeta bioavailability loss in multiple sclerosis. We investigated the reliability of radioimmunoprecipitation and cytopathic assays in detecting binding (BAbs) and neutralizing (NAbs) antibodies and the correlation of these antibodies to MxA mRNA production. Eleven percent of IFNbeta-treated patients showed a lack of MxA induction, with an inverse correlation between MxA mRNA and the presence of BAbs and NAbs. Some patients had contemporary MxA induction in the presence of high NAb titres, thus calling into question the reliability of cytopathic assay. Since anti-IFNbeta antibodies well correlated with MxA induction loss, MxA assay is an appropriate test to determine IFNbeta bioavailability.

Insights into SEN Virus Prevalence, Transmission, and Treatment in Community- Based Persons and Patients with Liver Disease Referred to a Liver Disease Unit

To document the prevalence and routes of transmission of SEN virus (SEN-V) in community-based individuals and patients referred to a liver disease unit, stored serum samples obtained from 160 Canadian Inuit and 140 patients with liver disease were tested for SEN-V DNA by polymerase chain reaction. In the community-based population, SEN-V was present in 57 (36%) of 160 persons. SEN-V-positive individuals tended to be younger and were more often male. Liver enzyme levels and serologic markers for hepatitis A and B viruses were similar in SEN-V-positive and SEN-V-negative individuals. SEN-V was present in 30 (21%) of the 140 patients with liver disease. Age, sex, risk factors for viral acquisition, prevalence of symptoms, and liver biochemical and histological findings were similar in SEN-V-positive and SEN-V-negative patients. These results indicate that SEN-V infection is a common viral infection in both healthy individuals and patients with chronic liver disease, that transmission likely occurs via nonparenteral routes, and that SEN-V infection is not associated with higher rates of or more-severe liver disease in persons with preexisting liver disease.

Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients.

The anti-α4 monoclonal antibody natalizumab inhibits lymphocyte extravasation into the central nervous system and increases peripheral T and B lymphocytes in multiple sclerosis patients. To investigate whether the lymphocyte accumulation was due to a higher lymphocyte production, an altered homeostasis, or a differential transmigration of lymphocyte subsets through endothelia, T-cell receptor excision circles and kappa-deleting recombination excision circles were quantified before and after treatment, T-cell receptor repertoire was analyzed by spectratyping, and T- and B-lymphocyte subset migration was studied using transwell coated with vascular and lymphatic endothelial cells. We found that the number of newly produced T and B lymphocytes is increased because of a high release and of a low propensity of naïve subsets to migrate across endothelial cells. In some patients this resulted in an enlargement of T-cell heterogeneity. Because new lymphocyte production ensures the integrity of immune surveillance, its quantification could be used to monitor natalizumab therapy safety.

Newly produced T and B lymphocytes and T-cell receptor repertoire diversity are reduced in peripheral blood of fingolimod-treated multiple sclerosis patients

Background: Fingolimod inhibits lymphocyte egress from lymphoid tissues, thus altering the composition of the peripheral lymphocyte pool of multiple sclerosis patients. Objective: The objective of this paper is to evaluate whether fingolimod determines a decrease of newly produced T- and B-lymphocytes in the blood and a reduction in the T-cell receptor repertoire diversity that may affect immune surveillance. Methods: Blood samples were obtained from multiple sclerosis patients before fingolimod therapy initiation and then after six and 12 months. Newly produced T and B lymphocytes were measured by quantifying T-cell receptor excision circles and K-deleting recombination excision circles by real-time PCR, while recent thymic emigrants, naive CD8+ lymphocytes, immature and naive B cells were determined by immune phenotyping. T-cell receptor repertoire was analyzed by complementarity determining region 3 spectratyping. Results: Newly produced T and B lymphocytes were significantly reduced in peripheral blood of fingolimod-treated patients. The decrease was particularly evident in the T-cell compartment. T-cell repertoire restrictions, already present before therapy, significantly increased after 12 months of treatment. Conclusions: These results do not have direct clinical implications but they may be useful for further understanding the mode of action of this immunotherapy for multiple sclerosis patients.

Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient.

Progressive multifocal leukoencephalopathy (PML) usually occurs in patients with severe immunosuppression, hematological malignancies, chronic inflammatory conditions or receiving organ transplant. Recently, PML has also been observed in patients treated with monoclonal antibodies. By taking advantage of the availability of samples from a multiple sclerosis (MS) patient treated with natalizumab, the antibody anti-α4 integrin, who developed PML and was monitored starting before therapy initiation, we investigated the fate of T and B lymphocytes in the onset of PML. Real-time PCR was used to measure new T- and B-cell production by means of T-cell receptor excision circle (TREC) and K-deleting recombination excision circle (KREC) analysis and to quantify transcripts for CD34, terminal-deoxynucleotidyltransferase, and V pre-B lymphocyte gene 1. T- and B-cell subsets and T-cell heterogeneity were measured by flow cytometry and spectratyping. The data were compared to those of untreated and natalizumab-treated MS patients and healthy donors. Before therapy, a patient who developed PML had a low TREC and KREC number; TRECs remained low, while KRECs and pre-B lymphocyte gene 1 transcripts peaked at 6 months of therapy and then decreased at PML diagnosis. Flow cytometry confirmed the deficient number of newly produced T lymphocytes, counterbalanced by an increase in TEMRA cells. The percentage of naive B cells increased by approximately 70% after 6 months of therapy, but B lymphocyte number remained low for the entire treatment period. T-cell heterogeneity and immunoglobulins were reduced. Although performed in a single patient, all results showed that an immune deficit, together with an increase in newly produced B cells a few months after therapy initiation, may predispose the patient to PML. These findings indicate the TREC/KREC assay is a potential tool to identify patients at risk of developing PML and may provide insights into the immunological involvement of monoclonal antibody-associated therapies.

Renewal of the T-cell compartment in multiple sclerosis patients treated with glatiramer acetate

The immunomodulating activity of glatiramer acetate on T-cells of multiple sclerosis patients has only been partially clarified. The objective of this work was to investigate whether glatiramer acetate modifies thymic release of newly produced T-cells and the peripheral composition of the T-cell repertoire. T-cell receptor excision circles, thymic naive (CD4+CD45RA+CCR7 +CD31+) T helper cells, and central (CD4+CD45RA -CCR7+) and effector (CD4+CD45RA-CCR7 -) memory T-cells were evaluated in 89 untreated patients, 84 patients treated for at least 1 year, and 31 patients beginning treatment at the time of inclusion in the study and then followed-up for 12 months; controls were 81 healthy donors. The T-cell repertoire was analysed in selected samples. The percentage of thymicnaive T helper cells was diminished in untreated patients, but rose to control values in treated subjects; a decrease in central memory T-cells was also observed in treated patients. Follow-up patients could be divided into two subgroups, one showing unmodified thymicnaive T helper cells and T-cell diversity, the other in which the increased release of new T-cells was accompanied by modifications of the T-cell repertoire. Glatiramer acetate modifies the peripheral T-cell pool by activating a thymopoietic pathway of T-cell release that leads to a different setting of T-cell diversity and, likely, to a dilution of autoreactive T-cells.

Modulation of IFNAR1 mRNA expression in multiple sclerosis patients.

Interferon-beta receptor (IFNAR) is composed of 2 subunits, IFNAR1 and IFNAR2, the latter of which is expressed as functional (IFNAR2.2), non-functional (IFNAR2.1) and soluble (IFNAR2.3) isoform. Real-Time PCR analysis of mRNA for all IFNAR components in multiple sclerosis patients naïve for therapy and undergoing long-term treatment with interferon-beta shows that IFNAR1 mRNA level is lower than in healthy controls. If long-term treated patients are divided according to the production of mRNA for Myxovirus protein-A, a marker of interferon-beta bioactivity, IFNAR1 mRNA reaches the values observed in controls only in Myxovirus protein-A-induced patients. Since chronic cell stimulation by interferon-beta induces IFNAR protein down-regulation, we suggest that the increase of IFNAR1 mRNA might serve as a mechanism for counterbalancing the loss of protein receptor, enhancing, at least in this sub-group of patients, cell responsiveness to interferon-beta.