Giovanna Corda

Azacitidine improves the T-cell repertoire in patients with myelodysplastic syndromes and acute myeloid leukemia with multilineage dysplasia

Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia show several immunological abnormalities. In this clinical setting, by combining flow cytometry and CDR3 spectratyping we monitored the kinetic of the T-cell repertoire during Azacitidine treatment, in order to explore its potential ability to reverse the immune derangement typical of these disorders. We firstly demonstrated by flow cytometry an increase in both CD4+ and CD8+ T-cell frequencies after starting treatment. Moreover, when monitored by spectratyping our patients showed significant changes in their T-cell receptor (TCR) CDR3 profiles, which were much more evident in helper T-cells. In fact, the frequency of BV (beta variable) subfamilies showing a skewed CDR3 profile significantly decreased from baseline to the following evaluations in CD4+ T-cells (81% vs. 70%). This pattern was even more pronounced in patients responding to Azacitidine (90% vs. 61%). Our data show that the overall derangement of the T-cell repertoire detectable in patients with MDS and AML with multilineage dysplasia gradually improves during Azacitidine treatment. These findings therefore suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these hematologic disorders.

T-cell receptor repertoire analysis in monozygotic twins concordant and discordant for type 1 diabetes.

Several data suggest that stochastic rearrangements of the TCR could play a pathogenic role in both disease predisposition and protection in type 1 diabetes (T1D). As twin sets offer an enormous potential in evaluating the role of genetic and environmental factors in susceptibility to disease, the main goal of this study was to assess whether the degree of sharing of the expressed TCR repertoire of twin pairs discordant for T1D differs from that of disease concordant pairs. We performed our analysis in 5 pairs of monozygotic twins, 3 of which were concordant and 2 discordant for T1D, by combining flow cytometry and CDR3 spectratyping on both CD4+ and CD8+ T-cells. Our data show that TCR repertoires show increased level of concordance within each twin pair, especially in CD8+ cells, in terms of mean BV expression levels on flow cytometry as well as of CDR3 patterns and frequencies of skewed or oligoclonal BV subfamilies on spectratyping. It is worth noting that the degree of similarity among twins seems to be independent of concordance or discordance for T1D. Our findings seem to suggest that in monozygotic twins with T1D the TCR repertoire is influenced by genetic factors more than by the presence of the autoimmune disorder itself.

Derangement of the T-cell repertoire in patients with B-cell non-Hodgkin's lymphoma

Although a number of studies suggest that different immune pathways may play a role in the pathogenesis of non‐Hodgkins lymphomas (NHL), the shape of the T‐cell compartment has been only superficially explored in these patients. In our study, we analyzed the peripheral T‐cell receptor (TCR) repertoire and the distribution of different T‐cell subsets – including regulatory T cells (Treg) – in 30 patients with NHL, by combining flow cytometry and spectratyping. We first demonstrated by flow cytometry an increased frequency of expanded T‐cell subpopulations expressing the same TCR beta variable (BV) subfamilies in CD8+ cells from NHL patients when compared with healthy controls, beside a higher frequency of Treg. Moreover, NHL patients were characterized by a higher percentage of BVs showing a skewed CDR3 profile both in CD4+ and CD8+ cells when analyzed by spectratyping. Our data suggest that the T‐cell branch of the immune system of patients with B‐cell NHL is deeply deranged, as witnessed by the increased degree of activation and skewing of their TCR repertoire along with the higher frequency of Treg.

Quantitative and functional abnormalities of total T lymphocytes in relatives of patients with Hodgkin's disease

Seven patients, long-term survivors of Hodgkins disease, and 24 of their relatives (parents, siblings and children), together with normal controls were studied for percentages, absolute counts and mitogen-proliferative responses by means of monoclonal antibodies, E rosette technique and in vitro cultures with PHA, ConA and PWM. The aim of the study was to ascertain whether the impaired cell-mediated immunity of Hodgkins patients was also present in relatives in order to elucidate the still debated etiology of the defect and of the disease (congenital? environmental? infectious?). The results show that both Hodgkins patients and their relatives have a significant decrease of total T cells (as T3+, T11+ and E rosette-forming cells) in peripheral blood and a significant impairment of polyclonal responses to all the mitogens employed. The Leu-7+ cells (i.e. a consistent amount of natural killer cells) are significantly increased only in the Hodgkins patients but not in their relatives. The T cell subpopulations (T4 and T8), B cells and monocytes do not show any difference between the patients, their relatives and normal controls. Our results seem to support, at least in part, the presence of a common defect of T cell lineage both in patients and in their relatives, but its etiology still remains uncertain (genetic? environmental?).

Study of the T-cell receptor repertoire by CDR3 spectratyping

The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR. This review will run through the most relevant studies which have tried to dissect the TCR repertoire usage in patients with different immune-mediated and infective diseases as well as solid or haematologic malignancies.

Epstein–Barr virus infection is associated to patients with multiple myeloma and monoclonal gammopathy of undetermined significance

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant disorder associated with a progression to multiple myeloma (MM). MM is a plasma cell malignancy with a chronic lymphoproliferative disorder. Epstein–Barr virus (EBV) has been associated with different types of lymphoma. The aim of this study was to evaluate whether there is an association between MM, MGUS, and EBV in patients by serological and molecular methods. The results showed only a serological IgG response against EBNA-1 in MGUS and MM patients, not against viral capsid antigen (VCA) EBV antigen. Molecular analyses display a significant EBV DNA in premalignant and malignant plasma cell disorders; in particularly we observed an overexpression of latent EBV LMP2A gene in MM and MGUS patients. These data display a possible contribute of EBV, during viral latent cell cycle, to MM cell survival through a possible deregulation of RAS pathway induced by a latent membrane protein 2A (LMP2A) of EBV. MM is a plasma cell malignancy which develops in the bone marrow and, when symptomatic, is clinically characterized by renal failure, immunosuppression with repeated infections, cytopenia, and bone lesions. The pathogenesis of this disorder is dominated by a variety of genomic abnormalities often related to genetic instability but also the complex interactions between malignant plasma cells and their microenvironment play a fundamental role.[1] MGUS is a premalignant plasma cell disorder associated with a 1% per year risk of progression to MM. It is defined by a serum monoclonal protein <30 g/L, <10% clonal plasma cells in the bone marrow and the absence of endorgan damage that can be attributed to the plasma cell proliferative disorder.[2] The potential role for EBV in lymphomagenesis was first advocated in the ‘African’ form of Burkitt lymphoma. Since then EBV has been also associated with the development of other lymphomas, including classical Hodgkin lymphoma, diffuse large B-cell lymphoma and natural killer/T-cell lymphoma.[3] Much less is known about the possible role of EBV in the disease history of patients with MM. In fact, apart from few case reports describing the onset of possibly EBV-related plasmacytomas in strongly immunodeficient patients,[4–9] a single study has tried to systematically address this scenario. In paraffin-embedded bone marrow biopsies DNA of EBV was detected slightly more frequently in MM patients than in age-matched controls.[10] In the present study, the possible role of EBV in the pathogenesis of MM was extensively evaluated by serological and molecular analysis in patients with MM and MGUS. A case–control study was performed on samples from 34 MM patients (16 males and 18 females, mean age 65.5 ± 7.11), 25 subjects with MGUS (12 were males and 13 females; mean age 67.0 ± 8.17) and 25 age and sex matched healthy controls (HCs; 14 males and 11 females; mean age 64.6 ± 7.5). The diagnosis of MM and MGUS was performed according to international guidelines.[2] Among MM patients according to Durie and Salmon classification 9%, 15%, and 76% were in Stage I, II, and III respectively while 74% and 26% were in Stage A and B. According to the International Scoring System 15%, 47%, and 38% were in Group I, II, and III, respectively. 60%, 32%, 4%, and 4% of the subjects were characterized by IgG-kappa, IgG-lambda, IgM-kappa, and IgA-lambda, respectively. All patients and donors had given informed consent. The study had been approved by the Local Ethics Committee. Presence and levels of IgG antibodies against EBV antigen-specific EBNA-1 and VCA were evaluated in sera using commercially available kit anti-EBV EBNA and VCA IgG ELISA (Bio-Rad, ref. 807017 and 807019). Detection of EBV genome in peripheral blood mononuclear cells (PBMCs) derived from samples previously analyzed for EBNA-1 and VCA antibodies response was attained by selective amplification of the qualitative and quantitative EBV DNA

Evidence of a skewed T-cell repertoire in patients with light chain amyloidosis.

Keywords: light-chain amyloidosis; T-cell receptor repertoire; regulatory T-cells; flow cytometry; spectratyping; CDR3