Silvana Bonfigli

Patients with myelodysplastic syndromes display several T-cell expansions, which are mostly polyclonal in the CD4+ subset and oligoclonal in the CD8+ subset

OBJECTIVE Immune dysregulation plays a role in the pathophysiology of myelodysplastic syndromes (MDS), as T-cell clones seem to be involved in the inhibition of hematopoietic precursors. The purpose of this study was to analyze the T-cell receptor (TCR) repertoire of MDS patients, focusing on the third complementarity determining region (CDR3) pattern of their CD4(+) and CD8(+) lymphocyte expansions. MATERIALS AND METHODS The study involved 30 patients and 15 age-matched controls. The beta-variable (betaV) subfamily flow-cytometry analysis was performed on peripheral CD4(+) and CD8(+) T-cells. Spectratyping TCR-CDR3 analysis was carried out on isolated helper and cytotoxic T lymphocytes after immunomagnetic separation and reverse-transcriptase polymerase chain reaction. RESULTS We first identified by flow cytometry an increased frequency of expanded betaVs in both CD4(+) and CD8(+) T-cells in MDS patients. We then showed, by spectratyping, that the CDR3 profile was mostly Gaussian in their CD4(+) T cells, whereas CD8(+) T cells usually showed skewed or oligoclonal CDR3 regions. When we compared spectratyping and flow-cytometry findings in each patient, we showed that most CD4(+) lymphocyte expansions detected by flow cytometry had Gaussian CDR3 profiles, whereas most CD8(+) expansions were oligoclonal. CONCLUSION We confirm that in MDS patients the TCR-betaV repertoire is overall extremely contracted, especially in cytotoxic T cells. This pattern is mainly determined by selective proliferations of both helper and cytotoxic T cells, which are, however, mostly polyclonal in the former and oligoclonal in the latter. Such a difference, possibly related to the different human leukocyte antigen restriction, could reflect the selective involvement of cytotoxic T cells either in the anti-tumor immune surveillance or in an autoreactive aggression toward hematopoietic precursors.

Cytogenetic and hematological spontaneous remission in a case of acute myelogenous leukemia

Abstract:  Several cases of spontaneous remission (SR) interrupting the invariably progressive course of untreated acute myeloblastic leukemia (AML) have been reported so far. We shall add to this series the hematological and cytogenetic SR occurring in a 72‐yr‐old man affected by AML following myelodysplastic syndrome. At diagnosis cytogenetic analysis showed the 48, xy, del (6) (p22‐pter), +13, +14 karyotype. Owing to a lobar pneumonia, the chemotherapy was deferred and a broad spectrum antibiotic therapy was established. Supportive care included red cells and platelet transfusions and low‐dose corticosteroid. Two months later, after the pneumonia had completely disappeared, a complete remission, lasting about 5 months, was documented on bone marrow morphological and cytogenetical examination, although some degree of myeloid dysplasia persisted. Possible mechanisms of the various SRs described during the course of AML are discussed with a review of the literature.

Patients with Early-Stage Myelodysplastic Syndromes Show Increased Frequency of CD4+CD25high+CD127low Regulatory T Cells

Regulatory T cells (Treg) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed Treg frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25high+CD127low Treg than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal 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.

Patients with myelodysplastic syndromes show reduced frequencies of CD4+ CD8+ double‐positive T cells

Even though the expression of CD4 and CD8 on thymocytes is considered mutually exclusive, CD4 + CD8 + double-positive T cells (DP) represent a small subset of T lymphocytes that have been described in the peripheral blood of normal individuals, as well as in some pathological conditions. In particular, an agedependent accumulation of monoclonal DP has been shown in elderly healthy subjects. From the functional point of view, DP are able to act as differentiated effector memory cells with specific antiviral functions.

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.

T-cell receptor repertoire in healthy Sardinian subjects

The T-cell receptor (TCR) Vbeta gene usage of CD4+ and CD8+ T-cell subpopulations was evaluated by flow cytometric analysis and by CDR3 spectratyping in healthy subjects belonging to Sardinian population, which is ethnically homogeneous and genetically distant from all other Italian and Caucasoid groups. As described in healthy Caucasian subjects, we found a nonrandom Vbeta gene usage and in some Vbeta families a significant skewed reactivity toward CD4+ T cells. Moreover, different subjects showed expansions in some Vbeta subfamilies, mainly in the CD8+ T cells. By CDR3 spectratyping analysis we found a significantly higher degree of skewness in the TCR Vbeta repertoire of CD8+ than in that of CD4+ T cells. The similarity found in the TCR Vbeta gene usage between the population examined and other Caucasoid groups suggest that the shape of the TCR repertoire is more influenced by rearrangement processes than ethnic background. However, genetic polymorphisms may condition the expression levels of some Vbetas, determining the variability of the TCR repertoire between different populations. Finally, the profound perturbations evidenced in the CD8+ T cell subpopulation could be related to a different response to the antigenic stimulation between CD8+ and CD4+ T lymphocytes.

TCRBV20S1 polymorphism does not influence the susceptibility to type 1 diabetes and multiple sclerosis in the Sardinian population.

Among the different T-cell receptor (TCR) BV20S1 polymorphisms, nucleotide substitution at position 524 results in the introduction of a stop codon, whose potential functional relevance is still unknown. We have recently showed in Sardinian subjects the most elevated allele frequency ever reported worldwide for this “null allele” (0.44). As this variant generates a gap in the TCR repertoire, this preliminary finding prompted us to further analyze the role of this polymorphism in the susceptibility to type 1 diabetes (T1D) and multiple sclerosis (MS), which are extremely common in this population. With this aim, we evaluated the influence of the TCRBV20S1 polymorphism by assessing it with the transmission disequilibirum test (TDT) in 652 T1D and 616 MS families, without detecting any significant difference. We conclude that the high frequency of this null allele in Sardinia is not directly related to the high incidence of these autoimmune diseases observed in this founder population.

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.

Efficacy of rituximab in autoimmune hemolytic anemia associated with splenic marginal zone lymphoma

Autoimmune manifestations including autoimmune hemolytic anemia (AIHA) are often described in association with splenic marginal zone lymphoma (SMZL) [1]. Moreover, AIHA has been recently reported as a potential risk factor of poor outcome for SMZL patients [2]. Among the different available therapeutic options, rituximab monotherapy has been recently shown to be extremely effective in SMZL [3]. On the other hand, this monoclonal antibody plays a key role in the treatment of steroid-refractory AIHA [4]. More specifically, although rituximab has been recently included among the possible strategies to treat AIHA secondary to chronic lymphocytic leukemia [5], very few cases describing its use in the course of SMZL-related AIHA have been described so far [6].