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.

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-Hodgkin's 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.

Patients with early-stage myelodysplastic syndromes show increased frequency of CD4+CD25+CD127(low) regulatory T cells.

Regulatory T cells (T(reg)) 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 T(reg) 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+CD25(high)+CD127(low) T(reg) 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.

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.

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.

High frequency of the TCRBV20S1 null allele in the Sardinian population.

Single nucleotide polymorphisms (SNPs) in the T-cell receptor (TCR) gene segments might play a role in shaping the TCR repertoire. Three polymorphisms have been described for the TCRBV20S1 gene segment, one of which is responsible for a nucleotide substitution at position 524, resulting in the introduction of a stop codon. Individuals homozygous for this inactivating polymorphism ("null allele") are unable to express TCRBV20 gene products. Using DNA restriction digestion analysis, we investigated the frequency of this polymorphism in 111 healthy Sardinian subjects. Inhabitants of the Mediterranean island of Sardinia are considered to represent a genetically isolated population. Our analyses revealed an incidence of 19.8% of homozygosity for the null allele, corresponding to an allele frequency of 0.45. Such an incidence, significantly higher than the one detected in 83 non-Sardinian Caucasians (6%), is the most elevated so far reported in the literature. BV20 is a single member subfamily and the null allele produces a gap in the potential TCR repertoire. Therefore, it is possible that an undetermined selective pressure could have played a role in determining the high frequency of this inactivating polymorphism in Sardinians. Alternatively, this finding could be related to a founder effect in this ancient island population.

Long-lasting fever of unknown origin preceding the diagnosis of intravascular lymphomatosis: a further case stimulates some remarks.

Neurological and skin involvements usually dominate the clinical presentation of intravascular lymphomatosis (IL), while fever is the most frequent general sign. However, an onset only characterized by fever of unknown origin (FUO) has been rarely reported. We would like to describe a further case of IL, which presented a long-lasting FUO before the diagnosis. At admission, physical examination detected hepatosplenomegaly without lymph nodes enlargement or dermatological or neurological abnormalities. Significant laboratory data included severe anemia, leukopenia, thrombocytopenia, and increased serum LDH. Moreover, a chest CT evidenced bilateral multiple pulmonary infiltrates and pleural effusion. After the development of proteinuria, a diagnosis of large B-cell intravascular lymphoma was made with a renal biopsy 10 months after the onset of the clinical manifestations. So far, more than 100 cases of IL have been reported and the diagnosis often turned out to be difficult, as clinical signs did not point to a lymphoproliferative disorder. This report confirms that FUO is not only frequently associated with IL but that it even marks the real onset of the disease. We are then tempted to conclude that undiagnosed fever is not so rare in IL and if we call it FUO, it is only because diagnosis is necessarily elusive and hence time-consuming.

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.