T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are circular DNA segments generated in T and B cells during their maturation in the thymus and bone marrow. These circularized DNA elements persist in the cells, are unable to replicate, and are diluted as a result of cell division, thus are considered markers of new lymphocyte output. The quantification of TRECs and KRECs, which can be reliably performed using singleplex or duplex real-time quantitative PCR, provides novel information in the management of T- and B-cell immunity-related diseases. In primary immunodeficiencies, when combined with flow cytometric analysis of T- and B-cell subpopulations, the measure of TRECs and KRECs has contributed to an improved characterization of the diseases, to the identification of patients’ subgroups, and to the monitoring of stem cell transplantation and enzyme replacement therapy. For the same diseases, the TREC and KREC assays, introduced in the newborn screening program, allow early disease identification and may lead to discovery of new genetic defects. TREC and KREC levels can also been used as a surrogate marker of lymphocyte output in acquired immunodeficiencies. The low number of TRECs, which has in fact been extensively documented in untreated HIV-infected subjects, has been shown to increase following antiretroviral therapy. Differently, KREC number, which is in the normal range in these patients, has been shown to decrease following long-lasting therapy. Whether changes of KREC levels have relevance in the biology and in the clinical aspects of primary and acquired immunodeficiencies remains to be firmly established.

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.

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. Cancer Res; 70(18); 7084–92. ©2010 AACR.

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.

Background: Assessment of T-cell diversity, besides giving insights about the molecular basis of tumor antigen recognition, has clinical implications since it provides criteria for evaluating antigen-specific T cells clinically relevant for spontaneous and vaccine-induced anti-tumor activity. Melan-A is one of the melanoma antigens most frequently recognized by peripheral and tumor-infiltrating lymphocytes in HLA-A2+ melanoma patients. Many clinical trials involving anti-tumor vaccination have been conducted using modified versions of this peptide.Methods: We conducted an in-depth characterization of 210 T-cell receptor beta chain (TRB) clonotypes derived from T cells of HLA-A2+ melanoma patients displaying cytotoxic activity against natural and A27L-modified Melan-A peptides. One hundred and thirteen Melan-A-specific clonotypes from melanoma-free subjects, 199 clonotypes from T-cell clones from melanoma patients specific for melanoma antigens other than Melan-A, and 305 clonotypes derived from T cells of HLA-A2+ individuals showing unrelated specificities, were used as control. After sequence analysis, performed according to the IMGT definitions, TRBV and TRBJ usage, CDR3 length and amino acid composition were compared in the four groups of clonotypes.Results: TRB sequences of Melan-A-specific clonotypes obtained from melanoma patients were highly heterogeneous, but displayed a preferential usage of few TRBV and TRBJ segments. Furthermore, they included a recurrent "public" amino acid motif (Glycine-Leucine-Glycine at positions 110-112-113 of the CDR3) rearranged with dominant TRBV and TRBJ segments and, in one case, associated with a full conservation of the entire TRB sequence.Conclusion: Contrary to what observed for public anti-Melan-A T-cell receptor alpha motifs, which had been identified in several clonotypes of both melanoma patients and healthy controls, the unexpectedly high contribution of a public TRB motif in the recognition of a dominant melanoma epitope in melanoma patients may provide important information about the biology of anti-tumor T-cell responses and improve monitoring strategies of anti-tumor vaccines.