High-throughput RNA sequencing of the T cell receptor alpha and beta chains for simultaneous clonality and biological analyses in Sezary syndrome.

BACKGROUND: Previous investigations pointed out a role for antigen stimulation in Sezary syndrome (SS). High-throughput sequencing of the T cell receptor (TR) offers several applications beyond diagnostic purposes, including the study of T cell pathogenesis. METHODS: We performed high-throughput RNA sequencing of the TR alpha (TRA) and beta (TRB) genes focusing on the complementarity-determining region 3 (CDR3) in 11 SS and one erythrodermic mycosis fungoides (MF) patients. Five psoriasis patients were employed as controls. Peripheral blood CD4+ cells were isolated and RNA sequenced (HiSeq2500). High-resolution HLA typing was performed in neoplastic patients. RESULTS: Highly expanded predominant TRA and TRB CDR3 were only found in SS patients (median frequency: 94.4% and 93.7%). No remarkable CDR3 expansions were observed in psoriasis patients (median frequency of predominant TRA and TRB CDR3: 0.87% and 0.69%, p < 0.001 compared to SS). CDR3 almost identical to the predominant were identified within each SS patient and were exponentially correlated with frequencies of the predominant CDR3 (R2 = 0.918, p < 0.001). Forty-six different CDR3 were shared between SS patients displaying HLA similarities, including predominant TRA and TRB CDR3 in one patient that were found in other three patients. Additionally, 351 antigen matches were detected (Cytomegalovirus, Epstein-Barr, Influenza virus, and self-antigens), and the predominant CDR3 of two different SS patients matched CDR3 with specificity for Influenza and Epstein-Barr viruses. CONCLUSIONS: Besides detecting clonality, these findings shed light on the nature of SS-related antigens, pointing to RNA sequencing as a useful tool for simultaneous clonality and biological analysis in SS.

Allelic diversity of MICA gene and MICA/HLA-B haplotypic variation in a population of the Murcia region in southeastern Spain.

Major histocompatibility complex class I-related chain A (MICA) is located at 46 kb centromeric of HLA-B. It is highly polymorphic and interacts with NKG2D, its receptor on the surface of NK, Tgammadelta and T CD8 lymphocytes. Data on MICA polymorphism in different populations are still limited. Our aim was to establish allelic diversity of MICA gene and linkage disequilibrium with HLA-B in our population. DNA was obtained from 154 unrelated healthy individuals from the Murcia region in southeastern Spain. HLA-B genotyping was performed using polymerase chain reaction (PCR)-sequence-specific oligonucleotide probes and allele-specific PCR-sequence-specific primers, and MICA genotyping by using PCR-sequence-specific oligonucleotide probes. A total of 19 MICA alleles were detected on this study. MICA*008 was the most frequent allele (25.3%), followed by MICA*002 (16.1%), MICA*004 (14.9%), MICA*001 (7.8%), MICA*009 and MICA*016 (7.1%), and MICA*010 (4.6%). Eleven alleles had frequencies of <1%. In the haplotype analysis, MICA*008-B*0702 was found to be the most common, followed by MICA*004-B*4403 and MICA*001-B*1801, MICA*002-B*3501, MICA*008-B*4402, MICA*004-B*4901, MICA*008-B*0801, and MICA*002-B*3801. The frequency of MICA*010-B*1501, MICA*008-B*1302, MICA*015-B*4501, and MICA*008-B*4001 was remarkable inasmuch as these two last haplotypes have not been reported in Spanish population. Indeed, MICA*016 linked to B*1402 has also not been reported in the literature. In conclusion, the allelic diversity in our population is similar to other Caucasian populations; however we found a series of less frequent alleles, in addition to as-yet-undescribed haplotypic associations in other populations of Caucasian origin.

Restricted T cell receptor repertoire in CLL-like monoclonal B cell lymphocytosis and early stage CLL.

Analysis of the T cell receptor (TR) repertoire of chronic lymphocytic leukemia-like monoclonal B cell lymphocytosis (CLL-like MBL) and early stage CLL is relevant for understanding the dynamic interaction of expanded B cell clones with bystander T cells. Here we profiled the T cell receptor ß chain (TRB) repertoire of the CD4+ and CD8+ T cell fractions from 16 CLL-like MBL and 13 untreated, Binet stage A/Rai stage 0 CLL patients using subcloning analysis followed by Sanger sequencing. The T cell subpopulations of both MBL and early stage CLL harbored restricted TRB gene repertoire, with CD4+ T cell clonal expansions whose frequency followed the numerical increase of clonal B cells. Longitudinal analysis in MBL cases revealed clonal persistence, alluding to persistent antigen stimulation. In addition, the identification of shared clonotypes among different MBL/early stage CLL cases pointed towards selection of the T cell clones by common antigenic elements. T cell clonotypes previously described in viral infections and immune disorders were also detected. Altogether, our findings evidence that antigen-mediated TR restriction occurs early in clonal evolution leading to CLL and may further increase together with B cell clonal expansion, possibly suggesting that the T cell selecting antigens are tumor-related.