Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+thymocyte stage

The natural CD25 + FOXP3 + regulatory T cell (Treg) population is generated as a distinct lineage in the thymus, but the details of Treg development in humans remain unclear, and the timing of Treg commitment is also contested. Here we have analyzed the emergence of CD25 + cells at the CD4 + CD8 + double positive (DP) stage in the human thymus. We show that these cells share T cell receptor repertoire with CD25 + CD4 single-positive thymocytes, believed to be committed Tregs. They already have a fully demethylated FOXP3 enhancer region and thus display stable expression of FOXP3 and the associated Treg phenotype. Transcriptome analysis also grouped the DP CD25 + and CD4 CD25 + thymocytes apart from the CD25 - subsets. Together with earlier studies, our data are consistent with human Treg commitment already at the DP thymocyte stage. We suggest that the most important antigens and signals necessary for human Treg differentiation may be found in the thymic cortex.Peer reviewe

Peripheral differentiation patterns of human T cells

Long-term T-cell memory is dependent on the maintenance of memory T cells in the lymphoid tissues, and at the surface interfaces that provide entry routes for pathogens. However, much of the current information on human T-cell memory is based on analyzing circulating T cells. Here, we have studied the distribution and age-related changes of memory T-cell subsets in samples from blood, mesenteric LNs, spleen, and ileum, obtained from donors ranging in age from 5 days to 67 years of age. Our data show that the main reservoir of polyclonal naive cells is found in the LNs, and the resting memory subsets capable of self-renewal are also prominent there. In contrast, nondividing but functionally active memory subsets dominate the spleen, and especially the ileum. In general, the replacement of naive cells with memory subsets continues throughout our period of observation, with no apparent plateau. In conclusion, the analysis of lymphoid and nonlymphoid tissues reveals a dynamic pattern of changes distinct to each tissue, and with substantial differences between CD4(+) and CD8(+) compartments.Peer reviewe

Characterization of human T cell receptor repertoire data in eight thymus samples and four related blood samples

T cell receptor (TCR) is a heterodimer consisting of TCR alpha and TCR beta chains that are generated by somatic recombination of multiple gene segments. Nascent TCR repertoire undergoes thymic selections where non-functional and potentially autoreactive receptors are removed. During the last years, the development of high-throughput sequencing technology has allowed a large scale assessment of TCR repertoire and multiple analysis tools are now also available. In our recent manuscript, Human thymic T cell repertoire is imprinted with strong convergence to shared sequences [1], we show highly overlapping thymic TCR repertoires in unrelated individuals. In the current Data in Brief article, we provide a more detailed characterization of the basic features of these thymic and related peripheral blood TCR repertoires. The thymus samples were collected from eight infants undergoing corrective cardiac surgery, two of whom were monozygous twins [2]. In parallel with the surgery, a small aliquot of peripheral blood was drawn from four of the donors. Genomic DNA was extracted from mechanically released thymocytes and circulating leukocytes. The sequencing of TCR alpha and TCR beta repertoires was performed at ImmunoSEQ platform (Adaptive Biotechnologies). The obtained repertoire data were analysed applying relevant features from immunoSEQ (R) 3.0 Analyzer (Adaptive Biotechnologies) and a freely available VDJTools software package for programming language R [3]. The current data analysis displays the basic features of the sequenced repertoires including observed TCR diversity, various descriptive TCR diversity measures, and V and J gene usage. In addition, multiple methods to calculate repertoire overlap between two individuals are applied. The raw sequence data provide a large database of reference TCRs in healthy individuals at an early developmental stage. The data can be exploited to improve existing computational models on TCR repertoire behaviour as well as in the generation of new models. (C) 2021 The Authors. Published by Elsevier Inc.Peer reviewe

Human thymic T cell repertoire is imprinted with strong convergence to shared sequences

A highly diverse repertoire of T cell antigen receptors (TCR) is created in the thymus by recombination of gene segments and the insertion or deletion of nucleotides at the junctions. Using next-generation TCR sequencing we define here the features of recombination and selection in the human TCR alpha and TCR beta locus, and show that a strikingly high proportion of the repertoire is shared by unrelated individuals. The thymic TCRa nucleotide repertoire was more diverse than TCR beta, with 4.1 x 10(6) vs. 0.81 x 10(6) unique clonotypes, and contained nonproductive clonotypes at a higher frequency (69.2% vs. 21.2%). The convergence of distinct nucleotide clonotypes to the same amino acid sequences was higher in TCRa than in TCR beta repertoire (1.45 vs. 1.06 nucleotide sequences per amino acid sequence in thymus). The gene segment usage was biased, and generally all individuals favored the same genes in both TCR alpha and TCR beta loci. Despite the high diversity, a large fraction of the repertoire was found in more than one donor. The shared fraction was bigger in TCR alpha than TCR beta repertoire, and more common in in-frame sequences than in nonproductive sequences. Thus, both biases in rearrangement and thymic selection are likely to contribute to the generation of shared repertoire in humans.Peer reviewe

T cell receptor diversity in the human thymus

A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCR alpha and TCR beta gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3 x 10(6) unique TCR beta and 0.5 x 10(6) TCR alpha sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3 x 10(6) unique TCR beta sequences and 3.7 x 10(6) TCR alpha sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70 x 10(6) unique TCR beta sequences and 60 to 100 x 10(6) TCR alpha sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Long-lived Memory T Lymphocyte Responses After Hantavirus Infection

Puumala virus (PUUV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS), which is an important public health problem in large parts of Europe. We examined the memory cytolytic T lymphocyte (CTL) responses in 13 Finnish individuals who had HFRS between 1984 and 1995. In seven of these donors, we detected virus-specific CTL responses against the PUUV nucleocapsid (N) protein after in vitro stimulation with PUUV. Six novel CD8+ CTL epitopes were defined on the N protein and were found to be restricted by various HLA alleles including A2, A28, B7, and B8. This is the first demonstration of PUUV-specific CTL responses in humans, and the first identification of CTL epitopes on PUUV. In addition, this study provides one of the few characterizations of a human antiviral memory T cell response, without the complicating issues of virus persistence or reinfection. Interferon (IFN)-γ ELISPOT analysis showed that memory CTL specific for these epitopes were present at high frequency in PUUV-immune individuals many years after acute infection in the absence of detectable viral RNA. The frequencies of PUUV-specific CTL were comparable to or exceeded those found in other viral systems including influenza, EBV and HIV, in which CTL responses may be boosted by periodic reinfection or virus persistence

Heterologous boosting of nonrelated toxoid immunity during acute Puumala hantavirus infection

Persistence of immune memory in humans is a crucial yet poorly understood aspect of immunology. Here we have studied the effect of Puumala hantavirus infection on unrelated, pre-existing immune memory by studying T cell- and antibody responses against toxoid vaccine antigens of diphtheria, tetanus and pertussis in a cohort of 45 patients. We found that tetanus- and pertussis-specific IgG concentrations elevate during acute Puumala virus infection. Increase in vaccine IgG was associated with proliferation of heterologous T cells. Interestingly, increases in tetanus-specific IgG persisted a year after the infection while pertussis-specific IgG declined rapidly; a difference in IgG kinetics resembling the difference seen after vaccination against tetanus and pertussis. These results suggest that persistence of immune memory is facilitated by heterologous boosting of old memory during memory formation against newly encountered antigens. They also show that different toxoid antigens may be treated differently. Our study gives new insight into how immune memory formation may alter pre-existing immune memory, and also shows that heterologous immunity may have an impact on vaccination outcomes. (C) 2021 Elsevier Ltd. All rights reserved.Peer reviewe

Identifying the inheritable component of human thymic T cell repertoire generation in monozygous twins

We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.Non peer reviewe