Microbial exposure drives polyclonal expansion of innate γδ T cells immediately after birth.

Starting at birth, the immune system of newborns and children encounters and is influenced by environmental challenges. It is still not completely understood how γδ T cells emerge and adapt during early life. Studying the composition of T cell receptors (TCRs) using next-generation sequencing (NGS) in neonates, infants, and children can provide valuable insights into the adaptation of T cell subsets. To investigate how neonatal γδ T cell repertoires are shaped by microbial exposure after birth, we monitored the γ-chain (TRG) and δ-chain (TRD) repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of TRG and TRD sequences that were shared by all children from Europe and Africa. These were primarily public clones, characterized by simple rearrangements of Vγ9 and Vδ2 chains with low junctional diversity and usage of non-TRDJ1 gene segments, reminiscent of early ontogenetic subsets of γδ T cells. Further profiling revealed that these innate, public Vγ9Vδ2+ T cells underwent an immediate TCR-driven polyclonal proliferation within the first 4 wk of life. In contrast, γδ T cells using Vδ1+ and Vδ3+TRD rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1+ and Vδ3+TRD sequences in the majority of African children. In summary, we show how dynamic γδ TCR repertoires develop directly after birth and present important differences among γδ T cell subsets.

The transcription factor c-Myb primes CD4+CD8+ immature thymocytes for selection into the iNKT lineage.

Type I invariant NKT cells (iNKT cells) are a subset of alphabeta T cells characterized by the expression of an invariant alpha-chain variable region 14-alpha-chain joining region 18 (V(alpha)14J(alpha)18) T cell antigen receptor (TCR) alpha-chain. The iNKT cells derive from CD4(+)CD8(+) double-positive (DP) thymocytes, and their generation requires a long half-life of DP thymocytes to allow V(alpha)14-J(alpha)18 rearrangements, expression of glycolipid-loaded CD1d on DP thymocytes, and signaling through the signaling-activation molecule SLAM-adaptor SAP pathway. Here we show that the transcription factor c-Myb has a central role in priming DP thymocytes to enter the iNKT lineage by simultaneously regulating CD1d expression, the half-life of DP cells and expression of SLAMF1, SLAMF6 and SAP.

Persisting fetal clonotypes influence the structure and overlap of adult human T cell receptor repertoires.

The diversity of T-cell receptors recognizing foreign pathogens is generated through a highly stochastic recombination process, making the independent production of the same sequence rare. Yet unrelated individuals do share receptors, which together constitute a "public" repertoire of abundant clonotypes. The TCR repertoire is initially formed prenatally, when the enzyme inserting random nucleotides is downregulated, producing a limited diversity subset. By statistically analyzing deep sequencing T-cell repertoire data from twins, unrelated individuals of various ages, and cord blood, we show that T-cell clones generated before birth persist and maintain high abundances in adult organisms for decades, slowly decaying with age. Our results suggest that large, low-diversity public clones are created during pre-natal life, and survive over long periods, providing the basis of the public repertoire.

Staying innate: transcription factor maintenance of innate lymphoid cell identity.

Innate and adaptive lymphocytes are characterized by phenotypic and functional characteristics that result from genomic rearrangements (in the case of antigen-specific B and T cells) coupled with selective gene expression patterns that are generated in a context-dependent fashion. Cell-intrinsic expression of transcription factors (TFs) play a critical role in the regulation of gene expression that establish the distinct lymphoid subsets but also have been proposed to play an ongoing role in the maintenance of lineage-associated transcriptional signatures that comprise lymphocyte identity. This is the case for CD19(+) B cells that require Pax5 expression throughout their lifespan, as well as for diverse T-helper subsets that have specialized immune functions. Innate lymphoid cells (ILCs) comprise diverse effectors cells that differentiate under TF control and have critical roles in the early stages of immune responses. In this review, ILC development is reviewed and the requirement for persistent TF expression in the maintenance of transcriptional signatures that define ILC identity is explored.

The RAG2 C terminus suppresses genomic instability and lymphomagenesis.

Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans and in mice. Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical non-homologous end joining for proper repair, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination, is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes (Rag2(c/c) mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2(c/c) p53(-/-) mice, unlike Rag1(c/c) p53(-/-) and p53(-/-) animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from Atm(-/-) mice. We show that, like ATM-deficiency, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in Rag2(c/c) p53(-/-) and Atm(-/-) mice.

A role for cohesin in T-cell-receptor rearrangement and thymocyte differentiation.

Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.

Persistent cytotoxic T lymphocyte expansions after allogeneic haematopoietic stem cell transplantation: kinetics, clinical impact and absence of STAT3 mutations.

Peripheral expansion of cytotoxic T lymphocytes (CTL) derived from the graft in the initial stages of allogeneic haematopoietic stem cell transplantation (alloHSCT) immune recovery is a well-known physiological event. The description of symptomatic large granular lymphocyte leukaemia in this setting may generate uncertainty, mostly in those cases in which the CTL expansion (CTLe) persists beyond the early transplantation period. We aimed to assess the nature of CTLe during the post-alloHSCT period in 154 adult patients with a long-term surveillance. We studied the longitudinal kinetics of those expansions, their relationship to clinical events, and their phenotypic and molecular features, including recently reported CTL leukaemia-STAT3 mutations. Persistent relative CTLe cases are frequent (49%), related with thymoglobulin prophylaxis (P ≤ 0·001), acute graft-versus-host disease (GVHD, P = 0·02), and reduced intensity conditioning (P = 0·04). Absolute CTLe are scarce (9%) and related to chronic GVHD. T cell receptor rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor.

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR ß-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αß T-cell lineage differentiation. Whereas αßTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR ß-chain (TCRß) following successful TCR ß-gene rearrangement. Here we provide the basis of pre-Tα-TCRß assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRß mirrored that mediated by the Cα-Cß domains of the αßTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) ß domain through residues that are highly conserved across the Vß and joining (J) ß gene families, thus mimicking the interactions at the core of the αßTCR's Vα-Vß interface. Disruption of this pre-Tα-Vß dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR ß-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.

Reconstructing immune phylogeny: new perspectives.

Numerous studies of the mammalian immune system have begun to uncover profound interrelationships, as well as fundamental differences, between the adaptive and innate systems of immune recognition. Coincident with these investigations, the increasing experimental accessibility of non-mammalian jawed vertebrates, jawless vertebrates, protochordates and invertebrates has provided intriguing new information regarding the likely patterns of emergence of immune-related molecules during metazoan phylogeny, as well as the evolution of alternative mechanisms for receptor diversification. Such findings blur traditional distinctions between adaptive and innate immunity and emphasize that, throughout evolution, the immune system has used a remarkably extensive variety of solutions to meet fundamentally similar requirements for host protection.

Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.

Frequency and prognostic value of cutaneous molecular residual disease in mycosis fungoides: a prospective multicentre trial of the Cutaneous Lymphoma French Study Group.

BACKGROUND: Monoclonal T-cell receptor (TCR) rearrangement is detected in 57-75% of early-stage mycosis fungoides (MF) at diagnosis. A retrospective study showed molecular residual disease (MRD) in 31% of patients in complete clinical remission (CR) after 1 year of treatment. OBJECTIVES: To confirm the frequency of MRD at 1 year and to determine its prognostic value for further relapse. METHODS: Patients with T1-, T2- or T4-stage MF were prospectively included in this multicentre study. At diagnosis, clinical lesions and healthy skin were biopsied. After 1 year of topical treatment, previously involved skin of patients in CR was biopsied for histology and analysis of TCR-γ gene rearrangement. The results were compared with the clinical status each year for 4 years. RESULTS: We included 214 patients, 133 at T1, 78 at T2 and three at T4 stage. At diagnosis, 126 of 204 cases (61·8%) showed TCR clonality in lesional skin. After 1 year, 83 of 178 patients (46·6%) still being followed up were in CR and 13 of 63 (21%) showed MRD. At 4 years, 55 of 109 patients (50·5%) still being followed up were in CR and 44 of 109 (40·4%) were in T1 stage. MRD did not affect clinical status at 4 years (CR vs. T1/T2, P = 1·0; positive predictive value 36·4%; negative predictive value 67·6%). CONCLUSIONS: T-cell clonality at diagnosis and MRD at 1 year are not prognostic factors of clinical status at 4 years.

T-cell receptor gene rearrangement studies using the GeneScan technique as an adjunct to the histopathological diagnosis of mycosis fungoides.

T-cell receptor gene rearrangement studies are considered to be an adjunct to the histopathological diagnosis of mycosis fungoides (MF). Fluorophore-coupled primers in polymerase chain reactions followed by fragment analysis with a capillary electrophoresis device (GeneScan analysis) have been recently advocated and widely used. This study was performed to assess T-cell receptor γ-gene rearrangement GeneScan as an adjunct to the histopathological diagnosis of MF. We studied 163 cases. The rates of clonality using this technique were found to be 22/37 (59.5%) in early patch MF, 30/46 (65.2%) in more advanced stages, 13/34 (38.2%) in inconclusive cases, and 10/46 (21.7%) in benign inflammatory dermatoses. Our results support the histopathological criteria for the diagnosis of early patch MF, because of the close rates of clonality obtained in early patch MF with subtle histopathological findings, and in the more advanced subtypes of MF with more overt and specific histopathological criteria.

A stepwise epigenetic process controls immunoglobulin allelic exclusion.

During the differentiation of T and B cells, immune-receptor loci in the genome must be made sterically accessible so that they can undergo rearrangement. Here, we discuss how this is carried out by the stepwise removal of epigenetic repression mechanisms - such as later-replication timing, heterochromatization, histone hypo-acetylation and DNA methylation - in a manner that initially favours one allele in each cell. We propose that this mechanism of allelic exclusion might also be the basis for the generation of gene diversity in other systems.

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists.

HIV infection results in a decrease in circulating CD4(+) T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4(+) T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis.

Increased concentration of T-cell receptor rearrangement excision circles (TREC) in peripheral blood in Graves' disease.

BACKGROUND: T-cell receptor rearrangement excision circles (TREC) are circular DNA molecules generated during T-cell maturation in the thymus. Recent studies suggested that a decreased TREC concentration in peripheral blood may be a general feature of autoimmunity. Our purpose was to assess the TREC concentration in Graves' disease (GD). METHODS: TREC concentration was assessed by real time PCR in DNA samples isolated from peripheral blood leucocytes among younger (n = 94, age range 6-29 years) and older patients with GD (n = 93, age range 57-80 years) and age-matched controls (n = 206). RESULTS: TREC concentration decreased with age in all subjects, but it was significantly higher in GD compared with controls (P = 9·4 × 10(-10) ). TREC concentration was higher (P = 0·0038) in hyperthyroid (n = 78) than euthyroid (n = 82) patients with GD, but in both groups, it remained increased relative to controls (P = 2·2 × 10(-11) and P = 4·4 ×10(-7) , respectively). CONCLUSIONS: Patients with GD, particularly those with hyperthyroidism, have increased concentration of TREC which may suggest increased rather than decreased thymic activity. Thus, GD does not follow the paradigm suggested for other autoimmune disorders which links autoimmunity with thymic senescence.

Modern management of primary T-cell immunodeficiencies.

The study of human T-cell PIDs with Mendelian inheritance has enabled the molecular characterization of important key functions and pathways in T-cell biology. In most cases, T-cell PIDs become apparent as combined T- and B-cell deficiencies. Severe combined immunodeficiencies (SCIDs) are characterized by a complete lack of T-cell development and, in some cases, a developmental block in other lymphoid lineages and manifest within the first year of life. Combined immunodeficiency syndromes (CIDs) result from hypomorphic mutations in typical SCID associated genes or from partial defects of T-cell development and manifest later in childhood by increased susceptibility to infection often combined with disturbances in immune homeostasis, e.g., autoimmunity and increased incidence in lymphoproliferation. The discovery of mutations and characterization of the cellular changes that underlie lymphocyte defects and immune dysregulation have led to novel, specific, successful therapies for severe diseases which are often fatal if left untreated. Over the last few years, impressive progress has been made in understanding the disease mechanisms of T-cell immunodeficiencies and in improving the long-term outcomes of potentially curative treatments, including gene therapy.

Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations.

Mice deficient in the DNA damage sensor P53 display normal T cell development but eventually succumb to thymic lymphomas. Here, we show that inactivation of the TCR beta gene enhancer (E beta) results in a block of T cell development at stages where recombination-activating genes (RAG) are expressed. Introduction of the E beta mutation into p53-/- mice dramatically accelerates the onset of lethal thymic lymphomas that harbor RAG-dependent aberrant rearrangements, chromosome 14 and 12 translocations, and amplification of the chromosomal region 9A1-A5.3. Phenotypic and genetic analyses suggest that lymphomas emerge through a normal thymocyte development pathway. These findings provide genetic evidence that block of lymphocyte development at stages with RAG endonuclease activity can provoke lymphomagenesis on a background with deficient DNA damage responses.

53BP1 facilitates long-range DNA end-joining during V(D)J recombination.

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

Epigenetic aspects of lymphocyte antigen receptor gene rearrangement or 'when stochasticity completes randomness'.

To perform their specific functional role, B and T lymphocytes, cells of the adaptive immune system of jawed vertebrates, need to express one (and, preferably, only one) form of antigen receptor, i.e. the immunoglobulin or T-cell receptor (TCR), respectively. This end goal depends initially on a series of DNA cis-rearrangement events between randomly chosen units from separate clusters of V, D (at some immunoglobulin and TCR loci) and J gene segments, a biomolecular process collectively referred to as V(D)J recombination. V(D)J recombination takes place in immature T and B cells and relies on the so-called RAG nuclease, a site-specific DNA cleavage apparatus that corresponds to the lymphoid-specific moiety of the VDJ recombinase. At the genome level, this recombinase's mission presents substantial biochemical challenges. These relate to the huge distance between (some of) the gene segments that it eventually rearranges and the need to achieve cell-lineage-restricted and developmentally ordered routines with at times, mono-allelic versus bi-allelic discrimination. The entire process must be completed without any recombination errors, instigators of chromosome instability, translocation and, potentially, tumorigenesis. As expected, such a precisely choreographed and yet potentially risky process demands sophisticated controls; epigenetics demonstrates what is possible when calling upon its many facets. In this vignette, we will recall the evidence that almost from the start appeared to link the two topics, V(D)J recombination and epigenetics, before reviewing the latest advances in our knowledge of this joint venture.