Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci.

The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here, we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Ig kappa and Tcr alpha J gene segments and Igh and Tcr beta J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination.

Single-cell analysis can define distinct evolution of tumor sites in follicular lymphoma.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

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.

Trends in TREC values according to age and gender in Chinese children and their clinical applications.

T cell receptor excision circles (TRECs) are small circularized DNA elements produced during rearrangement of T cell receptor (TCR) genes. Because TRECs are fairly stable, do not replicate during mitosis, and are not diluted during division of naïve T cells (Dion et al. [1]), they are suitable for assessing the number of newly formed T cells (Ping and Denise [2]). In this study, we detected TRECs in 521 healthy Chinese children aged 0-18 years in different clinical settings. The TRECs decrease with aging and show lower levels in preterm and low birth weight (BW) babies compared to those in full-term infants, while the preterm babies can also show comparable levels of TRECs when they have a gestation age (GA)-matched BW. We found a strong correlation between TRECs and peripheral CD4 naïve T cell numbers, which was age-related. We also analyzed the TRECs in different PIDs. Since T cell defects vary in PIDs, TREC levels change inconsistently. For example, in Wiskott-Aldrich syndrome (WAS), combining the level of TREC with lymphocyte subsets can help to distinguish subtypes of disease.Conclusion: We established the reference value range for TRECs by evaluating children below 18 years old in China, which could be used to screen for PIDs during early life. What is Known: • The TREC levels are decreased with age, and there is a positive correlation between TRECs and the numbers of naïve T cells. What is New: • This is the largest study to determine TREC reference levels in healthy Chinese pediatric, we provide solid data showing a correlation between CD4 naïve T cell counts and TREC levels according to age. We point out the GA matched BW is need to be considered during the SCID newborn screening. We are the first group showed that TREC levels can help clinician distinguish different WAS phenotype.

Inventories of naive and tolerant mouse CD4 T cell repertoires reveal a hierarchy of deleted and diverted T cell receptors.

Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3+ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.

Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.

Cytoskeletal actin dynamics are crucial for the activation of T-cells. Immortalised Jurkat T-cells have been the model system of choice to examine and correlate the dynamics of the actin cytoskeleton and the immunological synapse leading to T-cell activation. However, it has remained unclear whether immortalised cellular systems, such as Jurkat T-cells can recapitulate the cytoskeletal behaviour of primary T-cells. Studies delineating the cytoskeletal behaviour of Jurkat T-cells in comparison to primary T-cells are lacking. Here, we employ live-cell super-resolution microscopy to investigate the cytoskeletal actin organisation and dynamics of living primary and immortalised Jurkat T-cells at the appropriate spatiotemporal resolution. Under comparable activation conditions, we found differences in the architectural organisation and dynamics of Jurkat and primary mouse and human T-cells. Although the three main actin network architectures in Jurkat T-cells were reminiscent of primary T-cells, there were differences in the organisation and molecular mechanisms underlying these networks. Our results highlight mechanistic distinctions in the T-cell model system most utilised to study cytoskeletal actin dynamics.

CloneRetriever: An Automated Algorithm to Identify Clonal B and T Cell Gene Rearrangements by Next-Generation Sequencing for the Diagnosis of Lymphoid Malignancies.

BACKGROUND: Clonal immunoglobulin and T-cell receptor rearrangements serve as tumor-specific markers that have become mainstays of the diagnosis and monitoring of lymphoid malignancy. Next-generation sequencing (NGS) techniques targeting these loci have been successfully applied to lymphoblastic leukemia and multiple myeloma for minimal residual disease detection. However, adoption of NGS for primary diagnosis remains limited. METHODS: We addressed the bioinformatics challenges associated with immune cell sequencing and clone detection by designing a novel web tool, CloneRetriever (CR), which uses machine-learning principles to generate clone classification schemes that are customizable, and can be applied to large datasets. CR has 2 applications-a "validation" mode to derive a clonality classifier, and a "live" mode to screen for clones by applying a validated and/or customized classifier. In this study, CR-generated multiple classifiers using 2 datasets comprising 106 annotated patient samples. A custom classifier was then applied to 36 unannotated samples. RESULTS: The optimal classifier for clonality required clonal dominance ≥4.5× above background, read representation ≥8% of all reads, and technical replicate agreement. Depending on the dataset and analysis step, the optimal algorithm yielded sensitivities of 81%-90%, specificities of 97%-100%, areas under the curve of 91%-94%, positive predictive values of 92-100%, and negative predictive values of 88%-98%. Customization of the algorithms yielded 95%-100% concordance with gold-standard clonality determination, including rescue of indeterminate samples. Application to a set of unknowns showed concordance rates of 83%-96%. CONCLUSIONS: CR is an out-of-the-box ready and user-friendly software designed to identify clonal rearrangements in large NGS datasets for the diagnosis of lymphoid malignancies.

Generation of self-reactive, shared T-cell receptor α chains in the human thymus.

The T-cell receptor (TCR) repertoire is generated in a semistochastic process of gene recombination and pairing of TCRα to TCRß chains with the estimated total TCR diversity of >108. Despite this high diversity, similar or identical TCR chains are found to recur in immune responses. Here, we analyzed the thymic generation of TCR sequences previously associated with recognition of self- and nonself-antigens, represented by sequences associated with autoimmune diabetes and HIV, respectively. Unexpectedly, in the CD4+ compartment TCRα chains associated with the recognition of self-antigens were generated in significantly higher numbers than TCRα chains associated with the recognition of nonself-antigens. The analysis of the circulating repertoire further showed that these chains are not lost in negative selection nor predominantly converted to the regulatory T-cell lineage. The high abundance of self-reactive TCRα chains in multiple individuals suggests that the human thymus has a predilection to generate self-reactive TCRα chains independently of the HLA-type and that the individual risk of autoimmunity may be modulated by the TCRß repertoire associated with these chains.

CD8-positive cutaneous lymphoproliferation associated with large granular lymphocyte leukemia in a patient with X-linked agammaglobulinemia.

Patients with primary immunodeficiency are at increased risk for malignancy, especially hematologic neoplasms. This paper reports a unique case of a 47-year-old man with X-linked agammaglobulinemia who presented with progressive asymptomatic violaceous papules and plaques on his face, hands, and trunk for 1 year. Skin biopsies revealed deep, nodular infiltrates of histiocytes and CD8-positive lymphocytes, with a CD4:CD8 ratio of 1:10. Laboratory studies showed cytopenias. Flow cytometry in the skin, blood, and bone marrow (BM) showed a CD3+/CD8+/CD57+ large granular lymphocyte population. BM biopsy showed 30% involvement with these atypical T-cells. T-cell gene rearrangement studies of skin, blood, and BM revealed identical T-cell clones. He was diagnosed with T-large granular lymphocyte leukemia (T-LGLL) with an associated CD8+ cutaneous lymphoproliferation. Skin involvement was suspected to represent infiltration by T-LGLL. However, co-existence of two lymphoproliferative disorders (LPDs), T-LGLL and CD8+ granulomatous LPD, remains a possibility. In general, cutaneous infiltrates associated with LGLL are rare and poorly understood. It has been suggested that they are markers of poor prognosis. Our case report describes skin, blood, and BM findings in an immunosuppressed patient with T-LGLL in detail. These findings have not yet been reported and their significance requires further investigation.

Primary Cutaneous B-Cell Lymphoma Complicated by Clonal T-Cell Populations: A Diagnostic Dilemma.

ABSTRACT: Cutaneous lymphomas, both B-cell and T-cell, are not uncommonly seen in the skin, but those lymphomas exhibiting clonality for both B-cell and T-cell populations are scarce. Characterization of dual receptor rearrangement as primary composite lymphoma versus primary lymphoma with reactionary response is complex and often a challenge that goes unrecognized. In this study, we report a unique case of T-cell gene rearrangement positivity complicating the diagnosis of primary cutaneous low-grade B-cell lymphoma along with a review of reported cases containing dual receptor rearrangement to identify trends among final diagnostic decisions. As one might guess, for cutaneous lymphomas presenting with clonality for both T-cell and B-cell receptors, diagnosis can be difficult and confusing because the differential is broad. The literature suggests the majority of these cases may be cutaneous composite lymphomas. However, immunohistochemical, clinical, and histomorphologic features must all be assessed for an accurate diagnosis, which is critical for proper prognosis and therapy.

Hypopigmented Mycosis Fungoides: A Clinical and Histopathology Analysis in 9 Children.

BACKGROUND: Hypopigmented mycosis fungoides (HMF) is an uncommon variant of mycosis fungoides. AIMS: To study the clinical and histopathology presentation in children with HMF. METHOD: We reviewed 9 children diagnosed with HMF. The clinical data were collected and analyzed. RESULT: Eight boys and 1 girl were included, with a median onset age of 7.4 year old and median age of diagnosis of 10.5 year old. Multiple hypopigmented patches were observed in all patients, and 5 patients exhibited multiple scaly erythema at the center of hypopigmented patches. Histopathology showed atypical lymphocytes with hyperchromatic, irregular, and cerebriform nuclei, infiltrated in the epidermis and dermis. Pautrier's microabscesses was noted in 6 of 9 patients, and papillary dermal fibroplasia was noted in 6 of 9 patients. CD8 predominance was detected in 4 of 6 patients. Four patients were simultaneously subjected to skin biopsy on hypopigmented patches and scaly erythema simultaneously. Compared with hypopigmented specimens, erythema biopsy detected deeper and denser infiltration of atypical lymphoid cells in 3 of 4 patients, higher CD4+/CD8+ ratio in 4 of 4 patients, more CD5 loss in 2 of 4 patients, and more CD7 loss in 2 of 4 patients. TCR gene monoclonal rearrangement was detected in 2 of 5 patients. Narrowband ultraviolet B phototherapy was applied in 7 patients. One of 7 patients achieved complete response, and 6 of 7 patients achieved partial response. No recurrence was noted with the median follow-up period of 6 months. CONCLUSION: HMF could occur in young patients, with indolent and benign course. HMF could gradually seem as scaly erythema based on hypopigmented patches. The histopathology indicated a more advanced stage of the scaly erythema lesions than hypopigmented patches.

Pseudolymphomatous Atypical Fibroxanthoma.

Atypical fibroxanthoma is a rare mesenchymal skin tumor of intermediate malignancy that typically occurs on sun-damaged skin of elderly patients. Histologically, it is composed of pleomorphic cells with hyperchromatic nuclei and abundant cytoplasm, commonly arranged in a spindle cell pattern. Different histologic variants have been described during the past years. We present a case of atypical fibroxanthoma containing a dense inflammatory infiltrate, which in conjunction with the existence of immunoblast-like and Reed-Sternberg-like neoplastic cells could be misinterpreted as a lymphoid neoplasm. Immunohistochemical studies revealed strong positivity of tumor cells for CD10 and negativity for cytokeratins, p63, p40, S100, SOX10, ERG, actin, desmin, B and T-cell markers, BCL6, CD15, and CD30. The inflammatory infiltrate contained a mixed reactive T- and B-cell population with negative T-cell receptor and immunoglobulin heavy rearrangements. We discuss the differential diagnosis of this entity in which clinical, immunohistochemical, and molecular features are essential to avoid the diagnosis of a lymphoproliferative disease.

Clinicopathologic, immunophenotypic, and molecular analysis of subcutaneous panniculitis-like T-cell lymphoma: A retrospective study in a tertiary care center.

BACKGROUND: Subcutaneous panniculitis-like T-cell lymphoma (SPTL) as strictly defined by World Health Organization-European Organization for Research and Treatment of Cancer classification is a rare cytotoxic α/ß T-cell lymphoma, characterized by primary involvement of subcutaneous tissue mimicking panniculitis. OBJECTIVES: To describe the clinicopathologic, immunophenotypic, and molecular features of SPTL. METHODS: A 10-year retrospective study of 18 patients diagnosed with SPTL was thoroughly reviewed according to clinicopathology, immunophenotype, and T-cell receptor (TCR) gene rearrangement. RESULTS: Of the 18 patients, 16 patients were definitely diagnosed with SPTL. The median age was 26 years (ranged 14-53 years) with female predominance. Most patients presented with prolonged fever and subcutaneous nodules and/or plaques, usually located on lower extremities. 37.5% of patients had hemophagocytic syndrome. The main histopathology was lobular panniculitis with rimming of atypical lymphocytes highlighted by CD3+, CD8+, Beta-F1+, granzyme B+, and Ki-67 (50%-90%). Monoclonal TCR gene rearrangement was found in 50% of patients and upper extremities involvement indicated a poor prognosis. CONCLUSION: The correlation between clinicopathologic and immunophenotypic study is the most helpful method to give a precise diagnosis of SPTL. Rimming of CD8+ atypical lymphocytes highlighted by high Ki-67 index is highly specific for the diagnosis of SPTL.

Comparison between flow cytometry and standard PCR in the evaluation of MRD in children with acute lymphoblastic leukemia treated with the GBTLI LLA - 2009 protocol.

Minimal residual disease (MRD) monitoring is of prognostic importance in childhood acute lymphoblastic leukemia (ALL). The detection of immunoglobulin and T-cell receptor gene rearrangements by real-time quantitative PCR (RT-PCR) is considered the gold standard for this evaluation. However, more accessible methods also show satisfactory performance. This study aimed to compare MRD analysis by four-color flow cytometry (FC) and qualitative standard PCR on days 35 and 78 of chemotherapy and to correlate these data with patients' clinical characteristics. Forty-two children with a recent diagnosis of ALL, admitted to a public hospital in Brazil for treatment in accordance with the Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI LLA-2009), were included. Bone marrow samples collected at diagnosis and on days 35 and 78 of treatment were analyzed for the immunophenotypic characterization of blasts by FC and for the detection of clonal rearrangements by standard PCR. Paired analyses were performed in 61/68 (89.7%) follow-up samples, with a general agreement of 88.5%. Disagreements were resolved by RT-PCR, which evidenced one false-negative and four false-positive results in FC, as well as two false-negative results in PCR. Among the prognostic factors, a significant association was found only between T-cell lineage and MRD by standard PCR. These results show that FC and standard PCR produce similar results in MRD detection of childhood ALL and that both methodologies may be useful in the monitoring of disease treatment, especially in regions with limited financial resources.

Thymic Atrophy: Experimental Studies and Therapeutic Interventions.

The thymus is essential for T cell development and maturation. It is extremely sensitive to atrophy, wherein loss in cellularity of the thymus and/or disruption of the thymic architecture occur. This may lead to lower naïve T cell output and limited TCR diversity. Thymic atrophy is often associated with ageing. What is less appreciated is that proper functioning of the thymus is critical for reduction in morbidity and mortality associated with various clinical conditions including infections and transplantation. Therefore, therapeutic interventions which possess thymopoietic potential and lower thymic atrophy are required. These treatments enhance thymic output, which is a vital factor in generating favourable outcomes in clinical conditions. In this review, experimental studies on thymic atrophy in rodents and clinical cases where the thymus atrophies are discussed. In addition, mechanisms leading to thymic atrophy during ageing as well as during various stress conditions are reviewed. Therapies such as zinc supplementation, IL7 administration, leptin treatment, keratinocyte growth factor administration and sex steroid ablation during thymic atrophy involving experiments in animals and various clinical scenarios are reviewed. Interventions that have been used across different scenarios to reduce the extent of thymic atrophy and enhance its output are discussed. This review aims to speculate on the roles of combination therapies, which by acting additively or synergistically may further alleviate thymic atrophy and boost its function, thereby strengthening cellular T cell responses.

Minimal residual disease (MRD) detection using rearrangement of immunoglobulin/T cell receptor genes in adult patients with acute lymphoblastic leukemia (ALL).

MRD detection with allele-specific oligonucleotide-quantitative polymerase chain reaction (ASO-qPCR) and using clone-specific immunoglobulin/T cell receptor rearrangements is considered as a powerful prognostic factor in acute lymphoblastic leukemia (ALL). In the present study, we evaluated an ASO-qPCR assay for MRD quantification in peripheral blood (PB) samples of adult patients with ALL. DNA was isolated from PB samples of patients with newly diagnosed ALL. They were first investigated by multiplex-PCR assay to identify V/J usage. An ASO-qPCR technique was then applied for 2.5-year monthly MRD quantification for detection of patient-specific Ig/TCR receptor rearrangements as a molecular target. From 98 patients who were diagnosed as ALL, 72 (73.5%) were enrolled in the present study for MRD detection. MRD was successfully quantified in patients with 1-month interval time. MRD level at the end of induction therapy up to day 88 was the only significant prognostic factor. Regarding MRD level, patients were categorized into two groups of low and high-risk. 2.5-year OS in all three time points (days 28, 58 and 88) were significantly lower in high-risk group (P < 0.008). The results of the 2.5-year MRD detection indicate that MRD level at the end of induction up to about 6 months after the first diagnosis was associated with clinical outcome. This study may highlight the usefulness of PB and the definitions of cut-off level for early prediction of relapse and for stratifying ALL patients. Short-interval time points and frequent PB sampling to monitor MRD level is suggested for early clinical relapse prediction and clinical management of the disease.

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.