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While human autopsy samples have provided insights into pulmonary immune mechanisms associated with severe viral respiratory diseases, the mechanisms that contribute to a clinically favorable resolution of viral respiratory infections remain unclear due to the lack of proper experimental systems. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining successful resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication, histopathological manifestations of lung disease and loss of AT2 program, as reported in human COVID-19 patients. Infection resolution is associated with the activation of a limited number of hematopoietic subsets, including inflammatory monocytes and non-canonical double-negative T-cells with cytotoxic functions, which are highly enriched in viral RNA and dissipate upon infection resolution. Activation of specific human fibroblast and endothelial subsets also elicit robust antiviral and monocyte chemotaxis signatures, respectively. Notably, systemic depletion of human CD4+ cells, but not CD3+ cells, abrogates infection resolution in fLX and induces persistent infection, supporting evidence that peripheral CD4+ monocytes are important contributors to SARS-CoV-2 infection resolution in lung tissues. Collectively, our findings unravel a comprehensive picture of the immunological events defining effective resolution of SARS-CoV-2 infection in human lung tissues, revealing markedly divergent immunological trajectories between resolving and fatal COVID-19 cases., Competing Interests: The authors have declared that no conflict of interest exists.

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H + NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56 dim and CD56 bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2 -/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2 -/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Sepsis induces immune alterations, which last for months after the resolution of illness. The effect of this immunological reprogramming on the risk of developing cancer is unclear. Here we use a national claims database to show that sepsis survivors had a lower cumulative incidence of cancers than matched nonsevere infection survivors. We identify a chemokine network released from sepsis-trained resident macrophages that triggers tissue residency of T cells via CCR2 and CXCR6 stimulations as the immune mechanism responsible for this decreased risk of de novo tumor development after sepsis cure. While nonseptic inflammation does not provoke this network, laminarin injection could therapeutically reproduce the protective sepsis effect. This chemokine network and CXCR6 tissue-resident T cell accumulation were detected in humans with sepsis and were associated with prolonged survival in humans with cancer. These findings identify a therapeutically relevant antitumor consequence of sepsis-induced trained immunity., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets-Th1, Th2, Th17, and regulatory T-was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Despite significant advances, the eradication of cancer remains a clinical challenge which justifies the urgent exploration of additional therapeutic strategies such as immunotherapies. Human peripheral Vγ9Vδ2 T cells represent an attractive candidate subset for designing safe, feasible and effective adoptive T cell transfer-based therapies. However, following their infiltration within tumors, γδ T cells are exposed to various regulating constituents and signals from the tumor microenvironment (TME), which severely alter their antitumor functions. Here, we show that TGF-β, whose elevated production in some solid tumors is linked to a poor prognosis, interferes with the antigenic activation of human Vγ9Vδ2 T cells in vitro . This regulatory cytokine strongly impairs their cytolytic activity, which is accompanied by the induction of particular phenotypic, transcriptomic and metabolic changes. Collectively, these observations provide information for better understanding and targeting the impact of TME components to regulate the antitumor activity of human T cell effectors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rafia, Loizeau, Renoult, Harly, Pecqueur, Joalland and Scotet.)

Innate lymphoid cells (ILCs) are transcriptionally and functionally similar to T cells but lack adaptive antigen receptors. They play critical roles in early defense against pathogens. In this review, we summarize recent discoveries of ILC progenitors and discuss possible mechanisms that separate ILCs from T cells. We consider mechanisms of lineage specification in early ILC development and also examine whether differences exist between adult and fetal ILC development., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

T cells and innate lymphoid cells (ILCs) share expression of many key transcription factors during development and at mature stage, resulting in striking functional similarities between these lineages. Taking into account ILC contribution is thus necessary to appreciate T cell functions during immune responses. Furthermore, understanding ILC development and functions helps to understand T cells. Here we provide methods and protocols to isolate pure populations of multipotent precursors to T cells and innate lymphoid cells (ILCs) from adult mouse bone marrow, using flow cytometric sorting. These include precursors to all lymphocytes (viz., LMPPs and ALPs) and multipotent precursors to ILCs that have been recently refined (viz., specified EILPs, committed EILPs, and ILCPs)., (© 2023. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Regulatory elements activate promoters by recruiting transcription factors (TFs) to specific motifs. Notably, TF-DNA interactions often depend on cooperativity with colocalized partners, suggesting an underlying cis-regulatory syntax. To explore TF cooperativity in mammals, we analyze ∼500 mouse and human primary cells by combining an atlas of TF motifs, footprints, ChIP-seq, transcriptomes, and accessibility. We uncover two TF groups that colocalize with most expressed factors, forming stripes in hierarchical clustering maps. The first group includes lineage-determining factors that occupy DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprises ∼30 SP, KLF, EGR, and ZBTB family members that recognize overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking reveal that USFs impart accessibility to colocalized partners and increase their residence time. Mammalian cells have thus evolved a TF superfamily with overlapping DNA binding that facilitate chromatin accessibility., Competing Interests: Declaration of interests S.P. and L.V. are employees of Astra Zeneca and may own stock or stock options., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Innate-like T cells display characteristics of both innate lymphoid cells (ILCs) and mainstream αβ T cells, leading to overlapping functions of innate-like T cells with both subsets. In this review, we show that although innate-like T cells are probably present in all vertebrates, their main characteristics are much better known in amphibians and mammals. Innate-like T cells encompass both γδ and αβ T cells. In mammals, γδ TCRs likely coevolved with molecules of the butyrophilin family they interact with, whereas the semi-invariant TCRs of iNKT and mucosal-associated invariant T cells are evolutionarily locked with their restricting MH1b molecules, CD1d and MR1, respectively. The strong conservation of the Ag recognition systems of innate-like T cell subsets despite similar effector potentialities supports that each one fulfills nonredundant roles related to their Ag specificity., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Innate lymphoid cells (ILCs) are a family of immune cells that possess similar functions as T cells. We review steps of central ILC development in the bone marrow of adult mice and discuss recent evidence for peripheral ILC development suggesting extramedullary sites of ILC development. We also assess the contribution of development during different phases of life towards shaping the composition of the adult ILC pool. Finally, we briefly review the local cues that lead to heterogeneity of ILCs between tissues. We propose that tissue-resident ILC progenitors may economically allow tissues to elicit rapid expansion of specific ILC types that are needed based on the nature of antigenic assaults in tissues., (© 2022. The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.)

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

TCF1 plays a critical role in T lineage commitment and the development of αβ lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8 + T cells that possessed distinct characteristics including expression of CD62L, T cell factor 1 (TCF-1), and Eomesodermin; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1 + pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8 + T cells, coupled with elevated expression of multiple inhibitory receptors including programmed cell death 1 (PD-1), lymphocyte activating gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD5, and CD160. Genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62L hi TCF-1 + subset and subsequent CD8 + T cell memory. Although central memory phenotype CD8 + T cells were formed in the absence of these cells, subsequent memory CD8 + T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8 + T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8 + T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8 + T cell precursor pool may help reconcile models of the developmental origin of long-term CD8 + T cell memory., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Despite recent significant progress in cancer immunotherapies based on adoptive cell transfer(s)(ACT), the eradication of cancers still represents a major clinical challenge. In particular, the efficacy of current ACT-based therapies against solid tumors is dramatically reduced by physical barriers that prevent tumor infiltration of adoptively transferred effectors, and the tumor environment that suppress their anti-tumor functions. Novel immunotherapeutic strategies are thus needed to circumvent these issues. Human peripheral blood Vγ9Vδ2 T cells, a non-alloreactive innate-like T lymphocyte subset, recently proved to be a promising anti-tumor effector subset for ACT-based immunotherapies. Furthermore, new cell engineering tools that leverage the potential of CRISPR/Cas technology open astounding opportunities to optimize their anti-tumor effector functions. In this review, we present the current ACT strategies based on engineered T cells and their limitations. We then discuss the potential of engineered Vγ9Vδ2 T cell to overcome these limitations and improve ACT-based cancer immunotherapies., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The transcription factor TCF-1 (encoded by Tcf7 ) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7 . We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7 -expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs., (Copyright © 2020 Harly, Kenney, Wang, Ding, Zhao, Awasthi and Bhandoola.)

In this issue of JEM, Hosokawa et al. (https://doi.org/10.1084/jem.20190972) establish that transcription factor Bcl11b regulates almost completely distinct sets of genes in T cell precursors and ILC2s. To understand how this occurs, they identify multiple levels of functional regulation for Bcl11b that are used differently by T cell precursors and ILC2s., (© 2019 Harly and Bhandoola.)

Interactions between thymic epithelial cells (TEC) and developing thymocytes are essential for T cell development, but molecular insights on TEC and thymus homeostasis are still lacking. Here we identify distinct transcriptional programs of TEC that account for their age-specific properties, including proliferation rates, engraftability and function. Further analyses identify Myc as a regulator of fetal thymus development to support the rapid increase of thymus size during fetal life. Enforced Myc expression in TEC induces the prolonged maintenance of a fetal-specific transcriptional program, which in turn extends the growth phase of the thymus and enhances thymic output; meanwhile, inducible expression of Myc in adult TEC similarly promotes thymic growth. Mechanistically, this Myc function is associated with enhanced ribosomal biogenesis in TEC. Our study thus identifies age-specific transcriptional programs in TEC, and establishes that Myc controls thymus size.

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor-product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1 + ) early innate lymphoid progenitors (EILPs), which we named 'specified EILPs' and 'committed EILPs'. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate.

Small numbers of hematopoietic stem cells (HSCs) balance self-renewal and differentiation to produce the diversity and abundance of cell types that make up the blood system. How nutrients are recruited to support this massive differentiation and proliferation process remains largely unknown. The unique metabolism of adult HSCs, which rely on glycolysis and glutaminolysis, suggests a potential role for the post-translational modification O-GlcNAc as a critical nutrient signal in these cells. Glutamine, glucose, and other metabolites drive the hexosamine biosynthetic pathway (HBP) ultimately leading to the O-GlcNAc modification of critical intracellular targets. Here, we used a conditional targeted genetic deletion of the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), to determine the consequences of blocked O-GlcNAc cycling on HSCs. Oga deletion in mouse HSCs resulted in greatly diminished progenitor pools, impaired stem cell self-renewal and nearly complete loss of competitive repopulation capacity. Further, early T cell specification was particularly sensitive to Oga deletion. Loss of Oga resulted in a doubling of apoptotic cells within the bone marrow and transcriptional deregulation of key genes involved in adult stem cell maintenance and lineage specification. These findings suggest that O-GlcNAc cycling plays a critical role in supporting HSC homeostasis and early thymocyte development.

Competing Interests: LAR is a current paid employee at Public Library of Science.

Innate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Concordantly, ILCs express many transcription factors known to be important for T cell effector function. ILCs develop from lymphoid progenitors in fetal liver and adult bone marrow. However, the identification of ILC progenitor (ILCP) and other precursors in peripheral tissues raises the question of whether ILC development might occur at extramedullary sites. We discuss central and local generation in maintaining ILC abundance at peripheral sites., (Published by Elsevier Ltd.)

T cell development is orchestrated by transcription factors that regulate the expression of genes initially buried within inaccessible chromatin, but the transcription factors that establish the regulatory landscape of the T cell lineage remain unknown. Profiling chromatin accessibility at eight stages of T cell development revealed the selective enrichment of TCF-1 at genomic regions that became accessible at the earliest stages of development. TCF-1 was further required for the accessibility of these regulatory elements and at the single-cell level, it dictated a coordinate opening of chromatin in T cells. TCF-1 expression in fibroblasts generated de novo chromatin accessibility even at chromatin regions with repressive marks, inducing the expression of T cell-restricted genes. These results indicate that a mechanism by which TCF-1 controls T cell fate is through its widespread ability to target silent chromatin and establish the epigenetic identity of T cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Early innate lymphoid progenitors (EILPs) have recently been identified in mouse adult bone marrow as a multipotential progenitor population specified toward innate lymphoid cell (ILC) lineages, but their relationship with other described ILC progenitors is still unclear. In this study, we examine the progenitor-successor relationships between EILPs, all-lymphoid progenitors (ALPs), and ILC precursors (ILCps). Functional, bioinformatic, phenotypical, and genetic approaches collectively establish EILPs as an intermediate progenitor between ALPs and ILCps. Our work additionally provides new candidate regulators of ILC development and clearly defines the stage of requirement of transcription factors key for early ILC development., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2 neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2 neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2 neg γδ T cells that could be involved in an adaptive stress surveillance.

The cellular and molecular events that drive the early development of innate lymphoid cells (ILCs) remain poorly understood. We show that the transcription factor TCF-1 is required for the efficient generation of all known adult ILC subsets and their precursors. Using novel reporter mice, we identified a new subset of early ILC progenitors (EILPs) expressing high amounts of TCF-1. EILPs lacked efficient T and B lymphocyte potential but efficiently gave rise to NK cells and all known adult helper ILC lineages, indicating that they are the earliest ILC-committed progenitors identified so far. Our results suggest that upregulation of TCF-1 expression denotes the earliest stage of ILC fate specification. The discovery of EILPs provides a basis for deciphering additional signals that specify ILC fate.

T follicular helper (TFH) and T helper 1 (Th1) cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5. Furthermore, TCF1 was intrinsically required for the TFH cell response to viral infection; in the absence of TCF1, the TFH cell response was severely compromised, and the remaining TCF1-deficient TFH cells failed to maintain TFH-associated transcriptional and metabolic signatures, which were distinct from those in Th1 cells. Mechanistically, TCF1 functioned through forming negative feedback loops with IL-2 and Blimp1. Our findings demonstrate an essential role of TCF1 in TFH cell responses to viral infection., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In human beings, as well as in most non-human primates, the major peripheral γδ T cell subset, which accounts several percent of the whole lymphoid cells pool in adults, carries an heterodimeric TCR composed of Vγ9 and Vδ2 chains. Vγ9Vδ2 T cells are specifically and strongly activated by small organic pyrophosphate molecules termed phosphoantigens (phosphoAg). These low molecular weight compounds are metabolites that are produced by either microbes or endogenously, as intermediates of the mammalian mevalonate pathway, and can accumulate intracellularly during cell stress like transformation or infection. Despite the characterization of numerous natural and synthetic phosphoAg, the mechanism(s) underlying the unique and specific antigenic activation process induced by these compounds remains poorly understood. Activation is both TCR- and cell-to-cell contact-dependent, and results of previous studies have also strongly suggested a key contribution of membrane-associated molecules of primate origin expressed on target cells. The recent identification of B7-related butyrophilin (BTN) molecules CD277/BTN3A, and more precisely their BTN3A1 isoforms, as mandatory molecules in the phosphoAg-induced recognition of target cells by Vγ9Vδ2 T cells opens important opportunities for research and applications in this field. Here, we review the unusual and complex antigenic reactivity of human Vγ9Vδ2 T cells. We highlight the recent advances in our understanding of this process, and propose a model that integrates the type I glycoprotein BTN3A1 and its intracellular B30.2 domain as a physical intermediate implicated in the detection of dysregulated intracellular levels of phosphoAg and the sensing of cell stress by Vγ9Vδ2T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that utilize the unique functional potential of this major γδ T cell subset.

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

In humans, the majority of peripheral blood γδ T cells expresses Vγ9Vδ2 T-cell receptors (TCR) and recognize nonpeptidic phosphorylated antigens. In contrast, most tissue-derived γδ T cells, which are located mainly in spleen and epithelia, preferentially use Vδ1 or Vδ3 chains paired with diverse Vγ chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vδ2(-) γδ T cells remains limited. In an attempt to address this important issue, we characterized the specificity of a monoclonal antibody (mAb 256), screened for its ability to specifically inhibit cytolytic responses of several human Vδ2(-) γδ T-cell clones against transformed B cells. We show that mAb 256 does not target a TCR ligand but blocks key interactions between non-TCR molecules on effector γδ T cells and ILT2 molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classic and nonclassic major histocompatibility complex (MHC) class I molecules, blockade of MHC class I/ILT2 interactions using MHC class I- or ILT2-specific mAbs and ILT2-Fc molecules inhibited tumor-induced activation of Vγ8Vδ3 T-cell clones. Therefore, this study describes a new cytotoxic T lymphocyte activation pathway involving MHC class I engagement on γδ T cells.

Vgamma9Vdelta2 T lymphocytes are a major human gammadelta T cell subset that react against a wide array of tumor cells, through recognition of phosphorylated isoprenoid pathway metabolites called phosphoantigens. Immunotherapeutic protocols targeting Vgamma9Vdelta2 T cells have yielded promising, yet limited, signs of antitumor efficacy. To improve these approaches, we analyzed the effects on gammadelta T cells of IL-21, a cytokine known to enhance proliferation and effector functions of CD8(+) T cells and NK cells. IL-21 induced limited division of phosphoantigen-stimulated Vgamma9Vdelta2 T cells, but did not modulate their sustained expansion induced by exogenous IL-2. Vgamma9Vdelta2 T cells expanded in the presence of IL-21 and IL-2 showed enhanced antitumor cytolytic responses, associated with increased expression of CD56 and several lytic molecules, and increased tumor-induced degranulation capacity. IL-21 plus IL-2-expanded Vgamma9Vdelta2 T cells expressed higher levels of inhibitory receptors (e.g., ILT2 and NKG2A) and lower levels of the costimulatory molecule NKG2D. Importantly, these changes were rapidly and reversibly induced after short-term culture with IL-21. Finally, IL-21 irreversibly enhanced the proinflammatory Th1 polarization of expanded Vgamma9Vdelta2 T cells when added at the beginning of the culture. These data suggest a new role played by IL-21 in the cytotoxic and Th1 programming of precommitted Ag-stimulated gammadelta T cells. On a more applied standpoint, IL-21 could be combined to IL-2 to enhance gammadelta T cell-mediated antitumor responses, and thus represents a promising way to optimize immunotherapies targeting this cell subset.