Significance Despite their enormous potential for diversity (in excess of 10 ¹⁵ theoretical receptor specificities), the human γδ T-cell repertoire is dominated by a specific subset expressing the ...T-cell receptor containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2) known to react to a set of pathogen-derived small molecules (phosphoantigens). Overrepresentation of this restricted set of γδ T cells in adults has been thought to reflect an antigen-specific selection process resulting from postnatal exposure to pathogens. However, we demonstrate here that restricted Vγ9Vδ2 cells with preprogrammed effector function represent the predominant γδ T-cell subset circulating in human fetal blood. This observation suggests that, despite developing in a sterile environment, the human fetal γδ T cell repertoire is enriched for pathogen-reactive T cells well before pathogen exposure.
γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl- d -erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1 ⁺ and Vδ3 ⁺ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated.
The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in ...utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth.
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•Human fetuses in 2nd trimester show T cell diversity with effector-memory phenotype•Fetal organs show diverse bacterial genera that can be cultured and propagated•Bacterial structures with mucin-like threads are visualized in 14-weeks EGA fetal gut•Fetal bacteria induce syngeneic memory T cell activation in fetal mLN T cells
Analysis of human fetal tissues and the placenta in the 2nd trimester of gestation identifies live bacterial strains that are able to induce the activation of memory T cells in the fetal mesenteric lymph node, thus providing insights into early life immunity.
The naked mole-rat (Heterocephalus glaber) is a long-lived rodent species showing resistance to the development of cancer. Although naked mole-rats have been reported to lack natural killer (NK) ...cells, γδ T cell-based immunity has been suggested in this species, which could represent an important arm of the immune system for antitumor responses. Here, we investigate the biology of these unconventional T cells in peripheral tissues (blood, spleen) and thymus of the naked mole-rat at different ages by TCR repertoire profiling and single-cell gene expression analysis. Using our own TCR annotation in the naked mole-rat genome, we report that the γδ TCR repertoire is dominated by a public invariant Vγ4-2/Vδ1-4 TCR, containing the complementary-determining-region-3 (CDR3)γ CTYWDSNYAKKLF / CDR3δ CALWELRTGGITAQLVF that are likely generated by short-homology-repeat-driven DNA rearrangements. This invariant TCR is specifically found in γδ T cells expressing genes associated with NK cytotoxicity and is generated in both the thoracic and cervical thymus of the naked mole-rat until adult life. Our results indicate that invariant Vγ4-2/Vδ1-4 NK-like effector T cells in the naked mole-rat can contribute to tumor immunosurveillance by γδ TCR-mediated recognition of a common molecular signal.
Most human blood γδ cells are cytolytic TCRVγ9Vδ2
+
lymphocytes with antitumor activity. They are currently investigated in several clinical trials of cancer immunotherapy but so far, their tumor ...infiltration has not been systematically explored across human cancers. Novel algorithms allowing the deconvolution of bulk tumor transcriptomes to find the relative proportions of infiltrating leucocytes, such as CIBERSORT, should be appropriate for this aim but in practice they fail to accurately recognize γδ T lymphocytes. Here, by implementing machine learning from microarray data, we first improved the computational identification of blood-derived TCRVγ9Vδ2
+
γδ lymphocytes and then applied this strategy to assess their abundance as tumor infiltrating lymphocytes (γδ TIL) in ∼10,000 cancer biopsies from 50 types of hematological and solid malignancies. We observed considerable inter-individual variation of TCRVγ9Vδ2
+
γδ TIL abundance both within each type and across the spectrum of cancers tested. We report their prominence in B cell-acute lymphoblastic leukemia (B-ALL), acute promyelocytic leukemia (M3-AML) and chronic myeloid leukemia (CML) as well as in inflammatory breast, prostate, esophagus, pancreas and lung carcinoma. Across all cancers, the abundance of αβ TILs and TCRVγ9Vδ2
+
γδ TILs did not correlate. αβ TIL abundance paralleled the mutational load of tumors and positively correlated with inflammation, infiltration of monocytes, macrophages and dendritic cells (DC), antigen processing and presentation, and cytolytic activity, in line with an association with a favorable outcome. In contrast, the abundance of TCRVγ9Vδ2
+
γδ TILs did not correlate with these hallmarks and was variably associated with outcome, suggesting that distinct contexts underlie TCRVγ9Vδ2
+
γδ TIL and αβ TIL mobilizations in cancer.
Abstract
Developmental thymic waves of innate-like and adaptive-like γδ T cells have been described, but the current understanding of γδ T cell development is mainly limited to mouse models. Here, we ...combine single cell (sc) RNA gene expression and sc γδ T cell receptor (TCR) sequencing on fetal and pediatric γδ thymocytes in order to understand the ontogeny of human γδ T cells. Mature fetal γδ thymocytes (both the Vγ9Vδ2 and nonVγ9Vδ2 subsets) are committed to either a type 1, a type 3 or a type 2-like effector fate displaying a wave-like pattern depending on gestation age, and are enriched for public CDR3 features upon maturation. Strikingly, these effector modules express different CDR3 sequences and follow distinct developmental trajectories. In contrast, the pediatric thymus generates only a small effector subset that is highly biased towards Vγ9Vδ2 TCR usage and shows a mixed type 1/type 3 effector profile. Thus, our combined dataset of gene expression and detailed TCR information at the single-cell level identifies distinct functional thymic programming of γδ T cell immunity in human.
Cytomegalovirus (CMV) is a leading infectious cause of morbidity in immune-compromised patients. γδ T cells have been involved in the response to CMV but their role in protection has not been firmly ...established and their dependency on other lymphocytes has not been addressed. Using C57BL/6 αβ and/or γδ T cell-deficient mice, we here show that γδ T cells are as competent as αβ T cells to protect mice from CMV-induced death. γδ T cell-mediated protection involved control of viral load and prevented organ damage. γδ T cell recovery by bone marrow transplant or adoptive transfer experiments rescued CD3ε-/- mice from CMV-induced death confirming the protective antiviral role of γδ T cells. As observed in humans, different γδ T cell subsets were induced upon CMV challenge, which differentiated into effector memory cells. This response was observed in the liver and lungs and implicated both CD27+ and CD27- γδ T cells. NK cells were the largely preponderant producers of IFNγ and cytotoxic granules throughout the infection, suggesting that the protective role of γδ T cells did not principally rely on either of these two functions. Finally, γδ T cells were strikingly sufficient to fully protect Rag-/-γc-/- mice from death, demonstrating that they can act in the absence of B and NK cells. Altogether our results uncover an autonomous protective antiviral function of γδ T cells, and open new perspectives for the characterization of a non classical mode of action which should foster the design of new γδ T cell based therapies, especially useful in αβ T cell compromised patients.
IL‐23, highly produced by neonatal DC, promotes the generation of neonatal Vγ9Vδ2 T cells expressing a range of cytotoxic mediators and producing IFN‐γ and IL‐17
The immune system in early life is ...regarded as immature. However, the IL‐12 family member IL‐23 is highly produced upon TLR stimulation by neonatal DCs. Human adult Vγ9Vδ2 T cells can be stimulated specifically via their TCR by phosphoantigens (as the pathogen‐derived HMB‐PP) or agents and infections that lead to their endogenous accumulation (as the aminobisphosphonate zoledronate). As increasing evidence indicates that γδ T cells are especially important in early life, we investigated the effect of IL‐23 on neonatal Vγ9Vδ2 T cells stimulated via their TCR. Zoledronate induced clear proliferation and IFN‐γ production in neonatal Vγ9Vδ2 T cells. In contrast, HMB‐PP did not elicit a distinct response unless at high concentrations. Addition of IL‐23 to zoledronate enhanced the expression of IFN‐γ and generated a distinct, IFN‐γ‐negative, neonatal Vγ9Vδ2 T cell population producing IL‐17. Furthermore, IL‐23 significantly enhanced the expression of a range of cytotoxic mediators (perforin, granzymes, granulysin). Although the costimulatory effect of IL‐23 on IFN‐γ and cytotoxic mediators was also observed within adult Vγ9Vδ2 T cells, the induction of an IL‐17+IFN‐γ– subset was unique to neonatal Vγ9Vδ2 T cells. In conclusion, neonatal DC‐derived IL‐23 combined with specific TCR signaling drives the generation of neonatal Vγ9Vδ2 T cells equipped with a range of cytotoxic mediators and distinct subpopulations producing IFN‐γ and IL‐17.
The co-inhibitory programmed death (PD)-1 signaling pathway plays a major role in the context of tumor-specific T cell responses. Conversely, it also contributes to the maintenance of peripheral ...tolerance, as patients receiving anti-PD-1 treatment are prone to developing immune-related adverse events. Yet, the physiological role of the PD-1/PDL-1 axis in T cell homeostasis is still poorly understood. Herein, we show that under steady-state conditions, the absence of PD-1 signaling led to a preferential expansion of CD8+ T cells in the liver. These cells exhibit an oligoclonal T cell receptor (TCR) repertoire and a terminally differentiated exhaustion profile. The transcription factor EOMES is required for the clonal expansion and acquisition of this differentiation program. Finally, single-cell transcriptomics coupled with TCR repertoire analysis support the notion that these cells arise locally from liver-resident memory CD8+ T cells. Overall, we show a role for PD-1 signaling in liver memory T cell homeostasis.
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•PD-1 controls the homeostasis of hepatic memory CD8 T cells•PD-1 blockade leads to an expansion of liver-resident CD8 T cells•These cells are oligoclonal and display an exhaustion profile•The transcription factor EOMES plays a central role in this process
The physiological role of the PD-1 axis remains unclear. Le Moine et al. show that interfering with this pathway leads to a preferential clonal expansion of liver-resident CD8+ T cells. These results could help to understand the mechanisms involved in immune-related adverse events in patients under checkpoint inhibitors.
Mistletoe (Viscum album L.) is used in German-speaking European countries in the field of integrative oncology linking conventional and complementary medicine therapies to improve quality of life. ...Various companies sell extracts, fermented or not, for injection by subcutaneous or intra-tumoral route with a regulatory status of anthroposophic medicinal products (European Medicinal Agency (EMA) assessment status). These companies as well as anthroposophical physicians argue that complex matrices composed of many molecules in mixture are necessary for activity and that the host tree of the mistletoe parasitic plant is the main determining factor for this matrix composition. The critical point is that parenteral devices of European mistletoe extracts do not have a standard chemical composition regulated by EMA quality guidelines, because they are not drugs, regulatory speaking. However, the mechanism of mistletoe's anticancer activity and its effectiveness in treating and supporting cancer patients are not fully understood. Because of this lack of transparency and knowledge regarding the matrix chemical composition, we undertook an untargeted metabolomics study of several mistletoe extracts to explore and compare their fingerprints by LC-(HR)MS(/MS) and
H-NMR. Unexpectedly, we showed that the composition was primarily driven by the manufacturer/preparation method rather than the different host trees. This differential composition may cause differences in immunostimulating and anti-cancer activities of the different commercially available mistletoe extracts as illustrated by structure-activity relationships based on LC-MS/MS and
H-NMR identifications completed by docking experiments. In conclusion, in order to move towards an evidence-based medicine use of mistletoe, it is a priority to bring rigor and quality, chemically speaking.
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting ...in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire.
, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)
CD4
CD8
double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8
SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.
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