Publications by authors named "Anneke Wilharm"

11 Publications

  • Page 1 of 1

Maturation of the neonatal oral mucosa involves unique epithelium-microbiota interactions.

Cell Host Microbe 2021 02 6;29(2):197-209.e5. Epub 2021 Jan 6.

Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University, Jerusalem, Israel. Electronic address:

Postnatal host-microbiota interplay governs mucosal homeostasis and is considered to have life-long health consequences. The intestine monolayer epithelium is critically involved in such early-life processes; nevertheless, the role of the oral multilayer epithelium remains ill defined. We demonstrate that unlike the intestine, the neonate oral cavity is immensely colonized by the microbiota that decline to adult levels during weaning. Neutrophils are present in the oral epithelium prenatally, and exposure to the microbiota postnatally further recruits them to the preamble neonatal epithelium by γδT17 cells. These neutrophils virtually disappear during weaning as the epithelium seals. The neonate and adult epithelium display distinct turnover kinetics and transcriptomic signatures, with neonate epithelium reminiscent of the signature found in germ-free mice. Microbial reduction during weaning is mediated by the upregulation of saliva production and induction of salivary antimicrobial components by the microbiota. Collectively, unique postnatal interactions between the multilayer epithelium and microbiota shape oral homeostasis.
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http://dx.doi.org/10.1016/j.chom.2020.12.006DOI Listing
February 2021

Microbiota-dependent expansion of testicular IL-17-producing Vγ6 γδ T cells upon puberty promotes local tissue immune surveillance.

Mucosal Immunol 2021 01 30;14(1):242-252. Epub 2020 Jul 30.

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal.

γδT cells represent the majority of lymphocytes in several mucosal tissues where they contribute to tissue homoeostasis, microbial defence and wound repair. Here we characterise a population of interleukin (IL) 17-producing γδ (γδ17) T cells that seed the testis of naive C57BL/6 mice, expand at puberty and persist throughout adulthood. We show that this population is foetal-derived and displays a T-cell receptor (TCR) repertoire highly biased towards Vγ6-containing rearrangements. These γδ17 cells were the major source of IL-17 in the testis, whereas αβ T cells mostly provided interferon (IFN)-γ in situ. Importantly, testicular γδ17 cell homoeostasis was strongly dependent on the microbiota and Toll-like receptor (TLR4)/IL-1α/IL-23 signalling. We further found that γδ17 cells contributed to tissue surveillance in a model of experimental orchitis induced by intra-testicular inoculation of Listeria monocytogenes, as Tcrδ and Il17 infected mice displayed higher bacterial loads than wild-type (WT) controls and died 3 days after infection. Altogether, this study identified a previously unappreciated foetal-derived γδ17 cell subset that infiltrates the testis at steady state, expands upon puberty and plays a crucial role in local tissue immune surveillance.
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http://dx.doi.org/10.1038/s41385-020-0330-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790758PMC
January 2021

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

Proc Natl Acad Sci U S A 2020 08 20;117(31):18649-18660. Epub 2020 Jul 20.

Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany.

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 () and δ-chain () repertoires of peripheral blood T cells in newborns, infants, and young children from Europe and sub-Saharan Africa. We identified a set of and 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- 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 rearrangements did not significantly expand after birth. However, different environmental cues may lead to the observed increase of Vδ1 and Vδ3 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.
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http://dx.doi.org/10.1073/pnas.1922588117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414158PMC
August 2020

TCR repertoire analysis reveals phosphoantigen-induced polyclonal proliferation of Vγ9Vδ2 T cells in neonates and adults.

J Leukoc Biol 2020 06 17;107(6):1023-1032. Epub 2020 Feb 17.

Institute of Immunology, Hannover Medical School, Hannover, Germany.

The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis, termed phosphoantigens (pAgs). Vγ9Vδ2 T cells are first detected at midgestation and show postnatal expansion. Interestingly, neonatal Vγ9Vδ2 T cells display a higher TCR repertoire diversity with more public clonotypes and lower pAg responsiveness than in adults. Notably, it is not known whether postnatal changes occur by TCR-dependent reactivity to pAg exposure. Here, we applied next-generation sequencing of γδ TCR repertoires to understand potential differences in the pAg-mediated response of neonatal and adult Vγ9Vδ2 T cells at the level of the expressed γδ TCR. We observed a polyclonal pAg-induced response of neonatal and adult Vγ9Vδ2 T cells, albeit neonatal γδ T cells showed less in vitro pAg responsiveness. Neonatal Vγ9Vδ2 T cells displayed a less pronounced bias for Jδ1 usage and a more frequent use of Jδ2 or Jδ3 that remained stable after pAg exposure. In addition, public and private Vδ2 TRD clones took part in the polyclonal pAg-induced response in neonates and adults. In conclusion, adult and neonatal Vγ9Vδ2 T cells both undergo polyclonal pAg-induced proliferation, whereas especially adult Vγ9Vδ2 T cells display a high stability at the level of the expressed TCR repertoire.
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http://dx.doi.org/10.1002/JLB.1MA0120-427RRDOI Listing
June 2020

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Eur J Immunol 2019 Oct;49(10):1457-1973

Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Single-Cell Transcriptomics Identifies the Adaptation of Scart1 Vγ6 T Cells to Skin Residency as Activated Effector Cells.

Cell Rep 2019 06;27(12):3657-3671.e4

Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany. Electronic address:

IL-17-producing γδ T cells express oligoclonal Vγ4 and Vγ6 TCRs, mainly develop in the prenatal thymus, and later persist as long-lived self-renewing cells in all kinds of tissues. However, their exchange between tissues and the mechanisms of their tissue-specific adaptation remain poorly understood. Here, single-cell RNA-seq profiling identifies IL-17-producing Vγ6 T cells as a highly homogeneous Scart1 population in contrast to their Scart2 IL-17-producing Vγ4 T cell counterparts. Parabiosis demonstrates that Vγ6 T cells are fairly tissue resident in the thymus, peripheral lymph nodes, and skin. There, Scart1 Vγ6 T cells display tissue-specific gene expression signatures in the skin, characterized by steady-state production of the cytokines IL-17A and amphiregulin as well as by high expression of the anti-apoptotic Bcl2a1 protein family. Together, this study demonstrates how Scart1 Vγ6 T cells undergo tissue-specific functional adaptation to persist as effector cells in their skin habitat.
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http://dx.doi.org/10.1016/j.celrep.2019.05.064DOI Listing
June 2019

Styk1 is specifically expressed in NK1.1 lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.

Eur J Immunol 2019 05 18;49(5):686-693. Epub 2019 Feb 18.

Institute of Immunology, Hannover Medical School, Hannover, Germany.

Innate T cells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αβ iNKT, and γδ NKT cell lineages. To investigate the role of Styk1 in the development and function of NK1.1 innate T-cell subsets, we generated and analyzed a novel Styk1 mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKT cells and in thymic, but not in peripheral invariant αβ iNKT cells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKT cells, and thymic αβ iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.
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http://dx.doi.org/10.1002/eji.201848033DOI Listing
May 2019

Mutual interplay between IL-17-producing γδT cells and microbiota orchestrates oral mucosal homeostasis.

Proc Natl Acad Sci U S A 2019 02 28;116(7):2652-2661. Epub 2019 Jan 28.

Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University, 9190501 Jerusalem, Israel;

γδT cells are a major component of epithelial tissues and play a role in tissue homeostasis and host defense. γδT cells also reside in the gingiva, an oral tissue covered with specialized epithelium that continuously monitors the challenging dental biofilm. Whereas most research on intraepithelial γδT cells focuses on the skin and intestine epithelia, our knowledge on these cells in the gingiva is still incomplete. In this study, we demonstrate that even though the gingiva develops after birth, the majority of gingival γδT cells are fetal thymus-derived Vγ6 cells, and to a lesser extent Vγ1 and Vγ4 cells. Furthermore, we show that γδT cells are motile and locate preferentially in the epithelium adjacent to the biofilm. Vγ6 cells represent the major source of IL-17-producing cells in the gingiva. Chimeric mice and parabiosis experiments indicated that the main fraction of gingival γδT cells is radioresistant and tissue-resident, persisting locally independent of circulating γδT cells. Notably, gingival γδT cell homeostasis is regulated by the microbiota as the ratio of Vγ6 and Vγ4 cells was reversed in germ-free mice, and their activation state was decreased. As a consequence, conditional ablation of γδT cells results in elevated gingival inflammation and subsequent alterations of oral microbial diversity. Taken together, these findings suggest that oral mucosal homeostasis is shaped by reciprocal interplays between γδT cells and local microbiota.
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http://dx.doi.org/10.1073/pnas.1818812116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377488PMC
February 2019

Genetic models reveal origin, persistence and non-redundant functions of IL-17-producing γδ T cells.

J Exp Med 2018 12 19;215(12):3006-3018. Epub 2018 Nov 19.

Institute of Immunology, Hannover Medical School, Hannover, Germany

γδ T cells are highly conserved in jawed vertebrates, suggesting an essential role in the immune system. However, γδ T cell-deficient mice display surprisingly mild phenotypes. We hypothesized that the lack of γδ T cells in constitutive mice is functionally compensated by other lymphocytes taking over genuine γδ T cell functions. To test this, we generated a knock-in model for diphtheria toxin-mediated conditional γδ T cell depletion. In contrast to IFN-γ-producing γδ T cells, IL-17-producing γδ T cells (Tγδ17 cells) recovered inefficiently after depletion, and their niches were filled by expanding Th17 cells and ILC3s. Complementary genetic fate mapping further demonstrated that Tγδ17 cells are long-lived and persisting lymphocytes. Investigating the function of γδ T cells, conditional depletion but not constitutive deficiency protected from imiquimod-induced psoriasis. Together, we clarify that fetal thymus-derived Tγδ17 cells are nonredundant local effector cells in IL-17-driven skin pathology.
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http://dx.doi.org/10.1084/jem.20181439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279411PMC
December 2018

Human papillomavirus oncoproteins induce a reorganization of epithelial-associated γδ T cells promoting tumor formation.

Proc Natl Acad Sci U S A 2017 10 10;114(43):E9056-E9065. Epub 2017 Oct 10.

Laboratory of Cellular and Molecular Immunology, GIGA Research, University of Liège, 4000 Liège, Belgium;

It has been shown that γδ T cells protect against the formation of squamous cell carcinoma (SCC) in several models. However, the role of γδ T cells in human papillomavirus (HPV)-associated uterine cervical SCC, the third-leading cause of death by cancer in women, is unknown. Here, we investigated the impact of γδ T cells in a transgenic mouse model of carcinogenesis induced by HPV16 oncoproteins. Surprisingly, γδ T cells promoted the development of HPV16 oncoprotein-induced lesions. HPV16 oncoproteins induced a decrease in epidermal Skint1 expression and the associated antitumor Vγ5 γδ T cells, which were replaced by γδ T-cell subsets (mainly Vγ6 γδCCR2CCR6) actively producing IL-17A. Consistent with a proangiogenic role, γδ T cells promoted the formation of blood vessels in the dermis underlying the HPV-induced lesions. In human cervical biopsies, IL-17A γδ T cells could only be observed at the cancer stage (SCC), where HPV oncoproteins are highly expressed, supporting the clinical relevance of our observations in mice. Overall, our results suggest that HPV16 oncoproteins induce a reorganization of the local epithelial-associated γδ T-cell subpopulations, thereby promoting angiogenesis and cancer development.
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http://dx.doi.org/10.1073/pnas.1712883114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664550PMC
October 2017
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