T cell responses upon infection display a remarkably reproducible pattern of expansion, contraction, and memory formation. If the robustness of this pattern builds entirely on signals derived from ...other cell types or if activated T cells themselves contribute to the orchestration of these population dynamics—akin to bacterial quorum regulation—is unclear. Here, we examined this question using time-lapse microscopy, genetic perturbation, bioinformatic predictions, and mathematical modeling. We found that ICAM-1-mediated cell clustering enabled CD8+ T cells to collectively regulate the balance between proliferation and apoptosis. Mechanistically, T cell expressed CD80 and CD86 interacted with the receptors CD28 and CTLA-4 on neighboring T cells; these interactions fed two nested antagonistic feedback circuits that regulated interleukin 2 production in a manner dependent on T cell density as confirmed by in vivo modulation of this network. Thus, CD8+ T cell-population-intrinsic mechanisms regulate cellular behavior, thereby promoting robustness of population dynamics.
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•T cell clusters are communication hubs for coordination of population dynamics•T cells can regulate their own population dynamics akin to quorum regulation•T cell quorum regulation incorporates two nested antagonistic feedback circuits•Loop dominance of feedback circuits is controlled by local T cell density
Zenke et al. show that dynamics of activated CD8+ T lymphocytes are shaped by intercellular communication in a manner akin to quorum regulation. Specifically, a network of nested antagonistic feedback circuits functions to sustain or curtail T cell expansion based on cellular density, thereby promoting robustness of T cell population dynamics.
Upon infection, antigen-specific CD8⁺ T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We ...tracked the progeny of individual mouse CD8⁺ T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8⁺ T cell heterogeneity and demonstrates that reproducibility of CD8⁺ T cell responses is achieved through population averaging.
Intercellular communication is crucial for collective regulation of cellular behaviors. While clustering T cells have been shown to mutually control the production of key communication signals, it is ...unclear whether they also jointly regulate their availability and degradation. Here we use newly developed reporter systems, bioinformatic analyses, protein structure modeling and genetic perturbations to assess this. We find that T cells utilize trogocytosis by competing antagonistic receptors to differentially control the abundance of immunoregulatory ligands. Specifically, ligands trogocytosed via CD28 are shuttled to the T cell surface, enabling them to co-stimulate neighboring T cells. In contrast, CTLA4-mediated trogocytosis targets ligands for degradation. Mechanistically, this fate separation is controlled by different acid-sensitivities of receptor-ligand interactions and by the receptor intracellular domains. The ability of CD28 and CTLA4 to confer different fates to trogocytosed ligands reveals an additional layer of collective regulation of cellular behaviors and promotes the robustness of population dynamics.
Rapid intracellular transport and secretion of cytotoxic granules through the immunological synapse requires a balanced interaction of several proteins. Disturbance of this highly regulated process ...underlies familial hemophagocytic lymphohistiocytosis (FHL), a genetically heterogeneous autosomal-recessive disorder characterized by a severe hyperinflammatory phenotype. Here, we have assigned FHL-5 to a 1 Mb region on chromosome 19p by using high-resolution SNP genotyping in eight unrelated FHL patients from consanguineous families. Subsequently, we found nine different mutations, either truncating or missense, in
STXBP2 in twelve patients from Turkey, Saudi Arabia, and Central Europe.
STXBP2 encodes syntaxin binding protein 2 (Munc18-2), involved in the regulation of vesicle transport to the plasma membrane. We have identified syntaxin 11, a SNARE protein mutated in FHL-4, as an interaction partner of STXBP2. This interaction is eliminated by the missense mutations found in our FHL-5 patients, which leads to a decreased stability of both proteins, as shown in patient lymphocytes. Activity of natural killer and cytotoxic T cells was markedly reduced or absent, as determined by CD107 degranulation. Our findings thus identify a key role for STXBP2 in lytic granule exocytosis.
Variants of uncertain significance (VUS) in
CTLA4
are frequently identified in patients with antibody deficiency or immune dysregulation syndromes including, but not limited to, patients with ...multi-organ autoimmunity and autoinflammation. However, to ascertain the diagnosis of CTLA4 insufficiency, the functional relevance of each variant needs to be determined. Currently, various assays have been proposed to assess the functionality of
CTLA4
VUS, including the analysis of transendocytosis, the biological function of CTLA4 to capture CD80 molecules from antigen presenting cells. Challenges of this assay include weak fluorescence intensity of the internalized ligand, poor reproducibility, and poor performance upon analyzing thawed cells. In addition, the distinction of pathogenic from non-pathogenic variants and from wild-type
CTLA4
, and the classification of the different VUS according to its level of CTLA4 dysfunction, would be desirable. We developed a novel CD80-expressing cell line for the evaluation of CD80-transendocytosis and compared it to the published transendocytosis assay. Our approach showed lower inter-assay variability and better robustness regardless the type of starting material (fresh or thawed peripheral mononuclear cells). In addition, receiver operating characteristic analysis showed 100% specificity, avoiding false positive results and allowing for a clear distinction between pathogenic and non-pathogenic variants in
CTLA4
-variant carriers. With our transendocytosis assay, we assessed the pathogenicity of 24 distinct
CTLA4
variants from patients submitted to our diagnostic unit. Significantly impaired transendocytosis was demonstrated for 17
CTLA4
variants, whereas seven variants tested normal. In conclusion, our upgraded transendocytosis assay allows a reliable assessment of newly identified variants in
CTLA4
.
Highlights • Upon antigen encounter, proliferating T cells diversify in function and phenotype. • Lineage tracing demonstrates that the output of individual T cells is highly variable. • ...Reproducibility of T cell responses is due to the averaging of disparate single cell behaviors. • Variability in T cell output may be driven by cell-intrinsic or cell-extrinsic mechanisms.
Information regarding the prevalence of infectious diseases (IDs) in child and adolescent refugees in Europe is scarce. Here, we evaluate a standardized ID screening protocol in a cohort of ...unaccompanied refugee minors (URMs) in a municipal region of southwest Germany.
From January 2016 to December 2017, we employed a structured questionnaire to screen a cohort of 890 URMs. Collecting sociodemographic information and medical history, we also performed a standardized diagnostics panel, including complete blood count, urine status, microbial stool testing, tuberculosis (TB) screening, and serologies for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The mean age was 16.2 years; 94.0% were male, and 93.6% originated from an African country. The most common health complaints were dental problems (66.0%). The single most frequent ID was scabies (14.2%). Of the 776 URMs originating from high-prevalence countries, 7.7% and 0.4% tested positive for HBV and HIV, respectively. Nineteen pathogens were detected in a total of 119 stool samples (16.0% positivity), with intestinal schistosomiasis being the most frequent pathogen (6.7%). Blood eosinophilia proved to be a nonspecific criterion for the detection of parasitic infections. Active pulmonary TB was identified in 1.7% of URMs screened. Of note, clinical warning symptoms (fever, cough >2 weeks, and weight loss) were insensitive parameters for the identification of patients with active TB. Study limitations include the possibility of an incomplete eosinophilia workup (as no parasite serologies or malaria diagnostics were performed), as well as the inherent selection bias in our cohort because refugee populations differ across Europe.
Our study found that standardized ID screening in a URM cohort was practicable and helped collection of relevant patient data in a thorough and time-effective manner. However, screening practices need to be ameliorated, especially in relation to testing for parasitic infections. Most importantly, we found that only a minority of infections were able to be detected clinically. This underscores the importance of active surveillance of IDs among refugees.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder characterized by defective Fas signaling, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ+ CD4− CD8− ...double-negative T (DNT) cells. Although their phenotype resembles that of terminally differentiated or exhausted T cells, lack of KLRG1, high eomesodermin, and marginal T-bet expression point instead to a long-lived memory state with potent proliferative capacity. Here we show that despite their terminally differentiated phenotype, human ALPS DNT cells exhibit substantial mitotic activity in vivo. Notably, hyperproliferation of ALPS DNT cells is associated with increased basal and activation-induced phosphorylation of serine-threonine kinases Akt and mechanistic target of rapamycin (mTOR). The mTOR inhibitor rapamycin abrogated survival and proliferation of ALPS DNT cells, but not of CD4+ or CD8+ T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation by DNT cells. Importantly, increased mitotic activity and hyperactive mTOR signaling was also observed in recently defined CD4+ or CD8+ precursor DNT cells, and mTOR inhibition specifically reduced these cells in vivo, indicating abnormal programming of Fas-deficient T cells before the DNT stage. Thus, our results identify the mTOR pathway as a major regulator of lymphoproliferation and aberrant differentiation in ALPS.
•ALPS DNT cells and their putative precursors reveal high proliferative activity in vivo, which is associated with hyperactive mTOR signaling.•Rapamycin therapy controls mitotic activity and abnormal differentiation of ALPS DNT cells and reduces CD4+ or CD8+ precursor DNT cells.
Here, we present a protocol to examine the mechanisms underlying the intercellular transfer of transmembrane molecules, termed trogocytosis, and the fate of transferred molecules. We describe the ...steps needed from T lymphocyte isolation, via co-culture with cells expressing the ligand of interest, to cell harvest and subsequent staining for flow cytometry and confocal microscopy. Furthermore, we showcase critical parameters and pitfalls, which allow easy adaptation of the protocol to investigate trogocytosis of various cell surface receptors in different cell types.
For complete details on the use and execution of this protocol, please refer to Zink and Rohr.1
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•Detailed protocol for studying T cell trogocytosis by flow cytometry and microscopy•Provision of strategies to distinguish between donor and recipient cells•Comprehensive overview on principles and pitfalls for assay optimization•Easy adaptability of assay to study trogocytosis by other cell types
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Here, we present a protocol to examine the mechanisms underlying the intercellular transfer of transmembrane molecules, termed trogocytosis, and the fate of transferred molecules. We describe the steps needed from T lymphocyte isolation, via co-culture with cells expressing the ligand of interest, to cell harvest and subsequent staining for flow cytometry and confocal microscopy. Furthermore, we showcase critical parameters and pitfalls, which allow easy adaptation of the protocol to investigate trogocytosis of various cell surface receptors in different cell types.
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