CTLA-4 is required for CD4+Foxp3+ regulatory T (Treg) cell function, but its mode of action remains incompletely defined. Herein we generated Ctla-4ex2fl/flFoxp3-Cre mice with Treg cells exclusively ...expressing a naturally occurring, ligand-independent isoform of CTLA-4 (liCTLA-4) that cannot interact with the costimulatory molecules CD80 and CD86. The mice did not exhibit any signs of effector T cell activation early in life, however, at 6 months of age they exhibited excessive T cell activation and inflammation in lungs. In contrast, mice with Treg cells completely lacking CTLA-4 developed lymphoproliferative disease characterized by multi-organ inflammation early in life. In vitro, Treg cells exclusively expressing liCTLA-4 inhibited CD80 and CD86 expression on dendritic cells (DC). Conversely, Treg cells required the extra-cellular part of CTLA-4 to up-regulate expression of the co-inhibitory molecule PD-L2 on DCs. Transcriptomic analysis of suppressed DCs revealed that Treg cells induced a specific immunosuppressive program in DCs.
•The cell-extrinsic functions of CTLA-4 are dispensable for regulating CD80/86 expression on DCs.•Regulatory T cells use CTLA-4 to induce the co-inhibitory molecule PD-L2 on dendritic cells.•Regulatory T cells induce a specific genetic program in suppressed dendritic cells.•CTLA-4 is required to restrict the size of the regulatory T cell pool.
Objectives
Population‐level measures of seropositivity are critical for understanding the epidemiology of an emerging pathogen, yet most antibody tests apply a strict cutoff for seropositivity that ...is not learnt in a data‐driven manner, leading to uncertainty when classifying low‐titer responses. To improve upon this, we evaluated cutoff‐independent methods for their ability to assign likelihood of SARS‐CoV‐2 seropositivity to individual samples.
Methods
Using robust ELISAs based on SARS‐CoV‐2 spike (S) and the receptor‐binding domain (RBD), we profiled antibody responses in a group of SARS‐CoV‐2 PCR+ individuals (n = 138). Using these data, we trained probabilistic learners to assign likelihood of seropositivity to test samples of unknown serostatus (n = 5100), identifying a support vector machines‐linear discriminant analysis learner (SVM‐LDA) suited for this purpose.
Results
In the training data from confirmed ancestral SARS‐CoV‐2 infections, 99% of participants had detectable anti‐S and ‐RBD IgG in the circulation, with titers differing > 1000‐fold between persons. In data of otherwise healthy individuals, 7.2% (n = 367) of samples were of uncertain serostatus, with values in the range of 3‐6SD from the mean of pre‐pandemic negative controls (n = 595). In contrast, SVM‐LDA classified 6.4% (n = 328) of test samples as having a high likelihood (> 99% chance) of past infection, 4.5% (n = 230) to have a 50–99% likelihood, and 4.0% (n = 203) to have a 10–49% likelihood. As different probabilistic approaches were more consistent with each other than conventional SD‐based methods, such tools allow for more statistically‐sound seropositivity estimates in large cohorts.
Conclusion
Probabilistic antibody testing frameworks can improve seropositivity estimates in populations with large titer variability.
Correctly classifying low‐titer antibody responses is challenging using conventional assay cutoffs. To address this issue, we trained suitable probabilistic learners to assign likelihood of seropositivity. These more quantitative methods improve seroprevalence estimates and have potential application to the clinical setting.
Natural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage ...specification and differentiation remain poorly understood. Here, we demonstrate that the ubiquitin-modifying enzyme TNFAIP3/A20, an upstream regulator of T cell receptor (TCR) signaling in T cells, is an essential cell-intrinsic regulator of NKT differentiation. A20 is differentially expressed during NKT cell development, regulates NKT cell maturation, and specifically controls the differentiation and survival of NKT1 and NKT2, but not NKT17, sublineages. Remaining A20-deficient NKT1 and NKT2 thymocytes are hyperactivated in vivo and secrete elevated levels of Th1 and Th2 cytokines after TCR ligation in vitro. Defective NKT development was restored by compound deficiency of MALT1, a key downstream component of TCR signaling in T cells. These findings therefore show that negative regulation of TCR signaling during NKT development controls the differentiation and survival of NKT1 and NKT2 cells.
Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease ...(NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD.
Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by a chronic inflammatory process often associated with comorbid asthma. In this study, we analyzed the transcriptomes of single T ...helper (T
) cells from nasal polyps of patients with CRSwNP and validated these findings using multiparameter flow cytometry. Polyp tissue contained suppressive T regulatory (T
) cells, T
2 cells, type 2 innate lymphoid cells, and three transcriptionally distinct subsets of cytotoxic CD4
T cells (CD4
CTL). GATA3 expression was a feature of polyp T
cells, whereas T
2 cells highly expressed
,
, and
and were enriched for genes involved in lipid metabolism. Only a portion of polyp T
2 cells expressed the prostaglandin D2 receptor CRTH2, whereas a subpopulation of CD109
CRTH2
T
2 cells expressed mRNA for common inhibitor receptors including LAG3 and TIM3 and produced IL-10. Together, we resolved the complexity of T
cells in patients with CRSwNP, identifying several distinct clusters of CD4
CTL and a population of CD109
CRTH2
T
2 cells with putative regulatory potential.
IL-21 has antitumor activity through actions on NK cells and CD8(+) T cells, and is currently in clinical development for the treatment of cancer. However, no studies have addressed the role of ...endogenous IL-21 in tumor immunity. In this study, we have studied both primary and secondary immune responses in IL-21(-/-) and IL-21R(-/-) mice against several experimental tumors. We found intact immune surveillance toward methylcholanthrene-induced sarcomas in IL-21(-/-) and IL-21R(-/-) mice compared with wild-type mice and B16 melanomas showed equal growth kinetics and development of lung metastases. IL-21R(-/-) mice showed competent NK cell-mediated rejection of NKG2D ligand (Rae1beta) expressing H-2b(-) RMAS lymphomas and sustained transition to CD8(+) T cell-dependent memory against H-2b(+) RMA lymphomas. alpha-Galactosylceramide stimulation showed equal expansion and activation of NKT and NK cells and mounted a powerful antitumor response in the absence of IL-21 signaling, despite reduced expression of granzyme B in NKT, NK, and CD8(+) T cells. Surprisingly, host IL-21 significantly restricted the expansion of Ag-specific CD8(+) T cells and inhibited primary CD8(+) T cell immunity against OVA-expressing EG7 lymphomas, as well as the secondary expansion of memory CD8(+) T cells. However, host IL-21 did not alter the growth of less immunogenic MC38 colon carcinomas with dim OVA expression. Overall, our results show that endogenous IL-21/IL-21R is not required for NK, NKT, and CD8(+) T cell-mediated tumor immunity, but restricts Ag-specific CD8(+) T cell expansion and rejection of immunogenic tumors, indicating novel immunosuppressive actions of this cytokine.
Interleukin‐21 (IL‐21) is a common γ‐chain cytokine produced by T helper and natural killer T (NKT) cells. It has been shown to regulate the response of various lymphocyte subsets including NK, NKT, ...T and B cells. Owing to its potent anti‐tumor function in preclinical studies and its ability to induce cytotoxicity and interferon‐γ (IFN‐γ) production in NK and CD8 T cells, recombinant IL‐21 (rIL‐21) was fast‐tracked into early‐phase clinical trials of patients with various malignancies. In a phase 2a trial of patients with metastatic melanoma, we analyzed the frequency and function of NKT cells in patients receiving rIL‐21. NKT cells were present at a low frequency, but their levels were relatively stable in patients administered rIL‐21. Unlike our observations in NK and CD8 T cells, rIL‐21 appeared to reduce IFN‐γ and TNF production by NKT cells, whereas it enhanced IL‐4 production. It also modulated the expression of cell surface markers, specifically on CD4− NKT cells. In addition, an increase in CD3+CD56+ NKT‐like cells was observed over the course of rIL‐21 administration. These results highlight that IL‐21 is a potent regulator of NKT cell function in vivo.
Cancer: Late‐stage melanoma patients respond to immunotherapy
Immunotherapy with a cytokine called interleukin‐21 (IL‐21) has shown modest efficacy in the treatment of malignant melanoma. However, IL‐21 therapy can rapidly and potently induce natural killer and cytotoxic T cell activation in patients, implying that this cytokine may yet be useful in stimulating the immune response to cancers. Jonathan Coquet, now at the Flemish Institute of Biotechnology in Ghent, Belgium, and his colleagues now report that IL‐21 therapy in patients with malignant melanoma modulates the function of NKT cells, a population with the potential to mediate the rejection of cancers. IL‐21 enhanced NKT cell granularity, and modulated the expression of cell surface receptors and cytokines produced by NKT cells. In particular, IL‐21 shifted the balance of Th1/Th2 cytokines produced by NKT cells. In addition, the authors note that a population of ‘NKT‐like’ cells is expanded by IL‐21 therapy and single out this population for further studies. These results demonstrate that IL‐21 therapy is a potent modulator of the function of immune cells including, NKT cells.
Naive CD4+ T cells differentiate into functionally diverse T helper (Th) cell subsets. Th2 cells play a pathogenic role in asthma, yet a clear picture of their transcriptional profile is lacking. We ...performed single-cell RNA sequencing (scRNA-seq) of T helper cells from lymph node, lung, and airways in the house dust mite (HDM) model of allergic airway disease. scRNA-seq resolved transcriptional profiles of naive CD4+ T, Th1, Th2, regulatory T (Treg) cells, and a CD4+ T cell population responsive to type I interferons. Th2 cells in the airways were enriched for transcription of many genes, including Cd200r1, Il6, Plac8, and Igfbp7, and their mRNA profile was supported by analysis of chromatin accessibility and flow cytometry. Pathways associated with lipid metabolism were enriched in Th2 cells, and experiments with inhibitors of key metabolic pathways supported roles for glucose and lipid metabolism. These findings provide insight into the differentiation of pathogenic Th2 cells in the context of allergy.
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•scRNA-seq resolves distinct subsets of T helper cells in response to HDM•Airway Th2 cells express over 100 distinct genes, including Il13, Plac8, and Igfbp7•ATAC-seq confirms scRNA-seq and identifies TF motifs enriched in Th2 cells•Airway Th2 cells are enriched for genes associated with lipid metabolism
Th2 cells play a pathogenic role in allergies such as asthma, yet a clear picture of their transcriptional profile in this setting is lacking. Tibbitt, Stark, et al. elucidate a distinct gene expression signature in Th2 cells responding to house dust mites and show that pathogenic airway Th2 cells are highly enriched for genes associated with lipid metabolism.
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