Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , b , GSEA using a previously defined TGFβ gene set applied to T cells ( a ) and the gene set ‘Li M200 antigen processing and presentation’ applied to monocyte clusters ( b ) depicted as both UMAP (left) and a dot plot (right). c , Schematic overview of T cell reactivation assays. d , Frequencies of overall activated (CD69 + ) and antigen-specific reactivated (CD137 + CD69 + and CD154 + CD69 + ) CD4 + or CD8 + memory T cells (T mem , CD45RO + ) from patients with MIS-C during the acute phase and at follow-up after symptoms resolved ( n = 8 patients and n = 5 different viral peptides). e , f , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells (T mem ; CD45RO + ) from healthy donors ( n = 6) treated with serum from patients with MIS-C ( e ; n = 7) or patients with severe COVID-19 ( f ; n = 5) with or without anti-TGFβ. Samples that were obtained more than 24 h after the start of treatment are colour-coded in yellow. Unpaired ( a , b ) or paired ( d – f ) two-tailed Mann–Whitney U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in Fig. . Cells were identified by size and granularity in a FSC-vs SSC plot, followed by doublet exclusion in an FSC-A vs. FSC-H plot. Dump + (DAPI, CD14 and CD19) + cells were also excluded. As CD3 is downregulated after T cell activation (SEB plot in second row), the gate was extended to include CD3 low CD45RO + cells. CD4 + epitope-specific T cells were identified as CD69 + CD154 + and CD8 + epitope specific T cells were identified as CD69 + CD137 + or as CD69 + CD154 + . SEB was used as a positive control for correct gating. b , Cell counts for CD69 + or CD69 + and CD154 + or CD137 + memory T cells from Fig. . c , Gating strategy used in flow cytometry of the T cell reactivity assays depicted in d. d , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from six children with a confirmed infection with SARS-CoV-2 during the acute phase and follow-up upon after resolution of symptoms. e , Frequencies of overall activated and antigen-specific reactivated cells of CD4 + and CD8 + memory T cells from healthy donors ( n = 6) treated with 50 ng ml −1 TGFβ1. f , Frequencies of TCRVβ21.3 + on total T cells were quantified by Flow cytometry over time after treatment start with IVIG and methylprednisolone. Horizontal lines indicate normal range (0.9-4.9% for CD8 + T cells; 1.5-4-7% for CD4 + T cells) of TCRVβ21.3 + T cells ( n = 25, children with MIS-C). g , Significantly regulated TRBV determined by TCR sequencing of activated T cells. Dots indicate the frequency of specific TRBV in each sample relative to all TCRs sequenced. h , Frequencies of TRAV gene associated to TRBV11-2 + T cells not depicted in Fig. . i , HLA-class I haplotyping and ( j-k ) HLA-class-II haplotyping of our MIS-C cohort ( n = 20 patients and n = 10 healthy controls including the 4 children used as a control for the scRNAseq experiments). Additionally, HLA-haplotyping from a previously published MIS-C cohort ( n = 7 patients and 9 controls) was included. l-m , Sorting strategy for Fig. . P -values for ( b + d-e + h ) were determined by paired two-tailed Mann-Whitney- U -tests.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Flow Cytometry, Activation Assay, Positive Control, Infection, Sequencing, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children

doi: 10.1038/s41586-025-08697-6

Figure Lengend Snippet: a , Schematic showing generation of virus-specific TCR libraries and comparison of virus-specific TCRs with MIS-C-specific TCRs. scTCR-seq, single-cell TCR sequencing. b , UMAP of 22,344 virus-specific T cells from donors restimulated with EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3) or measles ( n = 3) peptides, representing 18,010 sequenced TCRβ chains and 15,496 full TCRs. AdV-specific T cells were TCR-sequenced. Virus-specificities are colour-coded. c , TRVB11-2 + T cells superimposed on the UMAP in b . d , Gene expression superimposed on the UMAP of antigen-specific T cells, showing that most TRVB11-2 + T cells have a CD4 or CD8 cytotoxic phenotype (low: ICOS ; high: PRF1 , GZMB , LAMP1 ). e , TCR repertoires of EBV ( n = 5), CMV ( n = 5), SARS-CoV-2 ( n = 3), measles ( n = 3) and AdV ( n = 1) virus-specific T cells from healthy donors, analysed by ARTE . Heat map showing distribution of TRAV gene expression associated with TRBV11-2 -positive T cells in virus-specific and MIS-C T cells ( n = 11) T cells, compared with 6 wpi no MIS-C ( n = 4) and paediatric influenza ( n = 3) T cells. Unsupervised clustering was performed with the R package pheatmap. f , TCRVβ21.3 expression on memory T (T mem ) cells after stimulation with EBNA2 275–294 (left) or EBNA2 279–289 (right) peptides, analysed by ARTE. Frequencies of TCRVβ21.3 + in all CD4 + (top) and CD8 + (bottom) memory T cells and those with antigen-specific reactivation (CD154 + CD69 + ) from n = 7 donors. Flow cytometry gating is shown in Extended Data Fig. . Two-sided paired t -test.

Article Snippet: After stimulation, cells were stained with TotalSeq anti-human Hashtags as previously mentioned, followed by CD154 MACS enrichment according to the manufacturer’s protocol (CD154 MicroBead Kit, human; Miltenyi Biotec).

Techniques: Virus, Comparison, Sequencing, Gene Expression, Expressing, Flow Cytometry