Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting. B, Increase in total number of cells of known (KIF-P1 and -P2, SYT-T1, -T2, -P1, -P2) and newly identified (KIF-sc1 and -sc2) TCRs upon antigen-specific stimulation and CD137-enrichment with dominance of KIF-P1 and -P2 harboring high precursor frequency. The two newly identified KIF2C-TCRs were selected based on fold change of TCR frequency and highest absolute frequency in the stimulated sample. C, Assessment of antigen-specific IFN-γ-secretion for the two newly identified TCRs KIF-sc1 and -sc2 in comparison to the known TCR KIF-P2. Cytokine secretion was measured by IFN-γ-ELISA upon 24h of co-culture of TCR-tg T cells from one representative donor with Mel15-LCLs transgenic for the mutated KIF2C P13L minigene (mut mg) and the wildtype KIF2C minigene (wt mg) as well as pulsed for 2h at 37°C with the mutated and wildtype peptide (mut pep and wt pep). An irrelevant peptide (irr peptide), target cells (LCL only) or T cells alone (T cell only) served as negative controls. D, Frequency of KIF-sc1 and -sc2 in relation to the previously identified TCR-sequences identified by deep sequencing of the TCR-β-chain in intestinal (MInt) and lung metastases (MLung) as well as corresponding non-malignant draining lymph nodes (MInt-LN1, MInt-LN2 and MLung-LN) of patient Mel15. Non-td: non-transduced.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transgenic Assay, Sequencing

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A-C, Mel15 LCLs were pulsed with titrated peptide concentrations (2h, 37°C) and co- incubated with TCR-tg T cells with subsequent ELISA-based assessment of IFN-γ-secretion within 24h of co-culture (A). The cellular activation level was determined after 24h by FACS staining of the extracellular level of CD137 (B) and PD-1 (C) expression (reflected by geometric mean of all CD3 + CD8 + /TCRmu + cells). The mean for ELISA data is depicted for technical triplicates of one donor; triplicates from the same donor have been pooled prior to EC FACS- staining. E:T = 1:1 (15.000 tg T cells:15.000 tumor cells). D-G, EC FACS staining at different timepoints after co-culture setup displays temporal dynamics of T cell activation marker CD137 (D, E) and inhibitory receptor LAG-3 (F, G) for TCR-tg T cells upon co-culture with JJN3-B27 peptide-pulsed target cells. A weak (0.01 µM for peptide pulsing; D, F) versus a strong (1 µM for peptide pulsing; E, G) stimulus were compared. E:T = 1:1 (10.000 tg T cells:10.000 tumor cells). H, Annexin-V/PI-staining was employed for detection of activation induced cell death (AICD) after 20h of co-culture upon strong stimulation with 1µM mut-peptide pulsed Mel15 LCLs (early apoptotic = AnnexinV + PI - , late apoptotic = AnnexinV + PI + ). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). I, Representative FACS plot of a healthy donor of CTV-analysis for all TCRmu + cells depicted after 4 days of co-culture with 1µM mut-peptide pulsed Mel15 LCLs (colors were chosen according to Figure B-E; representative wt mg-control depicted in grey). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). For all co-cultures in D-I technical triplicates per donor were pooled prior to staining; the mean and SD for biological replicates from three different human donors are shown.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Activation Assay, Staining, Expressing, Marker, Control

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting of xenograft neoTCR-tumor rejection experiment with newly transduced T cells. B, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for mut mg-U698M-tumor- bearing NSG-mice comparing neoTCR-tg T cells to the irrelevant TCR 2.5D6 until day 20. Mean values and SDs for each group of mice display rejection dynamics (n=6). Parts of this dataset were already published before . In this renewed version, tumor rejection kinetics of KIF-sc1 and -sc2 analyzed in the same experiment are included. C, Kaplan-Meier-survival curve is displayed until day 20 for tumor-bearing mice injected with different neoTCR-tg T cells. D, schematic experiment setting of xenograft neoTCR-TIL-rechallenge experiment. E-G, Ex vivo restimulation of T cells derived from TIL products on day 21 after tumor explant (TIL-P) compared to newly transduced (NEW) TCR-tg T cells from the same human donor stained for CD137 (EC; E), IFN-γ (IC; F) and GzmB (IC; G); expression was analyzed using geometric mean of all CD3 + CD8 + /TCRmu + cells after 18h of co-culture. Mut-mg and wt-mg U698M cells were used as target cells in E:T = 1:1 (50.000 tg T cells:50.000 tumor cells). Mean and SD are shown for three experimental replicates. Statistical significance is calculated with one-way ANOVA and Tukey’s multiple comparison test (*p≤0.05, ****p≤0.0001). H, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for NSG-mice continuing the experiment until day 17 after second injection of in total 5x10 6 neoTCR-tg T cells (transduction rate of 55% equalized for all groups). For the TIL- P-groups, TIL-P from two mice per TCR (highest numbers of expanded neoTCR-tg T cells) were pooled. Mean values and SEMs for each group of mice display rejection dynamics (n=5 for experimental groups, n=3 for 2.5D6; due to achievement of humane endpoint criteria one 2.5D6- control mouse was sacrificed on day 13 and excluded from this graph). Statistical significance is calculated for the tumor area on day 17 with one-way ANOVA and Tukey’s multiple comparison test (***p≤0.001, ****p≤0.0001). I , Kaplan-Meier-survival curve is displayed for tumor-bearing mice injected with different TCR-tg T cells (n=5 for experimental groups, n=4 for 2.5D6 control). Survival of mice receiving TIL-P-KIF-P2 compared to TIL-P-KIF-sc1 was significantly prolonged (p=0.0019, Mantel-Cox test).

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Injection, Ex Vivo, Derivative Assay, Staining, Expressing, Co-Culture Assay, Comparison, Transduction, Control

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

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34 + hematopoietic cells while allowing the detection of tumorigenic expansion

doi: 10.3389/fbioe.2024.1388312

Figure Lengend Snippet: Prospective model for in vitro safety assessment of gene-edited human hematopoietic stem and progenitor cells (hHSPCs). The MOAB system aims to be used to test the expansion of cells bearing an unwanted protumorigenic mutation in gene-editing procedures. In case the test does not detect a significative expansion of cells, the autologous transplantation can be performed (top right), conversely if the test highlights a malignant phenotype, the cells cannot be employed for clinical applications and the autologous transplantation cannot be performed (Bottom, right). Created in BioRender.com .

Article Snippet: CD34 + HSPCs were isolated through magnetic cell separation using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec, Italy), MS Columns (Miltenyi Biotec, Italy), and a MACS Separator (Miltenyi Biotec, Italy).

Techniques: In Vitro, Mutagenesis, Transplantation Assay

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Stem cell transcriptome of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst MSC, hESC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Dendrogram showing hierarchical clustering of MSC, hESC and MSC-hiPSC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing MSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC (d) and MSC-hiPSC (e) is reported in the indicated tables [Fisher's exact test].

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Mesenchymal potential of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst different F-hiPSC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Principal Component Analysis (PCA) showing 3D visualization of Principal Component (PC) 1, PC2 and PC3 of differentially expressed genes for different F-hiPSC, MSC-hiPSC and hESC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing F-hiPSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC-hiPSC (d) and F-hiPSC (e) is reported in the indicated tables [Fisher's exact test]. f) Schematic of the differentiation protocol toward MSC-like cells. g) Representative images of adipogenic (A, scale bar is 50 µm), osteogenic (O, scale bar is 50 µm) and chondrogenic (C, scale bar is 400 µm) mesenchymal derivatives. h) Left panel: representative density plot showing CD45 + hematopoietic cells (P3) gated from total cells of the cobblestone area-forming cell assay; FSC-A, forward scatter area, a.u., arbitrary units. Right panel: representative histograms showing CD34 + hematopoietic progenitor subpopulation (purple) of CD45 + cells compared to unstained control (grey); the vertical axis represents event percentage count (Count%).

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test, Control