Journal: Journal of Human Immunity

Article Title: FAS-controlled T cells drive lymphoproliferation through glycolysis without effector differentiation

doi: 10.70962/jhi.20250233

Figure Lengend Snippet: DNT in ALPS are as highly activated and proliferative as acutely EBV-stimulated CD8 T cells. (A) Representative flow cytometry plots showing CD38, HLA-DR, Ki-67, and EOMES expression in DNT of an ALPS patient and in CD8 T cells of an individual with acute EBV infection. Summary graph showing percentages of HLA-DR + CD38 + cells among total CD8 T cells of 11 individuals with acute EBV, DNT of 5 untreated ALPS patients, and CD8 T cells of 5 HD. (B and C) Enrichment score plots for the Hallmark gene sets “E2F Targets” (B) (normalized enrichment score [NES]: ALPS-DNT vs. EBV-CD8 −2.01; ALPS-DNT vs. HD-CD8 2.98; EBV-CD8 vs. HD-CD8 3.38) and “mTORC1 Signaling” (C) (NES: ALPS-DNT vs. EBV-CD8 −1.44; ALPS-DNT vs. HD-CD8 1.71; EBV-CD8 vs. HD-CD8 2.21) generated with RNA-sequencing data of sorted ALPS-DNT (CD3 + TCRαβ + CD4 − CD8 − CD45RA + CD38 + ) from three untreated ALPS patients, activated CD8 T cells (CD8 + CD38 + ) from three individuals with acute EBV infection, and naïve and early-differentiated CD8 T cells (CD8 + CD28 + CD57 − ) from three HD. (D and E) Representative histogram and summary plot of flow cytometric quantification of HIF1α (D) and GLUT1 expression (E). The median fluorescence intensities (MFIs) were normalized to those of the respective CD4 subset to allow for comparisons across multiple acquisition dates. (F) Maximum biomass flux values (mmol/gDW/h) predicted by FBA across T cell models based on constraint settings derived from RNA-sequencing data of sorted ALPS-DNT, EBV-CD8, and HD-CD8 (three samples/population). In all figures, statistical comparisons were conducted using the one-way ANOVA test for multiple comparisons. Only statistically significant differences between subsets are shown (**P < 0.01, ***P < 0.001, and ****P < 0.0001). Bars and error bars indicate mean ± SD.

Article Snippet: RNA-sequencing steps included RNA isolation (Qiagen RNeasy microkit), RNA-quality analysis (2100 Bioanalyzer, Agilent Technologies), creation of barcoded RNA-sequencing libraries (from 100 ng total RNA with Ovation Human RNA-Seq system, Nugen), and depletion of rRNAs (insert-dependent adaptor cleavage method, Nugen).

Techniques: Flow Cytometry, Expressing, Infection, Generated, RNA Sequencing, Fluorescence, Derivative Assay

Journal: Journal of Human Immunity

Article Title: FAS-controlled T cells drive lymphoproliferation through glycolysis without effector differentiation

doi: 10.70962/jhi.20250233

Figure Lengend Snippet: Glycolysis and OXPHOS in ALPS-DNT and EBV-CD8. (A) Summary plot of basal ECAR values of all Seahorse experiments. (B and C) Expression levels of selected glycolysis-related enzymes (B) and OXPHOS-related genes (C) based on RNA-sequencing data from sorted ALPS-DNT (CD3 + TCRαβ + CD4 − CD8 − ), EBV-CD8 (CD3 + CD8 + CD38 + ), and HD-CD8 (CD3 + CD8 + ) from three ALPS patients, three individuals with acute EBV infection, and three HD. The relative expression (Z-score) of genes is shown and color coded according to the legend. Rows are scaled to have a mean of 0 and SD of 1. (D) Summary plot of basal OCR values of all Seahorse experiments. (E) In silico ATP production capacity expressed relatively to the ATP production capacity to baseline models. Hexokinase reaction blocking in silico affected only ALPS-DNT model ATP production capacity, while simulating anoxic conditions (O 2 uptake blocking) led only to a substantial loss in ATP production capacity in HD-CD8 models. This demonstrates the reliance of ALPS-DNT models on glycolysis and hexokinase activity in particular. (F) Kernel density plots for the glutathione synthetase flux distributions from uniform random flux sampling ( n = 10,000), showing the higher L-lactate secretion and hexokinase fluxes (expressed in mmol/gDW/h) in ALPS-DNT models in comparison to EBV-CD8 and HD-CD8 models. (G) Biomass production in silico under various inhibitor simulations relative to the baseline model without any inhibitors applied. Inhibitor simulations were performed by blocking the biochemical reactions associated with the inhibited enzyme. Missing bars represent failed biomass (e.g., zero biomass flux) production. In A and D, statistical comparisons were conducted using the one-way ANOVA test for multiple comparisons. Only statistically significant differences between subsets are shown (**P < 0.01, ***P < 0.001). Bars and error bars indicate mean ± SD.

Article Snippet: RNA-sequencing steps included RNA isolation (Qiagen RNeasy microkit), RNA-quality analysis (2100 Bioanalyzer, Agilent Technologies), creation of barcoded RNA-sequencing libraries (from 100 ng total RNA with Ovation Human RNA-Seq system, Nugen), and depletion of rRNAs (insert-dependent adaptor cleavage method, Nugen).

Techniques: Expressing, RNA Sequencing, Infection, In Silico, Blocking Assay, Activity Assay, Sampling, Comparison

Journal: Journal of Human Immunity

Article Title: FAS-controlled T cells drive lymphoproliferation through glycolysis without effector differentiation

doi: 10.70962/jhi.20250233

Figure Lengend Snippet: Extended data on HD FCT survival, metabolic features and effector molecule production. (A) Representative flow cytometry plots showing FC-DNT (CD38 + CD45RA + ) of an HD at day (d) 0 (immediately after blood withdrawal and PBMC isolation) and after 18 h of in vitro incubation with medium (Med), allogenic monocyte-derived DC at a ratio 1:1 plus IL-21 100 ng/ml and IL-10 100 ng/ml (Stim), or with the Stim condition plus 2DG or rapamycin (Rapa) at the indicated concentrations. (B) Summary graph showing FC-DNT (as % of total DNT) reduction in immunologically healthy individuals under treatment with rapamycin for 2 wk (2w). (C) Summary plot for 2-NBDG, MTG, TMRM, and CellROX median fluorescence intensities (MFIs) in FC-DNT of five immunologically healthy individuals under treatment with rapamycin normalized to the MFI of CD4 as in . Statistics were performed with one-way ANOVA test for multiple comparisons. (D) Transcriptional levels of selected effector molecules based on RNA-sequencing data from sorted ALPS-DNT (CD3 + TCRαβ + CD4 − CD8 − ), EBV-CD8 (CD3 + CD8 + CD38 + ), and HD-CD8 (CD3 + CD8 + ) from three ALPS patients, three individuals with acute EBV infection, and three HD. The relative expression (Z-score) of genes is shown and color coded according to the legend. Rows are scaled to have a mean of 0 and SD of 1. (E) Representative flow cytometry plots showing granzyme B, A, and K, perforin, IL-10, and IFNγ expression in FC CD4 and FC CD8 of one ALPS patient and summary plots of three different ALPS patients.

Article Snippet: RNA-sequencing steps included RNA isolation (Qiagen RNeasy microkit), RNA-quality analysis (2100 Bioanalyzer, Agilent Technologies), creation of barcoded RNA-sequencing libraries (from 100 ng total RNA with Ovation Human RNA-Seq system, Nugen), and depletion of rRNAs (insert-dependent adaptor cleavage method, Nugen).

Techniques: Flow Cytometry, Isolation, In Vitro, Incubation, Derivative Assay, Fluorescence, RNA Sequencing, Infection, Expressing

Journal: Journal of Human Immunity

Article Title: FAS-controlled T cells drive lymphoproliferation through glycolysis without effector differentiation

doi: 10.70962/jhi.20250233

Figure Lengend Snippet: Despite shared metabolic features with EBV-CD8, FCT do not show classical effector differentiation and function. (A) Expression levels of selected TFs based on RNA-sequencing data from sorted ALPS-DNT (CD3 + TCRαβ + CD4 − CD8 − ), EBV-CD8 (CD3 + CD8 + CD38 + ), and HD-CD8 (CD3 + CD8 + ) from three ALPS patients, three individuals with acute EBV infection, and three HD. The relative expression (Z-score) of genes is shown and color coded according to the legend. Rows are scaled to have a mean of 0 and SD of 1. (B) Representative flow cytometry plots and summary plots showing production of the indicated cytokines or effector molecules in FC-DNT of ALPS patients ( n = 3), EBV-CD8 ( n = 3), and HD-CD8 ( n = 3–9) after stimulation of total PBMC with PMA/ionomycin for 5 h and subsequent gating. Statistics with one-way ANOVA test for multiple comparisons (*P < 0.05, **P < 0.01, and ****P < 0.0001). (C) Enrichment score plot generated with RNA-sequencing data of the indicated sorted T cell types using the gene set of day 8 acute LCMV CD8 effector versus day 30 chronic (clone 13) LCMV-exhausted CD8 T cells published in ( GSE41867 ) (NES: ALPS-DNT vs. EBV-CD8: 0.70; ALPS-DNT vs. HD-CD8: 1.36; EBV-CD8 vs. HD-CD8: 1.33).

Article Snippet: RNA-sequencing steps included RNA isolation (Qiagen RNeasy microkit), RNA-quality analysis (2100 Bioanalyzer, Agilent Technologies), creation of barcoded RNA-sequencing libraries (from 100 ng total RNA with Ovation Human RNA-Seq system, Nugen), and depletion of rRNAs (insert-dependent adaptor cleavage method, Nugen).

Techniques: Expressing, RNA Sequencing, Infection, Flow Cytometry, Generated