cd4 effector memory t cells miltenyi biotech  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Post-COVID impairment of memory T cell responses to community-acquired pathogens can be rectified by activating cellular metabolism

doi: 10.64898/2025.12.31.697156

Figure Lengend Snippet: A.) Experimental design. B.) Statistical information about variance in pre– vs. post-COVID gene signatures are contained in principal components 25-31. C.) Isolating principal components 25-31 effectively separates samples based on pre– vs. post-COVID gene signatures. D.) Differential gene expression hits in pre– vs. post-COVID CD4 memory T cells. E.) Top 25 differentially expressed genes in pre– vs. post-COVID samples. F.) Gene set enrichment analysis (GSEA) shows significantly decreased transcription factor binding genes and elevated ATP synthase, oxidoreductase, and mitochondrial respirasome pathway genes in post-COVID CD4 memory T cells. Pre-hi: pathway expression higher in pre-COVID samples. Post-hi: pathway expression higher in post-COVID samples. G.) Significantly altered gene expression pathways enriched in mitochondrial function-related cellular processes in post-COVID relative to pre-COVID CD4 memory T cells. DEGs were calculated based on p adj =0.05 using a Bonferroni correction for multiple comparisons. Log 2 MeanDiff= mean of gene expression difference, log 2. N=16 matched pre– and post-COVID samples.

Article Snippet: CD4+CD45RO+ memory T cells were isolated by negative selection using the CD4 memory T cell isolation kit from Miltenyi Biotec.

Techniques: Gene Expression, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Post-COVID impairment of memory T cell responses to community-acquired pathogens can be rectified by activating cellular metabolism

doi: 10.64898/2025.12.31.697156

Figure Lengend Snippet: A.) Baseline levels of active mitochondrial mass (MTT), mitochondrial electron flux (TMRM), and mitochondrial reactive oxygen species production (SOX) in immune cell subsets are unchanged pre– and post-COVID. B.) VZV antigen-specific (AIM+) CD4 memory T cell numbers are unchanged after COVID, while AIM+ CD8 memory T cell numbers are elevated. C-D.) Bimodal phenotype of mitochondrial ROS and mitochondrial flux post-COVID in VZV antigen-specific memory T cells. A majority of study subjects produced less mitochondrial ROS and displayed lower electron flux post-COVID. E.) Levels of SA antigen-specific memory T cells are unchanged post-COVID. F-G.) Deficiency in electron flux and mitochondrial ROS production in SA antigen-specific memory T cells in a majority of subjects. H.) Unchanged percentages of IAV antigen-specific memory T cells post-COVID. I-J.) Similar to VZV and SA, bimodal phenotype of mitochondrial ROS and mitochondrial flux is evident post-COVID in IAV antigen-specific memory T cells. K.) Heatmap of mitochondrial ROS and mitochondrial flux expression across all samples. L.) Flow diagram showing that 92.8% of subjects had ROS and/or flux deficiency to at least one set of antigens. Graphs show mean ±SEM. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001 by paired Student’s t test or Wilcoxon test from n=28 individuals, matched samples. C, F, I: representative data from 1/28 individuals; dashed histogram represents FMO.

Article Snippet: CD4+CD45RO+ memory T cells were isolated by negative selection using the CD4 memory T cell isolation kit from Miltenyi Biotec.

Techniques: MTT Mitochondrial, Produced, Expressing

Journal: bioRxiv

Article Title: Post-COVID impairment of memory T cell responses to community-acquired pathogens can be rectified by activating cellular metabolism

doi: 10.64898/2025.12.31.697156

Figure Lengend Snippet: A.) Metabolic enzyme targets quantified in flow cytometry (MetFlow). Specific targets are in red boxes. B-C.) Post-COVID elevations in HK1 (glycolysis) and ACAC (fatty acid synthesis) and decreased expression of ATP5a (OXPHOS) in memory T cells after VZV antigen stimulation. D-E.) Higher HK1 expression in SA antigen-stimulated CD4 memory T cells post-COVID. F-G). Elevated HK1 expression and TKT expression (both glycolysis enzymes) in IAV-stimulated memory T cells after COVID. H-I.) Connectivity between metabolic pathways (% enzyme or activation marker positive memory T cells) is lost post-COVID in both CD4 (H) and CD8 (I) memory T cells. The loss of connectivity is more pronounced in CD4 memory T cells. Blue arcs: positive correlation; red arcs: negative correlation. J.) Eigenspectrum neural network analysis of statistical connections within CD4 and CD8 memory T cells finds a group-specific pre– vs. post-COVID signature. K.) Feature distribution map differentiates pre– vs. post-COVID memory T cell phenotypes. Blue: post-COVID; red: pre-COVID. The top 13 features explain the majority of statistical variation between pre– and post-COVID groups (overlay). Filled histograms: post-COVID; open histograms: pre-COVID. Graphs show mean ±SEM. *p<0.05, **p<0.01 by paired Student’s t test in n=24 samples. H, I: chord diagrams of Spearman correlations in % of memory T cells positive for the specified marker. J, K: data from B-I used to generate feature maps using Boltzmann Brain analysis. Feature maps show clear segregation of biomarkers differentiating pre-COVID (CD137 lo , HLA-DR hi , PD1 lo , GLUT1 hi ) from post-COVID (CD137 hi , HLA-DR lo , PD1 hi , GLUT1 lo ) T cell states.

Article Snippet: CD4+CD45RO+ memory T cells were isolated by negative selection using the CD4 memory T cell isolation kit from Miltenyi Biotec.

Techniques: Flow Cytometry, Expressing, Activation Assay, Marker

Journal: bioRxiv

Article Title: Post-COVID impairment of memory T cell responses to community-acquired pathogens can be rectified by activating cellular metabolism

doi: 10.64898/2025.12.31.697156

Figure Lengend Snippet: A.) Decreased glycolysis (GLUT1, HK1) but elevated fatty acid synthesis (ACAC) and phosphor-mTOR in SA and IAV antigen-specific memory T cells post-COVID. B.) Deficits in glycolysis and fatty acid oxidation in VZV antigen-specific CD4 memory T cells can be partly rescued by modulation of mitochondrial complex I (Met, dotted filled histogram) or complex III (Ubq, dotted open histogram). C.) Expression of CPT1A, HK1, and ATP5a in VZV-specific CD4 memory T cells is partly rescued by exposure to Met or Ubq. Data are representative of n=16 samples. D.) Eigenspectrum neural network of VZV antigen-specific CD4 T cells after discovery of a pre– vs. post-COVID (“Group”) MetFlow signature. Bar to the right represents 1D UMAP representation of data where blue dots are pre-COVID samples and red dots are post-COVID samples. Red and blue windows represent areas with the greatest feature divergence between pre– and post-COVID VZV-specific CD4 memory T cells. Feature maps in 5E were derived from information contained within these windows. E.) Left: Feature distribution map of pre– vs. post-COVID VZV-specific CD4 memory T cells showing higher glycolysis (GLUT1, HK1), lower immunosuppression (pMTOR), and higher fatty acid oxidation (CPT1A) in pre-COVID samples relative to post-COVID samples. Right: Feature distribution map of post-COVID VZV-specific T cells treated with Met or Ubq shows higher expression of glycolytic enzymes (HK1), activation markers (CD134, HLA-DR), and lower pMTOR expression compared to untreated T cells. Graphs show mean ±SEM. *p<0.05, **p<0.01 by paired Student’s t test in n=16 samples.

Article Snippet: CD4+CD45RO+ memory T cells were isolated by negative selection using the CD4 memory T cell isolation kit from Miltenyi Biotec.

Techniques: Expressing, Derivative Assay, Activation Assay