macs miltenyi kits  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A The miCLIP workflow for identification of high-confidence crosslinks: (1) primary human CD8 + T cells subjected to in vitro activation with aCD3/aCD28 beads; (2) RNA enrichment and experimental controls, including m 6 A immunoprecipitation without UV irradiation (noUV) and input RNA samples; (3) SDS‒PAGE purification of RNA fragments crosslinked to the anti-m 6 A antibody followed by RNA isolation; (4) library preparation; (5) iCLIP analysis for determination of uniquely mapped crosslinks; (6) determination of peaks, i.e., regions with high density of crosslinks; (7) ‘positionally enriched k-mer analysis’ (PEKA) to extract sequences in high-confidence (‘thresholded’) miCLIP crosslinks. B Hierarchical clustering of 5-mers based on standardised PEKA scores at ‘thresholded sites’; here, crosslinks and Clippy peaks were used for PEKA. The median PEKA score of each miCLIP or Input group (noAct, Day1 or Day5) was calculated for all 5-mers, and the unique sets of the 20 most enriched and variant 5-mers were plotted on the heatmap. C Metagene distribution of GLORI-determined m 6 A counts at miCLIP sites (top) and number-matched control sites randomly selected from the 3’UTR (bottom), centred on RRACH, WWAWW and CRACH pentamers; GLORI counts show the effect of the METTL3 inhibitor at each position, and represent the average across two CD8 + T cell replicates. D Metagene distribution of GLORI-determined m 6 A counts centred on miCLIP-ARE sites that contain nearby RRACH motifs (left) or lack RRACH motifs (right), showing the effect of the METTL3 inhibitor. Stratification by distance highlights the spatial relationship between m 6 A deposition and RRACH proximity. The WWAWW + RRACH sites refer to miCLIP-ARE sites containing at least one RRACH motif within ≤ 50nt. E Density plot showing the motif-specific distribution of PEKA crosslinks in the miCLIP and Input samples around scaled mRNA regions. For all panels, CD8 + T cells from healthy donors were isolated; not activated (noAct; n = 4), Day1 after activation (Day1; n = 5), Day5 after activation (Day5; n = 4), Mock ( n = 12), noUV ( n = 3). Panel ( A ) was created in BioRender. Foskolou, I. (2026) https://BioRender.com/ h18sinc. Source data are provided as a Source Data file.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were thawed, and CD8 + T cells were isolated using a CD8+ isolation kit (CD8 + MicroBeads, 130-045-201, Miltenyi biotec).

Techniques: In Vitro, Activation Assay, Immunoprecipitation, Irradiation, Purification, Isolation, Variant Assay, Control

Journal: Nature Communications

Article Title: Meta-unstable mRNAs in activated CD8 + T cells are defined by interlinked AU-rich elements and m 6 A mRNA methylation

doi: 10.1038/s41467-025-67762-w

Figure Lengend Snippet: A Actinomycin D experiments showing % of remaining mRNA in CD8⁺ T cells treated with DMSO or METTL3inhibitor. Mean ± SD; n = 3 (one donor provided material for 0,2 h). Mixed-effects model (REML), p -values indicate treatment effects. Donors were processed in parallel. Representative results from three independent experiments ( IL7R , TNF ). B mRNA levels IL7R ( n = 5), CCL4 ( n = 5), CD69 ( n = 4), CD28 ( n = 4), TCF7 ( n = 3), ZFP36L1 ( n = 3), TNF ( n = 4), IFNG ( n = 4) in CD8⁺ T cells treated with METTL3inhibitor. Fold change relative to DMSO (grey line). Reference gene 18S. Mean ± SD; paired two-tailed t tests on ΔCt values. For TNF , one Grubbs-identified outlier is displayed but excluded from statistics. C , D Representative flow cytometry plots illustrating IL7Rα gating ( C ) and quantification of IL7Rα⁺CD8⁺ T cells ( D ) ( n = 10). Paired two-tailed t tests. E , F Representative histogram ( E ) and quantification ( F ) of IL7Rα median fluorescence intensity (MFI) in IL7Rα⁺ cells treated with METTL3inhibitor relative to DMSO (grey line) ( n = 10). Mean ± SD; one-sample two-tailed t test. G GFP MFI in T cells transduced with GFP-3’UTR constructs (S3H) after treatment with FTO inhibitor followed by PMA/ionomycin activation relative to DMSO ( n = 3). One-way ordinary ANOVA. H , I IFNγ (H) and TNFα (I) protein levels measured by ELISA in DMSO (0 μM) or FTOinhibitor treated cells ( n = 3). RM one-way ANOVA. J , K Representative amplification curves (ΔRn) and Ct quantification for reference ( J ) and miCLIP-ARE ( K ) amplicons in control (grey) and METTL3-KO (pink) samples ( n = 3). Mean ± SD. L Fold-change expression (ΔΔCt) for the miCLIP-ARE site in METTL3-KO and FTO-KO cells relative to control (grey line). Mean ± SEM; unpaired two-tailed t test comparing METTL3-KO and FTO-KO; paired t test for control vs METTL3-KO on ΔCt values; ( n = 3). M – P mRNA levels ( M ) and GFP protein levels ( N – P ) in CD8 + T cells transduced with GFP-3′UTR constructs (S3O). mRNA levels normalised to 3′UTR- TNF_ WT (grey line) ( n = 10); one-way ANOVA with Holm–Šídák correction ( M ). Representative plots of resting or PMA/ionomycin-activated cells ( N ). GFP MFI quantification in activated cells ( O , P ) ( n = 15); paired two-tailed t tests. Box-and-whisker plots (min–max, median, 25th–75th percentiles). All n values reflect distinct donors. Source data are provided as a Source Data file.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were thawed, and CD8 + T cells were isolated using a CD8+ isolation kit (CD8 + MicroBeads, 130-045-201, Miltenyi biotec).

Techniques: Two Tailed Test, Flow Cytometry, Fluorescence, Transduction, Construct, Activation Assay, Enzyme-linked Immunosorbent Assay, Amplification, Control, Expressing, Whisker Assay

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: In vivo CRISPR screen identifies target gene Glul . (A) Growth curve for subcutaneous MC38-Ova tumors in mice treated intraperitoneally with PBS (black) or DON (red). The asterisk indicates treatment start date (day 18 postinjection). n = 10 per treatment group. (B) Kaplan-Meier survival curve for mice in panel A. (C) Experimental scheme of in vivo CRISPR screen. (D) Representative volcano plot showing enriched and depleted guides in the recovered TIL compared with injected cell population. Statistical analysis performed by MAGeCK, representative for 7 biological replicates over 2 independent experiments. (E) Schematic of 1:1 target validation HKP1-Ova tumor model. (F) Representative flow plots showing relative populations of cells transduced with Glul gRNA ( Glul KO) or NTC gRNA that were injected (input), recovered from lungs without tumors, or recovered from HKP1-Ova tumors (left to right). (G) Quantification of ratio of recovered cells expressing Glul vs NTC gRNA from HKP1-Ova tumors (n = 7) or no tumor control lungs (n = 3). A one-sample t test used to determine if recovered ratios were significantly different than expected (input) ratio (dashed line). (H) Lung mass as a proxy for tumor burden. n = 10 per group. (I) Representative flow plots showing TCF-1 + population, gated on live CD8 + T cells, from mice treated with NTC or GS KO mice (left to right). n = 10 per group, quantified in panel J. (K) PD-1 geometric mean florescence intensity (MFI) of recovered CD8 + T cells, as measured by flow. Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P ≤ 0.0001. Log-rank (Mantel-Cox) test used in panel B. Mann-Whitney test used in panels H, J, and K.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: In Vivo, CRISPR, Injection, Biomarker Discovery, Transduction, Expressing, Control, Labeling, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: T cell specific GS deletion has no detrimental effects at baseline. (A) Breeding scheme for generation of T cell–specific GS KO vs WT littermate control mice. (B) Glul mRNA levels of WT vs GS KO CD8 + T cells activated in vitro, as measured via bulk RNA sequencing and expressed as normalized counts following DESEQ2 analysis. n = 5 biological replicates. (C) Western blot showing GS and vinculin expression in cell lysates from activated WT vs GS KO CD8 + T cells. (D) Live thymocytes and (E) splenocytes recovered from adult (8 wk) mice. Data from 2 experiments with 6 to 7 biological replicates each. (F) CD8 + and CD4 + T cells, reported as percentage of live CD45 + cells, recovered from WT vs GS KO spleens. n = 7–8 biological replicates. (G) Viability and activation of recovered CD8 + T cells from WT vs GS KO spleens, as measured by %CD69 + /CD62L − , following 24, 48, and 72 h of activation in vitro with plate-bound anti-CD3/CD28. (H) CD8 + T cell proliferation following ex vivo activation for 48 or 72 h, measured by CTV staining. Representative plot (left) and quantified proliferation index (right) with 3 biological replicates. (I) Geometric mean fluorescence intensity (gMFI) of activated WT vs GS KO CD8 + T cells showing mitochondrial mass (MitoTracker Green) and mitochondrial membrane polarization (TMRE), normalized to mitochondrial mass. Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P ≤ 0.0001. Mann-Whitney test used in panels D to I.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Control, In Vitro, RNA Sequencing, Western Blot, Expressing, Activation Assay, Ex Vivo, Staining, Fluorescence, Membrane, Labeling, MANN-WHITNEY

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: Pharmacologic GS inhibition alters cell proliferation while enhancing cytolytic capacity. (A) Isolated CD8 + T cells activated in 0, 5, 15, or 30 mM MSO in vitro for 72 h. Live cell count, viability, and forward scatter (FSA) all measured via flow cytometry. n = 3 biological replicates. (B) Live cell count and percent CD62L − /CD69 + after 72 h of in vitro activation in 0, 5, or 10 mM MSO. n = 3 biological replicates. (C) Representative CTV histograms and replication index quantification on cells from panel B. n = 3. (D) MitoTracker Green and TMRE geometric MFI (gMFI) of cells activated for 72 h with increasing doses of MSO. TMRE normalized to mitochondrial content. n = 3. (E) Representative flow plots for TNFα and IFNγ production upon restimulation by cells activated in 0, 5, or 10 mM MSO, quantified in panel F. n = 3. Statistically significant results are labeled. * P < 0.05, ** P < 0.01. One-way analysis of variance with Dunnett’s multiple comparisons test used in panels A to D and F.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Inhibition, Isolation, In Vitro, Cell Characterization, Flow Cytometry, Activation Assay, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: GS inhibition and KO increase glutamate and mitochondrial respiration. (A) Illustration showing glutamine utilization pathways. (B) Intracellular levels of glutamate and glutamine in activated CD8 + T cells from WT or GS KO mice. n = 5 biological replicates. (C) Glutamate and glutamine levels in activated CD8 + T cells treated for 48 h with MSO or vehicle control (PBS). n = 4 biological replicates. (D) Heatmap displaying log2 fold change of metabolites in WT vs GS KO CD8 + T cells from panel B. (E) OCR tracing from MitoStress Test performed on activated CD8 + T cells from WT or GS KO mice. Inhibitor injections labeled and marked with dashed lines. Each line represents a biological replicate, with 5 to 6 technical replicates each. (F) Quantification of basal respiration, maximal respiration, and spare respiratory capacity (SRC) of data shown in panel E. n = 3 biological replicates. (G) OCR tracing from MitoStress Test performed on activated CD8 + T cells injected with either MSO or PBS. Dashed lines indicate time at which labeled inhibitors were injected. Each line represents a biological replicate, with n = 5-6 technical replicates. (H) Quantification of basal respiration, maximal respiration, and spare respiratory capacity (SRC) of data shown in panel G. n = 3 biological replicates. Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001). Unpaired t test used in panels B to F, paired t test in panel G. AA, antimycin A (complex III inhibitor); Rot, rotenone (complex I inhibitor).

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Inhibition, Control, Labeling, Injection

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: GS inhibition or KO increase cellular resistance to ROS. (A) Schematic showing pathway of glutamine fueling GSH antioxidant pools. Glutamine is converted to glutamate intracellularly, which is then used in the synthesis of GSH. GSH is oxidized into GSSG upon neutralization of cellular ROS by GSH peroxidase. (B) Cellular ROS as measured by CellROX staining on flow cytometry for anti-CD3/CD28 in vitro activated CD8 + T cells from WT or GS KO mice. Representative histogram (left) and quantification (right). n = 6 biological replicates. (C) Mitochondrial ROS as measured by MitoSOX staining for WT vs GS KO CD8 + T cells (left, n = 4) and cells activated in increasing doses of MSO (right, n =3). (D) Glutathione (reduced and oxidized) levels in activated CD8 + T cells from WT vs GS KO mice. GSH = reduced glutathione. GSSG = oxidized glutathione. n = 4 biological replicates. (E) Viability as measured by flow cytometry of activated CD8 + T cells from WT vs GS KO mice, following 3-h incubation with indicated concentrations of hydrogen peroxide. Viability was normalized to viability of cells cultured without hydrogen peroxide. n = 4 replicates. (F) Normalized viability for WT vs GS KO CD8 + T cells incubated with 1,000 μM hydrogen peroxide, with and without supplementation with 1 mM GSH. n = 4 biological replicates. Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P ≤ 0.0001). Unpaired t test used in panels B to D, 1-way analysis of variance in with multiple comparisons in panels C and F, and 2-way analysis of variance in panel E.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Inhibition, Neutralization, Staining, Flow Cytometry, In Vitro, Incubation, Cell Culture, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: GS KO enhances the memory transcriptional profile and recall ability of CD8 + T cells. (A) Normalized enrichment score of top 5 differentially expressed hallmark pathways for WT (black) vs GS KO (pink) CD8 + T cells following GSEA. n = 5 biological replicates. (B) GSEA plot for Ivanova hematopoiesis stem cell long-term gene set (right, P < 0.0001) and heatmap showing normalized mRNA levels for genes included in set (left). (C) Volcano plot showing log fold change and −log10( P value) for pdcd1 , tcf7 , tox , lag3 , slamf6 , and havcr2 mRNA, as measured by bulk RNA sequencing and analyzed with DESeq2. Dotted line corresponds to a P value of 0.05. (D) Schematic showing timeline for Lm -Ova rechallenge experiment. (E) Ratio of GS KO to NTC OT-I T cells in mice infected with Lm- Ova on day 5 (acute response) and 35 (secondary recall response). The dotted line represents the ratio of GS KO: NTC cells at time of adoptive T cell therapy the day prior to Lm- Ova infection (0.909). (F) Schematic showing methodology for MC38-Ova tumor rechallenge experiment. (G) Ratio of GS KO to NTC OT-I T cells in mice rechallenged with MC388-Ova tumors. Dotted line represents ratio of GS KO:NTC cells on the day of adoptive T cell therapy (1.005). Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001). Paired Wilcoxon test in panel E), Wilcoxon matched-pairs signed rank test used in panel E), repeated measures 1-way analysis of variance with multiple comparisons in panel G).

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: RNA Sequencing, Infection, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: GS inhibition enhances CD8 + T cells infiltration and improves tumor control in vivo. (A) Schematic showing experimental protocol for results in panels B to H, n = 10 biological replicates. (B) Lung mass as a proxy for tumor burden. (C) Quantification of total bioluminescence immediately prior to sacrifice. (D) Images used for the quantification in panel C). (E) Percentage of infiltrating CD8 + T cells in tumors from PBS- vs MSO-treated mice, reported as percentage of CD45 + cells. (F) Percentage of PD-1 + CD8 + T cells, as measured by flow. (G) Representative flow plots showing IFNγ and TNFα production from recovered TILs following 3-h restimulation with PMA and ionomycin. (H) Percentage of IFNγ and TNFα double positive population. Statistically significant results are labeled. * P < 0.05, ** P < 0.01. Unpaired t test used in panels B, C, E, F, and H.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Inhibition, Control, In Vivo, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Glutamine synthetase deficiency enhances CD8 T cell survival and stress resilience in the tumor microenvironment

doi: 10.1093/jimmun/vkaf250

Figure Lengend Snippet: GS inhibition enhances adoptive T cell therapy efficacy in breast cancer model. (A) Schematic of experimental protocol for results in panels B to J. (B) Growth curve for tumors in mice treated with MSO (purple) vs PBS (black). n = 8 for both groups at start of experiment, 4 mice (3 PBS and 1 MSO) required euthanasia prior to study endpoint. (C) Tumor mass at study endpoint for mice treated with MSO vs PBS. n = 5–7 replicates. (D) Growth curve for PyMT tumors in Rag1 −/− mice treated with MSO (purple) vs PBS (black). n = 6 biological replicates each. (E) Representative flow plots showing IFNγ and TNFα production from recovered TILs following 3-h restimulation with PMA and ionomycin. n = 5–7 replicates, quantified in panel F. (G) Percentage of infiltrating CD8 + T cells in tumors from PBS- vs MSO-treated mice, reported as percentage of CD45 + cells. n = 5–7. (H) Percentage of PD-1 + CD8 + T cells, as measured by flow. n = 5–7. (I) Representative flow plots showing TOX + and TCF-1 + populations, gated on live CD8 + T cells and quantified in panels J and K. n = 5–7. Statistically significant results are labeled. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P ≤ 0.0001). Unpaired t test used in panels B, F to H, J, and K.

Article Snippet: Tumor cells were then passed through a 70 μm filter and tumor-infiltrating CD8 + T lymphocytes (TILs) were subsequently extracted using Miltenyi CD45 or CD8 isolation kits (130-110-618 and 130-116-478) per the manufacturer’s instructions.

Techniques: Inhibition, Labeling