Journal: Journal of Translational Autoimmunity

Article Title: Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

doi: 10.1016/j.jtauto.2024.100258

Figure Lengend Snippet: IL-7 induced the development of PD-1 high CXCR5- Tph-like cells from human PB CD4 T cells. (A) PBMCs from healthy human subjects were cultured with or without 50 ng/ml of IL-7 for 14 days. Representative dot plots showing the expression of PD-1 and CXCR5 on CD4 T cells are shown. The right panel (CD4 in RA joint) indicates the staining pattern of CD4 T cells isolated from RA joint. (B) The frequency (left) and cell number in a culture well (right) of PD-1 high CXCR5 - Tph-like cells induced with varying concentration of IL-7. (C) The frequency and number of PD-1 high CXCR5 - Tph-like cells induced from PBMC of HC and RA are shown (n = 6). (D) CTV-labelled PBMCs were cultured either with IL-7 (upper panels) or in plate wells precoated with anti-CD3 and anti-CD28 mAbs (lower panels). Expression of PD-1 and CTV staining of CD4 T cells cultured for the indicated days is shown. Induction of PD-1 high CXCR5 - Tph-like cells from PD-1-depleted (E) or CXCR5-depleted (F) PBMCs by IL-7. Left panels (d0) indicate the expression of PD-1 and CXCR5 on CD4 T cells before and after negative selection. ∗p < 0.05.

Article Snippet: Flow cytometric cell sorting of the cultured PD-1 high CTV low CD4 T cells, CD45RA + RO − naive and CD45RA − RO + memory CD4 T cells in PB, CD19 + CD27 + memory B cells in PB, PD-1 high CXCR5 - CD4 T (RA-Tph) cells in RASF was performed on a FACSAria Fusion cell sorter (BD Biosciences).

Techniques: Cell Culture, Expressing, Staining, Isolation, Concentration Assay, Selection

Journal: Journal of Translational Autoimmunity

Article Title: Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

doi: 10.1016/j.jtauto.2024.100258

Figure Lengend Snippet: Phenotypical and functional similarities between the IL-7-induced Tph-like (IL-7-Tph) cells and Tph cells in RA joint (RA-Tph cells). (A) Expression of surface molecules (ICOS, CD69, HLA-DR, CXCR3, and CCR6) on IL-7-Tph (n = 3) and RA-Tph cells (n = 3) are compared in histograms. Gray histograms indicate the staining with isotype controls. Graphs indicate the mean percentage of positive cells in PD-1 high CD4 T cells (n = 3). (B) Representative dot plots of cytokine production by the IL-7-Tph and RA-Tph cells with (lower panels) or without (upper panels) stimulation with PMA and ionomycin are shown. Graphs indicate the mean percentage of cytokine positive cells in PD-1 high CD4 T cells (n = 3). (C) Induction of plasmablasts (CD27 + CD38 + ) and IgG production from memory B cells cocultured with IL-7-Tph cells(n = 6). Representative dot plots of CD38 and CD27 expression on B cells are shown in left panels. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: Flow cytometric cell sorting of the cultured PD-1 high CTV low CD4 T cells, CD45RA + RO − naive and CD45RA − RO + memory CD4 T cells in PB, CD19 + CD27 + memory B cells in PB, PD-1 high CXCR5 - CD4 T (RA-Tph) cells in RASF was performed on a FACSAria Fusion cell sorter (BD Biosciences).

Techniques: Functional Assay, Expressing, Staining

Journal: Journal of Translational Autoimmunity

Article Title: Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

doi: 10.1016/j.jtauto.2024.100258

Figure Lengend Snippet: Gene expression analysis of the IL-7-Tph cells. (A) Heatmap showing hierarchical clustering by DEGs among sorted naïve CD4 T (n = 3), IL-7-Tph (n = 3), and RA-Tph cells (n = 3). (B) Venn diagrams showing the number of the upregulated genes overlapped in IL-7-Tph and RA-Tph cells compared to naive CD4 T cells. (C) Volcano plots showing DEGs between IL-7-Tph and naive CD4 (upper), RA-Tph and naive CD4 (middle), and IL-7-Tph and RA-Tph (lower) cells. (D) GSEA analysis showing the pathway of genes upregulated in (C). (E) Expression levels of MAF , PRDM1 , and PRDM1/BCL6 ratio in IL-7-Tph and RA-Tph cells by qRT-PCR analysis (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Flow cytometric cell sorting of the cultured PD-1 high CTV low CD4 T cells, CD45RA + RO − naive and CD45RA − RO + memory CD4 T cells in PB, CD19 + CD27 + memory B cells in PB, PD-1 high CXCR5 - CD4 T (RA-Tph) cells in RASF was performed on a FACSAria Fusion cell sorter (BD Biosciences).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Translational Autoimmunity

Article Title: Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

doi: 10.1016/j.jtauto.2024.100258

Figure Lengend Snippet: Signalling requirements for the development of IL-7-Tph cells. The frequency (left) and cell number in a culture well (right) of PD-1 high CTV low cells after culturing PBMC with IL-7 in the presence or absence of anti-DR mAb (A) or CTLA-4 Ig (B) is shown (n = 9). (C) Effect of adding anti-IL-2 mAb and TGF-β was examined (n = 4). (D) Representative dot plots showing the induction of IL-7-Tph cells from either naïve (upper panels) or memory (lower panels) CD4 T cells as depicted by the expression of CD45RA and PD-1 (d0 and d14 left). Expression of CXCL13 and IL-21 in PD-1 high CTV low cells after stimulation was shown in the right panels. (E) The effect of adding anti-DR mAb or CTLA-4 Ig on the induction of IL-7-Tph cells from naive (left) or memory (right) CD4 T cells (n = 5). ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Flow cytometric cell sorting of the cultured PD-1 high CTV low CD4 T cells, CD45RA + RO − naive and CD45RA − RO + memory CD4 T cells in PB, CD19 + CD27 + memory B cells in PB, PD-1 high CXCR5 - CD4 T (RA-Tph) cells in RASF was performed on a FACSAria Fusion cell sorter (BD Biosciences).

Techniques: Expressing

Journal: Journal of Translational Autoimmunity

Article Title: Homeostatic signals, including IL-7 and self-MHC recognition, induce the development of peripheral helper T cells, which are enriched in the joints of rheumatoid arthritis

doi: 10.1016/j.jtauto.2024.100258

Figure Lengend Snippet: Multiple mechanisms account for the accumulation Tph cells in the joint of RA. (A) Experimental system for the chemotaxis assay. (B) The frequency of PD-1 high CTV low IL-7-Tph cells migrated toward wells with or without RA SF (n = 5). (C) Representative dot plots showing the development of IL-7-Tph cells with or without RASF. (D) The frequency of PD-1 high CTV low IL-7-Tph cells induced with or without RA SF (n = 6). (E) Representative dot plots showing the expression of CXCL13 and IL-21 in the IL-7-Tph cells induced with RASF after stimulation. (F) PCA analysis on the gene expression of IL-7-Tph cells with or without RA SF, RA-Tph, and naive CD4 T cells (n = 3). (G) Representative dot plots showing the presence and absence of PD-1 high CXCR5- Tph cells in RA and OA joints, respectively. (H) Representative dot plots showing the development of IL-7-Tph cells from CD4 T cells in OA joints. Right panels show the expression of CXCL13 and IL-21 in PD-1 high CTV low cells after stimulation.

Article Snippet: Flow cytometric cell sorting of the cultured PD-1 high CTV low CD4 T cells, CD45RA + RO − naive and CD45RA − RO + memory CD4 T cells in PB, CD19 + CD27 + memory B cells in PB, PD-1 high CXCR5 - CD4 T (RA-Tph) cells in RASF was performed on a FACSAria Fusion cell sorter (BD Biosciences).

Techniques: Chemotaxis Assay, Expressing

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: IFN-γ, IL-4, and IL-17A secretion by PBMCs treated with nebivolol or carvedilol. PBMC samples were not activated or activated with anti-CD3/anti-CD28/anti-CD2(Act) for 4 days with nebivolol (Neb) or carvedilol (Carv) in the concentration of 10 μM as compared to the equivalent dilution of vehicle control (VC). The (A) IFN- γ, (B) IL-4, and (C) IL-17A levels in cell culture supernatants were measured by ELISA. (D) The cell viability of the cells was measured by trypan blue. Pooled data are expressed as mean ± SEM of seven to nine independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests was performed. *<0.05, **<0.01, ***p<0.001, and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Concentration Assay, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: IFN-γ, IL-4, IL-17A, and IL-10 secretion by memory Th cells treated with nebivolol. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days, and the (A) IFN-γ, (B) IL-4, (C) IL-17A, and (D) IL-10 levels in cell culture supernatants were measured by ELISA and expressed as fold change compared to the Act group, which was set to 1.0 (dotted line). Pooled data are expressed as mean ± SEM of 10 independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. *<0.05 and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Intracellular cytokine staining and expression of transcription factor genes associated with Th1, Th2, and Th17 in memory Th cells treated with nebivolol. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days, and the samples were stained for intracellular cytokines with or without nebivolol treatment and analyzed by flow cytometry. Representative dot plots are shown for CD4 and each of IFN-γ, IL-4, and IL-17A antibodies on memory Th lymphocytes. (A) Non-activated cells, (B) activated cells, (C) activated cells plus nebivolol, (D) vehicle control. (E) The proportion of memory Th cells expressing IFN-γ is shown as the percentage of IFN-γ + CD4 + T cells. (F) The proportion of CD4 + T cells expressing IL-4 is shown as the percentage of IL-4 + CD4 + T cells. (G) The proportion of CD4 + T cells expressing IL-17A is shown as the percentage of IL-17A + CD4 + T cells. Expression of (H) TBX21 , (I) GATA3 , and (J) RORC in RNA extracted from memory Th cells, shown as the relative amounts normalized to housekeeping RNA and compared to the Act group, which was set to 1.0 (dotted line). Pooled data are expressed as mean ± SEM of five independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. *<0.05, **<0.01, and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Staining, Expressing, Flow Cytometry, Control, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Nebivolol inhibits naive Th cell shift toward the Th17 phenotype, in contrast to terbutaline which augments the shift. (A) Expression of ADRB1 –3 in RNA extracted from naive CD4 + T cells shown as the relative amounts normalized to housekeeping RNA. (B) Naive Th cells were activated with polarizing cytokines and blocking antibodies (Th17) with treatment with nebivolol (Neb) or terbutaline (Terb) for 7 days, and a representative of IL-17A levels in cell culture supernatants was shown. (C) RNA expression of the Th17 cell-specific transcriptional factor RORC differentiated Th17 cells shown as the relative amounts normalized to housekeeping RNA and compared to shifted Th17 cells, which was set to 1.0 (dotted line). (D) A representative of overlapping histogram and (E) pooled data of intracellular cytokine staining shown for CD4 + IL-17A + cell percentage in polarized Th17 cells after treatment with nebivolol or terbutaline. Pooled data are expressed as mean ± SEM of five independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. *<0.05, **<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Expressing, Blocking Assay, Cell Culture, RNA Expression, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: T-cell proliferation and viability were measured by flow cytometry assay. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days. Representative histograms of the proliferation of memory Th cells in cultures with different groups including (A) activated cells overlayed with non-activated cells, (B) activated cells plus nebivolol overlayed with activation-only condition, and (C) activated vehicle control overlayed with activation-only cells. (D) The proliferation index of memory Th cells in culture with activation and nebivolol. (E) The division index of pooled data. (F) The percentage of alive memory CD4 + , 7AAD − T cells with or without treatment for 5 days gated on all cells in a dot plot by staining with 7AAD in cultures with different groups. Pooled data are expressed as mean ± SEM of four independent biological experiments. One-way ANOVA followed by Tukey’s multiple comparison tests. ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Activation Assay, Control, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Expression of ADRB1–3 in RNA extracted from enrichment memory Th cells after 5 days with or without treatment. Memory Th cells were not activated or activated with anti-CD3/anti-CD28/anti-CD2 for 5 days. (A) Expression of ADRB1 – 3 in RNA extracted from enrichment memory Th cells shown as the relative amounts normalized to housekeeping RNA. Pooled data are expressed as mean ± SEM of 10 independent biological experiments. (B) Expression of ADRB2 at mRNA levels from memory Th cells with or without treatment after 5 days of culture, shown as the relative amounts normalized to housekeeping RNA and compared to the Act group. The data of this figure are representative of three experiments. (C) Representative Western blot data of equal amounts of protein from the cell lysates were shown for β 2 -AR and GAPDH as a loading control. (D) A representative band intensity of five biological experiments was quantified and shown corrected to the loading control as a ratio. The bars show the mean ± SEM. ANOVA followed by Tukey’s multiple comparison tests. *<0.05 and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Nebivolol did not stimulate phospho-Ser133-CREB (p-CREB) in memory Th cells. Blasted memory Th cells activated with anti-CD3/anti-CD28/anti-CD2 for 15 min in conditions of non-activated cells, activated cells, activated cells plus nebivolol, and activated cells plus terbutaline. (A) Representative Western blot data of equal amounts of protein from the cell lysates were shown for p-CREB and α-tubulin as a loading control. (B) Band intensity was quantified and shown corrected to the loading control as a ratio, with data pooled from three independent biological experiments. (C) Memory Th cells were activated for 5 days, with nebivolol plus or minus H89. Representative IL-17A ELISA data for the effect of PKA in nebivolol’s signaling pathway by using H89 as a PKA inhibitor. ANOVA was followed by Tukey’s multiple comparison tests. *<0.05, **<0.01, and ****p<0.0001. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: The β 2 -adrenergic biased agonist nebivolol inhibits the development of Th17 and the response of memory Th17 cells in an NF-κB-dependent manner

doi: 10.3389/fimmu.2024.1446424

Figure Lengend Snippet: Nebivolol inhibited the phosphorylation of NF-κB p65 in memory Th cells. Blasted memory Th cells were activated with anti-CD3/anti-CD28/anti-CD2 for 15 min in conditions of non-activated cells, activated cells, and activated cells plus nebivolol. (A) Representative Western blot data of equal amounts of protein from the cell lysates were shown for p-NF-κB p65 and total NF-κB p65 as a loading control. (B) Band intensity was quantified and shown corrected to the loading control as a ratio, with data pooled from four independent biological experiments. ANOVA was followed by Tukey’s multiple comparison tests. *<0.05. ns, non-significant.

Article Snippet: Naive (CD3 + CD4 + CD45RA + CD45RO − ) and memory (CD3 + CD4 + CD45RA − CD45RO + ) T cells were isolated from peripheral blood mononuclear cells (PBMCs) in the recommended medium (PBS containing 2% FBS and 1 mM of EDTA) by using EasySep ® Human Naive CD4 + T Cell Isolation Kit II and human memory CD4 + T-cell enrichment kit, respectively (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Comparison

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , Schematic presentation of intratumoral and peritumoral tissue biopsies from patients with breast cancer. b , c , Quantification of Na + ( b ) and K + ( c ) concentrations with ICP–OES in intratumoral and peritumoral biopsies from patients with breast cancer ( n = 9; mean ± s.e.m., two-tailed, paired Student’s t -test). d , Preranked GSEA of patients with breast cancer from TCGA. Running enrichment scores and sorted positions of the 1,956 significantly upregulated genes from the NaCl signature are shown. The NaCl signature was generated by transcriptomic comparison of CD8 + memory T cells cultured under high versus low NaCl conditions (top 60 significantly upregulated DEGs). P , significance of the enrichment (one-tailed test for positive enrichment); n 1 and n 2 , number of patients providing either solid tumor samples or healthy breast tissue samples, respectively. e , ScRNA-seq of intra- and peritumoral CD8 + T cells from patients with breast cancer ( n = 3) (GEO accession no. GSE114727 ) . The module score for the transcriptomic NaCl signature obtained from DEGs on direct transcriptomic comparison of CD8 + CD45RA − T cells from high compared with low NaCl conditions, as described in d , was tested in intra- and peritumoral CD8 + T cells. CD8 + T cells were identified using marker gene expression.

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Two Tailed Test, Generated, Comparison, Cell Culture, One-tailed Test, Marker, Expressing

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , PCA projection after mRNA-seq of bulk human CD8 + CD45RA − T cells stimulated with CD3 and CD28 mAbs for 5 d under high and low NaCl conditions. The nos. 1–3 represent the individual blood donors. b , mRNA-seq analysis as in a . DEGs were identified using DESeq2 Wald’s test. c , d , ScRNA-seq of human CD8 + CD45RA − T cells stimulated with CD3 and CD28 mAbs for 3 d under high and low NaCl conditions.The UMAP shown is a representation visualizing the distribution of cells according to their respective treatment condition ( c ) and according to Leiden clustering ( d ). e , Proportion of CD8 + CD45RA − T cells from high and low NaCl conditions within the Leiden clusters after scRNA-seq as in c . Bray–Curtis dissimilarity testing was between high NaCl clusters (2, 3, 10, 11) and low NaCl clusters (1, 4–9, 12–14) ( P = 1.5 × 10 −17 , Wilcoxon’s rank-sum test). f , GSEA showing the top 15 GO terms among all biological processes. g , Module scores for the indicated gene sets comparing CD8 + CD45RA − T cells from high and low NaCl conditions after scRNA-seq (Wilcoxon’s rank-sum test). h , Phospho-low cytometry of CD8 + CD45RA − T cells stimulated as in a after TCR crosslinking with anti-CD3 mAbs and anti-mouse IgG F(ab′) 2 ( n = 3; mean ± s.e.m., two-way ANOVA with Fisher’s least significant difference (LSD)). gMFI, geometric mean fluorescence intensity. i , ScRNA-seq analysis of CD8 + CD45RA − T cells stimulated as in a (Wilcoxon’s rank-sum test). j , Ca 2+ flux measurement after expansion of human CD8 + CD45RA − T cells with CD3 and CD28 mAbs for 5 d in low and high NaCl conditions by flow cytometry. A representative plot shows the Ca 2+ flux ratio in different NaCl conditions after anti-CD3 crosslinking F(ab′) 2 . Dot plots show the area under the curve (AUC) during baseline, after F(ab′) 2 and the peak value of Ca 2+ flux ratio after F(ab′) 2 . Data present the mean ± s.e.m. from individual donors ( n = 5, one-way ANOVA with Tukey’s multiple-comparison test). a.u., arbitrary units; RFU, relative fluorescence units. k , l , Spectral flow cytometric analysis of CD8 + CD45RA − T cells stimulated as in a ( n = 14 ( k ), n = 8 ( l ), mean ± s.e.m., two-tailed, paired Student’s t -test). m , Spectral flow cytometry. The differences in the percentages of cells positive for the shown markers were visualized by z -score ( n = 4; * P < 0.05. two-tailed, paired Student’s t -test). The asterisk is located on the treatment side (high or low NaCl) that shows significant upregulation.

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Cytometry, Fluorescence, Flow Cytometry, Comparison, Two Tailed Test

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , GSEA for effector- and stemness-associated genes was performed after a transcriptomic comparison of bulk human CD8 + CD45RA − T cells stimulated with CD3 and CD28 mAbs for 5 d under high and low NaCl conditions. b , RNA velocity analysis after scRNA-seq of human CD8 + CD45RA − T cells stimulated with CD3 and CD28 mAbs for 3 d under high and low NaCl conditions. The velocities are shown in UMAP embedding. Cells are color coded according to the treatment condition as indicated. c – e , ScRNA-seq and analysis of the module scores of the indicated gene sets ( c , effector genes; d , cytokine activity; e , positive regulation of T cell mediated cytotoxicity) for human CD8 + CD45RA − T cells stimulated with CD3 and CD28 mAbs for 3 d under high and low NaCl conditions ( n = 1; Wilcoxon’s rank-sum test). f , Intracellular cytokine staining and flow cytometric analysis of human CD8 + CD45RA − memory T cells after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions. Left, representative experiment; right, cumulative data ( n = 13; mean ± s.e.m., two-tailed, paired Student’s t -test). g , ELISA of cell culture supernatants from human CD8 + memory T cells stimulated for 5 d with CD3 and CD28 mAbs ( n = 6; mean ± s.e.m., two-tailed, paired Student’s t -test). h , Flow cytometric analysis of human CD8 + CD45RA − T cells performed after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions. Left, representative experiment; right, cumulative data. Data present the mean ± s.e.m. ( n = 17; two-tailed, paired Student’s t -test). i – k , Intracellular cytokine staining and flow cytometric analysis ( i , perforin; j , TNF; k , IL-2) of human CD8 + CD45RA − T cells after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions and restimulation with PMA/ionomycin for 5 h. Left, representative experiment; right, cumulative data. Data present the mean ± s.e.m. ( n = 25 ( j ), n = 18 ( k ); two-tailed, paired Student’s t -test).

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Comparison, Activity Assay, Staining, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , Enrichment by overrepresentation analysis for all 45 upregulated KEGG pathways (with Benjamini–Hochberg-adjusted q value ≤ 0.05) using significantly upregulated DEGs ( P ≤ 0.05, log 2 (fold-change) ≥ 0.5) from the bulk transcriptomic comparison of human CD8 + CD45RA − T cells stimulated for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions. b , Luminometric assessment of ATP production in human CD8 + CD45RA + and CD8 + CD45RA − cells, which were stimulated as described in a , normalized to ATP per cell ( n = 3; mean ± s.e.m., two-tailed, paired Student’s t -test). c , d , Real-time analysis of the ECAR ( c ) and OCR ( d ) by human CD8 + CD45RA − T cells using a Seahorse Extracellular Flux Analyzer. The dotted lines show the time point of addition of the indicated substances. Left, representative experiment with technical replicates; right, cumulative quantification with individual healthy donors ( n = 7; mean ± s.e.m. two-tailed, paired Student’s t -test). e , g , i , n , Flow cytometric analysis of human CD8 + CD45RA − T cells. Left, representative experiment; right, cumulative quantification ( n = 7 ( e ), n = 9 ( g ), n = 9 ( i ), n = 4 ( n ); mean ± s.e.m., two-tailed, paired Student’s t -test). f , Expression of the indicated genes encoding glucose transporters in a transcriptomic comparison of bulk human CD8 + CD45RA − T cells stimulated for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions ( n = 3). h , Significantly enriched terms of GO-annotated, mitochondrial biological processes after overrepresentation analysis of upregulated genes from the bulk transcriptomic comparison of human CD8 + CD45RA − T cells stimulated as in a . The q values show P -value adjustment for multiple-test correction. Term redundancy was reduced using REVIGO ( http://revigo.irb.hr , similarity parameter = 0.5). j , k , Transmission electron microscopy, number of mitochondria ( j , n = 632 for low NaCl, 373 for high NaCl conditions) and cristae per mitochondrion ( k , n = 18 for low NaCl, 21 for high NaCl) (two-tailed, unpaired Student’s t -test). The data represent two independent experiments. l , Nontargeted metabolic profiling (metabolome analysis) of CD8 + CD45RA − T cells stimulated as in a ( n = 4 individual blood donors (matched samples); one-way ANOVA). m , o , ScRNA-seq analysis of cells cultured as in a ( m , SLC7A5 ; o , FABP5 ) one biological replicate (Wilcoxon’s rank-sum test). p , Phospho-flow analysis of CD8 + CD45RA − T cells cells stimulated as in a after TCR crosslinking for the indicated time points ( n = 3; mean ± s.e.m., two-way ANOVA with uncorrected Fisher’s LSD; * P < 0.05). q , Preranked GSEA. One-tailed permutation test for positive enrichment is based on an adaptive multilevel split Monte Carlo scheme (R package FGSEA).

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Comparison, Two Tailed Test, Expressing, Transmission Assay, Electron Microscopy, Cell Culture, One-tailed Test

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , Flow cytometric analysis of the membrane potential of human CD8 + CD45RA − T cells after stimulation for 5 d with CD3 and CD28 mAbs under high and low NaCl conditions in the presence or absence of ouabain treatment for 1 h before analysis ( n = 3; mean ± s.e.m., one-way ANOVA with Fisher’s LSD test). b , ATPase gene expression. Bulk mRNA-seq analysis of CD8 + CD45RA − T cells stimulated under high and low NaCl conditions with CD3 and CD28 mAbs for 5 d. Multiple test-adjusted P value and log 2 (fold-changes) according to DESeq2 test statistics are given over all transcriptome data ( n = 3). c , Colorimetric analysis of the Na + /K + -ATPase activity of CD8 + CD45RA − T cells treated under high and low NaCl conditions as in b in the presence or absence of ouabain ( n = 5; two-tailed, paired Student’s t -test). d , e , Flow cytometric analysis of intracellular K + ( d ) and Na + ( e ) for cells treated as in a . rel, relative. f , Calcium flux analysis with flow cytometry as in Fig. before and after TCR crosslinking of CD8 + CD45RA − T cells stimulated as in a (one-way ANOVA). g , Phospho-flow cytometry of CD8 + CD45RA − T cells stimulated as in a (one-way ANOVA). h , ELISA of 1-h cell culture supernatants on day 5 of CD8 + CD45RA − T cells stimulated as in a ( n = 3; mean ± s.e.m., one-way ANOVA with Fisher’s LSD test). i , Overview of molecular mechanism of NaCl-induced T cell hyperactivation.

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Membrane, Expressing, Activity Assay, Two Tailed Test, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , Real-time killing assay with nucleofected MART-1-specific T cells and A375 melanoma cell target cells at a 1:1 ratio under high and low NaCl conditions using the xCELLigence technology. Left, the normalized cell index; middle, the specific lysis; right, the cumulative quantification of 3T cell donors ( n = 3 experiments; mean ± s.e.m.; two-way ANOVA, * P < 0.05). b , Murine ROR1 CAR T cells generated and cultured for 48 h under high and low NaCl conditions and then cocultured with ROR1-expressing target cells at a 10:1 ratio. Antigen-specific lysis of Panc02-ROR1 cells by CD8 + CAR T cells was determined at different time points ( n = 3 independent experiments; mean ± s.d., two-way ANOVA). c , Experimental design. d , The tumor growth curves of subcutaneous tumors. Tumor growth was normalized to the tumor size on the day of CD8 + T cell injection ( n = 7 (PancOVA), n = 6 (PancOVA + low NaCl control (CTL)), n = 6 (PancOVA + high NaCl CTL); mean ± s.e.m. two-way ANOVA with Tukey’s honestly significant difference (HSD), multiple-comparison test). e , f , Flow cytometric analysis of intratumoral CD45.2 + CD8 + T cells 72 h after T cell transfer ( n = 6 ( e ), n = 5 ( f ); mean ± s.d., two-tailed, unpaired Student’s t -test). g , ScRNA-seq and module score calculation for T cell cytotoxicity genes obtained from published reports , , validated with genes from GO:0001916 ( P = 0.01). Intratumoral CD8 + T cells are shown from 56 patients with pancreatic cancer (from accession nos. GSE155698 , GSE111672 , GSE154778 , GSM4293555 and PRJCA001063 ) , integration of all cells: 10.5281/zenodo.6024273. CD8 + T cells were categorized into cells with a high and low NaCl signature based on the NaCl signature obtained from scRNA-seq of CD8 + CD45RA – T cells treated under high versus low NaCl concentrations (top 60 upregulated DEGs; Supplementary Table ; cutoff defined as module score ≥0 and <0 for high versus low NaCl signature, respectively; Wilcoxon’s rank-sum test). h , i , Kaplan–Meier tumor-free survival probability of patients from TCGA database diagnosed with pancreatic cancer. Patients were subgrouped by computing an optimal cutoff for NFAT5 ( h ) and ATP1A1 ( i ) expression. TPM values were normalized toward overall survival outcome. Number of patient samples: pancreatic cancer: n = 72 for NFAT5 high, n = 9 for NFAT5 low; n = 41 for ATP1A1 high; n = 40 for ATP2A2 low; significance of survival differences was determined using the Peto–Peto algorithm with the surv_pvalue function (method = ‘S1’) as implemented in the R package survminer.

Article Snippet: Nuclear and cytosolic extracts from CD8 + CD45RA − T cells after stimulation with CD3 and CD28 mAbs were separated using the Nuclear Extraction kit (Abcam).

Techniques: Lysis, Generated, Cell Culture, Expressing, Injection, Control, Comparison, Two Tailed Test