Journal: Nature immunology

Article Title: γδ T cells producing interleukin-17A regulate adipose regulatory T cell homeostasis and thermogenesis

doi: 10.1038/s41590-018-0094-2

Figure Lengend Snippet: a, Representative flow cytometry plots of γδ T cells from the epididymal WAT stromal vascular fraction (eWAT SVF). K denotes thousand. b, Frequency of γδ T cells across various adipose tissue depots as a percentage of CD3ε+ T cells in male eWAT (n ≥ 4 mice per group). Each symbol represents an individual mouse; small horizontal lines indicate the mean. c, Flow cytometry of lymphocytes in the blood, liver, spleen, and adipose tissue of CD45.1+ and CD45.2+ congenic C57BL/6 parabiotic pairs joined at 6 weeks of age and analyzed 2 weeks later (left); and frequency pie charts of CD45.1+ and CD45.2+ CD8+ and γδ T cells (right). d, Frequency of Vδ1+, Vδ2+, and Vδ3+ γδ T cells (% of CD45+PI− cells) in peripheral blood (PBMCs) or from matched omental fat from patients before bariatric surgery. Data are representative of three experiments (a,b,d; mean ± s.e.m. in b) or one experiment (c).

Article Snippet: Anti-TCR Vδ1 (REA173) and anti-TCR Vδ2 (123R3) were purchased from Miltenyi Biotec.

Techniques: Flow Cytometry

Journal: Cells

Article Title: Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins

doi: 10.3390/cells11213449

Figure Lengend Snippet: Frequency of Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and in healthy subjects. ( A ) Distribution of percentages of total lymphocytes, T (CD3+), Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy subjects. Shown is mean ± SD. ( B ) Representative dot-plots showing the frequency of Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and healthy subjects. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: After the incubation, the cells were harvested, washed with FACS buffer and surface stained using the following fluorochrome-conjugated monoclonal antibodies: APC-Cy7-conjugated anti-human CD45 (clone REA747), PerCP-Vio700-conjugated anti-human CD3 (clone REA613), FITC-conjugated anti-human Vδ1 (clone REA173), or Vδ2 (clone REA771) (Miltenyi Biotec).

Techniques:

Journal: Cells

Article Title: Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins

doi: 10.3390/cells11213449

Figure Lengend Snippet: Phenotypic analysis of circulating Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and in healthy subjects. Distribution of ex vivo memory subsets of Vδ1+ ( A ) and Vδ2+ ( C ) T lymphocytes based on the expression of CD27 and CD45RA in hospitalized and recovered COVID-19 patients, compared to healthy donors. Median is shown. Healthy donors were represented as triangle, COVID-19 patients as circle and COVID-19 recovered as rhombus. Representative dot-plots showing Vδ1+ ( B ) and Vδ2+ ( D ) T lymphocyte memory subset distribution in hospitalized, recovered, and healthy subjects. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: After the incubation, the cells were harvested, washed with FACS buffer and surface stained using the following fluorochrome-conjugated monoclonal antibodies: APC-Cy7-conjugated anti-human CD45 (clone REA747), PerCP-Vio700-conjugated anti-human CD3 (clone REA613), FITC-conjugated anti-human Vδ1 (clone REA173), or Vδ2 (clone REA771) (Miltenyi Biotec).

Techniques: Ex Vivo, Expressing

Journal: Cells

Article Title: Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins

doi: 10.3390/cells11213449

Figure Lengend Snippet: Analysis of exhaustion-marker expression by circulating Vδ1+ and Vδ2+ T lymphocytes in hospitalized and recovered COVID-19 patients and healthy subjects. Expression of TIM-3 and PD-1 on Vδ1+ ( A ) and Vδ2+ ( B ) T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy donors. Shown are percentage ( left panels ) and MFI ( right panels ) ± SD. ( C ) Representative dot-plots of exhaustion-marker expression by Vδ1+ and Vδ2+ T lymphocytes from hospitalized and recovered COVID-19 patients, compared to healthy donors. ( D ) Frequency of exhausted Vδ1+ and Vδ2+ T cells per 1 × 10 6 T lymphocytes. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After the incubation, the cells were harvested, washed with FACS buffer and surface stained using the following fluorochrome-conjugated monoclonal antibodies: APC-Cy7-conjugated anti-human CD45 (clone REA747), PerCP-Vio700-conjugated anti-human CD3 (clone REA613), FITC-conjugated anti-human Vδ1 (clone REA173), or Vδ2 (clone REA771) (Miltenyi Biotec).

Techniques: Marker, Expressing

Journal: Cells

Article Title: Phenotypical and Functional Alteration of γδ T Lymphocytes in COVID-19 Patients: Reversal by Statins

doi: 10.3390/cells11213449

Figure Lengend Snippet: Expression of pro-inflammatory cytokines by Vδ1+ T lymphocytes in hospitalized and recovered COVID-19 patients, compared to healthy subjects. Cumulative histograms representing the expression of pro-inflammatory cytokines by Vδ1+ T lymphocyte. Shown are percentage ( left panels ), MFI ( central panel , log 10 scale), and iMFI ( right panels ) ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: After the incubation, the cells were harvested, washed with FACS buffer and surface stained using the following fluorochrome-conjugated monoclonal antibodies: APC-Cy7-conjugated anti-human CD45 (clone REA747), PerCP-Vio700-conjugated anti-human CD3 (clone REA613), FITC-conjugated anti-human Vδ1 (clone REA173), or Vδ2 (clone REA771) (Miltenyi Biotec).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy

doi: 10.3389/fimmu.2023.1197630

Figure Lengend Snippet: IL-17-expressing cells are differently modulated in the skin lesion and in the circulation of melanoma patients developing vitiligo during anti-PD-1 therapy. (A) Representative images of the immunohistochemistry (panels a to d) and immunofluorescence (panels e, f) analysis performed using antibodies directed against CD56 (panels a–c), IL-17 (panels d–f) and TCR alpha V 7.2 (panels g–i) on normal skin biopsies and on biopsies from patients affected by vitiligo without melanoma (vitiligo) and who developed vitiligo during immunotherapy (vitiligo DI). Magnification 200x. (B) Quantitative analyses of immunohistochemical staining. The mean value + standard error of the mean (SEM) of the cell count obtained for five different fields is shown, ***p<0.05 as assessed by Kruskal-Wallis test.

Article Snippet: For immunofluorescence, 4-µm sections were dewaxed, rehydrated, and after quenching endogenous peroxidase with 3% bovine serum albumin in 1x phosphate buffer, achieving antigen retrieval and blocking non-specific binding sites, sections were incubated with the monoclonal antibody anti-TCR V alpha 7.2 FITC conjugated (cod.130-123-929, Miltenyi Biotech; 1:50) and incubated for 1 hour at 37°C in a humid chamber.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Cell Counting

Journal: Frontiers in Immunology

Article Title: Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy

doi: 10.3389/fimmu.2023.1197630

Figure Lengend Snippet: Expression of the immune cell subsets in the primary melanoma and metastases of patients developing or not vitiligo during anti-PD-1 therapy. (A) Representative images of immunohistochemistry (panels a to i) and immunofluorescence (l–n) obtained using anti-CD25 (a–c), anti-CD56 (d–f), anti-IL-17A (g–i), and anti-TCR alpha V 7.2 (j–l) in primary melanoma and metastasis from patients who developed (vitiligo) or not vitiligo (not vitiligo) during therapy. A–I 100x magnification, panel L-N 200X magnification. Magnification 200x. (B) Quantitative analyses of immunohistochemical staining. The mean value + standard error of the mean (SEM) of the cell count obtained for five different fields is shown, *p<0.05 as assessed by Kruskal-Wallis test.

Article Snippet: For immunofluorescence, 4-µm sections were dewaxed, rehydrated, and after quenching endogenous peroxidase with 3% bovine serum albumin in 1x phosphate buffer, achieving antigen retrieval and blocking non-specific binding sites, sections were incubated with the monoclonal antibody anti-TCR V alpha 7.2 FITC conjugated (cod.130-123-929, Miltenyi Biotech; 1:50) and incubated for 1 hour at 37°C in a humid chamber.

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Magnesium transporter 1 (MAGT1) deficiency causes selective defects in N- linked glycosylation and expression of immune-response genes

doi: 10.1074/jbc.RA119.008903

Figure Lengend Snippet: Mg2+ regulates glycosylation and plays a unique role in T-cell–mediated immunity, especially in immune protection against EBV. A, flow cytometry histograms of surface expression of NKG2D, CD70, HLA-DR, and CD5 in cycling T cells from HCs cultured either in cRPMI for 5 days, dRPMI for 5 days, or cells that were cultured in dRPMI for 3 days followed by the addition of Mg2+ (0.5 mm) back into the dRPMI for 2 days. B, quantification of the MFI in A. Error bars represent the standard error of the mean of eight independent experiments, and p values were calculated with a paired t test. C, representative Western blot analysis of NKG2D, CD70, TCR-β, and β-actin in cycling T cells from a HC as described in A. Numbers at left indicate kDa standards. Glycosylation patterns are shown at right: fully-glycosylated (2); partially-glycosylated (1); and unglycosylated (0). Data are mean of eight (A and B) or are representative of three (C) independent replicates. D, schematic diagram for killing pathway with NK cells (top) and EBV-721.221(target; bottom) cells. E, flow cytometry analysis of surface expression of NKG2D in EBV-specific NK cells from a HC with cRPMI and dRPMI. F, quantification of the MFI in F. G, percent lysis of autologous EBV-LCLs by EBV-specific NK cells from HC with a different dose of the Mg2+ with significance determined by one-way ANOVA. n indicates the number of independent samples. H, quantification from G. Data are representative of three independent biological replicates. NS, nonsignificant.

Article Snippet: The following antibodies were used: MAGT1 (Mer catalog no. 17-1, 1:50 dilution, produced by Merck); TUSC3 (Mer 85 catalog no. 38-1, affinity-purified, produced by Merck) ( Fig. S6 ) 8 ; β-actin (ab20272, Abcam); NKG2D (D-20, sc-9621, Santa Cruz Biotechnology); tubulin (AA2, 05-661, Merck Millipore); TCR-β (H-197, sc-9101, Santa Cruz Biotechnology); CERS2 (A303-193A, Bethyl Laboratories, Inc.); ribophorin I (C-15, sc-12164, Santa Cruz Biotechnology); COP1 (PA1-061, Thermo Fisher Scientific); EEA1 (C45B10, 3288 Cell Signaling); STT3B (HPA036646, Sigma); OST 48 (Santa Cruz Biotechnology); Na,K-ATPase α1 (D4Y7E, 23565, Cell Signaling).

Techniques: Glycoproteomics, Flow Cytometry, Expressing, Cell Culture, Western Blot, Lysis

Journal: Frontiers in Immunology

Article Title: T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

doi: 10.3389/fimmu.2025.1509855

Figure Lengend Snippet: (A) Schematic representation of the patient 3-TCR constructs synthesized and cloned for functional analyses. Human constant domains were replaced by murine homologous sequences. V-(D)-J gene segments of α- and β-chains and CDR3 sequences are shown. Red labels highlight differences between TCR-V1, -V2, and V3. Key data leading to the selection of the four T cell clonotypes for functional characterization are detailed in the adjacent table. (B) Recognition of patient 3 tumor cells by TCR-T cells as determined by IFN-γ ELISpot assay. Tumor cell suspension after thawing was sufficient for only one experiment. (C) Original ELISpot well-scans showing the tumor-response of TCR-V4-T cells. (D) Recognition of HLA-A2-matched allogeneic NSCLC cell line LC-MZ-16 by TCR-V4-T cells (summary of four independent experiments). OKT-3 mAb was used for unspecific activation. Pan-HLA class I mAb W6/32 blocked tumor-recognition in all experiments.

Article Snippet: TCR-T cells were tested for activation-induced cytolytic responses by coincubation with target cells (1:1) overnight followed by staining with anti-murine TCR constant domain antibody (FITC, CL075F, Origene) and anti-CD107a mAb (PE-Cy5, clone H4A3, BD Biosciences, Heidelberg, Germany).

Techniques: Construct, Synthesized, Clone Assay, Functional Assay, Selection, Enzyme-linked Immunospot, Suspension, Activation Assay

Journal: Frontiers in Immunology

Article Title: T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

doi: 10.3389/fimmu.2025.1509855

Figure Lengend Snippet: TCR-T cells transduced with P3-TCRs V1, V2, V3 recognize the naturally processed and presented KRAS Q61H-peptide 55-64 (ILDTAGHEEY). (A) Identification of the KRAS Q61H-peptide as target antigen of TCR-V2-T cells. ELISpot analysis testing TCR-T cells against monoallelic P3-HLA transduced K562 cells pulsed with 96 candidate neoantigen peptides identified by WES- and RNA-Seq. Only K562/HLA-A*01:01 cells were recognized when pulsed with several synthetic peptides in a cross-reactive manner, though less strong as the KRAS Q61H-peptide. (B) K562/HLA-A*01:01 cells were recognized by TCR-V1, TCR-V2, and TCR–V3-transduced CD4- and CD8-positive TCR-T cells when pulsed with mutant KRAS-peptide 55-64 (color code of the legend used for all figures). (C) K562/HLA-A*01:01 cells pulsed with titrated doses ILDTAGHEEY were recognized by TCR-T cells with high functional avidity (EC50<10nM). (D, E) Recognition of KRAS Q61H-mutated, HLA-A*01:01-transduced cell line NCI-H460/HLA-A*01:01 in comparison to wildtype NCI-H460 by CD8-positive (D) and CD4-positive (E) TCR-T cells. (F) Flow cytometry showing degranulation (CD107a) as surrogate for lytic activity of TCR-V1, TCR-V2, and TCR-V3-expressing CD8-positive TCR-T cells upon co-culture with NCI-H460/HLA-A*01:01. Corresponding results for CD4-positive TCR-T cells and the experiment gating strategy are shown in Supplementary Figure S8 . Lytic activity and cytokine release of all TCR-T cell cultures showed overlapping results. All ELISpot experiments were done in duplicates or triplicates. Results shown in (B, D, E) are derived from three independent experiments. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: TCR-T cells were tested for activation-induced cytolytic responses by coincubation with target cells (1:1) overnight followed by staining with anti-murine TCR constant domain antibody (FITC, CL075F, Origene) and anti-CD107a mAb (PE-Cy5, clone H4A3, BD Biosciences, Heidelberg, Germany).

Techniques: Transduction, Enzyme-linked Immunospot, RNA Sequencing, Mutagenesis, Functional Assay, Comparison, Flow Cytometry, Activity Assay, Expressing, Co-Culture Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: T-cell receptors identified by a personalized antigen-agnostic screening approach target shared neoantigen KRAS Q61H

doi: 10.3389/fimmu.2025.1509855

Figure Lengend Snippet: Key resources table.

Article Snippet: TCR-T cells were tested for activation-induced cytolytic responses by coincubation with target cells (1:1) overnight followed by staining with anti-murine TCR constant domain antibody (FITC, CL075F, Origene) and anti-CD107a mAb (PE-Cy5, clone H4A3, BD Biosciences, Heidelberg, Germany).

Techniques: Plasmid Preparation, Amplification, Software, CRISPR, Binding Assay