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Coculture of sorted T-cell populations and Mtb -infected macrophages: release of effector molecules <xref ref-type= a " width="100%" height="100%">

Journal: Infection and Immunity

Article Title: Polycytotoxic T cells mediate antimicrobial activity against intracellular Mycobacterium tuberculosis

doi: 10.1128/iai.00297-24

Figure Lengend Snippet: Coculture of sorted T-cell populations and Mtb -infected macrophages: release of effector molecules a

Article Snippet: The following antibodies were used for flow cytometry: CD3-PerCP (clone SK7, BD Biosciences), CD8-BV605 (clone RPA-T8, BioLegend), NKG2A-FITC (clone REA 110, Miltenyi), NKG2A-PE (clone REA 110, Miltenyi), NKG2C-APC (clone REA 205, Miltenyi), granzyme B-Pacific Blue (clone GB11, BioLegend), GZMB-APC (clone GRB05, Invitrogen), perforin-PE-Cy7 (cloneB-D48, BioLegend), granulysin-Alexa488 (cloneRB1, BD Biosciences), granulysin-PE (cloneDH2, eBioscience), and CD107a-PE (BD Biosciences).

Techniques: Infection

NKG2C + and NKG2A + T-lymphocytes inhibit the growth of intracellular Mtb and contain a higher frequency of P-CTLs. ( A ) Macrophages were infected with Mtb , coated with an anti-CD3 antibody, and cocultured with sorted T-cell subsets for 24 h. Lysates were plated in serial dilutions and the number of CFU was determined after 14 days of incubation. The number of CFU was determined and related to the number of CFU in the culture without T cells (defined as “1.0”). The graph shows the individual values of 13 independent donors (horizontal line: median). Statistical analysis was performed using the Wilcoxon test, two-tailed. ( B ) PBMCs from 30 donors were stained for CD3, CD8, NKG2A, NKG2C, granzyme B, perforin, granulysin, and Live/Dead and analyzed by flow cytometry. The Boolean gating function in FlowJo was used to determine combinatorial expression of granzyme B, perforin, and granulysin within all subsets. The dot plot illustrates the identification of P-CTLs using a subsequent gating strategy. The full gating strategy is depicted in S3. ( C ) The figure shows the frequency of P-CTL in all 30 donors investigated. Statistical comparison was performed with the Wilcoxon test, two-tailed. ( D ) Heatmap presenting the correlation between CTL-subsets and n -fold Mtb growth using Spearman’s rank correlation test ( n = 12). R -values are depicted in the squares. CFU, colony-forming units; Mtb , Mycobacterium tuberculosis , P-CTL, polycytotoxic T-lymphocytes; PBMC, peripheral blood mononuclear cell.

Journal: Infection and Immunity

Article Title: Polycytotoxic T cells mediate antimicrobial activity against intracellular Mycobacterium tuberculosis

doi: 10.1128/iai.00297-24

Figure Lengend Snippet: NKG2C + and NKG2A + T-lymphocytes inhibit the growth of intracellular Mtb and contain a higher frequency of P-CTLs. ( A ) Macrophages were infected with Mtb , coated with an anti-CD3 antibody, and cocultured with sorted T-cell subsets for 24 h. Lysates were plated in serial dilutions and the number of CFU was determined after 14 days of incubation. The number of CFU was determined and related to the number of CFU in the culture without T cells (defined as “1.0”). The graph shows the individual values of 13 independent donors (horizontal line: median). Statistical analysis was performed using the Wilcoxon test, two-tailed. ( B ) PBMCs from 30 donors were stained for CD3, CD8, NKG2A, NKG2C, granzyme B, perforin, granulysin, and Live/Dead and analyzed by flow cytometry. The Boolean gating function in FlowJo was used to determine combinatorial expression of granzyme B, perforin, and granulysin within all subsets. The dot plot illustrates the identification of P-CTLs using a subsequent gating strategy. The full gating strategy is depicted in S3. ( C ) The figure shows the frequency of P-CTL in all 30 donors investigated. Statistical comparison was performed with the Wilcoxon test, two-tailed. ( D ) Heatmap presenting the correlation between CTL-subsets and n -fold Mtb growth using Spearman’s rank correlation test ( n = 12). R -values are depicted in the squares. CFU, colony-forming units; Mtb , Mycobacterium tuberculosis , P-CTL, polycytotoxic T-lymphocytes; PBMC, peripheral blood mononuclear cell.

Article Snippet: The following antibodies were used for flow cytometry: CD3-PerCP (clone SK7, BD Biosciences), CD8-BV605 (clone RPA-T8, BioLegend), NKG2A-FITC (clone REA 110, Miltenyi), NKG2A-PE (clone REA 110, Miltenyi), NKG2C-APC (clone REA 205, Miltenyi), granzyme B-Pacific Blue (clone GB11, BioLegend), GZMB-APC (clone GRB05, Invitrogen), perforin-PE-Cy7 (cloneB-D48, BioLegend), granulysin-Alexa488 (cloneRB1, BD Biosciences), granulysin-PE (cloneDH2, eBioscience), and CD107a-PE (BD Biosciences).

Techniques: Infection, Incubation, Two Tailed Test, Staining, Flow Cytometry, Expressing, Comparison

Classification of CD3 + CD8 + CTL-subsets <xref ref-type= a " width="100%" height="100%">

Journal: Infection and Immunity

Article Title: Polycytotoxic T cells mediate antimicrobial activity against intracellular Mycobacterium tuberculosis

doi: 10.1128/iai.00297-24

Figure Lengend Snippet: Classification of CD3 + CD8 + CTL-subsets a

Article Snippet: The following antibodies were used for flow cytometry: CD3-PerCP (clone SK7, BD Biosciences), CD8-BV605 (clone RPA-T8, BioLegend), NKG2A-FITC (clone REA 110, Miltenyi), NKG2A-PE (clone REA 110, Miltenyi), NKG2C-APC (clone REA 205, Miltenyi), granzyme B-Pacific Blue (clone GB11, BioLegend), GZMB-APC (clone GRB05, Invitrogen), perforin-PE-Cy7 (cloneB-D48, BioLegend), granulysin-Alexa488 (cloneRB1, BD Biosciences), granulysin-PE (cloneDH2, eBioscience), and CD107a-PE (BD Biosciences).

Techniques: