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Increased cystatin F levels correlate with decreased cytotoxicity of cytotoxic T cells

,

Milica Perisic Nanut

Nanut, Milica Perisic

,

Sabotic, Jerica

,

et

03 mars 2019

Increased cystatin F levels correlate with decreased cytotoxicity of cytotoxic T cells's Cover Image

Radiology and Oncology

Édition 53 (2019): Edition 1 (Mars 2019)

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Catégorie d'article: Research Article

Publié en ligne: 03 mars 2019

Pages: 57 - 68

Reçu: 08 déc. 2018

Accepté: 05 janv. 2019

DOI: https://doi.org/10.2478/raon-2019-0007

Mots clés
cystatin F, cysteine cathepsins, TALL-104, TGFβ, ionomycin, anergy

© 2019 Mateja Prunk, Milica Perisic Nanut, Jerica Sabotic, Urban Svajger, Janko Kos, published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Expression of cystatin F in TALL-104 cells and human CD8+ T cells. (A) Representative western blot experiment showing expression of the monomeric and dimeric form of cystatin F in unstimulated and stimulated TALL-104 cells and human CD8+ T cells. Both, TALL-104 and human CD8+ T cells, were stimulated with anti-CD3/anti-CD28 antibody coated beads. Multiple bands correspond to differently glycosylated forms of cystatin F.21 (B) Quantification of western blot data was performed in Image Lab software. Signals for cystatin F were first normalized to β-actin signal and TALL-104 control sample intensity was set to 1 arbitrary unit (AU). Relative intensities of other bands were calculated accordingly. Error bars represent s.e.m between three separate experiments.
** p ≤ 0.01, statistical analysis was performed for total cystatin F levels.
ctrl = control; pCTL = primary human cytotoxic T cells: stim = stimulated; — Expression of cystatin F in TALL-104 cells and human CD8+ T cells. (A) Representative western blot experiment showing expression of the monomeric and dimeric form of cystatin F in unstimulated and stimulated TALL-104 cells and human CD8+ T cells. Both, TALL-104 and human CD8+ T cells, were stimulated with anti-CD3/anti-CD28 antibody coated beads. Multiple bands correspond to differently glycosylated forms of cystatin F.21 (B) Quantification of western blot data was performed in Image Lab software. Signals for cystatin F were first normalized to β-actin signal and TALL-104 control sample intensity was set to 1 arbitrary unit (AU). Relative intensities of other bands were calculated accordingly. Error bars represent s.e.m between three separate experiments. ** p ≤ 0.01, statistical analysis was performed for total cystatin F levels. ctrl = control; pCTL = primary human cytotoxic T cells: stim = stimulated;

Cytotoxicity of TALL-104 after TGFβ or ionomycin treatment is reduced. (A) TALL-104 cells were treated with 100 pM TGFβ for 24 hours or 0.5 μM ionomycin for 16 hours. Cytotoxicity against NK-sensitive targets K-562 cells and NK-resistant targets Raji cells was measured by a 4 hou r calcein-AM release assay at different effector-to-target cell ratios. Lytic units (LU 30/106) were calculated using the inverse number of TALL-104 cells needed to lyse 30% of target cells × 100. Error bars represent SD between triplicates. ANOVA was used for statistical analysis, *** p ≤ 0.001 and ****p ≤ 0.0001. (B) Cytotoxicity of control, TGFβ and ionomycin treated TALL-104 cells against K-562 and Raji target cells was measured by a 4 hour calcein-AM release assay at different effector-to-target cell ratio. During the assay the Ca2+ chelator EGTA or EGTA with excess Ca2+ was added. Error bars represent SD between triplicates.
ctrl = control; ion = ionomycin — Cytotoxicity of TALL-104 after TGFβ or ionomycin treatment is reduced. (A) TALL-104 cells were treated with 100 pM TGFβ for 24 hours or 0.5 μM ionomycin for 16 hours. Cytotoxicity against NK-sensitive targets K-562 cells and NK-resistant targets Raji cells was measured by a 4 hou r calcein-AM release assay at different effector-to-target cell ratios. Lytic units (LU 30/106) were calculated using the inverse number of TALL-104 cells needed to lyse 30% of target cells × 100. Error bars represent SD between triplicates. ANOVA was used for statistical analysis, *** p ≤ 0.001 and ****p ≤ 0.0001. (B) Cytotoxicity of control, TGFβ and ionomycin treated TALL-104 cells against K-562 and Raji target cells was measured by a 4 hour calcein-AM release assay at different effector-to-target cell ratio. During the assay the Ca2+ chelator EGTA or EGTA with excess Ca2+ was added. Error bars represent SD between triplicates. ctrl = control; ion = ionomycin

TGFβ and ionomycin increase cystatin F protein levels. (A, B) TALL-104 cells were stimulated with anti-CD3/anti-CD28 antibody coated beads in the presence or absence of 100 pM TGFβ. (C, D) TALL-104 cells were treated with 0.5 μM ionomycin for 16 hours and stimulated with 10 nM PMA and 1μM ionomycin. (E, F) Human CD8+ T cells were treated with 0.5 μM ionomycin for 16 hours and stimulated with anti-CD3/anti-CD28 antibody coated beads. (A, C, E) show representative western blots for cystatin F, while (B, D, F) show quantification of 3 (B, D) or 2 (F) independent experiments. Quantification was performed in Image Lab software. Signals for cystatin F were first normalized to β-actin (B, F) or GAPDH (D) signal and TALL-104 control sample intensity was set to 1 arbitrary unit (AU). Relative intensities of other bands were calculated accordingly. Data are presented as mean ± s.e.m. * p ≤ 0.05, ** p ≤ 0.01, statistical analysis was performed for total cystatin F levels.
ctrl = control; ion = ionomycin; pCTL = primary human cytotoxic T cells; stim = stimulated — TGFβ and ionomycin increase cystatin F protein levels. (A, B) TALL-104 cells were stimulated with anti-CD3/anti-CD28 antibody coated beads in the presence or absence of 100 pM TGFβ. (C, D) TALL-104 cells were treated with 0.5 μM ionomycin for 16 hours and stimulated with 10 nM PMA and 1μM ionomycin. (E, F) Human CD8+ T cells were treated with 0.5 μM ionomycin for 16 hours and stimulated with anti-CD3/anti-CD28 antibody coated beads. (A, C, E) show representative western blots for cystatin F, while (B, D, F) show quantification of 3 (B, D) or 2 (F) independent experiments. Quantification was performed in Image Lab software. Signals for cystatin F were first normalized to β-actin (B, F) or GAPDH (D) signal and TALL-104 control sample intensity was set to 1 arbitrary unit (AU). Relative intensities of other bands were calculated accordingly. Data are presented as mean ± s.e.m. * p ≤ 0.05, ** p ≤ 0.01, statistical analysis was performed for total cystatin F levels. ctrl = control; ion = ionomycin; pCTL = primary human cytotoxic T cells; stim = stimulated

Expression of cathepsins C, H and L in TALL-104 cells and human CD8+ T cells and their co-localisation with cystatin F. (A-C) TALL-104 and pCTLs were analysed for cathepsins C, H and L expression by western blot. GAPDH or β-actin staining was used to show protein loading. (D, E) Co-localisation of cystatin F with cathepsins C, H and L was studied by immunofluorescence microscopy in TALL-104 (D) and pCTLs (E). Cystatin F (green) and cathepsin C (red) co-localisation is shown in first row, cystatin F (red) and cathepsin H (green) in second row and cystatin F (red) and cathepsin L (green) in third row. Bars represent 10 μm. (F) TALL-104 cell lysates were immunoprecipitated with cystatin F antibody and analysed by western blot with anti-cathepsin C and H antibodies. (G) Proximity ligation experiment for cystatin F-cathepsin C interaction in TALL-104 cells and pCTLs. Signals were quantified in BlobFinder software. Bars represent 10 μm.
ctrl = control; cysF = cystatin F; IP = immunoprecipitation; pCTL = primary human cytotoxic T cells — Expression of cathepsins C, H and L in TALL-104 cells and human CD8+ T cells and their co-localisation with cystatin F. (A-C) TALL-104 and pCTLs were analysed for cathepsins C, H and L expression by western blot. GAPDH or β-actin staining was used to show protein loading. (D, E) Co-localisation of cystatin F with cathepsins C, H and L was studied by immunofluorescence microscopy in TALL-104 (D) and pCTLs (E). Cystatin F (green) and cathepsin C (red) co-localisation is shown in first row, cystatin F (red) and cathepsin H (green) in second row and cystatin F (red) and cathepsin L (green) in third row. Bars represent 10 μm. (F) TALL-104 cell lysates were immunoprecipitated with cystatin F antibody and analysed by western blot with anti-cathepsin C and H antibodies. (G) Proximity ligation experiment for cystatin F-cathepsin C interaction in TALL-104 cells and pCTLs. Signals were quantified in BlobFinder software. Bars represent 10 μm. ctrl = control; cysF = cystatin F; IP = immunoprecipitation; pCTL = primary human cytotoxic T cells

Activities of cathepsins C, H and L and granzyme B in TALL-104 cells. Activities of cathepsins C (A, E), H (B,F), L (C,G) and granzyme B (D, H) in TALL-104 whole cell lysates after TGFβ or ionomycin treatment. Error bars represent SD between triplicates. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.
ctrl = control; ion = ionomycin; stim = stimulated; TGF = transforming growth factor beta — Activities of cathepsins C, H and L and granzyme B in TALL-104 cells. Activities of cathepsins C (A, E), H (B,F), L (C,G) and granzyme B (D, H) in TALL-104 whole cell lysates after TGFβ or ionomycin treatment. Error bars represent SD between triplicates. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. ctrl = control; ion = ionomycin; stim = stimulated; TGF = transforming growth factor beta

Langue:: Anglais

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Sujets de la revue:: Médecine, Médecine clinique, Médecine interne, Hématologie, oncologie, Radiologie

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