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

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

Radiology and Oncology
Volume 53 (2019): Issue 1 (March 2019)
By: Mateja Prunk,  Milica Perisic Nanut,  Jerica Sabotic,  Urban Svajger and  Janko Kos  
Open Access
|Mar 2019

Figures & Tables

Figure 1

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;

Figure 2

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

Figure 3

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

Figure 4

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

Figure 5

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
DOI: https://doi.org/10.2478/raon-2019-0007 | Journal eISSN: 1581-3207 | Journal ISSN: 1318-2099
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Language: English
Page range: 57 - 68
Submitted on: Dec 8, 2018
|
Accepted on: Jan 5, 2019
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Published on: Mar 3, 2019
Published by: Association of Radiology and Oncology
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
cystatin F,
cysteine cathepsins,
TALL-104,
TGFβ,
ionomycin,
anergy
Related subjects:
Medicine,
Clinical medicine,
Internal medicine,
Haematology, oncology,
Radiology

© 2019 Mateja Prunk, Milica Perisic Nanut, Jerica Sabotic, Urban Svajger, Janko Kos, published by Association of Radiology and Oncology
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

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