Additional file 1: Figure S1. of Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: therapeutic implications

Showing MCL-1 mRNA levels do not change in Her2-positive BC cells under hypoxic conditions. Figure S2 showing a Bcl-2 is expressed in both Mcl-1wt/wt and Mcl-1Δ/null MEFs. Whole-cell extracts were analyzed by immunoblotting with indicated antibodies. Immunoblotting for Erk2 confirmed equal protein loading. b EU-5346 does not change Mcl-1 mRNA expression in Her2-positive BC cells under hypoxic conditions. Figure S3 showing Mcl-1 is a potential therapeutic target not only in Her2-positive, but also in Luminal A-like and TNBC cells under hypoxic conditions. (PDF 1580 kb

Arginine-independent cytotoxicity of tumor antigen specific T cells.

<p><b>A</b> Expanded MART-1_{aa26–35*A27L} specific CD8⁺ T cells of 3 HD were preincubated for 24 h in the presence (+Arg, 1000 µM) or absence (−Arg) of arginine and subsequently stimulated with MART-1_{aa26–35*A27L} peptide or control (irrelevant) peptide pulsed T2 cells for 4 h in a [⁵¹Cr]-Chromium release assay. Specific lysis of peptide loaded T2 cells upon incubation with MART-1_{aa26–35*A27L} specific CD8⁺ T cells in various E:T ratios (1∶1–20∶1) with/without arginine was determined in duplicates. Shown is the antigen specific lysis in % which is the specific lysis investigated with MART-1_{aa26–35*A27L} loaded T2 cells minus specific lysis with control peptide loaded T2 cells. <b>B</b> Quantitative determination of antigen specific T cells was performed by MART-1_{aa26–35*A27L} tetramer analysis and is displayed as % of CD8⁺CD3⁺ T cells (analyzed before separation into the +Arg/−Arg groups) together with the results for the [⁵¹Cr]-Chromium release assay for each individual experiment.</p

Impact of arginine depletion on activation of tumor antigen specific T cells analyzed by intracellular IFN -γ staining.

<p>CD8⁺ MART-1_{aa26–35*A27L} specific T cells of 5 HD were expanded and then restimulated by coincubation with MART-1_{aa26–35*A27L} (black bars) or irrelevant control peptide (white bars) loaded T2 cells either in the presence (+Arg, 1000 µM) or absence (−Arg) of arginine. After 24 h, cells were fixed, permeabilized and stained for intracellular IFN-γ as well as extracellular CD3, CD8, CD28, CD45RA and CCR7. After fixation cells were analyzed by flow cytometry. <b>A</b> The fractions of IFN-γ⁺ T cells are demonstrated relatively to the corresponding activation with MART-1_{aa26–35*A27L} peptide pulsed T2 cells in the presence of arginine, which was set as 100%. In all HD except of HD 12 the percentage of IFN-γ⁺ T cells was significantly reduced in the absence of arginine (p<0.05). <b>B</b> An exemplary intracellular IFN-γ flow cytometry analysis (from HD 10) is demonstrated. The numbers in the quadrants show the frequency of IFN-γ⁺ cells as a fraction of gated CD8⁺CD3⁺ lymphocytes. <b>C</b> IFN-γ⁺ CD8⁺CD3⁺ T cells express the CD28⁺ CD45RA^−/low CCR7⁺ phenotype of antigen experienced T cells. An exemplary flow cytometry analysis (from HD10) after activation with MART-1_{aa26–35*A27L} peptide loaded T2 cells is shown.</p

Impact of arginine depletion on cytotoxic capacity, cytokine secretion and proliferation of human CD8⁺ CMV antigen specific T cells expanded from the natural repertoire.

<p>Expanded CMV pp65_aa495–503 specific T cells of 2 HD (•,▴) and a CMV pp65_aa495–503 specific T cell clone (▪) were activated with pp65_aa495–503 peptide pulsed K562-A2 cells. <b>A</b> Antigen specific tumor cell cytotoxicity was analyzed by [⁵¹Cr]-Chromium release assay at various E:T ratios and peptide concentrations (100 nM, 10 nM, 1 nM) in the presence (–––) or absence (- - -) of arginine. In parallel, expanded CMV pp65_aa495–503 specific T cells were incubated with pp65_aa495–503 peptide pulsed K562-A2 target cells for 48 h (<b>B–D</b>) or 120 h (<b>E</b>) at an E:T ratio of 20∶1. Supernatant was harvested and concentrations of IFN-γ (<b>B</b>), granzyme B (<b>C</b>) and perforin (<b>D</b>) were determined by ELISA. Data in <b>A–D</b> are representative of 3 different experiments with a total of 5 independent HD. <b>E</b> Reduced proliferation of CMV pp65_aa495–503 specific T cells in the absence (−Arg) compared to presence (+Arg, 1000 µM) of arginine, as determined by CFSE staining, gated on all CD3⁺ T cells within the assay mixture. One representative experiment (total: 3) is shown in (E).</p

Arginine-independent T cell functions: chemotaxis and calcium flux.

<p><b>A</b> Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. <b>B</b> Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.</p

Tumor antigen specific secretion of IFN-γ and granzyme B by human CD8⁺ T cells is regulated by the extracellular arginine concentration, whereas perforin secretion is independent of arginine.

<p>Expanded MART-1_{aa26–35*A27L} specific CD8⁺ T cells of 3 HD were activated with MART-1_{aa26–35*A27L} peptide (black bars) or control peptide (white bars) loaded T2 cells in medium with different concentrations of arginine (0, 5, 20, 150 and 1000 µM) in ELISPOT assays. After 24 h supernatants were harvested to determine granzyme B/perforin release by ELISA and IFN-γ was detected on ELISPOT membranes. <b>A</b> Results of ELISPOT assays are shown as mean of triplicates. The corresponding ELISPOT membranes of activation with MART-1_{aa26–35*A27L} peptide loaded T2 cells are shown below the diagrams. <b>B</b> Granzyme B/perforin concentration in supernatants of ELISPOT assays of 2 HD determined by ELISA.</p

Unimpaired cytotoxicity and perforin secretion but severely suppressed proliferation and effector molecule secretion of retrovirally transduced CMV specific human CD8⁺ T cells.

<p>Human primary CD8⁺ T cells retrovirally transduced with a TCR specific for the CMV peptide pp65_aa495–503 (▪) or mock transduced T cells (▴) were activated with K562-A2 cells pulsed with the cognate pp65_aa495–503 peptide (100 nM) in the presence (–––) or absence (- - -) of arginine at the indicated E:T ratios (incubation time 4 h). T cell mediated, antigen specific tumor cell cytotoxicity was assessed by [⁵¹Cr]-Chromium release assay (<b>A</b>). In parallel, identical T cell-K562-A2 co-cultivation assays were set up at an E:T ratio of 20∶1 for 48 h (<b>B–E</b>). Antigen specific T cell proliferation was assessed by [³H]thymidine incorporation for additional 16 h (<b>B</b>) while IFN-γ (<b>C</b>), granzyme B (<b>D</b>) and perforin (<b>E</b>) concentration were measured in the 48 h supernatant by ELISA. −Arg: no arginine; +Arg: 1000 µM arginine. Data in <b>A–E</b> are representative of 2 different experiments with a total of 4 different TCR constructs and 3 independent HD.</p