15 research outputs found
Methylcholanthrene-Induced Sarcomas Develop Independently from NOX2-Derived ROS
<div><p>Reactive oxygen species (ROS) produced by the inducible NADPH oxidase type 2 (NOX2) complex are essential for clearing certain infectious organisms but may also have a role in regulating inflammation and immune response. For example, ROS is involved in myeloid derived suppressor cell (MDSC)- and regulatory T cell (T<sub>reg</sub>) mediated T- and NK-cell suppression. However, abundant ROS produced within the tumor microenvironment, or by the tumor itself may also yield oxidative stress, which can blunt anti-tumor immune responses as well as eventually leading to tumor toxicity. In this study we aimed to decipher the role of NOX2-derived ROS in a chemically (by methylcholanthrene (MCA)) induced sarcoma model. Superoxide production by NOX2 requires the p47<sup>phox</sup> (NCF1) subunit to organize the formation of the NOX2 complex on the cell membrane. Homozygous mutant mice (NCF1*<sup>/</sup>*) have a functional loss of their super oxide burst while heterozygous mice (NCF1*<sup>/+</sup>) retain this key function. Mice harboring either a homo- or a heterozygous mutation were injected intramuscularly with MCA to induce sarcoma formation. We found that NOX2 functionality does not determine tumor incidence in the tested MCA model. Comprehensive immune monitoring in tumor bearing mice showed that infiltrating immune cells experienced an increase in their oxidative state regardless of the NOX2 functionality. While MCA-induced sarcomas where characterized by a T<sub>reg</sub> and MDSC accumulation, no significant differences could be found between NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice. Furthermore, infiltrating T cells showed an increase in effector-memory cell phenotype markers in both NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice. Tumors established from both NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice were tested for their <i>in vitro</i> proliferative capacity as well as their resistance to cisplatin and radiation therapy, with no differences being recorded. Overall our findings indicate that NOX2 activity does not play a key role in tumor development or immune cell infiltration in the chemically induced MCA sarcoma model.</p></div
Growth of MCA induced tumors is not affected by NCF1 mutation.
<p>(A) NCF1*<sup>/</sup>* (n = 6) and NCF1*<sup>/+</sup> (n = 10) mice were tested for their ability to generate superoxide through stimulation of NOX2 with PMA. (B) NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice (n = 54, 27 per group) were injected with 125 μg MCA dissolved in 25 μl of corn oil. Tumor incidence was followed through palpation of the injected muscle site. (C) Tumors were resected from MCA injected NCF1*<sup>/</sup>* (n = 6) and NCF1*<sup>/+</sup> (n = 6) mice and passaged once in WT C57Bl/6 mice prior to transplantation of 5<sup>3</sup> mm tumor into four WT C57Bl/6 (n = 48) mice in which (Ci) survival (Cii) and tumor growth were monitored.</p
T cells are not affected differently in NCF1*<sup>/</sup>* compared to NCF1*<sup>/+</sup> tumor bearing mice with regard to their CD4/CD8 ratio, activation status or memory phenotype.
<p>(A) T cells were gated as singlet cells that were not stained for dead cell marker but positive for CD3. (B) The ratio of CD4+ to CD8+ was gated in blood, spleen and tumor in NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice, with the dotted line representing the ratio found in WT C57Bl/6 spleenocytes. (C) Activation marker CD69 was stained on blood, spleen and tumor resident T cells. (D) Blood, spleen and tumor resident T cells were stained for CD62L as well as CD44 to indicate their memory phenotype.</p
MCA induced tumors accumulate immune regulatory cells.
<p><b>(</b>A) MDSCs as characterized by CD11b+ and GR1+ were gated from splenocytes or tumor samples as a percentage of live cells. (B) Percentage of live splenocytes that are MDSCs in tumor bearing NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice compared to WT mice. (C) Percentage of live cells which are MDSC in tumors from both NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice. (D) Percent FoxP3+ cells of CD4+ cells in spleens of tumor bearing mice compared to WT mice. (E) Percent T<sub>reg</sub> cells in the blood, spleen and tumor of MCA tumor bearing mice.</p
Non-functional NOX2 does not change susceptibility to cisplatin or radiotherapy.
<p>(Ai) Cells were seeded in E-plates and treated with 5μg/ml of cisplatin at log phase growth. (Aii) Rate of cell death after addition of cisplatin was compared between NCF1 tumor cell lines, derived from MCA induced tumor in NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> mice. (B) NCF1 tumor cell lines were treated with 10 gray and morphology of the cells was followed for three days. (Ci) 100 tumor cells were seeded in plates and treated with 2 gray and followed over 6 or 9 days. Metabolic activity was measured with XTT as an indication of survival with treatment over control (Cii) indicating survival.</p
Oxidative state does not differ in immune cell subsets or tumors from NCF1*<sup>/</sup>* and NCF1*<sup>/+</sup> tumor bearing mice.
<p>(A) Cells were gated on live followed by CD45 positive both CD3 negative followed by CD11b positive and GR1 positive for MDSC, CD45 positive CD3 positive indicated T cells and CD45 negative live cells were considered tumor cells. (B) DHR-123 MFI of MDSCs as well (C) T cells and (D) tumor cells were determined in NCF1*<sup>/</sup>* (n = 4) and NCF1*<sup>/+</sup> (n = 5) tumor bearing mice.</p
Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress
<div><p>The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA<sup>+</sup> and their memory/effector CD45RO<sup>+</sup> T cell counterparts in the presence and absence of low dose hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA<sup>+</sup> and CD45RO<sup>+</sup> T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.</p> </div
Functional classification of differentially expressed proteins.
<p>RA− and RO− indicate the profiling results for the respective untreated T cell subsets, RA+ and RO+ for that of H<sub>2</sub>O<sub>2</sub>-treated T cell subpopulation as outlined in the Material and methods section. Proteins are listed by their protein entry names.</p
Representative cell viability in response to treatment with low doses of H2O2.
<p>A. Representative FACS data for untreated vs. H<sub>2</sub>O<sub>2</sub>-exposed (5 µM} T cell subsets isolated from one healthy donor are shown. Cell viability (gate) was assessed by changes of the forward scatter (FSC-A) profile and staining with 7-AAD.. B. Mean values for specific H<sub>2</sub>O<sub>2</sub>-induced cell death are shown (n = 5). Bars represent the standard error mean.</p
DataSheet_1_Induction treatment in high-grade B-cell lymphoma with a concurrent MYC and BCL2 and/or BCL6 rearrangement: a systematic review and meta-analysis.docx
Background and aimHigh-grade B cell lymphomas with concomitant MYC and BCL2 and/or BCL6 rearrangements (HGBCL-DH/TH) have a poor prognosis when treated with the standard R-CHOP-like chemoimmunotherapy protocol. Whether this can be improved using intensified regimens is still under debate. However, due to the rarity of HGBCL-DH/TH there are no prospective, randomized controlled trials (RCT) available. Thus, with this systematic review and meta-analysis we attempted to compare survival in HGBCL-DH/TH patients receiving intensified vs. R-CHOP(-like) regimens.MethodsThe PubMed and Web of Science databases were searched for original studies reporting on first-line treatment in HGBCL-DH/TH patients from 08/2014 until 04/2022. Studies with only localized stage disease, ≤10 patients, single-arm, non-full peer-reviewed publications, and preclinical studies were excluded. The quality of literature and the risk of bias was assessed using the Methodological Index for Non-Randomized Studies (MINORS) and National Heart, Lung, and Blood Institute (NHLBI) Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. Random-effect models were used to compare R-CHOP-(like) and intensified regimens regarding 2-year overall survival (2y-OS) and 2-year progression-free survival (2y-PFS).ResultsAltogether, 11 retrospective studies, but no RCT, with 891 patients were included. Only four studies were of good quality based on aforementioned criteria. Intensified treatment could improve 2y-OS (hazard ratio [HR]=0.78 [95% confidence interval [CI] 0.63-0.96]; p=0.02) as well as 2y-PFS (HR=0.66 [95% CI 0.44-0.99]; p=0.045).ConclusionsThis meta-analysis indicates that intensified regimens could possibly improve 2y-OS and 2y-PFS in HGBCL-DH/TH patients. However, the significance of these results is mainly limited by data quality, data robustness, and its retrospective nature. There is still a need for innovative controlled clinical trials in this difficult to treat patient population.Systematic review registrationhttps://www.crd.york.ac.uk/prospero, identifier CRD42022313234.</p