Apoptosis Induced via Gamma Delta T Cell Antigen Receptor “Blocking” Antibodies: A Cautionary Tale

Mechanistic studies contribute greatly to our understanding of γδ T cell (γδTc) biology, aiding development of these cells as immunotherapeutic agents. The antibody blocking assay is an accepted method to determine the receptors involved in γδTc killing of tumor targets. Effectors and/or targets are preincubated with microgram quantities of monoclonal antibodies (mAb), often described by commercial sources to be useful for blocking assays. We and others have used such assays extensively in the past, correlating decreases in cytotoxicity against specific targets with involvement of the blocked receptor(s). However, we wondered whether other mechanisms might be at play beyond cytotoxicity inhibition. Indeed, administration of certain “blocking” mAb to the γδ T cell antigen receptor (γδTCR) induced γδTc death. Upon further investigation, we discovered that γδTc underwent apoptosis triggered by incubation with mAb to the γδTCR. This effect was specific, as no apoptosis was observed when αβ T cells (αβTc) were incubated with these mAb. Apoptosis was further potentiated by the presence of interleukin (IL)-2, often included in cytotoxicity assays; however, exogenous interleukin-2 (IL-2) did not contribute significantly to γδTc cytotoxicity against breast cancer cell lines. Here, we have investigated the usefulness of four mAb for use in blocking assays by assessing blocking properties in conjunction with their propensity to induce apoptosis in cultured primary human γδTc. We found that the 5A6.E9 clone was usually a better alternative to the commonly used B1 (or B1.1) and 11F2 clones; however, some variability in susceptibility to apoptosis induction was observed among donor cultures. Thus, viability assessment of primary effector cells treated with mAb alone should be undertaken in parallel with cytotoxicity assays employing blocking antibodies, to account for cytotoxicity reduction caused by effector cell death. Previous findings should be reassessed in this light

Stoichiometry and intracellular fate of TRIM-containing TCR complexes

<p>Abstract</p> <p>Background</p> <p>Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The αβTCR includes the antigen-binding TCRαβ heterodimer as well as the signal transducing CD3εγ, CD3εδ and ζ₂subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.</p> <p>Results</p> <p>We used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing αβTCR is TCRαβCD3ε₂γδζ₂TRIM₂. Smaller αβTCR complexes possess a TCRαβ CD3ε₂γδζ₂stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the αβTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3ε colocalizes with ζ and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with ζ. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the γδTCR.</p> <p>Conclusions</p> <p>Small αβTCR complexes have a TCRαβ CD3ε₂γδζ₂stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε₂γδζ₂TRIM₂. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.</p

Celastrol attenuates angiotensin II-induced cardiac remodeling by targeting STAT3

RATIONALE: Excessive Ang II (angiotensin II) levels lead to a profibrotic and hypertrophic milieu that produces deleterious remodeling and dysfunction in hypertension-associated heart failure. Agents that disrupt Ang II-induced cardiac dysfunction may have clinical utility in the treatment of hypertension-associated heart failure. OBJECTIVE: We have examined the potential effect of celastrol-a bioactive compound derived from the Celastraceae family-on Ang II-induced cardiac dysfunction. METHODS AND RESULTS: In rat primary cardiomyocytes and H9C2 (rat cardiomyocyte-like H9C2) cells, celastrol attenuates Ang I-induced cellular hypertrophy and fibrotic responses. Proteome microarrays, surface plasmon resonance, competitive binding assays, and molecular simulation were used to identify the molecular target of celastrol. Our data showed that celastrol directly binds to and inhibits STAT (signal transducer and activator of transcription)-3 phosphorylation and nuclear translocation. Functional tests demonstrated that the protection of celastrol is afforded through targeting STAT3. Overexpression of STAT3 dampens the effect of celastrol by partially rescuing STAT3 activity. Finally, we investigated the in vivo effect of celastrol treatment in mice challenged with Ang II and in the transverse aortic constriction model. We show that celastrol administration protected heart function in Ang II-challenged and transverse aortic constriction-challenged mice by inhibiting cardiac fibrosis and hypertrophy CONCLUSIONS: Our studies show that celastrol inhibits Ang II-induced cardiac dysfunction by inhibiting STAT3 activity

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (\u3c100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer. © 2012 Quail et al

Characterization of colon cancer cells: a functional approach characterizing CD133 as a potential stem cell marker

<p>Abstract</p> <p>Background</p> <p>Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures.</p> <p>Methods</p> <p>We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated <it>in vivo</it>.</p> <p>Results</p> <p>All five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480.</p> <p>Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously.</p> <p>Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts.</p> <p>A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model.</p> <p><it>In vivo </it>growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones.</p> <p>Conclusions</p> <p>Our data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target.</p

Integral Roles for Integrins in γδ T Cell Function

Integrins are adhesion receptors on the cell surface that enable cells to respond to their environment. Most integrins are heterodimers, comprising α and β type I transmembrane glycoprotein chains with large extracellular domains and short cytoplasmic tails. Integrins deliver signals through multiprotein complexes at the cell surface, which interact with cytoskeletal and signaling proteins to influence gene expression, cell proliferation, morphology, and migration. Integrin expression on γδ T cells (γδTc) has not been systematically investigated; however, reports in the literature dating back to the early 1990s reveal an understated role for integrins in γδTc function. Over the years, integrins have been investigated on resting and/or activated peripheral blood-derived polyclonal γδTc, γδTc clones, as well as γδ T intraepithelial lymphocytes. Differences in integrin expression have been found between αβ T cells (αβTc) and γδTc, as well as between Vδ1 and Vδ2 γδTc. While most studies have focused on human γδTc, research has also been carried out in mouse and bovine models. Roles attributed to γδTc integrins include adhesion, signaling, activation, migration, tissue localization, tissue retention, cell spreading, cytokine secretion, tumor infiltration, and involvement in tumor cell killing. This review attempts to encompass all reports of integrins expressed on γδTc published prior to December 2017, highlights areas warranting further investigation, and discusses the relevance of integrin expression for γδTc function

Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia

Interactions between immune and tumor cells in the tumor microenvironment (TME) often impact patient outcome, yet remain poorly understood. In addition, the effects of biophysical features such as hypoxia [low oxygen (O2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (γδTcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety of γδTc immunotherapy and increased circulating γδTc levels correlate with improved patient outcome. Yet, the function of γδTc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O2 conditions. We have found increased infiltration of γδTc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences γδTc function. In vitro, the cell density of expanded primary healthy donor blood-derived human γδTc decreased in response to hypoxia (2% O2) compared to normoxia (20% O2). However, the secretion of macrophage inflammatory protein 1α (MIP1α)/MIP1β, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by γδTc were increased after 40 h in hypoxia compared to normoxia concomitant with the stabilization of hypoxia inducible factor 1-alpha protein. Mechanistically, we determined that natural killer group 2, member D (NKG2D) on γδTc and the NKG2D ligand MHC class I polypeptide-related sequence A (MICA)/B on MCF-7 and T47D breast cancer cell lines are important for γδTc cytotoxicity, but that MIP1α, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of γδTc such that exposure for 48 h increased cytotoxicity of γδTc against breast cancer cells that were maintained in normoxia; conversely, breast cancer lines incubated in hypoxia for 48 h prior to the assay were largely resistant to γδTc cytotoxicity. MICA/B surface expression on both MCF-7 and T47D remained unchanged upon exposure to hypoxia; however, ELISAs revealed increased MICA shedding by MCF-7 under hypoxia, potentially explaining resistance to γδTc cytotoxicity. Despite enhanced γδTc cytotoxicity upon pre-incubation in hypoxia, these cells were unable to overcome hypoxia-induced resistance of MCF-7. Thus, such resistance mechanisms employed by breast cancer targets must be overcome to develop more effective γδTc immunotherapies

Functional Plasticity of Gamma Delta T Cells and Breast Tumor Targets in Hypoxia

Interactions between immune and tumor cells in the tumor microenvironment (TME) often impact patient outcome, yet remain poorly understood. In addition, the effects of biophysical features such as hypoxia [ low oxygen (O-2)] on cells within the TME may lead to tumor evasion. Gamma delta T cells (gamma delta Tcs) naturally kill transformed cells and are therefore under development as immunotherapy for various cancers. Clinical trials have proven the safety gamma delta Tc immunotherapy and increased circulating gamma delta Tc levels correlate with improved patient outcome. Yet, the function of gamma delta Tc tumor infiltrating lymphocytes in human breast cancer remains controversial. Breast tumors can be highly hypoxic, thus therapy must be effective under low O-2 conditions. We have found increased infiltration of gamma delta Tc in areas of hypoxia in a small cohort of breast tumors; considering their inherent plasticity, it is important to understand how hypoxia influences gamma delta Tc function. In vitro, the cell density of expanded primary healthy donor blood-derived human gamma delta Tc decreased in response to hypoxia (2% O-2) compared to normoxia (20% O-2). However, the secretion of macrophage inflammatory protein 1 alpha (MIP1 alpha)/MIP1 beta, regulated on activation, normal T cell expressed and secreted (RANTES), and CD40L by gamma delta Tc were increased after 40 h in hypoxia compared to normoxia concomitant with the stabilization of hypoxia inducible factor 1-alpha protein. Mechanistically, we determined that natural killer group 2, member D (NKG2D) on gamma delta Tc and the NKG2D ligand MHC class I polypeptide-related sequence A (MICA)/B on MCF-7 and T47D breast cancer cell lines are important for gamma delta Tc cytotoxicity, but that MIP1 alpha, RANTES, and CD40L do not play a direct role in cytotoxicity. Hypoxia appeared to enhance the cytotoxicity of gamma delta Tc such that exposure for 48 h increased cytotoxicity of gamma delta Tc against breast cancer cells that were maintained in normoxia; conversely, breast cancer lines incubated in hypoxia for 48 h prior to the assay were largely resistant to gamma delta Tc cytotoxicity. MICA/B surface expression on both MCF-7 and T47D remained unchanged upon exposure to hypoxia; however, ELISAs revealed increased MICA shedding by MCF-7 under hypoxia, potentially explaining resistance to gamma delta Tc cytotoxicity. Despite enhanced gamma delta Tc cytotoxicity upon pre-incubation in hypoxia, these cells were unable to overcome hypoxia-induced resistance of MCF-7. Thus, such resistance mechanisms employed by breast cancer targets must be overcome to develop more effective gamma delta Tc immunotherapies

Imaging Human Breast Tumours in Different Species: How Human Are They?

In a gedankenexperiment, we pose the philosophical question as to whether human breast cancer cells or tissues can still be considered human after transplantation into another species. Alongside medical research images illustrating xenotransplantation, we provide descriptions of how tissues were prepared for imaging. In addition, we discuss how such models enable further understanding of cancer and provide invaluable tools for testing new therapies.Dans un gedankenexperminent nous posons la question philosophique de savoir si les tissus et cellules de cancer du sein humain peuvent toujours être considérés comme humains après qu’ils ont été transplantés dans une autre espèce. En plus de photographies de recherche médicale illustrant la xénotransplantation, nous offrons des descriptions expliquant comment des tissus ont été préparés pour la visualisation. Nous débattons en outre de la manière dont de tels modèles permettent une meilleure compréhension du cancer et offrent des outils inestimables pour tester de nouvelles thérapeutiques