DIFFERENTIAL SUSCEPTIBILITY OF CARDIAC CELL POPULATIONS TO IN VITRO IMMUNE INJURY

Interleukin-6 (IL-6) is a pleiotropic cytokine responsible for many different processes including the regulation of cell growth, apoptosis, differentiation, and survival in various cell types and organs, including the heart. Recent studies have indicated that IL-6 is a critical component in the cell-cell communication between myocytes and cardiac fibroblasts. In this study, we examined the effects of IL-6 deficiency on the cardiac cell populations, cardiac function, and interactions between the cells of the heart, specifically cardiac fibroblasts and myocytes. To examine the effects of IL-6 loss on cardiac function, we used the IL-6 −/− mouse. IL-6 deficiency caused severe cardiac dilatation, increased accumulation of interstitial collagen, and altered expression of the adhesion protein periostin. In addition, flow cytometric analyses demonstrated dramatic alterations in the cardiac cell populations of IL-6 −/− mice compared with wild-type littermates. We observed a marked increase in the cardiac fibroblast population in IL-6 −/− mice, whereas a concomitant decrease was observed in the other cardiac cell populations examined. Moreover, we observed increased cell proliferation and apoptosis in the developing IL-6 −/− heart. Additionally, we observed a significant decrease in the capillary density of IL-6 −/− hearts. To elucidate the role of IL-6 in the interactions between cardiac fibroblasts and myocytes, we performed in vitro studies and demonstrated that IL-6 deficiency attenuated the activation of the STAT3 pathway and VEGF production. Taken together, these data demonstrate that a loss of IL-6 causes cardiac dysfunction by shifting the cardiac cell populations, altering the extracellular matrix, and disrupting critical cell-cell interactions.

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Differential susceptibility of the alpha(1)-adrenergic receptor subtypes to antidepressant drugs action: in vitro study

10.26226/morressier.5971be83d462b80290b53017 ◽

2017 ◽

Author(s):

Justyna Kusmierczyk

Keyword(s):

Adrenergic Receptor ◽

Antidepressant Drugs ◽

Differential Susceptibility ◽

In Vitro Study ◽

Vitro Study ◽

Receptor Subtypes

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Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

Current Stem Cell Research & Therapy ◽

10.2174/1574888x14666190123161447 ◽

2019 ◽

Vol 14 (4) ◽

pp. 305-319 ◽

Cited By ~ 4

Author(s):

Marietta Herrmann ◽

Franz Jakob

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Progenitor Cell ◽

Adult Stem Cells ◽

Current Knowledge ◽

Cell Populations ◽

Surface Markers ◽

Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

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Loss of 111Indium as indicator of platelet injury

Blood ◽

10.1182/blood.v58.2.350.350 ◽

1981 ◽

Vol 58 (2) ◽

pp. 350-353 ◽

Cited By ~ 18

Author(s):

JH Joist ◽

RK Baker

Keyword(s):

Small Molecules ◽

Adenine Nucleotides ◽

Human Platelets ◽

Metabolic Energy ◽

Platelet Factor ◽

Platelet Protein ◽

Threshold Rate ◽

Immune Injury ◽

Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

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108 MCY-M11, a CAR-PBMC cell product transiently expressing a mesothelin targeted mRNA CAR, exhibits desirable functional and immune phenotype attributed to sustained antitumor immunity in vitro

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0108 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A120-A120

Author(s):

Sashi Kasimsetty ◽

Himavanth Gatla ◽

Dhana Chinnasamy

Keyword(s):

T Cells ◽

Tumor Cells ◽

Antitumor Immunity ◽

Memory T Cells ◽

Cell Populations ◽

Epitope Spreading ◽

Antitumor Response ◽

Cell Product

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

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PMN-MDSCs Enhance CTC Metastatic Properties through Reciprocal Interactions via ROS/Notch/Nodal Signaling

International Journal of Molecular Sciences ◽

10.3390/ijms20081916 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1916 ◽

Cited By ~ 20

Author(s):

Marc L. Sprouse ◽

Thomas Welte ◽

Debasish Boral ◽

Haowen N. Liu ◽

Wei Yin ◽

...

Keyword(s):

Cancer Patients ◽

Receptor Expression ◽

Cell Populations ◽

Nodal Signaling ◽

Breast Cancer Patients ◽

Tumoricidal Activity ◽

Notch Activation ◽

Notch1 Receptor

Intratumoral infiltration of myeloid-derived suppressor cells (MDSCs) is known to promote neoplastic growth by inhibiting the tumoricidal activity of T cells. However, direct interactions between patient-derived MDSCs and circulating tumors cells (CTCs) within the microenvironment of blood remain unexplored. Dissecting interplays between CTCs and circulatory MDSCs by heterotypic CTC/MDSC clustering is critical as a key mechanism to promote CTC survival and sustain the metastatic process. We characterized CTCs and polymorphonuclear-MDSCs (PMN-MDSCs) isolated in parallel from peripheral blood of metastatic melanoma and breast cancer patients by multi-parametric flow cytometry. Transplantation of both cell populations in the systemic circulation of mice revealed significantly enhanced dissemination and metastasis in mice co-injected with CTCs and PMN-MDSCs compared to mice injected with CTCs or MDSCs alone. Notably, CTC/PMN-MDSC clusters were detected in vitro and in vivo either in patients’ blood or by longitudinal monitoring of blood from animals. This was coupled with in vitro co-culturing of cell populations, demonstrating that CTCs formed physical clusters with PMN-MDSCs; and induced their pro-tumorigenic differentiation through paracrine Nodal signaling, augmenting the production of reactive oxygen species (ROS) by PMN-MDSCs. These findings were validated by detecting significantly higher Nodal and ROS levels in blood of cancer patients in the presence of naïve, heterotypic CTC/PMN-MDSC clusters. Augmented PMN-MDSC ROS upregulated Notch1 receptor expression in CTCs through the ROS-NRF2-ARE axis, thus priming CTCs to respond to ligand-mediated (Jagged1) Notch activation. Jagged1-expressing PMN-MDSCs contributed to enhanced Notch activation in CTCs by engagement of Notch1 receptor. The reciprocity of CTC/PMN-MDSC bi-directional paracrine interactions and signaling was functionally validated in inhibitor-based analyses, demonstrating that combined Nodal and ROS inhibition abrogated CTC/PMN-MDSC interactions and led to a reduction of CTC survival and proliferation. This study provides seminal evidence showing that PMN-MDSCs, additive to their immuno-suppressive roles, directly interact with CTCs and promote their dissemination and metastatic potency. Targeting CTC/PMN-MDSC heterotypic clusters and associated crosstalks can therefore represent a novel therapeutic avenue for limiting hematogenous spread of metastatic disease.

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Self-sparing of long-term in vitro-cloned or uncloned cytotoxic T lymphocytes.

Journal of Experimental Medicine ◽

10.1084/jem.164.3.962 ◽

1986 ◽

Vol 164 (3) ◽

pp. 962-967 ◽

Cited By ~ 19

Author(s):

M F Luciani ◽

J F Brunet ◽

M Suzan ◽

F Denizot ◽

P Golstein

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Cytotoxic T Cell ◽

Cell Populations ◽

Con A ◽

T Cell Clones ◽

Cell Clones

At least some long-term in vitro-cultured cytotoxic T cell clones and uncloned cell populations are able, in the presence of Con A, to lyse other cells, to be lysed by other cells, but not to lyse themselves. This as-yet-unexplained result may have implications as to the mechanism of T cell-mediated cytotoxicity.

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SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

Journal of Experimental Medicine ◽

10.1084/jem.140.1.1 ◽

1974 ◽

Vol 140 (1) ◽

pp. 1-18 ◽

Cited By ~ 175

Author(s):

Melvyn Greaves ◽

George Janossy ◽

Michael Doenhoff

Keyword(s):

B Cells ◽

B Lymphocytes ◽

Human Lymphocytes ◽

Culture Conditions ◽

Pokeweed Mitogen ◽

Cell Populations ◽

T And B Cells ◽

Activated T Cells ◽

Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

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