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Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic

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Abstract

Objectives

IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).

Methods

Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured.

Results

IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p < 0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection.

Conclusions

This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

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Abbreviations

TMV:

Tumor-derived microvesicles

ANXV:

Annexin V

PBMC:

Peripheral blood mononuclear cells

CHX:

Cycloheximide

BSA:

Bovine serum albumin

FBS:

Fetal bovine serum

MMP:

Mitochondrial membrane potential

AICD:

Activation-induced cell death

CTL:

CD8+ cytotoxic T lymphocytes

TCR:

T cell receptor

FLIP:

Cellular caspase 8 (FLICE)-like inhibitory protein

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Acknowledgments

This study was supported in part by NIH grant PO1 CA109688 to TLW.

Conflict of interest

None of the authors have any conflict of interest.

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Authors and Affiliations

  1. Research Pavilion at the Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Suite 1.27, Pittsburgh, PA, 15213-1863, USA

    Malgorzata Czystowska, Miroslaw J. Szczepanski, Marta Szajnik & Theresa L. Whiteside

  2. IRX Therapeutics, Inc, 1 Bioscience Park Drive, Farmingdale, NY, 11735, USA

    Karen Quadrini, Harvey Brandwein & John W. Hadden

Authors
  1. Malgorzata Czystowska
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  2. Miroslaw J. Szczepanski
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  3. Marta Szajnik
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  4. Karen Quadrini
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  5. Harvey Brandwein
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  6. John W. Hadden
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  7. Theresa L. Whiteside
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Corresponding author

Correspondence to Theresa L. Whiteside.

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Czystowska, M., Szczepanski, M.J., Szajnik, M. et al. Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic. Cancer Immunol Immunother 60, 495–506 (2011). https://doi.org/10.1007/s00262-010-0951-9

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  • DOI: https://doi.org/10.1007/s00262-010-0951-9

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