Multiplex Human Cytokine ELISA Kit (M1/M2/MDSC Cytokines)

INTENDED USE

This multiplex ELISA kit for M1/M2/MDSC cytokines is designed for semi-quantitative and simultaneous determination of cytokines relevant to the proliferation of Myeloid Derived Suppressor Cells (MDSCs) and their differentiation toward M1 or M2 phenotype.  The kit simultaneously determines granulocyte macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ)  interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), monocyte chemotactic and activating factor (MCAF, also known as MCP-1), and tumor necrosis factor-α (TNF-α) in cell culture supernatant and other biological samples.  In combination with other Anogen quantitative cytokine ELISA kits, the M1/M2/MDSC cytokine multiplex ELISA kit is expected to be useful for the investigation of the relationship of cytokine expression and MDSC induced inmmunosuppression in various disease models.  

The kit is intended FOR LABORATORY RESEARCH USE ONLY and should not be used in any diagnostic or therapeutic procedures. 

INTRODUCTION

Myeloid derived suppressor cells (MDSCs) are myelogenous cells that are capable of negatively regulating T cell immunity.  Increased numbers of MDSCs are found in pathological conditions such as malignancy, transplantation, chronic infection and inflammation.  MDSCs cannot be classified by a standard leukocyte linage marker since MDSCs are comprised of various myeloid originated cells at immature status including myeloid progenitor cells, immature monocytes, dendritic cells, and granulocytes.   MDSCs in human are broadly defined as lin(-/low)CD33(+) HLA-DR(-)CD11b(+) cells with altered enzyme and cytokine profile and immunosuppressive function.  While CD33 (+) and CD11b (+) denote myeloid origin in human, Gr-1(+) and CD11b (+) define myeloid origin in MDSCs in mouse.

In chronic inflammation caused by cancer, the interaction between tumor cells and MDSCs causes MDSCs to expand and increase its potential in T cell inhibition (Sevko et al. 2013).  MDSCs have been recognized as one of the major mechanisms for tumor evasion from host immunity and are recently evaluated as target for cancer treatment (Sinha et al. 2005).

Cytokines are believed to play a critical role in MDSC development and differentiation.  GM-CSF and IL-6 have been shown to stimulate MDSC expansion in vivo and in vitro ((Lechner et al. 2010, Morales et al. 2010).  T-helper 2 cytokines, IL-4 and IL-13, are the major polarization signals for MDSC to differentiate toward the more T-cell inhibitory M2 phenotype of MDSCs (Bronte et al. 2003, Sinha et al. 2005).  Additionally, interleukin 4 receptor alpha (IL-4Ra), the common receptor for IL-4 and IL-13, has been found to be up-regulated in MDSCs (Mandruzzato et al. 2009).  One of the main characteristics of M2 MDSCs is the up-regulation of IL-10 and down-regulation of IL-12 (Bunt et al. 2007).  IL-10 inhibits cell immunity by decreasing the secretion of T helper 1 type cytokines and the expression of MHC class II antigens and co-stimulatory molecules.  It has also been postulated that M2 MDSC-mediated T- cell inhibition is the consequence of increased production of arginases and reactive oxygen species by MDSCs (Zea et al. 2005, Rodriguez et al. 2006) and the enzymes secreted by MDSCs block the synthesis of zeta chain in T-cell receptor complex and sequester cystine and limit the availability of cysteine to T-cells.  Signal transduction through calcium binding protein S100A8/A9 and signal transduction and activator of transcription (STAT) is likely implicated in MDSC activities (Zhao et al. 2012). 

M2 MDSCs inhibit effector T cells but promote Regulatory T cells.  The increased expression of cytokines and chemokines such as VEGF, MCP-1 and MIF in the tumor microenvironment is believed to promote the infiltration of MDSCs and stimulate tumor angiogenesis and metastasis (Bellamy et al. 2001, Huang et al. 2007, and Simpson et al. 2013).

IFN-γ is a potent activator for MDSCs to develop into the more tumoricidal and virucidal M1 phenotype.  IFN-g and other M1 polarizing signals up-regulate IL-12 and TNF-α in M1 MDSC.  M1-polarized MDSCs express elevated signature markers such as inducible Nitric Oxide Synthase (iNOS), nitric oxide (NO), TNF-α, IFN-γ (Yang et al. 2013).  Nitric oxide (NO) and TNF-α  play important roles in clearing bacterial, and certain fungal, viral, and parasitic invasions as well as in the necrosis of specific tumors and nitric oxide (NO).

This ELISA assay is a 3.5 hour solid phase immunoassay readily applicable to measure the levels of eight cytokines relevant to the generation and differentiation of MDSCs in cell culture supernatant, and other biological fluids.  It showed no cross reactivity with other proteins.

CITATIONS  

1. Yagnik G, Rutowski MJ, Shah SS, Aghi MK. Stratifying nonfunctional pituitary adenomas into two groups distinguished by macrophage subtypes. Oncotarget 2019; 22(10):2212-2223.