immunomodulatory properties of mesenchymal stem cells

Mesenchymal stem cells inhibit natural killer–cell proliferation, cytotoxicity, andcytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2

Grazia Maria Spaggiari et al

BY: Shreya AhujaRoll no. 3

Immunomodulatory properties of Mesenchymal Stem Cells

STEM CELLS• What are Stem cells• Types of Stem cells • Mesenchymal Stem cells

A stem cell is an undifferentiated cell of a multicellular organism which is capable of giving rise to indefinitely more cells of the same type, and from which certain other kinds of cell arise by differentiation.

They show three major characteristics:

1.Unspecialized, capable of forming many cell types2.Proliferation and self renewal for long periods of time3.Differentiation potential by asymmetric division

What are Stem cells?

Types of Stem cells

MESENCHYMAL STEM CELLSA non-hematopoietic adult multipotent stem cell

Mesenchymal cells primarily give rise to:

1.Osteocytes2.Chondrocytes3.Adipocytes

BACKGROUND OF THE RESEARCH WORK

• Graft-versus-Host Disease (GvHD)• NK cells in immune response• Immunomodulatory properties of

mesenchymal stem cells

Graft-versus-Host Disease (GvHD)• Common complication following allogeneic tissue

transplantation. Commonly associated with Stem cell or Bone Marrow transplant but term used for other forms of tissue grafts too

• Nausea, Vomiting, Diarrhea, Weight loss, Bacterial infections etc.

NK CELLS How do they pull the trigger??

• Part of innate arm of immune system• Cytotoxic proteins within secretory lysosomes – granzymes and

perforin• Recognize aberrant target cell – absence of MHC I molecule• Participate in allorejection directly or by Antibody

dependent cytotoxicity. Stimulate TH cells by IFN-γ

secretions

Immunomodulatory Properties of Mesenchymal Stem Cells

• MSCs do not follow the ‘rules’ of immune rejection – instead suppress the activation and proliferation of immune cells of host

• Positive expression of MHC I on cell surface – Not recognized as foreign by NK cells

• Negative for MHC II and co-stimulatory molecules like CD40, CD80, CD86, CD45– Suppress T-cell activation

• MSC’s known to interfere with differentiation, maturation and function of Dendritic cells (APC’s)

• Expression of HLA-G5 suppresses allogeneic T-cell proliferation and induces expansion of Tregs

• IL-10, TGF-β, HGF, PGE-2, IDO, NO – responsible for

immunomodulatory properties of MSCs

EXPERIMENTAL SETUP TO STUDY THE

INHIBITORY ACTIVITY OF MSCs ON NK CELLS

1. Isolation and characterization of NK cells2. Isolation and characterization of MSCs3. Co-culture of MSCs and NK cells to study the

inhibitory effect of MSCs on receptor expression of NK cells

4. Effect of MSCs on cytotoxicity of NK cells5. Inhibition of cytokine production by NK cells

in the presence of MSCs6. Mechanisms underlying MSC mediated

inhibition of NK cells – Role of IDO and PGE-2

Isolation and Characterization of NK cells

T-Cells, B-cells, NK cells, MSCs and other non-granulocyte immune cells

• Cytofluorometric analysis done to check the purity of isolated NK cells

• Purified NK cells were cultured for up to 7 days in RPMI 164 (Roswell Park Memorial Institute) medium supplemented with 10% fetal calf serum (FCS), IL-2 to obtain short-term activated polyclonal NK cells

Cytofluorometric analysis

Isolation and Characterization of MSCs

MSCs – Trophic Factor Pool• Medium used for culturing stem cells contains the secretome of

the growing stem cells and is called conditioned medium (CM)• MSCs are known to secrete a number of trophic factors in the

culture medium: Transforming Growth Factor β Vascular Endothelial Growth Factor

Insulin like Growth Factor – 1 (IGF-1)

Fibroblast Growth Factor (FGF)

Hepatocyte Growth Factor (HGF)

Keratinocyte Growth Factor (KGF)

Prostaglandin E2 and many more….

Trophic factors for repair work in -vivo

Co-culture of MSCs and NK cells• NK cells plated with MSCs at 4:1 NK/MSC ratio (experimentally

determined) - MSC-mediated inhibition of NK-cell proliferation.• A series of MSC populations derived from different donors was

used in allogeneic combination with NK cells.

• CONTROL: 1 mM 1-methyl-tryptophan - inhibitor of IDO activity 5µM NS-398 - inhibitor of PGE2 synthesis

10µg/mL anti–TGF-β neutralizing monoclonal antibody

Trophic factors

Cytofluorometric analysis of NK Cells Inhibitory effects of MSCs on surface expression of receptors on NK Cells

Surface markers on NK cells cultured in IL-2 medium

Freshly isolated population of NK cells

NK Cells in culture for 6 days in the absence of MSCs

NK Cells in culture for 6 days in the presence of MSCs

Cytotoxicity of NK CellsNK Cells

iDC, Leukemia cell lines, Neuroblastoma cell lines

Results for cytotoxicity of NK CellsNK cells, when cultured alone could efficiently lyse all targets. On the contrary, when effector cells were cultured in the presence of MSCs, a strongly reduced killing capability was detected.Gray – NK cells cultured alone in IL-2, Black – NK cells grown in presence of MSCs

(leukemia cell line)

(Neuroblastoma cell line)

Cytokine production by NK Cells• Analysis of INF-γ production by NK cells

Results for cytokine secretion by NK cells• Together with cytolytic activity, cytokine production is another main NK-

cell function• NK cells cultured in IL-2 alone produced IFN- γ as a consequence of the

interaction with FO-1 cells, whereas the same cells cultured in the presence of MSCs were much less responsive to stimulation.

NK – FO1 (Neg. control)

NK + FO1 - MSC NK + FO1 + MSC

Inhibitory effect exerted by MSCs on NK cells can affect different aspects of NK-cell activation and function, ranging from

proliferation to cytotoxic activity and cytokine production. BUT WHAT IN MSCs IS CAUSING THIS TO OCCUR?

MSC mediated IDO and PGE-2 secretion affect NK cell proliferation and cytotoxicity

• NK cells co-cultured with MSCs as described before• H3-thymidine uptake method for proliferation assay

Results:I.NK cells underwent proliferation upon IL-2–induced activationII.Strong inhibition of cell proliferation in the presence of MSCsIII.Neither NS-398 nor 1-M-Trp alone had any substantial effect. However, simultaneous blocking of IDO and PGE2 could almost completely restore the NK-cell proliferation (synergistic effect)IV.Similar results obtained for cytotoxicity of NK cells which was restored almost completely in the absence of both IDO and PGE-2

(IDO)(PGE-2)

(leukemia cell line)

(Neuroblastoma cell line)

THERAPEUTIC APPLICATIONS OF MESENCHYMAL STEM CELLS

Graft vs Host Disease• MSC - ‘immunologically privileged’ cell population – inhibit

immune cells proliferation, suppress an immune response against foreign grafts, tissue-repair ability

• Can be used in allogeneic hematopoietic graft transplantations

• Example: 9-year-old boy with severe acute GVHD of the gut and liver, developed diarrhea (20 times daily) and a high concentration of bilirubin. Four days after an intravenous infusion of MSCs, the frequency of diarrhea fell to twice daily and there was a decline in total bilirubin

Allogeneic MSCs in tissue regeneration and repair

• Critical Limb Ischemia – MSC therapy for regeneration of necrotic blood vessels

• Liver Cirrhosis – Regeneration and repair of necrotic hepatic tissue

• Osteogenesis imperfecta (a genetic disorder) caused by deficiency in the production of type I collagen, the major structural protein in bone, resulting in bone fragility and growth deficiency. Significant increase in body length and in the bone mineralization in children with severe OI after infusions of purified bone marrow MSCs

• Duchenne muscular dystrophy in mice, human MSCs isolated from the synovial membrane, promoted the regeneration of skeletal muscle tissue

AND MANY MANY MORE

BIBLIOGRAPHY• Grazia Maria Spaggiari et al (2008) Mesenchymal stem cells inhibit

natural killer–cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood Journal 111(3):1327-1333

• Marlies EJ Reinders and Martin J Hoogduijn (2014) NK Cells and MSCs: Possible Implications for MSC Therapy in Renal Transplantation. J. Stem Cell Res Ther. 4(2)

• Cíntia de Vasconcellos Machado, Paloma Dias da Silva Telles, Ivana Lucia Oliveira Nascimento (2013) Immunological characteristics of mesenchymal stem cells. Rev Bras Hematol Hemoter. 35(1):62-7

• Xin Wei et al (2013) Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacologica Sinica. 34: 747–754

• www.stemcells.nih.gov

http://www.stemcells.nih.gov/

immunomodulatory properties of mesenchymal stem cells

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