Mesenchymal stem cells were found to inhibit natural killer cell proliferation, cytotoxicity, and cytokine production through the roles of indoleamine 2,3-dioxygenase and prostaglandin E2. An experiment co-cultured purified natural killer cells and mesenchymal stem cells, finding that mesenchymal stem cells suppressed natural killer cell receptor expression, cytotoxicity against tumor cells, and interferon-gamma production. This inhibitory effect was found to be mediated by indoleamine 2,3-dioxygenase and prostaglandin E2 secreted by mesenchymal stem cells.

1. Mesenchymal stem cells inhibit natural
killer–cell proliferation, cytotoxicity, and
cytokine production: role of indoleamine
2,3-dioxygenase and prostaglandin E2
Grazia Maria Spaggiari et al
BY: Shreya Ahuja
Roll no. 3
Immunomodulatory properties of
Mesenchymal Stem Cells

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

3. 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 types
2.Proliferation and self renewal for long periods of time
3.Differentiation potential by asymmetric division
What are Stem cells?

4. Types of Stem cells

5. MESENCHYMAL STEM CELLS
A non-hematopoietic adult multipotent stem cell
Mesenchymal
cells primarily
give rise to:
1.Osteocytes
2.Chondrocytes
3.Adipocytes

6. BACKGROUND OF THE
RESEARCH WORK
• Graft-versus-Host Disease (GvHD)
• NK cells in immune response
• Immunomodulatory properties of mesenchymal
stem cells

7. 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.

8. 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

9. 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

10. EXPERIMENTAL SETUP
TO STUDY THE
INHIBITORY ACTIVITY OF
MSCs ON NK CELLS

11. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

12. 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

13. Cytofluorometric analysis

14. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

15. Isolation and Characterization of MSCs

16. 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

17. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

18. 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

19. 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

20. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

21. Cytotoxicity of NK Cells
NK Cells
iDC, Leukemia cell lines,
Neuroblastoma cell lines

22. Results for cytotoxicity of NK Cells
NK 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)

23. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

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

25. 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?

26. 1. Isolation and characterization of NK cells
2. Isolation and characterization of MSCs
3. 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 cells
5. Inhibition of cytokine production by NK cells in
the presence of MSCs
6. Mechanisms underlying MSC mediated inhibition
of NK cells – Role of IDO and PGE-2

27. 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 activation
II.Strong inhibition of cell
proliferation in the presence of MSCs
III.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)

28. THERAPEUTIC APPLICATIONS OF MESENCHYMAL STEM CELLS

29. 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

30. 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

32. 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