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Demonstration of immunomodulatory properties for the human MuStem cell population, a promising candidate
for cell therapy of muscular dystrophies
Judith Lorant, Marine Charrier, Rafael Contreras Lopez, Christophe Blanquart, Blandine Lieubeau-Teillet, Cindy Schleder, Isabelle Leroux,
Tejedor Gauthier, Candice Babarit, Yann Pereon, et al.
To cite this version:
Judith Lorant, Marine Charrier, Rafael Contreras Lopez, Christophe Blanquart, Blandine Lieubeau-Teillet, et al.. Demonstration of immunomodulatory properties for the human MuStem cell population, a promising candidate for cell therapy of muscular dystrophies. 26th Annual Congress of the ESGCT, Oct 2018, Lausanne, Switzerland. Human Gene Therapy, 29 (12), 2018. �hal-01927456�
ü hMuStem cells directly
inhibit sorted T-cell proliferation in a dose dependent manner
ü hMuStem cells equally inhibit CD4+ and CD8+ lymphocyte
proliferation and activation in PBMC population
ü Inhibitory capacity of hMuStem cells is similar to those of BM-MSC
Inhibition of T-lymphocyte proliferation and activation
Modulation of CD4
+T-lymphocyte subsets
Modulation of T-modulating molecules under
pro-inflammatory conditions
0 20 40 60 80 100 120 % o f P ro lif erat io n **** **** CD4+ CD8+ 0 20 40 60 80 100 120 CD2 5 + c e lls (% of P B M C a lone ) ** ** *** CD4+ CD8+ 0 1 2 3 % o f cel ls CD4+ IL10+ * CD4+ IL10+ 0.0 0.5 1.0 1.5 2.0 % o f cel ls CD4+ FoxP3+ * ** CD4+ FoxP3+ 0 1 2 3 4 5 % o f cel ls CD4CD4+ IFNγ+ IFN+g+ 0.0 0.5 1.0 1.5 2.0 % o f cel ls CD4+ IL17+ CD4+ IL17+ CD80 CD86 N a tive co n d iti o n TN F a /IF N g st im u la te d CD4+ cells CD8+ cells 1 10 100 1000 10000 100000 PG E2 s e c re tio n (p g /1 0 6 c e lls ) * 0 2 4 6 8 10 NO c o n c e n tra tio n (µ M) CD4+ cells CD8+ cellsMLR assay using T-cells as responder and allogeneic irradiated PBMC as stimulator cells. Statistical analysis Kruskal-Wallis test. *p<0,05
Cytometry analysis of native or stimulated hMuStem cells (Isotype control in bright and membrane marker in deep colour)
ü hMuStem cells constitutively express class I MHC
ü hMuStem cells do not
express the co-stimulatory molecules CD80 and CD86 whatever the conditions
Analysis of CD4+ and CD8+ cell proliferation and activation by flow cytometry (CTV and CD25 labelling respectively). Statistical analysis Kruskal-Wallis test. **p<0,01, ****p<0,0001
Cytometry analysis of CD4+ T-lymphocytes subsets. Statistical analysis Mann-Whitney test. *p<0,05, **p<0,01
ü hMuStem cells induce formation of CD4+ IL10+ (Tr1-like) and CD4+
FoxP3+ (T regs-like) lymphocyte to a similar or a lesser extent that
BM-MSC
ü As BM-MSC, hMuStem cells have no impact on pro-inflammatory IFNg+ and IL17+ subsets
Quantification of PGE2 and NO in hMuStem cells supernatant (ELISA and NO dosage). Statistical analysis Mann-Whitney test. *p<0,05
BM -MS C hM uS tem iNOS
Cytometry analysis of lymphocyte proliferation (CTV labelling) with or without PGE2 (Indomethacin) and iNOS (L-NAME) inhibitors . Statistical analysis Mann-Whitney test. *p<0,05, ***p<0,001, ****p<0,0001
ü iNOS inhibition partially removes the inhibitory effect of hMuStem cells but not those of BM-MSC ü PGE2 inhibition partially removes the inhibitory
effect of hMuStem cells. Same results was obtained with BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC 1 10 100 1000 10000 100000 PG E2 s e c re tio n (p g /1 0 6 c e lls ) Native IFNγ-stimulated TNFα-stimulated 1 10 100 1000 10000 100000 PG E2 s e c re tio n (p g /1 0 6 c e lls ) Native IFNγ-stimulated TNFα-stimulated 1 10 100 1000 10000 100000 PG E2 s e c re tio n (p g /1 0 6 c e lls ) Native IFNγ-stimulated TNFα-stimulated
ü hMuStem cells natively secrete PGE2 and respond to TNFa stimulation
ü hMuStem cells constitutively express iNOS, unlike BM-MSC
ü hMuStem cells secrete NO independently of pro-inflammatory stimulation PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PBMC alone PBMC + hMuStem PBMC + BM-MSC PDL1 PDL2 ICAM1 N a tive co n d iti o n TN F a /IF N g st im u la te d
ü hMuStem cells express T-cell inhibitory molecules PDL1
and PDL2 after
pro-inflammatory stimulation
ü hMuStem cells express the adhesion molecule ICAM1
and respond to
pro-inflammatory stimulation 0 20 40 60 80 100 120 % o f P ro lif erat io n *** Indomethacin - + - + - +
Impact of hMuStem cells on T-lymphocyte features
MHC I T cells 16:16:1 4:4:1 1:1:1 1:1:2 0 10 20 100 200 % o f M L R-st imu lat ed T -cel l p ro fil erat io n
T cells : PBMC : MuStem cells * 0 20 40 60 80 100 120 % o f P ro lif erat io n * L-NMMA - + - + - + 0 20 40 60 80 100 120 % o f P ro lif erat io n * - + - + - + L-NMMA 0 20 40 60 80 100 120 % o f P ro lif erat io n **** - + - + - + Indomethacin
Demonstration of immunomodulatory properties for the human MuStem cell
population, a promising candidate for cell therapy of muscular dystrophies
Marine Charrier
1,2,3, Judith Lorant
1, Rafael Contreras Lopez
4,5, Christophe Blanquart
6, Blandine Lieubeau
7, Cindy Schleder
1, Isabelle
Leroux
1, Gautier Téjédor
4, Candice Babarit
1, Yann Pereon
8, Patricia Luz-Crawford
5, Guillaume Lamirault
2, Farida Djouad
4and Karl Rouger
1ESGCT Congress, 16-19 October 2018 – Lausanne, Switzerland
Introduction
Over the last eighteen years, the identification of stem cells in adult tissue opened new opportunities in cell-based therapy strategy. However, allogeneic cell transplantation protocols are highly limited by graft rejection. To overcome this issue, long-term immunosuppression (IS) are classically used, resulting in improved cell engraftment but also major adverse effects. Recently, many in vitro studies demonstrated pleiotropic immunomodulatory properties for adult stem cells, especially mesenchymal ones that have been shown to modulate the behavior of many immune cells through paracrine secretion or direct contact. These features could increase their ability to engraft in allogeneic recipient despite the lack of strong IS, thus improving their therapeutic efficiency. In addition, delivery of cells with immune privilege behavior may be beneficial in the context of degenerative disorders to limit chronic inflammation that characterizes tissues and interfere with the repair process.
In the lab, we isolated muscle-derived stem cells (termed MuStem cells) from healthy dogs and demonstrated that their systemic delivery in dystrophic dogs submitted to continuous IS lead to muscle regeneration and long-term clinical status stabilization. Interestingly, an IS restricted to the transplantation period was shown to be sufficient to sustain their transplantation benefits and to prevent host immunity response in allogeneic context, suggesting a possible immune privilege behaviour for the hMuStem cells. Recently, human MuStem cells were isolated and characterized as exhibiting in vitro/in vivo myogenic potential, positioning them as a promising candidate for muscle-dedicated regenerative medicine.
The aim of the present study is to explore the immunological-related features of hMuStem
cells and more specifically the interaction with T-cell features and the complement system
activation, two key effectors of allograft rejection.
Materials & Methods
Conclusions
1 PAnTher INRA UMR703, École nationale vétérinaire, agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), Nantes, F-44307, France ; 2 INSERM UMR1087/CNRS
UMR6291, Institut du Thorax, Nantes, F-44007, France ; 3 Université de Nantes, Nantes, France ; 4 INSERM UMR1183, IRMB, Hôpital Saint-Eloi, Montpellier, F-34295, France ; 5 Laboratoire d’Immunologie Cellulaire et
Moléculaire, Centre d’investigation biomédicale, Faculté de Médecine, Université des Andes, Santiago, Chile ; 6 INSERM UMR1232, CRCINA, Nantes, F-44007, France ; 7 IECM, INRA, USC 1383, Oniris, UBL, Nantes,
F-44307, France; 8 Centre de Référence Maladies Neuromusculaires AOC, Laboratoire d’Explorations Fonctionnelles, Centre Hospitalier Universitaire Hôtel Dieu, Nantes, F-44093, France
hMuStem cell isolation and culture: Human muscle-derived cells were isolated from skeletal muscles of 9 to 15-year-old patients free of known muscle
disease. To isolate hMuStem cells, MDC were submitted to a modified version of preplating protocol initially described by Rouger et al., 2011. hMuStem cells were then cultivated on coated-plastic flasks in proliferation medium containing 15% SVF, PSF and human recombinant growth factors. For pro-inflammatory stimulation, 70% confluent cells were cultured 24 to 48h in medium supplemented with 50 ng/mL of TNFa or/and IFNg. Four distinct batches of hMuStem cells were used.
Lymphocyte immunosuppression assay: Either hMuStem cells or bone marrow mesenchymal stem cells (BM-MSC) were cultured with Cell Trace
Violet (CTV)-labelled allogeneic peripheral blood mononuclear cells (PBMC) (1:10 ratio, stem cells:PBMC), during 2 to 3 days, under phytohaemagglutinin (PHA) stimulation. Analysis of T-lymphocyte proliferation, activation and regulatory profile were performed by flow cytometry. For inhibitory experiments, either Indomethacine or NG-monomethyl-L-arginine (L-NMMA) were added to the co-cultures at 0,1mM and 1mM, respectively. For Mixed Lymphoycte Reaction (MLR), irradiated (35Gy) hMuStem cells were added in graded ratio to human CD3+T-lymphocytes and allogeneic irradiated PBMC co-culture. After 5 days, T-cell proliferation was evaluated by 3H-thymidine uptake (0,925 µBq/mL).
In vitro complement-mediated hemolysis: Sheep red blood cells were incubated with 15% human serum as source of complement and (i) without
hMuStem cell supernatant (positive control), (ii) with native hMustem cell supernatant or (iii) with Factor H-depleted hMuStem cell supernatant. Lysis of red blood cells was measured by OD reading at 405nm.
Evaluation of hMuStem cells immune phenotype: hMuStem cell supernatants were collected after 48h of culture in native or stiumaled condition and
stored at -20 C. Prostaglandin E2 (PGE2) and Factor H secretions were measured by ELISA, NO concentration was measured using total nitrite detection kit (Cayman Chemical) according to manufacturing procedures. FACS analysis was made by counting at least 15.000 events after CD55, CD59, CD80, CD86, ICAM1, PDL1 and PDL2 immunolabeling.
Impact of hMuStem cells
on complement activation
Expression of complement-inhibitory molecules
Inhibition of complement-mediated
hemolysis and factor H implication
CD55 1 10 100 1000 Fa c tor H s e c re tion ( pg/1 0 6 c e lls ) Positive control 0 20 40 60 80 100 120 % o f Hemo lysi s Undepleted Factor H-depleted * + hMuStem cells conditionned medium * Positive control 0 20 40 60 80 100 120 % o f Hemo lysi s Undepleted Factor H-depleted * + hMuStem cells conditionned medium *
Cytometry analysis of native hMuStem cells (Isotype control in white and membrane marker in grey)
ü hMuStem cells express CD55 that is
known to prevent C3 convertase assembly and to accelerate formed convertase dissociation 1 10 100 1000 10000 100000 PG E2 s e c re tio n (p g /1 0 6 c e lls ) Native IFNγ-stimulated TNFα-stimulated
Quantification of Factor H secretion by ELISA. Hemolysis assay using undepleted or Factor H-depleted hMuStem cells supernatant. Statistical analysis Mann-Whitney test. *p<0,05
ü hMuStem cells secrete Facteur H that inhibits alternative pathway by preventing C3 convertase assembly
ü hMuStem cells partially inhibit complement mediated-lysis throught Factor H secretion whom depletion results in hemolysis restoration
CD59
Schematic representation of the complement cascade that lead to MAC assembly and target cell destruction
MuStem
cells
exhibit
interesting
immunomodulatory
properties
that
could favor their efficient engraftment in
dystrophic muscle tissue context.
Schematic summary
hMuStem cells are able to modulate lymphocyte feature - By inhibiting lymphocyte proliferation under MLR activation
- By specifically inhibiting both CD8+ and CD4+ lymphocyte proliferation
and activation in PBMC population - By promoting T-regulatory profile
- hMuStem cells act on T-lymphocytes through paracrine factors
PGE2 secretion
NO production (disctinctly from BM-MSC)
- hMuStem cells display a membrane profile that suggest a possible direct interaction with lymphocyte
Inhibitory molecules PDL1 and PDL2 Adhesion molecule ICAM1 expression
Lack of co-stimulatory markers CD80 and CD86 expression
hMuStem cells are able to inhibit complement pathway - In a paracrine manner, by factor H secretion
- hMuStem cells express the complement inhibitory molecule CD55 and CD59 that suggest a possible direct impact
CD59
Factor H CD55
Factor H
Model for inhibitory action of complement
cascade by hMuStem cells
Poster ID : P109
ü hMuStem cells express CD59 that bind to
C8 and/or C9, preventing membrane attack complexe (MAC) formation
This work was supported by a grant from the FEDER-FLS 2014-2020. It was realized in the context of the IHU-Cest project that received French Government financial support managed by National Reasearch Agency via the investment of the future program ANR-10-IBHU-005. The IHU-Cesti project was also support by Nantes Metropole and the Pays de la Loire Region.
