Expansion of natural CD8+Tregs for cell therapy

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HAL Id: inserm-02159875

https://www.hal.inserm.fr/inserm-02159875 Submitted on 19 Jun 2019

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Expansion of natural CD8+Tregs for cell therapy

Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, Stéphanie Kilens, Johanna Zoppi, Elodie Autrusseau, Audrey Donnart, Véronique Nerrière-Daguin, Frédérique Bellier-Waast, Eric Charpentier, et al.

To cite this version:

Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, Stéphanie Kilens, Johanna Zoppi, et al.. Expansion of natural CD8+Tregs for cell therapy. Labex IGO, Apr 2018, Nantes, France. �inserm-02159875�

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Tregs play a critical role in the control of immune responses and tolerance induction; however our understanding of CD8+ _{Tregs is limited while they are particularly promising for therapeutic}

application. We previously reported that CD8+CD45RClow Tregs use IFNγ and IL-34 to suppress donor responses in a rat model of allotransplantation (Guillonneau et al, JCI, 2007; Bezie et al, JCI, 2015). Here, we aim to characterize human CD8+ _{Tregs to assess their potential for cell therapy in transplantation.}

•All cells were isolated from blood of healthy volunteers.

•Expansion process: CD8+_CD45RClow _{Tregs and CD4}+_CD25high_CD127- _{Tregs were sorted by Facs Aria and plated with allogeneic APCs (Tregs:APC ratio 1:4) or in presence of anti-CD3 (OKT3 clone, coated, 1µg/ml) and anti-CD28 (CD28.2}

clone, soluble, 1µg/ml) mAbs in RPMI 1640 medium supplemented with AB serum 10%, IL-2 (1000U/ml) and IL-15 (10ng/ml). Cells were stimulated again with mAbs at day 7, and cytokines were freshly added at day 7, 10 and 12. IS were added at day 0 and day 7 when indicated.

•Proteomic analysis: T cells were stimulated 7h with PMA and ionomycin including 4h with BFA before cytokines staining and were analyzed by flow cytometry.

•Transcriptomic analysis: Tregs were sorted, expanded or not for 14 days, and analyzed by 3’DGE RNA-sequencing as compared to non-expanded freshly sorted Tregs.

•Suppression assay: CD4+_CD25- _{T cells were sorted by FACS Aria, labeled with CFSE and stimulated by allogeneic APCs, or cDCs, or pDCs, in presence or absence of CD8}+_CD45RClow _or high _{T cells, or CD4}+_CD25high_CD127- _{Tregs. 50µg/ml of}

blocking Abs or isotypic controls were added at day 0 when indicated. 0.4µm pore size Transwell membrane were used to separate Tregs from Teff. Proliferation was quantified by CFSE dilution in DAPI-_CD4+_{T cells after 5 days co-culture.}

•Cytotoxicity assay : Apoptosis was assessed by DAPI/Annexin V staining after 15h culture of Tregs with target cells.

•Skin transplantation model: NSG mice were grafted with 1cm2 _{human skin and injected 4 to 6 weeks later with 5x10}6 _{human PBMCs ± a range of expanded CD8}+_{Tregs. Allogeneic rejection was assessed by macroscopic observation.}

•GVHD model : NSG mice were 1,5Gy-irradiated and injected with 1.5x107 _{PBMCs ± a range of expanded CD8}+_Tregs.

Expansion of natural CD8

+

Tregs for cell therapy

C

ELL THERAPY USING EXPANDED CD8+TREGS INHIBITS TRANSPLANT REJECTION

B

ACKGROUND

M

ATERIALS AND METHODS

CD8+CD45RCLOW T CELLS ARE NATURAL TREGS ACTING THROUGH IFNγ

,

IL

-

34

,

TGFβ and IL-10 SECRETION

We report here existence of highly suppressive human CD8+_CD45RClow_{Tregs expressing Foxp3 and producing IFNg, IL-10, IL-34 and TGFb to mediate their suppressive activity. We have shown that}

CD8+_CD45RClow_{Tregs were as efficient as canonical CD4}+_CD25high_CD127low_{Tregs for suppression of allogeneic immune responses in vitro. Robustly expanded CD8}+_{Tregs displayed a specific gene}

signature and were able to delay human skin rejection and GVHD development in humanized mice. These results open new possibilities in cell therapy in transplantation and by extension in other diseases.

C

ONCLUSIONS + ± Allogeneic APCs T-cell depleted PBMCs CD8 +_CD45RClow or CD8+_CD45RChigh Suppressor cells CFSE-labeled CD4+_CD25-_{T cell} Effector cells 5days Donor 2 CD8+_CD45RClow T cells Donor 1 APC αCD3/28 mAbs IL2 1000U/ml IL15 10ng/ml 14 days 4:1 2nd _{stim D7}

Y

APC CD3 100 101 102 103 104 _ns /CD28 F o ld e x p a n s io n ( lo g ) 1:0 1:10 1:50 1:100 -60 -40 -20 0 20 40 60 80 100 necrosis, n=4 late apoptosis, n=4 early apoptosis, n=4

target : Treg ratio

% s p e c if ic l y s is o f a ll o g e n e ic A P C s R e la ti v e p ro p o rt io n o f d iv id in g C D 4 + C D 2 5 - T c e ll s APC CD3 0 20 40 60 80 100 ** /CD28 Graft survival D0 D-30 NSG mice 5x106 _PBMCs i.v. transfer PBMC isolation Human healthy volunteer 14 days Tregs expansion skin 0 25 50 75 100 0 1 2 3 PBMC n=12 PBMC+Tregs 1:1, n=9 PBMC+Tregs 1:2, n=5 PBMC+Tregs 1:4 n=5 * *

Days after PBMC injection

g ra ft r e je c ti o n s c o re

Days post PBMC injection

P e rc e n t s u rv iv a l 0 25 50 75 100 125 0 25 50 75 100 PBMC n=12 PBMC : Tregs 1:1 n=9 PBMC : Tregs 1:2 n=5 PBMC : Tregs 1:4 n=5 * * expanded Tregs fresh Tregs isotype f CTLA4 PD1

IL-10 IL-34 IFNγ

LAG3 CD39 ICOS

TGFβ CTLA4

TIM3 Foxp3

CD8+CD45RClowTregs were

expanded up to 2000 fold in 14 days in presence of allogeneic APCs or anti-CD3/28 mAbs, high dose of IL-2 and IL-15.

Expansion process

increased suppressive

activity (a) but not

cytotoxicity (b) of Tregs.

Expanded CD8+_{Tregs showed increased}

expression of Foxp3 and Tregs

associated molecules at proteomic (a) and transcriptomic (b) levels.

CD45RC marker is differentially represented in blood CD8+T cells

in healthy volunteers (a, overlay of 5 individuals). CD45RClow

cells include most of Foxp3+ cells (b). Low methylation of CpG

sites suggest a stable expression of Foxp3 (c).

Foxp3+_CD8+_CD45RClow _{T cells exclusively express IL-10, IL-34,}

GITR and TGFβ and low level of IFNγ (d).

CD45RC # ev en ts is o ty p e g IFN  R g IFN  iso ty p e b TGF  _Iso ty p e IL 3 4  0 20 40 60 80 100 *** n = 2 1 ** n = 2 2 n = 2 3 ** n = 1 0 n = 1 0 ** n = 6 n = 6 re la ti v e p ro p o rt io n o f d iv id in g C D 4 + C D 2 5 - T c e ll s

In contrast to CD8+CD45RChigh T cells, CD8+CD45RClow T cells

efficiently inhibited responder T cells proliferation in response

to allogeneic APCs (a) as efficiently as classical CD4+Tregs (b).

Blocking of IFNγ or its receptor, TGFβ or IL-34 partially restored effector T cells proliferation proving their involvement in Tregs function (a).

Transfer of expanded CD8+Tregs significantly inhibited rejection of human skin graft induced by

human allogeneic PBMCs injection in NSG mice, in a dose dependent manner, as monitored by graft rejection score (a) and survival (b).

Peripheral CD8+_Tcells (a) (b) (a) (b) (a) (b) (a) (b) (a) (b) ➢ 2. Function ➢ 4. Phenotype ➢2 . Function ➢ 5. SOT ➢ 3. Mechanisms ➢ 6. GVHD ➢ 1. Identification ➢ 1. Expansion process Low/- High

Representative dot plot

Foxp3

CD45RC

CD3+_CD8+ _cells _CD3+_CD4+ _cells

- + - +

Isotype control

Foxp3-_CD45RClow_CD8+_{T cells}

Foxp3+_CD45RClow_CD8+_{T cells}

IL-34 IFNγ GITR TGFb

IL-10 Representative histograms 1 :0 1 6 :1 8 :1 4 :1 2 :1 1 :1 1 :2 0 20 40 60 80 100 CD8+CD45RChigh, n=4 CD8+CD45RClow/-, n=4 effector:suppressor ratio n o rm a li z e d p ro li fe ra tio n o f C D 4 + C D 2 5 - T c e ll s **** effector:suppressor ratio n o rm a li z e d p ro li fe ra ti o n o f C D 4 + C D 2 5 - T c e ll s 1 :0 1 6 :1 8 :1 4 :1 2 :1 1 :1 0 25 50 75 100 CD4+Tregs, n=10 CD8+Tregs, n=10 p = 0 .0 8 4 4

Physical separation of Tregs from target cells by transwell membrane showed the importance of contact with APCs for Tregs to suppress (b), and particularly with pDCs (c).

Representative histograms CFSE Transwell Co-culture # ev ent s w/o CD8+ _Treg w/ CD8+ _Tregs (b) (c) 0 50 100

Methylation rate (centile)

(c)

(a)

effector: suppressor ratio

n o rm a li z e d p ro li fe ra ti o n o f C D 4 + C D 2 5 - T c e ll s 1 :0 8 :1 4 :1 2 :1 1 :1 0 20 40 60 80 100 cDC, n=3 APC, n=3 pDC, n=3

**

*

Weight loss D0 D-1 1,5 Gy irradiation NSG mice 1,5.107 _PBMCs i.v. transfer PBMC isolation Human healthy volunteer 14 days CD8+_CD45RC low/-Tregs expansion

Days after PBMC injection

% b o d y w e ig h t 0 25 50 75 100 80 85 90 95 100 105 110 PBMC, n=26 PBMC : CD8+Tregs 1:1, n=12 PBMC : CD8+Tregs 1:2, n=10 ** CD8+Tregs, n=4

Transfer of expanded CD8+Tregs significantly GVHD

development induced by human allogeneic PBMCs injection in NSG mice, as monitored by body weight loss (c).

➢ 3. IS supplementation expansion score s u p p re s s io n s c o re 0 5 10 15 20 0.0 0.5 1.0 1.5 2.0 2.5 Rapa-CsA Rapa-NT Rapa-MPr Rapa-Tacro Rapa-Rapa CsA-Rapa CsA-MPr MPA-MPA Rapa-MPA CsA-CsA CsA-Tacro no IS

Rapamycine addition in culture medium for at least the first 7 days improved Tregs yield and function.

IS added from day 0 to 7 - day 7 to 14

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Séverine Bézie

1,2,3

_{, Dimitri Meistermann}

1,2,4

_{, Laetitia Boucault}

1,2,3

_{, Stéphanie Kilens}

1,2

_{, Johanna Zoppi}

1,2

_{, Elodie}

Autrusseau

1,2,3

_{, Audrey Donnart}

5

_{, Véronique Nerrière-Daguin}

1,2,3

_{, Frédérique Bellier-Waast}

6

_{, Eric Charpentier}

5

_,

Franck Duteille

6

, Laurent David

1,2,7

, Ignacio Anegon

1,2,3

and Carole Guillonneau

1,2,3

1_{Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes, Nantes, France}

2_{Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France}

3_{LabEx IGO “Immunotherapy, Graft,Oncology”, Nantes, France}

4 _{Laboratoire des Sciences du Numérique de Nantes (LS2N) UMR6004, Université de Nantes, Nantes, France}

5 _{INSERM UMR1087, CNRS UMR6291, Université de Nantes, l’institut du thorax, Nantes, France}

6_{Chirurgie Plastique Reconstructrice et Esthétique, CHU Nantes, Nantes, France}

7_{INSERM UMS 016, SFR Francois Bonamy, iPSC core facility, CNRS UMS 3556, Université de Nantes, CHU de Nantes, Nantes, France}