Expanded human CD8+CD45RClowFoxp3+ Tregs secreting IFNγ, IL-10, IL-34 and TGFβinhibit human transplant rejection

(1)

HAL Id: inserm-02159985

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Expanded human CD8+CD45RClowFoxp3+ Tregs secreting IFNγ, IL-10, IL-34 and TGFβinhibit human

transplant rejection

Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, Stephanie Kilens, Johanna Zoppi, Elodie Autrusseau, Audrey Donnart, Véronique Daguin,

Frédérique Bellier-Waast, Eric Charpentier, et al.

To cite this version:

Séverine Bézie, Dimitri Meistermann, Laetitia Boucault, Stephanie Kilens, Johanna Zoppi, et al.. Expanded human CD8+CD45RClowFoxp3+ Tregs secreting IFNγ, IL-10, IL-34 and TGFβinhibit human transplant rejection. FOCIS, Jun 2017, Chicago, United States. �inserm-02159985�

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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

+

_CD45RC

low

_{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 in comparison to CD4}

+

_CD25

high

_CD127

-

_{Tregs .}

•All cells were isolated from blood of healthy volunteers.

•Culture: 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.

•Proteomic analysis: T cells were stimulated 7h with PMA and ionomycin including 4h with BFA before cytokines staining and were analyzed by flow cytometry. Apoptosis was assessed by DAPI/Annexin V staining after 15h culture. •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 in presence or absence of CD8}+_CD45RClow_or high _{T cells, or CD4}+_CD25high_CD127- _{Tregs. Secretion assay kits from}

Miltenyi were used to sort CD8+_CD45RClow _{Tregs on IFNg and IL-10 expression. 50µg/ml of blocking Abs or isotypic controls were added at day 0 when indicated. Proliferation was quantified by CFSE dilution in DAPI}-_CD4+_{T cells after 5}

days co-culture.

•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.}

Expanded human CD8

+

_CD45RC

low

_Foxp3

+

_Tregs

secreting IFNγ, IL-10, IL-34 and TGFβ inhibit

human transplant rejection

Séverine Bézie

1,2

_{, Dimitri Meistermann}

1,2

_{, Laetitia Boucault}

1,2

_{, Stéphanie Kilens}

1,2

_{, Johanna Zoppi}

1,2

_{, Elodie Autrusseau}

1,2

_{, Audrey}

Donnart

4

_{, Véronique Daguin}

1,2

_{, Frédérique Bellier-Waast}

3

_{, Eric Charpentier}

4

_{, Franck Duteille}

3

_{, Laurent David}

1,2

_{, Ignacio Anegon}

1,2

and Carole Guillonneau

1,2

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

4 _{INSERM UMR1087, CNRS UMR6291, Université de Nantes, l'institut du thorax, Nantes, F-44000 France;}

C

ELL THERAPY USING EXPANDED CD8

+

TREGS INHIBITS TRANSPLANT REJECTION

B

ACKGROUND

M

ATERIALS AND METHODS

CD8

+

CD45RC

LOW

T CELLS ARE NATURAL TREGS ACTING THROUGH IFNγ

,

IL

-

34 ,

TGFβ and IL-10 SECRETION

We report here existence of highly suppressive human CD8

+

_CD45RC

low

_{Tregs expressing Foxp3 and producing IFN}

_g

_{, IL-10, IL-34 and TGF}

_b

_{to mediate their suppressive activity. We show that}

CD8

+

_CD45RC

low

_{Tregs are superior to canonical CD4}

+

_CD25

high

_CD127

low

_{Tregs for suppression of allogeneic immune responses in vitro and in vivo to delay human skin rejection in humanized mice.}

Robustly expanded CD8

+

_{Tregs displayed a specific gene signature. These results open new possibilities in cell therapy in transplantation and by extension in other diseases.}

C

ONCLUSIONS

1:0 16 :1 _8:1 _4:1 _2:1 _1:1 _1:2 _1:4 0 20 40 60 80 100

unstim. CD8+CD45RClow Tregs, n=3 stim. CD8+CD45RClow Tregs, n=3

*** effector:suppressor ratio rel a ti ve p ro p o rt io n o f di vi d in g C D 4 + CD2 5 - T c e ll s CD45RClow _CD8+_{T cells} CD45RChigh _CD8+_Tcells No Treg + ± Allogeneic APCs

T-cell depleted PBMCs CD8+CD45RC_or low CD8+_CD45RChigh Suppressor cells CFSE-labeled CD4+_CD25-_{T cell} Effector cells 5days CFSE prolif #event s % F o x p 3 + c e lls i n CD 45 R C hi /lo C D 8 + T c e ll s low high 0 5 10 15 20

****

Representative dot plot

Foxp3 CD4 5RC IL-34 IFNγ TGFb IL-10 Isotype control

Foxp3-_CD45RClow_CD8+_{T cells}

Foxp3+_CD45RClow_CD8+_{T cells}

Representative histograms 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 aCD3 100 101 102 103 104 ns /CD28 Fol d e x pa n si o n (l og ) 1:0 1:10 1:50 1:100 -60 -40 -200 20 40 60 80 100 necrosis, n=4 late apoptosis, n=4 early apoptosis, n=4

target : Treg ratio

% s p e ci fi c ly s is of a lloge ne ic A P C s Re la ti ve p ro p o rt io n o f di vi di ng C D 4 + CD2 5 - T c e ll s APC aCD3 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

gr a ft r e je c ti o n s c o re

Days post PBMC injection

Pe rc e n t s u rv iv al 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

+

_CD45RC

low

_{Tregs 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 were significantly more efficient than expanded CD4}

+

_{Tregs to inhibit}

rejection of human skin graft induced by human allogeneic PBMCs injection in NSG mice, as

monitored by graft rejection score (a) and survival (b).

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).

CD45RC

low

_{cells include most of Foxp3}

+

_{cells (b).}

CD45RC #event s is o ty p e g IFN_a Rg IFN_a is o ty p e b TGF a _Iso ty p e IL3 4 a 0 20 40 60 80 100

***

n= 21

**

n= 22 n= 23

**

n= 10 n= 10

**

n= 6 n= 6 re la ti v e p ro p o rt io n o f di vi d in g C D 4 + CD2 5 - T c e ll s

In contrast to CD8

+

_CD45RC

high

_{T cells, CD8}

+

_CD45RC

low

_{T cells}

efficiently inhibited responder T cells proliferation in response

to allogeneic APCs (a) in a dose dependant manner (b).

Foxp3

+

_CD8

+

_CD45RC

low

_{T cells exclusively express IL-10, IL-34,}

and TGFβ and low level of IFNγ.

Blocking of IFNγ or its receptor, TGFβ or IL-34 partially restored

effector T cells proliferation proving their involvement in Tregs

function.

effector : suppressor ratio

re la ti ve p ro p o rt io n o f di vi di ng C D 4 + CD2 5 - T c e ll s 1 :0 1 6 :1 8 :1 4 :1 2 :1 1 :1 0 20 40 60 80 100

IFNg+IL10+CD8+Tregs n=10

***

CD4+CD25+CD127lowTregs n=6

CD8

+

_CD45RC

low

_Tregs

secreting IL-10 and IFNγ

were

significantly

more

efficient to inhibit T cell

allo-response

than

CD4

+

_Tregs.

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)

(a)

(b)

Ø

2. Function

Ø

3. Phenotype

Ø

2. Phenotype

Ø

3 . Function

Ø

4. SOT

Ø

4. Mechanisms

Ø

5. Relevance

Ø

5. Relevance

Ø

1. Definition

Ø

1. Expansion protocol

Graft survival D0 D-30 NSG mice 5.106 _PBMCs i.v. transfer PBMC isolation Human healthy volunteer 14 days CD8+ _or CD4+_{Tregs expansion} skin 0 25 50 75 100 0 1 2 3 PBMC n=12

Days after PBMC injection

gr a ft r e je c ti on s c or e PBMC : CD8+Tregs 1:4 n=5 *** PBMC : CD4+Tregs 1:4 n=3 *

Days post PBMC injection

Pe rc e n t s u rv iv a l 0 25 50 75 100 0 25 50 75 100 PBMC n=12 PBMC : CD8+ Tregs 1:4 n=5 * PBMC : CD4+Tregs 1:4 n=3