Terapia cellulare con

Linfociti T Regolatori

Marco Romano, PhD student

Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale (DIMES)

Università di Bologna

Ospedale Sant’Orsola – Malpighi

Via Massarenti, 9

40138 Bologna

GRAFT REJECTION

(A) Alloantigen presentation via the direct, semi-direct and indirect pathways following organ transplantation,

and (B) the relative intensity of each antigen-presentation pathway during the post-transplantation (post-Tx)

period. Sagoo et al 2010.

Chronic GRAFT VERSUS HOST DISEASE

phatogenesis

Bruce R. Blazar et al Nature Reviews Immunology 12, 443-458 June 2012

Common Immunosuppressive Drugs

Treg Discovery

FOXP3+ regulatory T cells in the human immune system S. Sakaguchi Nature Reviews Immunology 10, 490-

500 (July 2010)

“How regulatory T cells work”. Vignali D., Lauren W. Collison and Creg J. Workman – Nat Rev Immunol 2008

How Regulatory T cells work

Donor Tregs

In vivo expansion

Ex-vivo

expansion

Infusion

Infusion

Clinical strategy

Infusion of freshly

isolated cells

Background

• First-in-man clinical results of the treatment of patients with graft

versus host disease with human ex vivo expanded

CD4+CD25+CD127- T regulatory cells. Trzonkowski P. et al Clin Immunol. 2009 Oct;133(1):22-6.

• Infusion of ex vivo expanded T regulatory cells in adults transplanted

with umbilical cord blood: safety profile and detection kinetics. Brunstein CG et al. Blood. 2011 Jan 20;117(3):1061-70.

• Donor Regulatory T Cells Infusion in Patients With Chronic Graft-

versus-host Disease

ClinicalTrials.gov Identifier: NCT01903473.

Treg will be administered fresh. A dose of 0.5 x106 Treg/kg should be ideally administered.

PI: Baron F. University Hospital of Liege

Background

• Trial of Regulatory T-cells Plus Low-Dose Interleukin-2 for

Steroid-Refractory Chronic Graft-versus-Host-Disease. ClinicalTrials.gov Identifier: NCT01937468 . PI: John Koreth, MD, Dana-Farber

Cancer Institute

• Therapy of type 1 diabetes with CD4+CD25highCD127- regulatory T cells prolongs survival of pancreatic islets — Results of one year follow-up. N. Marek-Trzonkowska. Clinical Immunology Volume 153, Issue 1, July 2014, Pages 23–30

• ONE STUDY ongoing…

• Thril (ClinicalTrials.gov Identifier: NCT02166177) ongoing...

HD

Lymphocyte

%

CD4+CD25+

%

CD4+CD25high

%

CD4+CD25high FoxP3+

%

1 38,6 3,1 0,7 0,6

2 29 4,7 0,9 0,5

3 22,5 4,7 0,6 0,3

4 29 4,8 0,9 0,3

5 30 3 0,8 0,5

6 32 5,7 0,9 0,3

7 22,4 3 0,7 0,6

8 27,6 3,3 0,6 0,4

mean 28,89 4,04 0,76 0,44

St. Dev 5,21 1,06 0,13 0,13

Peripheral Blood analysis: Healthy Donors

Percentages are expressed as proportion of white blood cells

Patients

Lymphocyte

%

CD4+CD25+

%

CD4+CD25high

%

CD4+CD25high FoxP3+

%

1 39 9,7 1,5 0,9

2 14 2 0,4 0,2

3 24,5 3,6 0,7 0,4

4 28,6 7 1 0,7

5 23 7,5 0,9 0,6

6 15 2,6 0,3 0,3

7 20 3,7 0,6 0,5

8 18 5,6 0,7 0,4

9 30 6,8 0,8 0,4

10 20 5,9 1 0,9

11 15,5 7 0,6 0,5

12 4,9 4 0,8 0,8

mean 21,04 5,45 0,78 0,55

St. Dev 8,86 2,29 0,31 0,23

Peripheral Blood analysis: patients in waiting list for liver transplantation

Percentages are expressed as proportion of white blood cells

Circulating Tregs: HD Vs Patients

HD

Liver

Pts

0

2000

4000

6000

8000

10000

WB

C/u

L

HD

Liver

Pts

0

10

20

30

40

50

Tre

gs/u

L

Phenotypic evaluation

Cryopreservation

Treg function

Tregs purification

Phenotyphic characterization

Gating strategy used to characterize Treg cells pre and post thawing

Cryopreservation’ strategy

Medium: 50% Plasma AB;

40% CliniMacs Buffer

10% DMSO

Cells were cryopreserved using Planer Kryo 560-16

Yeld

Post-thawing0

20

40

60

80

100Patients

Healthy Donors

%

Yield

A

C

B

A: only stimulus

B: Treg/Tresp 1/4

C: Treg/Tresp 1/10

Suppression assay Method

ModFit LTTM 3.1Verify software house

Co-colture (4 days) of

autologous Tregs with CD4+

CD25- (Tresp) stimulated with

anti-CD3 and anti CD28

(5ug/ml) and labelled with

CFSE.

stim

ulus

 

Treg/

Tresp

1/2

5

Treg/

Tresp

1/1

0

Treg/

Tresp

1/4

0.0

0.2

0.4

0.6

0.8

1.0

*

***

Pro

life

rati

on

(Fo

ld-c

ha

ng

e)

FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp

cultured in the presence of increasing ratios of Tregs and the” upper generation proliferation index” of

CTR colture.

Function of freshly isolated and thawed

Tregs from healthly donors

Stim

ulus

Treg/

Tresp

1/2

5

Treg/

Tresp

1/1

0

Treg/

Tresp

1/4

0.0

0.2

0.4

0.6

0.8

1.0***

Pro

life

rati

on

(Fo

ld c

ha

ng

e)

Fresh Thawed

CD4+CD25+FOXP3+

86,14 %

CD4+CD25+ FOXP3+

89,60

CD4+CD25+127 low

92,43 %

CD4+CD25+127 low

94,43 %

Thawed

STIM

ULU

S

1/10

Tre

g/Tre

sp

1/4

Treg/

Tresp

1/2

Treg/

Tresp

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Pro

life

rati

on

(Fo

ld-c

ha

ng

e)

STIMULU

S

Treg/

Tresp

1/2

5

Treg/

Tresp

1/1

0

Treg/

Tresp

1/4

0.0

0.2

0.4

0.6

0.8

1.0

**

**

Fresh

Pro

life

rati

on

(Fo

ld-c

ha

ng

e)

Function of freshly isolated and thawed Tregs

from patients in waiting list for liver

transplantation

CD4+CD25+ FOXP3+

91,93 %

CD4+CD25+FOXP3+

85,93 %

CD4+CD25+127 low

95,43 %

CD4+CD25+127 low

92 %

FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp cultured in

the presence of increasing ratios of Tregs and the” upper generation proliferation index” of CTR colture.

Tregs expansion

Treg Expansion is based on Beads pre-loaded with CD3 and CD28 antibodies

(Miltenyi, Germany) at a bead-to-cell ratio of 4:1. Best expansion is achieved using

medium supplemented with recombinant Interleukin-2 and Rapamicyn

(Miltenyi, Germany)

CD3

CD28

Treg cell MACSiBead

+ rhIL-2 (1000 U/mL)

Rapamicyn (100nM)

DAY 0 7 14 21

IL-2

2

1 x 106 Tregs

Second stimulation Third stimulation

Infusion/

criopreservation

CD8+ depleted

CD25+ enriched

Manufacture

Protocol

One Study

CD8+ depleted

CD25+ enriched

5 Feedings

Medium 50%

increase

Rapa

IL-2 feed 1-4

Cryopreservation

Stimul 1 Stimul 2 Stimul 3 Final Harvest

Phenotype >60%

Bead removal <100b/3x106

cells

Viability >70%

Potency >60%

Sterility

Endotoxin

Mycoplasma

TexMACS +5%hAB

100nM Rapamycin

1000U/ml IL-2

ExpAct Treg beads CD3/28

5 Feedings

Medium 50%

increase

Rapa

IL-2 feed 3-4

5 Feedings

Medium 50%

increase

Rapa

IL-2 feed 1-4

Day -49 Day -13 Day 0 Day +5

Blood procurement

Manufacture Day 0

Final harvest

Tx

DP infusion

Storage of frozen DPDP manufactureTransport Max 24h

Manufacture Day -1

Day 0 Day +37 Day +92Month 3

Starting material

procurement

Manufacture Day 0

Final harvest

Tx

DP infusion

Storage of frozen DPDP manufactureTransport Max 24h

Kidney

Liver

Manufacture Timelines

Why RAPAMYCIN

Immunoregulatory functions of mTOR inhibition . Nat Rev Immunol 2009; Angus W. et al.

DAY 0

DAY 7

DAY 1

4

DAY 2

1

1.0×10 05

1.0×10 06

1.0×10 07

1.0×10 08

1.0×10 09

1.0×10 10

Cell

nu

mb

er

DAY 0

DAY 7

DAY 1

4

DAY 2

1

0.1

1

10

100

1000Donor A

Donor B

Donor C

FO

LD

IN

CR

EA

SE

Tregs Expansion from

Healthy Donors

Phenotypic evaluation of rapamycin

expanded Tregs from HD

DAY 0

DAY 7

DAY 1

4

DAY 2

1

50

60

70

80

90

100

110

CD4+CD25+

CD4+ CD25+ FOXP3+

CD4+CD25+CD127-

%

DAY 0

DAY 7

DAY 1

4

DAY 2

1

0.0

0.5

1.0

1.5

2.0

2.5

5

10

15CD8+ cells

Th17 cells

CD19+ cells

CD14+ cells

CD3+ CD56+ cells%

Function of rapamycin expanded Tregs from

healthy donors

FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp

cultured in the presence of increasing ratios of Tregs and the” upper generation proliferation index” of

CTR colture.

Stim

ulus

1/10

Treg/T

resp

1/4

Treg/T

resp

1/2

Treg/T

resp

0.0

0.2

0.4

0.6

0.8

1.0

**

*

Pro

life

rati

on

(Fo

ld-c

ha

ng

e)

Next step…

Treg stability in the presence of 2 pro-inflammatory milieu:

A: IL-2, IL-1β, IL-6, TGF-β. B: IL-2, IL-21, IL-23, TGF-β.

IL-17 and IFN-γ

production

CTLA-4, GITR and

CD39

TSDR analysis

Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of

human CD4+CD25+FOXP3+ T regulatory cell subpopulations. Scottà et al. Haematologica. 2013

Aug;98(8):1291-9.

Evaluation of

Impact of immunosuppressive regimen on ex

vivo expanded human regulatory T cells

Cristiano Scottà, Giorgia Fanelli, Julie Hoong, Marco Romano, Mitalee Sukthankar, Giuliana

Guggino, Henrieta Fazekasova, Ratnasothy Kulachelvy, Pablo Becker, Ben Afzali, Robert Lechler

and Giovanna Lombardi

Question:

Can immunosuppressive drugs affect Treg viability,function and trafficking?

Tacrolimus Tacrolimus is used after allogeneic organ transplant to reduce the activity of the patient's immune system and lower the risk of organ rejection. Tacrolimus inhibits calcineurin and prevents the dephosphorylation of NF-AT. Thus it negatively affects both T-lymphocyte signal transduction and IL-2 transcription.

Prednisolone Prednisolone irreversibly binds with glucocorticoid receptors. Its interaction with DNA modifies gene transcription. It induces synthesis of some proteins, and inhibit synthesis of others. Regulation of gene suppression leads to systemic suppression of inflammation and immune response.

Mycophenolate Mofetil (MMF) - MPA

MMF reversibly inhibits inosine monophosphate dehydrogenase (IMPDH) in purine biosynthesis which is necessary for the growth of T cells and B cells. It is extensively used in transplant medicine to suppress T and B cells from attacking donor’s graft.

Immunesuppressive Drugs

Treg Viability in the Presence of Immunosuppressants

Reagent Amount

IL-2 20 IU/ml

anti-CD3/CD28 beads 1:2 (Stimulation)

Rapamycin 100 nM

Methyl-Prednisolone

(mPred)

0-4 ug/ml

Mycophenolate (MPA) 0-2.5 ug/ml

Tacrolimus (Tacro) 0-20 ng/ml

0

1:25

6

1:12

81:

641:

321:

16 1:8

1:4

1:2

Max

0

20

40

60

80

100

Dilution from highest conc

Via

ble

cell

(%

)

In vivo drug distribution

Tacrolimus * : Erythrocyte fraction 70-80%

Plasma fraction 20%

Leukocytes 1%

Mycophenolate * * : Albumin bound ~ 97%

*Zahir et al. The Drug Monit 2004, * * Bullingham et al. Clinical Pharm 1998

Tacrolimus

00.

080.

160.

310.

621.

25 2.5 5 10 20

0

20

40

60

80

100EC50: 2ng/ml

ng/ml

Via

ble

cell

(%

)

MPA

00.

080.

160.

310.

621.

25 2.5

0

20

40

60

80

100EC50: 1ug/ml

ug/ml

Via

ble

cell

(%

)

methyl-Prednisolone

0

0.01

50.

030.

060.

120.

25 0.5 1 2 4

0

20

40

60

80

100EC50: 0.5ug/ml

ug/ml

Via

ble

cell

(%

)

Dose Response Curves to Derive the Concentration of

Immunosuppressant

Immunosuppressants Can Reduce the Proliferative

Ability of Treg Preparations after 5 Days of Culture

CTR

TAC

MPA

mPr

IS M

ix

1

10

100

1000

10000

TAC

CTR

MPA

mPr

IS Mix

**

**

*N

um

ber

of

cell

s (

x10

3)

0

20

40

60

80

100

Pro

life

rati

on

(%

)CTR

TAC

MPA

mPr

IS M

ix

0

20

40

60

80

1001:10

1:3

1:1

Pro

life

rati

on

(%

)Tregs Maintain Full Suppressive Capacity

after 5 days of Culture with Drugs

FOXP3

CD

25

1

0

1000

2000

3000

4000

CTR

MPA

mPr

TAC

IS Mix

CD25 ExpressionM

FI

1

0

500

1000

1500

FOXP3 Expression

MFIIS Mix

(5d)

IS Mix

(5d)

97.3%

98.8%

88.4%

87.4%

HD1

HD2

Immunosuppressants Do Not Affect FOXP3

Expression after 5 Days of Culture

CD39

GIT

R

HLA

-DR

CD95

PD-L

1

CTLA

4

ICOS

TGFbet

a-LA

P

0

20

40

60

80

100

CTR

TAC

MPA

mPr

IS Mix

Nu

mb

er

of

cell

s (

%)

Functional Treg Molecules are not Affected

by Immunosuppressants (5 days)

Acknowledgement

Laboratorio Terapia cellulare, istituto

Seragnoli:

•Francesca Ulbar

•L. Catani

•S. Rizzi,

•M. Motta,

•E. Dan

•D. Sollazzo,

•M. Arpinati

•Prof. RM Lemoli

Immuneregulation Lab

Cristiano Scottà

Prof. Giovanna Lombardi

Prof. Robert Lechler

Giorgia Fanelli

Julie Hoong

Estefania Nova Lamperti

Ratnasothy Kulachelvy

Giuliana Guggino

Ben Afzali

Reuben McGregor

Pablo Becker