utilizzo dei linfociti t regolatori nel trapianto da

UTILIZZO DEI LINFOCITI T REGOLATORI NEL TRAPIANTO DA DONATORE APLOIDENTICO

Mauro Di Ianni

SIES, Discutiamone Insieme, Firenze 20 Novembre 2014

CD8+ effector

Types of regulatory T cells

CD4+CD25+CD45RA+

CD4+CD25+CD45RO+

CD4+CD25+ regulatory T cells preserve graft versus-tumor-activity while inhibiting graft-versus host disease

after bone marrow transplantation

Edinger et al., Nat Med 2003, 9:1144-1150

Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative

control of immune responses

Sakaguchi et al., Ann Rev Immunol, 2004, 22:531-562

The impact of regulatory T cells on T-cell immunity following hematopoietic cell

transplantation

Nguyen et al., Blood 2008, 111:945-953

Donor type CD4+CD25+ regulatory T cells suppress lethal acute graft-versu-host disease

after allogeneic bone marrow transplantation

Hoffmann et al., J Exp Med, 2002, 196:389-399

Lessons from animal models

Adoptive transfer of naturally arising CD4+CD25+

regulatory T cells (Tregs), when coinfused with

conventional T lymphocytes (Tcons), prevents GvHD,

while favoring immune reconstitution

Nguyen et al. Blood 2007, 109:2646-2656

In vivo dynamics of regulatory T-cell trafficking and survival predict effective

strategies to control graft-versus-host disease following allogeneic transplantation

Rebuilding post-transplant immunity

Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered

with regulatory T cells for GvHD prevention

Gaidot et al. Blood 20010, 117:2975-2983

FOXP3

GITR

CTLA-4

CD 62L

CD 39

DONOR

mismatched HSC transplant

RECIPIENT

expansion of

alloantigen

specific Tregs

Treg lymph node APCs

IL 2

activation of

specific donor

Tregs for recipient

alloantigens

migration of

alloantigen

specific Tregs

Tregs

Skin

Gut

Lung

Bone

marrow

CD45RO+ 100%

Nguyen et al. Blood 109, 2646, 2007

Alloantigen

specific Treg

Alloantigen

specific Tcon

Recipient

dendritic cell

Alloantigen-specific Tregs

act in an antigen-specific

manner in vivo during their

effector phase, thus …..

, …

… they control activation and

early proliferation of alloreactive

donor T cells via intereaction

with APCs in priming sites.

Conversely they do not cross

inhibit pathogen-specific Tcon

expansion and response.

First “check point” in lymph nodes

Second “check point”

in peripheral tissues

(skin, gut, liver,lung)

Alloantigen specific Tregs control Tcon alloreactivity

T reg cell Isolation • Magnetic bead separation: Significant enrichment of the CD4+CD25+

FoxP3+Treg cells

• Cell Sorting techniques: Sort CD25 high positive population; significant loss

of FoxP3+ cells.

Sort CD127-/CD4+CD25+ cells; isolating the majority of FoxP3+ cells within an apheresis sample

• Major limitation:

Paucity of T reg cells in the peripheral blood

Expansion of T reg cells may be necessary

1st step:

Depletion of

CD8+/CD19+cells

2ndstep:

Enrichment of

CD25+ cells

Selection of

CD4+CD25+Cells

Selection and Characterization of CD4+CD25+ Regulatory T Cells

Cells (x109) 1060 (540-1370) 280 (202- 390)

%CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1)

N° cells (x 106) 330 (221-1020) 256 (185.6-365.4)

%CD4CD25high 0.3 (0.12- 0.89) 33.6 (14.4-39.6)

N° cells (x 106) 36.12 (19.98 - 84) 68.6 (20.9-143)

Starting fraction Final fraction

CD

25

CD127

CD4

FoxP3

Gate on CD4CD25+high

Gate on CD4CD25+

Leukapheresis product

Fox P3+ cells

92.05 ± 15 %

Di Ianni et al., Exp. Hematology 2008

Di Ianni et al., Clinical and Exp Immunology 2009

Di Ianni et al., Blood 2011

Di Ianni et al., Best Prac Res Clin Haematol 2011

Di Ianni et al., Transfusion Apheresis Science 2012

CD45RO

CD45RA

88.0% 85.9%

9.6% 8.2% 61.3%

31.8%

0.0% 0.3%

Final Cell Fraction

Murine T lymphocytes lacking CCR7 can generate attenuated GvHD besides robust

GvL responses

CCR7 negative Tregs strongly protect against lethal GvHD

Final Cell Fraction

CD4/CD25 CD4/CD25 CD4/CD25bright CD4/CD25bright

84.2% 93.2%

CD62L

SS

Lin

CD39

SS

Lin

64.9% 95.7%

SS

Lin

CD49d

37.7% 27%

GITR

SS

Lin

33.4% 58.1%

cyCD152

SS

Lin

15.1% 33.8%

Gate on CD4+CD25+ Gate on CD4+CD25+

CD127

Fo

xP

3

96% 3%

Fo

xP

3CD45RA

95% 4%

T regulatory cells characteristics

0

20

40

60

80

100

T resp + Tregs 1:2 T resp + Tregs 1:1 T resp + Tregs 1:0,1

% o

f In

hib

itio

n

Mean SD

T resp + Tregs 1:2 72,4 8,9

T resp + Tregs 1:1 60,8 20,5

T resp + Tregs 1:0,1 25,6 19,1

Tregs inhibit MLR in a dose dependent way

0

10

20

30

40

50

60

70

80

90

T resp allo + T regs T resp auto + T regs

% In

hib

itio

n

HLA identity between Tregs and Tcons is required

for maximun suppression of alloresponses

Donor Tregs do not prevent graft rejection in mismatched transplant

Tregs from a third party donor might not prevent GvHD after HSCT

Alloantigen APCs pre-incubation enhances Treg-mediated immunosuppression

Freshly selected polyclonal

T-reg cells were

co-cultured in the presence

of allogeneic APCs for 48 hours.

Cells were plated under

limiting dilution conditions and

proliferating T cell clones counted.

CD127 expression inversely correlates with suppressive capacity

Inhibition of xenogenic acute GvHD in vivo (NOD/scid IL-2 Rγnull mice)

% f

ree o

f G

vH

D

▀ CD25- (Group I)

▲Tregs (Group II)

▀ CD25- & Tregs (Group III) 0

20

40

60

80

100

1 3 5 7 9 11 13 15 17 19 21

%CD45

Bone marrow Spleen Liver Skin

Days +4 +12 +4 +12 +4 +12 +4 +12

Group I

(Tcon)

5.5 77.4 70.3 91.6 8.1 46 0 7.5

Group II

(Tregs)

3 13.8 80 20.8 1.7 2.8 0 0

Group III

(Tcon+Tregs)

3.1 46.5 47 71.2 3.7 3.75 0 0

Stable composition according to:

CD25 expression -35-40% high

-55-60% int

-5-10% low

FoxP3 expression

-90%

CD127 expression

<20%

Function:

-in vitro inhibition of MLR

-inhibition is enhanced by 48 hrs APC exposure

-in vivo prevention of GvHD

-MHC identity is required for suppression

Next step: clinical application

Characteristics of immunoselected Tregs

Sept 2008-Oct 2009

CD34+ No

po

st-tra

nsp

lan

t

imm

un

osu

pp

ressio

n

T regs

Tcons

days

Treg and Tcon adoptive immunotherapy in haplo HSCT

for patients with high risk AL

2x106/kg

10x106/kg

1x106/kg

Thymoglobuline

3-7.5 mg/kg;

day-21 before Tregs

8 Gy TBI

Thiotepa

Fludarabine

Jan 2012

May 2010-Jan 2011

Alemtuzumab 20mg/sqm;

day-21 before Tregs

8 Gy TBI

Thiotepa

Fludarabine

8 Gy TBI

Thiotepa 4 mg/kg/day

Cyclophosphamide 35 mg/kg/day

Fludarabine 40 mg/sqm/day

1st clinical trial*

2nd clinical trial**

In vivo activation

and expansion

of alloantigen

specific Tregs

* Di Ianni et al., Blood 2011; **Martelli, Di Ianni et al., Blood 2014

Demographics

No of patients 45

Median age (years, range) 40 (20-59)

Gender (male/female) 21/24

Disease and status at transplant

Acute myeloid leukemia 33

CR1 18

≥CR2 15

Acute lymphoid leukemia

12

CR1 8

≥CR2

4

# 25 in the 1st trial (Sept 2008-Oct 2009)

# 20 in the 2nd trial (May 2010

>500 >10000

10

20

30

ANC/l

Days p

ost

BM

T

>25000 >500000

20

40

60

PLT/mm3

Day

s p

ost

BM

T

PRIMARY ENGRAFTMENT

43/45 (95%)

Acute Graft vs Host Disease

Grade ≥II 6/43 (13%)

0 60 120 180 240

0

450

900

Recovery of CD4+ T cells

days post transplant

cell

s/μ

l

Treg Haplo

Standard Haplo

Pattern of immunoreconstitution

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10 11 12

Sp

ectr

aty

pe c

om

pexit

y S

co

reC

om

ple

xit

y s

co

re

months post transplant

Spectratyping

Months after transplant

0 2 4 6 8 10 12

1

10

100

1000

10000

0 2 4 6 8 10 12

1

10

100

T-reg Haplo (Campath1+ ATG)

(n=20)

0 2 4 6 8 10 12

1

10

100

1000

10000

Standard Haplo (n=150)

Pro

life

rati

ng

CD

4+

path

og

en

-sp

ecif

icT

cells i

n 1

06 c

ell

s

Pro

life

rati

ng

CD

8+

path

og

en

-sp

ecif

icT

cells i

n 1

06 c

ell

s

0 2 4 6 8 10 12

1

10

100

Reconstitution of pathogen-specific T cells after

HLA-haploidentical transplantation

ASP

Cand

CMV

ADV

HSV

VZV

Toxo

1

10

100

1000

10000

1

10

100

Donors


0

50

100

0

10

20

1 2 3 4 5 6 7-12 >12 months

CMV reactivation episodes Evaluable Patients

100

28

96

50

82

34

75

22

67

9

56

9

48

1 1

29

20

3

19

6

18

1

18

2

17

1

17

0 0

2

Sta

nd

ard

Ha

plo

T

reg

Ha

plo

CMV reactivations

17 17

Naive and Memory CD4+ and CD8+ reconstitution

1 3 6 9 120

50

100

150

200

250


CD

4+C

D45R

A+cell

s/

l

1 3 6 9 120

500

1000

1500


CD

4+C

D45R

O+cell

s/

l

1 3 6 9 120

100

200

300

400

500


CD

8+C

D45R

A+cell

s/

l

1 3 6 9 120

500

1000

1500

2000

2500


CD

8+C

D45R

O+cell

s/

l

Naive and Memory CD4+ and CD8+ Reconstitution

Fr1-JH Fr1-JH

Fr2-JH

Fr3-JH

Fr2-JH

Fr3-JH

Day + 30 post BMT Day + 90 post BMT

Reconstitution of peripheral blood B cell number, B cell repertoire

and seric levels of immunoglobulins

30 60 90 1200

250

500

750

1000

1250

Days post BMT

CD

20/

l

IgG IgA IgM

3 months

6 months

733 (80-1530) 31 (1-72) 71 (17-229)

692 (411-876) 44(8-111) 63 (45-140)

0

5

10

15

20

25

0

15

30

45

60

Donor 1 2 3 4 5 6 7 8 9 10 11 12


0

5

10

15

20

25

0

15

30

45

60

Donor 1 2 3 4 5 6 7 8 9 10 11 12

Donor 1 3 6 12

cell

s/c

mm

cell

s/c

mm

% a

llo

rea

cti

ve

NK

clo

ne

s

% a

llo

rea

cti

ve

NK

clo

ne

s

“Standard” haplo Lysis of KIR ligand-mismatched targets

“T-reg” haplo

Treg immunotherapy did not impair regeneration of

donor vs recipient alloreactive NK cell repertoires

(single) KIR2DL1+

(single) KIR2DL2/3+

(single) KIR3DL1+

C2 mismatch

C1 mismatch

Bw4 mismatch

Donor 1 3 6 12


0 1 2 3 4 5

0.0

0

.2

0.4

0

.6

0.8

1

.0

Cumulative incidences of transplant-related mortality P

rob

ab

ilit

y

Years post transplant

All the patients

CTX in the conditioning

Anti-T antibodies

in the conditioning

0 1 2 3 4 5


0,0

0,2

0,4

0,6

0,8

1,0 P

rob

ab

ilit

y

Disease-free survival

All the patients

Anti T antibodies

In the conditioning

Cyclophosphamide

In the conditioning

Is the graft vs leukemia effect maintained

in patients who received adoptive

immunotherapy with Tregs and Tcons?

FOXP3+ Tregs are indispensable for the maintenance of

immune homeostasis. However, as a double-edged sword,

they can also suppress antitumor immune responses

Treg and Tcon adoptive immunotherapy

in full haplotype mismatched HSCT

A crucial point

Trenado A, Charlotte F, Fisson S, et al. Recipient-type specific CD4+CD25+ regulatory T cells favor

immune reconstitution and control graft-versus-host disease

while maintaining graft-versus-leukemia.

J Clin Invest. 2003;112(11):1688-1696.

Edinger M, Hoffmann P, Ermann J, et al. CD4+CD25+ regulatory T cells preserve graft-versus-tumor

activity while inhibiting graft-versus-host disease

after bone marrow transplantation.

Nat Med. 2003;9(9):1144-1150

Studies in mouse models of mismatched HSCT

0 50 100 1500

20

40

60

80

100

Days

% s

urv

ival

Leuk. + T cons = no leuk, GvHD

Leuk. + T regs/Tcons = survival

w/o leukemia and GvHD

Leuk. + T regs = leukemia

Leukemia

AML primary

cells (7x106)

T cells

(3x106)

Clearance of human leukemia by human

T regs + T cons in immunodeficient (NSG) mice

Martelli, Di Ianni et al., Blood 2014

0

50

100

% l

ys

is

0 50 100 1500

20

40

60

80

100

days

0 50 100 1500

20

40

60

80

100

% s

urv

iva

l Clearance of human leukemia by human

T regs + T cons in immunodeficient (NSG) mice

ALL

Burkitt-like cell line

days


Pro

bab

ility

Cumulative Incidence of Relapse in High Risk AL Patients

Treg and Tcon adoptive immunotherapy in full haplotype mismatched HSCT

0.05 at median follow-up of 46 months

0 1 2 3 4 5 6

0.0

0

.2

0.4

0

.6

0.8

1

.0

P=0.03

0 5 10 15 20

0.0

0

.2

0.4

0

.6

0.8

1.0

0.0

0

.2

0.4

0

.6

0.8

1

.0

0 1 2 3 4 5

0.0

0

.2

0.4

0

.6

0.8

1

.0

0 1 2 3 4 5

Pro

bab

ilit

y

0.0

0

.2

0.4

0

.6

0.8

1

.0

Non-NK allo

NK allo


0 5 10 15 20


Pro

bab

ilit

y

Non-NK allo

NK allo

Non-NK allo

NK allo

Standard Haplo T cell depleted

ALL ALL

Treg Haplo

AML AML

Non-NK allo

NK allo

Cumulative Incidence of Post-transplant Relapse

The GvL Effect

What are the underlying mechanism/s?

2) No post-transplant pharmacological immunosuppression

1)High number of infused Tcons (1x106/kg)

which is non-specific immunologically,

only partially successful in preventing GvHD

and may compromise the T-cell induced GvL effect.

3) Tregs suppress GvHD at the periphery and unleash

the GVL effect in the bone marrow Ruggeri et al., ASH meeting 2014

Adoptive immunotherapy with naturally occurring

polyclonal Tregs is a feasible option in

HLA-haploidentical HSCT since this strategy

controls alloreactivity of a significant number of

coinfused Tcons with low incidence of GvHD

favors immune reconstitution and reduces the risk

of CMV reactivation

is associated with a powerful GvL effect

nTregs for future protocols: which separation and/or expansion strategy?

CD4/CD25/CD45RA is the strongest

immunosuppressive nTreg

and the more stable in culture (Edinger et al, Blood 2006)

Separation and infusion Negative selection with CD8,CD19 and

CD45RO followed by positive selection

with CD25 (CD45RO beads need to be validated)

Separation and in vitro expansion -Polyclonal expansion

(anti-CD28+anti CD3 +IL-2 ± rapamycine)

-Antigen-specific expansion

(more effective than polyclonal)

-Expansion of genetically modified Tregs (TCR with known specificity)

Advantage: no in vitro manipulation

Disadvantage: low Treg number?

Advantage: high Treg number

Disadvantage: in vitro manipulation

Clinical protocol

Phenotype

Immunosuppressive assay

TSDR

Mouse model

Translational Research in Full-Haplotype Mismatched Transplant

UNIVERSITY OF PERUGIA

HEMATOLOGY AND IMMUNOLOGY SECTION

Head: Brunangelo Falini

Alessandra Carotti

Loredana Ruggeri

Adelmo Terenzi

Antonio Pierini Maria Speranza Massei

Elena Urbani

Fabiana Topini

Antonella Tosti

Franca Falzetti

Mauro Di Ianni Lucia Amico

Tiziana Zei

Roberta Iacucci

Beatrice Del Papa

Debora Cecchini

Alain Bell

HSCT Unit and Transplantation

Immunology

Head: Andrea Velardi Department of Immunology

Weizmann Institute of Science

Head: Yair Reisner

Massimo Fabrizio Martelli

Professor Emeritus

utilizzo dei linfociti t regolatori nel trapianto da

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