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Firenze, 20 Novembre 2014

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Implicazioni cliniche dei linfociti T regolatori nel setting allotrapiantologico

[mario.delia@policlinico.ba.it]

Mario Delia

U.O.: Ematologia con Trapianto

Policlinico

Bari

T regolatorie (CD4+CD25highFoxp3+) Foxp3 positività condizione necessaria per conferire attività soppressiva alle CD4+CD25+ peripheral T regs

Base teorica della deficienza T regolatoria in: LES, diabete tipo I, epatite autoimmune, aplasia midollare

T regulatory function + T effector function:

Funzione: soppressione della risposta immune e mantenimento della tolleranza (inibizione del priming→expansion→function T conv)

CD4+CD25+ CD4+CD25highFoxp3+

CD4+CD25+

T regs

% Peripheral blood (PB) sani

<10 dei CD4

PB post allo

≥10 dei CD4

Graft content

≈ 10x10e6/kg recipient

Immunosuppressive mechanisms of Tregs CD4+CD25highFoxp3+ CD127low CD62L+

Ustun C et al Blood 2011;118:5084-5095

Regulatory T cells in animal models

T regs phenotype Results

Hoffmann P et al. J Exp Med 2002

CD4+CD25high The balance (1:1) of donor-type CD4+ CD25high T regs and conventional CD4+/CD25- T cells can determine the outcome of aGvHD

Edinger M et al. Nat Med 2003

CD4+CD25+ CD4+CD25+ regulatory T cells preserve GvL while inhibiting GvHD

Nguyen V. et al. Blood 2006

CD4+CD25high

Foxp3+

Tregs attenuate GvHD

Gaidot A. et al. Blood 2011

CD4+CD25high

CD62L+

GVHD prevention with T regs is associated with improved early immune reconstitution

T regs and immunological recovery

Model of MHC-mismatched HCT to assess the effect of Tregs

on GvHD, engraftment, and immune reconstitution

BM BM+ T cons TCD/BM+T cons+ T

regs(1:1)

Nguyen V et al. Blood 2008;111:945-953

Abrogration of GvHD

Preservation of thymic and peripheral lymph

node architecture

Accelerated donor lymphoid reconstitution

animal models

Accelerazione donor lymphoid reconstitution

Edinger Nat Med 2003 VH Nguyen Blood 2008

40 gg dopo allo

animal models

gg dopo allotransplant

Challenge intraperitoneale di CMV

VH Nguyen Blood 2008

recovery anti CMV:

animal models

Regulatory T cells in allo SCT

Results

Miura Y et al.

Blood 2004

T-cell replete

Foxp3 mRNA expression was significantly

decreased in GvHD

Rezvani K et al.

Blood 2006

T-cell depleted A low CD4+FOXP3+ T cell count early after SCT

(day 30) was associated with an increased risk of

GvHD

CSA does not affect recovery of T regs

Matthews K et al.

Haematologica

2009

RIT+

Alemtuzumab

Imbalance of effector and regulatory CD4 T cellsGvHD

GvHD(T eff/T regs 41:1) No GvHD 12:1

Rieger et al.2006

Fondi et al. 2009

Reduced numbers of CD4+ Tregs in tissue

affected by GvHD

human setting

21 pz T depl – Rezvani

Rezvani Blood 2006

T regs/aGVHD

human setting:

peripheral bòlood

45 patients

HLA mismatched (MAC /RIT) without T-cell depletion

GvHD prophylaxis: MAC------TAC+MTX+MPRD+MMF

RIT-------- TAC+MPRD

Fujioka T et al Bone Marrow Transplant 2012;1-6

Second week

after HSCT

T regs/aGVHD

human setting:

peripheral bòlood

T regs/recovery of CMV-specific CD8+ T cells

Allo PBSCT ( 46 pts 100% MRD 80% AML 80% CR 100% MAC )

GvHD prophylaxis: CSA+MTX

Pastore D et al BBMT 2011;17:550-7

CMV -

CMV +

HC

MV

-sp

eci

fic

CD

8+ /

uL

1° month 2° month 3° month 6° month

2 0

8

1

12

1

human setting:

peripheral bòlood

T regs/recovery of

CMV-specific CD8+ T cells

Circulating T regs in patients with (CMV+) and without (CMV-) CMV

infection/disease

T re

g/u

L

CMV -

CMV +

* p<0.001

2 MONTHS 3 MONTHS

3

15

6

22

T regs reconstitution of functional immunity

human setting:

peripheral bòlood

T regs/recovery of CMV-specific CD8+ T cells

Correlation between Tregs(CD4/CD25high/Foxp3+) and CMV-

specific CD8+ T cells

2 months 3 months

r=.61 r=.72

human setting:

peripheral bòlood

T regs in the graft

58 patients

Allogeneic PBSCT from HLA-identical siblings (MAC /RIT)

The mean number of Tregs: 9.1 x106/kg bw (range 0.7 to 33)

Donor-derived Treg might be of particular significance for the development of acute GVHD after myeloablative SCT using HLA-identical sibling donors

Wolf D et al Transplantation 2007; 83:1107-1113

Incidence of aGvHD Overall survival Incidence of relapse

Tumor Clearance

Microbial Immunity

Autoimmunity

Transplant rejection

Graft-versus host disease

Allergy

Self-tolerance

Transplantation tolerance

Tumor Progression

Microbial Persistence

Teff Treg Self-tolerance

Immunocompetence

Sheng Cai

CD3/ Tregs ratio in donor graft Balance of effector T cells and donor regulatory cells

CD3/Tregs ratio in donor graft Balance of effector T cells and donor regulatory cells

Pastore D et al BBMT 2012;18:5887-93

CD3/ Tregs ratio in donor graft

Cellular composition of allograft

CD3/ Tregs ratio in donor graft

The median gCD3/Tregs R was 18 (range, 8-250)

Cellular composition of allograft

CD3/ Tregs ratio in donor graft

Curva ROC per stabilire un valore soglia di graft CD3/Tregs ratio

correlabile con aGVHD

CD3/Tregs ratio =36

Sensitivity 71%

Specificity 94%

AUC: 0.79

graftCD3/Tregs <36: aGVHD graft CD3/Tregs ≥36: aGVHD

CD3/ Tregs ratio in donor graft

CMV infection/disease

LR: 15%

HR: 50%

CD3/ Tregs ratio and ATG

Median value of circulating peripheral blood Tregs in patients

receiving rATG and those not receiving rATG

Median value of circulating CMV-specific CD8+ T cells in patients

receiving rATG and those not receiving rATG

Low CD3/Treg ratio

CD3/ Tregs ratio in donor graft

Correlation between gTregs and peripheral blood Tregs

Correlation between gCD3/Tregs ratio and peripheral blood Tregs

r=0.36;P=0.03 r=0.38;P=0.02

CD3/ Tregs ratio in donor graft

E sulla sopravvivenza?

Delia M et al. BBMT2013 Mar;19(3):495-9.

New ways to separate GvHD and GvL after alloSCT: T regs? Kotsiou E et al Br J Haematol 2013;160:133-145

Delia M et al. BBMT2013 Mar;19(3):495-9.

Multivariata su outcome

Qestioni ancora pending?

1. Differenza Tregs tra GVHD

acuta e cronica

2. Interferenza con ‘drugs’

trapiantologici

Rezvani Blood 2006

Chronic graft-versus-host disease is associated with increased numbers of peripheral blood CD4+CD25high regulatory T cells – Clark Blood 2004

non è tanto questione di numeri (29% cGVHD; 34% aGVHD), ma di funzione fase specifica!

1

2

3

2

Clark Blood 2004

% T regs rispetto a CD4 (valore assoluto)

<30% (<0.3α T regs)

30 % (0.3α T regs)

>30% (>0.3α T regs)

GVHD

a 30 gg si si no

a >100 gg no si si

Ipotizzando un allotrapiantato con valore (α) CD4, muterà il rischio di GvHD a seconda della fase

T regs e

1. Ciclosporina (3-5 mg/kg/die) – Profilassi GVHD

2. ATG: (F-rATG: 20 mg/kg on day -3,-2,-1 (dose totale 60 mg/kg; rATG 1 mg/kg on day -3, 3.25 mg/kg on days -2 and -1 (dose totale 7.5 mg/kg) – Profilassi GVHD

3. Steroide (0.5-2 mg/kg/die) – Terapia aGVHD

Ciclosporina

Zeiser Blood 2006

► L’attività T regolatoria richiede la presenza di IL-2 (CD25: IL2 receptor)

► La calcineurina dependent production di IL-2 inibita da CsA

►↓ attività soppressoria T reg (sequenza non valida per Micofenolato e Rapamicina)

Rezvani Blood 2006

LOW DOSE (target plasma level 100-200 ug/ml)

10/21 T-depleti

Feng X et al. Blood 2008;111:3675-3683

We show here that in vitro culture of normal human peripheral blood mononuclear cells (PBMCs) with a low-dose rATG resulted in marked expansion of functional Treg by converting CD4+CD25− T cells to CD4+CD25+ T cells. hATG did not expand but rather decreased Treg.

ATG

Steroide►Depressione funzionale T linfocitaria►accentuata dalla linfodeplezione (ATG )►antagonizzata da Tregs

Ozdemir Blood 2002 Morita-Hoshi BMT 2007

Hakki Blood 2003

È una questione di dose (valutazione a 3 mesi post allo)

E di dose cumulativa (1-month period) ≤10 mg/kg >10 mg/kg

In multivariata correlano col mancato recovery immunologico: Higher steroids p=.01 BM stem cells p=.03 Low CD8 p=.04 No correlazione per ATG o donor

Steroide

Grazie per

l’attenzione