Discutiamone insieme
Firenze, 20 Novembre 2014
Boscolo Hotel Astoria
Implicazioni cliniche dei linfociti T regolatori nel setting allotrapiantologico
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