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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
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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+
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T regs
% Peripheral blood (PB) sani
<10 dei CD4
PB post allo
≥10 dei CD4
Graft content
≈ 10x10e6/kg recipient
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Immunosuppressive mechanisms of Tregs CD4+CD25highFoxp3+ CD127low CD62L+
Ustun C et al Blood 2011;118:5084-5095
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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
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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
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Accelerazione donor lymphoid reconstitution
Edinger Nat Med 2003 VH Nguyen Blood 2008
40 gg dopo allo
animal models
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gg dopo allotransplant
Challenge intraperitoneale di CMV
VH Nguyen Blood 2008
recovery anti CMV:
animal models
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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
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21 pz T depl – Rezvani
Rezvani Blood 2006
T regs/aGVHD
human setting:
peripheral bòlood
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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
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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
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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
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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
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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
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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
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CD3/Tregs ratio in donor graft Balance of effector T cells and donor regulatory cells
Pastore D et al BBMT 2012;18:5887-93
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CD3/ Tregs ratio in donor graft
Cellular composition of allograft
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CD3/ Tregs ratio in donor graft
The median gCD3/Tregs R was 18 (range, 8-250)
Cellular composition of allograft
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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
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CD3/ Tregs ratio in donor graft
CMV infection/disease
LR: 15%
HR: 50%
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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
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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
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CD3/ Tregs ratio in donor graft
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E sulla sopravvivenza?
Delia M et al. BBMT2013 Mar;19(3):495-9.
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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.
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Multivariata su outcome
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Qestioni ancora pending?
1. Differenza Tregs tra GVHD
acuta e cronica
2. Interferenza con ‘drugs’
trapiantologici
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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
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% 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
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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
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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
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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
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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
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Grazie per
l’attenzione