molecules and mechanisms of the graft versus leukemia (gvl
TRANSCRIPT
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Molecules and mechanisms of the graft versus leukemia (GVL) effect
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• University of Manitoba Faculty of Medicine Archives
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The “Goldenberg” Rules
• We are fortunate to have the opportunity to participatein research. Do not squander the opportunity --commit yourself to a life-time of learning
3. Treasure the time you have for research, it is special
2. Patients and professors can teach us much, if we payattention
4. Use correct grammar!!
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Immunologic Non Identity Contributes To The Efficacy Of Allogeneic HCT
Horowitz, Blood 1990
Twins
HLA id sibs
+ GVHD
CML > CLL, low grade lymphoma, myeloma > AML, ALL
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• Full donor chimerism• No circulating malignant cells
Myeoloablative Conditioning (Age <50)
Success rate ~ 30 - 60% in AML/ALL/MDS, 80% CML
• Toxicity• GVHD• Relapse
• Mixed hematopoietic chimerism• Circulating malignant cells• CSA/MMF immunosuppression
Nonmyeloablative Conditioning (Age up to 70)
Success rate ? (~50% for indolent leukemias (CLL), follicular lymphoma
• GVHD• Relapse
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Tolerance
Donor T Cells
Donor Cells
Genetic Polymorphism Results in the Display of Unique Peptides by Cell Surface MHC Molecules
• nonsynonymous SNPs• 5’ “untranslated regions”• differential gene expression• peptide splicing/reassortment
Recipient Cells
Activation
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• Antigens are “foreign” to donor T cells• T cell responses are of high avidity• T cell responses may be multivalent
• Responses typically involve both CD8 and CD4 T cells
Minor H Antigens - Characteristics of EffectiveTargets for Cancer Immunotherapy
Can the GVL effect for acute leukemias be augmented and separated from GVHD?
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Tissue Expression of Minor H Antigens May Provide ABasis for Segregating GVHD and GVL Responses
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Isolation of T Cells Specific For Recipient Minor H AntigensAfter In Vivo Priming
γ-irradiated APC (PBMC or leukemia)from the recipient pre transplant
Assay for cytolytic activityClone reactive T cell lines and characterize T cell clonesfor phenotype, HLA restricting allele, tissue expression
PBMC from recipientpost transplant
T Cell Clone
B-LCL
PHA-Blasts
Fibroblasts
Target Cells
% L
ysis
0
10
20
30
40
50
60
70
80
SKH-13 DJG-24
Donor Recipient Donor Recipient
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ClonogenicLeukemic Progenitors
Leukemic Stem Cell
SelfRenewal
CD34+CD38-
375 cGYAML +/- CTL
25
50
Control MHAg CTL
% Engraftment
Engraftment
• Bonnet D. et al. Proc Natl Acad Sci 1999
Part 1. Lost in Translation
“Hematopoietic Lineage”-Restricted Minor H Antigen
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Adoptive Transfer of Minor H Antigen-Specific T Cell Clones To TreatPost Transplant Leukemic Relapse
Outcome - Post transplant Relapse• Enroll patients with advancedleukemia (>2nd CR, refractorydisease)
• Isolate CD8+ T cell clones fromrecipients early post transplant
• Select clones that lyse recipienthematopoietic cells (includingleukemia) BUT NOT skin fibroblasts
• QC testing & cryopreservation
• Treat patients at relapse withescalating doses of T cells
• Evaluate toxicity, T cell migrationand persistence, and antitumoractivity
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CTX *
-2 1 4 11 21 28 ≥35
CTL Infusions IL-2CTL Infusions
Day
3.3x107 3.3x108 3.3x109 3.3x109 3.3x109 3.3x109
• Early TRM or relapse -- 32% of enrolled patients • 7 patients relapsed and received T cell infusions• 3 patients relapsed and had T cells available - not treated • 6 patients have T cells available and remain at risk
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UPN-15652 - primary refractory Ph+ ALL
- MRD SCT with Cy/TBI
- Isolated minor H antigen-specific CTL clones
- Relapse 7 months post SCT (>90% blasts)
- Cytoreduction with Mitoxantrone/VP16
0 10 20 30 40 50 60 70
Recipient LCL
RecipientFibroblasts
LeukemicBlasts
Donor LCL
DonorFibroblasts
% Specific Lysis
CTL Clone 11C6
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T Cells
x 103
0
5
10
15
20
25
30
35
-11 -5 1 7 13 19 25 31 37 43 49 57
WBC
Blasts
Day
*Skin GVHD(PCR -ve for CTL)
Toxicity: * Lung Toxicity
Blasts in BM 90% >15%
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Transferred CTL Localize At Site of Tissue Injuryand Modulate TcR Expression
Pre-Inf
4 hr PostT Cells
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MP
2 days PostT Cells
Modulation of Vβ 13 TcR Expression in bronchoalveolar lavage fluid
Detection of CTL by clone-specific PCR
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Blasts in BM 90% >15% <5%
x 103
0
5
10
15
20
25
30
35
-11 -5 1 7 13 19 25 31 37 43 49 57
WBC
Blasts
Day
T Cells
*Skin GVHD(PCR -ve for CTL)
Toxicity:
Methylprednisolone
* Lung Toxicity
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Post T cell infusions - Remission
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Post chemotherapy - Relapse
UPN15652
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No GVHD,toxicity
<5% CR80%90%#3 ALL
OutcomePost T CellsPost CTXPrePatient
Bone Marrow BlastsUPN 19492
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+ – PBL M PBL M PBL M
3 4 6Infusion #
Clone-specificTcR V beta PCR
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Results
• Toxicity
• 3/7 patients (2 lung, 1 GVHD)
• Transfer efficiency and migration
• high levels of transferred T cells in the blood and bonemarrow (>8% of PBMC), t1/2 of transferred cells in theblood ~ 7 days
• *Efficacy
• 4/6 evaluable patients achieved CR, including 2 withpersistent disease after chemotherapy.
• 6/7 patients subsequently progressed 4 -15 monthsafter therapy, 1 patient remains alive >39 months
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• Isolation of minor H antigen specific T cells from patients transplanted for advanced leukemia is feasible but problematic
• Separation of GVL effect from toxicity cannot be achieved by selecting clones based on in vitro cytotoxicity against non-hematopoietic targets
3. Adoptively transferred CD8+ T cell clones exhibit limited persistence in vivo
Mechanisms responsible may include:• high antigen load - activation induced cell death• inadequate T helper responses/prosurvival cytokines• intrinsic defect due to culture and/or differentiation• other
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Part 2. Back to Basics
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cDNA Library
pEAK10 Plasmid Vector
Plasmid VectorEncoding HLA-Allele
COS Cells
−
+
−
−
IFN γ(ELISA)
CTL Clone
Identification of the Genes that Encode Minor H Antigens
Recipient EBV-LCL
mRNA For mHAg
cDNA
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UDP Glycosyltransferase 2 Family, Polypeptide B17 (UGT2B17)Encodes a Minor H Antigen Presented by HLA A29
Construct I (52-513)
Construct II (493-993)Construct III (493-564)
ATGTAA
TAA
ATG TAA
ATG (−)(+)(+)
UGT2B17 cDNA 1 52 1644 21072H9
1 383 2335 2958cDNA
Peptide concentration (nM)
Spe
cific
lysi
s (%
)
5060
40302010
00 10-5 10-4 10-3 10-2 10-1 100 101 103102 104
Recognition of Donor LCLPrecultured with Synthetic Peptide HLA-A29 Anchors
1 2 3 4 5 6 7 8 9 E Y
V L L A D A V N P C G E L L A E L L N I P F L Y
CTL Clone PL-8
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UGT2B17
GAPDH
Unrelated HLA-A29+ LCLRecipient LCL
DonorLCL Ag+ Ag+ Ag- Ag-
Northern Blot Analysis of Total RNA
Immunogenicity of UGT2B17 Results from Differential Transcription in Recipient versus Donor Cells
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SSP-PCR Primers exon 1 (b)
SSP-PCR Primers exon 1 (a)
PCR Primers for5’ upstream
UGT2B17 gene 1 2 3 4 5 6Exon
- 370 - 30
4431
400 751
1394 1594SSP-PCR Primers for exon 6
52 16441365775535 5641 2107
ATG TAGEpitope5' 3'
Probe for Northern
Unrelated HLA-A29+ LCLRecipient LCL
DonorLCL Ag + Ag + Ag − Ag −
exon 1 (a)
5’ upstream
GAPDH
exon 1 (b)
exon 6UGT2B17
• 11% ofCaucasiandonors aredeficient inUGT2B17
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Tissue Expression MattersSSP-PCR for UGT2B17 cDNA from Human Tissues
UGT2B17
GAPDH
Hea
rt
Bra
inP
lace
nta
Lung
Live
r Ske
leta
l mus
cle
Kid
ney
Pan
crea
sS
plee
n
Thym
usP
rost
ate
Test
isO
vary
Sm
all i
ntes
tine
Col
on
• the recipient had acute GI/liver GVHD and cGVHD involving the GI tract
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241 336
MPHSPLGSMPEIRDNSPEPNDPEEPQEVSSTPSDKKGKKRKRGIWSTPKRRHKKKSLPRGTASSRHGIQKKLKRVDQVPQKKDDSTCNSTVETRAQ MSTPKRRHKKKSLPRGTASSRHGIQKKLKRVDQVPQKKDDSTCNSTVETRAQ
MPHSPLGSMPEIRDNSPEPNDPEEPQEVSSTPSDKKGKKRKRGIWSTPKRRHKKKSLPRGTASSR
STPKRRHKKKSLPRGTASSR
+++
DRN-7 CTL Recognize A Minor H Antigen Presented By HLA -A3And Encoded by the SP110 gene
-20
A/G G/G A/G G/G G/G A/G A/A G/G A/G A/G G/G
020406080
100
DRN CAN SEB ABB WGB RLB MDK TM WAS JDS JR
% S
peci
fic L
ysis
PCR
-RFL
P
Genotype
CTL Clone DRN-7
Donor SP110 - G
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0 500 1000 1500 2000 2500
STPKRRHKKKSLPRGTASSR
IFN-gamma (pg/ml)
Non-Electroporated
Electroporated
>2000
(SP110286-305)
What is the SP110 epitope?
• 20 mer peptide containing G-->R substitution sensitized donor LCL for recognition by CTL but only at high concentrations (>10 μg/ml)
• No synthetic 8 - 12 mer peptide comprised within the 20 mer sequence could be identified that sensitized target cells for recognition by the CTL clone
• Consistent with a requirement for a posttranslational modification for epitope generation
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Does the Proteasome Generate The SP110 Epitope?
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STPKRRHKKKSLPRGTASSR
But ---- STPKSLPRGT did not senstitize target cells for recognition
0 1 2 3 4 5 6 7 8
Interferon gamma (ng/ml)
STPK + SLPRGTASSR
STPK + SLPRGTASSR
Is the SP110 epitope a product of peptide splicing?
Electroporated
PulsedSLPRGTASSRSTPK
E or P
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HLA A3 binding motif
_ L _ _ _ _ _ _ K
STPK SLPRGT SLPRGTSTPK
STPKRRHKKKSLPRGTASSR
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-10
0
10
20
30
40
50
60
70
80
90
0.01 0.1 1 10 100 1000
[Peptide] nM
% S
peci
fic L
ysis
SLPGGTSTPK
SLPRGTSTPK
STPKSLPRGT
Peptide Splicing With A Twist -- Rearrangement of SP110Encoded Peptides Creates The DRN-7 Epitope
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The Putative DRN-7 Epitope SLPRGTSTPK Co-elutes WithThe Naturally Processed Epitope
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01020304050607080
Donor LCL
Recip-LCL
RecipT Cells
RecipFibroblasts
HLA-A3+LCL
HLA-A3+ CLL
Target Cell
% Lysis
CloneKSN-7A7
1.5 Kb
PANE-1 transcript k
-10
0
10
20
30
40
50
60
70
80
90
100
110
1.E-03 1.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03
[Peptide], nM
% S
peci
fic L
ysis
RVWDLPGVLK on GAO LCLRVWDLPGVLK on T2-A3SLPRGTSTPK on GAO LCLSLPRGTSTPK on T2-A3
PANE-1 transcript k -- a B-lineage minor H antigen
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8.4 Kb
PANE1transcript c
PANE1transcript k
1.5 Kb
PANE1transcript c
PANE1transcript k
A
M G R V W D L P GaggcaggccacggagatacctcgtggaaggaggaacagcaagtgcATGGGCCGAGTGTGGGACTTGCCTGGT V L K TGTGCTCAAGgtctggcaggcaggcctgggtggctgcagcacaatagcaaagggcgggttggggtggagaaatcaggaatggtggggctggatcagtggagcctggtggggagctgctggagggcttggggccagggagggatgtggcctgacttgggttttaaatggacccctctggctgccgggaggagagtggactgaagggaaactagggcagaaatagagagacaggtctggaggctctttctttctttctttctttctttctttttctttctttctttctttctttctttctttctttctttttctttctttctttctttctttttctttctttttctttctttttctttctttttctttcttttttgagacagacactgggcagcatcgtgttcttccagcggacgtgtctagaacatctgtgcccgggcctagagcagtcttgtccctctctggccccacagctcccctgggcccattcctttttgatggcaccttcttatctgtggtgagggccacccgctgcccctccctccttcctatccctgtatcttctgctccctggtcttgagcatctggggcccgagctgcctgggcattggttggaagtagccctgattcagagctcagctctggagtcactagctgggtgaccctagacagtttgttcgacctagccagccttgttaagccttcatttcctcttgagcaggaggtcaggctctggatgtctccggaggttgtttgaaggattgggcaagctgaggcagttggaagctactctgaggtgtggcatgaatgagggcgtggctgtggtgggcagtggtgacctgagctacccccaagtatccgcccccagccctgctctggcagcccctgcagctgctctgggtaagggagcagggccagcattctatctcactgctgttct V E G F R A T M A Q R L V R V L Q I CctgctctccccgcaggtGGAAGGCTTTAGGGCCACCATGGCGCAGCGCCTGGTGCGCGTGCTGCAGATCTGT A G H V P G V S A L N L L S L L R S S E G P S LGCTGGCCACGTGCCCGGTGTCTCAGCTCTGAACCTGCTGTCCCTGCTGAGAAGCTCTGAGGGCCCCTCCCTG E D L *GAGGACCTGTGAGGGTGGCTGGCCCCTGGGCTGCCCCTTCTCATGGCTTCGTGCTGACTCCATAAACATTCTCTGTTGAGGATGTCCAGTCAGGGCTTGACAGGCCCAGGCTCAGCCCGCCGTGGCTGGGAAGGTTCCCTGCAGTGCCAGTGCTGCAGCAGGGAGAGCTGGGCAGAAGCAGCGAGGGGGCCCAGCTGGCGAGACTGTAGCCCCCTCCCACTCCCACACTCACTCTTGCAGAGCCTGTGTCTTTAAGCAGCTGGCGTGTTACATCTCCATTTAAGGTTTCCTTTGAACAAAAGGTCTGTGGCTAAAAAAAGTTTAAAAATCaaaaaaaaaaaaaaaaaaaaaaaaa
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Bra
inH
eart
Lung
Live
rK
idne
ySk
elet
al M
us.
Pros
tate
Test
isO
vary
Panc
reas
Stom
ach
Smal
l Int
estin
eC
olon
Res
t. C
D19
+
Act
. CD
19+
EB
V-L
CL
PANE-1 is selectively expressed in B-lineage cells
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Expression of PANE-1 by q-PCR
0 20 40 60 80 100 120
Brain
Heart
Lung
Liver
Kidney
Pancreas
Small Intestine
Colon
Prostate
Testis
Ovary
Skeletal Muscle
Spleen
Thymus
Resting CD19
Activated CD19
CLL -1
CLL -2
CLL -3
CLL -4
CLL -5
CLL -6
CLL -8
CLL -9
Tis
sue S
ou
rce
Relative Level of Expression
Transcript cTranscript k
118124330776361126344978
221
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Minor Hantigen
HLArestriction
Gene/chromosome Peptide sequence Tissue distribu tion Identi fication technique 1
HA-125 HLA A201 KIAA0223/19 p13 VLHDDLLEA Hematopoieti c HPLC with mass spectometry
HA-126 HLA B60 KIAA0223/19 p13Chromosome
KECVLHDDL Hematopoieti c Polymorphi c peptide s creening
HA-227,28 HLA A201 MYOG1/7 YIGEVLSV Hematopoieti c HPLC with mass spectometry
HA-329 HLA A1 Lbc oncogene/15q24-25 VTEPGTAQY Ubiquitou s HPLC with mass spectometry
HA-830 HLA A201 KIAA0020/9 RTLDKVLEV Ubiquitou s HPLC with mass spectometry
HB-131,32 HLA B44 Chromosome 5q32 EEKRGSLHVW Hematopoieti cesp B cell leukemias
cDNA expression cloning
UGT2B17 33 HLA 2902HLA B4403
UGT2B17 /4q13 AELLNIPFLY Ubiquitou s cDNA expression cloning
BCL2A134 HLA A24 BCL2A1/15q2 4.3 DYLQYVKQI Hematopoieti c Genetic linka ge analysis
BCL2A134 HLA B4403 BCL2A1/15q2 4.3 KEFEDDIINW Hematopoieti c Genetic linka ge analysis
HY B7 (SMCY)35 HLA B702 SMCY SPSVDKARAEL Ubiquitou s HPLC with mass spectometry
HY A2 (SMCY)36 HLA A201 SMCY FIDSYICQV Ubiquitou s HPLC with mass spectometry
HY A1 (DFFRY)37 HLA A101 DFFRY IVDCLTEMY Ubiquitou s HPLC with mass spectometry
HY B60 (UTY)38 HLA B60 UTY RESEESVSL Ubiquitou s cDNA expression cloning
HY B8 (UTY)39
HY A2 (UTY)HLA B8HLA A2HLA A2
UTYUTYUTY
LPHNHTDLYLQQNTHTLLLIADNPQL
Ubiquitou s, Highlevels in AML
cDNA expression cloning
HY DQ5 (DBY)40 HLA DQ5 DBY HIENFSDIDMGE Ubiquitou s cDNA expression cloning
HY DRB341 HLA DRB3 RPS4Y VIKVNDTVQI Not reported cDNA expression cloning
C22orf18 HLA A3 C22orf18 RDWDLPGVLK B-cell (CLL) HPLC/mass spectrometry
SP110 HLA A3 SP110 SLPRGTSTPK** Hematopoieiti c, ifninducible
CDNA expression cloning
LYSE 95-4- F4 HLA A2 LYSE 95-4- F4 SNP (L H) Not determined(Renal cell Ca)
Genetic linka ge analysis
Molecularly Characterized Minor Histocompatibility Antigens
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CCR-7 CD28
Ag
TCM
TEM
CCR-7 CD28
CD28
Effector Memory
CD45RO
CCR-7 CD28
CD62L
Naive
CD45RA
Part 3 - A Naïve Answer (?)
• GVHD is common after unmodified allogeneic HCT requiring the administration of immunosuppression, poor platform for immunotherapy to augment GVL effect
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Chen B et al. Blood 2004Xystrakis E. et al Eur J Immunol 2004Zhang Y et al J Immunol 2005
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9.2%
CMV Fibroblasts
IFN-γ
CD
8
In the absence of priming of the donor to alloantigens, the overwhelming majority of alloreactivity should reside in the naïve T cell pool
CCR-7 CD28
Ag
TCM
TEM
CCR-7 CD28
CD28
Effector
CD45RO
CCR-7 CD28
CD62L
Naive
<1% of TcR diversity pathogen-specific(CMV, EBV, HSV)
>99% of TcRdiversity (Arstila etal. Science, 1999)
CD45RA
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Direct Analysis of Minor H Antigen AlloreactivityIn Naïve and Memory Subsets of CD8+ T Cells
• 5 HLA identical sibling pairs
• Purified naïve (Tn -- CD45RA, CD62L+) and memory (Tm -- CD45RO) T cells
• Limiting dilution assay using purified Tn and Tm cells as responder cells, recipient dendritic cells as the APC, and IL12/IL15
• Recipient and donor DC or CD40L B cells astarget cells, validation of positive wells
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CD45RO
100 101 102 103 104
72604.169
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104
72604.173
100 101 102 103 104
100 101 102 103 104
72604.175
100 101 102 103 104
100 101 102 103 104
72604.171
100 101 102 103 104 100 101 102 103 104CD45RA APC
72604.174
99
98
41
74
94
CD62
L
CD45RA
CD62
LCD
27
100 101 102 103 104
95
CD28
Naïve T Cell Purification
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Frequency of Minor H Antigen Specific CD8 T cellsCD45RA vs CD45 RO
40.00
60.00
80.00
100.00
10000 100000 1000000 10000000Number of CD8 T Cells
Frequency of Minor H Antigen Specific CD8 T cells CD45RA vs CD45 RO - Validation Assay
40.00
60.00
80.00
100.00
10000 100000 1000000 10000000
Number of CD8 T Cells
• T cell clones specific for broadly expressed and lineage restricted minor H antigens
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Stem Cells +Memory T cells
Conditioning Decreased posttransplant immunosuppression
Predicted Outcome:• rapid immunologic recovery• few infections• little or no GVHD
How to restore the GVL effect?- improved platform for immunotherapy- vaccinate donors to elicit lineage restricted minor H
antigen specific memory T cellsOR
- T cells specific for leukemia associated or minor Hantigens isolated from naïve donor T cells could be
adoptively transferred
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Pilot trial of naïve T cell depletion to reduce GvHD
• HLA id siblings (acute leukemia, donor parity/transfusion hx)
• high stem cell dose
• CD34 selection, CD62L depletion (removes all naive and central memory cells) -- administer defined dose of memory T cells
• FK506/MTX initially, if reduction in GvHD, test FK506 alone (MTX may inhibit homeostatic proliferation of memory T cells and delay immune reconsititution to pathogens)
• End points- engraftment - GVHD- relapse - immune reconstitution - infections (CMV, EBV, HSV, aspergillus)
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Donor Recipient
• Genotyping for relevant minor H antigens based on HLA type
• Vaccination of the donor to one or more antigens
• Preparation of stem cell graft (naïve T cell depletion)
• Vaccination of recipient to boost leukemia associated minor H antigen-specific T cell responses
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Edus H. WarrenMarc GavinJeff MitoMichele Brown
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Nathalie VigneronBenoit van den Eynde(Ludwig Institute, Brussels)
Tony BricknerVic Engelhard(University of Virginia)
Warren Shlomchik(Yale University)
MarieBleakleyAudrey MollerupThomas ManleyLarry AndersonTory Yamamoto
Tetsuya Nishida