TOLERANCE VACCINE- WHERE ARE WE? UNITED NATIONS-GENEVE H L Trivedi, F.R.C.P. (C) Director,...
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Transcript of TOLERANCE VACCINE- WHERE ARE WE? UNITED NATIONS-GENEVE H L Trivedi, F.R.C.P. (C) Director,...
TOLERANCE VACCINE- WHERE ARE WE?
UNITED NATIONS-GENEVE
H L Trivedi, F.R.C.P. (C)
Director, Professor,
G. R. Doshi & K.M. Mehta
Institute of Kidney Diseases & Research Centre,
Dr H L Trivedi
Institute of Transplantation Sciences,
Civil Hospital Campus, Ahmedabad, INDIA
What is a Vaccine ?
A vaccine is a biological preparation that
improves immunity to a particular disease. A
vaccine typically contains a small amount of
an agent that resembles a microorganism. ...
Tolerance Vaccine In Transplantation
• Tx pts –stable grafts-↓pCTL frequencies at 3 mths post-Tx
• May be permanent
• Tubular cells can induce specific anergy
• Chimerism can lead to graft acceptance
Ophir E, Reisner Y. Induction of tolerance in organ recipients by hematopoietic stem
cell transplantation. Int Immunopharmacol.2009 Jun;9(6):694-700.Epub 2009 Jan 16
• Veto - An authoritative prohibition or rejection of a proposed or
intended act
• What is a Veto Cell ?
• The term “VETO” relates to the ability of cells to neutralize
cytotoxic T-lymphocyte precursors directed against their antigen
• Veto Activity: capacity to specifically suppress cytotoxic T cell
precursors directed against antigens of themselves, not against third
party antigens (Miller,1980)
Tolerance vaccine of Veto Cells
Muraoka S, Miller RG. Cells in bone marrow and in T cell colonies grown from
bone marrow can suppress generation of cytotoxic T lymphocytes directed against
their self antigens. J Exp Med. 1980;152: 54-71.
• Cells within the human CD34+ population as well as their
immediate early myeloid progeny, are endowed with potent veto
activity
• Thus it is possible to harvest more veto cells on culturing of CD34+
cells for short term and make a vaccine from these cells with other
cells
• These cells should survive, multiply, block/ delete/ functionally
inactivate effector cells
How Can We Make a Vaccine of Veto Cells ?
Gur H, Krauthgamer R,Berrebi A, Nagler A, Klein T, Martelli M, Tabililo A and
Reisner Y. Tolerance induction by megadose hematopoietic progenitor cells:
expansion of veto cells by short-term culture of purified human CD34+ cells. Blood.
2002; 99: 4174-4181
Types And Sources Of Veto Cells
BM CELLS
CD34+ CD 33-
LYMPHOCYTE
SUBPOPULATION
CD34+ CD 33+
CD 34- CD 33+
Activated NK cells
Activated CD4+ T-cells
Activated CD8+ T-cells
Activated B-cells
SOURCES :
BM, Thymus, spleen, LN, Fetal Liver
In-vitro expansion
Mechanism Of Action Of Veto Cells
Effector cell Veto cell
Fas L
MHC
CD 8+
TCR
α-3
Fas
MHC 1
Apoptosis
Reduction of FADD like Il-1-converting enzyme-inhibitory protein
O hr 24 hr 48 hr 72 hr
FLIP
Salient Features
• Clonal stimulation with stem cell cocktail
• Clonal deletion with Bortezomib (Proteasome inhibitor)
with adjuvant MP, ATG, Rituximab
• Chimerism and veto cell associated tolerance
• Kidney transplantation without conventional
immunosuppression
Donor-Healthy relative with compatible blood groupInformed consent and study forms approved by IRB
• N=22, M: F: 20 : 2, • Mean Age (years): 28.3 (16-53)• Donor profile: HLA mis-match A/B/DR: 2.08 ± 1.02.
Parents: 17, Sibs: 4, Son: 1
Cross gender donors: 16• Mean CD34+ infused: 2 X 106/kgBW (range: 0.14-9.3)• Basic DS:
CGN : 17
Lupus Nephritis : 01
FSGS : 01
Vasculitis : 01
Primary IgAN : 01
MPGN : 01
Patient and Donor Profile
Generation and expansion of veto cells from CD34+ cells
• 100 ml BM aspirated from donor PSIC under LA after cytokine stimulation
(G-CSF, 600 g s.c.x 2 days)
• BM collected in T.C. flask with specific media for BM containing an early
acting cytokine mixture including SCF,TPO and Flt3 ligand
• In vitro expansion- CO2 incubator for 7-8 days at 37ºC under humidified
condition with 5% CO2 95% O2 , media changed every other day
• On 7/ 8th day cells were harvested and checked for sterility, viability and counts
under FACScan for yield
• Portal infusion of recipient on 8th day
• Second deletion cycle with Bortezomib to abrogate all antibodies
• Transplantation without immunosuppression with favorable immune response
• Follow-up (mths): 14.9 (10.4-19.6)
• Mean S. Cr. (mg%): 1.7 ± 0.6(0.9-3.2)
• Rejections -3 (13.6 %) (T/B/Suspicious)
Immunosuppression: Mean SCr (mg%)
Nil: 3 - 1.22 ± 0.16
Pred only: 10 1.6 ± 0.64
Tac./MMF : 8 1.97 ± 0.67
Clinical Results
n=22
CD34+/33- CD34+/33+ CD34-/33+
BM
Beginning
0.38%
(0.03-1.08%)
0.17%
(0.01-0.88%)
0.67%
(0.05-2.19%)
CBM
End
0.70 %
(0.08-2.85)
0.53%
(0.05-2.61%)
0.82%
(0.08-2.21%)
Rise in Veto
Cells
2 fold
p= 0.006
3.1 fold
p= 0.001
1.2 fold
p= NS
Lab ResultsVeto Cell Expansion In Cultured BM
n=22
CD34+/33- CD34+/33+ CD34-/33+
before 0.04 ± 0.03 0.02 ± 0.01 2.47 ± 1.84
9 months later
0.09 ± 0.08 0.29 ± 0.42 6.62 ± 3.09
Statistical
Significance
P =0.009 P =0.07 P= 0.09
Post Tx Status of Veto Cells In Renal Allograft Recipients
Lab Results
CD34+/33-: 0.13 CD34+/33+:0.14CD34-/33+: 0.20
BM: Before in-vitro expansion CBM: Post in-vitro expansion
CD34+/33-: 0.22 CD34+/33+:3.69CD34-/33+: 7.13
Pt.: Before infusion Pt.: Post-Tx- in vivo expansion
CD 4+: 36.98 %CD8+: 60.75 %
CD 4+: 43.17 %CD 8+: 54 %
CD34+/33-: 0.15 CD34+/33+:0.13CD34-/33+: 0.03
CD34+/33-: 0.36 CD34+/33+:0.38CD34-/33+: 8.56
H & E, X 100 PAS, X 100
S. P., 38, F, Basic Ds: DPLN, Donor: father, HLA match:4/6, Tx dt: 20th Apr,09, Surveil. Bx- 6 mths posttx., SCr: 0.8 mg%, No drugs
A., 23,M, Basic Ds: CGN, Donor: Mother, HLA match:3/6, Tx dt: 7th Apr,’09, Surveil. Bx- Normal, SCr: 1.48 mg%
Peripheral blood LH. chimerism: 6.8 % at 6 mths posttx, IS- 5 mg Pred/day
PBC BY FISH
CDB36: RN, 19/M (father) Tx: 12 June ‘09 3 June 2010
A B C DRB1 DQ DRB345 Bw
PT 1132(1
9) 18 51(5) 7 14 2 3 6(1) 2 51 52 4 6
DN 1 11 5 40 - 1415 (2) - 6(1) - 51 - 4 6
19
POD: 356
5/<5/75/<5/7
7/5/10FCM -/-7/5/10FCM -/-
MP (x4)
Prednisone
MP (x4)MP (x1)
MP (x4) +IVIg (x6)
DSA
MCDB11: SG, 36/M (brother)Tx: 8 Dec ‘08
A B Cw DRB1 DRB345 DQ Bw
PT 1 11 51 (5) 60(40) 3 - 8 13 52 - 1 7(3) 4 6DN 1 11 51 (5) 60(40) 3 - 8 13 52 - 6(1) 7(3) 4 6
POD: 51810 May 2010
20
12/10/15FCM -/+
12/10/15FCM -/+
5/<5/7FCM -/-5/<5/7FCM -/-
MP(x3)
MP(x4)
Biopsy
Prednisone
MP(x4)
Veto Cell Activity Noted As Absence of MLR
Positive MLRG0 / G1=2.48 %
AfterNegative MLR
G0 / G1=96.18%
73.68%2.48%
1.08%
Discussion
• Stem cell dose escalation is one way to overcome immune
rejection of incompatible transplant
• Studies suggest that veto cells within CD34+ progenitors
mediate facilitating effect to overcome major genetic barriers
and enable rapid and durable engraftment of mismatched
transplants without GVHD
• FACScan study revealed that the predominant phenotype of
CD34+/33- cells used at initiation of the culture was replaced
at the end of a culture by cells expressing phenotype of early
myeloid phenotypes such as CD34+/33+ and CD34-/33+ Cont…
Discussion
• What are the benefits due to ?
• Bortezomib with other deletional agents ?
• Stem cells and Veto cells ?
• It is the combination of both
• Stable graft function
• Normal surveillance Bx of graft
• Existence of long lasting peripheral blood chimerism
• Persistence of Veto cell counts
• Persistence of Veto activity (Negative MLR)
Conclusion• Transplantation without conventional immunosuppression is now
a reality at Ahmedabad using veto cells and clonal deletion
• Human CD34+ cells in short term cultures are usually associated
with a significant loss of self-renewal capacity, our cells have still
retained this self-renewal capacity and also achieved
differentiation into myeloid phenotype
• More veto cells can be harvested by culturing of human CD34+
cells for short term with an early-acting cytokine mixture
including SCF,TPO and Flt-3 ligand
• This vaccine will open up new doors to treat other autoimmune
disorders if treated on time