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

Our Experience With Veto Cells

Study Design - Oct ’08- ‘09

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

Sufferings have no age, gender, religion, race or color. Let this research give the gift of good healthy life to all

allograft recipients across the globe!