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RIP 16April2010

Research in Progress Seminar, STI
by

Eefje Dons

on 21 April 2010

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Transcript of RIP 16April2010

Research in Progess Seminar
Starzl Transplantation Institute, University of Pittsburgh Delayed development of anti-A, B, and Gal antibodies in immunosuppressed infant baboons Eefje M. Dons, MD
Dr. Cooper's laboratory
16 April 2010 Background Hypothesis Study design Results Group 1 (n=6) : Controls Group 2 (n=2): Artery Tx, no IS Group 3 (n=4) : AB-I or Pig Tx + IS Group 4 (n=2) : IS only, no Tx Overall Conclusions Blood groups A and B, as well as Gal[alpha]1,3Gal (Gal) are carbohydrate antigens expressed on RBC, vascular endothelium and certain other cells.

Natural anti-A/B or anti-pig antibodies (Abs) begin to develop during the first months of life in infant humans and baboons

Natural antibody production is considered to be T cell independent (Ohdan et al. Transplantation 2000;69:910-913)
Human infants: anti-A/B antibody development Fong et al. Transfusion 1974; 14(6):551-559 In humans: ABO-incompatible Tx in adults: preformed IgM Abs cause hyperacute rejection

ABO-incompatible heart Tx in infants - before the development of natural Abs - leads to absence of production of anti-A/B Abs; B-cell tolerance is achieved
Fan et al. Nat Med 2004; 10(11):1127-1133 Relevance for Xenotransplantation: Anti-pig antibodies are primarily directed against carbohydrate antigens on porcine cells

We investigated whether development of anti-pig antibodies in infants is similar to anti-A/B antibody production We have previously studied the development of IgM and IgG Ab binding to WT and GTKO pig PBMC (FCM) Rood et al. Transpl Int 2007;20:1050-1058 Infant humans: Infant baboons: Similar to ABO-incompatible Tx, if an infant receives a WT pig transplant, before the development of natural antibodies, then those anti-pig antibodies will NEVER develop, and B-cell tolerance may result.

We investigated this by carrying out aortic patch transplants in infant baboons, age 3 months
Donor Carotid Artery
AB-incompatible baboon (AB-I)
Wild-type pig (pig)

Recipient Abdominal Aorta
Non-immunosuppressed (No IS): Sensitization?
Immunosuppressed (IS): Development of Abs?
Aortic patch transplants in infant baboons, age 3 months Anti-A/B and Gal antibodies (ELISA) Group 1 (n=6), Controls Binding to WT pig PBMC (FCM) Group 1 (n=6), Controls WBC and CD4+ T cell count Group 1 (n=6), Controls Group 1 (n=6) Conclusions Gradual increase in anti-A/B IgM antibodies; detectable by 3-4 months. No increase in anti-A/B IgG (ELISA).

More rapid increase in anti-Gal IgM (ELISA) and anti-pig IgM binding to WT PBMC (FCM). No increase in anti-Gal IgG.

WBC count and CD4+ T cell count remain steady throughout the first year.
Anti A/B and anti-Gal antibody levels Group 2 (n=2) Binding to WT pig PBMC (FCM) Group 2 (n=2) Group 2 (n=2) MLR Group 2 (n=2) Conclusions We have confirmed that sensitization occurs in the infants that received artery patch grafts without immunosuppressive therapy

The sensitization was only to the specific donor, i.e., to the incompatible AB blood type (in the baboon that received an allograft) or to the pig antigens (in the baboon that received a wild-type pig graft)

The sensitization was shown by antibody- and cellular responses
IgG IgM IgM IgG Anti-A/B and anti-Gal antibody levels (ELISA) Group 3 (n=4) Development of natural Abs (ELISA) Group 3 (n=4) WBC and CD4+ T cell count versus controls Group 3 (n=4) Group 3 (n=4) MLR Group 3 (n=4) Conclusions During IS, IgM production was lower than in controls. This was irrespective of donor tissue origin (AB-I / pig)

Several months after IS discontinuation: IgM antibodies began to develop to both A / B and pig, but those were NOT specific to the donor

No IgG development to either A/B or pig Ag irrespective of donor origin Group 3 Conclusions (2) All Group 3 infants had overall reduced Ab production compared to age-matched controls

This was related to the IS rather than presence and type of a specific graft In view of this conclusion, we investigated the effect of IS alone on the development of natural Abs

IS only, from 1 – 8 months of age
309: antiCD154mAb 25 mg/kg/wk
209: CTLA4-Ig 25 mg/kg/wk

No other therapy (e.g., ATG / MMF) Anti-A/B and anti-Gal antbodies (ELISA) Group 4 (n=2) Binding to WT pig PBMC (FCM) Group 4 (n=2) Group 4 (n=2) CD4+ T cell count Group 4 (n=2) MLR Group 4 (n=2) Conclusions antiCD154mAb:
While receiving IS: no development of IgM Abs
After discontinuation of IS: slow development of IgM Abs
No IgG development

CTLA4-Ig:
Little effect on natural Ab production Treatment with antiCD154mAb seems to inhibit natural Ab production

This observation questions the assumption that development of natural Abs is T cell independent Group 3 Complication: Collagenous Colitis All 4 infants developed features of CC within the same time period of 6 weeks

1 infant died, 1 was euthanized, 2 recovered

In humans: etiology unknown. Relation with autoimmune disorders. Has been described in relation to Tx

In baboons: described in one case

Timing and close proximity of all 4 animals suggests an infectious origin
Thank You!

Dr Cooper and lab:
Burcin Ekser
Cassandra Long
Claudia Montoya
Corrin Ezzelarab
Gabriel Echeverri
Hidetaka Hara
Mohamed Ezzelarab Rotterdam - Dr. J.N.M. IJzermans
DLAR – Dr. R. Wagner
Edmonton - Drl L.J. West
Oklahoma – Dr. R. Wolf Immunosuppressive regimen Induction: ATG

Maintenance:
anti-CD154mAb 25 mg/kg/wk i.v.
MMF 50-120 mg/kg/x2day p.o.

Start: 3 months of age
End:12 months (n=1) or 8 months (n=3)

Gp 2 Gp 3 Gp 3 Gp 2 Gp 3 Gp 3 Group 3 (n=4) B cell count versus controls
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