Novel non-HLA antibodies and rejection Duska Dragun Nephrology, Charité Campus Mitte, Humboldt University Berlin

Antibody mediated acute rejection The issue is not trivial, humoral rejection causes 30% of acute rejections and 10% of graft failures Pathogenesis donor-specific antibodies against HLA class I and class antigens (C4d+) „non-HLA“ antibodies against unknown targets„non-HLA“ antibodies against unknown targets

Subpopulation of patients with „unclear“ rejection severe pathology: endarteritis or fibrinoid necrosis 16/16 cases negative for HLA class I and class II DSA no hereditary or autoimmune thrombophilia negative serology for autoimmune disorders

Dragun, D. et al. N Engl J Med 2005;352: Inferior survival in patients with non-HLA antibody rejection

HLA DSA-HLA DSA+ n 1613 donor Living donor 7/165/13 Male 8/164/13 First kidney graft 7/165/13 Cold ischemia [h] 10.3 (1.6 – 13.3) 13.2 (1.8 – 16.2) HLA-mismatches 3 (0 – 5)3 (2 – 6) Age at transplantation [years] 34.8 ( ) 43.6 ( ) Time transplantation to rejection [days] 4 () 4 (2-1217)9 (3-680) Demographic data

Clinical manifestations and biopsies HLA DSA-HLA DSA+ Severe hypertension 16/160/13 5/1613/13 Histology: C4d positive

Interesting detail from patients´medical history: „During my pregnancy, 25 years ago, I also had very high blood pressure and I almost lost my baby.“ Our index case 50-years old female recipient of the first, zero mismatch “full-house” kidney transplant with primary graft function (creatinine 1.0 mg/dl at postoperative day 3) develops refractory vascular rejection with BP 240/160 mm Hg. Why is this patient rejecting? Autoimmune serology is negative; CMV negative No hereditary thrombophilia.

J Clin Invest 103: , 1999

Patients´IgG Bioassay for Ang II Typ1–Rezeptor (AT1R) agonistic antibodies

HLA neg (before Tx) HLA neg (rejection) HLA pos (before Tx) HLA pos (rejection) C4d+ Bioassay results for Ang II type 1–receptor (AT1R) activity

IgG activate Angiotensin II Type 1 Receptor Ang IIAT1R-AA+Losartan+PD Increase in BPM

AT1R agonistic activity in transplant nephrectomies AT1R-AA Serum AT1R-AA Niere Losartan Increase in BPM

Dragun, D. et al. N Engl J Med 2005;352: Losartan + PPH improve graft survival in AT1R-AA+ patients

Influence of losartan + PPH on AT1R activity Dragun, D. et al. N Engl J Med 2005;352:

IgG1IgG2IgG3IgG Increase in beat number/min AT1R IgG subclass activity

Ang II type 1 receptor Binding sites of AT1R activating IgG1 and IgG3 Ingelfinger, J. R. N Engl J Med 2005;352:

Incidence of AT1R-AA positive rejection 279 kidney transplantations in 4.5 years (Jan 1st 2000 – July 31st 2004) 119 rejection episodes in 83 patients 23 refractory to steroids (19.3% of all rejections)

Incidence of AT1R-AA positive rejection 23 refractory to steroids (19.3% of all rejections) 9 episodesHLA-DSA pos 39.1% of refract. rejections 9 episodes HLA-DSA pos 39.1% of refract. rejections 10 episodesAT1R-AA pos 10 episodes AT1R-AA pos 43.5% of refract. rejections 4 HLA-DSA neg/ AT1R-AA neg 7.6% of all rejections 8.4% of all rejections

ECs, VSMCs Interdisciplinary „bedside to bench“ approach

ERK ½ phosphorylation coronary endothelial cells coronary VSMC

NF-kB

C4d+ Mutant promoter Tissue factor is a target of NF-kB and AP-1 AT1R-AA+ Cell promoter transfection experiments

TF vor PPH + LosartanTF nach PPH + Losartan Tissue Factor expression in patients´ biopsies

Kochs´postulate study to investigate pathogenic role of AT1R-AA in transplant vascular pathology passive transfer of human IgG with AT1R agonistic activity

F344 donor Lew recipient Orthotopic life-supporting functional NTx rat model AT1-AA c telemetry device implant. 7 d. pre NTx NTx, osmotic mini-pump 7 d. post NTx allograft morphology Telemetry follow-up Control IgG

AT1R-AA induce vascular rejection AT1R-AA+ human IgG AT1R-AA- human IgG

Colocalization of AT1R-AA (human IgG) with AT1R AT1R-AA+ human IgGAT1R-AA- human IgG human IgG AT1R

AT1R-AA induce hypertension Dragun, D. et al. N Engl J Med 2005;352:

1.The agent (AT1-AA) should be present in all cases but not in controls 2.The agent (AT1-AA) must be isolated from the cases and studied in cells 3.The agent (AT1-AA) transferred into a healthy laboratory animal, should cause the disease 4.The agent should be re-isolated from the experimental disease Do AT1R-AA fulfill Koch´s postulates?

Apoptosis?Permeability? AT1R-AA Putative mechanism of action of AT1R-AA ECs VSMCs AT1R AT1R-AA MAPKs (Erk 1/2) AT1R-AA MAPKs (Erk 1/2) NF-  B, AP-1 TF TF Thrombotic Angiopathy! MCP-1 Mø RANTES CTL Effector cell infiltration!

Max-Delbrück-Centre Berlin Gerd WallukatNephrology, Charité Campus Buch Ralf Dechend Dominik N. Müller Ralph Plehm Jan Hinrich Bräsen Friedrich C. LuftPharmacology, Charité Campus Mitte Ulrich Kintscher Thomas UngerNephrology, Charité Campus Mitte Diana Eckert Melina Nieminen-Kelhä Lutz Fritsche Klemens Budde Hans-H. Neumayer HLA Laboratory Charité Constanze SchönemannPathology, Charité Campus Mitte Birgit Rudolph CNRS, Strasbourg Johan Hoebeke AT1R-AA collaborators