Transplant Immunology & Renal Allograft Rejection Shehzad Rehman, MD Assistant Professor Division of Nephrology, Hypertension and Renal Transplantation University of Florida

HLA: Introduction Main barrier to transplantation is Major Histocompatibility Complex (MHC) MHC in humans is known as Human Leukocyte Antigen (HLA) Short arm of chromosome 6 Inherited as 2 haplotypes (half-sets), one from each parent

Types of HLA Class I and class II Class I: A, B, C Class II: DR, DP, DQ Important for transplant: A, B, DR Numbered sequentially Typical: A2, A23; B7, B13; DR3, DR7

Human Chromosome 6 2 types of HLA Class I HLA Class II HLA Slide courtesy Dr. Casey

Human Leukocyte Antigen Class I HLA expressed on all nucleated cells presents antigen to cytotoxic CD8(+) T cells which induce cell death Class II HLA expressed only on antigen-presenting cells (APCs) presents to helper CD4(+) T cells which activate macrophages, B cells, effector T cells APC: dendritic cells, macrophages, B-cells However, don’t be confused that B cells have class I and II Ag and T cells only have class I Ag.

Mechanisms of Rejection Cellular and antibody-mediated components Two stages Sensitization stage Effector stage Two kinds of response Amnestic response Anamnestic response

Sensitization Stage T-Cell receptors (TCRs) recognize foreign antigens Two signals needed: Signal 1: HLA interacts with TCR (CD3) Signal 2: Costimulation: CD28 with CD80 or CD86; or CD40 with CD40L Absence of costimulation may lead to clonal anergy

Sensitization: Direct Pathway Donor APCs carried over on graft present antigens to host T-cells Usual APCs are dendritic cells Important and dominant in early, acute rejection processes

Sensitization: Indirect Pathway Host APCs ingest and present allopeptides to host T-cells Lead to T-cell proliferation Important in late/chronic processes Also important in antibody-mediated rejection processes

Second Messenger Systems Inositol phospholipid → IP3 & DAG This leads to intracellular Ca2+ release Ca2+-calmodulin complexes form, triggering second messenger systems Calcineurin → NFAT PKC NFκB These bind to the IL-2 gene and promote IL-2 transcription via mRNA

Effector Stage IL-2 and other cytokines promote inflammation and proliferation Signal 3 β-chemokines cause macrophage infiltration Growth factors cause fibrosis and vascular damage Final common pathway is cell apoptosis

Halloran, N Eng J Med; 351:3715, 2004

Classification Acute rejection can be classified based on various patterns: Time of onset: early vs. late Pathogenetic mechanisms: antibody-mediated or cellular Clinical/histological features: hyperacute, accelerated, acute, chronic.

Hyperacute Rejection Irreversible antibody-mediated rejection, occurring in minutes-to-hours. Preformed donor-specific antibodies. Can also be due to ABO-incompatibility. Immediate antigen-antibody binding with complement activation. Very rare in the post-crossmatch era. Histology: intense engorgement of the glomerular and peritubular capillaries with RBC clumping and fibrin thrombi.

Afferent arteriolar thrombosis in a case of hyperacute rejection

This small artery in a patient with hyperacute rejection is almost occluded by a fibrin thrombus

Accelerated Rejection Usually due to presensitization. Presents several days after transplantation. Usually represents an anamnestic reponse, resulting in rapid production of antibodies after exposure. Rejection starts after antibodies reach a critical level. Can be avoided with attention to crossmatch.

Acute Rejection Most commonly seen clinically. Can happen anytime, but 75% are seen within the first 3 months. 10-20% of develop AR in first 12 months. T-cell-mediated (cellular), B-cell or antibody-mediated, or both. Classified according to Banff Schema (Solez et al., Am J Transplant, 2008).

Acute Cellular Rejection Generally T-cell mediated. Activated via alloantigen-dependent mechanisms (classic antigen-presentation and 3-signal model). Can also be via non-alloantigen-dependent pathway (innate immunity: TLRs and NK cells).

Acute Cellular Rejection Borderline changes: Either tubulitis with <25% parenchymal inflammation or infiltration with 1-4 cells/tubule Banff I: tubulointerstitial inflammation only with >25% parenchymal involvement. IA: moderate tubulitis (5-10 cells/tubule). IB: severe tubulitis (>10 cells/tubule).

Moderate interstitial mononuclear inflammation affecting 25-50% of the sampled parenchyma (grade i2)

Banff IA: Moderate tubulitis with 5-10 mononuclear cells per tubular cross section

Banff IB: Severe tubulitis with greater than 10 mononuclear cells per tubular cross section.

Acute Cellular Rejection Banff II: vascular rejection IIA: Mild-to-moderate intimal arteritis (<25% luminal involvement in at least 1 artery) IIB: Severe intimal arteritis (>25% luminal involvement) Banff III: severe vascular rejection Transmural arteritis and/or arterial fibrinoid changes and smooth muscle necrosis.

Banff IIA: Mild intimal arteritis with rare subendothelial mononuclear cells.

Banff IIB: Moderate intimal arteritis with numerous subendothelial mononuclear cells.

Banff III: Severe intimal arteritis with transmural inflammatory infiltration, fibrinoid change and myocyte injury.

Treatment Borderline: based on clinical and pathological picture. Some advocate no treatment. Banff IA: generally steroid pulse. Banff IB: steroid pulse and/or ATG. Banff II/III: ATG Banff II & III may also require treatment for possible concurrent AMR.

Antibody-Mediated Rejection Antibodies can target the capillary endothelium, leading to complement fixation. This causes inflammation and endothelial injury  capillary endothelialitis Complement activation leads to increased presence of terminal products of complement cascade  C4d. Can uncommonly be due to non-anti-HLA antibodies.

Antibody-Mediated Rejection According to Banff, diagnosis of AMR requires: Presence of circulating donor-specific antibodies. C4d+ Morphologic evidence of acute tissue injury: ATN-like minimal inflammation Capillary and/or glomerular inflammation (PTC/G) Arteritis (v3) If either C4d or DSA are absent, Banff classifies as “suspicious for AMR”.

Aggregates of mononuclear inflammatory cells in dilated peritubular capillaries, scored as ptc3 

Transplant glomerulitis with infiltrating mononuclear inflammatory cells (arrows) within capillary loops

Acute humoral rejection in a human renal allograft Acute humoral rejection in a human renal allograft. PAS staining shows the presence of a sparse interstitial infiltrate of neutrophils, together with edema.  C4d deposition in peritubular and glomerular capillaries. An immunohistochemical stain using polyclonal rabbit antibodies that are specific for C4d shows C4d deposition.

C4d staining: immunohistochemistry vs C4d staining: immunohistochemistry vs. immunocytochemistry/immunofluorescence

Treatment Treatment of AMR is not well defined. Options include: ATG IVIG Plasmapheresis Rituximab Newer options include agents under study e.g. bortezomib, eculizumab.

Chronic Rejection The term “chronic rejection” was previously used to describe all manner of chronic changes in the allograft, on the assumption that they were immune-mediated. As non-immune factors became known, the term “chronic rejection” has fallen out of favor, to be replaced by chronic allograft nephropathy, and now, interstitial fibrosis/ tubular atrophy.

Chronic Rejection Some features remain consistent with chronic immune injury: Transplant glomerulopathy with double-contouring Certain vascular changes (disruptions of the elastica, inflammatory cells)

Chronic transplant glomerulopathy with numerous double contours (arrows) in glomerular basement membranes

Implications of Rejection Certain clinical features have important implications in acute rejection. Presence of rejection. Grafts have a longer half-life in patients who never had an acute rejection (Hariharan, N Engl J Med, 2000; Flechner, Transplantation, 1996). If fully reversible, may not have a deleterious effect on graft function in the long-term (Opelz, Transpl Proc, 1997; CTS 6-year data): 60% vs. 75% for treatment for rejection. 73% vs. 75% for those whose S/Cr returned to normal (<1.5 mg/dL)

Implications of Rejection Number of rejections (Dickenmann, Transpl Int, 2002; Pascual, N Engl J Med, 2002). Better with single episode than with 2 or more episodes. Timing of rejection (Sijpkens, Transplantation, 2003). 10-yr death-censored graft survival 86% for AR <3 months vs. 45% for AR >3 months Severity of rejection. Correlation between Banff grade and outcome.

Implications of Rejection Data presented at ATC 2011 suggested interstitial inflammation is a worse prognosticator. Newer data suggested vascular rejection may not carry the same grim prognosis in modern era. Worse prognosis for AMR.