Management of

Liver Transplant Rejection

Dr Palepu B Gopal MD,FRCA,CCST,FICCM,FCCM

Consultant Critical Care Medicine

Hyderabad

Complications Of Organ Transplantation

1- Rejection

2-Malignancy

Causes of Allograft Failure Primary Nonfunction – slightly more

common in Living Donors Vascular Complications – 10% of patients

Hepatic Artery Thrombosis/StricturePortal Vein Thrombosis/StrictureHepatic Vein Thrombosis/Stricture

Biliary Complications – Donors after Cardiac DeathLiving Donors Anastomotic vs nonanastomotic strictures

Causes of Allograft Failure- Rejection Antibody Mediated Rejection – hours

to days 10-20% Acute Rejection

Risk 1st 3months>1st year>subsequent years

Chronic Rejection – a primary RF is prior episodes of Acute Rejection.

Acute vs Chronic – ○ time course○ pattern of liver enzyme

abnormalities○ response to therapy

Transplant Rejection – Issues for Critical Care

Single organ dysfunction of organ under Rejection

MOF related to single organ dysfunction

Extended Immunosuppression

Infections

Drug Interaction

Toxicity

Sepsis and MOF

Drug Toxicity

Renal Failure

Immunology andEtiopathogenesis

of Rejection

Rejection

Causes reduction or withdrawal of immunosuppressants, erratic absorptionpoor compliance

Poor prognosis Rejection in Hep C TxHigh risk for Ac rejection Age less than 30 years Tx for an autoimmune liver disease

ALDChild’s A at the time of TxInterferon for Hep C

Low Risk for Ac Rejection Age more than 60 yearsTx for Alcoholic liver diseaseChild’s class C at the time of Tx

Immunospp. Regimen high-dose steroids response 80%Others require antilymphocyte therapyLess rejection with tacrolimus than

cyclosporine regimens14% rejection rate with Tacro and

sirolimus combination

Late Hepatic Allograft Dysfunction : Wiesner et al. Liver Transpl 2001

Late Hepatic Allograft DysfunctionRussell H. Wiesner and K.V. Narayanan MenonLiver Transplantation, Vol 7, No 11, Suppl 1 2001

Types of Transplant

Autograft is self-tissue transferred from one body site to another in the same individual.

Isograft is tissue transferred between genetically identical individuals.

Allograft is tissue transferred between genetically different members of the same species.

Xenograft is tissue transferred between different species

Immunology of Transplant Rejection

Components of the Immune system involved in graft Rejection :1) Antigen presenting cells –

○ Dendritic cells○ Macrophages○ Activated B Cells

2) B cells and antibodies – ○ Preformed antibodies○ Natural antibodies○ Preformed antibodies from prior sensatization○ Induced antibodies

3) T cells

4) Other cells – ○ Natural killer cells○ T cells that express NK cell – associated Markers○ Monocytes/Macrophages

Recognition of Alloantigens

Direct Presentation Recognition of an intact MHC molecule displayed by donor APC

in the graft Basically, self MHC molecule recognizes the structure of an

intact allogeneic MHC molecule Involves both CD8+ and CD4+ T cells.

Indirect Presentation

Donor MHC is processed and presented by recipient

APC

Basically, donor MHC molecule is handled like any other

foreign antigen

Involve only CD4+ T cells.

Antigen presentation by class II MHC molecules.

Activation of Alloreactive T cells and Rejection of Allografts

Donor APCs migrate to regional lymph nodes and are recognized by the recipient’s TH cells.

Alloreactive TH cells in the recipient induce generation of TDTH cell and CTLs then migrate into the graft and cause graft rejection.

Activation of Alloreactive T cells and Rejection of

Allografts( )SENSATIZATION

Passenger leukocyte

.Class II MHCantigen

2IL HT HT

HTHT

Donar kidney

CTLDTHTCTL

DTHT

LYMPH NODEEFFECTOR

Role of Cytokines in Graft Rejection IL – 2, IFN – , and TNF - are important mediators of graft rejection.

IL – α promotes T-cell proliferation and generation of T –

Lymphocytes.

IFN - is central to the development of DTH response.

TNF - has direct cytotoxic effect on the cells of graft.

A number of cytokines promote graft rejection by inducing expression

of class – I or class – II MHC molecule on graft cell.

The interferon (α, and ), TNF – α and TNF - all increases class – I

MHC expression, and IFN - increases class – II MHC expression as

well.

Effector Mechanisms of Allograft Rejection - Types

Hyperacute Rejection Acute Rejection Chronic Rejection

Hyperacute Rejection Characterized by thrombotic occlusion of the graft Begins within minutes or hours after anastamosis Pre-existing antibodies in the host circulation bind

to donor endothelial antigens Activates Complement Cascade

1.Preformed Ab2.complement activation3.neutrophil margination4.inflammation, 5.Thrombosis

Acute Rejection Vascular and parenchymal injury mediated by T

cells and antibodies that usually begin after the first week of transplantation if there is no immunosuppressant therapy

Incidence is high (30%) for the first 90 days

T-cell, macrophage

and Ab mediated,

myocyte and endothelial

damage,

Inflammation

Chronic Rejection Occurs in most solid organ transplants

HeartKidneyLungLiver

Characterized by fibrosis and vascular abnormalities with loss of graft function over a prolonged period.

Macrophage – T cell mediated

Concentric medial hyperplasia

Chronic DTH reaction

Graft vs. Host Disease

Caused by the reaction of grafted mature T-cells in the marrow inoculum with alloantigens of the host

Acute GVHDCharacterized by epithelial cell death in the

skin, GI tract, and liver Chronic GVHD

Characterized by atrophy and fibrosis of one or more of these same target organs as well as the lungs

Clinical Picture and

Diagnosis

Wyatt (2010) Histopathology 57, 333–341

Biopsy

Causes of Liver Test Abnormalities in Asymptomatic Recipient

Allograft parenchymal damageImmune-mediated disease

rejection and de novo AIH)Recurrent Liver disease Drug toxicity

immunosuppressive drugsAlcohol and other toxinsDe novo infection

Bacterial, Viral (de novo HBV andHCV)

Space-occupying lesion De novo or recurrent NAFLD

Biliary damageBiliary strictures , anastomotic strictures, Hep A StrictureBiliary stones/cast syndromeRecurrent PSC

Vascular diseaseHepatic artery thrombosisPortal or hepatic vein thrombosis

Metabolic disease of GraftGilbert’s syndrome

Nonhepatic diseaseHemolysis – indirect bilirubinBone disease raised ALP

Nonhepatic diseaseCeliac diseaseDiabetes

Time to first rejection episode among adult liverrecipients who rejected following liver transplantation.

Late Hepatic Allograft DysfunctionRussell H. Wiesner and K.V. Narayanan MenonLiver Transplantation, Vol 7, No 11, Suppl 1 (November), 2001

Prevention and

Management of Rejection

Immunotherapy is truly a treatment that delays the inevitability of graft rejection.

Rejection Episodes can be treated by the one of the following treatments:

Corticosteroid

Polyclonal and Monoclonal Antibodies

Antiproliferatives

Alternative Therapies

Xenotransplantation

Tissue Engineering

Stem Cell Research

Induction of Tolerance

Episodic Treatment of Rejection

Recommendations for Prevention and Tt of

Rejection

Immunosuppressants recipients should be prescribed

and monitored by expertis in that area

The choice of agents is multi-factorial, and no one

regimen can be recommended for any patient

(grade 2, level A)

Reviewed at least every 6 months and modified as

required

Goal of minimizing long term toxicities (grade 1, level

B).

Rejection can be diagnosed only on the basis of liver

biopsy classified according to Banff criteria (grade

1, level A)

Long-Term Management of the Successful Adult Liver Transplant: 2012 Practice Guideline by the ASSLD and AST. Michael R. Lucey: LIVER TRANSPLANTATION 19:3-26, 2013

Clinical Immunosuppression

Two types of immunosuppression are used in transplantation: Induction and Maintenance immunosuppresion

Adverse side-effects that includehigh incidence of opportunistic infection transplant-related malignancies in patients

Major goal of immunosuppression

Identify the optimal balance of therapy Effective prevention of allograft rejection Minimized Adverse effects, infection & malignancies

A cute cellular rejection (ACR) is a very commonevent that occurs in utpo 70% of orthotopicl ivertransplant (OLT) recipients within the first year.l Thehighest incidence is around 7 to 10 days posttransplantation.ACR occurs i n 10% to 70% of patients,depending on the primary immunosuppression used,the indication for the transplantation, and the definitionof rejection.histologic features of portali nfiltrate and endotheliitis, which suggest mildACR, but without biochemical graft dysfunction, oftenresolves without adjuvant immunosuppression.3So, liver biopsies should be delayed until patientsdevelop biochemical graft dysfunction, unexplainedfever, or other surrogate markers of rejection.

Liver Transplantation, Vol8, No 12 (December), 2002:pp 1147-1 153

Immunosuppressants

Timeline for the introduction of immunosuppression medications.Immunosuppression in Liver Transplantation. Post et al. LiverTransplantation, Vol 11, No 11,2005: pp 1307-1314

Immunosuppressive Agents

Immunosuppression can be achieved

in by

1. Surgical ablation

2. Total Lymphoid Irradiation

3. Immunosuppressive drugs

Immunosuppressive Drugs

Three main immunosuppressant drugs

Cyclosporins act by inhibiting T-cell

activation, thus preventing T-cells from

attacking the transplanted organ

Azathioprines disrupt the synthesis of DNA

and RNA and cell division

Corticosteroids such as prednisolone

suppress the inflammation associated with

transplant rejection

Organ Specific Immunosuppressant Classification

Kidney transplants

○ Basiliximab in combination with cyclosporin and corticosteroids

Kidney transplants

○ Daclizumab in combination with cyclosporin and corticosteroids

Kidney, liver and heart transplants

○ muromonab CD3 (Orthoclone OKT3) along with cyclosporin

Liver transplants Tacrolimus

○ under study for kidney, bone marrow, heart, pancreas, pancreatic island cell, and small bowel transplantation.

Induction TherapyDef: prophylactic application of periop antibodies in addition to baseline immunosuppression. (immediate posttransplant)

The goal : induce hyporesponsiveness in the organ recipient toward the transplanted organ in order to prevent early post-transplant rejectionOnly T cells are inhibited and the rest of the patient's immune system would remain fully functional

Agents: Earlier nonspecific polyclonal antibodiesToday, however, murine monoclonal anti-lymphocyte antibodies, such as OKT3, are often usedAntimicrobials are a necessary component

Maintenance immunosuppression

Drugs administered to maintain immuno-suppression once recipients have recovered

from the operative procedure. Agents are either

Biological Monoclonal and polyclonal antibodies

Generally used in induction protocols Nonbiological agents

Mainstay of maintenance protocols.

corticosteroidsazathioprine cyclosporines

Maintenance TherapyMaintenance immunosuppression refers to the classic combination therapy (known clinically as Triple Therapy) to which transplant recipients usually adhere for the rest of their lives.

The Combination includes

a corticosteroid

a calcineurine inhibitor

an antiproliferative

The Goal Balance between underimmunosuppression and overimmunosuppression

Consider potential interactions

Calcineurin Inhibitors (CNIs)Calcineurine (cytokines activator ) Inhibitors are used in maintenance immunosuppression.Cyclosporin A From fungus Tolypocladium inflatum

selective suppression of CMI via inhibition of T-cell activation

standard formulation (Sandimmune) emulsion depends on bile flow, GI motility & food intake

Unpredictable bioavailabilityMicroemulsion in lipophilic solvent (Neoral)

Predictable bioavailabilityDrug InteractionsNephrotoxicity – up to 20% post OLT Renal

failureNeurological manifestations – 10-28%hyperkalemia, hypomagnesemia,

hyperglycemiaHypertension, gingival hyperplasia and

hirsutism

Immunosuppression in Liver Transplant. Post et al. LiverTransplantation, 2005

Tacrolimus

From fungus Streptomyces tsukubaensis in Japan

Tacrolimus is a microlide antibiotic

100 times more potent than CyA

Inhibits calcineurin

TAC absorption uninfluenced by presence of bile

Lower first year rejection rate better with TAC

compared to CyA

Hepatitis C infected patients on TAC have longer

graft survivalImmunosuppression in Liver Transplantation. Post et al. LiverTransplantation, Vol 11, No 11,2005: pp 1307-1314

Post-transplant diabetes mellitus

Nausea, vomiting, diarrhea

Hyperkalemia

Tremor

Hypertension

Hypomagnesemia

Headache

Renal dysfunction

Common Side Effects of Tacrolimus

Sirolimus Binds to same immunophilin as Tac (FKBP12) but

with a different mechanism of action

○ blocks response of T and B Cell Activation by cytokines

Theoretical (lab based) antineoplastic and antifungal effects.

Early excitement about renal protective effect- subsequent studies have not confirmed this

Black Box warning – possible increased risk of Hepatic Artery Thrombosis in immediate post-OLT setting

Recent study of switch from CNI to SRL suggests possible increased mortality

Currently using in those intolerant to CNIs, and in some patients for theoretical antineoplastic and renoprotective actions

Hepatology. 2010 Oct;52(4)

Sirolimus – Adverse Effects

• Anemia

• Hypercholesterolemia

• Hypertriglyceridemia

• Leukopenia

• Hyperlipidemia

• Interstitial lung disease

• Thrombocytopenia

• Peripheral edema

• Wound dehiscence

• Hepatic Artery Thrombosis

CorticosteroidsPrednisone and Methylprednisolone

Corticosteriods Maintenance immunosuppression

Teatment of acute rejection

Diffuse into cells and bind to specific cytoplasmic receptors progress to the nucleus, inhibit the transcription of the genes of the cytokines

Block T cell, APC and Macrophase cytokines

IL-1, IL-6, IL-2, INF-γ , and TNF-α

Non-specific anti-inflammatory agents acutely reduce lymphocytes & monocytes

Pretransplant bolus dose of methylprednisolone 500 to 1,000 mg, followed by rapid taper over next few weeks to minimal dose, i.e., 25-50 mg/day.

Common Side Effects of CorticosteroidsHypertension

Mental status changes

Lipid abnormalities

Impaired wound healing

Hyperglycemia

Cushingoid syndrome

Ulcers

Myopathy

Osteoporosis

Fluid retention

Cataracts

Antiproliferative Agents / Antimetabolites

Mycophenolate Mofetil, Azathioprine, Sirolimus

Antiproliferative Block the proliferative phase of ACR

Maintenance immunosuppression

Treatment of rejection

Azathioprine Mercaptopurine derivative & antagonises purine metabolism

Significant myelosuppression and hepatotoxicity

Use decreased dramatically

Mycophenolate mofetil and Mycophenolic acid

Mycophenolate Mofetil (MMF)

Rapidly hydrolyzed in the blood to its active form: MPA

MPA inhibits enzyme IMPDH in the pathway of purine biosynthesis

Activated lymphocytes are selectively inhibitedNausea, vomiting, diarrhea, Pancytopaenia

Antibody Induction Antithymocyte Globulin – induction/rejection.

Polyclonal antilymphocyte globulin – multiple epitopes on T cell receptor – lead to apoptosis of T-cells

ATGAM (of equine origin) Thymoglobulin (of rabbit origin)

Monoclonal anti T-Cell antibodies – induction/rejectionMuromonab-CD3 (OKT3) – binds CD3 Antigen

on T-Cell receptor – inactivates adjacent T-Cell – leads to rapid drop in T-Cells

IL-2 Receptor Antibodies – inductionBasiliximab (Simulect) daclizumab (Zenapax).

Monoclonal AntibodiesMuromonab-CD3 (Orthoclone OKT3®)

Used in LTx in 1987 for prophylaxis against acute cellular rejection and later to reduce CNI exposure and treatment of steroid-resistant rejection.OKT3 use today is much lower, with many centers preferring treatment regimensfor acute cellular rejection to increasing TAC blood levels and then adding corticosteroid boluses if rejection is still present Daclizumab (Zenapax®)Interleukin-2 Receptor Antagonist (Basiliximab, Simulect®)Monoclonal Antibodies are used in early rejection prophylaxis and treatment of rejection.Monoclonal antibodies are antigen-specific immunosuppressants that will reduce immune response to alloantigens of the graft while preserving the response to alloantigens to unrelated antigens.These agents are specific to blocking T-cell activation, resulting in rapid depletion of T cells from the circulation by binding of antibody coated T cells to Fc receptors on phagocytic cells. The most recently FDA approved monoclonal antibodies are the IL-2 receptor antagonists genetically engineered to possess both human and murine antibody sequences. The chimerization of these antibodies is an attempt to decrease the immunogenicity of the agent. Other monoclonal antibody-based drugs are still in clinical trials for FDA approval.

Monoclonal Antibodies

Muromonab-CD3 is the first type of murine monoclonal antibody directed against the ε chain of the CD3 molecule (an integral part of the T Cell Receptor complex) and modulates the receptor and inactivates T-cell function blocking both naïve T cells and CTLs. This results in rapid depletion of T cells from circulation and cytokine release. This antibody is used to treat acute rejection and steroid resistant rejection.

Basiliximab is a chimeric (70% human and 30% murine) monoclonal antibody utilized in the prevention of acute organ rejection. This monoclonal antibody has a specificity and high affinity for the α subunit of the interleukin (IL)-2 receptor (IL-2Rα, also known as CD25 or Tac) preventing IL-2 from binding to the receptor on the surface of activated T cells. By acting as an IL-2Ra antagonist, Basiliximab inhibits IL-2-mediated activation and proliferation of T cells, the critical step in the cascade of cellular immune response of allograft rejection. Therefore, Basiliximab has a long half-life of approximately 7-12 days and saturates the IL-2 receptor for up to 59 days. Due to its high percentage of humanization in its antibody sequences the occurrence and acuteness of adverse effects is significantly lower when used with standard immunotherapy.

Monoclonal Antibodies

Muromonab – CD3 Daclizumab is a similar agent to Basiliximab, but is a more humanized IgG monoclonal antibody (90% human and 10% murine). It also binds to and inhibits the α-subunit of IL-2 receptor and thus works in a manner similar to Basiliximab. Daclizumab is able to saturate the IL-2 receptor twice as long as Basiliximab. Because it is more humanized, there are less side effects associated with Daclizumab.

Side Effects – HAMA One of the major problems affecting monoclonal therapy is the side effects associated with the HAMA (Human anti-Murine Antibody) response and serum sickness. These are both directly caused by the structure of the antibodies. Laboratories routinely use murine antibodies as a starting point for monoclonal antibodies.

A counteractive measure includes humanizing (or chimerizing) the antibody. Because the only immunologically offensive portion of the antibody is the constant region, recombinant techniques can be used to splice and replace the constant region of the murine antibody with that of a characteristic human antibody. Current drugs have been developed that are more favorably humanized. They are also shown to be more effective with less adverse side effects. Further chimerization and other improvements in antibody therapy are active fronts of current research.

This poses a problem: human immune system is able to identify the murine antibody as non-self and eliminate the treatment from circulation. This renders the monoclonal therapy ineffective.

Immunosuppressive Therapy

Monoclonal antibodies To suppress the activity of subpopulation of T-cells. To block co-stimulatory signals. Ab to the CD3 molecule of TCR (T cell receptor) complex results in

a rapid depletion of mature T-cells from the circulation. Ab specific for the high-affinity IL-2 receptor is expressed only on

activated T-cell, blocks proliferation of T-cells activated in response to the alloantigens of the graft.

To treat donor’s bone marrow before it is transplanted. Molecules present on particular T-cells subpopulation may also be

targeted for immunosuppressive therapy. Antibody to CD4 shown to prolong graft survival. Ab specific for implicated cytokine can prolong the survival of graft.

Polyclonal AntibodiesAntithymocyte globulin-equine (Atgam®)Antithymocyte globulin-rabbit (RATG, Thymoglobulin®)Polyclonal Antibodies are used in early rejection prophylaxis, treatment of rejection.Polyclonal antibodies are directed against lymphocyte antigensinstead of the single-specificity of the monoclonal antibodies, these anitlymphocyte antibodies are directed against multiple epitopes.Antithymocyte globulin is a polyclonal antibody derived from either horses (Atgam®) or rabbits (Thymoglobulin®). The agents contain antibodies specific for many common T cell surface antigens including CD2, CD3, CD4, CD8, CD11a, CD18. The antithymocyte globulin binds lymphocytes that display the surface antigens previously listed. This effectively depletes T-cell concentration in the body through complement-dependent cytolysis and cell mediated opsonization following with T cell clearance from the circulation by the reticuloendothelial system (RES). Side effects can includefirst-dose effect (cytokine release syndrome) and is related to cytokines release by these lymphocytes upon their demise. fever, chills, tachycardia, gastrointestinal disturbances,bronchospasm, and fluctuations of blood pressure,which all can be ameliorated by pretreatment with corticosteroids, diphenhydramine, and acetaminophen.20

Summary

CsA, Tac or Sirolimus are the backbone of maintenance immunosuppresion

Addition of other agents (Steroids, MMF, Azathioprine) can be used to decrease risk of rejection or allow for lower doses of the primary agents.

50% of post-OLT deaths are directly/indirectly related to immunosuppressive medications.

Reading McGuire BM et al. Long-term Management of

the Liver Transplant Patient: Recommendations for the Primary Care Doctor American Journal of Transplantation 2009; 9: 1988–2003

Post DJ. Immunosuppression in Liver Transplantation. Liver Transplantation, 2005; 11: 1307-1314