tx immunobiology
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Mohammad ShihataUniversity of Alberta
IntroductionIntroduction
Transplant Immunobiology has established itself as a scientific discipline to study themechanisms by which recipient rejects or
l f i llaccepts a transplant from a geneticallydifferent donor
Tx ImmunobiologyTx Immunobiology
In the early history of transplantation five separate disciplines of investigators separate disciplines of investigators approached the problem of graft rejection.these are Surgeons, Tumor specialists, g , p ,Mendelian geneticists, Biologists and Immunologists.
HistoryHistory 1958 ‐ First HLA antigen described 1959 ‐ Radiation attempted 1961 ‐ Azathioprine available 1962 ‐ Azathioprine & glucocorticoids combined 1966 ‐ Direct cross match b/w donor lymphocytes & recipient serum
HistoryHistory 1978 – cyclosporine trials 1981‐ successful use of monoclonal antibodies for acute
j i (OKT )rejection (OKT 3) 1990s ‐MMF replaced AZT, Sirolimus,IL R MAb IL‐2R MAb
TransplantationTransplantation Autotransplantation
Within one individual Isotransplantation
Between genetically identical individualsll l Allotransplantation Between individuals from the same species
Xenotransplantation Xenotransplantation Between different species
Basic ImmunologyBasic Immunology The ability of the immune system to recognize non‐self peptides is the what initiates the body’s specific defense mechanisms against invading organismsdefense mechanisms against invading organisms.
The same mechanisms are responsible for post The same mechanisms are responsible for post transplant rejection .
Specific Specific ‐‐ NonspecificNonspecific Non ‐specific inflammatory mechanisms:
Macrophages, dendritic cells. NK cells Granulocytes Complement Complement
Antigen‐specific immune response: T‐lymphocytes: TH TC T‐lymphocytes: TH, TC. B‐lymphocytes: Immunoglobulin, IG.
Basic ImmunologyBasic Immunology Cellular mediated immunity cell to cell contact result of mature T cell activity
l di d i i ib d Humoral mediated immunity antibody (immunoglobulin) production by mature B lymphocytes plasma cellslymphocytes plasma cells
The immune response to allograft usually involves both systemsy
TT‐‐ LymphocyteLymphocyte 70% of circulating lymphocyte. Mediators of cellular immunity Responsible for induction of humoral immunity Genetically programmed to bind specific cell bound
i b TCR ( T ll )antigens by TCR ( T cell receptor )
T T –– lymphocyte lymphocyte cont.cont.
All T cells have CD3 molecules on their surface : Intracellular signal transduction.CD % ( h l / I d ) CD4 60% ( helper/ Inducer )‐ Th1 IL ‐2, TNF ‐ γ cell mediatedTh IL IL h l ‐ Th2 IL ‐ 4, IL ‐ 5 humoral
CD8 30% ( Cytotoxic / Suppressor )
B B ‐‐ LymphocyteLymphocyte 20% of circulating Lymphocytes Can bind free antigens Activated Plasma cells Ab production BCR Ag specific ( IgM )
Antigen presenting cells (APC’s)Antigen presenting cells (APC’s) Natural Killer cells, PMNs, monocytes, macrophages All can pick up antigens & present them to T cells, stimulating an immune responsestimulating an immune response
Antigen presentationAntigen presentation Direct: Donor‐APC with donor‐MHC. Indirect: Recipient‐APC with donor‐MHC.
B‐CELL
T-CELLCD3
Ig Ig
TCR
Surface Ig
Complete Processed peptidep
molecule(conformational
epitope)
MHC11
2
peptide(linear epitope)
ANTIGEN PRESENTING CELL
MHC 2
2
Major Histocompatibility Complex (MHC)Major Histocompatibility Complex (MHC) Bind peptide fragments of foreign proteins and present them to T cellsG l f d h f h Gene clusters found on short arm of chromosome 6
Referred to as HLA (Human Leukocyte Antigen) antigensantigens
First discovered on human leukocytes
MHC classesMHC classesMHC Class I molecules
HLA A, B & C Found on all nucleated cells & platelets Each person inherits two Class I antigens from each parentparent
MHC Class II molecules HLA DP, DQ & DR Found on B cells, monocytes, macrophages & other antigen presenting cells (APC)
Each person inherits one Class II antigen from each Each person inherits one Class II antigen from each parent
GENETIC ORGANIZATION OF MHC
CD4 T-CELLCD3
CD8 T-CELLCD3
TCR
CD3
CD8
TCR
CD3
TCR CD4
AgAg
2 1
MHCCLASS II
11
2 2 CLASS IMHC 2m
3
ANTIGEN PRESENTING CELL ANTIGEN PRESENTING CELL
HistocompatibilityHistocompatibility ABO blood type Major Histocompatibility Complex (MHC) Determination of PRA Prospective HLA Cross‐Matching
Blood TypingBlood Typing Donor & recipient must be ABO compatible ABO blood type antigens are expressed in all body
i i l di d h li l lltissues including endothelial cells Most people have antibodies to the antigens they lack
fAs immunosuppression strategies for Heart and Lung transplantation evolve,M i h dMore aggressive approaches to expandthe donor pool are attempted, includingHi h PRA i i t d ABOHigh PRA recipients and ABOmismatched ( incompatible ) allografts .
Panel anel Reactive eactive Antibody (PRA)ntibody (PRA) Combines recipient’s serum with Ag containing cells taken from 60 different individuals, representing the
i l d lpotential donor pool. Reflected as a percentage which relates to presence of antibodies (recipient’s) to HLA moleculesantibodies (recipient s) to HLA molecules
The higher the PRA, the more “sensitized” the recipient is (i.e. more likely to have a positive cross‐p ( y pmatch/rejection)
High PRAHigh PRA Pregnancy Blood transfusions Exposure to allograft material ( e.g. CHD ) Prior transplant Assist Devices, ( e.g. LVAD ) ?
High PRA = “high risk” for rejection
HLA TypingHLA Typing Donor & Recipient HLA alleles HLA A, B & DR are the most important Each person has two alleles for each, constituting 6 antigens to compareZ i i h Si i h Zero antigen mismatch vs. Six antigen match
Impact on organ sharing
Types of RejectionTypes of RejectionHistologically and Immunologically categorized into three major types
Hyperacute – occurs within minutes to hours of release of clampp
Acute – usually occurs days to weeks after transplant
Chronic – occurs over months to years
Hyperacute RejectionHyperacute Rejection Irreversible Result of preformed circulating antibodies Very rare if cross‐match is negative Results in activation of complement
h b i / l i jthrombosis/vascular injury( Hemorrhage / ischemia ) graft loss
Hyperacute RejectionHyperacute Rejection If PRA is > 10%, prospective HLA matching is recommended.R i i Pl h i IVIG C ( Rx perioperative Plasmapheresis, IVIG, Cytoxan ( cyclophosphamide ).
Cobra venom factor to deplete Complements !! Cobra venom factor to deplete Complements !!
Acute RejectionAcute RejectionC ll di t d ( % ) Ab di t d Cell mediated ( 90 % ) or Ab mediated
Weeks to months after transplantation
Change in balance between Immunosuppression and host immunitypp y
Constitutional symptoms often present owed Constitutional symptoms often present, owed to cytokine release (TNF, IL‐1 & 2, etc.)
Morphologic grading of acute Morphologic grading of acute RejectionRejectionHeartGrade 1A: Focal aggregates of perivascular
activated lymphocytes; rarely interstitial foci
Grade 1B: Diffuse but sparse interstitialf d l hfoci;activated lymphocytes
Grade 2: One focus of perimyocytic –i d l h i h activated lymphocytes with
myocyte damage
Morphologic grading of acute Rejection Morphologic grading of acute Rejection ( ( Heart cont. )Heart cont. )
Grade 3A: Multifocal areas of myocytedamage caused by activatedlymphocytes and eosinophils
Grade 3B: Borderline severe rejection
G d Diff i d ( i hil fGrade 4: Diffuse mixed (eosinophils, oftenneutrophils) infiltrate with
liti h h d tvasculitis, hemorrhage, and myocytenecrosis
Morphologic grading of acute rejection (Morphologic grading of acute rejection (Morphologic grading of acute rejection ( Morphologic grading of acute rejection ( Lung )Lung )Grade 1: Minimal perivascular and interstitial
mononuclear infiltratesGrade 2: Mild perivascular and interstitial
mononuclear infiltratesGrade 3: Moderate perivascular and
interstitial mononuclear infiltratesGrade 4: Severe perivascular and interstitial
mononuclear infiltrates
Chronic RejectionChronic Rejection Occurs gradually over months to years with progressive GRAFT function loss
Etiology not clearly known, but probably multi‐f t i l (i & i h i d factorial (immune & non‐immune mechanisms, drug toxicity, chronic ischemia, repeated bouts of acute rejection) j )
hhChronic RejectionChronic Rejection HEART: Allograft vaculopsthy HEART: Allograft vaculopsthy
LUNG : Bronchiolitis OblitransLUNG : Bronchiolitis Oblitrans
The lung is constantly exposed to the outerThe lung is constantly exposed to the outerenvironment which makes it sussiptable to immunomodulatory effects of Respiratoryy ff f p yviruses
Allograft Vasculopathy ( AV )Allograft Vasculopathy ( AV )Neointimal hyperplasia
BronchiolitisOblitrans
ImmunosuppressantsImmunosuppressants Steroids Calcineurin inhibitors Sirolimus Anti‐lymphocyte antibodies Anti‐IL‐2 receptor antibodies Anti‐proliferative agents Allo‐antibody modulation
According to SHLT registry 3‐ year survival after Heart Tx increased from 40% ( 1975 – 1981 ) to 70% ( 8 )( 1982 – 1994 )
In many regestries 10 year survival is only around 50% ( i.e. Current immunosuppressive therapy is far from ideal )far from ideal )
SteroidsSteroids Prevent all phases of T cell activation Block cytokine gene expression (IL‐1,2,3, 6, 15, IFN‐ & IFN IFN‐
Commonly used at high doses on induction Taper to 5‐10mg/day for maintenanceape to 5 0 g/day o a te a ce Commonly used as high dose (pulse) therapy for acute rejection
Side effectsSide effectsISHLT/UNOS thoracic registry ( in 1st year )
HTN 61% DM 16% Hyperlipidemia 26% Symptomatic OP 5% Cataract 2%
“ There is increasing evidence that steroidscan be weaned during the first year aftertransplantation in the majority of patientsp j y pwithout any impact on long term graft andpatient outcome “p
Calcineurin InhibitorsCalcineurin InhibitorsBinding of allo‐antigen to cell surface receptor signal transduction elevated cytosolic Ca++
activation of Calcineurin (a phosphatase) dephosphorylates nuclear regulatory proteins (NF‐AT) passage through nuclear membrane IL‐2, IL‐4, IFN‐, etc. transcription cytokine production
Must follow drug levels closely (12 hour trough) Must follow drug levels closely (12 hour trough)
Are inherently nephrotoxic due to vasoconstrictive Are inherently nephrotoxic due to vasoconstrictive properties
Tacrolimus is more potent than Cyclosporin ( 100 time greater in vitro inhibition of lymphocytes prolifration )
Side effectsSide effectscyclosporine tacrolimuscyc ospo e tac o us
hyperkalemia +++ +++ hyperlipidemia +++ +hyperlipidemia +++ +tremor +diabetogenesis + ++gingival hyperplasia +g g yp prenal insufficiency +++ ++ HTN ++ +hirsutism ++ +
PP‐‐450450 Inhibitors (increase levels) dil i il
Inducers (decrease levels)
if i‐ diltiazem>verapamil‐ ketoconazole >
fluconazole
‐ rifampin‐ isoniazid
h t i (Dil ti )fluconazole‐ erythromycin or
clarithromycin
‐ phenytoin (Dilantin)‐ carbamazepine
(Tegretol)y‐ cimetidine
(Tegretol)‐ phenobarbital
TOR inhibitor (sirolimus or “Rapamycin”TOR inhibitor (sirolimus or “Rapamycin”TOR inhibitor (sirolimus or “Rapamycin” TOR inhibitor (sirolimus or “Rapamycin” (Rapamune))(Rapamune))Macrolide antibiotic similar to tacrolimus Binds to FKBP like tacrolimus, but sirolimus/FKBP complex does not block calcineurin
Sirolimus/FKBP complex engages a regulatory protein called target of rapamycin (TOR)
TOR inhibition reduces cytokine dependent ll l lif i h G S h f ll cellular proliferation at the G1 to S phase of cell
division cycle
AntiAnti‐‐Lymphocyte antibodiesLymphocyte antibodies OKT3 ( anti CD3 MAb ) Thymoglobulin ( ATGAM / RATG ) Used only for induction or treatment of rejection, not for maintenance immunosuppression
OKT3 side effectsOKT3 side effects Cytokine release syndrome, life threatening“Fevers, chills/rigors, pulmonary edema,hypotention, bronchospasm “
nephrotoxicity, aseptic meningitis with encephalopathy
infections (esp. CMV), lymphoma
ATGAM side effectsATGAM side effects Thrombocytopenia Leukopenia Infection (esp. CMV) Unprededictability and variable efficacy
AntiAnti‐‐ILIL‐‐2 Receptor Antibodies2 Receptor Antibodies Daclizumab Basiliximab
In clinical trials
AntiAnti‐‐proliferative agentsproliferative agents mycophenolate mofetil (Cellcept) azathioprine (Imuran)
Mycophenolate Mofetil (Cellcept)Mycophenolate Mofetil (Cellcept) Reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH)IMDPH i i i l i h d h i f IMDPH is a critical enzyme in the de novo synthesis of purines & guanosine nucleotides
Lymphocytes rely on de novo pathway more than other Lymphocytes rely on de novo pathway more than other cells
MMFMMF Differs radically from activity of calcineurin inhibitors or sirolimusT i “d ” f i i i Target is “down‐stream” from antigen recognition or signal transduction
Essentially blocks the proliferation of lymphocytes Essentially blocks the proliferation of lymphocytes
AlloAllo‐‐antibody modulationantibody modulation Intravenous Immunoglobulin (IV Ig) Plasmapheresis
IV IgIV Ig Down‐regulates antibody production (anti‐HLA Ab) by plasma cellsI d i f B ll Induces apoptosis of B cells
Effective treatment for acute humoral rejection U d i l i d i i i Used in pre‐transplant immune desensitization protocols
PlasmapheresisPlasmapheresis Removes circulating antibodies non‐specifically Effective in treatment of acute humoral rejection Effective in pre‐transplant immune desensitization protocols
Alloantigen
OKT3 / Thymoglobulin
T Cell Receptor
A ti t d l i i
Cyclosporin / FK506
Activated calcinurin
Dephos. NFATp
IL-2 gene promotion
Glucocorticoids
IL-2 / IL-2 R
Daclizumab / BasiliximabDaclizumab / Basiliximab
Cell Cycle
Sirolimus
MMF
DNA syn / Prolifration
Non PharmacologicalNon PharmacologicalNon PharmacologicalNon Pharmacological Total Lymphoid Irradiation Photopheresis Apheresis ( plasma exchange )
Future DirectionsFuture DirectionsFuture DirectionsFuture Directions Selective immunoabsorption filtration Co receptors ( CD 154 CD 28 CTLA 4 ) Co‐receptors ( CD 154, CD 28, CTLA 4 )Ab
Our Protocol ( 1996Our Protocol ( 1996 ))Our Protocol ( 1996 Our Protocol ( 1996 ‐‐ )) Intra‐Operative‐methylprednisolone sodium p y psuccinate(Solumedrol), 10mg/kg just prior to release of aortic cross‐clamp.
Post‐Transplant‐ Solumedrol 2mg/kg IVq12h x 3 doses then 1mg/kg tapering by 2mg/day until
d b b dprednisone can be substituted.MMF 1000‐1500 mg bid PO. ATGAM (or equivalent RATG/OKT 3)10‐20mg/kg/day over 24 hours by continuos infusion until
l i l l i t t cyclosporine levels are in target range.
Following the discontinuation of ATGAM Solumedrol 2mg/kg/day Iv for threeATGAM,Solumedrol 2mg/kg/day Iv for threedoses.
P d i /k /d t ti 8 h t Prednisone 1mg/kg/day starting 48 hours postopand tapered by 2mg/day until at 0.3 mg/kg/day for heart transplant patient and 0 3mg/kg/day for heart transplant patient and 0.3mg/kg/day starting 48 hours postop for heart‐lung and lung transplant patients.p p
Cyclosporine 8‐10mg/kg/day in 2 divided doses PO/NG starting on the 2‐4 postop day depending on renal function‐trough level 350‐500 ng/ml in the first 3 function trough level 350 500 ng/ml in the first 3 months and then 200‐500 ng/ml depending on the renal function.
After 3 mth postop: MMF,Prednisone and Cyclosporine/Tacrolimus continue.I h f j i l i d In the event of no rejection cyclosporine dose maybe reduced to achieve trough level 200‐500ng/ml.500ng/ml.
StartUniversity of Alberta
Transplant Process for Heart Recipients
Is potentialrecipient *high risk ?No Yes
Potential donor identified
Obtain donor HLA typing(F HOPE if di t t d f
Potential donor identified
Proceed(No prospective XM
required. T-AHG XM on
Hi h i k
(From HOPE if distant donor, fromHLA lab if local donor)
required. T AHG XM onnext working day)
End Is the patientclinically stable
(Discussion betweensurgeon and
Yes
Yes
High risk = positive PRA (>15%) or regraftor recent transfusion
Does potentialrecipient have DSA? (If unsurecheck with Dr. Campbell (cell
940 7358) or HLA Lab(Pager 445 6708))?
surgeon andcardiologist)?
Recommend waitfor better match
No
transfusion Protocol A – 1
volume plasmapheresisfollowed by 2g/kg
Is DSAClass I?
Is Class IDSA detected
by AHG?
Yes
Yes No
No
End
Noy g/ g
IVIG pre transplant.
DSA = Donor specific
ib di
Proceed
Prospective flowXM required ASAP
(Transplant mayproceed before
Class II DSAIncreased
immunological riskProceed withProtocol A**
Very high riskDo NOT proceed
Proceed withProtocol A
Set up T-AHG andflow XM ASAP
(discuss timing with antibodiespXM results are
ready)Set up flow XM ASAP
(discuss timing withDr. Campbell)
End
THANK YOU
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