Tx immunobiology

1. Mohammad Shihata
University of Alberta

2. IntroductionIntroduction
Transplant Immunobiology has established
itself as a scientific discipline to study the
mechanisms by which recipient rejects or
l f i llaccepts a transplant from a genetically
different donor

3. 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.

5. 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

6. 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

7. TransplantationTransplantation
 Autotransplantation
 Within one individual
 Isotransplantation
 Between genetically identical individuals
ll l Allotransplantation
 Between individuals from the same species
 Xenotransplantation Xenotransplantation
 Between different species

8. 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 .

9. 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.

10. 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

11. 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 )

12. 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 mediated
Th  IL IL  h l ‐ Th2  IL ‐ 4, IL ‐ 5  humoral
 CD8 30% ( Cytotoxic / Suppressor )

13. B B ‐‐ LymphocyteLymphocyte
 20% of circulating Lymphocytes
 Can bind free antigens
 Activated  Plasma cells  Ab production
 BCR Ag specific ( IgM )

14. 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.

15. B‐CELL
 
T-CELLCD3
Ig Ig
TCR
Surface Ig
Complete
Processed
peptidep
molecule
(conformational
epitope)
MHC
11
 2
peptide
(linear
epitope)
ANTIGEN PRESENTING CELL
MHC 
2
2

18. Major Histocompatibility Complex (MHC)Major Histocompatibility Complex (MHC)
 Bind peptide fragments of foreign proteins and
present them to T cells
G 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

19. MHC classesMHC classes
MHC Class I molecules
 HLA A, B & C
 Found on all nucleated cells & platelets
 Each person inherits two Class I antigens from each
parentparent

20. 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

21. GENETIC ORGANIZATION OF MHC

22. CD4 T-CELL
CD3
CD8 T-CELL
CD3

 
TCR 
CD3
CD8
 
TCR 
CD3

TCR 
CD4
Ag
Ag

2 1
MHC
CLASS II
11
2 2
CLASS I
MHC 2m
3
ANTIGEN PRESENTING CELL ANTIGEN PRESENTING CELL

26. HistocompatibilityHistocompatibility
 ABO blood type
 Major Histocompatibility Complex (MHC)
 Determination of PRA
 Prospective HLA Cross‐Matching

27. 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

28. fAs immunosuppression strategies for
Heart and Lung transplantation evolve,
M i h dMore aggressive approaches to expand
the donor pool are attempted, including
Hi h PRA i i t d ABOHigh PRA recipients and ABO
mismatched ( incompatible ) allografts .

29. 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 p
match/rejection)

30. 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

31. 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 compare
Z i i h Si i h Zero antigen mismatch vs. Six antigen match
 Impact on organ sharing

32. Types of RejectionTypes of Rejection
Histologically 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

33. 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

34. 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 !!

35. Acute RejectionAcute Rejection
C 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.)

36. Morphologic grading of acute Morphologic grading of acute
RejectionRejection
Heart
Grade 1A: Focal aggregates of perivascular
activated lymphocytes; rarely
interstitial foci
Grade 1B: Diffuse but sparse interstitial
f d l hfoci;activated lymphocytes
Grade 2: One focus of perimyocytic –
i d l h i h activated lymphocytes with
myocyte damage

37. Morphologic grading of acute Rejection Morphologic grading of acute Rejection ( (
Heart cont. )Heart cont. )
Grade 3A: Multifocal areas of myocyte
damage caused by activated
lymphocytes and eosinophils
Grade 3B: Borderline severe rejection
G d Diff i d ( i hil fGrade 4: Diffuse mixed (eosinophils, often
neutrophils) infiltrate with
liti h h d tvasculitis, hemorrhage, and myocyte
necrosis

38. 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 infiltrates
Grade 2: Mild perivascular and interstitial
mononuclear infiltrates
Grade 3: Moderate perivascular and
interstitial mononuclear infiltrates
Grade 4: Severe perivascular and interstitial
mononuclear infiltrates

39. 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 )

40. 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 outer
environment which makes it sussiptable to
immunomodulatory effects of Respiratoryy ff f p y
viruses

41. Allograft Vasculopathy ( AV )Allograft Vasculopathy ( AV )
Neointimal hyperplasia

42. Bronchiolitis
Oblitrans

43. ImmunosuppressantsImmunosuppressants
 Steroids
 Calcineurin inhibitors
 Sirolimus
 Anti‐lymphocyte antibodies
 Anti‐IL‐2 receptor antibodies
 Anti‐proliferative agents
 Allo‐antibody modulation

44.  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 )

45. 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

46. Side effectsSide effectsISHLT/UNOS thoracic registry ( in 1st year )
 HTN 61%
 DM 16%
 Hyperlipidemia 26%
 Symptomatic OP 5%
 Cataract 2%

47. “ There is increasing evidence that steroids
can be weaned during the first year after
transplantation in the majority of patientsp j y p
without any impact on long term graft and
patient outcome “p

48. Calcineurin InhibitorsCalcineurin Inhibitors
Binding 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

49.  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 )

50. Side effectsSide effectscyclosporine tacrolimuscyc ospo e tac o us
hyperkalemia +++ +++
hyperlipidemia +++ +hyperlipidemia +++ +
tremor +
diabetogenesis + ++
gingival hyperplasia +g g yp p
renal insufficiency +++ ++
HTN ++ +
hirsutism ++ +

51. 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

52. 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

53. AntiAnti‐‐Lymphocyte antibodiesLymphocyte antibodies
 OKT3 ( anti CD3 MAb )
 Thymoglobulin ( ATGAM / RATG )
 Used only for induction or treatment of rejection, not
for maintenance immunosuppression

54. 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

55. ATGAM side effectsATGAM side effects
 Thrombocytopenia
 Leukopenia
 Infection (esp. CMV)
 Unprededictability and variable efficacy

56. AntiAnti‐‐ILIL‐‐2 Receptor Antibodies2 Receptor Antibodies
 Daclizumab
 Basiliximab
In clinical trials

57. AntiAnti‐‐proliferative agentsproliferative agents
 mycophenolate mofetil (Cellcept)
 azathioprine (Imuran)

58. 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

59. MMFMMF
 Differs radically from activity of calcineurin inhibitors
or sirolimus
T 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

60. AlloAllo‐‐antibody modulationantibody modulation
 Intravenous Immunoglobulin (IV Ig)
 Plasmapheresis

61. IV IgIV Ig
 Down‐regulates antibody production (anti‐HLA Ab)
by plasma cells
I 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

62. PlasmapheresisPlasmapheresis
 Removes circulating antibodies non‐specifically
 Effective in treatment of acute humoral rejection
 Effective in pre‐transplant immune desensitization
protocols

64. 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

65. 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

66. Our Protocol ( 1996Our Protocol ( 1996 ))Our Protocol ( 1996 Our Protocol ( 1996 ‐‐ ))
 Intra‐Operative‐ methylprednisolone sodium p y p
succinate(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.

67.  Following the discontinuation of
ATGAM Solumedrol 2mg/kg/day Iv for threeATGAM,Solumedrol 2mg/kg/day Iv for three
doses.
 P d i /k /d t ti 8 h t Prednisone 1mg/kg/day starting 48 hours postop
and 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

68.  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.

69.  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.

70. Start
University of Alberta
Transplant Process for Heart Recipients
Is potential
recipient *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, from
HLA lab if local donor)
required. T AHG XM on
next working day)
End Is the patient
clinically stable
(Discussion between
surgeon and
Yes
Yes
 High risk =
positive PRA
(>15%) or regraft
or recent
transfusion
Does potential
recipient have DSA? (If unsure
check with Dr. Campbell (cell
940 7358) or HLA Lab
(Pager 445 6708))?
surgeon and
cardiologist)?
Recommend wait
for better match
No
transfusion
 Protocol A – 1
volume
plasmapheresis
followed by 2g/kg
Is DSA
Class I?
Is Class I
DSA detected
by AHG?
Yes
Yes No
No
End
No
y g/ g
IVIG pre
transplant.
 DSA = Donor
specific
ib di
Proceed
Prospective flow
XM required ASAP
(Transplant may
proceed before
Class II DSA
Increased
immunological risk
Proceed with
Protocol A**
Very high risk
Do NOT proceed
Proceed with
Protocol A
Set up T-AHG and
flow XM ASAP
(discuss timing with
antibodies
p
XM results are
ready)
Set up flow XM ASAP
(discuss timing with
Dr. Campbell)
End

71. THANK YOU