Renal transplant immunology

Renal transplant immunology

1. Dept of Urology
Govt Royapettah Hospital and Kilpauk Medical College
Chennai

1

2. Professors:
Prof. Dr. G. Sivasankar, M.S., M.Ch.,
Prof. Dr. A. Senthilvel, M.S., M.Ch.,

Asst Professors:
Dr. J. Sivabalan, M.S., M.Ch.,
Dr. R. Bhargavi, M.S., M.Ch.,
Dr. S. Raju, M.S., M.Ch.,
Dr. K. Muthurathinam, M.S., M.Ch.,
Dr. D. Tamilselvan, M.S., M.Ch.,
Dr. K. Senthilkumar, M.S., M.Ch.
Dept Of Urology, KMC and GRH, Chennai 2

3. C.C. LITTLE & ERNEST TYYZER

1914

Genetic basis for transplantation
rejection

“Tumors transplanted between
genetically identical mice grew
normally, but tumors transplanted
between non-identical mice were
rejected and failed to grow”
Dept Of Urology, KMC and GRH, Chennai 3

4. PETER MEDAWAR

Role of the immune system in transplant reject

“Skin graft transplants in world war two victims
showed that skin transplants between
individuals had much higher rejection rates
then self-transplants within an individual”

“Suppressing the immune system delayed skin
transplant rejection”

1960 Nobel Prize
Dept Of Urology, KMC and GRH, Chennai 4

5. GEORGE SNELL (1930s) & PETER GORER
(1940s)

Individually isolated the genetic factors
that when similar allowed transplantation
between mouse strains

Naming them H and antigen II respectively

These factors were in fact one and the
same, and the locus was named H-2

Snell coined the term "histocompatibility“
Dept Of Urology, KMC and GRH, Chennai 5

6. JEAN DAUSSET (1950s)

Human version of the histocompatibility
complex

Snell, Dausset and Baruj Benacerraf -
1980 Nobel Prize
Dept Of Urology, KMC and GRH, Chennai 6

7. Dept Of Urology, KMC and GRH, Chennai 7

8. •Dendritic cells
•Macrophages
•Activated B Cells
Antigen presenting cells
B cells and antibodies
T cells
•Natural killer cells
•T cells that express NK cell – associated
Markers
•Monocytes/Macrophages
Other cells
Dept Of Urology, KMC and GRH, Chennai 8

9. ABO incompatibility
Major Histocompatibility complex
Preformed anti HLA antibodies
Dept Of Urology, KMC and GRH, Chennai 9

10. Initial & most important barrier

Rejected immediately due to the presence of circulating preformed anti-A
and/or anti-B antibodies.

Transplantation across ABO disparate blood groups
ABO blood group B or O may receive a kidney from an ABO A2 donor if their
anti-A antibody titer is low (IgG ≤ 1:2)
A2 antigen - less reactive & expressed in lower amounts on the surface of red blood
cells and tissue cells



Dept Of Urology, KMC and GRH, Chennai 10

11. RECIPIENT
BLOOD TYPE
COMPATIBLE
DONORS
O O
A A OR O
B B OR O
AB A, B, AB OR O
Dept Of Urology, KMC and GRH, Chennai 11

12. Anti-A/B antibodies are aimed to be below a certain threshold (less than
1:32) at the time of ABOi kidney transplantation and during the first 2 weeks
after surgery.

Thereafter, even a rebound of anti-A/B antibodies does not appear to harm
the kidney transplant, a phenomenon that is called accommodation
Dept Of Urology, KMC and GRH, Chennai 12

13. Plasmapheresis
Intravenous immunoglobulin (IVIG)
Splenectomy
Rituximab – anti CD20 antibody
Donor thrombocyte transfusion
Infusion of A or B trisaccharide, together with intensified immunosuppression
Dept Of Urology, KMC and GRH, Chennai 13

14. Dept Of Urology, KMC and GRH, Chennai 14

15. Cell surface glycoproteins
Function as peptide display molecules
Enable the immune system to distinguish between ‘self’ and ‘non-self’
Dept Of Urology, KMC and GRH, Chennai 15

16. Gene complex encoding Major Histocompatibility
complex proteins in humans

Chromosome 6p21.31

3.6 Mbp complex – over 200 genes

Regulates immune system

Highly polymorphic
Dept Of Urology, KMC and GRH, Chennai 16

17. Dept Of Urology, KMC and GRH, Chennai 17

18. Consist of a heavy chain with three domains
(alpha1, alpha2, alpha3) and an invariable
light chain called beta-2 microglobulin
Alpha3 domain anchors the molecule into
the cell, while the alpha1 and alpha2
domains form a peptide binding groove.
Beta 2-microglobulin, 12kDa, non-MHC
encoded, non-transmembrane, non covalently
bound to alpha chain
Alpha3 - Highly Conserved Among MHC I
Molecules; Interacts with CD8+ T
Cyt cell
Dept Of Urology, KMC and GRH, Chennai 18

19. •Class I MHC is found in almost all
nucleated cell.

•Presents endogenous antigen.

•The formed cleft can bind peptides of 8 -10
amino acids in a flexible extended
conformation.

•CD8 + T cell are responsive

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21. Dept Of Urology, KMC and GRH, Chennai 21

22. •Alpha-chain of 34kDa
•Beta-chain of 29kDa
•NO beta-2 microglobulin
•Peptide antigen in a groove formed from
alpha1 & beta1 chains
Dept Of Urology, KMC and GRH, Chennai 22

23. Expressed on antigen presenting cells
(mononuclear phagocytes, B lymphocytes,
dendriticcells) as well as some endothelial
cells and thymus epithelium.
Expressed on the endothelial cells of
glomeruli and peritubular capillaries
Presents exogenous antigen.
CD4+ T cells are responsive
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26. Co- dominant expression

Dept Of Urology, KMC and GRH, Chennai 26

27. Probability that a sibling has inherited the same two haplotypes as a brother
or sister - 25 %

Probability that he/she inherited either one – 50%

Probability that he/she inherited neither - 25 %

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28. Dept Of Urology, KMC and GRH, Chennai 28

29. Direct Presentation
•MHC molecule displayed by antigen-presenting cells
(APCs) in the graft is recognized by recipient T cells
without a need for host APCs.
Indirect Presentation
•Donor MHC molecules are captured and processed
by recipient APCs and then presented to T cells
Dept Of Urology, KMC and GRH, Chennai 29

30. Dept Of Urology, KMC and GRH, Chennai 30

31. Dept Of Urology, KMC and GRH, Chennai 31

32. Alloreactive CD4+ and CD8+ T cells that are activated by graft alloantigens
cause rejection by distinct mechanisms.

CD4+ helper T cells - differentiate into cytokine producing effector cells
 damage grafts by cytokine mediated inflammation (similar to a
delayed-type hypersensitivity (DTH) reaction)

CD8+ T cells - differentiate into cytotoxic T lymphocytes (CTLs)  kills
nucleated cells in the graft that express the allogeneic class I MHC
molecules.
CTLs also secrete inflammatory cytokines, which can contribute to graft
damage.
Dept Of Urology, KMC and GRH, Chennai 32

33. Most high-affinity alloantibodies are produced by helper T cell–dependent
activation of alloreactive B cells

The antigens most frequently recognized by alloantibodies in graft rejection
are donor HLA molecules, including both class I and class II MHC proteins.

Dept Of Urology, KMC and GRH, Chennai 33

34. Anti-HLA antibodies do not occur naturally (as do anti-ABO antibodies)

There are three ways that an individual can be exposed to HLA antigens and
subsequently develop anti-HLA antibodies
Blood product transfusions
Pregnancy
Tissue transplantation

Donor specific antibodies (DSAs), if present in high amounts, will
cause immediate (hyperacute) graft loss and if present in small
amounts will limit the survival of an allograft.
Dept Of Urology, KMC and GRH, Chennai 34

35. Donor harvest
Anastomosis of donor & recipient vessels
Recipient immune response to transplanted organ
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36. Donor harvest
Anastomosis of donor & recipient vessels
Recipient immune response to transplanted organ
Dept Of Urology, KMC and GRH, Chennai 36

37. Donor kidney – resident immune cells such as dendritic cells
Resident dendritic cells – dormant
Acquire phagocytic ability and mobility upon slightest injury to kidney
Events activating resident dendritic cells:
Low blood flow to the kidney
Anoxia
Deceased donor – rapid swings in blood pressure (early ht follow by hypotens
phase) due to catecholamine induced autonomic storm
Brain death  massive cytokine storm

“Donor kidney is highly activated even before removal ”
Dept Of Urology, KMC and GRH, Chennai 37

38. Ischemia / Anoxia induced death of donor kidney cells
Ischemic cells spill intracellular contents
Contains immunologically active molecule – “DAMAGE
ACTIVATED MOLECULAR PATTERNS” (DAMPs)
Receptors for DAMPs – in epithelial, mesenchymal &
endothelial cells within donor kidney (TLRs / NLRs)
Receptor ligand complex  sets off biochemical signalling
pathway  induce inflammatory signals & cell death
pathways
DAMP/TLR/NLR interactions  strong attractants for
recipient inflammatory cells
Dept Of Urology, KMC and GRH, Chennai 38

39. Dept Of Urology, KMC and GRH, Chennai 39

40. GREATER THE ISCHEMIC DAMAGE

MORE THE DAMP/TLR/NLR SIGNALING

MORE THE INFLAMMATORY CELLS PRESENTATION TO RECIPIENT

GREATER THE ACTIVATION OF RECIPIENT’S IMMUNE SYSTEM



Dept Of Urology, KMC and GRH, Chennai 40

41. Donor harvest
Anastomosis of donor & recipient vessels
Recipient immune response to transplanted organ
Dept Of Urology, KMC and GRH, Chennai 41

42. Recipient exposed to torrent of DAMPs/cytokines /
chemokines
Recipient immune cells vigorously infiltrate donor
tissue
Augements ischemia induced tissue injury
Activated donor dendritic cells migrate to T cell rich
regions of recipient’s lymph nodes
Donor dendritic cells – Recipient T cell interaction 
key initiating event of cellular rejection
T cells discriminate between ‘self’ and ‘nonself’ based
on the foreigness of HLA / peptide complex presented
Dept Of Urology, KMC and GRH, Chennai 42

43. Donor harvest
Anastomosis of donor & recipient vessels
Recipient immune response to transplanted organ
Dept Of Urology, KMC and GRH, Chennai 43

44. Dendritic cell HLA / peptide complex with T-cell receptor  several cell surface molecules
coalesce at the junction between the two cell types  “IMMUNOLOGICAL SYNAPSE ”
Immunological synapse – juxtaposed Tcell / T cell receptor / Dendritic cell (HLA/peptide) /
costimulatory / adhesion molecules
Provides the go signal to T cell
Immunological synapse – needs to be formed in order for the T cell to receive proper
combination of activation signals
Dept Of Urology, KMC and GRH, Chennai 44

45. IMMUNOLOGICAL SYNAPSE FORMATION
BIOCHEMICAL SIGNALLING TURNED ON
Activation of calcineurin
Activation of mitogen activated protein kinases
Genes transcribed  secretion of cytokines (IL2)
Dept Of Urology, KMC and GRH, Chennai 45

46. Regulates gene transcription, cell division, cell survival, cell death
IL2 – IL2 R on surface of T cells activates three different cytoplasmic
signalling cascades (block receptor interaction – daclizumab, basiliximab)
Mitogen activated protein
(MAP) kinase pathway
Phosphoinositide 3 kinase
(PI3K) pathway
Janus kinase/signal
transducers and activators
of transcription protein
pathway (JAK/STAT)
Dept Of Urology, KMC and GRH, Chennai 46

47. Remains activated as long as the synapse exists
The cells stays in contact for a longer period of time if a greater disparity
exists between HLA molecules of donor & recipient

Drugs aimed at disrupting this contact
Belatacept – CTLA4 Ig fusion protein
Alefacept – anti CD2 antibody

Dept Of Urology, KMC and GRH, Chennai 47

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49. Dept Of Urology, KMC and GRH, Chennai 49

50. ABO and Rh blood typing
Cross matching (Preformed
antibodies)
HLA typing
Dept Of Urology, KMC and GRH, Chennai 50

51. Serological typing
Molecular typing

Important : HLA A, B, DR
Dept Of Urology, KMC and GRH, Chennai 51

52. HLA TYPING
HLA typing of the donor kidney and
Recipient : expressed as “1-0-0-mismatch”
that corresponds to the pair of alleles
mismatched, respectively, at HLA-A, HLA-B
and HLA-DR.

These three antigens are the considered as
the most important ones in kidney
transplantation.

Influence of HLA-DR mismatching had the
most effect during the first six months post-
transplant while the maximal effect of HLA-B
mismatching occurred two years post-
transplant

HLA-A mismatches have a deleterious effect
on long-term graft survival
Dept Of Urology, KMC and GRH, Chennai 52

53. ADCC – Antibody dependent cell mediated cytotoxicity
CDC – Complement dependent cytotoxicity
Dept Of Urology, KMC and GRH, Chennai 53

54. A tray containing sera with antibodies to a multitude of known HLA alleles is
used
Recipient lymphocytes are introduced into the tray wells contacting sera,
complement and dye.
In tray wells where antibodies can bind to the antigens on the surface of
lymphocytes; complement is activated. This results in complement pathways
triggered resulting in cell death, ultimately allowing the dye to enter the cell.
Tray wells with significant cell death are then identified under phase contrast
microscopy.
Through a process of comparison and elimination of positive wells the HLA
type is assigned.
Dept Of Urology, KMC and GRH, Chennai 54

55. Dept Of Urology, KMC and GRH, Chennai 55

56. The key benefit of serologic typing is that results are available in a short
period.

This is particularly important in deceased donor renal transplantation

LIMITATION: Lack of sera with antibody specificities that are capable of
identifying the ever-growing number of HLA alleles

More advanced methods of typing currently available & serological typing has
fallen into disuse
Dept Of Urology, KMC and GRH, Chennai 56

57. Degree of cytotoxicity expressed as %PRA

% of lymphocytes in the cell panel which has undergone lysis as a result of
complement action

20% - minimum cut off for positive result

Dept Of Urology, KMC and GRH, Chennai 57

58. DNA-based HLA typing methods using molecular techniques
1.Sequence specific oligonucleotide probe hybridization(SSOP)
2.Sequence-specific primer amplification (SSP)
3.Sequencing-based typing (SBT)
4.Reference strand-based conformation analysis (RSCA)
5.Nextgeneration sequencing (NGS)
6.Short tandem repeat (STR) genotyping
Dept Of Urology, KMC and GRH, Chennai 58

59. In this approach extracted DNA from the subject is amplified in several wells.

Each well has primers that are complementary to specific HLA alleles.

In wells where DNA probes are complementary to the specific sequence of the HLA
molecule, an amplification product is formed.

This is then instilled into an agarose gel and undergoes electrophoresis where they
appear as a band.

HLA typing is then allocated by matching the primers of the amplification product
to DNA sequences
Dept Of Urology, KMC and GRH, Chennai 59

60. Amplified DNA is mixed with oligonucleotide probes that are complementary to
specific segments of the DNA of different alleles.

Unique HLA alleles are then identified using fluorescent tags.
Dept Of Urology, KMC and GRH, Chennai 60

61. The usual route for sensitisation
towards HLA antigens occurs in
three instances
•Pregnancy
•Post blood transfusion
•Prior transplantation
METHODS FOR HLA ANTIBODY
SCREENING
•Cytotoxic cell based
•Solid phase antibody screening –
employs soluble or recombinant
HLA molecules instead of
lymphocytes
•ELISA
•Microbead platform / single
antigen beads
Dept Of Urology, KMC and GRH, Chennai 61

62. Recipient serum + Donor lymphocytes in
individual wells along with complement &
dye

Serum with Ab + Donor lymphocyte 
complement activation  cell death 
uptake of dye

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63. Dept Of Urology, KMC and GRH, Chennai 63

64. Limitations: detects only complement binding antibodies

False positive: non HLA antibodies, Autoantibodies & nonspecific IgM
antibodies

False negative: low antibody titre ( as high levels are required for
complement mediated action)
Dept Of Urology, KMC and GRH, Chennai 64

65. Antibody levels vary with time due to new antigen exposures and
immunological sensitisation

Recently drawn recipient serum with donor lymphocytes – best

Positive T & B cell cross match  Ab against Class I & II
Positive B cell cross match  Ab against Type II antigens alone or low levels
of Ab to type I
Positive T cell cross match  technical error
Dept Of Urology, KMC and GRH, Chennai 65

66. Positive T cell cross match
•Poor outcome &
unacceptably high incidence
of acute graft rejection

•Desensitisation  persistent
positive cross match 
ABSOLUTE
CONTRAINDICATION
Positive B cell cross match
•Not as consistently
associated with humoral
rejection as positive T cell
cross matches
•Less significance in acute/
hyperacute rejection
•Still warrant desensitization
prior to transplant
Dept Of Urology, KMC and GRH, Chennai 66

67. Donor lymphocytes + recipient’s serum 
binds to donor specific antibodies

Quantified by detectors in impedence flow
cytometer

Dept Of Urology, KMC and GRH, Chennai 67

68. Measurement of
fluorescence intensity
as a ratio of the
control

Serial dilution of
serum & minimum
dilution which yields
negative result gives a
measurable estimate
Dept Of Urology, KMC and GRH, Chennai 68

69. Interpretation : negative CDC + Positive flow cytometry

Non- complement fixing antibodies
Non HLA antibodies
Low level of anitbodies

CDC negative + Positive T cell flow cytometry  significantly poorer absolute
5 year graft survival rates than those who were both negative
Dept Of Urology, KMC and GRH, Chennai 69

70. Recipient serum + Purified HLA molecules +
Enzyme conjugated
Uses HLA glycoprotein immobilized into microtitre
wells
Recipient’s serum is added
Specific antibodies bind to the epitopes available
Wash  anti IgG with a passenger reporter
molecule (alkaline phosphatase) is added which
combines with anti HLA antibody
Wash to remove unbound antibody
Substrate is added which after dephosphorylation
by reporter  color change
Dept Of Urology, KMC and GRH, Chennai 70

71. Beads labelled with fluorescein – impregnated with
different ratios of two flurochromes resulting in a signal
unique to specific bead

Each bead have one or more HLA molecule incorporated

Incubation of recipient serum with beads

Washed and incubated with a second antibody
(antihuman IgGantibody) labelled with phycoerthyrin

Two lasers are used to excite the fluorochorme of bead
and the phyoerythrin bound to Ab
Dept Of Urology, KMC and GRH, Chennai 71

72. Multiple synthetic microspheres with single given HLA antigen coating incubated with
recipient serum and recipient’s panel reactive antibodies are identified by Luminex
flow analyser

Based on the comparison of anti HLA antibodies of the recipient to donor HLA
antigens
E.g. Recipient’s PRA – MFI >10,000 : A2, A68, B57, DR1, DQ2
Donor HLA – A1, B57, C35, DP2, DQ23, DR 7

Predicts eventual cross match

Assist in rapid identification of suitable donor
Dept Of Urology, KMC and GRH, Chennai 72

73. Allorecognition is the first step of a series of
complex events that leads to T-cell activation,
antibody production, and allograft rejection
Matching of donor and recipient for MHC antigens
and for preformed HLA antibodies has been shown
to have a significant positive effect on graft
acceptance
Knowledge of the immune mechanisms is critical in
developing strategies to minimize rejection and in
developing new drugs and treatments that blunt
the effects of the immune system on transplanted
organs, thereby ensuring longer survival of these
organs
Dept Of Urology, KMC and GRH, Chennai 73

74. THANK YOU
Dept Of Urology, KMC and GRH, Chennai 74