1. Histocompatibility & kidney transplantation
A. Amirzargar PhD
Tehran University of Medical Sciences
Dept. of Immunology

2. Major histocompatibility complex

5. Polymorphism in MHC Class I genes
Variation >1% at a single genetic locus in a population of individuals
In the human population, over 4700 MHC class I alleles have been identified - some are null alleles, synonyms or differ in regions outside the coding region
2069
Class I A B C
polymorphisms
No of E F G
4721 alleles
(potential alleles)
(Jan. 2011)
1519
1016 10 46 22

6. Polymorphism in MHC Class II genes
Over 9000 human MHC class II alleles have been identified - some are null alleles, synonyms or differ in regions outside the coding region DR DP DQ DM DO
Class II A B1 A1
polymorphisms
No of B
966
2000 alleles
145
144 35 28 3 4 7 12 9

7. Tissue MHC class I MHC class II
B cells
Macrophages
Dendritic Cells
Thymus epithelium
Neutrophils
Hepatocytes
Kidney
Brain
Erythrocytes

13. Accessory molecule / cell interaction

15. Costimulatory blockade

16. PRETRANSPLANT MONITORING
1-ABO typing
2- Ab screening and Identification
3-HLA matching
4- Biochemical and infectious disease evaluation
5-Cardiovascular and pulmonary tests

17. Matching between Recepient And Donor
A- Tissue typing
Determined by 6 antigens located on cell surface encoded for by the HLA gen located on the short arm of chromosom 6.
Class I antigens (HLA-A and HLA-B) are expressed on the surface of most nucleated cells.
Class II antigen (HLA-DR) are expressed on surface of APC and activated lymphocytes.
These 6 antigens are refered to as major transplant antigens.
The match between donor and recepient can range from 0 to six.

18. Matching between Recepient And Donor
B- Cross matching
A laboratory test that determines weather a potential transplant recepient has preformed antibodies against the HLA antigens of the potential donor. (Donor Lymphocytest +Recepient Serum)
A Final CM is mandatory
C- Compatible ABO blood group.

19. HLA matching
It was first noted 40 years ago that HLA matching between donor and recipient was associated with better transplant and patient survival.
Matching for the class I HLA-A and HLA-B antigens influenced survival, but matching for the class II HLA-DR antigens had the most powerful effect.

20. HLA matching
Over the years, there has been an overall improvement in transplant survival
In solid organ transplantation, the effects of HLA matching reported are generally based on matching at the HLA-A, HLA-B, and HLA-DR loci

21. Survival of first cadaver renal allografts by HLA match grade.

22. The best transplant survival was achieved in transplants that had no mismatches at HLA-A, HLA-B, and HLA-DR (000 mismatch grade).

23. HLA-matching The emphasis on HLA matching is declining, because:
The overall difference in 10 yrs graft survival rates between the best and worst matched kidney txs remains at about 18%
Only 15% of candidates can expect to receive HLA-matched kidney because of extreme polymorphism of HLA

24. Little time to search for a HLA-matched in the case of deceased donors
The waiting list is growing more and more, especially with adding of older patients in recent yrs
Increasingly difficult and expensive to maintain the patients in optimal medical condition
A disadvantage of allocation based on HLA-matching is that patients with rare HLA types are difficult to transplant

25. HLA-matching The emphasis on HLA matching is declining, because:
Effective therapeutics for the successful treatment of AMR, utilizing IVIG either alone or in combination with plasmapheresis
Several IS agents available that provided more anti-B cell activity
Strategies for the successful removal or inactivation of DSA prior to Transplantation
If several medically suitable relatives are available, the decision for donation can be made on the basis of histocompatibility testing—an HLA-identical sibling being the ideal choice

26. HLA-matching Factors influencing the relevance of HLA-matching are:
Donor/recipient age, living or deceased donor, IS protocol, sex, cause of death, cold ischemia time, donor/recipient body size
Long-term outcome is better for a completely mismatched younger donor/recipient kidney compared to an HLA-matched older one
Results with HLA-mismatched living donors are generally superior to even well-matched cadaver donors, suggesting that the quality of the kidney and short preservation time outweigh the benefit of matching

27. HLA-matching
When stronger IS drugs were used, the HLA effect diminished
More detrimental effect of cold ischemia time than HLA-matching
Maybe the HLA-matching has more beneficial for long-term survival of deceased donor allograft
However in the case of sensitized patients, a zero HLA-mismatched organ may be the only option

28. Factors affecting TX survival
In a rigorous multivariate analysis of factors influencing the outcome of primary deceased donor transplants in a cohort of transplants performed in the United Kingdom from 1986 to 1993,
donor and recipient age,
waiting time to transplant,
diabetes in the recipient,
donor cause of death
cold ischemia time,
HLA mismatching

29. Allorecognition pathways
Presentation of foreign antigen to recipient T cells :
direct Direct pathway. Recognition of intact foreign MHC on donor APC primes CD4 and CD8 recipient T cells. CD4 cells then provide T cell help for the effector function of CD8 cells
direct response dominates the early posttransplant period
indirect Indirect pathway. Recipient APCs traffic through transplanted organs, phagocytose allogeneic MHC shed from foreign cells through cell necrosis and apoptosis and present the processed peptides in the context of self-MHC class II to MHC class II-restricted CD4 T cells.
Semi-direct pathway

30. Allorecognition

31. Direct and indirect allorecognition
dAPC
dAPC
rAPC
rCD8 T
rCD4 T
rCD4 T
P cell
B cell
Alloantibodies

32. Indirecrt pathway
Indirect alloantigen presentation results in alloresponses that are dominated by CD4 T cells.
As T cell help for B cells to class switch and differentiate into antibody secreting plasma cells is provided by CD4 T cells that recognize peptides derived from antigens internalized by B cell surface immunoglobulins
the presence of class-switched alloantibodies is indicative of help provided by indirect pathway T cells

33. Semi-direct pathway.
Cell-to-cell contact between donor and recipient APC
transfer intact membrane components including intact allo-MHC .
donor APC can release small vesicles, known as ‘exosomes’ containing intact MHC , which fuse with the membrane of recipient APCs .
Recipient APCs, now chimeric for MHC,
stimulate direct pathway CD4 and CD8 responses through intact foreign MHC
indirect responses through processing and presentation of peptides of foreign MHC acquired from necrotic and apoptotic cell material.
the same APC stimulates both CD4 and CD8 cells, linked help can occur.

34. Semi direct allorecognition
dendritic cells are able to acquire intact MHC–peptide complexes from other dendritic cells and endothelial cells and to present them to alloreactive T cells

35. Acute rejection appears within the first 3 posttransplant months and affects 30% of cadaveric transplants and 27% of transplants from living donors. Approximately 20% of patients with transplants experience recurrent rejection episodes.
Patients present with decreasing urine output, hypertension, rising creatinine, and mild leukocytosis. Fever, graft swelling, pain, and tenderness may be observed with severe rejection episodes.
The final diagnosis depends upon a graft biopsy.

36. Chronic Rejection
Usually apparent from 3 months onwards and detected clinically by gradual deteriation in graft fxn.
Factors associated with chronic rejection are both immunological + non-immunological.

37. Allorecognition

38. SENSITIZATION Routes of Sensitization
An individual can become sensitized to HLA alloantigens as a result of blood transfusion, pregnancy, or previous organ transplantation.
Transplantation of poorly HLA-matched kidneys can result in allosensitization to the mismatched HLA antigens.

39. In the United Kingdom, an audit of the national transplant waiting list showed that 20% of patients waiting for a first transplant were sensitized,
but 77% of patients waiting for a second transplant were sensitized.

40. Allosensitization
Approximately 20% of pregnant women produce HLA-specific antibodies to paternally derived fetal HLA antigens.
The use of erythropoietin for the treatment of patients with anemia has decreased the use of blood transfusion in renal patients with a consequent decrease in the number of patients becoming sensitized by this route.
idiopathic HLA antibodies may result from cross-reactivity with infectious agents and are usually IgM.

41. Antibody Detection and Specificity Definition
The aim of an antibody screening strategy is to determine whether the patient has developed HLA alloantibodies and, if so, the antibody class and specificity of the antibodies.
detection and definition of HLA antibodies , can be used to define precisely the specificity of HLA antibodies in serum samples and to elucidate a patient’s sensitization profile.

42. sensitization The sensitization profile :
the timing of appearance or disappearance of antibody reactivity,
The specificity and antibody class of HLA antibodies,
and the presence or absence of autoreactive antibodies.

43. presensitization
Kidney transplantation in patients with donor-specific sensitization has a significant detrimental impact on graft survival,
with most transplants undergoing hyperacute or acute humoral rejection.
Recipient antibodies against donor histocompatibility antigens bind to the vascular endothelium of the transplanted organ, which disrupts the intercellular junctions and causes release of cell surface heparin sulfate and loss of the antithrombotic state, leading to rapid uncontrollable activation of the thrombotic and complement cascades.
The resultant intravascular coagulation and interstitial hemorrhage can lead to graft destruction within minutes or hours after revascularization.

44. Living donor transplants fxn immediately after transplant
30% of cadaveric transplants have delayed graft fxn because of more prolonged ischaemic cold preservation. These pts need continued dialysis support until the kidney starts to fxn.

45. HLA-matching
Despite improvement in IS during the past 50 yrs ago, the more number of HLA-mismatches, the more incidence of immune complications
HLA-matching for the class l influenced survival, but for class ll had the most powerful effect
Matching for HLA-DQ has been variously reported as having either a beneficial effect or no effect on tx outcome
Matching for HLA-DP has an effect on the tx survival of regrafts, but not of first txs

47. Allosensitization
Routes of sensitization are blood transfusion, pregnancy, and previous organ transplantation
Then the immune system is stimulated in a helper T-cell dependent process → class switching from IgM to IgG. This has 2 implications:
Upon re-exposure, memory B-lymphocytes proliferate rapidly and differentiate into plasma cells that produce Abs → AMR(Ab mediated rejection) (even if the Ab titer has decreased to a level not detectable with current assays prior to tx )

48. Allosensitization (con.)
Approximately 20% of pregnant women produce HLA-specific Abs to paternally derived fetal HLA antigens
A study in UK showed that 20% of patients waiting for a first tx and 77% of patients waiting for a second tx were sensitized
The use of erythropoietin for the treatment of anemia has decreased the use of blood transfusion in renal patients with a consequent decrease of sensitized patients
The use of leukocyte-depleted blood would prevent allosensitization
There are some idiopathic HLA antibodies which may result from cross-reactivity with infectious agents and are usually IgM

49. Allosensitization (con.)
There is a casual link/risk/predictive factor between preexisting anti-HLA Abs and
Hyper acute rejection (which is obvious from 4 decades ago)
More rapid onset of CAN in the case of subclinical rejection
C4d-positive acute Ab-mediated rejection
Mixed cellular and humeral acute rejection and can be treated by IS
Chronic allograft function
Post-tx lymphoproliferative disease (skin cancer related to Anti-HLA-B)
Anti-class ll Abs found to appear at later times than class l and are associated with late graft function
Mechanism of injury by Anti-HLA Abs:
IgG Anti-HLA class l: complement lysis
Anti-HLA class ll: leukocyte activation
Anti-MICA: complement lysis of ECs

50. Ab Detection and Specificity Definition
To perform effective antibody screening, samples should be obtained regularly from patients on the waiting list, on at least a 3-monthly basis.
Information about the nature and timing of potential sensitizing events is important in a patient’s sensitization profile.
If sensitization occurs, additional samples are required (e.g., 14 and 28 days after a transfusion with blood products).

51. Technologies to elucidate patient sensitization profile
This profile enables the definition of unacceptable HLA-mismatches
Complement-dependent cytotoxicity (CDC)
The first technique for detection of complement-fixing IgG and IgM in PRA and in crossmatch test
Dithiothreitol (DTT) for differentiation between IgG and IgM
Presence/absence as well as the specificity definition is possible with CDC

52. Complement-Dependent Cytotoxicity
Complement-dependent cytotoxicity (CDC) was the first technique routinely used for HLA antibody detection and for the cross match test .
In this assay, lymphocyte target cells(lymphocytes isolated from peripheral blood, lymph node, or spleen)
are used to detect complement-fixing IgG1&3 and IgM antibodies present in patient’s serum samples after the addition of rabbit complement.

53. Lymphocytotoxic cross match test
Panel or donor lymphocytes are incubated with recipient serum in the wells of a microtiter (Terasaki) tray.
followed by the addition of rabbit complement.
After a second incubation period, vital stains (e.g., eosin-y or acridine orange and ethidium bromide) are added, and the wells are viewed using inverted or fluorescent (ultraviolet) microscopy to determine cell viability.

54. Addition of Lymphoflot

55. Centifuge for 20 minutes at 1000 x g without braking
Centifuge for 20 minutes at 1000 x g without braking. The lymphocytes will settle as a white ring at the boundary between the plasma and the Lymphoflot
White ring
of Lymphocytes

56. Isolation of Lymphocytes
Using a pipette, carefully transfer the ring to a fresh centrifuge tube,
fill the tube with Hanks Solution and mix.

57. Washing of Lyphocytes
Centrifuge for 10 minutes at 230 x g, pour off the supernatant, resuspend the sediment,
fill the tube with Hanks Solution and mix. Centrifuge again for 10 minutes at 110 x g.
Pour off the supernatant.
For HLA-ABC typing, resuspend the lymphocyte sediment in Lymphostabil.
The lymphocyte suspension should contain approximately lymphocytes/μl.

58. Performing MLCT Thaw the microplates MP at room temperature
- Carefully add 1 μl of the adjusted lymphocyte suspension to the HLA
reagent into each well just under the oil.
- incubate at room temperature ( °C) for:
HLA-ABC typing minutes
HLA-DR/DQ minutes

60. CDC

61. CDC
A high percentage of cell death above background levels is interpreted as a “positive cross match” with the potential to damage a transplanted kidney.
A positive CDC is C/I

62. Positive CDC due to auto-ab
During the 1970s, it emerged that not all lymphocytotoxic antibodies that cause a positive crossmatch are specific for donor histocompatibility antigens,
some antibodies display autoreactivity, causing in vitro lysis of the patient’s own cells in the CDC assay.

63. Auto-Ab
a positive crossmatch caused by non-HLA (autoreactive) lymphocytotoxic antibodies could be safely ignored , with transplant survival rates being equivalent to, transplants with a negative crossmatch.
these autoantibodies as polyreactive IgM, capable of low-affinity binding to multiple antigens owing to weak electrostatic interactions.

64. Determination of IgM ab
IgM antibodies can be differentiated from IgG antibodies by the use of dithiothreitol(DTT).
DTT reduces the disulfide bonds in the IgM pentamer and consequently renders negative a reaction due to IgM.
A positive cross match with IgM is safe
A positive cross match due to IgG is C/I

65. Panel Reactive Ab (PRA)
Serum samples are tested against panels of cells that can be “random” or alternatively can be “selected” to represent the spectrum of HLA types in the population.
The technique can be used for specificity definition, but the results are often expressed as the percentage of the panel to which the sample has reacted (% panel reactive antibody [%PRA]).

66. PRA its use is now strongly discouraged
because the figure entirely depends on the composition of the panel used for testing.
If a patient has a monospecific antibody to a specificity that is common in a population, and a random panel is used, the %PRA is high,
but if the panel has been carefully selected to cover rare and common specificities, the %PRA value for the same antibody may be low.
Values for %PRA cannot be compared between panels or laboratories.

67. Technologies to elucidate patient sensitization profile (con.)
%PRA (which is done by CDC) usage is now strongly discouraged because:
The figure entirely depends on the composition of the panel used for testing
Values for %PRA can not be compared between panels or laboratories
Only complement fixing Abs are detected
Both HLA and non-HLA Abs are detected
The sensitivity of test depends on the viability of the target cells
To increase the sensitivity of CDC:
Increase the incubation times
Augmentation with Antihuman Globulin (AHG)

68. limitations of the CDC technique
Only complement-fixing antibodies are detected,
the sensitivity of the technique depends on the viability of the target cells
the particular batch of rabbit complement
Both HLA and non-HLA antibodies are detected.
Although the use of DTT can differentiate IgM from IgG antibodies, this does not indicate the specificity of the antibody.
Reactivity resulting from an IgM HLA-specific antibody would be indistinguishable from reactivity of an IgM autoreactive antibody.

69. B cell cross match
Most studies did not differentiate between nondamaging (autoreactive) and potentially harmful (HLA specific) B lymphocyte–reactive antibodies.
The clinical interpretation of a B cell crossmatch result is impossible without definition of the specificity of the antibody;
most positive donor B cell crossmatches are not caused by HLA-DR–specific antibodies.
most positive B cell crossmatches are caused by non–HLA-specific, usually B cell–autoreactive antibodies
The B cell cross match Ab is not harmful to a transplant.

70. approaches used to increase the sensitivity of the CDC test
increasing the incubation times,
the wash technique,
- unbound serum is washed from the cell suspension before the addition of rabbit complement, removing the anti complement
factors in the serum.
- preferentially detect IgG antibodies because the lower affinity IgM antibodies become detached during the washing process.
augmentation with antihuman globulin

71. Crossmatch techniques and their clinical relevance (con.)
CDC crossmatch (con.)
CDC significantly reduces the incidence of hyper acute rejection
Poor outcome is seen in sensitized and regrafts despite –ve crossmatch
Predictive value of CDC crossmatch is 80%, the reason is:
↓ Ab levels below the threshold of CDC definition
Non complement fixing Abs (IgG2, IgG4)
The problem can be resolved by:
Extended post complement incubation time
Addition of Antihuman globulin (AHG)

72. Enzyme-Linked Immunosorbent Assays (ELISA)
The targets in an enzyme-linked immunosorbent assay (ELISA) are soluble HLA antigens coated onto plastic, and this is termed a solid-phase assay
These commercially available kits have immediate advantages over CDC in that the test does not rely on viable cells,
only HLA antibodies are detected.

73. Technologies to elucidate patient sensitization profile (con.)
ELISA, A solid phase assay (Ag coated plates)

74. ELISA for Ab sreening
The overall sensitivity of ELISA is greater than CDC.
Two different types of ELISA are routinely used
1- assays to detect the presence or absence of HLA antibodies that can be used as a prescreen of a patient’s serum sample
2- assays that are designed for antibody specification.
The assays have been shown to be reliable for the detection of IgG antibodies, but less so for the detection of IgM, probably because of the washing steps required and the lower affinity of IgM antibodies.

75. Ab screening strategy
Because many patients are unsensitized, first screening the samples is done by a sensitive method to detect presence/absence of HLA Abs
Then the specificity of positive samples is determined
Samples should be obtained regularly on at lest 3-monthly basis
If sensitization occurs, additional samples are required (eg. 14 and 28 days after a transfusion with blood products

76. ELISA metod
Antibody screening using solid-phase binding assays (enzyme-linked immunosorbent assay [ELISA]
1- Purified HLA proteins (either pooled HLA specificities or single antigen specificities) coated onto a solid phase (microtiter tray [ELISA] or microparticles [flow cytometry/Luminex]) are incubated with patient serum.
2- HLA-specific antibodies bind to the antigen-coated solid phase, and nonspecific antibodies are washed off.
3,-IgG HLA specific antibodies bound to the antigen-coated solid phase are detected using a conjugated (e.g., alkaline phosphatase [ELISA] or fluorescein isothiocyanate [FITC] [flow cytometry/Luminex]) anti–human IgG secondary antibody
and detected by colorimetric analyses (ELISA, e.g., using p-nitrophenyl phosphate)

77. ELISA

78. Technologies to elucidate patient sensitization profile (con.)
Advantages of ELISA over CDC:
Does not rely on viable cells
Only Anti-HLA abs are detected
More sensitivity of ELISA compared to CDC
To detect the presence/absence as well as for Ab specification
Reliable for IgG detection

79. Flow Cytometry
The original use of flow cytometry in antibody screening was as a test to determine the presence or absence of antibody.
Flow cytometry is more sensitive than CDC and primarily detects IgG antibodies; this offers the advantage that IgM autoreactive antibodies are not detected.
Although less frequent, however, IgG autoreactive antibodies will be detected by this method.

80. B screening by flowcytometry
Patient sera + donor lymphocyte + rabbit antihuman FITC conjugate
Positive control serum
Negative control serum

81. Technologies to elucidate patient sensitization profile (con.)
Flow cytometry, A solid phase assay (Ag coated microparticles)

82. Ab screening by flowcytometry

83. Technologies to elucidate patient sensitization profile (con.)
Flow cytometry advantages:
To detect Ab presence/absence as well as specification
More sensitive than CDC
Specific microplates coated with a single Ag are used to elucidate highly complex Ab profiles

84. Technologies to elucidate patient sensitization profile (con.)
Luminex, A solid phase assay
Uses multiplexed microparticles and allowes the simultaneous detection and specification of multiple Abs in a serum sample

86. Strategies for transplanting sensitized or likely sensitized patients
Ab removal (con.)
High-dose of IVIG for living and deceased donors when there is a preformed low-titer < 1/16
The main difficulty with high-dose IVIG is that not all patients respond and not all anti-HLA antibodies are suppressed equally
Plasmapheresis combined with low-dose IVIG for living donors in the case of higher titer > 1/16
allows for more consistent and universally reliable removal of anti-donor Ab
As a rule of thumb, each PP/IVIG treatment reduces the DSA titer by one dilution
Ab removal is slower In the case of:
higher number of mismatches and previous txs
Anti DRB3/4/5> Anti DRB1, DQB> Anti class l
+ve CDC> +ve flow, -ve CDC
Ab removal usually occurs before tx, so monitoring is necessary to indicate additional removal after tx