Transplantation Immunology

Transplantation Immunology
Anthony Warrens
Barts and The London School of Medicine and Dentistry
Queen Mary, University of London
a.warrens@qmul.ac.uk
Learning Outcomes
• An understanding of importance of the HLA system in
the rejection of transplanted organs (‘allorecognition’)
• The mechanisms by which the recipient immune system
recognises donor HLA and non-HLA antigens
• An understanding of the mechanisms underlying
immunological tolerance and how we believe they
currently apply to transplantation
• An understanding of the sequence of activation of the
CD4+ T lymphocyte and how our current
immunosuppressive drugs interfere with it
rejection of transplanted tissues
• The strength of the alloimmune response
and the HLA system
• Mechanisms of allorecognition
– Direct and indirect pathways
• Immunological tolerance in transplantation
• T cell activation and immunosuppression
• New developments: semi-direct recognition
• Mechanisms of rejection
and the HLA system
Strength of alloresponse
•The ‘strength’ of a primary alloresponse
is considerably greater than that against
conventional antigens
•This allows it to be seen in vitro in a
mixed lymphocyte response
HLA matching is beneficial
% grafts
surviving
100
90
80
0 MM (n=3958)
1 MM (n=6404)
70 2 MM (n=13285)
3 MM (n=18510)
4 MM (n=13941)
60 5 MM (n=7052)
6 MM (n=2238)
50
0
0 1 2 3 4 5
Years
Revision from Dr Stagg’s lecture: How a T cell is activated
T Cell APC
T Cell Receptor
(TCR) Major Histocompatibility
Complex (MHC)
Revision from Dr Stagg’s lecture:
The polygenic and polymorphic of MHC genes ensures multiple different MHC
molecules expressed, increasing the repertoire of peptides that can be presented
B B C A schematic of human MHC

A A C
MHC Class I molecules
Maternal MHC
DR DR DP DP DQ DQ A B C
MHC class II MHC class I
DR DR DP DP DQ DQ A B C
Paternal MHC
DR DQ
MHC Class II molecules

DP
HLA-A HLA-B HLA-C
Number of classical HLA alleles
and proteins described – Feb 2012
Class I HLA-A HLA-B HLA-C
Alleles 1,757 2,338 1,304
Proteins 1,290 1,795 946
Nulls 87 73 35
Class II DRA DRB (1- DQA1 DQB1 DPA1 DPB1

9)
Alleles 7 1,166 47 162 33 152
Proteins 2 873 29 113 16 131
Nulls 0 20 1 1 0 3
and the HLA system
Direct and indirect allorecognition
Direct allorecognition
CD8 CD4
IL-2
I II
allo APC
Strength of direct allorecognition
A B
Immunosuppression
Re-
The graft was not txn.
1/12
rejected: the graft was B
fully “antigenic” but
not “immunogenic”
no immunosuppression
Very slow rejection
Strength of direct allorecognition
A B
Immunosuppression
Re-
txn.
1/12
B
Mature dendritic cells
The graft was immediately rejected

Indirect allorecognition
A B
Immunosuppression
Re-
Slow rejection txn.
1/12
represents B
indirect recognition
no immunosuppression
Very slow rejection
Direct and indirect allorecognition
Indirect allorecognition
CD8 Direct allorecognition
CD4
I
allo APC
CD8 CD4
Shed IL-2
allogenei
c MHC
II
IL-2
Taken up and
processed by I II
auto APC
allo APC
Auto APC
Long-term patient survival after first adult heart only transplant in the
UK, 1 January 1996 – 31 December 2008
100
90
80
70
60
50
% Patient survival
40
Year of transplant
30 (Number at risk on day 0)
1996-1998 (708)
20
1999-2001 (501)
2002-2004 (387)
10 2005-2008 (429)
0 1 2 3 4 5 6 7 8 9 10
Years since transplant
Source: Transplant activity in the UK, 2009-2010, NHS Blood and Transplant
Long-term patient survival after first adult heart only transplant in the
UK, 1 January 1996 – 31 December 2008
100
Impact of direct allorecognition

90
80
70
60
50
Impact of indirect allorecognition
% Patient survival
40
Year of transplant
30 (Number at risk on day 0)
1996-1998 (708)
20
1999-2001 (501)
2002-2004 (387)
10 2005-2008 (429)
0 1 2 3 4 5 6 7 8 9 10
Years since transplant
Source: Transplant activity in the UK, 2009-2010, NHS Blood and Transplant
T cells with indirect
anti-donor allospecificity
104
p<0.05 *
1/frequency
105
106
CAN CAN Free
Hyporesponsiveness in patients :
Direct allorecognition is switched off
and the HLA system
“Switching off” immune
responsiveness
• Antigen- specific non-responsiveness
= immunological tolerance
• Preserves all other immune responses
– Superior to immunosuppression
• Does occur spontaneously in some
patients
• How can we induce it?
Main mechanisms of tolerance
• Deletion
– Central
– Peripheral
• Switching cells off (making them “anergic”)
• Active regulation
Treg families:
induced and naturally occurring
Induced in vitro and in vivo:
Anergic T cells
Tr1 Naturally occurring:
Th3 CD4+CD25+
CD8+CD28- NK-T cells
CD4+CD25-
CD4+CD25+ T cells
Normal Partially T cell
Mouse Thymectomy 3-5 depleted mouse
days after birth
Mouse has severe

polyautoimmune disease
including gastritis, thyroiditis,
oophritis and diabetes
CD4+CD25+ T cells
Normal Thymectomy 3-5

Mouse days after birth
Normal Mouse
Adoptive transfer of CD4 T cells that
express CD25 prevents the auto-
immunity caused by d3 thymectomy
CD4+CD25+ T cells
from normal animal
Regulatory cells in transplantation
Rendering cells ‘anergic’
• Probably encouraged by
immunosuppression
• If a T cell recognises antigen but does not
become activated
– It is rendered anergic
and the HLA system
Activating the Helper T Lymphocyte
Antigen-presenting cell (APC)
Ag B7 HLA
class II
Ag
T cell
CD28 receptor
Helper T Lymphocyte
HLA class II
B7
Ag
T cell receptor
CD28
Helper T Lymphocyte
HLA class II
B7
Ag
T cell receptor
CD28
Signal 1
Helper T Lymphocyte
HLA class II
B7
Ag
T cell receptor
CD28
Signal 1
Signal 2
Helper T Lymphocyte
HLA class II
B7
Ag
T cell receptor
CD28
Signal 1
Signal 2
Cytokine
gene
activation
Helper T Lymphocyte
HLA class II IL-2

B7
Ag
T cell receptor
CD28
Signal 1
Signal 2
Cytokine
gene
activation
Helper T Lymphocyte
HLA class II IL-2

B7
Ag IL-2 receptor
T cell receptor
CD28
Signal 1
Signal 2
Cytokine
gene
activation
Helper T Lymphocyte
HLA class II IL-2

B7
Ag IL-2 receptor
T cell receptor
CD28
Signal 1 Signal 3
Signal 2
Cytokine
gene Proliferation
activation
Helper T Lymphocyte
HLA class II IL-2

B7
X Ag
T cell receptor X IL-2 receptor
CD28
Signal 2
X Signal 1 X Signal 3
Cytokine
X
gene X
Proliferation
activation
Helper T Lymphocyte
and the HLA system
Semi-direct presentation
Semi-direct pathway
Recipient APC
Recipient T cell
Donor cell
Passage of MHC from donor cell (not necessarily dendritic cell) to recipient
APC
Human Neutrophils Acquire
SLA Class II from Porcine Endothelium0
2.29
%
SLA class II
0.60
Isotype %
control
CD16
Human Neutrophils Acquire
SLA Class II from Porcine
Endothelium
Semi-direct presentation
Figure 5: Functional consequences of acquisition. M1 cells were stained with CFSE and removed from cocultures by FACS sorting. This involved
selection of those cells that were both within the lymphocyte gate R1 (A) and CFSE negative, gate M2 (B). (C) shows that sorting on size and
CFSE ensured no contaminating CFSE+ cells post-sorting: the percentage represents all ungated cells and is comparable to that found by analysis
of CD4+ cells cultured alone, in the absence of M1 cells (D). Once isolated, the degree of acquisition was assessed by flow cytometry and
depicted in (E–I); background staining on naïve CD4+ cells is shown in D; for all figures HLA-DR is thick black, CD80 is thick gray and CD86 is
thin gray; isotype controls are given in thin black. To test stimulator function, the exposed T cells were irradiated before 3H-thymidine
proliferation assay with nonirradiated exposed T cells (J), fresh autologous CD4+ cells (K) or fresh allogeneic CD4+ cells (L). Dark black bars
represent the MLR and gray bars represent proliferation of irradiated stimulator cells only.
Game and Lechler 2005 AJT 5: 1614
Minor histocompatibility
antigens
• Induce a slower alloresponse
• Molecules presented to T cells (by MHC) that are
antigenically distinct
• Classical H-Y antigen
• In many cases antigen is cloned and the presented peptide
identified
♀
♀ ♂ accepted
♂ ♂
♂
♂♀ rejected
♀ X♀
and the HLA system
Classification of rejection
• Hyperacute
• Acute
• Chronic
• Hyperacute: minutes to hours

• Acute: one week to six months
occasionally later
• Chronic: months to years
• Hyperacute
• Acute
• Chronic
Defined on basis of time to occurrence

Actually very different mechanisms
Rejection: Immune-mediated
damage
Helper (CD4+) T lymphocyte
Cytotoxic (CD8+) Macrophage B lymphocyte

T lymphocyte
Antibodies
Relating mechanisms to rejection
processes - 1
Acute cellular rejection
Renal allograft
Helper T lymphocyte
Cytotoxic T Macrophage B lymphocyte

lymphocyte
Antibodies
Cell-mediated rejection
Relating mechanisms to rejection
processes - 2
Acute antibody-mediated rejection
Renal allograft
Helper T lymphocyte

lymphocyte
Antibodies
Acute antibody-mediated rejection
• Development of anti-donor antibodies after

transplantation
– or development of secondary response in sensitised
individual (accelerated acute rejection)
• May occur in combination with cell-mediated
rejection
• Diagnosis greatly helped by
– staining for C4d
– Opportunity to detect circulating donor-specific
antibodies (DSA)
Classical pathway of complement activation
Antibody-antigen complex
C1 Activated C1 C3
C2 C4b2a C4b2a3b
C4 C3 convertase C5 convertase
C4d
Covalent bonds with thio-ester groups on

endothelial cell surfaces and matrix components
C4d Immunofluorescence
Acute Cellular Rejection Acute Humoral Rejection
Page 61 © Imperial College London

Herzenberg et al (Vancouver) JASN, 13:234,2002
Tubulo-Interstitial Endarteritis
Page 62 © Imperial College London

Relating mechanisms to rejection - 3
Hyperacute rejection
Previous transplant
Previous transfusion
Renal allograft
Previous pregnancy
Helper T lymphocyte

lymphocyte
Antibodies
Antibody-mediated rejection
Endothelial
cells (EC)
Vessel lumen Coagulation
C’
Preformed Ab EC activation
and damage
Rejection
Definition: Recognition and destruction by recipient immune
system
Multiple non-
Immune-mediated damage
Immunological
Factors
Hyperacute Acute Chronic

rejection rejection graft
dysfunction
Learning Outcomes
• An understanding of importance of the HLA system in
the rejection of transplanted organs (‘allorecognition’)
• The mechanisms by which the recipient immune system
recognises donor HLA and non-HLA antigens
immunological tolerance and how we believe they
currently apply to transplantation
• An understanding of the sequence of activation of the
CD4+ T lymphocyte and how our current
immunosuppressive drugs interfere with it
rejection of transplanted tissues
Take-home messages
• The response to transplanted tissue and organs is
amongst the strongest immune response that exists
• In the early phase after transplantation, the recipient
immune system recognsies the major antigen stimulating
it (i.e. HLA) directly
• Later, that recognition is indirect whereby peptides
derived from donor HLA are presented by recipient HLA
on recipient APCs
• Immunological tolerance may result form a number of
mechanisms, but is usually incomplete and unreliable
• Current immunosuppression attacks sequential events in
T cell activation

Transplantation Immunology - Professor Anthony Warrens

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B B C A schematic of human MHC

MHC Class I molecules

MHC class II MHC class I

MHC Class II molecules

Class II DRA DRB (1- DQA1 DQB1 DPA1 DPB1

The graft was immediately rejected

Years since transplant

Impact of direct allorecognition

Years since transplant

Mouse has severe

Normal Thymectomy 3-5

HLA class II IL-2

HLA class II IL-2

HLA class II IL-2

HLA class II IL-2

• Hyperacute: minutes to hours

Defined on basis of time to occurrence

Cytotoxic (CD8+) Macrophage B lymphocyte

Cytotoxic T Macrophage B lymphocyte

Cytotoxic T Macrophage B lymphocyte

• Development of anti-donor antibodies after

Covalent bonds with thio-ester groups on

Page 61 © Imperial College London

Page 62 © Imperial College London

Cytotoxic T Macrophage B lymphocyte

Hyperacute Acute Chronic

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