1. Why is an organ rejected?
The mammalian immune system is an complex system developed in response to evolutionary stressors provided by co-existence with micro-organisms over millions of years.
The system is divided into:
Natural immunity: the nonspecific immune response
Macrophages, neutrophils, natural killer cells, cytokines, certain cellular receptors, and complement components
Adaptive immunity: the response to a specific antigen.
B cells, T cells

2. Why is an organ rejected?
In organ Tx, the principal target of the immune response to the graft are the Major Histocompatibility Complex (MHC) molecules expressed on the surface of donor cells (allo-MHC).
This is a form of adaptive immunity.

3. T cells recognize antigen in the form of peptide bound to MHC proteins.
B cells have IG receptors that can recognize the antigenic portions of intact molecules.

4. T Cells
T cells regulate the activities of B cells, T cells & other cells participating in immune responses.
They provide help for Ab production by B cells,
They are effectors of Ag-specific cell-mediated immunity (CMI).
CMI is important in the elimination of
Cells infected with intracellular pathogens (viruses, mycobacteria, some bacteria)
Cells exhibiting aberrant differentiation (eg, neoplasms).
Allogeneic cells (graft rejection).

5. Bretscher P, Cohn M: A theory of self-nonself discrimination
Bretscher P, Cohn M: A theory of self-nonself discrimination. Science 169: , 1970

6. Activated T cells → Clonal expansion → Effector Mechanisms
Mitogenic growth & differentiation factors. e.g IL-2.
Activated T cells → Clonal expansion → Effector Mechanisms

7. IL2 Allorocognition Costimulation IL-2 T cell activation
Clonal Expansion of
Alloreactive Cells
Effector Mechanisms
CD4 + T cells
IL2
IFN
CD8 + T cells
Memory
T cells
IL4, 5, 10, 13
Th2
Th1
B cells
Macrophages
Allo-Ab
Production
Apoptosis
Induction
Cell
Lysis
DTH

9. MHC class I

10. MHC class I molecules are found on almost all cell types except for RBCs
Intra-cytoplasmic peptides are presented together with class I MHC on the cell surface to CD8 T cells.
CD8 positive T cells induce cell lysis (by inducing apoptosis or active killing of cells)

11. MHC class II

12. Class II MHC is expressed only on interstitial dendritic cells, macrophages, & B cells and activated endothelial cells.
MHC II molecules bind peptides derived from extracellular (exogenous) proteins.
Exogenous proteins that have been endocytosed and fragmented are transferred to the cell surface and presented to CD4 T cells together with class II MHC molecules.

13. High levels of class II MHC are expressed on APCs
Dendritic cells: are the major "professional" APCs.
Monocyte/Macrophages: play a partial role in Ag presentation
B cells: express MHC II, required to receive T cell help. Although they do not activate resting naive T cells, they may stimulate memory T cells.
Vascular endothelial cells.

15. Other antigens mediating rejection Minor Histocompatibility Ags
NIH GUIDE, Volume 26, Number 24, July 25, 1997
These antigens appear to be MHC class I bound small peptides presented to - -T cells in an MHC-restricted manner.
Thus, mHAgs are recognized with the same degree of specificity as the major histocompatibility antigens
Most mHAgs result from nonsynonymous Single Nucleotide Polymorphisms (SNP), accounting for around 90% of sequence differences (Collins et al, 1998).
They elicit most commonly, MHC I restricted responses.
They donnot activate Ab production, though they need CD4+ activation for CD8+ help
ABO Antigens
Monocyte endothelial Antigens:
Rarely cause hypercute rejection despite ABO compability

16. Pathways of Allorcognition

17. Game DS, Lechler RI, Transplant Immunology, 2002 ,10(2) pp. 101-108(8)

18. Early Acute rejection
Normal
pathway

19. Ben M. Illigens et al, Human Immunology:2002, 63, 912–925

20. T cell activation and function Activation of calcineurin, NFAT, NF-kB
Signal 1
TK p651ck
Ph. of IC proteins
Signal 2
Activation of calcineurin, NFAT, NF-kB
Bretscher P, Cohn M: A theory of self-nonself discrimination. Science 169: , 1970

21. Lck
Fyn
ZAP-70
Cellular and Molecular Immunology (5th Ed.) Abbas AK, & Lichtman, Editor: Saunders, Philadelphia, 2005

22. Resting T cells express CD28, but resting antigen-presenting cells (APCs) do not express B7 molecules. Within six hours after activation, B7-2 is expressed by antigen-presenting cells and is available to bind to CD28, transmitting a costimulatory signal to the T cell. By 48 to 72 hours after activation, antigen-presenting cells also express B7-1, whereas T cells express the CTLA-4 inhibitory receptor. Both B7-1 and B7-2 can bind to either CD28 or CTLA-4, providing continued costimulation or a new inhibitory signal, respectively. Because CTLA-4 binds B7 molecules with a higher affinity than does CD28, its inhibitory interaction eventually predominates, leading to the termination of the immune response. The fusion protein CTLA-4–Ig can compete with CD28 and CTLA-4 for B7 binding, thus preventing costimulatory interactions.
6 hrs
IL-2 & other cytokines
Anergy or apoptosis
Sayegh MH, Turka LA NEJM, 1998, 338:

23. Resting antigen-presenting cells (APCs), which include B cells, macrophages, and dendritic cells, express CD40. When activated, T cells express CD40 ligand (CD40L). Interactions between CD40 ligand and CD40 are important in providing B-cell help to prevent apoptosis and induce immunoglobulin production and isotype switching. Activation of CD40 on APCs provides a signal for the induction of B7 molecules, particularly B7-2. CD40 ligand may act in T-cell costimulation by directly providing costimulation, by inducing B7, or by inducing other costimulatory ligands.
Sayegh MH, Turka LA NEJM, 1998, 338:

24. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

25. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

26. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

27. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins & adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

28. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

29. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

30. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand(D95)- Fas: T cell apoptosis and elimination

31. Accessory molecules of T cells
CD 28- B7: Production of IL-2 and other cytokines
CTLA4- B7: Inhibition of T cell stimulation [Abatacept (CTLA4-Ig)]
LFA-1- ICAM-1 (2, 3): Adhesion of T cells to APCs, endothelial cells and extracellular matrix proteins and adhesion-dependent lymphocyte functions
L-Selectin-CH ligands on HEV: Regulate migration of leukocytes to various tissues
CD44-matrix proteins: Retention of T cells to endothelium at sites of inflammation
CD40L- CD40:
important signals for Ab production
Activation of macrophages to destroy phagocytosed microbes
B7 expression on all APCs (role in co-stimulation)
Production of adhesion molecules & inflammatory cytokines by APCs → T- cell activation
Fas ligand (D95)- Fas: T cell apoptosis and elimination

32. Principle accessory molecules of T Lymphocytes
Name
Gene family
Cellular expression
Ligand
Role in T cell activation
Adhesion
Signaling
CD4 Ig
Class II T cells
Class II MHC +
CD8
Class I T cells
Class I MHC
CD28
>90% CD4+ T cells, ~50% D8+ T cells
B7-1 (CD 80) B7-2 (CD 86) -
CTLA-4
(CD 152)
Activated T cells
B7-1
B7-2
CD45R
Leukocytes
Unknown
CD2
(LFA-2)
>90% T cells (human), NK cells
LFA-3
LFA-1
Integrin
Leukocytes, platelets
ICAM-1
ICAM-2
ICAm-3
L-selectin
Selectin
Carbohydrate ligands on HEV
CD44
Lymphocytes, granulocytes
Matrix proteins
Cellular and Molecular Immunology (5th Ed.) Abbas AK, & Lichtman, Editor: Saunders, Philadelphia, 2005.

33. Alternative T-Cell Costimulatory Pathways in
Transplant Rejection and Tolerance Induction
(B7-1)
(B7-2)
Salama AD, Sayegh MH. Am J Transplant 2003; 3: 509–511

35. Intracellular events

37. Immunoreceptor Tyrosine Based Activating Motifs TCR complex Itams AP-1
CD3
Itams
LAT
LAT
SLP-76
Fyn
SLP-76
Grb2
Lck
ZAP-70 ζζ
PLC-γ1
CD4, CD8
Shc
Adaptor proteins:
Protein-Protein interaction
SLP-76
Lat
GRb
VAV
Sos
LAT
IP-3
DAG
SLP-76
Ras
Vav
PLC-γ1
Ca2+
MAP-K
Rho
Family
GTPase
calcineurin
p110
p85
dephosphorylate
Regulatoroy
Function
for Cytoskelton
PI-3 K
NF-ATp
NF-қB
Jun
AP-1
Fos
Isakov N - J Leukoc Biol 1997 Jan;61(1):6-16.

38. 1 2 3 4
1, Cyclosporine
2, Tacrolimus (FK506)
3, Rapamycin
4, Corticosteroid

39. Scan information about new immunological events
peripheral tissues L D
Toll-like
receptor T T T T T T T T
spleen,
lymph nodes,
Peyer's patches

40. Tolerance Central (Negative Selection): Peripheral:
Immature lymphocytes in the generative lymphoid organs (thymus for T cells and bone marrow for B cells) encounter only self Ags in high concentration.
These lymphyctes are killed or deleted.
Some of the T cells may develop into regulatory cells
→ Inhibition of immune response
Peripheral:
Important for maintaining unresponsiveness to those self Ags that are expressed in peripheral lymphoid tissues (spleen, lymph nodes, mucosal lymphoid)

41. Mechanisms of Tolerance
Deletion: Apoptotic cell death
Activation induced cell death:
Fas-FasL interaction
Binding if FAAD
Activation of caspase 8
Activation of effector caspases
Apoptosis
Passive cell death (death by neglect)
Loss of survival stimuli e.g. growth factors or costimulators
Increase mitochondrial permeability
Release of cytochrome c and then Apaf
Activation of caspase 9

42. Mechanisms of Tolerance…
Anergy: Functional inactivation without cell death
Ag presentation by APCs without costimulation (Signal 2 or B7-CD28)
Engagement of inhibitory receptors (CTLA-4 instead of CD28)
CTLA-4 Ig: Abatacept, LEA29Y (belatacept)
Antibodies against CD40L

43. Mechanisms of Tolerance…
Supression: by regulatory lymphocytes
These are CD4+ T cells expressing high levels of IL-2-R (CD25) but not other markers of activation
Inhibition of these cells may cause autoimmune disease
Clonal ignorance: Self Ags may be ignored by the immune system, i.e. lymphocytes encounter the Ag but fail to respond

44. Auchincloss H, Am J Transplant 2001;1(6-12)
The Thymic Deletional Mechanism
Exposure to donor antigens during fetal development achieved the same thymic deletional tolerance that occurs for many self antigens.
Engraftment of donor stem cells
Mixed bone marrow chimeras
Thymic deletion of T cells reactive to donor APCs
Re-poplulation of a new T cell repertoire

45. Auchincloss H, Am J Transplant 2001;1(6-12)
Peripheral Deletional Mechanisms
Removal donor-specific T cells after they have matured.
Mechanisms:
Veto phenomenon: involves the ability of a population of CD8 T cells to destroy attacking T cells that would otherwise kill them, via attachment to their MHC I molecules.
Cell death mediated through Fas Ligand,
Other forms of activation-inducedcell death (AICD)
Withdrawal of growth factors such as IL-2.
Peripheral Nondepleting Mechanisms
An active regulation of T cell responses by other (suppressor) T cells
Change in the character of the donor specific response

46. Auchincloss H, Am J Transplant 2001;1(6-12)
Although multiple mechanisms of tolerance induction may be combined, there are two hallmarks of a tolerance induction strategy that will ultimately be successful in clinical practice:
It will include the use of donor bone marrow (or stem cells) somewhere in the protocol,
It will eventually lead to easily measurable macrochimerism.

47. Graft rejection is usually initiated by CD4 helper T cells (TH) that bind peptides in complexes with MHC class II molecules on antigen-presenting cells. In the direct pathway of recognition, an MHC molecule on a foreign (allogeneic) cell, such as an antigen-presenting cell (allogeneic APC), binds to the helper T cell. In the indirect pathway, the foreign MHC molecule is processed into peptides that are presented to the helper T cell by one of the body's own antigen-presenting cells (self APC). In either case, activated CD4 helper T cells proliferate and secrete a variety of cytokines that serve as growth and activation factors for CD8 cytotoxic T cells (TC), B cells, and macrophages, which cause destruction of the graft by direct lysis of target cells, antibody production, and delayed-type hypersensitivity mechanisms, respectively.
Sayegh MH, Turka LA NEJM, 1998, 338: