1. Transplant Immunology

2. Transplantation and Immunology
Review of cytokines
Cells involved in alloreactivity
Cell to cell interaction
Major histocompatibility locus: transplant antigens
Clinical immunosuppression

3. Sources and Effects of Cytokines
TNF
MΦ, others
Proliferation of T and B cells; enhances T cell function, activates MΦ and PMNs
IFN-α
Leukocytes
Increases expression of MHC class I receptors, acute inflammatory response and macrophage activation
IFN-β
fibroblasts
Same as IFN-α
IFN-γ
TH1
Activates MΦ and NK cells, Promotes TH1 pathway
Upregulates MHC I/II
IL-1
Proliferation of T and B cells, fever, inflammation
IL-2
Promotes T-cell and activated B-cell proliferation, stimulates cytokine secretion by T-cells
IL-3
T cells
Stimulates pluripotent stem cells
IL-4
TH2
Promotes TH2 differentiation
B cell growth and differentiation
IGE production, mast cell growth factor
Inhibits secretion of proinflammatory cytokines
Il-5
T-cells, mast cells
Growth/differentiation of eosinophils
B cell proliferation
IL-6
MΦ, TH2
Induces fever, promotes B-cell maturation and differentiation, stimulates hypothalamic-pituitary-adrenal axis, induces hepatic production of acute phase proteins; levels are increased in sepsis and trauma
IL-8
MΦ, endothelial cells
Stimulates chemotaxis and oxidative burst by PMNs, high circulating levels associated with fatal outcome in sepsis
Helper T cells have two important functions: to stimulate cellular immunity and inflammation, and to stimulate B cells to produce antibody. Two functionally distinct subsets of T cells secrete cytokines which promote these different activities. Th1 cells produce IL-2, IFNg, and TNFb, which activate Tc and macrophages to stimulate cellular immunity and inflammation. Th1 cells also secrete IL-3 and GM-CSF to stimulate the bone marrow to produce more leukocytes. Th2 cells secrete IL-4, IL-5, IL-6, and IL-10, which stimulate antibody production by B cells.
T cells are initially activated as Th0 cells, which produce IL-2, IL-4 and IFNg. The nearby cytokine environment then influences differentiation into Th1 or Th2 cells. IL-4 stimulates Th2 activity and suppresses Th1 activity, while IL-12 promotes Th1 activities. Th1 and Th2 cytokines are antagonistic in activity. Th1 cytokine IFNg inhibits proliferation of Th2 cells, while IFNg and IL-2 stimulate B cells to secrete IgG2a and inhibit secretion of IgG1 and IgE. Th2 cytokine IL-10 inhibits Th1 secretion of IFNg and IL-2; it also suppresses Class II MHC expression and production of bacterial killing molecules and inflammatory cytokines by macrophages. IL-4 stimulates B cells to secrete IgE and IgG1. The balance between Th1 and Th2 activity may steer the immune response in the direction of cell-mediated or humoral immunity

4. IL-9
TH2
Promotes proliferation of activated T-cells
IL-10
Inhibits proinflammatory cytokines by MΦ
IL-11
Neurons, fibroblasts, epithelial cells
Increases platelet production, inhibits proliferation of enterocytes; increased levels in sepsis and DIC
IL-12 MΦ
Promotes differentiation of CD4 cells to TH1 cells, enhances IFN-γ secretion by TH1 cells and NK cells; implicated in inflammatory bowel disease
IL-13
IL-18
Costimulation of IL-12 of IFN-γ secretion by TH1 cells and NK cells

5. Cells involved in alloreactivity
Key components
T cells
B cells
Antigen presenting cells (APC)
Development of lymphoid system begins with pluripotential stem cells in liver and bone marrow of fetus. As fetus matures, bone marrow is primary site for lymphopoiesis .
Pre T cells migrate to thymus, where CD3+ cells mature and become “educated” to self
Learn to restrict to self MHC and learn tolerance to self antigens
Mature T cells then populate lymph nodes, spleen, gut

6. Development of tolerance occurs centrally and peripherally
Central tolerance occurs through clonal deletion in the thymus
Pre-T cells (CD3+) enter thymus, proliferate and become CD4+ and CD8+
Undergo “education” by self MHC class I or II

7. POSITIVE SELECTION NEGATIVE SELECTION
T cells that have a TCR receptor molecule with intermediate affinity for self MHC survive
If affinity too high or low, cells undergo apoptosis
NEGATIVE SELECTION
Occurs when T cells exposed to self antigens
Undergo apoptosis if react too strongly

8. Apoptosis Regulated cell death
Cell condenses, fragments, phagocytosis occurs
Occurs through Fas Ligand system
Fas- receptor expressed on activated T cells
Expression of Fas and FasL lead to apoptosis

9. Cell to Cell Interactions
APCs- dendritic cells and macrophages-
Bind antigen and present it to B and T cells
Protein antigens need to be digested by phagocytes before presented to lymphocytes for self and non self recognition by MHC

10. T cell activation
TCR- T cell receptor recognizes antigens only if presented as peptide/MHC complexes presented on surface of APCs
TCR (heterodimer) binds covalently with CD3 molecule
Foreign antigen causes conformational change; causes intracellular signaling
When T cell activated, TCRs decrease; IL-2/IL-2R release increases(thru phospholipase C activation)
T cell proliferates

11. FIGURE T-cell signaling through membrane inositol lipid metabolism. T-cell receptor (TCR)–associated protein tyrosine kinases activated by antigen presentation lead to the phosphorylation of phosphatidylinositol phospholipase C-γ1 (PI-PLC-γ1) as well as docking sites for PI-PLC-γ1 on the plasma membrane. PI-PLC-γ1, activated tyrosine phosphorylation, catalyzes the breakdown of membrane phosphatidylinositol 4, 5-bisphosphate (PIP2), generating inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of Ca2+ stored in the endoplasmic reticulum (ER), and DAG plus Ca2+ activate protein kinase C (PKC). Ca2+ and PKC both serve to activate other enzymes and eventually transcription factors. The symbol (P) refers to phosphorylated tyrosine. (From Abbas AK, Lichtman AH, Pober JS: T lymphocyte antigen recognition and activation. In Cellular and Molecular Immunology, 3rd ed. Philadelphia, WB Saunders, 1997.)

12. T cell activation also requires co-stimulatory signals (CD 28/B-7 interaction) Without this, T cell anergy results (basis for monoclonal Ab)

13. T cell effector functions
Cytotoxic T cells (CD8)- interact with MHC class I/peptide complexes and lead to cell lysis
Helper T cells (CD4)- recognize antigen in context of MHC class II molecules
Leads to cell mediated (TH1) or humoral (TH2) response

14. FIGURE T-helper (CD4+) cells can be divided into functionally distinct subsets (TH1 and TH2) based on their cytokine secretion profiles. Cytokines secreted by TH1 cells play a key role in cell-mediated immunity, whereas those produced by TH2 cells are important in B-cell stimulation and antibody production. An important feature of the TH1/TH2 cell paradigm is that cross-regulation of function between TH1 and TH2 cells occurs. Thus, for example, IFN-γ stimulates TH2 cells whereas IL-10 inhibits TH1 cells. Cyclosporine and tacrolimus (FK-506) are potent inhibitors of IL-2 and IFN-γ production by TH1 cells. There is evidence, however, that they may spare IL-10 (cytokine synthesis inhibitory factor) production by TH2 cells. ADCC, antibody-dependent cell-mediated cytotoxicity.

15. B lymphocytes IL-7- growth factor for pre B cells
IL-4, 5, 6 stimulate maturation and proliferation of B cells
Responsible for antibody mediated immune response against foreign antigen
Express immunoglobulin antibody on cell surface
One antigen specific antibody produced per mature B cell
Naïve B cells express IgD and IgM
After antigen stimulation, undergo isotype switching to produce IgG (memory B cells)

16. FIGURE 26-8. Phases of helper T-cell–dependent antibody responses
FIGURE Phases of helper T-cell–dependent antibody responses. Ig, immunoglobulin; FDCs, follicular dendritic cells. (From Abbas AK, Lichtman AH, Pober JS: T lymphocyte antigen recognition and activation. In Cellular and Molecular Immunology, 3rd ed. Philadelphia, WB Saunders, 1997.)

17. Macrophages Role of monocyte/macrophage Phagocytosis
Presentation of processed antigen to lymphocytes results in production of cytokines

18. MHC locus/transplant antigens
Major histocompatibility locus located on Chromosome 6
Produces Human Leukocyte Antigens (HLA)
Class I molecules are expressions of HLA-A, HLA-B, HLA-C
Class II molecules are expressions of HLA-DR, HLA-DQ, HLA-DP
Class III- contains mediators of immune function (TNF, heat shock protein)

19. Comparing MHC Class I and II
Properties Class I Class II
ANTIGENS HLA-A, B, C HLA-D-DR/Q/P
TISSUE On all nucleated cells involved in
DISTRIBUTION cells immune system
FUNCTIONS Endogenous Ag Exogenous Ag
presented to CD8 presented to
(cytotoxic) T-cells CD4 (T-helpers)

20. HLA-Typing: Prevention and Rejection
Must make graft less antigenic so host doesn’t reject it
Major strategy: minimize alloantigen differences between donor and host
ABO compatibility to prevent hyperacute rejection
HLA (tissue) typing: HLA-A, HLA-B, HLA-DR most important
More alleles matched, greater survival of graft in 1st year
Appears to only matter if 6 allele match

21. HLA-typing Serologic Molecular Cross match
Uses antigen specific serum to bind cells expressing antigen
Not very accurate
Molecular
PCR
Cross match
Uses flow cytometry to test for preformed antibodies

22. Rejection Three types Hyperacute- due to preformed Ab
ABO incompatibility
Minutes to hours
Prevented by screening
Result of prior pregnancy, transplant, blood transfusion
Cannot be treated with anti rejection meds
Mechanism- complement cascade; mediated by IgG
Characterized by rapid thromobosis/occlusion of graft vasculature

23. Hyperacute Rejection

24. Acute rejection Mediated by T lymphocytes
Occurs 1-3 weeks after transplantation without immunosuppression
Most common 3-6 months post transplantation but can occur anytime
Characterized by macrophage/lymphycyte infiltration

25. Acute Rejection Two forms Acute vascular rejection IgG response
Leads to lysis of endothelial cells and cytokine production
Endothelial necrosis
Acute cellular rejection
Necrosis of parenchymal cells due to infiltration of T cells and macrophages

26. Acute Rejection

27. Chronic rejection
Appears as fibrosis and scarring
Atherosclerosis in heart patients
BOOP in lung patients
“vanishing bile duct syndrome” in liver patients
Fibrosis, glomerulonephropathy in kidney patients
Due to ischemia and inflammation

28. Chronic Rejection Risk factors
Previous acute rejection ( risk with more episodes)
Inadequate immunosuppression
Initial delayed graft function
Donor factors (age, HTN)
Procurement issues (ischemia time, reperfusion injury)
Recipient issues (DM, HTN, infections)

29. Chronic Rejection

30. FIGURE Hyperacute rejection results when preformed antibodies bind to vascular endothelium and activate complement. In acute rejection, CD8+ T cells attack alloantigens in parenchyma and vessels. Chronic rejection has a multifactorial etiology but leads to smooth muscle proliferation and fibrosis. (Adapted from Abbas AK, Lichtman AH, Pober JS: Immune mechanisms of graft rejection. In Cellular and Molecular Immunology, 4th ed. Philadelphia, WB Saunders, 2000.)

31. Clinical immunosuppression
Risks of immunosuppression
Infection
Environmental pathogens and reactivation of host pathogens (CMV, EBV)
Requires prophylaxis with gangcyclovir; hep B vaccine, Bactrim to prevent PCP, UTI
Malignancy
No increase in lung, breast, prostate, colon, uterine CA
Skin tumors, cervical CA, Kaposi’s, lymphoma, other virus mediated tumors more common in Tx patients
Cardiovascular disease
Pretransplant risk factors (DM, HTN, CAD) amplified by immunosuppression
Pts need good pre-op workup

32. Objectives of Immunosuppression
Facilitate acceptance of the allograft
Specific
Low toxicity

33. Basic Strategies of Immunosuppression
Induction (High dose initial immunosuppression)
Facilitate graft acceptance
Minimize early rejection
Favor induction of tolerance
Maintenance therapy for chronic acceptance
Augmentation to reverse acute rejection.

34. Graft Rejection
Highly dependent on T cell activation and proliferation.
Signaling pathways and control points for entry into cell cycle are appropriate targets for immunsuppression.

35. Broad Mechanisms of Immunosuppression
T cell depletion. (induction)
Inhibition of T cell activation.
Block antigen binding.
Block accessory molecules.
Inhibition of IL-2 production.
Inhibition of T cell proliferation.
Inhibition of B cell proliferation.

36. Induction Agents- Thymoglobulin
Antithymocyte globulin- polyclonal sera produced when human lymphocytes injected into animals (rabbit or horse)
Interferes with cell-mediated reactions: allograft rejection, graft v host disease
Used in early post transplant period or to reverse ongoing rejection
Can cause anemia, thrombocytopenia
Allergic reaction most common problem

37. Induction Agents- Monoclonal Antibody
OKT3- engages CD3 TCR receptor
TCR receptor internalized by cell and no longer expressed on cell surface
T cells removed by spleen and reticuloendothelial cells
Less effective over time
Cytokine release syndrome

39. Induction Agents- IL-2R inhibitors
Basiliximab/ daclizumab (CD25 Ab)
Bind to IL-2 receptors without activating them
Cells can’t bind IL-2; can’t proliferate
Must be combined with other immunosuppressants
Well tolerated

40. Induction Agents- Rituximab- Anti-CD 20 Ab Campath 1H- Anti CD52 Ab
Treats humoral rejection due to B cell depleting ability
Campath 1H- Anti CD52 Ab
Acts against B/T cells, macrophages
Depletes lymphocytes for 2-6 months

41. Maintenance Agents Adrenal Corticosteroid- prednisone
Antiproliferative
Azathioprine
Mycophenolate mofetil
Leflunomide
T cell directed
Calcineurin inhibitors: cyclosporine, tacrolimus
Cell cycle arrest: sirolimus
Lymphocyte Sequestration- FTY720

42. Adrenal Corticosteroids
Suppresses transcription and secretion of IL-1, IL-6, TNF
Inhibits IL-2 production and binding of IL-2R
Blocks ability of macrophages to respond to signals such as migration inhibition and activation factors
Side effects
Cushingoid features
Ulcer/GI bleed
Diabetes
Avascular necrosis

43. Antiproliferative Agents Antimetabolites
Lymphocytes depend on de novo purine synthesis rather than salvage pathway
Azathioprine (6 mercaptopurine)
Blocks de novo purine synthesis, impedes T cell replication
Mycophenolate (Cellcept)
Blocks inosine monophosphate dehydrogenase (Rate limiting step for de novo sythesis)
Side effects- diarrhea, vomiting, bone marrow suppression, opportunitistic infections

44. Antiproliferative Agents Antimetabolites
Leflunomide
Blocks dihydro-orotate dehydrogenase (necessary for de novo pyrimidine synthesis)

45. Inhibitors of T cell Receptor Signaling
Cyclosporine-binds to cyclophilin
Complex binds/inhibits calcineurin
Blocks cytokine production, esp IL-2, but does not inhibit proliferation
P 450 metabolism
Side Effects
Gingival Hyperplasia
Infection
Nephrotoxicity
Hypertension
Tremors, Nightmares, Insomnia
Hirsutism
Fibrous Breast Tissue

46. Inhibitors of T cell Receptor Signaling
Tacrolimus (FK506, Prograf)
Also inhibits calcineurin, thus inhibiting IL-2 production
Inhibits production of cytotoxic T cells
Side effects: alopecia, risk of post transplant diabetes

47. Inhibitors of T cell Receptor Signaling
Sirolimus
Macrolide antibiotic
Causes cell cycle arrest
Blocks transduction of signals from IL-2R to nucleus rather than blocking cytokine gene expression

48. T cell Receptor Signaling Pathway

49. T cell Signaling: Calcineurin

50. Lymphocyte Sequestration
FTY720
Sequesters lymphocytes in Peyer’s patches
Can cause bradycardia

51. Treatment of Acute Rejection
Need prompt diagnosis from biopsy
Treat mild rejection with steroids
Thymoglobulin or OKT3 for severe rejection
CMV prophylaxis during treatment of acute rejection

52. Now for a Quick Review:

53. The correct terminology for a graft between genetically nonidentical members of the same species is:
Allogeneic graft
Autogeneic graft
Isogeneic graft
Syngeneic graft
Xenogeneic graft

54. Which of the following statements correctly characterize the genetic basis of histocompatibility?
A. Histocompatibility is determined by a series of genes inherited as a complex and subject to the mendelian rules that characterize recessive traits
B. Histocompatibility depeds in part on the inheritance of histocompatibility genes and in part of the inheritance of T-cell receptor genes
C. Major histocompatibility genes are polymorphic
D. Major histocompatibility genes are independently segregating and co-dominant
E. Histocompatibility is learned

55. Which of the following distinguish MHC classs I from MHC class II antigens?
A. MHC class I and class II antigens are encoded in different regions of the MHC complex
B. MHC class I antigens are expressed on specialized antigen presenting cells, whereas MHC class II antigens are expressed on all cells
C. MHC class I and class II are members of different supergene families
D. MHC class I are considered to be the major histocompatibility antigens and MHC class II the minor antigens
MHC class I is recognized by the CD8 glycoprotein, whereas MHC class II is recognized by the CD4 glycoprotein

56. Which of the following characterize the role of the major histocompatibility antigens in immune responses?
A. The major histocompatibility antigens are critical in antigen processing and presentation
B. Major histocompatibilty antigens contribute to the maturation of T cells in the thymus
C. T cells recognize only foreign antigens that are complexed with major histocompatibility antigens
D. Expression of major histocompatiblity antigens is increased in inflammation
E. Recognition of major histocompatibility antigens is critical to the development of tolerance

57. Which of the following statements correctly characterize the role of histocompatibility typing in transplantation?
A. Histocompatibility typing must be carried out before transplantation can be safely undertaken.
B. The “rules” of histocompatibility wre established shrotly after the advent of immunosuppressive therapy made transplantation feasible
C. Histocompatibility typing may involve serologic, cellular, and molecular procedures for typing
D. The role of histocompatibility matching in transplantation is controversial
E. The cross match test is carried out to determine whether a potential graft recipient has antibodies against the donor

58. Activation of T cells requires:
A. Stimulation of the antigen receptor
B. Stimulation of the MHC antigen
C. Co-stimulation (CD28/B7)
D. Anergy
E. CD3

59. Which of the following statements characterize the biology of allotransplantation?
A. The rejection response is systemic
B. The rejection response is learned
C. The rejection response involves a constellation of immunologic and enviromental factors
D. Allotransplantation evokes a cellular immune response
E. Allotransplantation evokes a humoral immune response

60. Allograft rejection may involve which of the following?
A. Helper T cells
B. Veto cells
C. Cytotoxicity
D. Cytokines
E. The Arthus reaction

61. Which of the following statements about allograft rejection are true?
A. In the absence of immunosuppression, the time and intensity of rejection of transplants between unrelated donors and recipients is highly variable.
B. Allograft rejection may be mediated by antibodies or by cells
C. Allograft rejection is thought to be caused by Th2 cells
D. Acute cellular rejection is the major cause for loss of clinical organ transplants
E. An individual with “tolerance” is unable to reject an allograft

62. The presence of donor reactive lymphocytotoxic antiboies in the serum of a potential kidney transplant recipient:
A. Can be detected by in vitro testing with recipient leukocytes and donor serum
B. Is a contraindication of kidney transplantation
C. Can be found in all male patients older than 20 years

63. Utilization of a living related donor instead of a cadaver donor is no longer an advantage in rental transplantation beause:
A. Public recognition of transplantation as a successful therapy has facilitated obtaining family permission of recovery of transplantable organs. Thus, there is no need to use related donors.
B. Cyclosporine therapy after cadaveric renal transplants has improved their outcome, which is now comparable to related-donor transplants
C. Modern preservation techniques can maintain viability of kidneys from cadaver donors for many hours, consistently allowing their function to be as good as that of kidneys from living donors
D. None of the above

64. As compared with the early immunosuppressive drugs (azathioprine, steriods, antilyphocyte serum), some newer agents have the following specific advantages:
A. Cyclosporine, which interferes with lymphokine production, exhibits neither bone marrow nor renal toxicity
B. Monoclonal antibody (OKT3) is more available and has greater specificity and few side effects than antilymphocyte serum
C. Tacrolimus (FK506) has properties similar to those of cyclosporine but is especially valuable for rescue of grafts that are failing on cyclosporine therapy
D. None of the above

65. Which of the following statements about posttransplantation malignancy is correct?
A. Certain immunosuppressive agents increase the incidence of malignancy whereas others do not
B. Those malignancies most commonly seen in the general population (breast, colon) are substantially more common in transplant recipients
C. Lymphoproliferative states and B-cell lymphomas are associated with Epstein-Barr virus
D. None of the above