Transplant Immunology

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

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

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

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

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

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

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

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

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

FIGURE 26-5. 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.)

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

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

FIGURE 26-7. 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.

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)

FIGURE 26-8. Phases of helper T-cell–dependent antibody responses FIGURE 26-8. 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.)

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

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)

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)

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

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

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

Hyperacute Rejection

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

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

Acute Rejection

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

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)

Chronic Rejection

FIGURE 26-12. 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.)

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

Objectives of Immunosuppression Facilitate acceptance of the allograft Specific Low toxicity

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.

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.

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.

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

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

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

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

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

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

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

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

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

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

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

T cell Receptor Signaling Pathway

T cell Signaling: Calcineurin

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

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

Now for a Quick Review:

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

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

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

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

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

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

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

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

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

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

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

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

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