Rejection of Foreign Tissue

1. Rejection of Foreign Tissue • Transfusion reactions are induced mainly by ABO incompatibilities • Transplant rejection is induced mainly by HLA incompatibilities (especially HLA-DR)

2. Transfusion Immunology • The ABO system • Landsteiner groups: A, B, AB, O • Blood type named for the ANTIGEN on the RBC surface, e.g., those with the A blood type have the A blood group carbohydrate on the surface of their RBCs • Humans possess natural isohemagglutinins (IgM antibodies) to blood group antigens not present on their own RBCs • Blood group carbohydrates are controlled by the A, B, and O alleles • The O allele is inactive and not expressed • H substance is precursor to the A and B carbohydrates

3. GlcNAc Gal GlcNAc Gal Fuc Precursor H substance GlcNAc Gal GalNAc GlcNAc Gal Gal Fuc Fuc A substance B substance

4. Landsteiner Blood Groups (ABO) Genotype AA, AO BB, BO AB OO Phenotype A B AB O Antibodies in serum Anti-B (aggl. B and AB RBCs) Anti-A (aggl. A and AB RBCs) None (don’t aggl. A, B, AB, or O) Anti-A, anti-B (aggl. A, B, and AB RBCs) ABO antigens are also found on vascular endothelium, where they may cause hyperacute graft rejection

5. B B O O B O A A AB AB AB AB A B AO AO BO BO A O AB AO BO OO B O A B B B A A AB AO AB AO A B AA AB AB BB A O AB AB BO BO • Can a man with blood type B father a child with blood type A when the mother has blood type A? • Can a man with blood type O father a child with blood type AB when the mother has blood type AB? • Can a man with blood type B father a child with blood type O when the mother has blood type A? O O B O A A AO AO AO AO O O BO OO BO OO A. Yes! (Both parents must be heterozygous). • No! • Yes! (The father must be heterozygous).

6. Rh Antigens (D, C, E) • Many distinct specificities • D antigen is most important, causing 90% of cases of HDNB • Presence or absence of the D antigen is indicated by (+) or (-), respectively, on ABO designation • Universal donor O– • Universal recipient AB+ Minor Blood Group Antigens • MN, Ss, Kell, Duffy

7. Transfusion Reactions • Hemolytic reactions • 1/40,000 units; death 1/100,000 units • Most serious kind • Result from ABO, Rh, or minor blood group incompatibilities, usually from clerical error • Occurs during or up to 2 hours after transfusion • Signs and symptoms • Fever • Chills • Low blood pressure – fainting or dizziness • Flank or back pain • Bloody urine • Complement-mediated intravascular hemolysis, histamine release • Vasodilation, hypotension, bronchial and smooth muscle contraction • Renal damage, DIC, shock, death

8. Transfusion Reactions • Treatment of hemolytic reactions • Stop transfusion immediately • Check labels for clerical error • Start normal saline drip to maintain IV access • Maintain adequate urine output with fluids and diuretics • Treat hypotension with low-dose dopamine to enhance renal perfusion

9. Transfusion Reactions • Febrile reactions (3-4% of transfusions) • Abs to mismatched minor blood group antigens • Arise after repeated transfusions • Could be first sign of a more serious reaction • Take seriously any temperature rise ≥ 1°C or 2°F • Allergic/anaphylactic reactions (1-2% of transfusions) • Mild urticaria to anaphylactic shock • Mediated by IgE to donor plasma proteins, including IgA • Mild reactions characterized by pruritus and urticaria; treat with antihistamines • Anaphylaxis is rare • Early signs: wheezing, GI upset, hypotension without fever • Treat with antihistamines and steroids; epinephrine for severe cases • Wash RBCs to remove IgA

10. Cross-Matching Blood Donors • Donor cells are mixed with recipient’s serum • If agglutination occurs, do not perform transfusion • Agglutination indicates that the recipient’s serum contains IgM isohemagglutinins to donor ABO carbohydrates • Donor cells are mixed with recipient’s serum AND anti-human IgG • This is the Coombs test to detect IgG hemagglutinins • If agglutination occurs, do not perform transfusion • Emergencies: use O–

11. Transplantation • Transfer of any tissue or organ other than blood • Donor and recipient must match at histocompatibility loci, or immunosuppressants must be used • The 30 major and minor histocompatibility gene loci cause rejection at different rates • It is most important that donor and recipient match at the major histocompatibility locus encoded by the HLA on chromosome 6 • Graft rejection is mainly a type IV hypersensitivity, with contributions by type II and type III

12. Autograft : One area to another, same person Isograft (syngraft): Between monozygotic twins Allograft: Between genetically different individuals of the same species Xenograft: Between different species xenograft between species e.g. pig to human

13. Human skin allograft 1st graft at 5 days 1st graft at 14 days First Set Rejection Second graft from same donor, day 7 Second Set Rejection Graft rejection shows specificity and memory

14. Cellular Basis of Graft Rejection • The most important T cell subset in graft rejection is the HLA-D (especially HLA-DR) restricted CD4+ TH subset • Sensitized to foreign class II molecules on the graft when passenger leukocytes (dendritic cells) of donor origin take up residence in the recipient's secondary lymphoid tissue • – Sensitized TH cells then produce the typical battery of cytokines that stimulate cell-mediated immunity and antibody production

15. Clinical Characteristics of Rejection • Density of HLA-D antigens on the graft determines how quickly the graft will be rejected • The rate of rejection also depends on the underlying effector mechanisms involved • Hyperacute rejection • Occurs within a few minutes to a few hours of transplantation • Always results in the loss of the graft • Type II hypersensitivity caused by preformed antibodies, usually to ABO antigens on vasculature of the graft

16. Clinical Characteristics of Rejection • Hyperacute rejection continued • Antibodies activate complement, followed by platelet activation and deposition • Swelling and interstitial hemorrhage in the transplanted tissue decrease blood flow • Thrombosis and fibrinoid necrosis • Fever and leukocytosis • Urine: scant, may contain various cellular elements such as erythrocytes • No therapy for successful prevention or termination of hyperacute rejection

17. Clinical Characteristics of Rejection • Acute rejection • Takes up to one month to occur • TH1 cells encountering foreign tissue antigens for the first time undergo clonal expansion and trigger Tc cells and macrophages that damage the grafted tissue

18. Clinical Characteristics of Rejection • Chronic rejection • Characterized by a progressive loss of function of the grafted organ over a period of months to years • Return of original problem necessitating the graft may be to blame • Walls of blood vessels in the graft thicken and become blocked • Low-grade cell-mediated rejection (type IV hypersensitivity) • Deposition of antibodies or antigen-antibody complexes (type III hypersensitivity) in vessels

19. Preventing Rejection • It is more important to match some HLA molecules than others • Kidney: avoid mismatches at HLA-DR, HLA-A, HLA-B • Stem cell transplants: avoid mismatches at HLA-DR, HLA-A, HLA-B, HLA-C • Clinical tests to reduce rejection risk • ABO compatibility is essential • Serological tissue typing • Polymerase chain reaction • Tissue cross-matching • Recipient’s serum is mixed with lymphocytes from the donor • Anti-human IgG/M labeled with a fluorescent dye is added • Fluorescent cells are counted by FACS (flow cytometry) • A high number of fluorescent cells indicates that this donor should NOT be used

20. Selection of Donors • One copy (haplotype) of the HLA genes is inherited en bloc from the mother, and the second copy is inherited en bloc from the father • The genes are expressed co-dominantly • Children will share 50% of their HLA alleles with each biological parent • There is a 25% chance that two fraternal siblings will be HLA-matched • HLA identical siblings are the first choice for donors • Half-matched relatives (parents) are the second choice for donors

21. Dad Mom DP DQ DR B C A Kid combos One copy of chromosome 6 is inherited en bloc from the mother, and the second copy of chromosome 6 from the father. The alleles present on one copy of chromosome 6 are termed the MHC haplotype of that chromosome.

22. Dad Mom Is it possible for the offspring of these parents to express each of the following HLA phenotypes? • DP3, DP4, DQ4, DQ9, DR1, DR4, B13, B47, C6, C7, A36, A69 • DP1, DP4, DQ2, DQ4, DR2, DR4, B8, B13, C6, C7, A1, A69 • DP1, DP3, DQ2, DQ7, DR2, DR3, B8, B62, C6, C8, A1, A3 • DP3, DQ7, DQ9, DR1, DR3, B47, B62, C6, C8, A3, A36 • DP3, DQ2, DQ4, DR2, DR4, B8, B13, C6, C8, A1, A3

23. Selection of Tissue Donors • The chance of finding two unrelated individuals who are exact HLA matches is extremely remote because MHC genes are highly polymorphic • It is estimated that in the population there are at least 350 alleles of HLA-A genes, 620 alleles of HLA-B genes, 400 alleles of DR genes, and 90 alleles of DQ genes

24. Immunosuppressive Therapy • Corticosteroids (prednisone) • Block cytokine gene expression • Cyclosporine and Tacrolimus (FK506) • Inhibits calcineurin and therefore NFAT • Suppresses IL-2 synthesis and IL-2 receptor expression • Prevents T cell and NK cell activation • Must give before transplantation as well as after; cannot suppress rejection after it starts • Rapamycin (Sirolimus) • Suppresses IL-2 receptor signaling, but not IL-2 synthesis

25. Immunosuppressive Therapy • Antimetabolites that interfere with DNA • Azathioprine - inhibits purine synthesis • Chlorambucil – alkylation of DNA • Cyclophosphamide – alkylation of DNA • Mycophenolate mofetil (MMF) - blocks guanine nucleotide synthesis, few side effects • Blockade of the B7 co-stimulatory molecule • Abatacept™ is a fusion protein composed of CTLA-4 linked to the Fc fragment of IgG • Competes with CD28 for B7 binding • Prevents T cell activation • Also used to treat RA

26. Immunosuppression Therapy • Total lymphoid irradiation for BMT • Recipient's thymus, spleen and lymph nodes are X-irradiated while shielding the bone marrow • Destroys mature T cells • Allogeneic bone marrow transplantation is performed immediately after irradiation • During recovery, bone marrow stem cells differentiate and repopulate the lymphoid tissue • The newly immunocompetent individual is now highly tolerant to the allograft • Recipient becomes chimeric, displaying both autologous and allogeneic histocompatibility antigens

27. Graft versus Host Disease • Donor T cells attack the tissues of the immunosuppressed recipient

28. Signs of Graft vs. Host Disease • Enlargement of spleen and liver • Skin rash, including palms and soles; exfoliative dermatitis • Severe diarrhea, vomiting, weight loss • Autoimmune hemolytic anemia • Fever • Hepatitis with hyperbilirubinemia • Jaundice Prevent by tissue typing, removal of T cells from graft with anti-lymphocyte antibodies, continued immunosuppression

29. Tolerance of the Allogeneic Fetus • The placenta does not display conventional class I or class II MHC, so it cannot be "seen" by T cells • The placenta displays a nonpolymorphic class I MHC molecule called HLA-G, which protects it from maternal NK cells by engaging the KIRs on the NK cells • The placenta expresses FasL, which kills T cells expressing Fas • The cells of the placenta secrete progesterone, -fetoprotein, and TGF, all of which are immunosuppressive • The placenta and decidua are resistant to complement-mediated damage because they express high levels of a C3 and C4 inhibitor called Crry • T cell responses to the fetus are blocked by low concentrations of intact tryptophan or high concentrations of tryptophan metabolites in the decidua