Phylogeny of the Immune System

1. Phylogeny of the Immune System Yufang Shi yufang.shi@umdnj.edu Evolution of Adaptive Immunity by M. F. Flajnik http://www.ivis.org/proceedings/ACVP/2004/Flajnik/IVIS.pdf http://www.youtube.com/watch?v=8-TUG084GW4

2. Phylogeny of the Animal Kingdom

3. Protozoa http://www.cartage.org.lb/en/themes/Sciences/Zoology/Animalclassification/Polygenetic/phylogenetictree/phylogenetictree.htm

4. Annelida Mollusca Arthropoda Echinodermata Chordata Body Organization Platyhelminthes Nematoda Cnidaria Parazoza; Sponge 3-germ layers No body cavity 2-germ layers Pseudocoelom Eucoelom

5. Kinship of Chordates Adaptive immunity http://cas.bellarmine.edu/tietjen/images/Evolution_of_chordata.gif

6. Lancelets Sea Squirt Shark Jawless Fish Lamprey Bonny fish

7. Big Events in the Evolution of the Immune System

8. Approaches • Compare with important evolution events/stages • Cellular analysis • Allograft recognition • Genome Analysis gene duplication domain shuffling Ag Receptors, MHC, TLR, Complements, Cytokines

10. "Big Bang" of gene duplication • Jawless fish to jawed fish • Make the formation of vertebra and tissue specificity possible • (form gene clusters such as HOX genes) • Mechanisms: • 1. Whole Genome Duplication (not fully supported by genome sequencing) • Tendon or segmental duplication • Continuous small scale gene duplication.

11. Phylogeny of Chordates and the Major Events in the Evolution of Adaptive Immunity * Genome Duplication: Large-scale gene duplication and subsequent reshuffling of exons lead the emergence of new genes TRENDS in Immunology Vol.25 No.2 February 2004

12. In jawless vertebrate (lamprey & hagfish), protochordate (amphioxus and sea squirt) and other invertebrates variable lymphocyte receptors, TCR-like protein, Lymphocyte -like cells （Nature, 2004) lamprey vertebrate V region-containing chitin-binding protein family, proto-MHC region (Nat Immuol; Nat Genet, 2002) amphioxus innate immunity related molecules, proto-MHC region. Protocytokines sea squirt invertebrate arthropod Immunoglobulin Domain mollusk Immunoglobulin Domain TLRs expansion and innate immunity (Science, 2006) sea urchin

13. Types of Immune Recognition

14. Self and non-self Recognition Every organism is capable of innate defense. • Exist in both animals and plants • In some plants • Plants developed more and longer lateral roots towards neighboring roots of different plants than towards other roots of the same plant. Het recognition. • Pollen recognition, self-incompatibility (prevent inbreeding):s-locus polymorphism • In animals • Define the characteristics of individual organisms from those of similar kind. • Identify self and prevent self-reactivity. Defend organisms from their predators • Recognize and destroy viruses, bacteria or infected cells. • **Recognize “Pathogen Associated Molecular Patterns (PAMPS)” through Pattern Recognition Receptors (PRRs) • **Major histocompatibility complex in higher animals.

15. Strategies of Immune Reaction in Invertebrates Pattern Recognition Receptors • Anti-microbial peptides • Interference RNA • Phagocytic cells/cytolytic cells • Lysozymes • Production of toxic oxygen and nitrogen metabolites • The most advanced defense mechanisms are: C3 and Toll receptors. • Critical for self-nonself discrimination, even exist in Amoeba • Complement in Cnidaria • Toll receptors are well described in Drosophila. Recognition of infecting micro-organisms • Activation of intracellular signaling cascades, especially the NFkB pathway, which leads to the expression of a vast array of antimicrobial effector molecules that attack microorganisms at many different levels.

16. Immunoglobulin Domain • An IgG molecule consists of a total of 12 immunoglobulin domains. Found in many other proteins that play key roles in the immune system. • Consist of a pair of β sheets, each built of antiparallel β strands. A single disulfide bond bridges the two sheets. • Most prevalent domains encoded by the human genome. Can be found in nemotodes. • The conserved beta barrel structures making up each immunoglobulin domain along with the beta turns and helical turns at the ends of the beta strands • Key for Ig, TCR, costimulation molecules and MHC

17. MHC Origin • Origin not known • Class II first? • Class I first as a result of a recombination between an immunoglobulin-like • C-domain and the peptide-binding domain of an P70 heat-shock protein • Not in jawless fish, but both class I and class II can be seen in shark. • Generation of polymorphism: • (1) a high mutation rate • (2) gene conversion or interlocus genetic exchange • (3) overdominant (balancing) selection • (4) frequency-dependent selection. • Maintenance of polymorphism: • (1) Pathogen-driven • (2) Non-pathogen-driven • inbreeding depression • mate selection • selective fertilization • selective abortion • 5. Allorecognition in invertebrates is not related to MHC

18. Generation of Antigen Receptor Diversity Most vertebrate animals use the way as humans. In all animals, antigen receptors require RAG proteins. TCR in most species uses similar rearrangement strategies. There are differences in the ways Ig molecules are generated. In chickens and rabbits, the recombined V region is diversified by gene conversion (one piece of DNA is donated from one chromosome to another) in the bursa of Fabricius (in chickens) or another intestinal lymphoid organs (in rabbits). Sheep, in its ileal Peyer's patch, generates diverse repertoire by somatic hypermutation of a fairly invariant recombined V region. Sharks have multiple copies of discrete VL-JL-CL and VH-DH-JH-CH cassettes, and activate rearrangement in different copies.

19. Evolution of the Adaptive Immune Response. • The evolution of adaptive immunity is one of the greatest biological mystery. • Adapt genes for MHC, immunoglobulin, RAG and TCR: • By the invasion of a putative immunoglobulin-like gene by a retroposon with the ability to undergo gene rearrangement. The T-cell receptor α, β, γ, and δ chain loci and the immunoglobulin H, κ, and λ loci • The Key is the RAG genes (lack introns), which was recently found to catalyze transposition events. It was found that the stretch of DNA containing the recombination signal sequences was being inserted into other DNA fragments!! • Adaptive immunity appears abruptly in jawed fish and thus can mount an adaptive immune response. • Hagfish and lampreys lack all signs of an adaptive immune system: no lymphoid tissue, no primary immune responses, and no immunological memory.

20. Major steps of the phylogeny of the immune system

21. Level 1: Cell or species-specific aggregation: plants, sponges and protozoans. Level 2: Specific immunorecognition/ immunoimcompatibility: Cnidaria; not MHC mediated; differentiated leucocytic cells exist Level 3: cell-mediated immunity with at least short-term memory: annelids, arthropods, echinoderms and possibly, mollusks. Level 4: Integrated cell and humoral antibody immunity: first evolved in advanced bony fishes. helper T cells and B cells capable of producing two or more molecular classes of antibodies thymus found in fish and amphibians Bursa of Fabricius in the reptilian-avian branch of the phylogenic tree Level 5: multiple classes and subclasses of immunoglobulins and heterogeneous T and B repertoires : birds and mammals.

22. Sponge Protozoa • Amoeba: Pattern recognition: Food particles, not other amoeba or own pseudopods Prototype of Macrophages? Mouse macrophage Amoeba http://cellmotility.genebee.msu.ru/images/foto/pic-3.jpg • Sponge: • 1. Separated cells aggregate only with their own kind. Thus are able to distinguish between "self" and "non-self," • 2. Phagocytes

23. Cnidaria • Clonal animals, such as sea anemones, can attack individuals belonging to different genetic clones while avoiding attacking individuals of the same clone • Phagocytes and Cnidocyste for defense. • C3 like molecules can be detected in some species Nematoda • Pattern Recognition • Constitutive components: anti-microbial or digestive peptides • Inducible Components: a TGFb-like pathway, a p38 kinase pathway, a programmed cell death pathway, and an insulin receptor-like pathway • Related to Stress?

24. Annelida In earthworms, the coelomocytes (leukocytes) located in the coelomic cavity. Humoral: agglutinins Arthropoda, Example: Drosophila • Defense response can be activated by TOLL (Activate NFkB) or Imd (a family of TNFR like molecules). • Primarily through the secretion of antimicrobial peptides. • Lectins and serine proteases in hemolymph function like complement and blood-clotting.  Coagulation in response to LPS (horseshoe crab) • Very beginnings of complement-mediated immunity as well as blood-clotting • molecules capable of making covalent linkages to pathogens called TEPs. Able to induce opsonization. Thus opsonization may be the oldest function of complements.

25. The Similarity between TOLL Receptors and TOLL-like Receptors • Drosophila Toll recognize fungi, 18–wheeler recognize bacteria • TOLL Like Receptors and IL-1 receptor in mammals share • strong homology to TOLL

26. Echinoderms • C3/C4/C5-like gene was discovered in sea urchin. Can be upregulated upon exposure to LPS.   Act primarily as an opsonin. • C2/Bf-like gene was also identified • The prototype of the alternative complement pathway. The alternative pathway is the oldest •   Therefore, the first appearance of a complement-mediated immune system.  Thus, possess effective phagocytosis. • TLR

27. Urochordates (sea squirt) • C3/C2 homologues have been identified and can opsonize. • The complement system seems to be more complete due to the existence of MASP, a component of the lectin pathway • (TNF) and IL-8 homologue was found. Amphioxus (lancelets) • The closest relative to the vertebrates.  • A C3-like gene and a C6-like gene were found. • C6 like protein is involved in the lysis of foreign pathogens • Some prototype cytokines: MIF, TNF, IRF • Death Domain • Lymphocyte like cells, V and C domains • Several TLRs

28. Jawless Fish (lampreys and hagfish) • most ancestral vertebrates.  • no rearranging antigen receptors or MHC genes have been found.  No Rag. V and C domains exist. • No adaptive immune system • Have a C3 homologue, a Bf/C2 homologue, and a MASP homologue (All vertebrates except jawless fish share the human complement systems) • Have CD59, an inhibitor of the lytic pathway of the complement system • No secondary lymphoid organ. May have lymphocytes, but since lymphocytes in all other vertebrates utilize rearranging antigen receptors, their role in lamprey immunity remains unclear. Immunization lead to increase in proteins in lymphocytes containing leucine-rich repeats (LRR). Existence of an adaptive immunity? (Nature. 2004 Jul 8;430(6996):174-80. )

29. Rearranging antigen receptors of jawless and jawed vertebrates. The lamprey mature VLR gene consists of the signal peptide (SP), N-terminal LRR (LRRNT), first 18-residue LRR (LRR1), variable number of 24-residue LRR (LRRV), a connecting peptide (CP), C-terminal LRR (LRRCT) and threonine/proline-rich stalk. Portions of LRRNT and LRRCT that are not encoded in the germline VLR are hatched. Gnathostome antibody genes are assembled via random joining of Ig gene fragments consisting of variable (V), diversity (D) and joining (J) elements, and Ig constant (C) domains. --Zeev Pancer, University of Maryland

30. Cartilaginous Fish • Oldest animals have Ig/TCR/MHC/Rag based adaptive immune response. • Their B cells resemble CD5+ B cells and T cells resemble gamma/delta T cells. • Mostly IgM; some IgNAR (new antigen receptor) and IgW • Heavy-chain genes in the cluster-type organization (V, 2 - 3 D, 1 J and C). Gene rearrangement seems to occur only within a cluster. High levels of N-region addition. • Three L chain isotypes, also in the cluster organization. • IgNAR is a covalently linked dimer without L chains. The V domains in the dimer are free and flexible and each V interacts with antigen. • TCR genes. Secondary lymphoid tissue has segregated into discrete T and B cell zones. TCR genes are in the translocon organization (not multicluster). • Polymorphic MHC class I/II molecules • No known cancer.

31. Ontogeny of thymus • During ontogeny, thymic epithelial cells are derived from the third endodermal pharyngeal pouch and the third ectodermal branchial cleft. • The ectodermal cells proliferate extensively to cover the endodermal cells. • Thus, cortex epithelial cells are ectodermal origin, while the medulla is endoderm derived.

32. Teleost Fish • IgM (in a tetrameric form). • Some species have IgD • No class switch. Alternative splicing leads to IgD from IgM. • Heavy-chain genes are in the same organization as primates/rodents. Light-chain genes are cluster organization like that in the cartilaginous fish (cluster organization is more primitive?). There 2 to 4 light-chain isotypes.

33. Amphibians • IgM and IgY (four C-domains) isotypes-IgY emerged in an immediate ancestor of the amphibians). • Start to have the isotype switch, • very low MHC class II polymorphism. • Some species such as Xenopus have high MHC polymorphism and robust T-dependent responses. • IgX in the intestine, mucosal immunity? IgM and IgX are present in thymectomized animals, while IgY production is strictly T-dependent. • Major changes during metamorphosis, prevention of autoimmunity?

34. Reptiles • IgM and IgY. • Turtles have a large number of V genes. Thus, the generation of diversity more like primates/rodents than birds. • All ectothermic vertebrates have no germinal centers. Thus no affinity maturation and no high affinity antibodies. • Need more studies.

35. The Endotherms: Birds and Mammals • Gut-associated lymphoid tissue (GALT) species: chickens, rabbits, sheep, cows, and pigs. A small number of V genes (only one in birds) is rearranged and then modified by gene conversion or somatic hypermutation. There are dedicated organs for B cell development: the bursa and appendix. • B cells are continually produced throughout life in the primates/rodents group, but only during early in development in the GALT species (bursa and appendix degenerate afterward). • In all jawed species, T cell development is conserved: all rearange the TCR gene families (α, β, γ, and δ). The GALT species have more g/d T cells

36. The Mammalian Type Adaptive Immune System • Ig and TCR as antigen receptors • RAG-Mediated Recombination • Primary and secondary lymphoid organs • MHC • Somatic mutation • Memory • Cytokines • Do not exist in invertebrates or jawless fish. Might start in placoderms.

37. Camelantibodies [VH-D-D-J-C]n [L-J-C]n, Shark IgNAR Chicken IgY