BM Lecture 8 - Evolution of Adaptive Immune Systems (AIS) – part II

1. BM Lecture 8 - Evolution of Adaptive Immune Systems (AIS) – part II The origins of lymphocytes and development of lymphocyte types. extant agnathans – lamprey & hagfish

2. The origins of lymphocytes and development of lymphocyte types. • 2 scenarios: • Lymphocyte-like cells evolved before agnathans diverged but for a • distinct type of adaptive immunity – gnathostomes use these cells • The gnathostomes may have inherited & modified these cells, or may • have independently developed T- & B-cells from their own innate cells • 2) Within gnathostomes lymphocytes gradually develop B & T traits • NK cells give rise to innate T-cells • Macrophages give rise to innate B-cells

3. Agnathans have an adaptive immune system that is analogous to ours Gary W. Litman & John P. Cannon; Nature 2009. 459: 784-786

4. Agnathan Variable Lymphocyte Receptors (VLRs) have leucine rich repeats (LRRs) inserted into an invariant gene cassette VLRs will prove to be analogous to our TcR and antibodies Seminars in Immunology 22 (2010): 25-33

5. An AID homolog inserts the LRRs into the centre of the gene to make one of 1014 possible functional VLRs Seminars in Immunology 22 (2010): 25-33

6. Backing up to Imin 371 Activation Induced Cytidine Deaminase (AID or AICDA) AID normally initiates Ab affinity maturation & isotype class switching It is an Ig gene mutator enzyme AID deaminatescytosines to make uracils in the DNA AID

7. somatic hypermutations 'repair' of Uracils in DNA leads to point mutations &/or breaks in strands of DNA (breaks can happen on both DNA strands leading to Double Strand Breaks - DSBs) AID initiated mutations can also be the starting points for Gene Conversions ….. leads to DSBs & class switching Nature Reviews Immunology5, 171-178 (2005)

8. Gene Conversion is non-reciprocal imprinting of DNA sequence - during meiosis it can make new MHC alleles - as shown here (this is why MHC pseudogenes are retained in the genome) donor sequence is not altered Gene conversion can be used to modify the V-element part of the VDJ exon during Ab affinity maturation in birds & some mammals (Lect 9)

9. The AID mediated ‘insertion’ of LRRs into the centre of the VLR gene appears to be through a gene conversion-like process Seminars in Immunology 22 (2010): 25-33

10. VLRs exist in membrane and secreted forms. Nature Reviews Genetics (2010) 11, 47-59

11. There are 2 VLR genes & types - both undergo allelic exclusion & both are expressed on lymphocyte-like cells. Only VLR-B expressing cells secrete VLRs Gary W. Litman & John P. Cannon; Nature 459, 784-786

12. x-section of juvenile lamprey: main immune organs include the: typhlosole & kidney which are both filled with lymphocyte-like cells Lymphocytes - black arrows RBCs - white arrows kidney typhlosole (intestinal infold) Seminars in Immunology 22 (2010): 25-33

13. Proposed that lymphocytes predated the evolution of adaptive immunity Nature Reviews Genetics (2010) 11, 47-59

14. There appear to be remnant VLRs in vertebrate genomes These two VLR-like genes were found in the zebrafish genome These appear to be 'pre-assembled' VLR versions of unknown function They suggest that some components of the VLR system existed when the agnathans diverged from the common ancestor to us Seminars in Immunology 22 (2010): 25-33

15. It’s still too soon to decide what gnathostomes inherited to develop their adaptive immune systems Agnathans and Gnathostomes may have independently developed such things as allelic exclusion, education and tolerance (the latter two haven’t yet been formally shown in agnathans) Because the agnathan AIS systems is a new discovery most past thought on the origins of gnathostome AIS presumed that lymphocytes started from scratch in gnathostomes (which may be somewhat true) That’s the premise we’ll follow next …..

16. There were lots of (unsuccessful) intermediates between the common ancestors of gnathostomes & the extant agnathans agnathans evolutionary window for AIS devel. last common ancestor with modern agnathans

17. Gnathostomes still have a number of innate lymphocyte types that represent good candidate precursors to B- and T-cells. These include: NK cells NK-T cells / T-cells B-1 B-cells Marginal Zone B-cells We’ll consider each in turn ….

18. An NK-like cell was proposed as a T-lymphocyte predecessor • Allo-recognition abilities through MHC regardless of Ag presentation NKG2D is an activating receptor that induces a controlled death to stressed cells, especially gut epithelium. infection or stress MIC-A/B (non-classical class I MHCs) NK with NKG2D Apoptosis & replacement of epithelial cell

19. The NK cell first develops a means to distinguish stressed self from healthy (expression of non-classical MHC) The NK cell can induce death (apoptosis) The MHC doesn’t present peptide (a derived process) The next elaboration is development of an innate NK/T-cell hybrid

20. From Imin 371 - NK cells are related to T-cells and 'hybrid' NK-T cells exist TcR / chains are invariant TcR recognize glycolipids in CD1d may act as anti-bacterial innate cell and/or as just a regulatory cell - they don't form memory cells also express NK cell markers NK-T release cytokines

21. Imagine the first TcR as being invariant (one each V & J?) and it may have originally functioned as a homodimer Like NKG2D it recognizes a non-classical MHC – CD1 but this non-classical MHC has bacterial lipids bound to its surface the precursor to peptide Ag presentation? The cells are further developing systems to distinguish self from non-self and are developing further effector responses to the latter?

22. NK-T cells & conventional T-cells share a precursor cell in development Nature Reviews Immunology4; 231-237 (2004)

23. from Imin 371 - another possible predecessor of 'conventional' T-cells / TcR T-cells are also classified as innate lymphocytes associated with epithelial surfaces - skin and gut some populations have limited VDJ repertoire, other popns diverse Variable region of / TcR more like Ig - for binding free Ag - proposed to also monitor for stressed epithelial cells

24. free or non- classical MHC I assoc. in body cavities in epithelial linings in blood/lymph syst. from imin 371

25. free or non- classical MHC I assoc. in body cavities in epithelial linings in blood/lymph syst. from imin 371 all regions of the body are protected by an innate lymphocyte type

26. B-cells appear to have a shared history with macrophages • Recent evidence: • B-cells & Macrophage have common precursors • (Nature 1992; 356:612-5 & Nature Immunol. 2001; 2:83-86) • Macrophages can be ‘reprogrammed’ into B-cells • (Cell 2004; 117:663-676) • Lower vertebrate B-cells can be professional phagocytes • (Nature Immunol. 2006; 7:1116-1124)

27. bl blM blMTB M T TB B Classical model of hematopoietic cell origins Classical model bl - blood - RBC & megakaryocyte M - Myeloid (monocyte/macrophage) B - B-cell T - T-cell presumed there was an early split to lymphoid & myeloid progenitors Adapted from: Trends in Immunology 27:169-175 2006

28. bl blM M M blMTB T MT M MTB M MB B Recent models of hematopoietic cell origins Adapted from: Trends in Immunology 27:169-175 2006 accepts a lymphoid/myeloid split, but the lymphoid retains myeloid potential in development, and under certain experimental conditions this should be reflected in stem & progenitor cell development in hematopoietic tissues

29. Other evidence that B-cells emerged as 'innate' T-independent cells (conventional) B-1 cells B-2 cells Marginal Zone B Restricted V-elements Renewal Mechanism Spontaneous Ig production Isotypes secreted Response to Carb Ag’s Requires T-help Memory Development Yes No Somewhat Self-renewal Marrow Long-lived Yes Rare Rare IgM>>IgG IgG>IgM IgM>IgG Yes possibly Yes No Yes Sometimes Little or none Yes ? PRR? PRR? Adapted from Janeway Fig. 7.41

30. Hypothesis based on known B-cell functions - Original B-cells derived from phagocytic myeloid lineage with several pattern recognition receptors. These had Ig with restricted number of variable regions - i.e. the Ab acted more like a pattern recognition receptor (PRR) These cells would be T-independent and the only ‘selection’ for effective V-elements would be through natural selection Original function of secreted Ab is in pathogogen neutralization, opsonization & possibly Complement activation (?)

31. Evolution of Adaptive immune systems within the gnathostomes … • probably layers and layers of regulation & redundancy had developed • perhaps some T-cell functional specialization? • are TH1, 2 & 3 the norm, the exception or derived? • The most obvious changes have happened to: • Antibodies & Humoral immunity • gene organization & # of isotypes & isotype forms • how Ab diversity is generated, and where it develops • Immune organs - both their presence or absence • what precise role each plays

32. A case in point - the Ig heavy chain locus is still changing ….. mice have 8 isotypes and humans have 9 Mouse IgH constant domain clusters J m d g3 g1 g2b g2a e a Human IgH ? ? J m d g3 g1 e a1 g g2 g4 e a2 duplication (intra-locus duplication)

33. The Ig locus started simple and got more complex with time … Version 1 - different isotypes encoded in separate Ig genes the shark multi-cluster gene arrangement Version 2 - different isotypes through alternative splicing -  vs.  V. 2.1 - different isotypes by alternative VDJ recombination bony fishes Version 3 - amphibians learn Class Switch Recombination

34. Sharks still have a very simple Ig gene organization …. but they are (somewhat) unique in having multiple Ig genes version 1 many Ig loci - some encoding unique isotypes singular or few V-, D- & J- V D J C V J C IgH IgL sharks sharks bony fishes mouse l (human l is V-JC-JC-JC-JC) This ancestral organization still evident in mammalian light chains

35. Intra-locus duplications led to the "Translocon" Ig gene organization with multiple V-, D-, and J-elements V1 - Vn D’s J’s C IgH IgL Bony Fishes, Amphibians, most (?) Mammals V1 - Vn J’s C Amphibians, Human & Mouse k Probably a lot easier to control one gene versus multiple Ig genes …..

36. Limited V-element usage - modified Translocon organization (generic) (pseudogenes!?) V1 - Vn V D’s J’s C IgH IgL Birds, some Mammals - cattle, swine, rabbits (pseudogenes!?) V1 - Vn V J’s C Birds - ducks, geese, chickens, others?

37. Mammalian Ig Isotype Diversity - the 5 major classes diversification of types allows specialization of function ….

38. A reminder of isotype diversity & secondary effector functions Kuby The multiple isotypes in mammals differentially facilitate such functions as: C' fixation mucosal or placental transport opsonization degranulation etc

39. Sharks had different isotypes … encoded on separate genes (version 1)! a simplified representation of heavy chain genes for: IgNAR IgMIgW (unique) (IgD - like?) V D J C probably specialization of function, but you're stuck with whatever isotype happened to have a VDJ that recognized Ag - can't "switch"!

40. Next up - bony fish & intralocus duplication of C domain exons leads to IgM & IgD which are the result of alternate splicing VDJ m1 m2 m3 m4 d1 d2 d3 d4 m-chain or d-chain transcripts maintains the Ag specificity while allowing a change in isotype … but it requires a very long pre-mRNA transcript - a limitation version 2 - alternate isotypes through alternative splicing of pre-mRNA

41. Some fish tried another type of "class switching" (version 2.1) alternative VDJ recombination changes the Ag specificity - to a point …but at least they were trying MJ Flajnik Nature Immunology6, 229 - 230 (2005)

42. For proper CSR we need more exons, switch sequences & AID Switch sequences - repetitive seq. of 2-5 kb Class Switch Recombination Overview version 3 excised all the components were probably close to in place even before the first steps onto land ….

43. Though fish don’t have conventional class switching their AID molecule is able to drive CSR in B-cells of AID-/- mice % IgG1 / GFP+ cells (i.e. class switched) mouse AID human AID chicken AID Zebrafish AID Catfish AID mock - no AID 10% 20% 30% 40% Wakae et. al. 2005, International Immunology

44. The apparent “pre-adaptation” of fish AID to drive CSR, before it evolved is because of pleiotropic functions of overlapping active domains on the protein. AID protein schematic: Class switching domain Hypermutation domain Nuclear localization domain Nuclear export domain Transport of AID in and out of nucleus - limits ectopic mutational damage to other genes

45. Evolution of Ig isotypes & likely derivations of C-domain exons lost? lost IgNAR ? IgM ? IgW IgA IgM IgE IgG IgD IgZ - lost IgX IgM IgY IgD? IgA IgM IgY (IgD) Lost? IgM IgD ? lost (lost in some) Sharks Bony Amphibian Birds Mammals & Rays Fishes class switch recombination

46. novel vertebrate Ig isotypes & forms: The major serum Ig of some birds (and turtles) exists in 2 forms these products of alternate splicing create potential problems IgY IgY(DFc) Truncated form can't be bound by Fc receptors so is of limited use.

47. Ig forms lacking an Ig light chain - one ancestral (?) and one derived? Shark NAR Camel IgG1 Camel IgG2 Both lost CH1 biotechnology benefits - phage display

48. Lymphoid organ evolution GALT Thymus Spleen Bone marrow lymph nodes Germinal Centres Germinal centres From: Origin & Evolution of the vertebrate immune system. In: Fundamental Immunology, W.E. Paul editor.