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Session 1.2 Chiorazzi

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Nicholas Chiorazzi

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Session 1.2 Chiorazzi

  1. 1. Immunobiology of chronic lymphocytic leukemia Nicholas Chiorazzi Departments of Cell Biology and of Medicine Albert Einstein College of Medicine and The Feinstein Institute for Medical Research North Shore – LIJ Health System
  2. 2. <ul><li>Ig V gene sequencing </li></ul><ul><ul><li>Rajendra Damle </li></ul></ul><ul><ul><li>Joy Yan </li></ul></ul><ul><ul><li>Bradley T. Messmer </li></ul></ul><ul><ul><li>Emilia Albesiano </li></ul></ul><ul><ul><li>Angelo Valetto </li></ul></ul><ul><ul><li>Fabio Ghiotto </li></ul></ul><ul><ul><li>Franco Fais </li></ul></ul>Collaborators Kanti R. Rai Steven L. Allen Jonathan E. Kolitz Matthew Kaufman Manlio Ferrarini Autoantigen reactivity Charles Chu Rosa Catera Manuela Woelfle Katerina Hatzi Zev Sthoeger Eric Meffre Maxime Herve Gregg Silverman
  3. 3. Chronic Lymphocytic Leukemia <ul><li>Most prevalent adult leukemia of the Western world </li></ul><ul><ul><li>15,340 new cases and 4,500 deaths in 2007 </li></ul></ul><ul><li>Disease of aging individuals (>50) with incidence </li></ul><ul><li>increasing with each subsequent decade </li></ul><ul><li>Usually affects men > women; Caucasian > African > Asian </li></ul>
  4. 4. <ul><li>Clinical courses of patients are heterogeneous and that </li></ul><ul><li>of an individual patient is unpredictable </li></ul><ul><ul><li>Some patients follow very benign courses living decades and dying with the disease, not from it </li></ul></ul><ul><ul><li>Other patients follow more malignant courses living only a few years after diagnosis, despite therapy </li></ul></ul>Clinical dilemma CLL remains an incurable disease Therefore, “wait and watch” / “wait and worry” approaches are taken by clinician and patient
  5. 5. Chronic Lymphocytic Leukemia Lymphocytosis seen in blood, but most leukemic cells are in non-vascular areas including bone marrow, lymph nodes, and spleen Usually detected upon routine blood workup as an elevated white cell or lymphocyte count (~3x10 10 total in blood) Clonal expansion of a CD5 + B lymphocyte with low surface immunoglobulin (Ig) Most clones express predominantly IgM isotype surface Ig, but ~7% are predominantly IgG or IgA, though in those cases IgM clonal relatives can be found
  6. 6. Smudge cell Granulocyte CLL cells CD19 CD5 CD23 Clonal disease of B lymphocytes smIg (BCR)
  7. 7. Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  8. 8. Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure
  9. 9. Ig molecules and their genes V H D J H C H Chromosome 14 Chromosome 2 V K J H C K Ig Genes Ig Molecule V Region Hinge Region C K COOH NH 2 -S - S- Fab Fc V K C H 1 V H C H 2 C H 3 V K J K V H D J H CDR3 CDR1 CDR2 FR4 FR3 FR2 FR1
  10. 10. CLL cells differ from normal CD5 + B cells by the overuse of certain autoreactive genes Fais et al. J Clin Invest 98: 1659, 1998
  11. 11. CLL clones differ in the degree of somatic mutations, especially in particular IgV genes V H Specific % Cases with Family V H Gene Mutations All cases - 50.7 1 - 33.3 1-69 10.0 3 - 66.7 3-07 90.0 4 - 41.2 4-34 55.0 Fais et al. J Clin Invest 98: 1659, 1998
  12. 12. Ig V H gene mutation status of CLL cells is an important prognostic indicator of outcome Damle et al. Hamblin et al. Blood 94: 1840, 1999 Blood 94: 1848, 1999 ≥ 2% mutation < 2% mutation ≥ 2% mutation < 2% mutation
  13. 13. Ig molecules and their genes V H D J H C H Chromosome 14 Chromosome 2 V K J H C K Ig Genes Ig Molecule V Region Hinge Region C K COOH NH 2 -S - S- Fab Fc V K C H 1 V H C H 2 C H 3 V K J K V H D J H CDR3 CDR1 CDR2 FR4 FR3 FR2 FR1
  14. 14. CLL clones are culled from the normal B-cell repertoire based on structural constraints of the B-cell antigen receptor
  15. 15. IgV gene segment recombination Heavy Chain Light Chain (  / λ ) V H (44) HC = V H x D x J H = 44 x 27 x 6 = 7,128 D (27) J H (6) J L (5/7) V L (46/36) V H DJ H rearrangement: ~1 : 7,000 = RAG mediated recombination
  16. 16. <ul><ul><li>~1% of CLL patients express a BCR with a V H 1-69 gene exhibiting very similar HCDR3s often comprised of the same V H -D-J H segments </li></ul></ul><ul><li>Widhopf et al . Blood 104: 2499-2504, 2004 </li></ul>
  17. 17. Ig V region gene segment recombination Heavy Chain Light Chain (  / λ ) V H (44) HC = V H x D x J H = 44 x 27 x 6 = 7,128  = V  x J  = 46 x 5 = 230  = V  x J  = 36 x 7 = 252 D (27) J H (6) J L (5/7) V L (46/36) V H DJ H / V L J L  rearrangement: ~1 : 3 x 10 6 = RAG mediated recombination
  18. 18. CLL cases with remarkably similar B-cell receptors Ghiotto et al . J Clin Invest 113: 1008 , 2004
  19. 19. IgV gene segment recombination Heavy Chain Light Chain (  / λ ) V H (44) D (27) J H (6) J L (5/7) V L (46/36) = RAG mediated recombination
  20. 20. Because of the differences that occur at the junctions when gene segments combine, the likelihood that the same V H DJ H - V L J L rearrangement with the same junctional characteristic would occur in two different B cells is even much more remote ≈ 1 / 1x10 8 – 1 : 1x10 12 Therefore, if the gene structure of the Ig variable region found in B-CLL cells from different patients is very similar or identical, then this must indicate a selective process of leukemogenesis that targets B cells with a given type of Ig V region.
  21. 21. CLL068 : CAR GG D YDYVWGSYR S N DAFDIWG CLLSMI  : CAR GG N YDY I WGSYR S N DAFDIWG CLL258 : CAR GG I YDYVWGSYR P N DAFDIWG aCLA*: CAR GG N YDY I WGSYR S N DAFDIWG CAR YYDYVWGSYRY DAFDIWG D3-16 J H 3 V H 1-69 Heavy chain sequence alignment CLL022 : CAR GG D YDYVWGSYR P N DAFDIWG  Natural autoantibody * aCLA = anti-cardiolipin ab Messmer et al . J Exp Med 2004; 200: 519-525
  22. 22. Almost 30% of patients with chronic lymphocytic leukemia carry stereotyped receptors Stamatopoulos et al . Blood 109:259-270, 2007 Murray et al . Blood 111:1524- 15 33 , 2008 >35% chance of fitting into a stereotypic set if U-CLL or if express a specific V H gene (1-69, 3-21, 4-39) associated with poor outcome
  23. 23. Take home messages 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes
  24. 24. Expression of recombinant CLL mAbs 293T HEK cell line Antibody purification using Protein G beads Wardemann et al . Science 301:1374, 2003 Immuno assay for quantification of CLL mAb 4-5 days of culture 1. Plasmid DNA carrying heavy and light chain Ig gene 2. Lipofectamine 2000 Reagent 3. Lipofectamine 2000 Reagent and DNA are mixed and incubated 4. Liposomes are added in 293T HEK cell culture Transfection of 293T HEK cells using Lipofectamine 2000
  25. 25. Herve et al . J Clin Invest 115:1636-1643, 2005
  26. 26. <ul><li>~20% of mAbs from CD5 + B cells from normal subjects </li></ul><ul><li>~75% of mAbs from CLL cells </li></ul><ul><ul><li>~90% from unmutated CLL </li></ul></ul><ul><ul><li>~60% from mutated CLL </li></ul></ul>Reactivity of recombinant mAbs with viable HEp-2 cells that were permeabilized to allow Ab entry [“Anti-cell antibodies”] Reactivity mainly with cytoplasmic structures and occasionally with nucleoli. Only one mAb from M-CLL patient reacted with nucleus in a homogeneous pattern Herve et al . J Clin Invest 115:1636-1643, 2005
  27. 27. Autoreactivities of individual CLL BCRs/mAbs <ul><li>V H 1-69/D3-16/J H 3 + V K 3-20: </li></ul><ul><li>Non-muscle myosin heavy chain IIA </li></ul><ul><li> ( Chu et al. Blood 112: 5122-5129, 2008 ) </li></ul><ul><li>2. V H 4-39/D6-13/J H 5 + V K 1D-39/J K 1 </li></ul><ul><li>Vimentin </li></ul><ul><li> ( Chu et al. Blood 112: 5122-5129, 2008 ) </li></ul><ul><li>3. 60% of U-CLL and 10% of M-CLL: </li></ul><ul><li>Apoptosis-associated autoantigens </li></ul><ul><li>( Catera et al. Mol Med 2008; 14: 665-674 ) </li></ul>
  28. 28. Herve et al . J Clin Invest 115:1636-1643, 2005
  29. 29. CLL068 : CAR GG D YDYVWGSYR S N DAFDIWG CLLSMI  : CAR GG N YDY I WGSYR S N DAFDIWG CLL258 : CAR GG I YDYVWGSYR P N DAFDIWG aCLA*: CAR GG N YDY I WGSYR S N DAFDIWG CAR YYDYVWGSYRY DAFDIWG D3-16 J H 3 V H 1-69 Heavy chain sequence alignment CLL022 : CAR GG D YDYVWGSYR P N DAFDIWG  Natural autoantibody * aCLA = anti-cardiolipin ab Messmer et al . J Exp Med 2004; 200: 519-525
  30. 30. mAb 068 binds 225KDa molecule C. Chu et al . Blood 112: 5122-5129, 2008
  31. 31. LC MS/MS identifies 225KDa band as non-muscle myosin heavy chain IIA (MYHIIA) C. Chu et al . Blood 112: 5122-5129, 2008
  32. 32. CLL mAb 068 co-localizes with pAbs to MYHIIA C. Chu et al . Blood 112: 5122-5129, 2008
  33. 33. Autoreactivities of individual CLL BCRs/mAbs <ul><li>V H 1-69/D3-16/J H 3 + V K 3-20: </li></ul><ul><li>Non-muscle myosin heavy chain IIA </li></ul><ul><li> ( Chu et al. Blood 112: 5122-5129, 2008 ) </li></ul><ul><li>2. V H 4-39/D6-13/J H 5 + V K 1D-39/J K 1 </li></ul><ul><li>Vimentin </li></ul><ul><li> ( Chu et al. Blood 112: 5122-5129, 2008 ) </li></ul><ul><li>3. 60% of U-CLL and 10% of M-CLL: </li></ul><ul><li>Apoptosis-associated autoantigens </li></ul><ul><li>( Catera et al. Mol Med 2008; 14: 665-674 ) </li></ul>
  34. 34. CLL mAbs react with apoptotic (not healthy) cells R. Catera et al . Mol Med 14: 665-674, 2008 Jurkat RAMOS Annexin V CLL014 DO13 C D Annexin V CLL014 DO13 A B 28.17 0.92 3.70 67.27 13.86 0.23 69.14 16.77 52.86 18.12 0.55 28.47 55.35 16.96 0.32 27.37
  35. 35. Apoptotic B and T cells are a source of autoantigens for CLL mAbs <ul><li>SUMMARY of the results: </li></ul><ul><li>More than 60% (18/28) of the mAbs tested reacted with these two cell types </li></ul><ul><li>15 of the 18 reactive mAbs used an unmutated V H gene, only 3 used a mutated V H gene </li></ul>R. Catera et al . Mol Med 14: 665-674, 2008
  36. 36. Antigens bound at the surface of apoptotic cells have translocated from intracellular compartments Cytox Orange Annexin V CLL 114 Merge Membrane blebs Apoptotic body without DNA Apoptotic body with DNA R. Catera et al . Mol Med 14: 665-674, 2008
  37. 37. MYHIIA is one of the intracellular antigens that translocates to the surface and is bound by CLL mAbs
  38. 38. Live Late apoptotic Early apoptotic Chu et al . Blood 2010 in press MEAC : M YHIIA e xposed a poptotic c ell
  39. 39. CLL 068 mAb binds to MEACs Chu et al . Blood 2010 in press Negative Apoptotic MEACs
  40. 40. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al . Blood 2010 in press
  41. 41. Take home messages 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  42. 42. Herve et al . J Clin Invest 115:1636-1643, 2005 Polyreactivity is a feature primarily of unmutated CLL cells
  43. 43. Ig V H gene mutation status of CLL cells is an important prognostic indicator of outcome Damle et al. Hamblin et al. Blood 1999; 94: 1840 Blood 1999; 94: 1848 ≥ 2% mutation < 2% mutation ≥ 2% mutation < 2% mutation
  44. 44. Binding well to MEACs correlates with poor patient survival Hi binding 99 months (n = 15) Lo binding ?? Months (n = 9) Chu et al . Blood 2010 in press
  45. 45. Unmutated 118 months (n = 18) Mutated ?? Months (n = 6) In this limited series, MEAC binding correlates better with patient survival than IGHV mutation status Chu et al . Blood 2010 in press
  46. 46. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al . Blood 2010 in press
  47. 47. Inferences <ul><li>1. MEACs may be a source of autoantigens </li></ul><ul><li>driving CLL disease </li></ul><ul><li>2. The origin of many CLL clones may be cells </li></ul><ul><li>that produce natural antibodies to MEACs </li></ul><ul><li>or other natural products of catabolism </li></ul><ul><ul><li>B1-like cells, MZ B cells? </li></ul></ul>3. If MEAC binding is a “better” predictor of patient survival than IGHV mutations status, is it because the former implies antigen-binding activity whereas the latter directly measures it ?
  48. 48. B-CLL evolution hypothesis MEACs MYHIIA+ Vimentin Filamin B Oxidation Chemical Modification M-CLL U-CLL Initiation Progression

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