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Session 1.2: Chiorazzi
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
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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
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. 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. 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. 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. 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. 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. 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. CLL clones are culled from the normal B-cell repertoire based on structural constraints of the B-cell antigen receptor
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
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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. CLL cases with remarkably similar B-cell receptors Ghiotto et al . J Clin Invest 113: 1008 , 2004
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. 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. 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. 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. 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. 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. Herve et al . J Clin Invest 115:1636-1643, 2005
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28. Herve et al . J Clin Invest 115:1636-1643, 2005
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. mAb 068 binds 225KDa molecule C. Chu et al . Blood 112: 5122-5129, 2008
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. CLL mAb 068 co-localizes with pAbs to MYHIIA C. Chu et al . Blood 112: 5122-5129, 2008
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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
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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. MYHIIA is one of the intracellular antigens that translocates to the surface and is bound by CLL mAbs
38. Live Late apoptotic Early apoptotic Chu et al . Blood 2010 in press MEAC : M YHIIA e xposed a poptotic c ell
39. CLL 068 mAb binds to MEACs Chu et al . Blood 2010 in press Negative Apoptotic MEACs
40. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al . Blood 2010 in press
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. Herve et al . J Clin Invest 115:1636-1643, 2005 Polyreactivity is a feature primarily of unmutated CLL cells
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. 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. 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. Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al . Blood 2010 in press
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48. B-CLL evolution hypothesis MEACs MYHIIA+ Vimentin Filamin B Oxidation Chemical Modification M-CLL U-CLL Initiation Progression
Editor's Notes
I would like to thank the organizers for giving me the opportunity to discuss our research results with you. This represents the work of not only myself but of many others including… Lu, Katerina, Rosa Steve and Kanti for clinical collaboration Nick for support, guidance and inspiration and others that I will acknowledge throughout the talk
This is a typical example of flow cytometry data showing that CLL 068 mAb reacts to MEACs. This is a negative control. This shows that CLL 068 binds to a subset of dead cells (AV-PE positive) and not live cells. This shows that CLL 068 binds only to MEACs (MYHIIA positive). And not other cells.
This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
Indeed binding well to MEACs correlates to poor patient survival (24 patients have survival data)… Hi binding has a median survival of 99 months (n=15), Whereas Lo binding has not reached median survival (n=9). This is statistically significant P<0.0087. This significance is better than that for UM versus Mut (P<0.06) in this patient cohort. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
IGHV mutation status versus patient survival (24 patients have survival data)… UM IGHV has a median survival of 118 months (n=18), Whereas Mut IGHV has not reached median survival (n=6). This is not quite statistically significant P<0.06. This significance is not as good as MEAC binding. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
These results lead to the following inferences…
Just to end with a MODEL cell death (CLL or other) leading to MEAC formation and exposure of MYHIIA and other epitopes observed by Rosa This could be important for the initiation of CLL or the ongoing stimulation that may be required for CLL growth and development.
![[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)