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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. Ig V gene sequencing Autoantigen reactivity
Rajendra Damle Charles Chu
Joy Yan Rosa Catera
Bradley T. Messmer Manuela Woelfle
Emilia Albesiano Katerina Hatzi
Angelo Valetto Zev Sthoeger
Fabio Ghiotto Eric Meffre
Franco Fais Maxime Herve
Gregg Silverman
Collaborators
Kanti R. Rai
Steven L. Allen
Jonathan E. Kolitz
Matthew Kaufman
Manlio Ferrarini
3. Chronic Lymphocytic Leukemia
Most prevalent adult leukemia of the Western world
15,340 new cases and 4,500 deaths in 2007
Disease of aging individuals (>50) with incidence
increasing with each subsequent decade
Usually affects men > women; Caucasian > African > Asian
4. Clinical dilemma
Clinical courses of patients are heterogeneous and that
of an individual patient is unpredictable
Some patients follow very benign courses living
decades and dying with the disease, not from it
Other patients follow more malignant courses
living only a few years after diagnosis, despite
therapy
CLL remains an incurable disease
Therefore, “wait and watch” / “wait and worry”
approaches are taken by clinician and patient
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 (~3x1010 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. Clonal disease of B lymphocytes
CD19
CD5
CD23
Smudge cell smIg (BCR)
CLL cells
Granulocyte
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
NH2
VK
CK
VH
Fab CDR3 CDR2 CDR1
CH1
Fc FR4 FR3 FR2 FR1 VH D JH CH
-S - S-
Hinge CH2
Region JH D VH
Chromosome 14
VK JH CK
CH3
Chromosome 2
COOH JK VK
Ig Molecule V Region Ig Genes
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
VH Specific % Cases with
Family VH 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 VH gene mutation status of CLL cells is an
important prognostic indicator of outcome
≥ 2% mutation ≥ 2% mutation
< 2% mutation < 2% mutation
Damle et al. Hamblin et al.
Blood 94: 1840, 1999 Blood 94: 1848, 1999
13. Ig molecules and their genes
NH2
VK
CK
VH
Fab CDR3 CDR2 CDR1
CH1
Fc FR4 FR3 FR2 FR1 VH D JH CH
-S - S-
Hinge CH2
Region JH D VH
Chromosome 14
VK JH CK
CH3
Chromosome 2
COOH JK VK
Ig Molecule V Region Ig Genes
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 (k/λ)
VH (44) D (27) JH (6) VL (46/36) JL (5/7)
= RAG mediated
recombination
HC = VH x D x JH = 44 x 27 x 6 = 7,128 VHDJH rearrangement: ~1 : 7,000
16. ~1% of CLL patients express a BCR with a VH 1-69
gene exhibiting very similar HCDR3s often
comprised of the same VH-D-JH segments
Widhopf et al. Blood 104: 2499-2504, 2004
17. Ig V region gene segment recombination
Heavy Chain Light Chain (k/λ)
VH (44) D (27) JH (6) VL (46/36) JL (5/7)
= RAG mediated
recombination
HC = VH x D x JH = 44 x 27 x 6 = 7,128
k = Vk x Jk = 46 x 5 = 230
VHDJH / VLJL rearrangement: ~1 : 3 x 106
l = Vl x Jl = 36 x 7 = 252
20. Because of the differences that occur at the
junctions when gene segments combine, the
likelihood that the same VHDJH- VLJL
rearrangement with the same junctional
characteristic would occur in two different B
cells is even much more remote
≈ 1 / 1x108 – 1 : 1x1012
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. Heavy chain sequence alignment
VH1-69 D3-16 JH 3
CAR YYDYVWGSYRY DAFDIWG
CLL068: CAR GG DYDYVWGSYRS N DAFDIWG
CLL258: CAR GG IYDYVWGSYRP N DAFDIWG
CLL022: CAR GG DYDYVWGSYRP N DAFDIWG
CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG
aCLA*: CAR GG NYDYIWGSYRS 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 111:1524-1533, 2008
>35% chance of fitting into a stereotypic set if U-CLL
or if express a specific VH 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
Transfection of 293T HEK cells using Lipofectamine 2000
1. Plasmid DNA 2.Lipofectamine 3. Lipofectamine 2000 4. Liposomes are added in
carrying heavy and 2000 Reagent Reagent and DNA are 293T HEK cell culture
light chain Ig gene mixed and incubated
Immuno assay for
quantification of
CLL mAb
4-5 days of culture
293T HEK cell line
Antibody purification using
Protein G beads
Wardemann et al. Science 301:1374, 2003
26. Reactivity of recombinant mAbs with viable HEp-2
cells that were permeabilized to allow Ab entry
[“Anti-cell antibodies”]
~20% of mAbs from CD5+ B cells from normal subjects
~75% of mAbs from CLL cells
~90% from unmutated CLL
~60% from mutated CLL
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. Autoreactivities of individual CLL BCRs/mAbs
1. VH1-69/D3-16/JH3 + VK3-20:
Non-muscle myosin heavy chain IIA
(Chu et al. Blood 112: 5122-5129, 2008)
2. VH4-39/D6-13/JH5 + VK1D-39/JK1
Vimentin
(Chu et al. Blood 112: 5122-5129, 2008)
3. 60% of U-CLL and 10% of M-CLL:
Apoptosis-associated autoantigens
(Catera et al. Mol Med 2008; 14: 665-674 )
29. Heavy chain sequence alignment
VH1-69 D3-16 JH 3
CAR YYDYVWGSYRY DAFDIWG
CLL068: CAR GG DYDYVWGSYRS N DAFDIWG
CLL258: CAR GG IYDYVWGSYRP N DAFDIWG
CLL022: CAR GG DYDYVWGSYRP N DAFDIWG
CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG
aCLA*: CAR GG NYDYIWGSYRS 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
32. CLL mAb 068 co-localizes with pAbs to MYHIIA
C. Chu et al. Blood 112: 5122-5129, 2008
33. Autoreactivities of individual CLL BCRs/mAbs
1. VH1-69/D3-16/JH3 + VK3-20:
Non-muscle myosin heavy chain IIA
(Chu et al. Blood 112: 5122-5129, 2008)
2. VH4-39/D6-13/JH5 + VK1D-39/JK1
Vimentin
(Chu et al. Blood 112: 5122-5129, 2008)
3. 60% of U-CLL and 10% of M-CLL:
Apoptosis-associated autoantigens
(Catera et al. Mol Med 2008; 14: 665-674 )
34. CLL mAbs react with apoptotic (not healthy) cells
A B
3.70 28.17 16.77 13.86
Annexin V
RAMOS
67.27 0.92 69.14 0.23
CLL014 DO13
C D
52.86 18.12 55.35 16.96
Annexin V
Jurkat
28.47 0.55 27.37 0.32
CLL014 DO13
R. Catera et al. Mol Med 14: 665-674, 2008
35. Apoptotic B and T cells are a source of
autoantigens for CLL mAbs
SUMMARY of the results:
- More than 60% (18/28) of the mAbs tested reacted with
these two cell types
- 15 of the 18 reactive mAbs used an unmutated VH gene,
only 3 used a mutated VH gene
R. Catera et al. Mol Med 14: 665-674, 2008
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. MEAC: MYHIIA exposed apoptotic cell
Late apoptotic
Live
Early apoptotic
Chu et al. Blood 2010 in press
39. CLL 068 mAb binds to MEACs
Negative Apoptotic MEACs
Chu et al. Blood 2010 in press
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. Polyreactivity is a feature primarily of unmutated CLL cells
Herve et al. J Clin Invest 115:1636-1643, 2005
43. Ig VH gene mutation status of CLL cells is an
important prognostic indicator of outcome
≥ 2% mutation ≥ 2% mutation
< 2% mutation < 2% mutation
Damle et al. Hamblin et al.
Blood 1999; 94: 1840 Blood 1999; 94: 1848
44. Binding well to MEACs correlates with poor
patient survival
Lo binding
?? Months (n = 9)
Hi binding
99 months
(n = 15)
Chu et al. Blood 2010 in press
45. In this limited series, MEAC binding correlates better
with patient survival than IGHV mutation status
Mutated
?? Months (n = 6)
Unmutated
118 months
(n = 18)
Chu et al. Blood 2010 in press
46. Many CLL mAbs bind MEACs
MEAC binding
Subset
Mutation
Chu et al. Blood 2010 in press
47. Inferences
1. MEACs may be a source of autoantigens
driving CLL disease
2. The origin of many CLL clones may be cells
that produce natural antibodies to MEACs
or other natural products of catabolism
B1-like cells, MZ B cells?
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. B-CLL Initiation
M-CLL
evolution U-CLL
hypothesis Progression
MEACs
MYHIIA+
Vimentin
Filamin B
Oxidation
Chemical
Modification
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.