se1.2 chiorazzi pc aecc_retreat_5-5-10_for_website

Immunobiology of Immunobiology of chronic lymphocytic leukemiachronic lymphocytic leukemia

Nicholas ChiorazziNicholas Chiorazzi

Departments of Cell Biology and of MedicineDepartments of Cell Biology and of Medicine Albert Einstein College of MedicineAlbert Einstein College of Medicine

andand

The Feinstein Institute for Medical ResearchThe Feinstein Institute for Medical Research

North Shore – LIJ Health SystemNorth Shore – LIJ Health System

Ig V gene sequencingIg V gene sequencing

Rajendra DamleRajendra DamleJoy YanJoy YanBradley T. MessmerBradley T. MessmerEmilia AlbesianoEmilia AlbesianoAngelo ValettoAngelo ValettoFabio GhiottoFabio GhiottoFranco FaisFranco Fais

CollaboratorsCollaborators Kanti R. RaiKanti R. Rai Steven L. AllenSteven L. Allen Jonathan E. KolitzJonathan E. Kolitz Matthew KaufmanMatthew Kaufman

Manlio FerrariniManlio Ferrarini

Autoantigen reactivityAutoantigen reactivity

Charles ChuCharles Chu Rosa CateraRosa Catera Manuela WoelfleManuela Woelfle Katerina HatziKaterina Hatzi Zev SthoegerZev Sthoeger Eric MeffreEric Meffre Maxime HerveMaxime Herve Gregg Silverman Gregg Silverman

Chronic Lymphocytic LeukemiaChronic Lymphocytic Leukemia

Most prevalent adult leukemia of the Western worldMost prevalent adult leukemia of the Western world 15,340 new cases and 4,500 deaths in 200715,340 new cases and 4,500 deaths in 2007

Disease of aging individuals (>50) with incidence Disease of aging individuals (>50) with incidence increasing with each subsequent decadeincreasing with each subsequent decade

Usually affects men > women; Caucasian > African > Asian Usually affects men > women; Caucasian > African > Asian

Clinical courses of patients are heterogeneous and thatClinical courses of patients are heterogeneous and thatof an individual patient is unpredictableof an individual patient is unpredictable

Some patients follow very benign courses living Some patients follow very benign courses living decades and dying with the disease, not from itdecades and dying with the disease, not from it

Other patients follow more malignant courses Other patients follow more malignant courses living only a few years after diagnosis, despite living only a few years after diagnosis, despite therapy therapy

Clinical dilemmaClinical dilemma

CLL remains an incurable diseaseCLL remains an incurable disease

Therefore, “wait and watch” / “wait and worry” Therefore, “wait and watch” / “wait and worry” approaches are taken by clinician and patientapproaches are taken by clinician and patient

Chronic Lymphocytic LeukemiaChronic Lymphocytic Leukemia

Lymphocytosis seen in blood, but most leukemic cells are Lymphocytosis seen in blood, but most leukemic cells are in non-vascular areas including bone marrow, lymph in non-vascular areas including bone marrow, lymph nodes, and spleennodes, and spleen

Usually detected upon routine blood workup as an Usually detected upon routine blood workup as an elevated white cell or lymphocyte count (~3x10elevated white cell or lymphocyte count (~3x101010 total total in blood)in blood)

Clonal expansion of a CD5Clonal expansion of a CD5++ B lymphocyte with low surface B lymphocyte with low surface immunoglobulin (Ig)immunoglobulin (Ig)

Most clones express predominantly IgM isotype surface Most clones express predominantly IgM isotype surface Ig, but ~7% are predominantly IgG or IgA, though in Ig, but ~7% are predominantly IgG or IgA, though in those cases IgM clonal relatives can be foundthose cases IgM clonal relatives can be found

Smudge cellSmudge cell

GranulocyteGranulocyte

CLL cellsCLL cells

CD19CD19CD5CD5CD23CD23

Clonal disease of B lymphocytesClonal disease of B lymphocytes

smIg (BCR)smIg (BCR)

Take home messagesTake home messages

1. CLL results from the non-random selection and 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid antigen receptors (BCRs) of restricted amino acid structurestructure

2. These BCRs can be poly- and auto-reactive, binding 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by natural as well as novel autoantigens generated by apoptosis and other catabolic processesapoptosis and other catabolic processes

3. The clinically-distinct subgroups differ in the retention 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical of polyreactivity being associated with worse clinical diseasedisease


1. CLL results from the non-random selection and 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid antigen receptors (BCRs) of restricted amino acid structurestructure

VVHH DD JJHH CCHH

Chromosome 14Chromosome 14


VVKK JJHH CCKK

Ig GenesIg GenesIg MoleculeIg Molecule V RegionV Region

HingeHingeRegionRegion

CCKK

COOHCOOH

NHNH22

-S - S--S - S-

FabFab

FcFc

VVKK

CCHH11

VVHH

CCHH22

CCHH33

VVKKJJKK

VV HHDDJJ HH

CDR3CDR3 CDR1CDR1CDR2CDR2

FR4FR4 FR3FR3 FR2FR2 FR1FR1

Ig molecules and their genesIg molecules and their genes

Specific VH Genes

Per

cen

tag

e

IgM+ CD5+ B-CLL cells

IgM+ CD5+ normal blood B cells

0

5

10

15

.3-3

0.3

.4-3

0.2

.4-3

0.4

VH

VM

W

.1-0

2.1

-03

.1-0

3b.1

-08

.1-1

8.1

-24

.1-4

6.1

-58

.1-6

9D

P-8

8

.2-0

5.2

-05

.2-7

0

.3-0

7.3

-08

.3-0

9.3

-11

.3-1

3.3

-15

.3-2

0.3

-21

.3-2

3.3

-30

.3-3

3.3

-48

.3-4

9.3

-53

.3-6

6.3

-73

.3-7

4D

P-5

8

.4-0

4.4

-04b

.4-2

8

.4-3

1.4

-34

.4-3

9.4

-59

.4-6

1D

P-6

7

.5-5

1

.6-0

1

.7-4

.1

p = 0.00096

p = 0.021

CLL cells differ from normal CD5CLL cells differ from normal CD5++ B cells B cells by the overuse of certain autoreactive genes by the overuse of certain autoreactive genes

Fais Fais et al.et al. J Clin Invest 98: 1659, 1998 J Clin Invest 98: 1659, 1998

CLL clones differ in the degree of somatic mutations, especially in particular IgV genes

VVHH Specific Specific % Cases with % Cases withFamilyFamily VVHH Gene Gene MutationsMutations

All casesAll cases - - 50.7 50.7

11 - - 33.3 33.3 1-691-69 10.0 10.0

33 - - 66.7 66.7 3-073-07 90.0 90.0

44 - - 41.2 41.2 4-344-34 55.0 55.0

Fais Fais et al.et al. J Clin Invest 98: 1659, 1998 J Clin Invest 98: 1659, 1998

Ig VIg VHH gene mutation status of CLL cells is an gene mutation status of CLL cells is an important prognostic indicator of outcomeimportant prognostic indicator of outcome

Damle Damle et al. et al. Hamblin Hamblin et al. et al.

Blood 94: 1840, 1999 Blood 94: 1840, 1999 Blood 94: 1848, 1999Blood 94: 1848, 1999

≥ ≥ 2% mutation2% mutation

< 2% mutation< 2% mutation



VVHH DD JJHH CCHH



VVKK JJHH CCKK

Ig GenesIg GenesIg MoleculeIg Molecule V RegionV Region

HingeHingeRegionRegion

CCKK

COOHCOOH

NHNH22

-S - S--S - S-

FabFab

FcFc

VVKK

CCHH11

VVHH

CCHH22

CCHH33

VVKKJJKK

VV HHDDJJ HH

CDR3CDR3 CDR1CDR1CDR2CDR2

FR4FR4 FR3FR3 FR2FR2 FR1FR1

Ig molecules and their genesIg molecules and their genes

CLL clones are culled from the normal B-cellCLL clones are culled from the normal B-cell

repertoire based on structural constraints ofrepertoire based on structural constraints of

the B-cell antigen receptorthe B-cell antigen receptor

IgV gene segment recombinationIgV gene segment recombination

Heavy ChainHeavy Chain Light Chain (Light Chain (//λλ))

VVHH (44) (44)

HC = VHC = VHH x D x J x D x JH H = 44 x 27 x 6 = 7,128= 44 x 27 x 6 = 7,128

D (27)D (27) JJHH (6) (6) JJLL (5/7) (5/7)VVLL (46/36) (46/36)

= RAG mediated = RAG mediated recombinationrecombination

VVHHDJDJHH rearrangement: ~1 : 7,000rearrangement: ~1 : 7,000

~1% of CLL patients express a BCR with a V~1% of CLL patients express a BCR with a VHH 1-69 1-69 gene exhibiting very similar HCDR3s often gene exhibiting very similar HCDR3s often comprised of the same Vcomprised of the same VHH-D-J-D-JHH segments segments

Widhopf Widhopf et alet al. Blood 104: 2499-2504, 2004 . Blood 104: 2499-2504, 2004

Ig V region gene segment recombinationIg V region gene segment recombination


VVHH (44) (44)

HC = VHC = VHH x D x J x D x JH H = 44 x 27 x 6 = 7,128= 44 x 27 x 6 = 7,128

= V= V x J x J = 46 x 5 = 230 = 46 x 5 = 230

= V= V x J x J = 36 x 7 = 252 = 36 x 7 = 252

D (27)D (27) JJHH (6) (6) JJLL (5/7) (5/7)VVLL (46/36) (46/36)


VVHHDJDJHH / V/ VLLJJLLrearrangement: ~1 : 3 x 10rearrangement: ~1 : 3 x 1066

CLLV GeneFamily

GermlineV Gene

PercentDifference

DGene

JGene

CLL 039 VH4VI

4-39O12

0.30.0

6-13-

5b2

CLL 057 VH4VI

4-39O12

0.70.0

6-13-

5b1

CLL 114 VH4VI

4-39O12

0.30.0

6-13-

5b1

CLL 202 VH4VI

4-39O12

0.70.0

6-13-

5b1

CLL 209 VH4?

4-39?

0.3?

6-13-

5b?

CLL cases with remarkably similar B-cell receptorsCLL cases with remarkably similar B-cell receptors

Ghiotto Ghiotto et alet al. J Clin Invest . J Clin Invest 113: 1008113: 1008, 2004, 2004

IgV gene segment recombinationIgV gene segment recombination


VVHH (44) (44) D (27)D (27) JJHH (6) (6) JJLL (5/7) (5/7)VVLL (46/36) (46/36)


Because of the differences that occur at the Because of the differences that occur at the junctions when gene segments combine, the junctions when gene segments combine, the likelihood that the same Vlikelihood that the same VHHDJDJHH- V- VLLJJLL

rearrangement with the same junctional rearrangement with the same junctional characteristic would occur in two different B characteristic would occur in two different B cells is even much more remotecells is even much more remote

≈ ≈ 1 / 1x101 / 1x108 8 – 1 :– 1 : 1x101x101212

Therefore, if the gene structure of the Ig variable Therefore, if the gene structure of the Ig variable region found in B-CLL cells from different region found in B-CLL cells from different patients is very similar or identical, then this patients is very similar or identical, then this must indicate a selective process of must indicate a selective process of leukemogenesis that targets B cells with a given leukemogenesis that targets B cells with a given type of Ig V region. type of Ig V region.

CLL068CLL068:: CAR CAR GGGG DDYDYVWGSYRYDYVWGSYRS NS N DAFDIWGDAFDIWG

CLLSMICLLSMI::CAR CAR GGGG NNYDYYDYIIWGSYRWGSYRSS NN DAFDIWGDAFDIWG

CLL258CLL258:: CAR CAR GGGG IIYDYVWGSYRYDYVWGSYRPP N N DAFDIWGDAFDIWG

aCLA*:aCLA*: CAR CAR GGGG NNYDYYDYIIWGSYRWGSYRSS NN DAFDIWGDAFDIWG

CAR YYDYVWGSYRY DAFDIWGCAR YYDYVWGSYRY DAFDIWGD3-16D3-16 JJHH33VVHH1-691-69

Heavy chain sequence alignmentHeavy chain sequence alignment

CLL022CLL022:: CAR CAR GGGG DDYDYVWGSYRYDYVWGSYRPP NN DAFDIWGDAFDIWG

Natural autoantibodyNatural autoantibody

**aCLA = anti-cardiolipin abaCLA = anti-cardiolipin ab MessmerMessmer et alet al. J Exp Med 2004; 200: 519-525. J Exp Med 2004; 200: 519-525

Almost 30% of patients with chronic lymphocytic leukemia Almost 30% of patients with chronic lymphocytic leukemia carry stereotyped receptorscarry stereotyped receptors

Stamatopoulos Stamatopoulos et alet al. Blood 109:259-270, 2007. Blood 109:259-270, 2007

Murray Murray et alet al. . Blood 111:1524-Blood 111:1524-15153333,, 20082008

>35% chance of fitting into a stereotypic set if U-CLL>35% chance of fitting into a stereotypic set if U-CLLor if express a specific Vor if express a specific VHH gene (1-69, 3-21, 4-39) gene (1-69, 3-21, 4-39) associated with poor outcomeassociated with poor outcome


2. These BCRs can be poly- and auto-reactive, binding 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by natural as well as novel autoantigens generated by apoptosis and other catabolic processesapoptosis and other catabolic processes

Expression of recombinant CLL mAbs

293T HEK cell line293T HEK cell lineAntibody purification using Antibody purification using

Protein G beadsProtein G beads

Immuno assay for Immuno assay for quantification of quantification of

CLL mAbCLL mAb

4-5 days of culture4-5 days of culture

1.1. Plasmid DNA Plasmid DNA carrying heavy and carrying heavy and light chain Ig genelight chain Ig gene

2.2.Lipofectamine Lipofectamine 2000 Reagent2000 Reagent

3.3. Lipofectamine 2000 Lipofectamine 2000 Reagent and DNA are Reagent and DNA are mixed and incubatedmixed and incubated

4.4. Liposomes are added in Liposomes are added in 293T HEK cell culture293T HEK cell culture

Transfection of 293T HEK cells using Lipofectamine 2000Transfection of 293T HEK cells using Lipofectamine 2000

Wardemann Wardemann et alet al. Science 301:1374, 2003 . Science 301:1374, 2003

Herve Herve et alet al. J Clin Invest . J Clin Invest 115:1636-1643, 2005115:1636-1643, 2005

~20% of mAbs from CD5~20% of mAbs from CD5++ B cells from normal subjects B cells from normal subjects

~75% of mAbs from CLL cells~75% of mAbs from CLL cells

~90% from unmutated CLL~90% from unmutated CLL

~60% from mutated CLL~60% from mutated CLL

Reactivity of recombinant mAbs with viable HEp-2Reactivity of recombinant mAbs with viable HEp-2

cells that were permeabilized to allow Ab entrycells that were permeabilized to allow Ab entry

[“Anti-cell antibodies”][“Anti-cell antibodies”]

Reactivity mainly with cytoplasmic structures and Reactivity mainly with cytoplasmic structures and occasionally with nucleoli. Only one mAb fromoccasionally with nucleoli. Only one mAb fromM-CLL patient reacted with nucleus in a M-CLL patient reacted with nucleus in a homogeneous patternhomogeneous pattern


Autoreactivities of individual CLL BCRs/mAbsAutoreactivities of individual CLL BCRs/mAbs

1.1. VVHH1-69/D3-16/J1-69/D3-16/JHH3 + V3 + VKK3-20: 3-20:

Non-muscle myosin heavy chain IIA Non-muscle myosin heavy chain IIA ((Chu et al. Blood 112: 5122-5129, 2008Chu et al. Blood 112: 5122-5129, 2008))

2. V2. VHH4-39/D6-13/J4-39/D6-13/JHH5 + V5 + VKK1D-39/J1D-39/JKK11

VimentinVimentin ((Chu et al. Blood 112: 5122-5129, 2008Chu et al. Blood 112: 5122-5129, 2008))

3. 60% of U-CLL and 10% of M-CLL: 3. 60% of U-CLL and 10% of M-CLL:

Apoptosis-associated autoantigens Apoptosis-associated autoantigens ((Catera et al. Mol Med 2008; 14: 665-674Catera et al. Mol Med 2008; 14: 665-674 ))

CLL068CLL068:: CAR CAR GGGG DDYDYVWGSYRYDYVWGSYRS NS N DAFDIWGDAFDIWG

CLLSMICLLSMI::CAR CAR GGGG NNYDYYDYIIWGSYRWGSYRSS NN DAFDIWGDAFDIWG

CLL258CLL258:: CAR CAR GGGG IIYDYVWGSYRYDYVWGSYRPP N N DAFDIWGDAFDIWG

aCLA*:aCLA*: CAR CAR GGGG NNYDYYDYIIWGSYRWGSYRSS NN DAFDIWGDAFDIWG

CAR YYDYVWGSYRY DAFDIWGCAR YYDYVWGSYRY DAFDIWGD3-16D3-16 JJHH33VVHH1-691-69

Heavy chain sequence alignmentHeavy chain sequence alignment

CLL022CLL022:: CAR CAR GGGG DDYDYVWGSYRYDYVWGSYRPP NN DAFDIWGDAFDIWG

Natural autoantibodyNatural autoantibody

**aCLA = anti-cardiolipin abaCLA = anti-cardiolipin ab MessmerMessmer et alet al. J Exp Med 2004; 200: 519-525. J Exp Med 2004; 200: 519-525

mAb 068 binds 225KDa moleculemAb 068 binds 225KDa molecule

C. Chu C. Chu et alet al. Blood 112: 5122-5129, 2008. Blood 112: 5122-5129, 2008

LC MS/MSLC MS/MSidentifies 225KDaidentifies 225KDaband as band as non-muscle non-muscle myosin heavy myosin heavy chain IIA chain IIA (MYHIIA)(MYHIIA)


CLL mAbCLL mAb 068 co-localizes with pAbs to MYHIIA068 co-localizes with pAbs to MYHIIA


Autoreactivities of individual CLL BCRs/mAbsAutoreactivities of individual CLL BCRs/mAbs

1.1. VVHH1-69/D3-16/J1-69/D3-16/JHH3 + V3 + VKK3-20: 3-20:

Non-muscle myosin heavy chain IIA Non-muscle myosin heavy chain IIA ((Chu et al. Blood 112: 5122-5129, 2008Chu et al. Blood 112: 5122-5129, 2008))

2. V2. VHH4-39/D6-13/J4-39/D6-13/JHH5 + V5 + VKK1D-39/J1D-39/JKK11

VimentinVimentin ((Chu et al. Blood 112: 5122-5129, 2008Chu et al. Blood 112: 5122-5129, 2008))

3. 60% of U-CLL and 10% of M-CLL: 3. 60% of U-CLL and 10% of M-CLL:

Apoptosis-associated autoantigens Apoptosis-associated autoantigens ((Catera et al. Mol Med 2008; 14: 665-674Catera et al. Mol Med 2008; 14: 665-674 ))

JurkatJurkat

RAMOSRAMOS

An

nex

in V

An

nex

in V

CLL014CLL014 DO13DO13

CC DD

An

nex

in V

An

nex

in V

CLL014CLL014 DO13DO13

AA BB28.17

0.92

3.70

67.27

13.86

0.2369.14

16.77

52.86 18.12

0.5528.47

55.35 16.96

0.3227.37

CLL mAbs react with apoptotic (not healthy) cellsCLL mAbs react with apoptotic (not healthy) cells

R. Catera R. Catera et alet al. Mol Med 14: 665-674, 2008. Mol Med 14: 665-674, 2008

Apoptotic B and T cells are a source of Apoptotic B and T cells are a source of autoantigens for CLL mAbsautoantigens for CLL mAbs

SUMMARY of the results:SUMMARY of the results:

- More than 60% (18/28) of the mAbs tested reacted with More than 60% (18/28) of the mAbs tested reacted with these two cell typesthese two cell types

- 15 of the 18 reactive mAbs used an unmutated V15 of the 18 reactive mAbs used an unmutated VHH gene, gene,

only 3 used a mutated Vonly 3 used a mutated VHH gene gene


Antigens bound at the surface of apoptotic cells Antigens bound at the surface of apoptotic cells have translocated from intracellular compartmentshave translocated from intracellular compartments

Cytox OrangeCytox Orange Annexin VAnnexin V CLL 114CLL 114 MergeMerge

Membrane blebsMembrane blebs

Apoptotic body Apoptotic body without DNAwithout DNA

Apoptotic body Apoptotic body with DNAwith DNA


MYHIIAMYHIIA is one of the intracellular antigensis one of the intracellular antigens

that translocates to the surface andthat translocates to the surface and

is bound by CLL mAbsis bound by CLL mAbs

Live

Late apoptotic

Early apoptotic

Chu Chu et alet al. Blood 2010 in press. Blood 2010 in press

MEAC: MYHIIA exposed apoptotic cellMEAC: MYHIIA exposed apoptotic cell

CLL 068 mAb binds to MEACsCLL 068 mAb binds to MEACs

NegativeNegative ApoptoticApoptotic MEACsMEACs


Many CLL mAbs bind MEACsMany CLL mAbs bind MEACsM

EA

C b

ind

ing

Subset

Mutation



3. The clinically-distinct subgroups differ in the retention 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical of polyreactivity being associated with worse clinical diseasedisease


Polyreactivity is a feature primarily of unmutated CLL cellsPolyreactivity is a feature primarily of unmutated CLL cells

Ig VIg VHH gene mutation status of CLL cells is an gene mutation status of CLL cells is an important prognostic indicator of outcomeimportant prognostic indicator of outcome

Damle Damle et al. et al. Hamblin Hamblin et al. et al.

Blood 1999; 94: 1840 Blood 1999; 94: 1840 Blood 1999; 94: 1848Blood 1999; 94: 1848





Hi binding99 months(n = 15)

Lo binding?? Months (n = 9)

Binding well to MEACs correlates with poor Binding well to MEACs correlates with poor patient survivalpatient survival

Binding well to MEACs correlates with poor Binding well to MEACs correlates with poor patient survivalpatient survival


UnmutatedUnmutated118 months118 months(n = 18)(n = 18)

MutatedMutated?? Months (n = 6)?? Months (n = 6)

In this limited series, MEAC binding correlates better In this limited series, MEAC binding correlates better with patient survival than with patient survival than IGHVIGHV mutation status mutation status


Many CLL mAbs bind MEACsMany CLL mAbs bind MEACsM

EA

C b

ind

ing

Subset

Mutation


InferencesInferences1. MEACs may be a source of autoantigens

driving CLL disease

2. The origin of many CLL clones may be cellsthat produce natural antibodies to MEACsor 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?

B-CLL evolution

hypothesis

B-CLL evolution

hypothesis

MEACs MYHIIA+ Vimentin Filamin B Oxidation Chemical Modification

M-CLLM-CLL

U-CLLU-CLL

InitiationInitiation

ProgressionProgression

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