L. MorettaL. Moretta

IstitutoIstituto GianninaGiannina Gaslini, Gaslini, GenovaGenova and University of and University of GenovaGenova

Le cellule Le cellule NaturalNatural Killer:Killer:dal laboratorio alla terapia di dal laboratorio alla terapia di

leucemie acute ad alto rischioleucemie acute ad alto rischio

Immune SystemImmune System

Innate Immunity

Adaptive (or Specific)Immunity

DefendingDefending cellscells of the of the Innate Innate ImmunityImmunity

VariousVarious cell cell typestypesspecializedspecialized in in differentdifferenteffectoreffector functionsfunctions

An infection and the response to it can An infection and the response to it can be divided into a series of stagesbe divided into a series of stages

Cells of the Innate Immunity:Cells of the Innate Immunity:The Unsung HeroesThe Unsung Heroes

Peter Parham, Nature, 423, May 2003

The fact that most people are not The fact that most people are not perpetually sick is testament to innate perpetually sick is testament to innate

immunity squelching most of the immunity squelching most of the infections that we contract.infections that we contract.

Natural killer Natural killer cell: a major cell: a major player of the player of the

innate immunityinnate immunity

NK cell functionsNK cell functions

CytotoxicityCytotoxicity -- Tumor or leukemia cell killingTumor or leukemia cell killing-- Killing of virus infected cellsKilling of virus infected cells-- DC editingDC editing-- ADCCADCC

Cytokine productionCytokine production -- Induction of inflammatory responsesInduction of inflammatory responses-- Regulation of adaptive immune responsesRegulation of adaptive immune responses-- Regulation of Regulation of hematopoiesishematopoiesis-- Induction of DC maturationInduction of DC maturation

Ist. G. Gaslini

NK cell NK cell activationactivation isis under the under the control of control of HLAHLA--ClassClassII--specificspecificinhibitoryinhibitory receptorsreceptors, , activatingactivating

receptorsreceptors and and theirtheir cellularcellular ligandsligands

Schematic representationof the main interactionsoccurring between normalnatural killer (NK) cells (expressing both HLA class I-specific inhibitoryreceptors and activatingreceptors) and potentialtarget cells.

HLA class I -specific inhibitory receptors

CLys80

allelesCAsn80

allelesalleles alleles

Moretta A et al, Annu Rev Immunol, 1996(modified)

A schematic representation of the human natural A schematic representation of the human natural killer (NK) receptor repertoire (killer (NK) receptor repertoire (autologousautologous setting)setting)

Ist. G. Gaslini

Harrison’s 16th editionPrinciples of Internal Medicine(Adapted from Moretta A et al, with permission)

In In anan allogeneicallogeneic settingsetting(e.g. (e.g. haploidenticalhaploidentical BM transplantation)BM transplantation) a a fractionfraction of of donordonor’’s s NK cells NK cells maymay express KIR express KIR thatthat are are notnot engagedengaged by the by the

HLAHLA--ClCl II allelesalleles of the of the recipientrecipient

Ist. G. Gaslini

NK

NK Lysis

leukemiccell

Stop !

NK

Stop !

alloreactiveNK cell

NK cells in the NK cells in the therapytherapy of of acute acute myeloidmyeloid leukemiasleukemias

(in the (in the haploidenticalhaploidenticalHemopoieticHemopoietic Stem Cell Stem Cell (HSC) transplantation)(HSC) transplantation)

In In haploidenticalhaploidentical ((TT--depleteddepleted) BMT NK ) BMT NK cells derive from donor CD34cells derive from donor CD34++ HSCHSC

NKdonorHSC

donor

NKdonor2 weeks 4 - 6 weeks

KIRCD94/NKG2A

“Alloreactive” NK cells

HaploidenticalHaploidentical BM transplantation: BM transplantation: clinical data in AML clinical data in AML (Perugia, 2002)(Perugia, 2002)

NONO YESYES5 5 –– years survival years survival probabilityprobability < 5%< 5% 60%60%

Graft rejectionGraft rejection 15.5%15.5% 0 %0 %

GvHDGvHD 13.7%13.7% 0 %0 %5 5 –– years probability of years probability of relapserelapse 75 %75 % 0 %0 %

Ruggeri et al, Science 2002

Presence of KIR/HLAPresence of KIR/HLA--ClCl I mismatch in I mismatch in the donor the donor vsvs recipient directionrecipient direction

RecentRecent advancesadvances in in haploidenticalhaploidentical HSCT HSCT toto treattreat high high riskrisk leukemiasleukemias in in

Pediatric Pediatric PatientsPatients

More precise More precise identificationidentification of the of the alloreactivealloreactive NK NK cell cell populationpopulation ((presencepresence of of activatingactivating KIRsKIRs))Donor Donor selectionselection ((basedbased on the on the sizesize of the of the alloreactivealloreactiveNK subset in NK subset in differentdifferent potentialpotential donorsdonors))IdentificationIdentification of of donordonor--derivedderived alloreactivealloreactive NK NK cell cell populationspopulations in in patientspatients after HSCTafter HSCTCorrelationCorrelation withwith the clinical the clinical outcomeoutcome, , particularlyparticularlyin high in high riskrisk ALLALL

(collaboration with F. Locatelli, Pavia)

Donor Donor selectionselectionin in haploidenticalhaploidentical

HSC transplantationHSC transplantation

In In anan allogeneicallogeneic settingsetting(e.g. (e.g. haploidenticalhaploidentical BM transplantation)BM transplantation) a a fractionfraction of of donordonor’’s s NK cells NK cells maymay express KIR express KIR thatthat are are notnot engagedengaged by the by the

HLAHLA--ClCl II allelesalleles of the of the recipientrecipient

Ist. G. Gaslini

NK

NK Lysis

leukemiccell

Stop !

NK

Stop !

alloreactiveNK cell

KIR specific for HLA-class I alleles

absent in patient's haplotype

KIR specific for patient's HLA-class I alleles + NKG2A

Double fluorescence analysis, using appropriate mAbscombinations, reveals the size of the alloreactive NK cell population (i.e. expressing only KIR that do not

recognize HLA-class I alleles of the patient)

DefinitionDefinition of the of the alloreactivealloreactive NK cell subset in NK cell subset in DonorDonor’’s NK cell s NK cell populationpopulation in GvH direction in GvH direction

Moretta et al, Imm Rev, 2008

DONORS

HLA-A

HLA-B

HLA-C

KIR phenotype of donor’s alloreactive NK cells

Patient ML2 A2 B15, 50 (Bw6)

Cw12, w6 (CAsn80, CLys80)

_

NK DONOR A (sibling)

A2 B15, 44 (Bw6,w4)

Cw12, w5 (CAsn80, CLys80)

KIR3DL1+ KIR2DL1-, 2/3-

NK DONOR B (unrelated)

A2, 3 B35, 44 (Bw6,w4)

Cw3, w5 (CAsn80, CLys80)

KIR3DL1+ KIR2DL1-, 2/3-

0

20

40

60

80

1 2 3

% 5

1 Cr-

releas

e

40:1 20:1 10:1

DONOR A

DONOR B

ML2

E:T

Identification of Identification of alloreactivealloreactive NK cells and NK cells and theirtheir cytolyticcytolytic effecteffect againstagainst ML2ML2

ConclusionsConclusions

The size of the The size of the alloreactivealloreactive NK cell subset NK cell subset parallels the degree of NK parallels the degree of NK cytotoxicitycytotoxicityagainst leukemic cells. This approach can against leukemic cells. This approach can be useful for selecting the most appropriate be useful for selecting the most appropriate NK cell donorNK cell donor

Donor Donor derivedderived, , functionalfunctionalalloreactivealloreactive NK cell NK cell populationspopulations

are are generatedgenerated and and persistpersist in in patientspatients after HSCTafter HSCT

DONORS HLA-A HLA-B HLA-C KIR phenotype of donor’sAlloreactive NK cells

HSCT RECIPIENTPatient PC

A2,31B35,44

(Bw6,w4)Cw4,w5(CLys80)

HSCT DONORMother DMF

A23,31B35,49

(Bw6,w4)Cw4,w7

(CLys80, CAsn80)

KIR2DL2/3+

KIR2DL1-, 3DL1-

KIR2

DL2

/3DONOR DMF

RECIPIENT PC(7mo post-t.)

KIR2DL1,3DL1,NKG2A

15 44

38

45 23

13

NK cells

Cytolytic activity of alloreactive NK cells in a case of Haplo-mismatched transplant

NK isolatedfrom:

0

20

40

60

80

100

1 2 340:1 20:1 10:1

E:T

BOB B-EBV (Bw4,CLys80)

% 51

Cr-r

elea

se

HSCT with CAsn80 (C1) as KIR ligand mismatch

UPN Diagn Status at Tx

HSCDonor aGvHD Outcome

Relevantinhibitory

KIR§

ActivatingKIR§

Alive, CR(+42 m) 2DL2/3

2DL2/3

2DL2/3

2DL2/3

5GR

CD10+ALL 2nd CR mother NO Alive, CR

(+ 8 m) NO 2DL2/32DS12DS23DS1

36% 34%

7MO

AML(M0) 1st CR sister NO

Relapsed(+ 3 m)Dead

NO 2DL2/32DS12DS23DS1

2% 3%

6LC JMML Disease

present mother Grade II Alive, CR(+ 8 m) NO 2DL2/3

2DS12DS23DS1

9% 69%

2DL2/3

Relapsed(+ 7 m)Dead

2DS23DS1

2DS2

2DS2

2DS23DS1

8JA

CD10+ALL 2nd CR brother NO Alive, CR

(+ 5 m) NO2DS12DS23DS1

5%

Alive, CR(+22 m)

Relapsed(+ 5 m)Dead

10%

Grade II

NO

Grade I

NO

Infections% Donor

Alloreactivesubset

% RecipientAlloreactive

subset

1CP

Ph+ALL 3rd CR mother NO 26% 54%

2%

15%

3%

2IL

CD10+ALL 2nd CR father CMV 6%

3AG

AML(M2) 3rd CR sister

BK cystitis, EBV,

Adenovirus, Measles

8%

4APa

AML(M0-1) 1st CR mother NO 15%

§ 2DS1, 2DS2 e 3DS1 are indicated when present in KIR genotype. 2DS3, 2DS4 e 2DS5, even though evaluated, are not indicated. Pende et al, Blood 2009

The The sizesize of the of the alloreactivealloreactiveNK cell subset NK cell subset correlatescorrelateswithwith the clinical the clinical outcomeoutcome

ALL and AML:ALL and AML:LeukemiaLeukemia--freefree survivalsurvival byby NK NK alloreactivityalloreactivity

0.00

0.25

0.50

0.75

1.00

0 1 2 3 4 5

YEARS AFTER HSCT

PRO

BA

BIL

ITY

(95%

CI) YES 73% (53-93)

NO = 42% (14-70)

P = 0.04

YES: N = 19; E = 5NO: N = 13; E = 7

Acute Lymphoblastic Leukemia

0.00

0.25

0.50

0.75

1.00

0 1 2 3 4 5

YES: N = 11; E = 7NO: N = 10; E = 7

NO = 24% (0-52)

YES 31% (2-60)

P = N.S.

YEARS AFTER HSCT

Acute Myeloid Leukemia

Pediatric Hematology / OncologyFondazione IRCCS Policlinico San MatteoUniversity of Pavia, Italy Updated January 31th, 2009

ConclusionsConclusions ((haplohaplo--HSCTHSCT))

Importance ofImportance of selectingselecting donorsdonors on the on the basisbasis of of the the sizesize of of theirtheir alloreactivealloreactive NK cell subsetNK cell subset

DonorDonor--derivedderived alloreactivealloreactive NK cells are NK cells are generatedgenerated and and maymay persistpersist forfor yearsyears in in patients patients receiving haploreceiving haplo--HSCTHSCT

CorrelationCorrelation betweenbetween sizesize of the of the alloreactivealloreactive NK NK cell subset and clinical cell subset and clinical outcomeoutcome

Dept of Exp Med,University of GenovaAlessandro MorettaSimona SivoriRoberta CastriconiEmanuela Marcenaro

IST, GenovaMaria Cristina MingariDaniela PendeMassimo VitaleGabriella PietraStefania Marcenaro

G. Gaslini Institute, GenovaCristina BottinoClaudia CantoniMichela FalcoGrazia Maria Spaggiari

S. Matteo Hosp. and University of Pavia, PaviaFranco LocatelliRita MaccarioDaniela Montagna