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Immunodeficiencies and Genetic Mutations Affecting NK Cells in HumansProf. Jordan Orange MD/PhD

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Immunodeficienciesand Genetic Mutations

Affecting NK Cells in Humans

1

Prof. Jordan Orange MD/PhDAssistant Professor of PediatricsUniversity of Pennsylvania School of MedicineChildren’s Hospital of Philadelphia

NK cellsNK cells are lymphocytes important in immune regulation and host defense that are capable of being specifically activated or inhibited after the ligation of germline-encoded receptors

LGL

• Cytotoxicity

2J.S. Orange

NK

K562

y y– Perforin-mediated

Antibody dependent (ADCC)– TRAIL/FAS-L-mediated

• Cytokine production (NK1/NK2)– IFN-γ, TNF, IL-5, IL-13

• Costimulation– CD40L (CD154)– OX40L

Activation receptor ligand Activation (lysis) receptor

NK cell function – inhibition

3

Class I MHC Inhibitory KIR

Orange and Ballas Clin. Immunol. 2006 118: 1-10



Activation (lysis) receptorActivation receptor ligand

NK cell activation – “missing self”

Granzymes

4

Inhibitory KIR

Orange and Ballas Clin. Immunol. 2006 118: 1-10

Perforin

NK cells in defense

• Important in anti-viral defense

– Especially herpes viruses

– Induced by type-I IFN

• Important in tumor surveillance

– Function in graft versus tumor in transplant

5

• Defective in a number of diseases

– Single gene immunodeficiencies

– Polygenic diseases

– As a result of therapies

• Rare isolated deficiencies

– Informative

– Clinically relevant

Measureand compare

Experimental release

E

T TE

Time

Detergent

T

51Cr

TE

Measuring cytotoxicity

6

TDetergent

Maximum “total” release

Media T

Background “spontaneous” release

E

T(Experimental release - spontaneous release)

% lysis = 100 X ---------------------------------------------------------(Total release - spontaneous release)



The immunological synapse

• Contact zone between an immune cell and the cell it is recognizing

• Formation of the supramolecular activation cluster (SMAC)• Functions in antigen presentation/recognition• Secretion

7

pSMAC

cSMAC

Secretory domain

The lytic NK cell immunological synapse

8

J.S. Orange

The lytic NK cell immunological synapse

912,000x 50,000x

J.S. Orange



Learning about NK cells from human single gene deficiencies

Insights into function and role in immunity

10

Insights into function and role in immunity

Wiskott-Aldrich syndrome

11

• WAS and NK cell-mediated defense– 1/3 of patient have severe herpesviral infections– 1/3 die from hematopoetic malignancies

• Historical studies of NK cell function in PBMC are variable

NK cell cytotoxicity is deficient in WAS NK cells

is (%

)

12

K56

2 ly

si



WASp function

13Pollard and Borisy, Cell 2004 112: 453-65

Orange, et al., Cell and Molecluar Life Sciences 2004 61: 2361-85

WASp accumulates with F-actin at the NK cell immunological synapse

Actin WASp PerforinDIC Overlay

14

81±4 72±4 50±6

DIC Perforin MergeF-actin CD11a

Formation of the cytolytic immunological synapse requires WASp/actin function

NK

xy

15DIC Perforin MergeF-actin CD11a

WASp-

NK*

*same with cytochalasin-D

xz

x

y



Surface receptor clustering and polarization of cytolytic granules

require WASp/actin function

WASp-dependent actin reorganization

16

Surface receptor clustering

Lytic granule polarization

Polarization of lytic granules to the cytolytic synapse requires microtubules, but SMAC formation does not

17

DIC Perforin MergeF-actin CD11a

x

z

y

x

NK + Colchicine

WASp-dependent actin reorganization

Polarization of lytic granules to the cytolytic synapse requires microtubules, but SMAC formation does not

18

Surface receptor clustering

Microtubule-dependent lytic granule polarization



Step 1,2 3 4-5 6-10 11-13 14,15 16,17 18,19 22,2320,21

Target cell

NK cell

Stage Recognition Effector Termination

Stages of lytic immunological synapse formation

19Activation receptor and ligand

Adhesion receptor and ligand

Tethering receptor and ligand

Plasma membrane

MTOC

Lytic granules (LG)

Centrosome

Accumulated F-actin

Lipid raft

F-actin cortex

ApproximationCellular tethering

Actin reorganizationReceptor clusteringRaft membrane clusteringActivation signalingMinus ended LG movement

Plus ended LG movement?LG transit through cortex

Adhesion LG docking LG priming

LG fusionLG release

Relative inactivityDownmodulation

Detachmentrecycling

MTOC polarizationMTOC anchoringActin clearance

AdhesionActivation signaling

Recognition

Steps in lytic immunological synapse formation

Plasma membrane

L ti l (LG)

Accumulated F-actin

Lipid raft

F-actin cortex

NK cell

Step:

Stage:

20

• Plus ended LG movement?• LG transit through cortex

Activation receptor and ligand

Adhesion receptor and ligand

Tethering receptor and ligand

MTOC

Lytic granules (LG)

Centrosome

Target cell

NK

21

EBV-BCL

J.S. Orange



Case presentation – Ihistory

• 5 yo male previously very healthy

• Had congestion and pharyngitis

– MD diagnosed strep pharyngitis and prescribed antibiotics

22

MD diagnosed strep pharyngitis and prescribed antibiotics

• 1wk later had fever to 40.0° at a family picnic

• Was taken to ER

• T=40.8°, BP 75/45, HR 148

– Had enlarged spleen on examination

Case presentation – II laboratory studies

• WBC=2,100 (2b, 27n, 52L, 3atL, 11m), PLT=80, HgB 7.9 • ALT=185 (12-42), AST=317 (20-64), GGT=23 (17-126), LDH 2391,

bili=0.1 (0.6-1.4)• Cr=0.3 (0.1-0.6), BUN=15 (2-19), • Fibrinogen=164 (172-471) D-dimer=0 84 (0 1-0 6) Ferritin=4280

23

Fibrinogen 164 (172 471), D dimer 0.84 (0.1 0.6), Ferritin 4280 (10-95), Lipase=70 (10-115),

• Na=137 (133-140), K=4.8 (4.1-5.8), Cl=112 (96-106), C02 18 (20-26), Glucose=63 (74-127), Ca=7.8 (9.2-10.5), phos=4.6 (4.8-8.2), Mg=2.2 (1.5-2.5),

• U/A 1+ protein, trace ketones, SG 1034(1003-1035)• CSF 9WBC, 6425RBC protein 114 (15-40), Glucose 66 (32-82)• CSF, blood and urine cultures obtained

Case presentation – IIIclinical course

• Admitted to PICU – given parenteral antibiotics • Developed critical thrombocytopenia and anemia • Blood cultures were negative; Bone marrow aspirate• Developed severe hypotension and respiratory failure • Patient expired after 4 days

24CD163 stain of spleen



Case presentation – IVNK cell function

Cytotoxicity

15

20

Patient

Control

25

%K

562

lysi

s

Effector to target cell ratio

0

5

10

0 10 20 30 40 50 60

HLH diagnostic guidelines

• Specific molecular diagnosis, or ≥5 of the following

– Fever

– Splenomegally

– Cytopenia affecting ≥2 out of 3 peripheral lineages

26Pediatr Blood Cancer 2007; 48: 124-31

– Hypertriglyceridemia and/or hypofibrinogenemia

– Hematophagocytosis

– No evidence of malignancy

– Elevated ferritin and/or sIL-2R

– Low or absent NK cell cytotoxicity

Management of HLH

• Immunosuppression

• HLH-94/2004 – dexamethasone, VP-16, CsA

27

• HLH-94 – 3-year survival 55%

• HSCT – success ~80%



Case presentation – Vdiagnosis: perforin deficiency (FEL2)

Control - MFI=91 Patient - MFI=8

Intracellular perforin FACS

28Perforin fluorescence

% o

f max

Critical deficiency of cytolytic synapse: hematophagocytic lymphohistiocytosis

• Example of abnormal antiviral inflammation

• Genetic inability to regulate the antiviral response

• Defect effects both NK cells and CTLs

29

– Timing of response suggests strong role for NK cells

• Abnormal function of the cytolytic immune synapse

• Illustrates the relevance of specific sequence in lytic immunological synapse formation

Model for HLH

Infected monocyte

VirusImmunologic infection

Lymphocyte

IL-12+

TNF+ IFN-γ

IFN-γ

x xx x

x x

x

30

monocyte

NK cell

DC

Monocyte

TNF+

IFN-α

γ

IL-12

TNF

IFN-γ

IL-12+

TNF+

x xx x

xx xx



Molecular defects leading to HLH

31

Steps in immune-mediated cytolysis

2

1

pSM

AC

AC

F-actin

Actinosome

KinesinCytolytic granule

MTOC

XIAP SAP X

Perforin

3

4 5

6

7 8

X X X XX X

32Target cellNK cell

cSM

ApS

MA

C

Microtubule

Adhesion receptor

Activation/adhesion receptor

Cell membrane

Activation receptor

RAB27aMUNC13-4 Syntaxin-11

123

4 5

6

7 89

XLYSTAP-3

X XX X

Single gene diseases with NK cell defects1978-2002

33J.S. Orange, Microbes and Infection 2002



Single gene diseases with NK cell defects

2002-2007

34J.S. Orange, Current Opinion in Allergy and Clinical Immunology 2006

/CD

16+

lym

phoc

ytes

CD

16+

lym

phoc

ytes

/ml

NK cell population: normal

35

Perc

ent C

D3-

CD

56+

Abs

olut

e C

D3-

CD

56+/

C

Adapted from: Comans-Bitter, WM, et al., J Pediatr 1997 130: 388

Isolated deficiencies of NK cells

Do not affect other components of immunity

36

J.S. Orange, Current Opinion in Allergy and Clinical Immunology 2006

Without a diagnosis…mechanism is hopeless



ANKD

37


CNKD

38


FNKD

39




What suggests NKD

• Herpes viral disease

– Unusually severe

– Unusually recurrent

40

• HPV disease

– Unusually severe

– Recalcitrant to therapy

• Viral susceptibility

• Rule out other potential explanations

Practical approach to NKD

Repeat

Clinical suspicion of NKD

R/O otherimmunodeficiency

NK cell FACS (CD3-/CD16+/CD56+)

AbsentPresentMake other Dx

C t t i it t ti

41

R/O secondary causeCytotoxicity testing

AbsentPresent

Still absent (x2)

ANKD, or CNKD

Impaired (x3)Present

Consider more advanced testing

(phenotype, cytokines)

FNKD

Cytotoxicity testing

Comprehensive approach to NKD

• Extensive phenotypic analysis (FACS)

– Developmental markers

– CD16 epitopes

• Ex vivo functions

42

Ex vivo functions

– Migration, adhesion, synapse formation

– ADCC

– Specific receptor functions

– Cytokine responsiveness

• In vitro development



Management of NKDIntervention Reported or theoretical benefit Risks

Antimicrobials Decrease incidence of disease Adverse drug reactionsMicrobial resistance

IVIG Decrease incidence of disease increase NK cell function?

Adverse drug reactionsInfections/neurodegenerationInconvenience

IL-2 Increase NK cell function Adverse drug reactionsAutoimmunity

Stem cell transplantation

Correction of defect Defect not correctedTransplant-related mortality or morbidity

43Orange, J.S., et al., J Clin Invest 2002 109:1501

J.S. Orange, in Up to Date, Rose, B.D. (ed) 2006 v14.1

Conclusions

• NK cells serve critical surveillance functions in host defense

• NK cells are defective in an number of single-gene primary immune deficiencies and are likely of clinical relevance

44

• Human genetic immunodeficiency advances the basic mechanistic understanding of NK cell function

• Isolated deficiencies of NK cells are mechanistically unclear but should be suspected in specific cases

45

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