NK Cells

Document Sample
NK Cells Powered By Docstoc
					                          NK Cells
           Experimental Basis of Immunology
                   January 17, 2007




W.H. Chambers, Ph.D.
G.17e Hillman Cancer Center
623-3218
chamberswh@msx.upmc.edu
                        I. Introduction

• Natural Killer (NK) Cells were first described in the early 1970’s by R.
  Herberman; R. Kiessling; and G. and E. Klein
• Defined as a functional entity, i.e. cell capable of recognizing and
  killing tumor cells without prior exposure
• Represent a component of the non-adaptive immune system
• Defined in the early 1980’s as having a large granular lymphocyte
  (LGL) morphology (Reynolds, et al., 1981)
• Represent a heterogeneous population of cells with diverse functions
• Can be best defined phenotypically as CD3-, CD16+, CD56+, CD122+,
  CD158+, CD161+
Innate capacity of lysis
Large granular lymphocytes
CD3-, CD16+, CD56+, CD122+, CD158+, CD161+
II. Pathway of NK Cell Differentiation: Topics


    • Differentiation of NK cells in the fetus
    • Differentiation of NK cells in adults
    • Terminal differentiation of mature
      NK cells
                NK Cell Differentiation

• Derive from, and require normal, intact bone marrow for
  functional maturation
• Represent one of the major lymphocyte populations [T, B,
  NK, NK-T] – ~5% of cells among PBLs
• Present in athymic [nude] mice and rats
• Present in scid mice, and in RAG-1 and RAG-2 knockout
  mice
• Can be distinguished from other lymphocytes by the
  absence of clonally distributed, receptors derived via gene
  rearrangements
 NK Progenitors: Fetus
                                                   Fetal thymus
                                                             p-T        T
Fetal liver                          Fetal blood             c-kit+
                                                             Thy-1+
                                                             CD25+
 HSC        CLP       c-T/NKP           p-T/NK     p-T/NK
                                                             CD161c-
            c-kit+    c-kit+            c-kit+     c-kit+
            Thy-1-    Thy-1-            Thy-1+     Thy-1+
            CD25-
            CD161c-
                      CD25-
                      CD161c-
                                        CD25-
                                        CD161c+
                                                   CD25-
                                                   CD161c+
                                                             p-NK       NK
                                                                        .. ..
                      CD19-                                  c-kit-
                      B220lo                                 Thy-1+/-
                                                             CD25-
                                                             CD161c+




Modified from Lian and Kumar, 2002
  Differentiation of NK Cells: In Vitro Requirements for
                  Growth and Maturation

- Stroma from normal animals
  estrogen- or strontium-treated mice have functionally impaired NK cells
  stroma from LTa-/- mice have functionally impaired NK cells

- Cytokines for growth and Differentiation
  c-kit ligand; IL7; Flt3 ligand [stem cell factor]; IL15

- Cytokines and direct contact with stroma are required for
   differentiation of phenotypically and functionally mature NK cells
   – LY49- NK cells develop in cultures with cytokines but no stroma
  NK Progenitors: Adult

        Bone marrow
                                                           stroma

         HSC         CLP             NKP            p-NK              NK
                                                                      ..   ..
                                                                                        NK
                                                                                        .. ..
                     Lin-            CD122+         CD122+           CD122+             CD122+
                     c-kitlo         CD161c-        CD161c+          CD161c+            CD161c+
                     Thy-1-          CD49b-         CD49b-           CD49b+             CD49b+
                     IL7R+                                           Ly49+              Ly49+
                     Sca-1lo


                                           Thymus
                                                              p-NK              NK
                                                                                .. ..
                                           p-T/NK

                                                              p-T                T         T




Modified from Lian and Kumar, 2002
               HSC

                                       pB
                      CLP
       MY       X
                                                            Bone Marrow
                             T/NKP


                     pT               NKP
                                                          NK
                                                          .. ..
                                                                                  Periphery
                                                 NK
                                                 .. ..
                                                                    CTX
                                      NK     .
                                      . ..               NK
                                                         .. ..               Enhanced
                                                                             CTX
                                                                   NK
                                  Stimulus
                                                                                    NK
                                                      Cytokines:
                                                       IFNg
                                                       GM-CSF           NK
                                                       TNFa                      NK
Modified from Yokoyama, et al, 2004
NK Progenitors: Adult
                                       Periphery

                                       Cytokines:

                        ..
                     NK                 IFNg
                     ..                 GM-CSF
                                        TNFa
                             NK
  Stimulus: Hrs
       IL2        Enhanced                      NK
                  CTX
       IL12
                                  NK
       IL15                                  NK
       IL23
       IL27
       IFNa, -b
NK Progenitors: Adult
                                             Periphery


                        NK
                        .. ..
                                NK
       Stimulus: Days                                           Cytokines:
                                                                 IFNg
           IL2                                         NK        GM-CSF
                                                                 TNFa
           IL12
                                      NK
           IL15                                       NK    Proliferation
           IL23                 Enhanced
           IL27                 CTX, with
                                broader specificity
           IFNa, -b
        Knockouts/Transgenics: Transcription Factors

Gene Deleted     Effect                          Reference
Ikaros           NK cells absent                 Georgopoulos, 1994
                                                 Wang, 1996
PU.1             NK cell number decreased,       Colucci, 2001
                 normal lytic function
Ets-1            NK cell number decreased,       Barton, 1998
                 decreased lytic function
Id2              NK cells decreased or absent,   Yokota, 1999
                 reduced lytic function          Ikawa, 2001
TCF-1            Altered acquisition of Ly49s    Held, 1999
                                                 Kunz,2001
IRF-1            NK cell number decreased,       Duncan, 1996
                 lytic function impaired         Ogasawara, 1998
IRF-2            NK cell number decreased,       Lohoff, 2000
                 lytic function impaired
           Knockouts/Transgenics: Receptors

Gene Deleted   Effect                         Reference
LTbr           NK cells severely decreased    Wu, 2001
LTa1b2         NK cells severely decreased,   Iizuka, 1999
               reduced lytic function         Smyth, 1999
                                              Ito, 1999
IL15Ra         NK cells severely decreased    Lodolce, 1998
IL2/15Rb       NK cells absent                Gilmour, 2001
                                              Suzuki, 1997
c-kit          NK cells decreased, impaired   Colucci, 2000
               lytic function
               Knockouts/Transgenics: Cytokines


Gene Deleted     Effect                         Reference
IL15             NK cells absent; no lytic      Puzanov, 1996
                 function                       Kennedey, 2000
Flt3-ligand      NK cells severely decreased,   McKenna, 2000
                 impaired lytic function
    NK Cell Differentiation Pathway: Informative Gene
             Knockout and Transgenic Mice

                       HSC >> CLP >> T/NKP >> NKP >> NK

Trnscrptn. Fctr./DBP      Ikaros                  Ets1    IRF-1
                          PU.1 (P)*               Id2    IRF-2
                                                         STAT5a/b (P)
                                                         MEF (P)
Cytokine/Rcptr.           Flt3L                          IL15
                                                          IL15Ra
                                                          IL2/IL15Rb
                                                          LTa/LTbR (P)
Sgnl. Trnsdcr.            Jak3        CD3e tg
                                      FceR1g tg
 III. NK Cell Function as Anti-tumor and
        Anti-Viral Effector Cells

• NK cells were initially described as being
  cells important for surveillance against
  tumor development, or more importantly,
  against tumor metastases
• NK cells were also found to be important as
  anti-viral effector cells, particularly against
  Herpes virus infection.
    NK cells and Anti-tumor Activity

•   What is the evidence of NK cell
    anti-tumor function?
      In vitro – many tumor cells are
      susceptible to lysis by NK cells
      depending upon how you assess killing (Kashii,
      Y., et al. J. Immunol. 163:5358-66 [1999]).
•   In vivo…..
Putative Evidence for Immunosurveillance by NK
   Cells Using Transplantable Tumor Models

•   Elimination of NK cells resulted in increased tumor growth
•   Elimination of NK cells resulted in increased numbers of
    metastastic lesions in lungs
•   Adoptive transfer of NK cells, into immunodeficient
    animals challenged with tumors, results in tumor clearance
    in metastases models
•   Best results almost always were derived in models of
    metastatic disease (Barlozzari, T., et al., J. Immunol.
    134:2783-2789, 1985)
    This evidence did not initially garner robust
    support for NK cell participation in immune
                surveillance – Why?
•   There has been a growing belief that transplantable tumor
    models have little value in assessing tumor immunity, and
    particularly for “immune surveillance” of tumors
•   The only report providing evidence for disease in
    individuals with reduced NK cells is for recurrent Herpes
    virus infections (Biron, C.A., et al., NEJM 322:1731-1735,
    1989)
•   Identification of receptors on NK cells with coordinate
    tumor cell ligand was lacking
    Studies Supporting Increased Incidence of
    Cancer in Immunosuppressed Individuals

• An 11 year follow-up study of immune function and
  cancer incidence in a general population of 3625
  individuals was carried out (Imai, K., et al., The Lancet
  356:1795-1799, 2000)
• Immune function, i.e. NK cell lytic activity, was assessed
  at baseline and cancer incidence
• Medium and high cytolytic function was associated with
  reduced cancer risk; low cytolytic function was associated
  with increased cancer risk
Support for NK Cells Providing a Mechanism for
        Immune Surveillance of Cancer

•   Families of NK cell receptors (e.g. NKG2s) with activating and
    inhibitory function have been defined
•   Tumor associated ligands similar to MHC Class I have been defined,
    e.g. Rae-1 [mice], MICA/B [humans]
•   Binding of MHC Class I and Class I-related proteins (e.g. Rae-1(a-e);
    ULBP-1, -2, -3; H60) by NKR has been demonstrated
•   In mice, binding of NKG2D to Rae1a (Cerwenka, A., et al., PNAS
    USA 98:11521-11526, 2001) or Rae1b (Diefenbach, A., et al., Nature
    413:165-171, 2001) has been demonstrated to activate anti-tumor lytic
    function
•   Human orthologs of Rae-1 genes, e.g. ULBP-1 also are bound by
    NKG2D; and this activates NK lytic function
   NK Cells as Anti-viral Effector Cells:
Evidence for a role as anti-virus effector cells

• Natural defects in NK cells
   Recurrent Herpes virus infections [Biron, 1989]
• Expansion of NK cells during viral infections
   LCMV infections
• Viral antigens as ligands for NK cell receptors
   ULBP1-4
NK-mediated Response to Virus Infection
    NK Cells as Anti-viral Effector Cells:
 Mechanisms of Evasion of NK Cell Function by Viruses

• Expression of virally encoded MHC class I protein
  homologs
• Selective modulation of MHC Class I expression by viral
  proteins
• Virus-mediated inhibition of activating receptor function
• Production of virally encoded cytokine-binding proteins or
  cytokine-receptor agonists
• Direct viral effects on NK cells – infection/envelope
  ligation of inhibitory receptors
        Virus-infected Cell                                NK Cell
           MHC ClassI                                         MHC Class I
           Homolog                                            Inhibitory    1
                                                              Receptor

             Selective                                        MHC Class I
             Expression                                       Inhibitory    2
                                                              Receptor
                                              Activating
                                              Receptor
                                              Antogonist
                                                              Activating
                                                              Receptor      3
Virus      Down Regulating
           Activating Ligand
                               Cytokine Binding
                               Protein

                                                              Cytokine
                                                              Receptor
                                                                            4
                                                              Cytokine
                                                              Receptor
                                 Cytokine
                                 Antogonist
                                                                            5
                                                              NK Cell
                                                              Infection
IV. NK Cell Recognition Receptors

•   “Missing Self” Hypothesis
•   Activation and Inhibition via Receptors
•   Recognition of “Self”
•   Recognition of Tumor Cells
•   Recognition of Virus-infected Cells
                “Missing Self” Hypothesis
•   NK cells do not require expression of MHC Class I determinants for
    recognition of target cells.
•   There is, in fact, an inverse relationship between expression of MHC
    Class I and susceptibility to lysis by NK cells, i.e. less Class I equals
    more lysis.
•   Led to the hypothesis* that NK cells surveyed the surface of target
    cells for “self”. If it was present, the cell was presumed to be normal
    and not lysed. If self was absent, as is often the case in tumor cells and
    virus-infected cells, NK cells could be activated to lyse the “abnormal”
    cell.

    *Ljunggren, H.G. and K. Karre, 1990. Immunology Today 11:237-244.
    Receptors in Innate and Adaptive Immunity

Characteristics                Innate   Adaptive
Specificity inherited in the   Yes      No
genome
Expressed by all cells of a    Yes      No
particular type
Trigger immediate response     Yes      No
Recognize broad classes of     Yes      No
pathogens
Encoded in multiple gene       No       Yes
segments
Require gene rearrangement     No       Yes
Clonal distribution            No       Yes
Able to recognize a wide       No       Yes
variety of molecular
structures
                 Recognition – NK cells
-   There is no evidence supporting clonally restricted recognition
    molecules expressed by NK cells, nor for recombinatorial events being
    important for development of an NK cell repertoire
-   NK cells recognize MHC determinants, but these structures, nor
    peptides expressed by MHC, are target antigens for activation of NK
    lytic function
-   Some NK cells express CD8 homodimers, but it is unclear whether
    binding to MHC Class I affects activation
-   NK cell recognition of targets involves a balance between inhibitory
    signals and activation signals
-   Receptor:ligand pairs providing inhibitory signals are fairly well
    defined
-   Receptor:ligand pairs providing activation signals are rapidly being
    defined
           NK Cell Gene Complex (NKC)
• The NKC is a genomic region, first described on NK cells, encoding
  structurally related receptors
• NKC maps to Chromosome 12p13, 6 and 4 in man, mouse and rat,
  respectively
• Type II integral membrane proteins with external domain similar to C-
  type (Ca++-dependent) lectins. However, they lack amino acid residues
  that coordinate binding of Ca++, and do not bind carbohydrates in the
  same manner as conventional C-type lectins. Can be expressed homo-
  or heterodimers.
• Highly conserved evolutionarily – found in sea squirt and several
  poxviruses
• Activating and inhibitory receptors for immune cells; can be either
  primary or co-stimulatory receptors.
      NK Cell Gene Complex (NKC)




- Contains genes encoding C type lectin related receptors
- Disease resistance elements mapped to this locus, e.g. Cmv1
- Conserved across species
    Human – Chromosome 12
    Mouse – Chromosome 6
    Rat – Chromosome 4
   Leukocyte Receptor Cluster (LRC)




LRC is a ~1 mb region located on chromosome 19q13.42
NK Cell Inhibitory Receptors: CLRR and KIR

  Name             Alternative Name[s]              Cellular Ligand     Viral Ligand
  p58.1                 KIR2DL1                     HLA-Cw2,4,5,6
  p58.2                 KIR2DL2                     HLA-Cw1,3,7,8
  p70                   KIR3DL1                     HLA-Bw4
  p140                  KIR3DL3                     HLA-A3, -A11
  p49                   KIR2DL4                     HLA-G
  LIR1                  ILT2/LILRB1                 HLA-G               HCMV-UL18
  LIR2                  ILT4/LILRB2                 HLA-F
  CD94*                 KLRD1                       HLA-E**
  NKG2A                 KLRC1/CD159A                HLA-E
  NKR-P1B, D            CD161B, D                   Clrb
  p40                   LAIR1                       ?
  IRC1                  IRp60/CMRF35H               ?
  p75AIRM1              Siglec-7                    Sialylated sugars


  *CD94 forms heterodimers with NKG2A, -C and –E
  **CD94/CD159A heterodimer is specific for HLA-E
                         Target Cell membrane




                  NH3

      IgV               IRp60


                                    NK Cell membrane

                                                Cytoplasm

          SHP-1




   COOH



                                Inhibition of lytic function
I/VxYxxL
                     ITIM

• Immunoreceptor tyrosine-based inhibitory motif
• Based upon the amino acid motif: I/VxYxxL
• Commonly expressed in signaling receptors in
  lymphocytes
• Recruits SHP-1/SHP-2 phosphatases
• Linked to inhibition of function in lymphocytes
                  NK Cell Activating Receptors

Name                   Alternative Name[s]       Cellular Ligand       Viral Ligand
NKp46                  Ly94/NCR1                 ?                     SV-HA, IV-HA
NKp30                  IC7/NCR3                  ?
NKp44                  Ly95/NCR2                 ?                     SV-HA, IV-HA
2B4                    CD244                     CD48
NTB-A                  KALI                      ?
NKp80                  KLRF1                     ?
CD16                   FcgRIII                   IgG
CD2                    LFA-2                     CD58, LFA-3
DNAM-1                 CD226                     PVR/CD155, Nectin-2/CD112
NKG2D                  D12S2489E/CD159D          MICA, MICB, MULT1 ULBP1-4
NKR-P1A                CD161A                    [IC-21]*
NKR-P1C                CD161C                    ?
NKR-P1F                CD161F                    Clrg
P40                    LAIR1                     ?
IRC1                   IRp60/CMRF35H             ?
p75AIRM1               Siglec-7                  Sialylated sugars


*Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells
             NK cell activating receptors

•   Loss of the inhibitory signal does not, in and of itself, provide signals
    to kill target cells
•   Some receptors able to activate NK cells to kill target cells have been
    defined – NKG2D, Ly49D, Ly49H, NKp30, NKp44, NKp46,
    CD161A
•   Some activating receptors are members of the C-type lectin [e.g.
    NKG2D] and IgSF [NKp30] superfamilies
•   IgSF members often referred to as KARs
•   Associate with an adaptor molecule [e.g. DAP12] containing an
    ITAM. Associate via a charged residue in the TM domain
•   Some ligands for activating receptors have been defined, e.g. RAE-1
    for NKG2D
        NH3



 IgC2
                  NKp46:SV-HA or IV-HA


IgC2



              FceR1g
                       CD3z


        R
            *D                   NK Cell membrane
                   I
                   T                     Cytoplasm
                   A
              I    M
              T   I
              A          ZAP70
                  T
   COOH       M   A
                         SYK
                  M

                  I
                  T
                  A
                  M




                                         Activation
NKp44:SV-HA or IV-HA
             NH3



       IgV



        DAP12



 K
     *D                    NK Cell membrane

                                   Cytoplasm
        I    I
        T    T
        A    A     ZAP70
        M    M     SYK

COOH




                                   Activation
NKp30:? [iDCs and some tumors]
                NH3



          IgV



            CD3z



    R
        *   D                 NK Cell membrane
            I   I
            T   T                     Cytoplasm
            A   A
            M   M
            I   I
   COOH               ZAP70
            T   T
            A   A
                      SYK
            M   M

            I   I
            T   T
            A   A
            M   M




                                      Activation
                           ITAM

• Immunoreceptor tyrosine-based activating motif
• Based upon the amino acid motif: …YxxL/Ix6-8YxxL/I…
• Serves as a signaling partner to transmembrane receptors with a
  charged residue in the transmembrane region which allows docking of
  signal transducers such as DAP12, CD3z-CD3z homodimers, CD3z-
  Fcer1g heterodimers
• Activation of cells either via PI3 kinase, or ZAP70 or Syk tyrosine
  kinases
                       NKG2D

• Single gene
• Distantly related to other NKG2 family members
• Alternatively spliced isoforms (short and long) in mice
• NKG2D-s and NKG2D-l, short from binds both DAP10
  and DAP12
• Expressed in NK cells, CD8+ cells and macrophages
NKG2D:MICA, MICB, ULBPs

   COOH                     COOH



      CTLD              CTLD




                        DAP10


                 R
                       *D                      NK Cell membrane

                               Y                        Cytoplasm
                               x
                                    PI3K
                               x
                               M

                                    Grb2

             NH3 NH3
                                   ERK1/2


                                   MAPK


                                           Cytokine secretion
                                           Cytotoxicity
      Ligands for NK Cell Activating Receptors
•   MICA, MICB: Stress-inducible molecules encoded within the human MHC,
    also can be induced by some infections. Normally expressed by
    gastrointestinal epithelium, but also by some epithelial, lung, breast, kidney,
    ovary, prostate and colon tumors, and by some melanomas. Transmembrane
    with a1, a2, and a3 domains; but do not associate with b2m and do not bind
    peptides.
•   ULBP1-4: 1-3 are GPI-linked, cell surface molecules which bind human
    cytomegalovirus UL-16; ULBP-4 is a cell surface molecule with
    transmembrane and cytoplasmic domains. ULBPs have a1 and a2 MHC
    Class I-like domains.
•   Rae1b: Retinoic acid inducible protein, in mice, that shares sequence
    homology with ULBPs. Expressed in early embryogenesis and in some
    tumors, but generally absent in normal tissues.
•   H60: Minor histocompatibility antigen expressed by Balb/c mice, target for
    alloreactivity responses by C57Bl/6 mice.
•   DCs: Known that NKp30 is required for recognition of immature DCs by
    activated NK cells.
•   IC-21: Known that rat CD161A is required for recognition of IC-21 tumor
    cells to mediate their lysis.
 Signal Transduction Pathway for NK cells*
                    (NKp44)                     (KIR2DL1)             (NKG2D)

                        (DAP12)




Modified from Vely and Vivier, 2005, www.stke.org/cgi/content/full/sigtrans;2005/292/cm6
 V. Non-adaptive vs. Adaptive Function

- Mediators of non-adaptive immunity
- Interface between non-adaptive and adaptive
   immunity –
  “Passive” interaction – antibody dependent cellular
  cytotoxicity
  “Active” interaction – reciprocal co-activation of NK cells
  and DCs to induce adaptive responses
- New hypothesis regarding NK cells as mediators of
   adaptive immunity is the topic of the journal club
   article
Interactions with Dendritic Cells to Promote
       Adaptive Immune Responses
VI. Therapeutic Applications of NK Cells
 Biological Response Modifiers
    IL2, IL12, IL15, IL21, IFNa, IFNg, IFNb, PolyI:C, b-glucan
 Adoptive Cellular Immunotherapy
    Freshly isolated NK cells – autologous/allogeneic-alloreactive
    BRM/Cytokine activated NK cells – autologous/allogeneic
    Long term established NK cell lines (NK-92)
 NK Cells as Vehicles for Delivery of Therapeutic Agents
    Chemotherapeutic agents - doxorubicin
    Cytokines – IL2

 Trials for:
 melanoma, renal cell carcinoma, lung carcinoma, ovarian cancer,
 Glioblastoma – variable results
            Utilization of Modified NK-92
   Modification of          NK-92               Control of in
  functional activity                          vivo Expansion

 IL2                    Targeting specific           NK-92-CD20
                          tumor types
Prolonged                                            In vivo control
             NK-92-Her2/neu        NK-92-CD19        of proliferation
in vivo
activity                NK-92-CD38                   through suicide
                                                     gene binding
  Epithelial tumors
   breast                  Myeloma           B-cell precursor
   ovarian                                   leukemia
               Improved cytolytic efficacy
               Accessibility to resistant tumors         Modified from Suck, G. 2006

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:32
posted:12/19/2011
language:English
pages:50