Natural Killer Cells: Development, Diversity, and Applications to Human Disease Dr. Michael A. Caligiuri

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1 Natural Killer Cells: Development, Diversity, November 26, 2008 The Ohio State University Comprehensive Cancer Center The James Cancer Hospital and Solove Research Institute Columbus, Ohio, USA 1 Human large granular lymphocytes or natural killer (NK) cells Monocyte NK cell NK cell T cell The role of cytokines in NK cell development Secondary lymphoid tissue and human NK cell development Human NK cell subsets Clinical application of NK cell receptor biology 2 Human natural killer cell development: 1993: what was known?? Bone marrow stroma CD34 + HPC IL-2 CD56 + NK cell But, is IL-2 the real growth factor for NK cells in vivo?? IL-2 -/- NK IL-2Rα -/- NK IL-2Rβ -/- NO NK IL-2Rγ c -/- NO NK 3 1

2 α HA IL-2Rαβγ IL-2 IL-15 γ c γ c IL-2Rα γ c IL-2/15Rβ IL-2/15Rβ IL-2/15Rβ Jak1 STAT3 Jak3 STAT5 Jak1 STAT3 Jak3 STAT5 Jak1 STAT3 Jak3 STAT5 4 IL-15 induces human NK cell differentiation from CD34+ HPCs CD34+ HPC IL-15 CD5 56 Cells (X 1000 ) CD56+ NK CD3 Medium IL-15 IL-15 -/- mice and IL-15Rα -/- mice lack NK cells 5 What are the other factors responsible for NK cell development? A clue: the CD56 bright human NK cell subset expresses the RTK c-kit CD D56-PE Negative control A BRIGHT NEG DIM B c-kit-fitc NEG DIM BRIGHT CD56-PE C BRIGHT NEG D BRIGHT NEG 6 2

3 KL or Flt3 ligand (FL) enhance IL-15-mediated development of CD56 + CD3 - NK cells from CD34 + HPCs in vitro CD56-PE KL/FL IL-15 KL/FL + IL-15 0% 92% 94% Fold Increase in Mononuclear Cells CD3-FITC Mononuclear cells Absolute Num. of CD56 + Cells (X 1000) CD56+ NK cells 0 Medium FL IL-15 FL + IL-15 0 Medium FL IL-15 FL + IL-15 7 How does KL or FL assist IL-15 in NK cell development?? φ FL KL IL L-2/15Rβ 0.01% 29% 7.2% CD34-FITC Both KL and FL induce expression of CD122 (IL-2/15Rb) protein on CD34+ HPCs making the CD34+ HPC more responsive to IL-15 8 How does KL (SCF) assist IL-15 in NK cell development? UNSTIM SCF IL2 SCF+IL2 Phospho-ERK1/2 Actin 9 3

4 Human natural killer cell development NK progenitor NK precursor Mature NK cell Flt3L IL-15 CD34 + CD38+ flt3+ c-kit+ KL CD34 + IL-2/15Rβ + c-kit+ (IL-2) CD56 + NK cells CD16+/- NKR+ c-kit+/- IL-2/15Rβ+ 10 Only CD34 dim CD45RA(+) HPC differentiate into CD56 bright NK cells CD34 I II III CD34 I II III CD45RA CD45RA IL2 or IL15 CD56 CD3, CD14 11 The CD56 bright NK cell precursor, now identified as CD34 dim CD45RA(+), has unique high expression of L-selectin and β 7 L-selectin I II III CD34 III β 7 integrin CD45RA III 12 4

5 The CD34 dim CD45RA(+)β 7 bright NK precursor is selectively enriched in human lymph nodes Peripheral blood ~6% Lymph node >95% I II CD34 III III CD45RA Freud, et al., Immunity, The CD34 dim CD45RA(+)β 7 bright NK precursor is selectively enriched in human lymph nodes and differentiates to a CD56 bright NK cell Lymph node >95% CD34 IL-15 IL-2 CD56 bright CD45RA CD3/14 14 CD56+ cells in the parafollicular T-cell rich regions of human lymph nodes Hypothesis: secondary lymphoid tissue might be the site of human NK development Fehniger, et al., Blood,

6 NK cells are in human lymph nodes and are CD56 bright FACS from fresh human lymph node 16 Putative stages of human NK cell differentiation in nodes/tonsils Human NK cell development occurs in secondary lymphoid tissues Freud, et al., J. Exp. Med., How NK cell development fits in human lymphopoiesis Tpro Thymus T cell NK/ Tpro Secondary lymphoid tissue IL-3 PHSC Ikaros CD34+ CD38- LSC CD34+ CD38+ CD10+ IL-7Rα flt3+ c-kit+ Id2? NKpro CD34+ IL-15Rβ- CD38+ flt3 c-kit CD7 Flt3L (KL) NKpre IL-15 +? CD34 dim CD45RA(+)β 7 bright NK cell CD56 bright CD2+ CD16+/- c-kit+/- Bpro Bone marrow B cell 18 6

7 IL-15 and human NK cell survival: low concentrations of IL-15 maintain human NK cell survival in serum free medium in vitro % viable NK cells ng/ml IL-15 (10pM) 0.01 ng/ml IL-15 (1pM) SFM Time (days) 19 Adoptive transfer of NK cells into IL-15-deficient mice WT + IL-15 -/- WT C57BL/6 mouse Purified NKs IL-15 -/- or control IL-15 +/- mouse Harvest cells 20 IL-15 is required for NK cell survival in vivo: adoptive transfer of NK cells into IL-15 KO mice Normal IL-15 -/- 0.12% 0.03% Day 1.5 Ly5.1-PE 0.11% 0% Day 5 CFSE 21 7

8 IL-15 and NK cell development and survival IL-15 is critical for NK cell development, and human NK development appears to occur in secondary lymphoid tissue IL-15 is required for NK cell survival in vivo, and so are dendritic cells Collectively, these data point towards IL-15 as a key mediator of NK cell homeostasis in vivo 22 IL-15 transgene Class I promoter (H-2D d ) 3 hgh gene IL-2SP/IL-15/FLAG 1. Multiple AUG s removed 3. C-terminus stabilization 2. Replacement of signal peptide 23 NK cell expansion in IL-15tg mice DX5 (NK) WT 4% 76% 51% IL15tg 29% CD3-FITC 3% WT 1% 39% IL15tg 12% Ly49D-FITC Abs. # NK Cel ls/ul blood Absolute NK # p < * IL-15tg Controls n = 71 n =

9 IL-15tg mice: 3-7 months: lymphoblastic leukemia Clinical signs and findings (20-30% of mice): Progressive alopecia Decreased activity, weight loss, labored breathing Massive hepatosplenomegaly Dramatic WBC elevation blood with massive lymphocytosis (see below) (mean 186,000/uL blood, range 47, ,000/uL blood, n = 22) Premature death (19 weeks) likely secondary to respiratory insufficiency Bone marrow 13% 64% Spleen 10% 46% Blood (476,000 WBC/uL) 10% 63% DX5-PE DX5-PE DX5-PE 10% 30% 21% CD3-FITC CD3-FITC CD3-FITC 25 IL-15tg mice: acute T-NK lymphoblastic leukemia CD3+ T Cells Cell number 53% TCR Vβ6 26 FLT3 AML/ALL ITD FL IL-15 NK progenitor CD34 + CD38+ flt3+ c-kit+ KL T-NK precursor CD34 dim IL-2/15Rβ + c-kit+ Mature NK cell CD56 + NK Cells CD16+/- NKR+ c-kit+/- IL-2/15Rβ+ 27 9

10 Summary of human NK cell subsets Peripheral blood lymphocytes CD56-P PE CD16-FITC CD56 bright CD56 dim Receptor CD56 bright CD56 dim CD16 Dim/neg bright IL-2Rα pos neg KIR < 10% > 85% L-selectin pos neg c-kit pos neg Effector functions ADCC Natural cytotoxicity Cytokine production Do NK cell subsets have unique immunoregulatory roles? 29?? 30 10

11 Macrophage and NK co-culture: LPS activation 1. Pathogen 2. Monokines Mφφ NK 3. IFN-γ NK cells provide critical IFN-γ for early monocyte/ macrophage control of infection 31 NK cell-derived IFN-γ in the innate immune response Early NK-derived IFN-γ is requisite for the elimination of intracellular pathogens and activation of monocytes Genetic deficiencies in NK cells lead to fatal viral infections Genetic deficiencies in IFN-γ lead to fatal infections by obligate intracellular pathogens 32 Macrophage and NK co-culture: LPS activation (2) 1. Pathogen 2. Monokines Mφ CD56 bright NK??? 3. IFN-γ CD56 dim NK 33 11

12 CD56 bright NK cells are the primary source of NK-derived IFN-γ Mono CD56 dim CD56 bright Mono + LPS * IFN-γ (pg/ml) 34 IL-12 + IL-15 IL-12 + IL-1β IL-12 + IL-18 Bright % % MFI = % 44.7% MFI = % MFI = Dim CD56 6-PE % MFI = IFN-γ-FITC 35 Contributions of the CD56 bright NK cells in the human immune response Lymph node 36 12

13 Emerging model for human NK cell subsets: innate immunoregulator, CD56 bright 37 Emerging model for human NK cell subsets: innate cytotoxic effector, CD56 dim Low cytokine production CD56 dim CD16 bright IL-2/15Rβ γ c KIR CD56 dim NK Cell PEN5/PSGL1 (~70%) NKRs +++ KIR + CD94/NKG2A Effector functions +++ADCC +++LAK +++natural cytotoxicity 38 How do inhibitory NKR work? Inhibitory signal in the absence of activating ligand blocks autologous NK killing activation of normal cells NK neg KIR 2DL2 Inhibitory R HLA-Cw3 SELF HPC Activating R 39 13

14 How does KIR work? KIR sees and engages MHC class I Negative signal via KIR blocks NK killing activation Leukemia cells that lack MHC class I are rare, as they are most likely killed by NK cells Certain infectious agents (e.g., CMV) can downregulate MHC class I to avoid T-cell immune surveillance; These agents would presumably be destroyed by NK cells 40 How do inhibitory NKR work? (2) Lack of negative signal (absent or defective MHC class I): NK cell kills tumor cell target if tumor expresses ligand to activating receptor KIR NK + kill Leukemia Target Lysis Activating Receptor Activating Ligand 41 Human NK cells have inhibitory receptors that bind to specific EPITOPES that are shared among some different MHC class I molecules, but not others HLA-Cw2, 4, 5, and 6 (Group 2 HLA-C epitopes) HLA-Cw1, 3, 7, and 8 (Group 1 HLA-C epitopes) KIR 2DL1 KIR 2DL

15 Human NK cells have inhibitory receptors that bind to specific EPITOPES that are shared among some different MHC class I molecules, but not others (2) Cw1 Cw3 Cw7 Cw8 KIR 2DL2 KIR 2DL2 KIR 2DL2 KIR 2DL2 43 Haplo-mismatch BMT Donor A24, B35, Cw2 A3, B62, Cw3 Recipient (leukemia) A24, B35, Cw2 A24, B35, Cw2 Donor has KIR on some NK cells that bind to Cw3 That KIR is called KIR 2DL2 KIR 2DL2 recognizes an epitope shared by group 1 HLA-C, that includes HLA-Cw1, 3, 7, and 8 If the recipient AML blast lacks group 1 HLA-C, and expresses a ligand to an activating receptor, lysis occurs 44 Haplo-mismatch BMT (2) KIR 2DL2 expressed on some donor NK cells recognizes an epitope shared by HLA-Cw1, 3, 7, 8 The AML blast from recipient expresses HLA-Cw2: no engagement of KIR 45 15

16 Haplo-mismatch BMT (3) Donor A24, B35, Cw2 A3, B62, Cw3 Recipient (leukemia) A24, B35, Cw2 A1, B14, Cw8 Donor has KIR on some NK cells that binds to HLA-Cw3 That KIR is called KIR 2DL2 KIR 2DL2 recognizes an epitope shared by group 1 HLA-C that includes HLA-Cw1, 3, 7, and 8 If the recipient AML blast expresses group 1 HLA-C, NO lysis 46 Haplo-mismatch BMT (4) KIR 2DL2 expressed on donor NK cells recognizes an epitope shared by HLA-Cw1, 3, 7, 8 The AML blast from recipient expresses HLA-Cw8: engagement of KIR Inhibitory KIR2DL2 (group 1 specific) HLA-Cw8 (group 1) Donor A24 B35 Cw2 A3 B62 Cw3 - no kill NK + Leukemia Target NO Lysis Activating Receptor Activating Ligand Recipient A24 B35 Cw2 A24 B35 Cw8 47 Haplo-mismatch BMT Haplo-mismatch BMT HLA-Cw2 (group 2) Inhibitory- KIR2DL2 (group 1 specific) Inhibitory- KIR2DL2 (group 1 specific) HLA-Cw8 (group 1) NK + Leukemia kill Target Activating Activating Receptor Ligand - NK no Leukemia kill Target + Activating Activating Ligand Receptor Donor A24 B35 Cw2 A3 B62 Cw3 Recipient A24 B35 Cw2 A24 B35 Cw2 Donor A24 B35 Cw2 A3 B62 Cw3 Recipient A24 B35 Cw2 A24 B35 Cw

17 60 AML patients transplanted in CR 1,0 Leukemia relapse 1.0 Event-free survival 0,8 No KIR mismatch (n = 31).8 KIR mismatch 0,6.6 0,4 p = p = ,2 KIR mismatch (n = 29).2 No KIR mismatch 0, years years Ruggeri et al., Science 2002, updated Role of NK cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation Conclusions In mismatched transplants, a clinical graft-versusleukemia effect, independent of T-cell-mediated graftversus-host disease, appears to positively impact on relapse rate when NK cell KIR epitope incompatibility is in the graft-versus-host direction Additional study of donor-versus-recipient NK cell alloreactivity in mismatched hematopoietic stem cell transplants has confirmed these results 50 Summary from today Monocyte NK cell NK cell T cell The role of cytokines in NK cell development Secondary lymphoid tissue and dhuman NK cell development Human NK cell subsets Clinical application of NK cell receptor biology 51 17

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