T and NK Cell Mediated Immunity in Chronic Myeloid Leukaemia

dissertationes scholae doctoralis ad sanitatem investigandam universitatis helsinkiensis 30/2016 METTE ILANDER T and NK Cell Mediated Immunity in Chronic Myeloid Leukaemia HEMATOLOGY RESEARCH UNIT DEPARTMENT OF CLINICAL CHEMISTRY AND HEMATOLOGY FACULTY OF MEDICINE AND HELSINKI UNIVERSITY HOSPITAL COMPREHENSIVE CANCER CENTER DEPARTMENT OF HEMATOLOGY DOCTORAL PROGRAMME IN CLINICAL RESEARCH UNIVERSITY OF HELSINKI

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CD45 CD3 CD16 CD56 CD56 unstimulated CD107a/b K562 stimulated

CD3 CD45 CD56 CD16 CD56 unstimulated K562 stimulated Granzyme B TNF-α/IFN-γ In study I,

International scale (%) 10 1 0.1 0.01 0.001 0.0001 MMR (IRIS) IFN Start 0.00001-42 -37-32 -27-22 -17-12 -7-2 3 8 13 18 23 28 33 38 43 IFN Stop IFN Immune cell analyses -42-37 -32-27 -22-17 -12-7 -2 3 8 13 18 23 28 33 38 43 IM -42-37 -32-27 -22-17 -12-7 -2 3 8 13 18 23 28 33 38 43

A NK cell proportion p= 0.0002 50 % of lymphocytes 40 30 20 10 Study III Study I Study II B % of CD56 DIM NK cells 0 Non-relapsingEarly RelapseLate Relapse IFN-ON IFN-OFF Healthy T315I Study III Study I Study II CD57 CD56 DIM NK cells p= 0.0226 100 90 80 70 60 50 40 30 20 10 0 Non-relapsingEarly RelapseLate Relapse IFN-ON IFN-OFF Healthy T315I εγ

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Adaptive markers Baseline % of lymphocytes A 10 5 EAT2- NK cells p= 0.0329 p= 0.0152 p= 0.4762 p= 0.1667 B % of lymphocytes 25 20 15 10 5 1 month EAT2- p= 0.0152 p= 0.0029 p= 0.1419 p= 0.2788 0 Non-relapsing Early Relapse Late Relapse 0 Non-relapsing Early Relapse Late Relapse % of lymphocytes C 20 15 10 5 PLZF- NK cells p= 0.1610 p= 0.4740 p= 0.0238 p= 0.7143 D % of lymphocytes 20 10 PLZF- p= 0.0232 p= 0.0270 p= 0.0115 p= 0.3761 0 Non-relapsing Early Relapse Late Relapse 0 Non-relapsing Early Relapse Late Relapse % of lymphocytes E 20 15 10 5 FCER1G- NK cells p= 0.2815 p= 0.3874 p= 0.0952 p= 0.9048 F % of lymphocytes 30 20 10 FCERG1- p= 0.1012 p= 0.0547 p= 0.0887 p= 0.5281 % of lymphocytes G 0 20 15 10 5 Non-relapsing Early Relapse Late Relapse SYK- NK cells p= 0.4941 p= 0.4740 p= 0.4762 p= 0.3333 H % of lymphocytes 0 20 10 Non-relapsing Early Relapse Late Relapse SYK- p= 0.1923 p= 0.0547 p= 0.3473 p= 0.7852 0 Non-relapsing Early Relapse Late Relapse Non-relapsing Early Relapse Late Relapse εγ 0

A Unstimulated K562 stimulation 8.6% 12.6% CD56DIMCD16neg CD56DIMCD16neg CD56 B % of CD56DIMCD16- NK cells CD16 50 40 30 20 10 0 CD56DIMCD16- TNFα/IFNγ secretion p < 0.0001 Non-relapsing Early Relapse Late Relapse IFN-ON IFN-OFF Healthy

A C % CCR7 + CD45RA - of CD4 + T-cells 80 60 40 20 0 p= 0.0093 T EM CD4 + T-cells healthy IFN-OFF IFN-ON % CCR7 - CD45RA + of CD4 + T-cells B D 20 15 10 5 0 p=0.0721 T EMRA CD4 + T-cells healthy IFN-OFF IFN-ON % CCR7 + CD45RA + of CD4 + T-cells 80 60 40 20 0 p=0.0489 Naive CD4 + T-cells healthy IFN-OFF IFN-ON % CCR7 + CD45RA - of CD4 + T-cells 80 60 40 20 0 p=0.1893 T CM CD4 + T-cells healthy IFN-OFF IFN-ON

Cytokine secretion of CD4+ T-cells 50 40 30 20 10 non-relapsing relapsing 0 0 10 20 30 40 50 NK-cell %

A 1500000 Granzyme B + CD4 + T cells p= 0.0202 B 50 40 Granzyme B + T-cells p=0.0145 /ml 1000000 500000 % TNF-α / IFN-γ+ 30 20 10 0 DA 6m IM 6m NI 6m 0 Healthy DA IM NI

120 100 80 B16.OVA alive 60 40 20 Näive OT-1 dasatinib pretreated Näive dasatinib pretreated OT-I B16.OVA only 5:1 10:1 E:T

Dasatinib concentration A 100 nm 0 M 10 µm 1 µm 100 nm 10 nm 1 nm 0 M Dasatinib cytotoxicity K562 B16.OVA B 0 50 100 150 Cell viability (%) Tumor sizes C 11 7 ** Days of treatment 8 4 1 Days of treatment 4 1 0 50 100 150 200 Tumor size (mm 2) 0 50 100 150 Tumor size (mm 2 ) Control Dasatinib Control + CD8, CD4, NK1.1 Dasatinib + CD8, CD4, NK1.1 Dasatinib + CD8, CD4 Control + IgG Dasatinib + IgG

PD-1 expressing CD8+ T cells Blood Dasatinib Control Dasatinib Tumor Control p= 0.06 0 20 40 60 80 100 % of CD8+ T cells

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