Challenges)facing)An./tumour) Immunotherapy)in)Myeloma)))

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1 Challenges)facing)An./tumour) Immunotherapy)in)Myeloma))) Immune Dysfunction Prof)Gordon)Cook) Leeds)Ins.tute)of)Cancer)&)Pathology) University)of)Leeds)

2 Impaired)immunity)in)MM) )clinical) % of Infection-related Admissions Admission & infection No therapy Bortezomib-Based Thalidomide-Based Lenalidomide-Based Neutropenic No therapy Bortezomib-Based Thalidomide-Based Lenalidomide-Based Normal ANC 354 FCE in n=320 with median age 65.5 (range 40-91) 25.7% of FCE are infection-related

3 The immuno-surveillance hypothesis Ehrlich 1909: suggested immune system surveys body, eliminating newly transformed cells Thomas & Burnet 1959: mice rejected transplanted syngeneic tumours proposed immunosurveillance hypothesis Cannot directly observe immune reaction to subclinical cancers

4 The)danger)hypothesis)and)tumours) Signal 1 only (TAA) DC T T cell tolerance Tumour Danger Signal 1 and 2 + Activated DC Heat shock proteins, RNA & DNA, IFN-α, cytokines T T T Activated T cells Tumour death Matzinger P, Nature, 1994, 369(6482), Fuchs EJ & Matzinger P, Semin Immunol, 1996, 8(5),

5 Immuno/edi.ng)Hypothesis:)The)3)E s) Dunn GP, Ann Rev Immunol 2004 ELIMINATION EQUILIBRIUM ESCAPE

6 Immune)Surveillance)in)MM?) ELIMINATION EQUILIBRIUM ESCAPE????? MGUS Asy MM MM

7 Th 1 )cells)are)a)well)characterized)helper)subset) Th1 IFN-γ IL-12 IL-4 Th2 IL-4 IL-5 IL-13 Naive T cell TGF-β TGF-β + IL-6 Treg IL-10 TGF-β Th17 IL-17A IL-17F IL-21 IL-22 Maniati et al., Oncogene 29, (2010).

8 CD4 + )Lymphopenia)in)MM) CD4 RTE (Abs) cells/µl Control p=0.026 MM RTE (Abs) cells/µl CONTROL p= MM

9 Immune)modula.on)in)Myeloma) T/cell) T Effec' FoxP3 +' HLA/G) T=cell' DendriDc'cell' Plasma)cell) MM'Plasma'Cell' Soluble'factors' e.g.'tgfβ,'il=10' FoxP3 +' SDmulatory' Soluble'Factors' Physical'contact' Inhibitory' Cook, Blood, 120 (10),

10 Th 1 )cells) T cell subsets MM - Th1 only 40 CD4 + T-cells Normal donors MGUS Asymptomatic MM - untreated MM-plateau

11 Immune)Synapse)Dysfunc.on) U266 PB B-cells

12 )MM/induced)T/cell)Suppression) MM=derived'TGF=β'strongly' suppresses't'cell'acdvadon'and' proliferadon' Treatment'of'T'cells'with'LAP' (TGF=β'latency'associated'PepDde)' restores't'cell'funcdon' TGF=β'from'MM'cells'inhibits'IL=2' signalling'pathways'but'not'il=15' signalling' IL=15'pre=treatment'restores'IL=2' responses'in'padent't'cells'(pb)' TGF-β M Cook G, Campbell JDM, et al, J Leuk Biol, 1999, 66, Campbell JDM, Cook G, et al, J Immunol, 2001, 167,

13 Trogocytosis) Brown et al, Blood, 2012, 120(10), 2055

14 T Reg )cells)and)the)immune)response) Th1 IFN-γ IL-12 IL-4 Th2 IL-4 IL-5 IL-13 FoxP3 + Il-10 + TGF-β Naive T cell TGF-β + IL-6 Treg FoxP3 + IL-10 TGF-β FoxP3 + TGFβ + Th17 IL-17A IL-17F IL-21 IL-22 Adapted from Maniati et al., Oncogene 29, (2010).

15 CD25 Naturally)occurring)T Reg )cells ) CD4 1.5% 8.3% FoxP3

16 Natural T Reg cells in Myeloma p<0.003 (Anova) Feyler et al, B J Haem, 2009, 144(5),

17 Myeloma cells expand nt Reg cells Control %CD4+ T-cells 60 p= Co-culture 0 D0 CD25 pos D7 Control. D7 Coculture Feyler et al, PLoS ONE, (5) pp. e35981 FoxP3 CFSE

18 Myeloma cells generate de novo T Reg cells from CD25 - CD4 + T-cells Day 7 Control Hi Lo Hi %FoxP3+CD4 T-cells CD25 Lo Feyler et al, PLoS ONE, (5) pp. e35981 FoxP3 Day 7 Co-culture

19 Functionality of tt Reg Cells IL P=0.03 pg/ml Feyler et al, PLoS ONE, (5) pp. e D0 CM D7 U266B alone D7 Control D7 Coculture

20 Myeloma-specific effect? CD4 + CD25 - T-cells & Cells Lines BM vs HMCL %CD4 + CD25 + FoxP control U266B ** * * JMI3 JJN3 KMS11 K562 1-way ANOVA p= Mel888 HeLa %CD4+CD25+FoxP control p=0.004 p< BM 1-way ANOVA, p< HMCL Feyler et al, PLoS ONE, (5) pp. e35981

21 The Generation of tumour-associated regulatory T- cells is contact dependent. D7 Transwell Experiments with U266B %CD4 pos Control Coculture Transwell 0 Control Coculture Feyler et al, PLoS ONE, (5) pp. e35981 Transwell

22 ICOS-L Blockade tt Reg cells CD25 FoxP3 80 ICOS-L blockade (MoAb) 88.1% ICOS %Inhibition FoxP µm Feyler et al, PLoS ONE, (5) pp. e35981

23 T H 17)cells)are)a)recently)described)helper)subset) Th1 IFN-γ IL-12 IL-4 Th2 IL-4 IL-5 IL-13 Naive T cell TGF-β TGF-β + IL-6 Treg IL-10 TGF-β Th17 IL-17A IL-17F IL-21 IL-22 Maniati et al., Oncogene 29, (2010).

24 Th 17 )cells)in)pb)/)age) T cell subsets in controls T cell subsets in normals by age - Th17 only % of CD4 positive cells % of CD4 positive cells Age R 2 = Th17 Th1 Th % 0.3% Th 17-1 IFNγ IL % Th 17

25 CD161)&)RORγt)expression)in)CD4 + )T/cell)subsets)in)health) ) CD161 RORγt IFNγ IL-17 CD161 in T cell subsets in normals p=ns 100 p=ns p= % of cells expressing CD Th17 Th1 Th17-1 Treg

26 Th 17 )cell)chemokine)receptor)expression) CCR6 CXCR3 IFNγ IL-17 CCR6 in T cell subsets in normals p=ns p=ns p< % of cells expressing CCR Th17 Th1 Th17-1 Treg

27 Th 17 )cells)in)myeloma) 2.0 Cell numbers - Th % 0.3% 0.9% % of CD4 positive cells IFNγ" IL Normal donors MGUS Asymptomatic MM - untreated MM-plateau Relapse

28 Th 17 )phenotype)in)myeloma) 80 p= Th17 phenotype - CD p= Th17 phenotype - CCR6 p= p= % Th17 cells 40 % of Th17 cells Normal donors MGUS Asymptomatic MM - untreated MM-plateau 0 Normal donors MGUS Asymptomatic MM - untreated MM-plateau

29 Cancer/induced)immunosuppresion) Tumour Cell Suppression" DC" Effector T cell" Survival" NKG2D TGF-β, VEGF, IL-6, HHV-8" Stromal cell" CD25 Induction" T Reg cell" γδ T-cell" NK cell" CD8 cell"

30 BM)Microenvironment)influences) an./mm)t/cell)cytotoxicity)(cam/ir) De Haart et al, Clin Cancer Res, 2013, 19(20), 1

31 Results)of)the)in#vitro)studies)&)in# What)does)it)mean?) vivo)observa.ons) Understanding)of)immuno/biology)of)the) disease) Influence)the)design)of)immunotherapy) strategies)

32 Immunotherapy)in)MM) ) Possibility)or)Probability?) 'Serotherapy' 'Cellular'Therapy' 'Pharmacological' 'Virotherapy'

33 Summary/1) Myeloma'plasma'cells'can'mediate'both' adapdve'and'innate'immune'dysfuncdon.' MM'cells'can'mediate'the'immuno=modulatory' effect'both'directly'and'via'microenvironment' cells.' IdenDfied'immune'deficiencies'may'in'part' relate'to'concurrent'therapy,'especially'steroids.' Novel'therapies'have'the'potenDal'not'only'to' be'tumoricidal'but'to'also'manipulate'the'host' immune'response.'

34 Summary/2) New'agents'in'MM'treatment'have'potenDal'to' reverse'immune'funcdonal'skewing.' PotenDal'for'targeDng'the'cyto=protecDve'(CAM= DR)'and'cyto=immunomodulatory'(CAM=IR)'effect' using'novel'approaches.' An'understanding'of'the'immuno=genomics'of'the' host'may'permit'more'adapdve'treatment' strategies' 'pharmaco(immunotherapy:0the0 drugable0immunome.'

35 Acknowledgements Transplant Immunology Group Clive Carter Chris Parrish Collaborators: Graham Cook University of Leeds Marie von Lillienfeld-Toal University of Bonn Sylvia Feyler Gina Scott The Friends of Leukaemia and Lymphoma Unit All patients and volunteers