Challenges)facing)An./tumour) Immunotherapy)in)Myeloma))) Immune Dysfunction Prof)Gordon)Cook) Leeds)Ins.tute)of)Cancer)&)Pathology) University)of)Leeds)
Impaired)immunity)in)MM) )clinical) % of Infection-related Admissions 40 30 20 10 0 Admission & infection 2011-13 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
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
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), 605-606 Fuchs EJ & Matzinger P, Semin Immunol, 1996, 8(5), 271-280
Immuno/edi.ng)Hypothesis:)The)3)E s) Dunn GP, Ann Rev Immunol 2004 ELIMINATION EQUILIBRIUM ESCAPE
Immune)Surveillance)in)MM?) ELIMINATION EQUILIBRIUM ESCAPE????? MGUS Asy MM MM
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, 5653-62 (2010).
CD4 + )Lymphopenia)in)MM) CD4 RTE (Abs) cells/µl 1500 1000 500 0 Control p=0.026 MM RTE (Abs) cells/µl 300 200 100 0 CONTROL p=0.0002 MM
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), 1966-67.
Th 1 )cells) T cell subsets MM - Th1 only 40 CD4 + T-cells 30 20 10 0 Normal donors MGUS Asymptomatic MM - untreated MM-plateau
Immune)Synapse)Dysfunc.on) U266 PB B-cells
)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 1 2 3 4 5 6 7 M Cook G, Campbell JDM, et al, J Leuk Biol, 1999, 66, 981-988. Campbell JDM, Cook G, et al, J Immunol, 2001, 167, 553-561.
Trogocytosis) Brown et al, Blood, 2012, 120(10), 2055
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, 5653-62 (2010).
CD25 Naturally)occurring)T Reg )cells ) CD4 1.5% 8.3% FoxP3
Natural T Reg cells in Myeloma p<0.003 (Anova) Feyler et al, B J Haem, 2009, 144(5), 686-695
Myeloma cells expand nt Reg cells Control %CD4+ T-cells 60 p=0.001 40 20 Co-culture 0 D0 CD25 pos D7 Control. D7 Coculture Feyler et al, PLoS ONE, 2012 7(5) pp. e35981 FoxP3 CFSE
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, 2012 7(5) pp. e35981 FoxP3 Day 7 Co-culture
Functionality of tt Reg Cells IL-10 60 P=0.03 pg/ml 40 20 Feyler et al, PLoS ONE, 2012 7(5) pp. e35981 0 D0 CM D7 U266B alone D7 Control D7 Coculture
Myeloma-specific effect? CD4 + CD25 - T-cells & Cells Lines BM vs HMCL %CD4 + CD25 + FoxP + 30 20 10 0 control U266B ** * * JMI3 JJN3 KMS11 K562 1-way ANOVA p=0.0015 Mel888 HeLa %CD4+CD25+FoxP3+ 30 20 10 0 control p=0.004 p<0.0001 BM 1-way ANOVA, p<0.0002 HMCL Feyler et al, PLoS ONE, 2012 7(5) pp. e35981
The Generation of tumour-associated regulatory T- cells is contact dependent. D7 Transwell Experiments with U266B %CD4 pos. 40 30 20 10 Control Coculture Transwell 0 Control Coculture Feyler et al, PLoS ONE, 2012 7(5) pp. e35981 Transwell
ICOS-L Blockade tt Reg cells CD25 FoxP3 80 ICOS-L blockade (MoAb) 88.1% ICOS %Inhibition 60 40 20 FoxP3 0 1 100 100 µm Feyler et al, PLoS ONE, 2012 7(5) pp. e35981
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, 5653-62 (2010).
Th 17 )cells)in)pb)/)age) T cell subsets in controls T cell subsets in normals by age - Th17 only 100 4 10 2 % of CD4 positive cells 1 0.100 % of CD4 positive cells 0 40 60 80 Age R 2 =0.04235-2 0.010 0.001 Th17 Th1 Th17-1 -4 15.9% 0.3% Th 17-1 IFNγ IL-17 0.9% Th 17
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=0.0018 % of cells expressing CD161 50 0 Th17 Th1 Th17-1 Treg
Th 17 )cell)chemokine)receptor)expression) CCR6 CXCR3 IFNγ IL-17 CCR6 in T cell subsets in normals p=ns p=ns p<0.0001 100 % of cells expressing CCR6 50 0 Th17 Th1 Th17-1 Treg
Th 17 )cells)in)myeloma) 2.0 Cell numbers - Th17 15.9% 0.3% 0.9% % of CD4 positive cells 1.5 1.0 0.5 IFNγ" IL-17 0.0 Normal donors MGUS Asymptomatic MM - untreated MM-plateau Relapse
Th 17 )phenotype)in)myeloma) 80 p=0.0121 Th17 phenotype - CD161 100 p=0.0116 Th17 phenotype - CCR6 p=0.0107 p=0.0194 60 % Th17 cells 40 % of Th17 cells 50 20 0 Normal donors MGUS Asymptomatic MM - untreated MM-plateau 0 Normal donors MGUS Asymptomatic MM - untreated MM-plateau
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"
BM)Microenvironment)influences) an./mm)t/cell)cytotoxicity)(cam/ir) De Haart et al, Clin Cancer Res, 2013, 19(20), 1
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)
Immunotherapy)in)MM) ) Possibility)or)Probability?) 'Serotherapy' 'Cellular'Therapy' 'Pharmacological' 'Virotherapy'
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.'
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.'
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