Understanding the T cell response to tumors using transnuclear mouse models Stephanie Dougan Dana-Farber Cancer Institute Boston, MA
Presenter Disclosure Information Stephanie Dougan The following relationships exist related to this presentation: No Relationships to Disclose 2
Melanoma: Responds well to immunotherapy (CTLA-4 and/or PD-1 blockade) Pancreatic ductal adenocarcinoma: Responds poorly to most therapies, notable for being one of few cancer types to fail immunotherapy 7% survival at 5 years
Transnuclear mice Advantages of transnuclear mice: 1) T cells are isolated directly from the tumor no time spent in tissue culture 2) TCR genes are in their endogenous loci, under physiological control no other genetic manipulations 3) Multiple, independent lines can be made against the same antigen 4) High-throughput 5) Rapid
TRP1 TN mice: models of CD8 T cell response to melanoma TRP1 low TRP1 high Two lines of TN mice derived from CD8 T cells specific for tyrosinase-related protein 1 (TAPDNLGYA) presented by H-2D b Dougan et al., Cancer Immunology Research 2013
Two lines of TN mice recognize the same TRP1 epitope, but differ in affinity TRP1 native peptide (conc.) TRP1 high TRP1 low t 1/2 = 41 min t 1/2 = 4.9 min Dougan et al., Cancer Immunology Research 2013
Both high and low affinity TRP1 cells can be cytotoxic in 3D tissue culture Cytotoxicity assay using matrigel co-culture of B16 cells with activated CD8 T cells Dougan et al., Cancer Immunology Research 2013
Upon activation, both high and low affinity CD8 T cells delay tumor growth in vivo Naive Peptide-activated Anti-CD3/CD28 activated Homeostatic expansion (RAG1-/-) Dougan et al., Cancer Immunology Research 2013
TRP1 TN cells transferred into RAG1-/- hosts and reisolated from tumors show markers of exhaustion Increased LAG3, PD-1, Tim3 and CTLA-4 expression Dougan et al., Cancer Immunology Research 2013
Candidate gene silencing leads to increased TRP1 accumulation in tumors DGKα LacZ DGKζ TRP1 TN cells (CD45.2) TRP1 high TRP1 low CD8 Collaboration with Kai Wucherpfennig
Silencing of Ppp2r2d enhances anti-tumor effect of TRP1 TN cells p=0.007 LacZ control Ppp2r2d Wet Weight (g) 1.0 0.8 0.6 0.4 0.2 0.0 LacZ Ppp2r2d Collaboration with Kai Wucherpfennig
How does TCR affinity affect activation and anti-tumor activity? T cell TCR Altered Peptide Ligands Native peptide: TAPDNLGYA Reference (M9): TAPDNLGYM K8: TAPDNLGKM Anchor Residues APC Peptide MHC I Mean fluorescence H-2Db 500 450 400 350 300 250 200 150 100 50 0 0 5000 10000 Peptide Dilution Factor M9 Nat K8 Negative
TRP1 high T cells activated by M9 or K8 delay growth of B16 4500 4000 Tumor Volume (mm3) 3500 3000 2500 2000 1500 1000 500 0 Adoptive Transfer 0 5 10 15 20 25 Days since tumor inoculation No treatment K8 high M9 high
K8 activation results in more long-term survivors Total number mice Long term survivors Survived rechallenge TRP1low K8 TRP1high K8 TRP1low M9 TRP1high M9 15 3 1/3 15 2 2/2 15 1 0/1 15 0 0 TRP1 tetramer Memory T cells in spleen 3 months after transfer CD8
K8 activated TRP1high and TRP1low cells remain PD-1 low TRP1high A1 native A3 K8 PD-1 TRP1low Cell trace violet A1 native A3 K8 PD-1 Cell trace violet
Summary for melanoma Both high and low affinity CD8 T cells are capable of mounting antitumor responses. TRP1 transnuclear mice are good models to study anti-tumor immunity. K8 activation leads to good control of tumor growth and induction of long-term memory. K8 activated CD8 cells are PD-1 low and have a unique transcriptional profile. Can we exploit the genes differentially regulated in K8 activated cells to enhance CD8 T cell memory?
Pancreatic ductal adenocarcinoma Poor prognosis Highly metastatic Hypovascular Dense stroma Poor drug delivery Very few T cell infiltrates (mostly Tregs)
Transnuclear mice from pancreatic tumor-infiltrating CD4 T cells Panc CD4a: TCR Vβ14+ Germline-transmitting chimera Normal frequency of Foxp3+ Tregs
PancCD4A cells specifically infiltrate pancreatic tumors accumulation % PancCD4A / total CD4+ Panc02 B16 draining LN Panc02 B16 TILs B16 melanoma Panc02 pancreatic tumor
Orthotopic injections of cancer cells directly into pancreas 609 SNPs ~30 SNPs
CD8 T cells are important in control of orthotopic Panc02 0.8 0.64 Tumor weight (g) 0.6 0.4 0.2 Rag2-/- B6 Tumor weight (g) 0.48 0.32 0.16 isotype CD4 depletion CD8 depletion 0 0
PancCD4a cells affect tumor growth only when given early PancCD4a cells given 5 days after tumor PancCD4a cells given 1 day after tumor 0.3 0.25 0.2 0.15 0.1 0.05 entire pancreas pancreatic tumor 0.2 0.15 0.1 0.05 0.05 0.04 0.03 0.02 0.01 0 untreated naive T cells Activated T cells 0 untreated naïve T cells 0 untreated activated T cells
Transferred PancCD4a cells proliferate in pancreas draining LN spleen pancreas dln tumor naïve PancCD4a Celltrace violet activated PancCD4a no T cells CD45.1
VHH Antibodies Readily expressed in bacteria Small size (15 kda) allows deep tissue penetration Rapidly cleared after injection No FcR or complement engagement or cross-linking Expressed with LPETG tag for modification by sortase In collaboration with Hidde Ploegh, Jessica Ingram and Michael Dougan
Alpaca antibody B3 is specific for PD-L1 Hidde Ploegh, Jessica Ingram and Michael Dougan
Pancreatic tumor cells express PD-L1 Panc02 KPC Cell lines Resected orthotopic tumors B3 (alpaca anti-pdl1)
Alpaca-derived anti-pd-l1 conjugated to IL-2 sortase Fluorescent Tags Biotin Protein linking Pancreatic cancer neoantigens adjuvant 18 F cytokines In collaboration with Hidde Ploegh, Jessica Ingram and Michael Dougan
Very low dose B3-IL2 decreases pancreatic tumor burden 0.3 0.25 isotype Weight (g) 0.2 0.15 0.1 ** * B3 B3IL-2 B3IL-2 +PancCD4A 0.05 0
Very low dose B3-IL2 allows infiltration of PancCD4a T cells CD25 CD4 gated on CD45.1 transferred cells
Immunotherapy for pancreatic cancer Anti-PDL1 alpaca antibodies can target immunotherapeutic agents to pancreatic tumors. Targeted IL-2 increases CD8 T cells, increases T cell trafficking to tumors, and decreases overall tumor size. Transnuclear PancCD4a cells can be used to monitor CD4 T cell trafficking and lineage choice. Intratumoral anti-cd40 generates systemic immune responses against nontreated tumors and may synergize with targeted radiation therapy.
Acknowledgements Paul Tyler Mariah Servos Patrick Bruck Eleanor Clancy-Thompson Cherry Ng Albert Li Hidde Ploegh (Whitehead) Rudolf Jaenisch (Whitehead) Kai Wucherpfennig (DFCI) Wilfred Ngwa (DFCI/BWH) Rajiv Kumar (DFCI) Michael Dougan (MGH) Jessica Ingram (Whitehead) Anirban Maitra (MDACC) Funding Claudia Adams Barr Award for Cancer Research Pancreatic Cancer Action Network Pathway to Leadership Award Melanoma Research Alliance Young Investigator Award HMS Shore Fellowship