Early growth response gene 2 (Egr2) is essential for T cell anergy and its targets define dysfunctional T cells in the tumor microenvironment Yan Zheng Dr. Thomas Gajewski Laboratory University of Chicago Presenter Disclosure Information Yan Zheng • The following relationships exist related to this presentation: – No relationship to disclose T cell anergy in the tumor context • Anergy defined as a hyporesponsive state induced by TCR engagement in the absence of costimulation • Indirect evidence suggests that T cell dysfunction in the tumor microenvironment is partially due to anergy – Minimal expression of B7-1/B7-2 costimulatory molecules in the tumor microenvironment – T cell dysfunction can be antigen-specific (Harlin et al., 2006) – Provision of B7-1, and homeostatic proliferation, prevent anergy and promote tumor rejection (Chen et al., 1992; Townsend et al., 1993, Kline et al., 2008) • Difficult to prove directly due to the lack of positive markers for this loss of function state • We previously showed that anergy is associated with defective Ras pathway activation, and is contributed to by diacylglycerol kinases (Fields et al., 1996; Zha et al., 2006) Model for DGK- in T cell anergy Anergy induction Rechallenge TCR TCR CD28 PLC- PLC- PIP2 PIP2 DAG IP3 DGK- DAG IP3 RasGRP RasGRP Calcium Calcium Ras Ras Calcineurin Calcineurin Erk Erk AP-1 NFAT AP-1 NFAT Egr2 DGK- IL-2 Zha et al Nature Immunol. 2006; Zheng et al EMBO Reports 2008 Adeno-Cre mediated Egr2 deletion in CARTg x Egr2fl/fl Th1 cell clones CARTg Egr2 fl/fl Adeno-Cre Egr2 T cells X CARTgx Egr2 fl/fl OVA+CFA Egr2 CARTg x Egr2 fl/fl Th1 clone or peripheral T cells OVA+APCs+ IL-2+IFN- Zha et al., Journal of Immunological Methods, 2008 Adeno-Cre mediated Egr2 deletion in CARTg x Egr2fl/fl Th1 cell clones cont. Egr2 qRT-PCR Immunoblot -5 4.0×10 EV Cre Control Anergic 3.0×10 -5 -5 EV EV Cre 2.0×10 Cre Egr2 1.0×10 -5 Expression level 0 Total Erk Control Anergic Egr2 deletion leads to resistance to anergy induction in vitro IL-2 fl/fl CAR Tg x Egr2 10 Control EV clones Control Cre EV and Cre 8 Ane rgic EV Ane rgic Cre 6 Anergy induction 4 No treatment (Control) (U/ml) IL-2 or 2 Plate-bound anti-CD3 (Anergic) 0 AntiCD28 AntiCD3/28 Rest 1-2 days In medium Rechallenge with plate- bound anti-CD3+anti-CD28 Might Egr2 be a “central regulator” of the anergic state? • Besides DGK-, several other genes encoding negative regulatory molecules have been reported to be upregulated in anergic T cells –DGK- – Cbl-b, GRAIL, Itch – Tob1, Deltex1 • Goal: to identify global transcriptional program regulated by Egr2 in anergic cells •Strategies: – QRT-PCR and ChIP assays for known candidates – Combine gene expression profiling of conditional Egr2-deleted T cells with ChIP-seq analysis of genes directly binding Egr2 to identify total program Egr2 directly regulates most of the known anergy associated genes: qRT-PCR DGK- Cbl-b Itch 4.0×10 -4 -5 -6 EV 1.2×10 EV 2.0×10 EV Cre Cre Cre 1.0×10 -5 3.0×10 -4 1.5×10 -6 8.0×10 -6 2.0×10 -4 6.0×10 -6 1.0×10 -6 4.0×10 -6 1.0×10 -4 5.0×10 -7 Expression level Expression level Expression level 2.0×10 -6 0 0 0 Control Anergic Control Anergic Control Anti-CD3 1D Tob1 Deltex1 1.5×10 -5 2.0×10 -7 EV EV Cre Cre 1.5×10 -7 1.0×10 -5 1.0×10 -7 5.0×10 -6 Similar results by 5.0×10 -8 Expression level Expression level ChIP assay 0 0 Control Anergic Control Anergic Model for Egr2 as central transcriptional regulator of T cell anergy Dominant Ca2+ signaling NFAT Egr2 DGK-,-, Cbl-b, Itch, Tob1, Deltex1 Inhibition of TCR/CD28-mediated activation Strategy to determine global Egr2-driven transcriptional program in anergic T cells CAR Tg x Egr2fl/fl T cells: EV versus Cre Treat cells: Control versus plate-bound anti-CD3 (Anergic) Affymetrix gene expression profiling ChIPseq with anti-Egr2 Merge datasets & Confirmatory qRT-PCR, ChIP assays 46 genes identified as targets of Egr2 by gene array x ChIP-SEQ in anergy New Egr2-dependent anergy associated genes Crtam Lag3 Chip Assay Crtam intron Lag3 core promoter 0.04 IP Egr2 0.25 IP Egr2 IP No Ab IP No Ab 0.20 0.03 0.15 0.02 0.10 Recovery ratio 0.01 Recovery ratio 0.05 0.00 0.00 Control Anergic Control Anergic Crtam Lag3 -5 -5 qRT-PCR 8.0×10 EV 5.0×10 EV Cre Cre 4.0×10 -5 6.0×10 -5 3.0×10 -5 4.0×10 -5 2.0×10 -5 2.0×10 -5 Expression level Expression level 1.0×10 -5 0 0 Control Anergic Control Anergic Can Egr2-driven cell surface molecules mark the anergic T cells in the tumor microenvironment ? • Crtam (Class-I-MHC-restricted T cell associated molecule) – A transmembrane protein, and expressed mainly on T cells and NK cells – Reported to maintain T cell polarity during the late phase of T cell activation, and T cell retention in lymph nodes (Yeh et al., Cell, 2008; Takeuchi et al., 2009). • Lag3 (lymphocyte-activation gene 3 ) – CD4-related transmembrane protein, and binds to MHC class II on APCs with higher affinity than CD4 – Deletion of Lag3 in T cells led to enhanced anti-tumor response (Grosso et al., 2007). PD1, Lag3 and Crtam are highly upregulated on CD8+ tumor-infiltrating lymphocytes (TILs) in the context of B16 melanoma Lag3+Crtam+ CD8+ TILs are defective in IL-2 production upon ex vivo stimulation Control Anti-CD3+28 Lag3+Crtam+ Lag3-Crtam- IL-2 CD8 Sorted Lag3+Crtam+ CD8+ TILs are hypoproliferative upon ex vivo stimulation 8000 DNLag3-Crtam- Lag3+Crtam+DP 6000 4000 2000 Proliferation (cpm)Proliferation 0 Control Anti-CD3+28 Anergy-associated genes are enriched in Lag3+Crtam+ CD8+ TILs Egr2 DGK Cbl-b 4 4 15 Splenocyte Lag3-Crtam- TIL 3 3 Lag3+Crtam+ TIL 10 2 2 5 1 1 Expression level Expression level Expression level 0 0 0 Conditional deletion of Eg2 in T cells leads to enhanced anti-tumor immune response and slowed tumor growth Tumor Size IFN- 500 80 WT WT ) KO 2 KO 400 60 300 40 200 20 100 Number of spot of Number Tumor areaTumor (mm 0 0 0 7 10 Medium SIY Time (day) Conclusions • Egr2 is a major transcriptional regulator of the anergic state • Egr2-deleted T cells are relative anergy-resistant in vitro and also to SEB in vivo (data not shown) • Combined gene expression profiling and ChIP-seq has identified the knowable Egr2 transcriptome in T cell anergy • New identified anergy-associated genes are surface markers • Crtam and Lag3 may identify the population of anergic T cells from the tumor microenvironment ex vivo Acknowledgments • Thomas Gajewski • Harinder Singh Laboratory Laboratory • Yuanyuan Zha • Chauncey Spooner • Robbert Spaapen • Gregory Driessens • Fred Locke • Albert Bendelac • Long Zhang Laboratory • Justin Kline • Rebecca Mathew • Mercedes Fuertes •MihirVohra • Michelle Gao • Seng-Ryong Woo Tumor-infiltrating CD8+ T cells (brown) in human melanoma are EGR2+ (blue) Implies that strategies to inhibit EGR2 pathway or target genes may have the potential to improve T cell function in tumor context 22 Egr2 deletion with Cre Adenovirus followed by adoptive transfer leads to resistance to superantigen induced anergy in vivo Egr2 deletion confirmation Rechallenge with SEB Control Anergic 20.00 PBS SEB * * EV 15.00 Cre 10.00 IL-2 (U/ml) IL-2 5.00 Cells 0.00 EV Cre Egr2 Infect with 3 daysAdopt 1 day I.P. mice 7 days Harvest spleen, and EV or Cre - transfer into with SEB rechallenge with adenovirus B6 mice PBS or SEB Egr2 deletion leads to resistance to superantigen induced anergy in vivo Egr2 deletion confirmation Rechallenge with SEB Unstimulated Stimulated 20.00 f/f f/f PBS SEB NS CD4-Cre Egr2 CD4-Cre Egr2 B6 B6 15.00 * * Cre 10.00 IL-2 (U/ml) IL-2 Egr2 5.00 0.00 Total Erk B6 CD4-Cre Egr2fl/fl Inject mice 7 days Harvest spleen, and intraperitoneally rechallenged with PBS or with SEB SEB DGK- triumphs Egr2 deletion and inhibits T cell activation Immunoblot IL-2 Proliferation 1.2×10 4 70.00 EV-EV EV-EV EV-DGK- EV-DGK- 4 60.00 Cre-EV 1.0×10 Cre-EV Cre-DGK- Cre-DGK- 50.00 8.0×10 3 40.00 6.0×10 3 30.00 3 IL-2 (U/ml) IL-2 4.0×10 20.00 Proliferation (cpm) 3 10.00 2.0×10 0.00 0 Anti-CD28 Anti-CD3/CD28 0 0.01 0.1 Anti-CD3 (g/ml) Egr2 directly regulates the most known anergy factors: ChIP DGK- Cbl-bpp Itch 0.20 IP Egr2 0.30 IP Egr2 0.05 IP Egr2 IP No Ab IP No Ab IP No Ab 0.25 0.04 0.15 0.20 0.03 0.10 0.15 0.02 0.10 Recovery ratio Recovery ratio 0.05 Recovery ratio 0.05 0.01 0.00 0.00 0.00 Control Anergic Control Anergic Control Anti-CD3 Tob1 Deltex1 0.012 0.50 IP Egr2 IP Egr2 IP No Ab IP No Ab 0.010 0.40 0.008 0.30 0.006 0.20 0.004 Recovery ratio Recovery ratio 0.10 0.002 0.00 0.000 Control Anergic Control Anergic Egr2 DGK 4 4 3 3 2 2 1 1 Expression level Expression level 0 0 Spleen DN TILs DP TILs Spleen DN TILs DP TILs Grail Cbl-b Itch 25 15 3 20 10 2 15 10 5 1 Expression level Expression level 5 Expression level 0 0 0 Spleen DN TILs DP TILs Spleen DN TILs DP TILs Spleen DN TILs DP TILs CCL1 Sema7A 41BB 500 60 150 400 40 100 300 200 20 50 Expression level Expression level Expression level 100 0 0 0 Spleen DN TILs DP TILs Spleen DN TILs DP TILs Spleen DN TILs DP TILs Nrgn Nrn1 BCL2L11 60 200 4 150 3 40 100 2 20 50 1 Expression level Expression level Expression level 0 0 0 Spleen DN TILs DP TILs Spleen DN TILs DP TILs Spleen DN TILs DP TILs New Egr2-dependent anergy associated genes A ChIP Assay CCL1 Crtam Sema7A CCL1 intron Crtam intron Sema7A core promoter 0.10 0.10 IP Egr2 0.04 IP Egr2 IP Egr2 IP No Ab IP No Ab IP No Ab 0.08 0.08 0.03 0.06 0.06 0.02 0.04 0.04 Recovery ratio Recovery ratio Recovery ratio 0.01 0.02 0.02 0.00 0.00 0.00 Control Anergic Control Anergic Control Anergic B Real-time RT-PCR CCL1 Crtam Sema7A -3 -5 2.5×10 -5 8.0×10 EV 8.0×10 EV EV Cre Cre Cre 2.0×10 -5 6.0×10 -3 6.0×10 -5 1.5×10 -5 4.0×10 -3 4.0×10 -5 1.0×10 -5 -3 -5 2.0×10 2.0×10 Expression level Expression level Expression level Expression 5.0×10 -6 0 0 0 Control Anergic Control Anergic Control Anergic C Protein expression CCL1 2 6.0×10 EV Cre 4.0×10 2 2.0×10 2 CCL-1 (pg/ml) CCL-1 0 Nonanergic Anergic T cell anergy and tumor conti.