4DN Regulation of T cell Exhaustion
Ansu Satpathy, M.D., Ph.D.
Assistant Professor, Department of Pathology
Parker Institute for Cancer Immunotherapy
Gladstone Institute of Genomic Immunology
Stanford University School of Medicine
Genome technologies to study cancer immunotherapy
Primary immune cells
Single-cell epigenomics
Genome engineering of immune cell therapies
Paired single-cell phenotype and TCR
Single-cell immune/tumor profiling
3D genome architecture
Immunotherapy: Breakthrough in Cancer Treatment
Nobel Prize 2018: James Allison and Tasuku Honjo
Non-responders: ~60-70%
Checkpoint blockade in basal cell carcinoma (BCC)
Chang ALS et al, JAAD, 2019
Pre-treatment
Post-treatment
Tumor
T cell
Antibody blockade
of inhibitory receptors
Tracking the T cell response with paired single-cell RNA/TCR-seq in BCC
~28,000 TCRs (~85%)
~33,000 single cells
Clonal expansion of CD8+ Tex cells after PD-1 blockade
CD8+ tumor-infiltrating T cells
x
Clonal expansion
Tumor specificity
T cell exhaustion
Single-cell RNA/TCR-seq
Post-therapy TILs have a distinct TCR repertoire compared to pre-therapy TILs
x
Yost et al, Nature Medicine, 2019
What are the molecular programs that underlie T cell exhaustion?
Wherry et al, Nat Immunology, 2011
High-throughput single-cell epigenomics in droplets
Jeff Granja, Grace Zheng, 10x Genomics team
Exhausted T cells exhibit a distinct epigenetic state
~5,000 unique sites
The TEx epigenetic state does not change after PD-1 blockade
CD8+ TEx
Summary 1: T cell exhaustion is a barrier to T cell immunotherapy
Nature Medicine, 2019
Nature Biotechnology, 2019
Science, 2021
Chimeric antigen receptor (CAR) and TCR T cell therapies for cancer
Majzner and Mackall, Nature Medicine, 2019
Monoclonal antibody
T cell receptor
Chimeric antigen receptor
CAR-T cells also become exhausted
Lynn et al, Nature, 2019
H3K27ac HiChIP: 3D enhancer-promoter contacts
Mumbach et al, Nat Genetics, 2017
HiChIP generates high-resolution 3D maps in primary T cells
25 Million Cells
1 Million Cells
50 Thousand Cells
HiChIP reveals regulatory mechanisms of T cell differentiation
~50% new enhancers
~50% new contacts
2,123 differential contacts
FOXP3
Altered chromosome conformation in exhausted CAR-T cells
Gennert et al, PNAS, 2021
Sandor et al, Unpublished
Genome-wide CRISPR screens for T cell exhaustion and persistence
1
2
Julia Belk, Winnie Yao, Nghi Ly, Unpublished
In vitro exhaustion recapitulates the in vivo epigenetic exhaustion footprint
92% of Tex epigenetic footprint induced by chronic TCR
With Santosh Vardhana
Epigenetic factors limit T cell persistence in vitro and in vivo
Epigenetic factors limit T cell persistence in vitro and in vivo
In vitro
In vivo
Epigenetic factor KO T cells preferentially maintain a progenitorTEx state
Epigenetically-reprogrammed T cells improve anti-tumor immunity
First-in-human CRISPR trial in engineered T cells
Stadtmauer*, Fraietta* et al, Science, 2020
Summary 2: Regulation of T cell exhaustion for cancer immunotherapies
Stadtmauer*, Fraietta* et al, Science, 2020
Belk et al, In Review, 2022
Acknowledgments
Howard Chang (Stanford)
Katie Yost
Kevin Parker
Max Mumbach
Crystal Mackall (Stanford)
Rachel Lynn
Evan Weber
Elena Sotillo
Katie Freitas
Carl June (UPenn)
Edward Stadtmauer
Joseph Fraietta
Avery Posey
Denis Migliorini
Robbie Majzner (Stanford)
Satpathy Lab
Quanming Shi
Frank Buquicchio
Julia Belk
Joy Pai
Bence Daniel
Stefanie Meier
Nghi Ly
Max Miao
Katalin Sandor
Winnie Yao
Caleb Lareau
Aditi Limaye
Kamir Hiam-Galvez
Markus Diehl
Andy Chen
Siva Kasinathan
Christie Chang
Vincent Liu
Chris McGinnis
Emma Yin
Theo Roth
Robert Stickels
Colin Raposo
Patrick Yan
Mollie Black
Takeshi Egawa (WashU)
Saran Raju
Yu Xia
Renee Wu
Sunnie Hsiung
Matt Hellman (MSKCC)
Andrew Chow
Anne Chang (Stanford)
Will Greenleaf (Stanford)
Jeff Granja
John Wherry (UPenn)
Josephine Giles
10x Genomics
Grace Zheng
Francesca Meschi
Geoffrey McDermott