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579-C Reinvigoration of progenitor-exhausted CD8 T cells by anti-CTLA-4 contributes to the sustained activity of combination checkpoint blockade
  1. Kevin Wang1,
  2. Paulina Coutifaris1,
  3. David Brocks2,
  4. Sabrina Solis3,
  5. Nicholas Han1,
  6. Sasikanth Manne1,
  7. Evgeny Kiner2,
  8. Chirag Sachar2,
  9. Sangeeth George4,
  10. Patrick Yan5,
  11. Melanie W Kiner6,
  12. Amy I Laughlin7,
  13. Shawn Kothari8,
  14. Josephine R Giles9,
  15. Divij Mathew9,
  16. Rheem Ghinnagow1,
  17. Cecile Alanio10,
  18. Ahron Flowers11,
  19. Wei Xu1,
  20. Daniel Tenney12,
  21. Xiaowei Xu1,
  22. Ravi K Amaravadi1,
  23. Giorgos C Karakousis1,
  24. Lynn M Schuchter11,
  25. Marcus Buggert13,
  26. E John Wherry9,
  27. Andy Minn1,
  28. Jeffrey Weber14,
  29. Tara Mitchell11,
  30. Ramin S Herati3 and
  31. Alexander Huang9
  1. 1University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
  2. 2Immunai Inc., New York, N, USA Y
  3. 3New York University Grossman School of Medicine, New York, NY, USA
  4. 4Bristol Myers Squibb Co Cambridge, Cambridge, MA, USA
  5. 5Stanford University, Stanford, CA, USA
  6. 6Icahn School of Medicine at Mount Sinai, New York, NY, USA
  7. 7Orlando Health Cancer Institute, Orlando, FL, USA
  8. 8Winship Cancer Institute of Emory University, Atlanta, GA, USA
  9. 9University of Pennsylvania, Philadelphia, PA, USA
  10. 10Institut Curie, Paris, Île-de-France, France
  11. 11Abramson Cancer Center, Philadelphia, PA, USA
  12. 12Bristol Myers Squibb, Princeton, NJ, USA
  13. 13Karolinska Institute, Solna, Sweden
  14. 14NYU Langone Medical Center, New York, NY, USA


Background Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. A better understanding of the cellular and molecular mechanisms of αPD-1 and αCTLA-4 individually, and in combination, will guide the development of safer and more effective combination immunotherapy strategies.

Methods We performed multimodal (scRNA + TCR + epitope) analysis across time in 32 stage IV melanoma patients treated with anti-PD-1, anti-CTLA-4, or anti-PD-1 + anti-CTLA-4 (combination) therapy. In order to understand the effect of checkpoint blockade on T cells at a single-clone resolution, we developed a novel algorithm Cyclone to track temporal clonal dynamics and their underlying cell states.

Results Anti-CTLA-4 induced more durable immune responses than anti-PD-1, whereas combination therapy mobilized greater and more sustained immune responses. Using Cyclone, we identified 6 clonotypic trajectories with distinct temporal patterns. These analyses revealed that checkpoint blockade induced waves of immune responses composed of distinct clonotypes that peaked at different timepoints. Combination therapy generated clonal effector and exhausted CD8 T cells (TEX) responses that peaked at 6–9 weeks after treatment. Focused analyses of TEX in additional cohorts identified that anti-CTLA-4 induced robust expansion and proliferation of progenitor TEX, which synergized with anti-PD-1 to generate a large and durable reinvigoration of TEX. Immune profiling of a cohort of patients that first received anti-CTLA-4, followed by anti-PD-1 revealed that these enhanced progenitor responses were largely due to anti-CTLA-4 therapy. The induction of progenitor TEX by anti-CTLA-4 were independently validated using samples collected from the Checkmate 238 clinical trial of adjuvant nivolumab versus ipilimumab in resectable melanoma.

Conclusions First, durable immune responses represent waves of immune responses generated by different T cell clones. Second, progenitor TEX induced by CTLA-4 blockade may contribute to durable immune responses through self-renewal and replenishing of the TEX pool.

Ethics Approval Patients were consented for blood collection under the University of Pennsylvania Abramson Cancer Center’s melanoma research program tissue collection protocol UPCC 08607, in accordance with the Institutional Review Board. For specimens from Checkmate 238, PBMC were obtained following informed consent under an IRB-approved protocol at NYU.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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