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253 PD1 and LAG3 converge to limit polyfunctionality and systemic immunity
  1. Lawrence Andrews1,
  2. Sasikanth Manne2,
  3. E John Wherry2,
  4. Creg Workman1 and
  5. Dario Vignali1
  1. 1University of Pittsburgh, Pittsburgh, USA
  2. 2University of Pennsylvania, Philadelphia, PA, USA


Background Targeting PD1 with monoclonal antibodies has yielded clinical success across a variety of tumor types, yet overcoming inhibitory receptor (IR)-mediated tolerance is essential to improve immunotherapeutic responses. LAG3 co-expresses with PD1 on CD8+ tumor-infiltrating T cells (TIL), signifying a highly exhausted phenotype, and dual PD1/LAG3 blockade in C57BL/6 mice enhances antitumor immunity. As CD8+ TIL is the dominant LAG3-expressing TIL population, it is hypothesized that PD1 and LAG3 synergizes to limit CD8+ TIL function controlling antitumor immunity.

Methods To understand the cellular and mechanistic basis for PD1/LAG3 synergy, conditional knock-in mice ”surgically dissect” Pdcd1 and/or Lag3 floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. These mice were crossed with the Pmel transgene to assess PD1 and/or LAG3-sufficient or deficient gp100-specific CD8+ T cell populations. CD8+ Pmel cells were isolated and adoptively transferred into C57BL/6 mice harboring a B16-gp100-overexpressing tumor to observe therapeutic benefit, or to assess T cell functionality within the tumor.

Results While little therapeutic benefit was observed with a prophylactic adoptive transfer of wild-type CD8+ Pmel cells into mice which then received B16-gp100 tumor, there was reduced tumor growth in mice receiving PD1-deficient CD8+ Pmel cells, which was further enhanced in mice receiving PD1/LAG3-deficient CD8+ Pmel cells with long-term tumor-free survival. Likewise, a therapeutic administration of PD1/LAG3-deficient CD8+ Pmel cells into mice once the tumor has been established showed an initial therapeutic benefit, with enhanced survival, that was not demonstrated with adoptive transfer of PD1 or LAG3-deficient, or wild-type, counterparts. Each PD1 and/or LAG3-sufficient or deficient CD8+ Pmel mice were differentially congenically marked to assess each of the four genotypes that can be adoptively transferred into the same host as a ”quad transfer” system. Recovery of these populations within the tumor show that the PD1/LAG3-deficient CD8+ Pmel cells out-compete PD1 or LAG3-deficient, or wild-type, counterparts due to enhanced proliferation (Ki67/BrdU). Furthermore, PD1/LAG3-deficient CD8+ T cells were more functional with increased IFNg and GzmB release observed by flow cytometry.

Conclusions Overall PD1 and LAG3 limit anti-tumor immune effects as removal of both IRs on a gp100 antigen-specific CD8+ T cell population results in reduced B16-gp100 tumor growth and enhanced survival in an adoptive transfer model, as a result of enhanced CD8+ TIL functionality and proliferation. These results provide striking evidence that the development of anti-LAG3 agents in the clinic would yield improved responses with anti-PD1.

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