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The extent of metalloproteinase-mediated LAG3 cleavage limits the efficacy of PD1 blockade
© Andrews et al. 2015
Published: 4 November 2015
Inhibitory receptors control immune responses preventing exacerbated T cell activation and the onset of autoimmunity; however, they also limit antitumor immunity. Enhanced co-expression of PD1 and LAG3 phenotypically mark functionally exhausted tumor-specific T cells, with dual PD1/LAG3 blockade synergistically limiting tumor growth in murine models. Like PD1, LAG3 expression is induced on activated T cells to negatively regulate T cell activation and proliferation and LAG3 is also required for maximal regulatory T (Treg) cell function. However, LAG3 expression and function is itself regulated by cell surface cleavage of the transmembrane domain connecting peptide by ADAM10 and ADAM17 metalloproteinase-disintegrins. This releases soluble LAG3, of which no biological function has been found to date. To investigate the impact of LAG3 cleavage on T cells within tumors, a non-cleavable LAG3 mouse (LAG3.NC) was generated in which exons 7 and 8 of Lag3, including the connecting peptide, is deleted in Cre-expressing cells. These exons are replaced and modified so that the connecting peptide is absent preventing LAG3 cleavage. LAG3.NC CD4Cre mice (with non-cleavable LAG3 expressed on all CD8+ and CD4+ T cells, including Tregs) and LAG3.NC E8ICre mice (restricted to CD8+ T cells only) exhibit enhanced expression of LAG3 on the respective T cell subsets in B16-F10 or MC38 tumors, co-expressing with PD1. Despite increased LAG3 expression, no difference in B16-F10 or MC38 tumor growth was observed in either LAG3.NC CD4Cre or LAG3.NC E8ICre mice compared with wild-type littermates. Upon therapeutic administration of anti-PD1 antibody (clone G4), MC38 tumor-bearing wild-type mice show significant tumor regression and 40% become tumor-free resulting in long-term survival. LAG3.NC CD4Cre mice were resistant to anti-PD1 therapy and succumb to tumor growth. However, anti-PD1 mediated tumor regression and long-term survival in LAG3.NC E8ICre mice. Analysis of re-stimulated CD8+ TILs isolated from LAG3.NC CD4Cre mice did not show enhanced IFN-gamma and TNF-alpha production following anti-PD1 therapy, which was observed with LAG3.NC E8ICre mice or wild-type littermates. Moreover, reduced proliferation was observed for all T cell subsets in LAG3.NC CD4Cre mice compared with LAG3.NC E8ICre and wild-type littermates following anti-PD1 treatment. As LAG3.NC CD4Cre, but not LAG3.NC E8ICre mice, are resistant to the favorable antitumor immune effects induced by anti-PD1, this suggests that enhanced LAG3 expression on CD4+ T cells or Tregs may act as a barrier to effective anti-PD1 immunotherapy. LAG3.NC mice crossed with Cre that restricts non-cleavable LAG3 to Tregs (Foxp3yfpiCre) or CD4+ T cells (ThPOKCre) are currently under analysis.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.