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RMAS Cell Clearance from Granyzme B Mice

Performed in collaboration with Tim Ley's Lab.
Granzyme B is important for the ability of NK cells and CD8+ T cells to kill their targets. However, we showed here that granzyme B-deficient mice clear both allogeneic and syngeneic tumor cell lines more efficiently than do wild-type (WT) mice. To determine whether regulatory T (Treg) cells utilize granzyme B to suppress immune responses against these tumors, we examined the expression and function of granzyme B in Treg cells. Granzyme B was not expressed in naive Treg cells but was highly expressed in 5%-30% of CD4+Foxp3+ Treg cells in the tumor environment. Adoptive transfer of WT Treg cells, but not granzyme B- or perforin-deficient Treg cells, into granzyme B-deficient mice partially restored susceptibility to tumor growth; Treg cells derived from the tumor environment could induce NK and CD8+ T cell death in a granzyme B- and perforin-dependent fashion. Granzyme B and perforin are therefore relevant for Treg cell-mediated suppression of tumor clearance in vivo.
Adoptively Transferred WT, but Not Gzmb-/- or Prf1-/-, Treg Cells Suppress RMAS Tumor Clearance in Gzmb-/- Mice. (A) 2 x 106 click beetle red luciferase-expressing RMAS cells were injected intraperitoneally into WT mice, various Gzm-/- mice, and Prf1-/- mice. Bioluminescence imaging was performed to serially assess whole-body tumor clearance in each mouse, with luciferase activity as a measure for tumor burden. Representative imaging time course of individual mice from each genotype is shown, with the same scale of photon flux indicating luciferase activity. (B) Summaries of tumor burdens for all genotypes are shown as mean ± SEM, after i.p. injection of 2 x 106 luciferase-expressing RMAS cells, with 4-5 mice assessed in each genotype. Data points are shown as mean ± SEM, and two-tailed t tests were used to determine statistical significance between WT mice and mice deficient for the indicated granzymes or perforin (*p < 0.05). (C) A summary of three independent adoptive transfer experiments, with nine mice assessed in each group. Abbreviations: WT, WT mice receiving only tumor cells, as a control for tumor outgrowth in these mice; Gzmb-/-, Gzmb-/- mice receiving only tumor cells, as a control to demonstrate the reduced tumor growth in these animals; Gzmb-/- + WT Treg, Gzmb-/- mice adoptively transferred with WT Treg cells, in addition to tumor cells; Gzmb-/- + Gzmb-/- Treg, Gzmb-/- mice adoptively transferred with Gzmb-/- Treg cells, in addition to tumor cells. (D) A summary of two independent adoptive transfer experiments is shown (n = 6 in each group). Abbreviation: Gzmb-/- + Prf1-/- Treg, Gzmb-/- mice adoptively transferred with Prf1-/- Treg cells, in addition to tumor cells. Data points are shown as mean ± SEM, and two-tailed t tests were used to determine statistical significance (*p < 0.05).
Reference:	Cao X, Cai SF, Fehniger TA, Song J, Collins LI, Piwnica-Worms DR, Ley TJ. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 2007; 27(4): 635-46.	PubMed Link

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