The unique roles of murine granzymes (Gzm) A and B for NK-dependent tumor cell killing and regulatory T cell control of NK cells

626 NK-dependent clearance of RMAS lymphoma and B16 melanoma has been demonstrated in NK cell-deficient mice ( Kim S et al, PNAS, 2000, 97:2731-6 ). To examine the roles of Gzm A and B in NK-mediated tumor clearance, RMAS and B16 cells were injected intravenously into WT mice and mice deficient for Gzm A, Gzm B, or Gzm AxB. The survival curves following RMAS injection are shown in Fig. 1; similar results were found with B16 cells. All Gzm AxB-deficient mice died within 6 weeks. Gzm A-deficient mice also displayed reduced survival. Surprisingly, Gzm B-deficient mice demonstrated significantly improved survival compared to WT mice. To understand the molecular and cellular basis, we examined the Gzm expression patterns at various times following tumor injection. Lymphocytes isolated from tumor-infiltrated organs, from spleens and lymph nodes were examined for Gzm A and B expression in NK cells and T cell subsets using 4-color flow cytometric analysis. Within NK compartment, Gzm A expression is constitutive and remains unchanged during the 1st 4 weeks. In contrast, very few Gzm B-expressing cells ( In vitro killing assays indicated that resting NK cells enriched from spleens of Gzm A-deficient mice exhibited diminished killing of RMAS cells, while Gzm B-deficient resting NK cells killed RMAS cells as efficiently as WT resting NK cells. These data suggest that Gzm A is a major effector in NK cells in the early stages of tumor challenge, while Gzm B plays a more important role later. Importantly, Gzm B (but not Gzm A) was detected in 10-25% of CD4 + /FoxP3 + regulatory T cells isolated from tumors, tumor-infiltrated lungs and livers between day 12 and 26, but Gzm B was not detected in regulatory T cells found in spleens or lymph nodes. Since Gzm B deficient mice clear RMAS and B16 more efficiently than WT mice, these data suggest that these tumors may somehow induce the expression of Gzm B in regulatory T cells that go on to kill tumor-specific NK cells. By recruiting and activating regulatory T cells that kill NK cells, these tumors could gain a survival advantage that would be lost in the setting of Gzm B deficiency.