In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells

Abstract

Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

Figure 1

Analysis of wild-type (C57BL/ 6) and the mutant mice gzmA^−/−, gzmB^−/−, gzmA×B^−/−, and perforin^−/− mice by PCR. Tail DNA from individual mice was analyzed by PCR amplification using the gzmA-, gzmB-, and perforin-specific primer pairs, as indicated in Materials and Methods.

Figure 2

⁵¹Cr-release (top) and ¹²⁵I-DNA release (bottom) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA^−/− (○), gzmB^−/− (□), gzmA×B^−/− (▪), and perforin^−/− (*) mice (pools of two spleens per mouse strain, A–D; (E) spleens of four individual gzmA×B^−/− (▪), one B6 mouse (•), and one perforin^−/− mouse (*) were activated in primary MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.

Figure 2

⁵¹Cr-release (top) and ¹²⁵I-DNA release (bottom) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA^−/− (○), gzmB^−/− (□), gzmA×B^−/− (▪), and perforin^−/− (*) mice (pools of two spleens per mouse strain, A–D; (E) spleens of four individual gzmA×B^−/− (▪), one B6 mouse (•), and one perforin^−/− mouse (*) were activated in primary MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.

Figure 3

⁵¹Cr-release from L1210.3 (top) and L1210.Fas (bottom) target cells induced by in vivo–derived alloreactive splenic effector cells from two individual (open and closed symbols) mutant or B6 mice in the absence (squares) and presence (circles) of EGTA. Mice were immunized intraperitoneally with 10⁷ PFU of vaccinia virus encoding K^d 6 d before removal of spleen. All assays were harvested after 6 h. All values are the mean lysis of triplicate samples at the four e/t values given. SEM never exceeded 3%.

Figure 4

⁵¹Cr-release (top) and ¹²⁵I-DNA release (bottom) from NPP-modified EL4 target cells by ex vivo–derived influenza virus A/WSN–immune Tc from mutant or B6 mice. B6 (•), gzmA^−/− (○), gzmB^−/− (□), gzmA×B^−/− (▪), and perforin^−/− (*) mice were infected intravenously with influenza virus A/WSN and their splenocytes (pool of three mice) were tested after 6 d for cytolytic (top) and nucleolytic (bottom) activities on NPP-modified EL4 target cells. Assay times were 2 and 4 h. All values are the mean lysis of triplicate samples at the four e/t values given. SEM never exceeded 3%.

Figure 5

⁵¹Cr-release (top) and ¹²⁵I-DNA release (bottom) from YAC-1 target cells by ex vivo–derived NK cells from mutant or B6 mice. B6 (•), gzmA^−/− (○), gzmB^−/− (□), gzmA×B^−/− (▪), and perforin^−/− (*) mice were either treated with poly I:C (20 h) or infected with SFV (2 d) and their splenocytes (poly I:C, pool of two spleens; SFV, pool of three spleens) were tested for cytolytic (top) or nucleolytic (bottom) activities on YAC-1 target cells. Assay times were 2 and 4 h. All values are the mean lysis of triplicate samples at three or four e/t values given. SEM never exceeded 3%.