To date, the in vivo function of GNLY has been inferred from in vitro assays (5
) and clinical correlates (6
). rGNLY is cytotoxic against a variety of microbes (13
) and tumor cells (5
); GNLY is coexpressed with perforin and granzymes in cytotoxic granules in CTL and NK cells (13
); and increased GNLY expression is associated with improved prognosis in cancer patients (6
). This study is the first direct demonstration that GNLY functions in vivo to enhance tumor-specific immunity.
GNLY homologues have been identified for pig (NK-lysin) (54
), cow (Bo-lysin) (55
), and horse (56
), but not for rodents (10
). Although gene disruption in mice has proven highly informative in defining the function of perforin (57
) and granzymes (58
), such experiments are not possible for GNLY. Therefore, we engineered a transgenic mouse to assess the in vivo effects of GNLY. Initial efforts used a TCR promoter and the human granzyme B promoter, both of which have been used previously to produce transgenic animals expressing proteins of interest (59
). However, no mRNA or protein was detected in mice using these promoters for either the 9- or 15-kDa forms of GNLY, suggesting that flanking and/or intronic sequences are required for expression.
GNLY is constitutively expressed in NK cells isolated from human PBMC or from GNLY+/− spleens. Although the relative amounts of GNLY mRNA in human and GNLY+/− NK cells are similar, there is substantially more GNLY protein in human than GNLY+/− NK cells, suggesting that GNLY protein expression is controlled in part at the level of translation. Moreover, only the 15-kDa form of GNLY is detectable in GNLY+/− mouse NK cells, whereas both the 9- and 15-kDa forms are present in nearly equal amounts in human NK cells. Nevertheless, NK cells from GNLY+/− mice are capable of expressing high levels of both forms of GNLY when activated by IL-15 (). This suggests that mice maintained in a relatively clean facility are not exposed to environmental Ags that induce NK cell activation, altering the pattern of expression of GNLY.
For humans and GNLY+/−
mice, GNLY is constitutively expressed by NK cells and inducible in T cells. We observed that splenocytes from GNLY+/−
mice activated with anti-CD3/CD28 show enhanced lysis of targets at early times and at low E:T ratios when assayed by FloKA. Additionally, GNLY+/−
mice survived longer than wild-type mice after challenge with the C6VL tumor. In contrast, splenocytes from GNLY+/−
mice activated with IL-15 did not show enhanced lysis of RMA-S tumor cells, and there was no difference in survival of GNLY+/−
mice challenged with this tumor in vivo. Thus, transgenic human GNLY plays a role in elimination of tumors by CD8+
T cells, but not by NK cells. This is especially interesting in light of previous in vitro data indicating that, although both human CD8+
T cells and NK cells express GNLY and kill Cryptococcus neoformans
, only the CD8+
T cells, and not the NK cells, use GNLY to kill this fungus (11
The BAC clone contains one partial gene (SnRNP assembly defective 1 homologue) and three complete genes SFTPB, GNLY, and basic helix-loop-helix transcription factor 6 (also known as HATH6 or ATOH8). The SnRNP assembly defective 1 homologue gene is truncated at the 5′ terminus, and therefore is not expressed in the transgenic mice. Using RT-PCR, we detected human SFTPB mRNA expression in the transgenic mice at a level similar to that of murine SFTPB. The main function of SFTPB is to lower the surface tension at the air-liquid interface in the alveoli of lung, suggesting that expression of human SFTPB in mice would not affect any of the parameters measured in our studies. In humans, expression of ATOH8 is restricted to infants <3 years of age (National Center for Biotechnology Information UniGene Hs.135569). Because all of our studies were conducted in adult mice, we do not expect expression of this gene to affect any of the parameters investigated.
The FloKA is more sensitive than the 51
Cr release assay and allows analysis of early steps in apoptosis (39
). EL4.F15 targets are defective in signaling through Fas, and thus, lysis is mediated chiefly by the granule exocytosis pathway (35
). Allospecific cultures from GNLY+/−
mice showed significantly enhanced cytolysis at early time points and low E:T ratios compared with nontransgenic littermates. In contrast, purified NK cells from GNLY+/−
and nontransgenic littermates show equivalent lysis of YAC-1 or RMA-S targets in the FloKA. These studies indicate that GNLY can play a role in allospecific cytotoxicity in vitro. The relatively minor effect of GNLY is most likely due to high expression of other cytolytic molecules such as perforin and granzymes. These molecules most likely overwhelm the measurements at higher E:T ratios and longer times in culture.
The identification and characterization of GNLY as an antimicrobial and tumoricidal product of T lymphocytes and NK cells suggest a broader, and perhaps more important, role for these cell types in the ongoing war against microbes and provide an additional effector mechanism against tumors (3
). Phagocytes, not lymphocytes, have generally been implicated as the important lines of defense against bacteria (21
). The roles of CTL and NK cells have been more narrowly confined to tumor and antiviral immunity and certain specific bacterial and parasitic infections (46
). GNLY has broad-based clinical relevance as a diagnostic (biomarker) and potential therapeutic in entities as diverse as transplant rejection, cancer, and infectious diseases (6
). GNLY may also be involved in the normal physiologic response that kills dysregulated cells (64
). Two clinical studies suggest that GNLY may be important in fighting cancer in patients. As noted above, Kishi et al. (6
) correlated GNLY expression with clinical outcome in cancer patients. More recently, colorectal cancer patients have prolonged survival associated with increased level of GNLY mRNA in effector T cells in tumors without early invasion (31
). Sekiya et al. (30
) used a murine lung cancer model to show that gene transfer of 9-kDa GNLY is therapeutic in vivo. In this study, we now show that GNLY expressed as a transgene in mice improves outcome for experimental lymphoma compared with wild-type controls. These studies suggest that GNLY or its derivatives may prove useful as new therapeutics with novel mechanisms of action and low toxicity.