PLRP2, together with lipid substrates, mediates indirect toxicity toward tumor cells
We questioned if the lipase, PLRP2, was directly or indirectly toxic towards tumor cells. We incubated recombinant PLRP2 with P815 cells, with or without triglyceride lipids in the presence or absence of the lipase cofactor, colipase, over a 48 hour period. Native purified pancreatic triglyceride lipase (PTL) was used as a control because it is a similar enzyme and is commercially available. Both PLRP2 and PTL were unable to damage P815 cells directly (). With the lipases and their cofactor, colipase, one might expect direct toxicity to be enhanced; however, direct toxicity was still absent (illustrated for PLRP2 in ). To monitor indirect toxicity (from lipid products), we added lipid substrate to the cellular assays together with the lipases. P815 viability was reduced when PLRP2 was combined with the triglyceride, trilinolein, and colipase ().
PLRP2 with triglyceride lipid and colipase mediated indirect toxicity toward tumor cells
JURKAT cells were also tested as tumor cell targets for direct and indirect cytotoxicity by PLRP2 or by PTL. JURKAT cells were similarly invulnerable to direct attack (in the absence of exogenous lipids) and susceptible to indirect toxic effects by either lipase plus cofactor when combined with lipid substrate (). PLRP2 alone (without cofactor but with triglyceride lipid) at 10 μg/ml mediated modest indirect cytotoxicity toward JURKAT cells (), which suggests that JURKAT cells are more sensitive than P815 cells.
We established that fatty acid products from the lipid substrates are potential toxic mediators. These lipases will release 2 or 3 molecules of linoleic acid from each molecule of trilinolein. The toxicity generated by each lipase in the presence of triglycerides was similar to the toxicity observed when P815 cells were incubated with high concentrations of linoleic acid for 48 hours (). The 1.5 mM trilinolein substrate in the assays and the activities of the lipases would be sufficient to produce the 100+ micromolar concentrations of fatty acids needed. Furthermore, addition of delipidated bovine serum albumin (BSA) at physiological concentrations blocked the toxic effects (not illustrated). The BSA found in serum can bind as much as 3.6 mM fatty acid (calculated at a buffering capacity of 7 molecules of fatty acid per molecule of albumin 24
Triglyceride-hydrolyzing lipases are secreted by IL-4-induced CTLs
The potential methods of PLRP2 (or other lipases) release from CTLs include constitutive secretion, a burst upon antigen receptor-stimulated exocytosis and/or prolonged secretion after the CTLs have encountered their antigens. To simulate antigen recognition, we treated the CTLs with either phorbol myristate acetate (PMA) combined with an ionophore (ionomycin) 25
or with anti-CD3ε covalently bound to beads 26
, a signaling protein associated with the T cell receptor for antigen. IL-4 induced T cells were stimulated with PMA and ionomycin or with DMSO as a solvent control for 4 hours in media with 3
H-triolein as a reactive substrate that was incorporated into lipid micelles. The cell-free supernatant was collected and tested for the release of radio-labeled fatty acids from 3
H-triolein. Supernatants from DMSO-treated CTLs displayed triglyceride lipase activity that was above background, indicating that there was some constitutive secretion of lipase (). Fetal calf serum in the media contributed lipase activity to the background (). This background hydrolysis of 3
H-triolein handicapped detection of the cellular lipases; however, it was necessary to retain fetal calf serum in these experiments in order to reduce the death of T cells. The CTL-mediated constitutive secretion of lipase compared to the media had a P<0.05 in two of three experiments, including the one illustrated. Therefore the CTLs constitutively secrete lipase activity. Stimulation with PMA and ionomycin or anti-CD3 (data not illustrated) failed to significantly increase lipase release over constitutive secretion in two of three experiments ().
CTLs secreted triglyceride lipase(s)
We gained additional information from these assays. Calibration with rPLRP2 as an internal standard indicated that there would be less than 30 ng equivalents of PLRP2 secreted per 106 IL-4 CTLs, assuming that all the secreted lipase activity was mediated by only PLRP2. CTLs grown in IL-2 also had constitutively secreted triglyceride lipase(s) but there was no evidence of stimulated release of lipase (data not illustrated).
IL-4 cultured CTLs lacked lipid-dependent cytotoxicity; however, CTLs induced by one exceptional lot of IL-4 had lipid-dependent cytotoxicity
To determine if the addition of the triglyceride, trilinolein, to CTL cytotoxicity assays would support lipid-dependent cytotoxicity, we performed two types of cytotoxic killing assays. One assay depended on 8 hour 51Cr-release and was substantially perforin dependent while the other assay utilized fluorescent P815 cells in a 48 hour tumor viability assay. CTLs from wild type (perforin-positive) mice induced with IL-4 mediated similar 8 hour cytotoxicity with or without lipid, regardless of the lot of IL-4 (data not illustrated). To favor detection of lipid-dependent cytotoxicity which could be covered up by perforin activity, we used CTLs from perforin deficient (perforin−/−) mice.
With perforin−/− CTLs without lipid, there were low levels of cytotoxicity at 4 hours, but moderate levels at 8 hours. All lots of IL-4 tested induced expression of PLRP2 mRNA in CTLs detectable by Q RT-PCR and by immunoblots (Alves et al., submitted). Despite the presence of PLRP2, cytotoxicity was not enhanced by the inclusion of lipid (). In fact, cytotoxicity was sometimes suppressed by the lipid, as indicated in . CTLs induced with IL-2 were also unable to mediate lipid-dependent cytotoxicity (data not illustrated).
CTLs mediated lipid-dependent cytotoxicity in 8 hour 51Cr-release assays only when they were induced with an exceptional lot of IL-4
However, perforin−/− CTLs, when induced with an exceptional lot of IL-4 (BD lot 60506), demonstrated lipid-enhanced cytotoxicity (). CTLs with this exceptional lot had lipid enhanced cytotoxicity at 8 hours () but not 4 hours (not shown). Since perforin-dependent cytotoxicity is readily observed within 4 hours, the data indicate that lipid-dependent cytotoxicity is much slower than perforin-dependent cytotoxicity.
Thus we obtained a clear indication of lipid-enhanced cytotoxicity with CTLs induced with one exceptional lot of IL-4 (which contained IL-4 and probably an additional factor). We continued our investigations with lot 60506 IL-4, without knowledge at the time that the lot was exceptional.
Flow cytotometry assays for long term lipid-dependent cytotoxicity, monitored by viable tumor cell recovery
Longer term assays, in which apoptotic killing mediated by WT and perforin−/− CTLs is equally effective (without lipid), were performed next. Trilinolein was added to 48 hour anti-CD3 redirected lysis of fluorescent eGFP-P815 cells using IL-2 and IL-4 induced CTLs. For reference, the viability of P815 cells was unaffected when the P815s were incubated with lipid alone. With most lots of IL-4, cytotoxicity was the same with or without lipid, mediated either by WT or perforin−/− CTLs. (See and a following section in which additional variables were added to stimulate lipid-dependent killing.)
With the exceptional lot of IL-4 (and only this lot of IL-4), viable P815 cells were reduced in the triglyceride-containing assays when compared to the assays without lipids (). Thus CTLs do have the potential to mediate lipid-dependent cytotoxicity. This lipid-dependent cytotoxicity was reproduced 3 times with WT CTLs and three times with perforin−/− CTLs (). The difference between lipid-dependent and lipid-independent cytotoxicity was slight but statistically significant for the WT cells. The triglyceride-enhanced cytotoxicity was absent in the long term killing assays using IL-2 induced WT CTLs ().
CTLs mediated lipid-dependent cytotoxicity in long term tumor viability assays only when they were induced with an exceptional lot of IL-4
CTL induced with the exceptional lot of IL-4 were also tested for lipid-dependent cytotoxicity in allogeneic killing assays in which the anti-CD3 antibody was omitted (). The BALB/c effectors and the P815 cells are both H-2d
but differ at minor histocompatibility loci27
. In the allogeneic system, these IL-4 induced cells displayed higher cytotoxic activity toward P815s when lipid was present (). In summary, CTLs induced with an exceptional lot of IL-4 acquired a novel lipid-dependent method for mediating tumor cell death.
The long term triglyceride-enhanced cytotoxicity by the CTLs induced with the exceptional lot of IL-4 was perforin-independent
Perforin is a primary mediator of the cytotoxic activity utilized by CTLs. Perforin deficient CTLs have dramatically less cytotoxic capacity than their wild type counterparts in short term assays 28
. We questioned if the triglyceride-enhanced cytotoxic activity was perforin dependent or independent using the long term killing assays with IL-2 or IL-4 induced CTLs from perforin−/−
CTLs induced with the exceptional lot of IL-4 had enhanced cytotoxicity upon the addition of triglyceride to the long term assays (). Perforin−/−
IL-2 induced CTLs showed no enhancement upon the addition of triglycerides (). The triglyceride-enhanced cytotoxicity was easier to detect with the perforin−/−
CTLs than with the WT CTLs (compare with ). The lipid-enhanced cytotoxicity was consistent in 3 experiments with perforin−/−
CTLs, using BD lot 60506 IL-4 ().
Triglyceride enhanced CTL activity was perforin-independent
The CTLs induced with the exceptional lot of IL-4 generated toxic products in the presence of lipid
To determine if the lipid-enhanced killing was mediated by toxic products produced from the lipid, CTLs were stimulated to undergo exocytosis in the presence or absence of triglycerides and cell-free supernatants were evaluated for toxicity. IL-4 induced CTLs were used because they had PLRP2 lipase expression (which might produce fatty acids to contribute to cytotoxicity). The IL-4 induced CTLs were generated from perforin−/− mice because anti-CD3 triggers the release of toxic concentrations of perforin that will kill some of the CTLs even though released perforin is very unstable. Exocytosis from these perforin−/− IL-4 induced CTLs was stimulated with anti-CD3 covalently coated beads for 48 hours. Following exocytosis, cell-free supernatants were collected and added to eGFP-P815 cells and the cells plus supernatants were incubated for 48 hours. Lipid was present under three conditions: (1) only during the initial 48 hours of CTL exocytosis (to test for lipase activity upon the lipid); or (2) the lipid was present only during the second 48 hours of incubation of the CTL supernatants with eGFP-P815s (to test for a released lipase that might remain active in the cell-free supernatants and later be able to convert lipids into toxic products). As a last condition (3), lipid was present during both the 48 hours of CTL exocytosis and during the incubation of the supernatants with the P815s. We found that P815 cell viability was reduced by the cell-free supernatants that were collected from CTLs undergoing exocytosis in the presence of triglycerides (, bars 3 and 4, numbering 1–4 from left to right). Bar 3 illustrates the loss of viability when lipid was present during only the initial CTL anti-CD3 stimulation. Bar 4 illustrates the loss of viability when lipid was present during both the CTL anti-CD3 stimulation and the incubation of the P815s with the cell-free supernatants.
CTLs induced with lot 60506 IL-4 and stimulated with anti-CD3 antibodies produced soluble toxic products in the presence of triglyceride lipids
The loss of viability was consistent with lipase and lipid-dependent cytotoxicity. When lipid was missing during CTL exocytosis, there was little subsequent effect on P815 viability (, bar 1). Inclusion of lipid in the P815 assays with the CTL-derived supernatants generated without lipid had no effect. The controls that represented 100% tumor cell viability for each bar in , were P815 cells exposed to supernatants from CTLs incubated without anti-CD3 antibody (with or without lipid). These supernatants of un-stimulated cells lacked cytotoxicity. Thus, we could generate toxic supernatants only from anti-CD3 treated CTLs and only when lipid was present during the initial anti-CD3 stimulation. We interpret this toxic activity as evidence that a released lipase may be able to generate toxic products from lipid.
We consumed all of lot 60506 IL-4 before we realized its unique qualities. The other lots of IL-4 did not support the production of soluble toxic products. These findings concerning the CTLs and their cell-free supernatants indicate that lipid-enhanced killing can be generated by CTLs.
Efforts to stimulate triglyceride-enhanced cytotoxicity
The triglyceride-enhanced cytotoxicity was missing when other lots of IL-4 were used to induce these cells (see ). Initially the potency of the additional lots was suspected to be lower than BD lot 60506 and to be unable to induce PLRP2. Multiple lots of IL-4, from different vendors, induced mRNA for PLPR2 (data not illustrated).
IL-4 is a growth factor for T cells, and, if the ineffective lots of IL-4 were less potent, then T cell growth would be reduced. However, the T cells growth rates from day 0 to day 3 were similar with each lot of IL-4, suggesting a separate factor was responsible for the triglyceride-enhanced killing (). The growth rates from day 3 (when the conA was removed) to day 5 were similar (not illustrated). IL-4 at the 500 units/ml in the assays with the various lots varied from 1.4to 3.5 ×10−9 M and the IL-4 receptors of T cells (comprised of the IL-4 alpha receptor chain with the common gamma cytokine receptor chain) have a Kd of 1.1×10−10 M, indicating that the receptors were saturated by each lot of IL-4. These results suggest that the critical variable was something other than IL-4 and also that this variable was effective in the presence of active IL-4.
The different lots of IL-4 had similar bioactivity as reflected by growth of CTLs
Attempts were also made to reproduce the triglyceride-enhanced toxicity by altering several variables (). Increasing the IL-4 concentration (of the ineffective lots of IL-4) was without effect. We questioned if lipopolysaccharide was responsible for the triglyceride-enhanced cytotoxicity, but reports from the vendors indicated that the effective lot 60506 had less LPS than the ineffective lots of IL-4. Following these results, we questioned whether lack of the triglyceride-enhanced toxicity could be caused by variation in type 1 responses in the CTL cultures. Both IL-12 and IFN-γ are the dominant cytokines associated with the generation of a type 1 response in naive T cells 29,30
. To reduce the type 1 polarizing effects of IFN-γ and IL-12, IFN-γ receptor−/−
splenocytes were induced with an ineffective lot of IL-4 in the presence or absence of 20 μg/ml IL-12 blocking antibodies. Again, these conditions were unsuccessful at reproducing the triglyceride enhanced killing. We also tested for effects of type 1 polarization by adding high concentrations of mouse IFN-γ during splenocyte activation with IL-4. However, these experiments were performed with ineffective lots of IL-4 and, as might be expected, the triglyceride-enhanced killing was still absent (). We tested if stimuli other than conA could activate triglyceride-enhanced cytotoxicity. We used plate bound anti-CD3/CD28 to activate splenocytes instead of conA activation. This induction method was also unsuccessful at reproducing the lipid-enhanced toxicity originally observed in the IL-4 induced CTLs ().
Another possible explanation for the triglyceride-dependent killing would be the coincident CTL expression of the cofactor that increases the activity of pancreatic lipases, a small protein termed colipase. Colipase increases both PTL and PLRP2 activity toward triglycerides 31,32,33
. We tested for the presence of colipase in lymphocytes by Taqman® RT-PCR, using pancreatic mRNA for calibration, and were unable to detect any mRNA for colipase in lymphocytes. Based on the detection threshold, the relative amount of colipase mRNA in CTLs was less than 1 × 10−6
that of the pancreas.
However, a search of the NCBI’s GEO Profiles Gene Expression Omnibus database indicated that under certain conditions T lymphocytes will express colipase. (See Geo Profiles: GDS993, GDS2649 and GDS1336.) We wondered if an unknown factor associated with the exception lot 60506 of Il-4 could have induced colipase. Since lot 60506 was consumed, direct information was impossible. As an alternative approach, we tested if the addition of purified colipase to CTLs induced with standard lots of IL-4 would support lipid-enhanced cytotoxicity. The colipase increased the triglyceride-enhanced cytotoxicity mediated by purified PTL (used as a control to indicate the efficacy of the colipase) but the colipase was without effect on the IL-4 induced CTLs. At this time, we can confidently report that PLRP2-positive CTLs induced with standard lots of IL-4 are unable to mediate the triglyceride-dependent cytotoxicity that was observed for recombinant PLRP2 with colipase and P815 cells above. In addition, the results associated with IL-4 lot 60506 indicate the existence of CTL-mediated triglyceride lipid-dependent cytotoxicity.