Upon performing a cytotoxicity study using NK and CD8+ T cells isolated from wildtype and gene knockout Black 6 mouse splenocytes, the knockout immune cells induce more cell death and granzyme B secretion when co-cultured with lewis lung carcinoma cells. This was measured by cytotoxicity assays and ELISpots. Interestingly, I polyclonally actived the immune cells ex vivo in culture but did not pre-activate them to tumor antigens while in the mouse.
Can anyone explain why I see differences in killing abilities and seemingly that the immune cells are able to recognize and kill the tumor cells? Would there be any way that the immune cells would recognize the tumor cells in vitro without pre-activation in vivo?
So you did a bulk activation of the mouse splenocytes and then used those stimulated cells in cytotoxicity assays? Or did you just use straight-up, untouched splenocytes in the assay?
What was the knockout? If you see differences between your knockout and your wild-type control, wouldn't it be reasonable to conclude that the difference is a result of knocking out whatever gene you knocked out?
I have some thoughts:
1) The tumor cells may be expressing self-antigens similar to the cancer testis antigens (MART-1, NY-ESO-1, etc) that generally are not centrally tolerated (as they're usually only expressed in a certain subset of tissues in early development) but can re-appear at high levels in tumors. Really any peripherally tolerated self-antigen could potentially have a cytotoxic T-cell that recognizes it - upon non-specific activation and expansion of the splenocyte CD8+ population, tolerance to these antigens would probably be broken and they could elicit reactivity from antigen-specific T-cells
2) The tumor antigens are cross-reactive with antigens that the mouse has previously been sensitized to. There have been some studies where previous exposure to one pathogen has resulted in a memory response that also provided protection against a second, unrelated pathogen in people who have never been exposed to that pathogen (PMID:23395677). I suppose a similar thing could be happening here
3) I've noticed in my own work that mouse CD8 T-cells tend to take on an LAK phenotype when they've been chronically exposed to too much IL-2 for too long in vitro (this is especially true if you're using human IL-2). I don't know what your activation method is, but I'd be careful with the hIL-2 dosing on those mouse T-cells.
I hope that helps.
Hi Govinda,
Yes, I isolated the splenocytes then activated them in vitro with 1000U/ML mouse recombinant IL-2 for NK and 50U/ML with CD3/CD28 beads for CD8+ T cells.
Hmm...it doesn't sound like you're overdosing your cells with IL-2, so there's probably no weird new LAK cells showing up in culture.
NK cells do have some natural antitumor activity (especially since many tumors have decreased MHC-I expression), so it doesn't surprise all that much that the NKs are killing the tumor cells in vitro.
Expanding the T-cells with the beads and low-dose IL-2 will selectively expand the CTL compartment (I don't think T-regs will grow out, they need high IL-2). So if there are T-cells with some antigen-specific reactivity to the tumor, they're probably being completely dis-inhibited by the expansion process and are free to kill the tumor in vitro.
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