Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes
N. Manjunath, P. Shankar, J. Wan, W. Weninger, M.A. Crowley, K. Hieshima, T.A. Springer, X. Fan, H. Shen, J. Lieberman, U.H. von Andrian
N. Manjunath, P. Shankar, J. Wan, W. Weninger, M.A. Crowley, K. Hieshima, T.A. Springer, X. Fan, H. Shen, J. Lieberman, U.H. von Andrian
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Article

Effector differentiation is not prerequisite for generation of memory cytotoxic T lymphocytes

Abstract

The lineage relationship between short-lived effector T cells and long-lived memory cells is not fully understood. We have described T-GFP mice previously, in which naive and early activated T cells express GFP uniformly, whereas cells that have differentiated into effector cytotoxic T cells selectively lose GFP expression. Here we studied antigen-specific CD8 T cell differentiation using T-GFP mice crossed to the TCR transgenic (Tg) mice P14 (specific for the lymphocytic choriomeningitis virus glycoprotein peptide, gp33-41). After activation with antigenic peptide, P14XT-GFP CD8+ T cells cultured in high-dose IL-2 developed into cells with effector phenotype and function: they were blastoid, lost GFP expression, expressed high levels of activation and effector markers, and were capable of immediate cytotoxic function. In contrast, cells cultured in IL-15 or low-dose IL-2 never developed into full-fledged effector cells. Rather, they resembled memory cells: they were smaller, were GFP+, did not express effector markers, and were incapable of immediate cytotoxicity. However, they mediated rapid-recall responses in vitro. After adoptive transfer, they survived in vivo for at least 10 weeks and mounted a secondary immune response after antigen rechallenge that was as potent as endogenously generated memory cells. In addition to providing a simple means to generate memory cells in virtually unlimited numbers, our results suggest that effector differentiation is not a prerequisite for memory cell generation.

Authors

N. Manjunath, P. Shankar, J. Wan, W. Weninger, M.A. Crowley, K. Hieshima, T.A. Springer, X. Fan, H. Shen, J. Lieberman, U.H. von Andrian

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Figure 1

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Effect of IL-2 and IL-15 on activated CD8 T cell proliferation and funct...
Effect of IL-2 and IL-15 on activated CD8 T cell proliferation and function. (a and b) All tested concentrations of IL-2 and IL-15 at 5 ng/ml or greater support cell proliferation. Cell proliferation was measured by viable cell counts (a) or BrdUincorporation (b) as described in Methods. In b, histograms of BrdU incorporation by CD8+ gated cells on day 3 of cytokine treatment are shown. Background staining obtained with isotype control is also shown for comparison. Similar results were seen for all treatment groups on each day for all 5 days tested (not shown). (c) IL-2 induces increased cell death compared with IL-15. Cell death was measured by flow cytometric analysis of non-gated cells after staining with propidium iodide (PI). Shown are dot plots after 3 days of cytokine treatment. (d) Both IL-2– and IL-15–treated cells acquire the ability for IFN-γ production. On day 7 following peptide stimulation and cytokine treatment, the cultures were restimulated with αCD3 for 6 hours, stained externally with αCD8 Cy 5, intracellularly with αIFN-γ Ab, and examined by flow cytometry. Less than 2% of unstimulated cells produced IFN-γ (not shown). (e) Peptide primed cells cultured with IL-2 at concentrations of 10 ng/ml or greater exhibit high levels of cytotoxicity, whereas cells cultured with IL-2 at or below 5 ng/ml and all tested concentrations of IL-15 exhibit greatly diminished levels of cytotoxicity. On day 7 after stimulation, cultures were tested for specific killing of gp33 peptide-pulsed EL-4 target cells by chromium-release assay.