Virus-induced natural killer cell lysis of T cell subsets

Abstract

In addition to direct anti-viral activity, NK cells regulate viral pathogenesis by virtue of their cytolytic attack on activated CD4 and CD8 T cells. To gain insight into which differentiated T cell subsets are preferred NK targets, transgenic T cells were differentiated in vitro into Th0, Th1, Th2, Th17, Treg, Tc1, and Tc2 effector cells and then tested for lysis by enriched populations of lymphocytic choriomeningitis virus (LCMV)-induced activated NK cells. There was a distinct hierarchy of cytotoxicity in vitro and in vivo, with Treg, Th17, and Th2 cells being more sensitive and Th0 and Th1 cells more resistant. Some distinctions between in vitro vs in vivo generated T cells were explainable by type 1 interferon induction of class 1 histocompatibility antigens on the effector T cell subsets. NK receptor (NKR)-deficient mice and anti-NKR antibody studies identified no one essential NKR for killing, though there could be redundancies.

Keywords: CD4; CD8; Cytotoxicity; Interferon; Lymphocyte; Lymphocytic choriomeningitis virus; Mouse; Natural killer; Pathogenesis; T cell.

Figure 1.. Differentiated T cell cultures.

OT-I (CD8-Tc1 and Tc2) and OT-II (CD4-Th0, Th1, Th2, Th17, and Treg) short term (4 day) transgenic T cell cultures were set up in the presence of cognate peptide and differentiation agents, as described in the Materials and Methods. Cells were stained either directly (-stimulation) or after re-stimulation with PMA and Ionomycin (+ stimulation) and analyzed by flow cytometry.

Figure 2.. Lysis of target cells by unfractionated spleen leukocytes.

Plots show 4-hour ⁵¹Cr-release cytotoxicity assays against YAC-1, P52, and NCTC929 (L-929) continuous cell lines and against cultured Th0, Th1, and Th2 differentiated effector T cell targets. Solid lines represent lysis by day 3 LCMV-induced C57BL/6 mouse spleen leukocytes, and dashed lines represent lysis by spleen leukocytes from uninfected mice.

Figure 3.. MACS column-enrichment of NK cells and lysis of differentiated T cell cultures.

NK cells were partially purified from spleen leukocytes from day 3 LCMV-infected C57BL/6 mice and tested in 4-hour ⁵¹Cr-release cytotoxicity assays against Th1 and Th2 cultures. A. Upper panels are low cytometry plots against NK1.1 (y-axis) and NKp46 (x-axis). Boxes in upper panels represent %NK1.1+ cells. B. Unfractionated cytotoxicity data are plotted at E:T = 100:1, whereas enriched and depleted samples are plotted at E:T =10:1.

Figure 4.. Hierarchy of killing of differentiated T cell cultures.

A. In a single experiment all studied effector T cell subset cultures were tested for susceptibility to killing by NK cell-enriched populations of LCMV day 3 spleen leukocytes (solid lines) or by the NK cell-depleted population (dotted lines). Data are from a 4-hour ⁵¹Cr-release cytotoxicity assay. B. Correlation of cell surface expression of class 1 D^b antigens, as analyzed by flow cytometry, with cytotoxicity (E:T=5:).

Figure 5.. Mechanisms of NK cell killing of T cell targets.

Lysis of T cell targets by NK cell-enriched populations of LCMV-induced day 3 leukocytes from C57BL/6 (panel A), perforin KO (panel B), or type 1 IFN R KO (panel C) mice. Panel D: Cold target competition against killing of Th2 and Treg cultures by NK cell-depleted LCMV day 3 leukocyte fractions.

Figure 6.. In vivo cytotoxicity assay against differentiated T cell cultures.

This depicts in vivo cytotoxicity assays against cultured differentiated CD4 T cells by displaying the cytotoxic values in individual mice from 2–3 experiments per T cell subset. Details of this assay are in the Materials and Methods section. A. Representative histograms showing the survival of cell populations in NK cell replete (control IgG-treated) vs. anti-NK1.1-depleted mice. The T cell subsets are identified by the concentration of the cell trace violet dye. B. Panel showing that the cytotoxicity against Treg, Th17, and Th2 cells was significantly greater than that against the Th0 and Th1 cells, as determined by Students t test (p<0.0001).

Figure 7.. Modulation of differentiated T cell effectors. A. IFN-induced protection of differentiated T cells in vitro.

A. Differentiated transgenic T cells were treated (or not) with 500 units/ml of IFNβ for 24 hrs and tested as targets of NK cell-enriched LCMV-induced day 3 spleen leukocytes in 4-hr ⁵¹Cr-release cytotoxicity assays. This experiment examined the Th0, Th1, Treg, and Tc1 cells, but only the first three are plotted, for better resolution of the graph. B. Expression of Class 1 D^b on Treg cells in vivo. Fox-P3-GFP mice were inoculated with LCMV, and on days 0, 3, and 5 post-infection their CD4-gated spleen lymphocytes were examined for co-expression of the FoxP3-GFP transgene and for staining with mAb to class 1 D^b.

Figure 8.. Lysis of differentiated T cell effectors by NK cell-enriched spleen leukocytes from day 3 LCMV-infected NKR mutant mice.

Panel A. Shows normal levels of lysis of T cell cultures by enriched NK cells from Day 3 LCMV-infected C57BL/6 control, dap 10/12 KO, and ncr mutant (NKp46-deficient) mice. Panel B. Shows killing by NK cell-enriched leukocytes from Day 3 LCMV-infected C57BL/6 control, DNAM1/NKG2D KO, and ncr mutant (NKp46-deficient) mice.

Figure 9.. Blockade of NK cell-mediated cytotoxicity against differentiated T cell cultures.

Panels A, B, and C represent cytotoxicity assays against Th0, Th1, and Th2 cultures by NK cell-enriched day 3 LCMV-induced spleen leukocyte populations in 4-hr ⁵¹Cr-release cytotoxicity assays, at E:T ratio = 5:1. These were added to all samples except those in the second column, which received NK-cell depleted leukocytes from day 3 LCMV-infected mice.. Except for the first two columns, additional agents were added to block the killing. These include mAb to NKR or combinations of anti-NKR mAb. The green hatched bar represents killing in the presence of a rat IgG1, k control mAb with no specificity to NK cells. *p< .05; **, p<.00.5