Antigen-dependent versus -independent activation of invariant NKT cells during infection

Abstract

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation.

Fig. 1. iNKT cells upregulated GFP in response to various antigenic lipids, including self-lipids

Expression of GFP (top) and CD69 (bottom) by splenic iNKT cells from B6 Nur77^gfp mice 18 h after injection with bone marrow-derived unpulsed DCs (cntrl) or BMDCs pulsed with αGalCer, OCH, or the self-lipids βGlcCer or iGb3 as described in Materials and Methods. Histograms are representative of eight independent experiments. Average relative mean fluorescence intensity (MFI) of GFP (normalized to that in CD4 T cells in each experiment) is indicated.

Fig. 2. MCMV infection induced iNKT cell cytokine production, but not GFP expression

Nur77^gfp B6 mice were infected with MCMV i.p. and tet⁺ liver iNKT cells were analyzed by flow cytometry 36 h later for GFP expression (A) and IFNγ production (B). Results show uninfected (gray), MCMV infected (red) and αGalCer treated animals 3 h after injection (black). Data are representative of two independent experiments, with at least 3 animals per time point. The histogram in (B) shows GFP expression of the IFNγ⁻ iNKT cell population (black) versus the IFNγ⁺ iNKT cell population (red) from an animal 36 h post infection. In graphs, each dot represents one mouse (Uninfected n = 6; 12 h post infection n = 3; 24 h post infection n= 3; 36 h post infection n= 6; 3 h post αGalCer injection n= 3). **P = 0.0026, percentage uninfected IFNγ⁺ iNKT cells versus percentage 36 h post infection IFNγ⁺ iNKT cells (unpaired two-tailed t-test).

Fig. 3. S. pneumoniae infection induced cytokine production and GFP expression

Nur77^gfp B6 mice were infected with S. pneumoniae i.v. and tet⁺ iNKT cells were analyzed by flow cytometry for GFP expression (A) and IFNγ production (B). Results shown are from uninfected mice (gray), mice 6 hr post S. pneumoniae infection (red) and αGalCer treated animals 2.5 h after injection (black). The histogram in (B) shows GFP expression in the IFNγ⁻ iNKT cell population (black) versus the IFNγ⁺ iNKT cell population (red) from 20 h post infection. Data show representative dot plots of two independent experiments with 6 animals per time point. For percentage of GFP⁺ iNKT cells graph (A), ***P =0.0006, uninfected versus 6 h post infection; and ***P =0.0007, uninfected versus 20 h post infection. For percentage of IFN-γ⁺ iNKT cells graph (B), *P = 0.0478, uninfected versus 6 h post infection; *P = 0.0483, uninfected versus 20 h post infection (all using unpaired two-tailed t-test).

Fig. 4. S. paucimobilis infection induced cytokine production and GFP expression

IL-4 reporter (KN2) Nur77^gfp B6 mice were infected with S. paucimobilis i.v. and tet⁺ iNKT cells were analyzed by flow cytometry 18 h post infectionfor GFP (A) and human CD2 expression (B). Histograms in (A) show GFP expression for uninfected (gray), 18 h infected (red) and 3 h αGalCer injected (black) animals. The histogram in (B) shows GFP expression on the hCD2-iNKT cell population (black) versus the hCD2⁺ iNKT cell population (red) from an animal 18 h post infection. Data show representative dot plots of two independent experiments with 4 to 6 animals per time point. **P = 0.0082, uninfected versus 18 h post infection (unpaired two-tailed t-test).

Fig. 5. S. typhimurium infection induced cytokine production, but not GFP expression

Nur77^gfp B6 mice were infected with S. typhimurium i.v. and tet⁺ iNKT cells were analyzed by flow cytometry directly ex vivo for GFP expression (A) and IFN-γ production (B). The histogram shows uninfected mice (gray), 20 h S. typhimurium infected (red) and 3 h αGalCer injected (black) animals. The histogram in (B) show GFP expression of the IFNγ⁻ iNKT cell population (black) versus the IFNγ⁺ iNKT cell population (red) from an animal 20 h post infection. Data show representative dot plots and histogram of four independent experiments with 4 to 6 animals per time point. For percentage of IFNγ⁺ iNKT cells graph (B), **P =0.0059, uninfected versus 4 h post infection; ***P <0.0001, uninfected versus 20 h post infection; ***P <0.0001, uninfected versus 72 h post infection (all with unpaired two-tailed t-test).

Fig. 6. iNKT cell cytokine production is CD1d independent during S. typhimurium infection

iNKT cells were purified from Nur77^gfp Vα14 TCR transgenic B6 mice and transferred into either WT hosts (iNKT → WT) or CD1d^−/− hosts (iNKT → CD1d^−/−). Mice were then infected with S. typhimurium i.v. and the transferred tet⁺ iNKT cells were isolated and analyzed by flow cytometry for intracellular IFNγ 20 h post infection. Dot plots (left) show representative data of iNKT cells isolated from an uninfected WT host (top, left), an uninfected CD1d^−/− host (bottom, left), an infected WT host (top, right) or infected CD1d^−/− host (bottom, right) from two independent experiments with 3 to 6 animals per group. *P = 0.0127, uninfected in WT host versus 20 h post infection in WT host; *P = 0.0381, uninfected in CD1d^−/− host versus 20 h post infection in CD1d^−/− host (unpaired two-tailed t-test).

Fig. 7. iNKT cell cytokine production is CD1d independent during TLR stimulation

Nur77^gfp B6 mice were injected with LPS (top panel) or CpG (bottom panel) i.v. and tet⁺ iNKT cells were analyzed by flow cytometry 4 or 36 h later, respectively, for GFP expression, CD69 expression, and IFNγ production. Representative histograms show GFP expression (left) or CD69 expression (middle) for uninjected (gray), LPS or CPG injected (red), or 2-3 h αGalCer injected animals (black) from two independent experiments with 3 animals per group. For percentage of IFNγ⁺ iNKT cells graph (right, top), **P = 0.0026, uninjected versus 4 h post LPS injection. For percentage of IFNγ⁺ iNKT cells graph (right, bottom), **P = 0.0092, uninjected versus 36 h post CpG injection (unpaired two-tailed t-test).