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Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells

Nature Immunology volume 14, pages 1146–1154 (2013)Cite this article

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A Corrigendum to this article was published on 18 February 2014

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Abstract

Invariant natural killer T cells (iNKT cells) can produce copious amounts of interleukin 4 (IL-4) early during infection. However, indirect evidence suggests they may produce this immunomodulatory cytokine in the steady state. Through intracellular staining for transcription factors, we have defined three subsets of iNKT cells (NKT1, NKT2 and NKT17) that produced distinct cytokines; these represented diverse lineages and not developmental stages, as previously thought. These subsets exhibited substantial interstrain variation in numbers. In several mouse strains, including BALB/c, NKT2 cells were abundant and were stimulated by self ligands to produce IL-4. In those strains, steady-state IL-4 conditioned CD8+ T cells to become 'memory-like', increased serum concentrations of immunoglobulin E (IgE) and caused dendritic cells to produce chemokines. Thus, iNKT cell–derived IL-4 altered immunological properties under normal steady-state conditions.

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Figure 1: BALB/c iNKT cells produce IL-4 in the steady state.
Figure 2: PLZF, RORγt and T-bet differentiate NKT1, NKT2 and NKT17 cells.
Figure 3: IL-4-producing NKT2 cells do not give rise to NKT1 cells.
Figure 4: Steady-state IL-4 is produced by NKT2 cells and is enhanced in the absence of T-bet.
Figure 5: Interstrain comparison of iNKT cell subsets; production of IL-4 by NKT2 cells is associated with memory-like CD8+ T cells.
Figure 6: IL-4-producing iNKT cells are stimulated by self ligands.
Figure 7: Steady-state IL-4 produced by iNKT cells influences B cells and thymic DCs.

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  • 28 October 2013

    In the version of this article initially published, the upper graph of Figure 1b was incorrectly duplicated as the lower graph. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank J. Ding and S. Perry for technical support; M. Mohrs (Trudeau Institute) for KN2 mice on the B6 and BALB/c background; D.B. Sant'Angelo (Rutgers University) for Alexa Fluor 488–conjugated antibody to PLZF; M. Mohrs and M. Kronenberg for discussions; and S. Hamilton for critical review of the manuscript. Supported by the US National Institutes of Health (R37-AI39560 to K.A.H.; RO1-AI075168 to S.C.J.; T32 HD060536 to K.L.H.; and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, for J.Z.) and the Cancer Research Institute (Y.J.L.).

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Authors and Affiliations

  1. The Department of Laboratory Medicine and Pathology, Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA

    You Jeong Lee, Keli L Holzapfel, Stephen C Jameson & Kristin A Hogquist

  2. Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Jinfang Zhu

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Contributions

Y.J.L. designed and did experiments, analyzed data and wrote the manuscript; K.L.H. did experiments and provided input for interpretation; J.Z. provided reagents and research interpretation; S.C.J. provided input for research design and interpretation; and K.A.H. conceptualized the research, directed the study, analyzed data and edited the manuscript.

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Correspondence to Kristin A Hogquist.

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Integrated supplementary information

Supplementary Figure 1 GATA-3, IRF-4 and surface marker expression profiles in subsets of iNKT cells.

(a) Thymic iNKT cells from 7 week-old B6 and BALB/c mice were intracellularly stained with GATA-3 and IRF-4. Expression on NKT1, NKT2 and NKT17 subsets, as defined in Fig. 2a is shown. Histogram plots of each subset were overlaid with that of DP thymocytes (grey). (b) iNKT cells from 7 week-old B6 or BALB/c mice were analyzed for the forward and side scatter profile with expression patterns of CD4, CD122, CD27 and NK1.1 or DX5. (c) Thymic iNKT cells from B6 and BALB/c KN2 mice were stained with PLZF, IL-17RB and hCD2. (d) Thymic iNKT cells from B6 and BALB/c mice were stained with PLZF, RORγt and IL-17RB. Representative data from three independent experiments are shown (a ∼ d).

Supplementary Figure 2 iNKT cell phenotype in spleen and liver.

(a) iNKT cells from spleen and liver of 7 week-old WT B6 and BALB/c mice were stained with PLZF, T-bet and RORγt. Representative data of five independent experiments are shown. (b) Statistical analysis of percent iNKT cells among total cells (left panel) or each iNKT subset among total iNKT cells (middle and right panels) in spleen and liver of 6 to 8 week-old mice is shown (n = 4~6). Each symbol represents an individual mouse and unpaired two tailed t-tests were used for comparison. Pooled data from five independent experiments. ** P<0.0001, * P=0.015; NS, not significant (P>0.05). (c) T-bet green reporter (T-betGFP) KN2 mice in B6 and BALB/c background were intravenously injected 5μg of α-GalCer and analyzed 3 hours later for cytokine secretion. Representative data from three independent experiments are shown.

Supplementary Figure 3 iNKT cell subsets in T-bet-deficient mice.

(a and b) Representative flow cytometry profile of thymic iNKT cells from 12 week-old mice and statistical analysis for 10-14 week-old mice using B6 WT (n = 13) Tbx21+/− (n = 16) and Tbx21−/− (n = 5) mice are shown. Pooled data from three independent experiments. (c) Mixed bone marrow chimeras were generated with equal or unequal ratio of donor bone marrow cells using B6 WT and B6 Tbx21−/− mice. Six weeks later mice were sacrificed and analyzed iNKT phenotype. (d) Statistical analysis of WT or Tbx21−/− iNKT cells in 6 week-old BM chimeras (n = 4 ∼ 5). Numbers indicate percentages of cells among total iNKT cells of WT or Tbx21−/− donor. Each Line indicates an individual mouse and paired two tailed t-tests were used. (e) Frequency of Eomes+ or CD44hiCXCR3+ cells among CD8SP thymocytes are shown for 6 week-old B6 WT (n = 15) Tbx21+/− (n = 15), and Tbx21−/− (n = 4) mice. Pooled data from five independent experiments. (f) Frequencies of Eomes+ or CD44hiCXCR3+ cells among CD8SP thymocytes from WT (n = 4) Tbx21+/− (n = 5), and Tbx21+/−Cd1d−/− (n = 3) CB6 mice are shown. Pooled data from two independent experiments. (g) Representative flow cytometry profile of CD8SP thymocytes from CB6 mice of indicated genotypes is shown. ***P<0.001, **P<0.01, *P<0.05; NS, not significant (P>0.05). Each symbol represents an individual mouse and one-way ANOVA was used for analysis (a, b, e and f).

Supplementary Figure 4 KLF2 deficiency facilitates the development of NKT2 cells.

(a) Thymic iNKT cells from 6 week-old KLF2 deficient and littermate control mice were analyzed for PLZF, T-bet and RORγt expression. (b) Numerical analysis of iNKT subsets of WT (n = 8) and Klf2−/− (n = 3) B6 mice is shown. Unpaired two tailed t-tests were used to compare each subset. Pooled result of three independent experiments.

Supplementary Figure 5 Revised model for iNKT cell differentiation.

(a) The currently held “linear stages” model supports a linear differentiation of iNKT cells from immature stage 1 cells to mature stage 3 cells. (b) Our data support a new transcription factor based model of iNKT subsets, where terminally differentiated cells producing distinct cytokines derive from a common precursor.

Supplementary Figure 6 IL-4-producing iNKT cells are stimulated by self ligands.

(a) Thymic iNKT cells from BALB/c KN2 mice were MACS enriched by depleting CD8 and CD24 positive cells, labeled with violet cell tracer (VCT) and intrathymically (IT) injected into WT or CD1d KO BALB/c hosts. Six days later, VCT positive donor iNKT cells were analyzed after enrichment of CD1d tetramer positive cells. Gates in right panel show percentage of PLZFhi NKT2 cells expressing hCD2. Unpaired two tailed t-tests were used to compare frequency of NKT2 cells in WT (n = 5) or CD1d−/− (n = 5) hosts. (b) Six to seven weeks old B6 (n = 6) and BALB/c (n = 6) mice were analyzed for TCR Vβ 2, 7 and 8 repertoire by flow cytometry. One-way ANOVA was used to compare the frequency of NKT1, NKT2 and NKT17 cells. Pooled results from two independent experiments. *** P<0.001, **P<0.01, * P<0.05. NS, not significant (P>0.05).

Supplementary Figure 7 Age-dependent kinetics of the NKT1, NKT2 and NKT17 lineages in B6 and BALB/c thymi.

(a) Number (left panel) and frequency among total thymocytes (right panel) of iNKT cells are shown. (b) Percentages of each iNKT subset among total iNKT cells are shown. (c) Frequency of NKT2 cells among total thymocytes (left), Eomes+ cells among CD8SP thymocytes (middle) and hCD2+ iNKT cells among total thymocytes (right) are shown. Data are represented as mean +/− SD at each time point. Pooled data of 12 independent experiments with 3~9 mice in each time point and a total of 38 B6 and 35 BALB/c mice. Unpaired two tailed t-tests were used to compare indicated time points. ***P<0.001, **P<0.01, * P<0.05; NS, not significant (P>0.05).

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Lee, Y., Holzapfel, K., Zhu, J. et al. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat Immunol 14, 1146–1154 (2013). https://doi.org/10.1038/ni.2731

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