Intestinal microbes affect phenotypes and functions of invariant natural killer T cells in mice

Abstract

Background & aims: Invariant natural killer T (iNKT) cells undergo canonical, Vα14-Jα18 rearrangement of the T-cell receptor (TCR) in mice; this form of the TCR recognizes glycolipids presented by CD1d. iNKT cells mediate many different immune reactions. Their constitutive activated and memory phenotype and rapid initiation of effector functions after stimulation indicate previous antigen-specific stimulation. However, little is known about this process. We investigated whether symbiotic microbes can determine the activated phenotype and function of iNKT cells.

Methods: We analyzed the numbers, phenotypes, and functions of iNKT cells in germ-free mice, germ-free mice reconstituted with specified bacteria, and mice housed in specific pathogen-free environments.

Results: Specific pathogen-free mice, obtained from different vendors, have different intestinal microbiota. iNKT cells isolated from these mice differed in TCR Vβ7 frequency and cytokine response to antigen, which depended on the environment. iNKT cells isolated from germ-free mice had a less mature phenotype and were hyporesponsive to activation with the antigen α-galactosylceramide. Intragastric exposure of germ-free mice to Sphingomonas bacteria, which carry iNKT cell antigens, fully established phenotypic maturity of iNKT cells. In contrast, reconstitution with Escherichia coli, which lack specific antigens for iNKT cells, did not affect the phenotype of iNKT cells. The effects of intestinal microbes on iNKT cell responsiveness did not require Toll-like receptor signals, which can activate iNKT cells independently of TCR stimulation.

Conclusions: Intestinal microbes can affect iNKT cell phenotypes and functions in mice.

Figure 1. Distribution and phenotype of intestinal iNKT cells

(A) Lymphocytes from the indicated sites were incubated either with αGalCer loaded or unloaded CD1d-tetramers, analyzed by flow cytometry and the frequencies of tetramer-positive cells within live TCRβ⁺CD44⁺CD8α⁻CD19⁻ cells are shown. (B, C) Relative percentage of iNKT cells within total live lymphocytes (B) and their expression of CD4 and NK1.1 (C), from indicated sites. The graphs summarize data from 3–5 independent experiments, with 6–9 samples per group. (D) Representative expression of CCR9 on iNKT cells derived from the spleen (tinted, in both panels), IEL (dashed) or LPL (black line) from the small or large intestine. The numbers in histograms denote the geometric mean values for CCR9 on iNKT cells.

Figure 2. Environmental influences on the responsiveness and Vβ-usage of iNKT cells

(A-C) C57BL/6 animals, purchased from either Taconic Farms (Tac) or Jackson Laboratory (Jax), were either analyzed within one week after delivery (top panels). Or alternatively, new-born offspring from Tac or Jax mice were co-housed from 2–5 days after birth until analysis 8–10 weeks later (lower panels). Relative frequency of iNKT cells (A) and their Vβ7-usage (B) in indicated organs is shown. Production of indicated cytokines by splenic iNKT cells 90min after i.v. injection of αGalCer was analyzed by intracellular staining (C). Representative data from four (top panels) or three (lower panels) independent experiments are shown. (D, E) Frequency of CD127⁺CD4⁻ iNKT cells in indicated organs (D) or from SI-LPL (E) from indicated mice. Representative data from three independent experiments are shown.

Figure 3. iNKT cells from germ-free animals are hyporesponsive

(A) Expression of CD69, CD25 and CD5 by iNKT cells from indicated organs derived from germ-free (GF) or specific-pathogen-free (SPF) housed Swiss Webster mice. (B) Expression of CD69 (left panel) and indicated cytokines (right panel) by splenic iNKT cells from GF or SPF housed Swiss Webster mice with or without αGalCer challenge in vivo (90min). The expression of CD69 following αGalCer increased on SPF derived iNKT cells 1.9fold (MFI), whereas the increase on GF derived iNKT cells was lower at 1.75fold (p_{(SPF +/- αGalCer vs GF +/- αGalCer)}= 0.004). (C) Splenocytes from GF and SPF Swiss Webster mice were co-cultured with αGalCer loaded RMA-CD1d cells for 4h and cytokine production by iNKT cells was analyzed by intracellular staining. (D) GF or SPF housed animals on the C57BL/6 background were injected with αGalCer and the cytokine production by splenic iNKT cells was analyzed 90min later. The graph summarizes data from two independent experiments, with 4–5 mice per group. (E) αGalCer-specific in vivo cytotoxicity in spleen 4h after injection of B cell targets into GF or SPF housed Swiss Webster mice. Representative data from two independent experiments are shown. (F) Relative percentage of iNKT cells within TCRβ⁺ live lymphocytes (left) and of CD127⁺CD4⁻ iNKT cells (right) from indicated organs of GF or SPF housed Swiss Webster animals. The graphs summarize data from three independent experiments, with 5–8 mice per group.

Figure 4. Bacterial products promote iNKT cell responsiveness in a TLR independent fashion

(A, B) C57BL/6J wild-type and MyD88^−/−Trif^Lps2/Lps2 animals were either mock treated or injected with αGalCer and 90min later the expression of indicated surface markers (A) and cytokines (B) by splenic iNKT cells was analyzed. (C, D) C57BL/6J wild-type and IL-12^−/− animals were either mock treated or injected with αGalCer and 90min later the expression of indicated surface markers (C) and cytokines (D) by splenic iNKT cells was analyzed. Representative data from two independent experiments are shown.

Figure 5. Bacterial exposure corrects the hyporesponsive phenotype of iNKT cells

(A) GF and SPF housed Swiss Webster mice were co-housed for four weeks and expression of CD69 in splenic iNKT cells was analyzed. The numbers in histograms denote the geometric mean values for CD69 on iNKT cells. (B–D) GF and SPF housed Swiss Webster mice were mock treated or intra-gastrically challenged with either S. yanoikuyae or E. coli bacteria as indicated. Four to five days later the expression of CD69 in splenic iNKT cells was analyzed and is represented as example histogram (B) or as summary (C). Furthermore, the relative frequency of Vβ7⁺ iNKT cells is shown (D). (E, F) Expression of CD69 (E) and indicated cytokines (F) by splenic iNKT cells from restricted-flora (RF) and SPF housed C57BL/6 mice with or without αGalCer challenge in vivo (90min). Representative data from two (A, E, F), three (D) or four (B, C) independent experiments are shown.