Normal development and function of invariant natural killer T cells in mice with isoglobotrihexosylceramide (iGb3) deficiency

Abstract

CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Valpha14-Jalpha18 gene segments in mice and Valpha24-Jalpha18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4(+)/CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vbeta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Fig. 1.

Metabolic pathways of globo- and isoglobo-series of GSL. iGb3 and Gb3, the initial members of isoglobo- and globo-series, respectively, are synthesized from lactosylceramide (LacCer) by addition of a galactose (Gal) sugar moiety in α1–3 or α1–4 linkage, respectively. Through subsequent addition of N-acetylgalactosamine (GalNAc), iGb4 and Gb4 are formed. Lysosomal breakdown (dashed arrows) of iGb4 and Gb4 is performed by β-hexosaminidases A and B, which both require a functional β-subunit (Hexb). Furthermore, through the action of iGb3S on iGb3 and Gb3, polyGalα1–3iGb3 and polyGalα1–3Gb3 structures are formed. For sake of clarity, other enzymes and members of these GSL series have been omitted.

Fig. 2.

Generation of iGb3S-deficient mouse. (A) Targeting strategy showing murine iGb3S WT locus, targeting vector and the targeted locus before and after Cre-mediated recombination together with relevant restriction sites and probes for Southern blot analysis. Coding and noncoding regions of exon 1–5 are marked by filled and nonfilled boxes, respectively. Homology arms are depicted as bold lines. Arrowheads show the position and orientation of PCR primers. Triangles flanking the PGK-gb2-neomycin selection cassette (neo) symbolize loxP sequences. (B) Southern blot analysis using 3′ external probe, as indicated in A, shows shortening of the diagnostic 22.8-kb-long NdeI fragment down to 13.7 kb after homologous recombination. (C) A replica of the Southern blot filter as used in B was hybridized with a probe specifically recognizing the coding part of exon 5 to demonstrate its deletion in iGb3S^−/− mice. (D) Cre-mediated deletion of the neomycin selection cassette was confirmed by PCR by using primers F2 and R2 as a shortening of the 2,044-bp-long product to 494 bp. neo/neo and −/−, targeted locus before and after Cre-mediated deletion of neomycin selection cassette, respectively. (E) Detection of iGb3S-specific mRNA by RT-PCR by using total RNA from lung, thymus, spleen, and kidney of iGb3S^+/− and iGb3S^−/− animals; GAPDH was used as a positive control.

Fig. 3.

HPLC analysis of GSL composition of DRG and thymus. Highly sensitive HPLC analysis [see Speak et al. (24) for details] demonstrates the absence of iGb3 in DRG of iGb3S^−/− mice compared with controls. iGb4 is the biosynthetic derivative of iGb3 in the isoglobo-series and is consequently also absent. An additional three peaks are absent in iGb3S^−/− DRG (labeled A–C) compared with controls. These peaks are sensitive to digestion with α1–3,6-galactosidase and are therefore most likely poly-Galα1–3 (i)Gb3 GSLs. There is no evidence for any of these species being present in iGb3S^+/− thymus, and no difference is seen between iGb3S^+/− and iGb3S^−/− thymus. Profiles are representative of three independent observations on three different animal pairs with equivalent protein quantities processed for the analysis.

Fig. 4.

iNKT population shows no deviations in number, development, and TCRVβ usage in iGb3S^−/− mice. (A) Thymocytes, splenocytes, and hepatic leukocytes from 8- to 10-week-old iGb3S^+/− and iGb3S^−/− mice were stained with anti-CD19 and -CD44 antibodies and with either αGalCer-loaded CD1d-tetramers to visualize iNKT population or unloaded CD1d-tetramers to define the amount of nonspecific binding. Plots were gated on lymphocytes. In case of spleen, CD19⁺ cells have been gated out. Numbers indicate the percentage of CD1d-tetramer⁺/CD44⁺ cells in the lymphocyte gate ± SEM; n = 5. (B) Thymocytes from 8-week-old iGb3S^+/− and iGb3S^−/− mice were stained with anti-NK1.1, -CD3, and -CD44 antibodies and αGalCer-loaded CD1d-tetramers to determine the amount of cells in immature (CD44⁻/NK1.1⁻), semimature (CD44⁺/NK1.1⁻), and mature (CD44⁺/NK1.1⁺) stage. Analysis was gated on CD3⁺/αGalCer-CD1d⁺ cells, and bars represent the percentage of cells with the depicted phenotype ± SEM; n = 4. (C) Splenocytes from 8- to 10-week-old iGb3S^+/− and iGb3S^−/− mice were stained with αGalCer-loaded CD1d-tetramers, anti-CD19 and -CD44, and either TCRVβ2, -7, or -8.2 antibodies. Analysis was gated on CD19⁻/αGalCer-CD1d⁺/CD44⁺ lymphocytes, and the bars represent the percentage of cells positive for the corresponding TCRVβ-chain ± SEM; n = 6. No significant differences could be seen between iGb3S^+/− and iGb3S^−/− mice in A–C.

Fig. 5.

In vivo response to αGalCer injection is unaffected in iGb3S^−/− mice. iGb3S^+/− and iGb3S^−/− mice (12 week old) were injected intravenously with 1 μg of αGalCer or vehicle. (A) Serum concentrations of indicated cytokines were measured 3 h after injection by cytometric bead array technology. iGb3S^−/− mice did not significantly differ in the cytokine response from controls. Depicted are means ± SEM; n = 5; ∗, P < 0.05. (B) Hepatic leukocytes were isolated 2 h after injection and stained with anti-CD3, anti-CD44, or αGalCer-loaded CD1d-tetramers and either CD69 or intracellularly with anti-IL-4 or anti-IFN-γ. Histograms were gated on CD3⁺/αGalCer-CD1d⁺/CD44⁺ lymphocytes. Filled curve, isotype control binding in the gate; dotted line, vehicle-injected control; bold gray line, iGb3S^−/−; thin black line, iGb3S^+/−. (C) Surface expression of CD86 was investigated in splenic DCs 12 h after injection. Histograms were gated on CD11c⁺/MHCII⁺ cells. Legend as in B. One representative image from three independent analyses is shown.