Wiskott-Aldrich syndrome protein is required for homeostasis and function of invariant NKT cells

Abstract

NKT cells comprise a separate T lineage expressing semi-invariant T cell receptors. Canonical invariant NKT (iNKT) cells specifically recognize lipid Ags presented by CD1d, a MHC class I-like molecule. iNKT cells function, in part, as initial responders to bacterial infection and play a role in immune surveillance and tumor rejection. The Wiskott-Aldrich Syndrome protein (WASp) serves as a crucial link between cellular stimuli and cytoskeletal rearrangements. Although we and others have identified a key role for WASp in homeostasis of T-regulatory and marginal zone B cells, little data exist regarding the role for WASp within the iNKT lineage. Analysis of WASp-expressing cell populations in heterozygous female WASp mice revealed a substantial selective advantage for WASp(+) vs WASp(-) iNKT cells. Although adult WASp-deficient (WASp(-/-)) mice had normal thymic and bone marrow iNKT numbers, we observed 2- to 3-fold reduction in the numbers of iNKT cells in the spleen and liver. This peripheral iNKT deficit is manifested, in part, due to defective iNKT homeostasis. WASp(-/-) iNKT cells exhibited reduced levels of integrin surface expression and decreased homing and/or retention within peripheral tissues in a competitive repopulation model. In addition, analysis of young mice showed that WASp is important for both maturation and egress of thymic iNKT cells. WASp(-/-) iNKT cells also exhibited a marked reduction in Ag-induced proliferation and cytokine production. Our findings highlight the crucial role for WASp in iNKT development, homeostasis, and activation, and identify iNKT dysfunction as an additional factor likely to contribute to the clinical features observed in WAS patients.

Figure 1. WASp⁺ iNKT cells exhibit a marked selective advantage

(A) Representative FACS plots showing gating strategies. (B) Combined data showing selection in NK and NKT, but not HSC compartments. Boxes underneath each graph denote statistical significance compared to HSC while bars at top of the graph represent statistical difference between the liver vs. other tissues. Data for each subset includes analysis of multiple (≥6) heterozygous carriers. (C) CD1d tetramers were used to identify iNKT cells in WASp-heterozygous carriers. Left panel shows selection in the thymus, comparing iNKT subsets vs. conventional thymic T cell subsets. Right panel shows selection of CD24⁻CD1d tetr⁺ iNKT cells in the peripheral tissues. Boxes underneath show statistical differences relative to HSC and bars within panel denote statistical differences in thymus vs. peripheral tissues. (D) Representative FACS analysis of one BM chimera. (E) Combined data showing WASp⁺ HSC engraftment and subsequent selection of WASp⁺ NK and NKT subsets. Stars represent statistical differences compared to HSC. Data are representative of 3 independent experiments with 2–4 mice/experiment. * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. WASp^−/− mice generate normal thymic iNKT cell numbers but exhibit a deficit in peripheral iNKT cells

(A) Representative FACS plot showing the gating strategy used to identify iNKT cell subsets in various tissues in WT vs. WASp^−/− mice. (B) WASp^−/− mice exhibit normal numbers of immature thymic iNKT cells. (C) Reduced mature iNKT numbers in WASp^−/− spleen and the liver. Error bars represent data from 3 independent experiments using 6, 8–12 wk old, animals/experiment. (D) Representative FACS data showing normal expression of CD44 and NK1.1 on splenic and thymic iNKT cells. At least ten animals were examined per genotype. * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. iNKT compartment composition in very young vs. older WASp^−/− mice

(A) Analysis of iNKT compartment in 15–16 day old WT and WASp^−/− mice. Cells were stained as in Fig. 2. Data represent summary of at least 6 mice per genotype. (B) Analysis of iNKT compartment in 6 month old WT and WASp^−/− mice showing data from 6 mice per genotype. (C) Analysis of CD69 expression in cells obtained from 6-month old WT and WASp^−/− mice. The MFI was normalized as described in materials and methods. Error bars represent standard deviation. * p<0.05, ** p<0.01, *** p<0.001.

Figure 4. WASp^−/− iNKT cells exhibit defects in antigen-induced proliferation

All FACS plots are representative of the 72hr timepoint. (A) A representative FACS plot of iNKT cells stimulated with 1ng/ml αGalCer. Shaded and open histograms represent control and stimulated cells, respectively. Right panel: Summary of CFSE data showing percentage of tetramer⁺ cells after stimulation (left) and dividing cells within the tetramer⁺ gate (right). (B) CD45.1⁺ (WT) and CD45.2⁺ (WASp^−/−) cells were mixed and co-cultured at 50:50 ratio and stimulated with 1ng/ml αGalCer. (C) Analysis of IL-15 induced proliferation in WT and WASp – deficient splenocytes. Results are representative of 3 independent experiments, 6 mice per experiment. * p<0.05, ** p<0.01, *** p<0.001.

Figure 5. WASp^−/− iNKT cells exhibit defects in antigen-triggered cytokine production

(A) A representative FACS plot showing IL-4 and IFNγ cytokine production in Tetramer⁺CD4⁺ iNKT cells stimulated with 10ng/ml αGalCer for 5 hours. (B) Combined results are shown for CD4⁺ (top right panel) and CD4⁻ (bottom right panel) iNKT subsets. Data shown are representative of one of 2 experiments. * p<0.05, ** p<0.01, *** p<0.001.

Figure 6. WASp^−/− iNKT cells exhibit reduced integrin expression and altered homeostasis in vivo

(A) Representative CD11a histogram on CD24⁻CD4⁺ splenic iNKT cells. Numbers in the upper left indicate CD11a median fluorescence intensity (MFI). Combined data from splenic iNKT and naïve CD4⁺ T cells are shown in the right panel. Similar data was obtained from thymus, bone marrow and liver (not shown). Results are representative of 2 independent experiments, 6 mice/experiment. (B) Analysis of CD11a expression on WASp⁺ and WASp⁻ splenic iNKT cells in carrier females. Results are representative of two experiments, 3–4 mice/experiment. (C) Analysis of Rag2^−/− mice transplanted with thymic iNKT cells. Upper left panel shows analysis of input cells. Lower left panels show analysis of tissues using CD45.1 (WT) or CD45.2 (WASp^−/−) and homeostatic proliferation was assessed using CFSE dilution (FACS histogram). Data shown are representative of one of 2 experiments. AU: Arbitrary Units. ** p<0.01, *** p<0.001.