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. 2008 Jul 15;181(2):907-17.
doi: 10.4049/jimmunol.181.2.907.

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway

Joy A Williams  1 , Joanne M Lumsden, Xiang Yu, Lionel Feigenbaum, Jingjing Zhang, Seth M Steinberg, Richard J Hodes
Affiliations

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway

Joy A Williams et al. J Immunol. .

Abstract

Invariant NKT (iNKT) cells are a population of TCRalphabeta-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.

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Figures

Figure 1
Figure 1
Decrease in NKT thymocytes in B7 DKO and CD28 KO mice. A. Thymocytes from B7 DKO (age 8−12 wk), CD28 KO (age 7−9 wk old) and littermate control mice were stained with anti-CD3 and anti-NK1.1 and analyzed by flow cytometry. B. Summary of analyses performed as in Panel A for B7 DKO and littermate controls (n=7 pairs) and CD28 KO and littermates controls (n=4 pairs). Data shown represent mean ± SE of each group. Thymic NKT cell frequencies in CD28 KO and B7 DKO mice are statistically different from those in wildtype mice using an exact Wilcoxon rank sum test (*, p < 0.05; **, p < 0.005).
Figure 2
Figure 2
Decreased number and frequency of iNKT thymocytes with aging in CD28 KO and B7 DKO mice. Thymocytes from wildtype, CD28 KO and B7 DKO mice were harvested at the indicated number of weeks post birth and stained with anti-CD3 and CD1tet. A. Representative flow cytometry dot plots for wildtype and B7 DKO mice at 8 weeks. B. and C. Thymocytes from a minimum of 3 mice per strain at each age were analyzed as shown in Panel A. With the exception of CD28 KO iNKT frequency at 3 weeks, where only 3 mice per group were analyzed, thymic NKT cell numbers and frequencies in CD28 KO and B7 DKO mice are statistically different from those in wildtype mice (p ranging from <0.05 to < 0.005) at all time points using an exact Wilcoxon rank sum test.
Figure 3
Figure 3
Decreased iNKT frequency in periphery of young (3−5 week) mice. Spleen and liver cells from wildtype and CD28 KO mice were harvested at the indicated ages (young mice, 3−5 weeks; old mice, 6−12 weeks) and stained with anti-CD3 and CD1tet (spleen) or anti-CD45, anti-CD3 and CD1tet (liver). Data shown represent a mean ± SE for each group (minimum of 9 mice per group). Spleen and liver NKT cell frequencies in young CD28 KO mice are statistically different from those in young wildtype mice using an exact Wilcoxon rank sum test (p < 0.005).
Figure 4
Figure 4
HSAhi CD1tet+ thymocytes are present at comparable levels in wildype and CD28 KO mice. A. Representative flow cytometry dot blots showing frequencies of HSAhi CD1tet+ thymocytes in wildtype, CD28KO, B7 DKO and β2M KO mice. CD1tet-enriched thymocytes were stained with anti-HSA, CD1tet, anti-CD3 and anti-NK1.1. CD1tet+ -gated thymocytes (left panel) were examined for expression of HSA and NK1.1 (right panel). B. iNKT frequencies in wildtype, CD28 KO and B7 DKO thymocytes prior to CD1tet enrichment. C. Frequency of HSAhiCD1tet+ thymocytes in wildtype, CD28KO and B7 DKO mice. Data shown in B and C represent mean ± SE of 5 separate experiments each using mice at 3.5−4.5 weeks with n=3 to 5 mice per strain for each experiment. B7 DKO mice were evaluated in only 2 of the 5 experiments, precluding statistical analysis. Thymic iNKT cell frequencies in CD28 KO are statistically different from those in wildtype mice using an exact Wilcoxon rank sum test (*, p < 0.05).
Figure 5
Figure 5
iNKT thymocytes from CD28 KO and B7 DKO mice are phenotypically less mature than wildtype iNKT thymocytes. Thymocytes from 4−5 week old wildtype, CD28 KO and B7 DKO mice were stained with CD1tet, anti-CD3, anti-CD4 and anti-NK1.1 (A) or CD1tetCD1tet, anti-CD3, anti-CD69 and anti-NK1.1 (B) and analyzed by flow cytometry. C. Total thymocytes from 4−5 week old wildtype and CD28 KO were treated with PMA (200 ng/ml) and ionomycin (300 ng/ml) for 5 hrs then stained with CD1tet and intracellular IL4 and IFN-γ. Data shown is representative of 3 separate experiments performed under similar conditions. Data shown in A-C represent mean ± SE of 3 to 6 mice in each group. The frequency of NK1.1+CD4−, NK1.1+CD4+, and NK1.1+CD69+ iNKT cells in CD28 KO and B7 DKO mice is statistically different from that in wildtype mice using an exact Wilcoxon rank sum test (two-tailed; *, p < 0.05; **, p < 0.005). The frequency of IFNγ+/IL-4+, and IFN-γ+ cells in CD28 KO and B7 DKO mice is statistically different from that in wildtype mice using an exact Wilcoxon rank sum test (one-tailed, consistent with our prior hypothesis (see text); *, p < 0.05).
Figure 6
Figure 6
Cell-autonomous and non-cell-autonomous roles for CD28 signaling during iNKT cell development. CD28 WT-CD28 KO mixed chimeras were made by reconstituting lethally irradiated B6.Ly5.2/Ly5.1 host mice with equal numbers of bone marrow cells from B6.Ly5.2 (CD28 WT) and B6.Ly5.1 (CD28 KO) mice. Thymocytes were analyzed 6−10 wk after reconstitution. Analyses of cell populations deriving from CD28 WT and CD28 KO donors were made by gating on each donor-derived population and determining the frequency of either CD4 SP, CD8 SP (A), CD4+CD25+ (B) or CD3+NK1.1+ (C) within the gated population. Data shown in A-C represent the mean ± SE; n=12 mice D. and E. show the frequencies of CD4+CD25+ thymocytes (D) or CD3+NK1.1+ (E) in individual chimeric mice. The frequency of CD4+CD25+ and NKT thymocytes in the CD28KO donor population is statistically different from that in the CD28WT donor by a Wilcoxon signed rank test (*, p < 0.05;**, p < 0.005; ***, p < 0.0005). F. A simple linear regression model for a straight line shows a significant correlation (r-squared = 0.64) between overall chimerism and the relative frequencies of NKT thymocytes within CD28WT and CD28KO donor populations.
Figure 7
Figure 7
Differential reconstitution of iNKT thymocytes in mice expressing CD28 transgenes at low and high levels. A. Thymocytes from 9−10 week old wildtype, CD28 KO, CD28WTlow, and CD28TLlow mice were stained with CD1tet, anti-CD3 and anti-NK1.1 and analyzed by flow cytometry. B. Thymocytes from 6−7 week old wildtype, CD28 KO, CD28WThigh, and CD28TLhigh mice were stained with CD1tet, anti-CD3 and anti-NK1.1 and analyzed by flow cytometry. Data shown represent mean ± SE of at least 3 mice in each group. Thymic iNKT cell frequencies in CD28WThigh mice are statistically different from those in wildtype mice using an exact Wilcoxon rank sum test (*, p < 0.05).
Figure 8
Figure 8
Patterns of B7.1 and B7.2 expression in B7 transgenic mice. A.and B. Thymocytes from 9−11 week old wildtype, B7 DKO and B7 transgenic mice were stained with anti-B7−2 (A) or anti-B7−1 (B) and CD4 and CD8. The mean fluorescence intensity (MFI) for each thymocyte subpopulation for each strain is presented as the MFI for that strain minus the MFI for the B7 DKO. C. and D. Collagenase-digested thymus preparations were stained with CD45, CD11c, UEA-1 and B7.2 (C) or anti-B7−1 (D). The MFI for each thymus cell subpopulation (CD45+, CD45neg, CD11c+ (CD45+), and UEA+ (CD45 neg )) for each strain is presented as the MFI for that strain minus the MFI for the B7 DKO. Data shown represent the mean MFI of 2−4 mice for each strain.
Figure 9
Figure 9
Effect of B7 transgene expression on iNKT development. A. Thymocytes from 9−11 week old wildtype, B7 DKO and B7 transgenic mice were stained with CD1tet, anti-CD3, anti-NK1.1 and analyzed by flow cytometry. All B7tg Lines are on a B7 DKO background. B. Thymocytes from 9−10 week old littermates from backcrosses of each of three B7 transgenic lines (Lines I2, E4 and 7) to CD28 KO mice were stained with anti-CD3 and anti-NK1.1. The CD28WT (CD28+/−) and CD28KO (CD28−/−) groups represent transgene negative littermates from the three crosses. The B7Tg/CD28KO group represents pooled data from B7 transgenic lines I2, E4 and 7 lines on a CD28−/− background. Data shown are the mean ± SE of 3 mice in each group (except Figure 9B, CD28WT where n=2).
Figure 10
Figure 10
Vβ chain usage in wildtype, B7 DKO and Line T59 thymic iNKT cells. CD8-depleted thymocytes from 6−12 week old wildtype, B7 DKO and Line T59 mice were incubated with CD1tet, anti-CD3 and anti-Vβ antibody. The percent Vβ+ was calculated from within CD1tet+ gated cells. Frequencies of Vβ2+ and Vβ7+ thymic iNKT cells in the Line T59 mice are statistically different from those in wildtype and B7 DKO mice using an exact Wilcoxon rank sum test (*, p < 0.05;**, p < 0.005; ***, p < 0.0005).
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