Pub graphs depict the mean SEM from 3 individual experiments (n=5 for every group)

Pub graphs depict the mean SEM from 3 individual experiments (n=5 for every group). and reduced cytotoxicity to Compact disc1d-expressing lymphoma cells. The impaired IL-4 creation by SAP-deficient 24 T cells was associated with reduced IRF4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. test for two groups. For three or more groups, one- or two-way ANOVA was performed with multiple comparisons, followed by Fishers LSD post-test comparisons. All statistical analyses were performed using GraphPad Prism software. Value of 0.05 was considered to be statistically significant. RESULTS The development of 24 T cells with NKT cell characteristics is dependent on CD1d-expressing hematopoietic cells A transgenic RPR-260243 mouse model (24Tg) expressing a CD1d-reactive TCR (V3.2/V9) was used to examine the developmental requirements of type II NKT cells. The self-lipid antigen(s) recognized by 24 TCR remain to be elucidated since it does not recognize any CD1d ligands examined thus far, including sulfatides and cellular phospholipids [38, 39]. We have previously shown that the development of 24 RPR-260243 transgenic T cells (hereafter referred to as 24 T cells), which exhibit an NKT cell phenotype (NK1.1+, CD122+, CD44hi), is CD1d-dependent [34]. As NK1.1+ 24 T cells (V3.2+ V9+ NK1.1+ cells) were virtually absent in 24Tg/CD1d?/? mice (Figure 1A), we used these markers to identify CD1d-selected 24 T cells in bone marrow chimera experiments. These experiments sought to determine whether the expression of CD1d on hematopoietic or non-hematopoietic cells is required for the development of 24 T cells with characteristics of NKT cells. Open in a separate window Figure 1 CD1d expression on hematopoietic cells is required for the development of 24 T cells with NKT cell characteristics(A) The development of NK1.1+24 T cells is CD1d-dependent. Thymocytes, splenocytes and liver lymphocytes from 24Tg RPR-260243 and 24Tg/CD1?/? mice were stained with mAbs to V3.2, V9 and NK1.1, and analyzed by flow cytometry. Bar graphs depict the mean SEM of the percentage (left), and absolute number (right) of V3.2+ V9+ NK1.1+ cells in the indicated organs of 24Tg (open bars, n=6) and Rabbit Polyclonal to IKK-gamma 24Tg/CD1?/? (solid bars, n=6) mice. **, test). Data shown are pooled from 5 individual experiments. (B) CD1d-expressing hematopoietic cells support the development of NK1.1+ 24 T cells. RAG?/? or CD1?/?/RAG?/? mice were reconstituted with bone marrow cells from 24Tg and 24Tg/CD1?/? mice. 5C6 weeks later, lymphocytes were isolated from the spleen and liver of recipient mice, stained with mAbs against V3.2, V9 and NK1.1, and analyzed by flow cytometry. The percentages of V3.2+ NK1.1+ cells in the lymphocyte gate for each experimental group of bone marrow chimeras are indicated in representative FACS plots. RPR-260243 Bar graphs depict the absolute number of NK1.1+ 24 T cells in each experimental group. Data shown represent the mean SEM from 4 to 6 6 for each group. *, test). (C, D) 24Tg/SAP?/? mice have increased numbers of DP thymocytes. (C) Representative dot plots show the percentage of DN, DP, CD4SP and CD8SP subsets in the thymus of 24Tg and 24Tg/SAP?/? mice. (D) Bar graph indicates the absolute number of various T cell subsets (n=8). *, test). (E, F) 24Tg/SAP?/? mice have decreased Nur77 expression. (E) The histograms show the expression of Nur77 in DP thymocytes from 24Tg (thick line) and 24Tg/SAP?/? mice (dotted line). (F) Bar graph depicts mean SEM of mean fluorescence intensity (MFI) of Nur77 expression RPR-260243 on DP thymocytes from 24Tg (n=3) and 24Tg/SAP?/? mice (n=3). *, test). (G, H) The proportion and total numbers of CD44hiNK1.1+ 24 T cells are decreased in the thymus of 24Tg/SAP?/? mice. (G) Representative dot plots show the percentages of CD44hiNK1.1+ cells within V3.2+V9+ gated cells in the thymus of indicated mice. (H) Bar graphs depict the percentage (left) and absolute number (right) of CD44+NK1.1+ in the thymus of 24Tg (n=5) and 24Tg/SAP?/? mice (n=5). *, test). (I) The histograms show the expression of CD24, CD62L and CD122 (black line) on V3.2+V9+ cells in the thymus of 24Tg and 24 Tg/SAP?/? mice. Data are representative of 3 independent experiments. SAP signaling was critical for the induction of Egr2 and PLZF expression as well as the development of NKT1/NKT2 subsets in 24 T cells Recent studies have suggested that SAP.