PMC

C3H-gld mice (14-week-old) or their age-matched C3H-wt mice were immunized i.p. with SEB (100 µg/mouse). Frequencies of SEB-reactive Vβ8⁺ T cells among the CD8 (A), CD4 (B), and DN (C) subsets in peripheral blood were determined on the indicated days. Similar results were obtained in lymph nodes and spleens when the experiment is terminated. Frequencies of Vβ6⁺ T cells were used as negative controls. Results show mean±SD from three mice per group.

Wild-type and gld mice (12-week-old) received two i.p. injections of BrdU during a 24-h period as described in . Peripheral T cells and IEL were isolated and stained with APC-TCRβ, PerCP-CD4/PerCP-CD8α, FITC-BrdU and PE- sdc1 specific antibodies. TCR⁺ cells were gated and percentage of BrdU^high in the DN or SP (CD4⁺+CD8⁺) subsets in wt (A) and gld (B) mice were determined. The graph below each panel shows mean±SEM from two independent experiments with 2–3 mice per each genotype. *, P<0.05; N.S., not significantly different.

(A) Single cell suspensions from spleens of gld mice or intestine (small and large intestines were combined) of gld or wt mice were stained for APC-TCRβ, PerCP-CD4, and PerCP-CD8α, and FITC-CD2, CD5 or B220. TCR⁺ cells were gated and expression of indicated molecules by DN and SP subsets were determined. Spleens of wt mice are not included in the analysis because of the paucity of DN T cells. (B) Similar distribution of Vβ expression by DN T cells. Peripheral T cells and IEL were isolated from 12-week-old C3H-gld/gld mice and surface-stained for TCR, CD4, CD8, and Vβ6, Vβ8, Vβ13 or Vβ14. After gating on TCR⁺ cells, the frequencies of different Vβs among DN, CD4 or CD8 T cells were determined. Data show mean±SD from one of two independent experiments. (C) Downregulation of sdc1 by TCR activation. Top panel: Analysis of TCR (left dot plot); and CD4 and CD8α expression (right dot plot) by freshly isolated DN cells prior to culture. The isolated cells were stained with TCR, CD4 and CD8 specific antibodies and their purity assessed by flow cytometry (>95% of isolated cells expressed TCR (left dot plot) and lacked CD4 and CD8 expression (middle dot plot). The histogram shows CFSE intensity in DN T cells before (day 0) and after (day 4) TCR activation. Bottom panel: Kinetics of sdc1 and CD62L downregulation by DN T cells in response to CD3/CD28 stimulation. Data from one of two independent experiments are shown.

T cells were isolated from the periphery and gut epithelium of wt or gld C3H mice of different ages as described in . Isolated cells from lymph nodes (LN), spleen, small intestine (SI), and large intestines were stained with APC-TCRβ, PerCP-CD4, PerCP-CD8α, anti-B220 and PE-sdc1 and analyzed by FACS. CD4 and CD8 T cells were included in one subset (CD4 and CD8) by simultaneously staining samples with PerCP-conjugated anti-CD4 and PerCP-conjugated anti-CD8 mAbs. This allowed us to compare directly SP (CD4⁺ and CD8⁺) and DN (CD4⁻CD8⁻) TCR⁺ subsets. Frequencies of sdc1⁺ DN T cells in the periphery and gut epithelium of wt (A) and gld (C) mice are shown. Numbers indicate the percentage of positive cells in each quadrant. Sdc1⁺ cells in the upper right quadrants in the lymph nodes and spleen of 13- and 21- to 24-week-old gld mice were CD4 T cells (not shown). Percentages of sdc1⁺ DN T cells relative to total T cells in the periphery and gut epithelium of wt (B) and gld (D) mice are shown. DN T cells isolated from the gut epithelium expressed B220 ( and data not shown). Results are expressed as mean±SD from three independent experiments with two to three mice per group. LN, Lymph nodes, SI, small intestine, Li, large intestine.

(A) Comparison of the transcript profiles of DN T cells and autologous SP T cells (combined CD4 and CD8 T cells) isolated from inguinal and axillary lymph nodes of gld mice as described in . The insets show purity and hallmarks properties of DN T cells including expression of TCR and B220 and lack of CD4 and CD8 on their surface. The Minus versus Average (MvA) plot shows intensity log-ratio M = log₂ (DN/SP) versus mean log-intensity A = [(log₂ (DN)+log₂ (SP)]/2. Out of 32,000 transcripts examined, expression of 160 genes was at least 3-fold higher in DN than in SP T cells. Highly expressed genes including junctional adhesion molecules, hydrolyase enzymes, and apoptotic death molecules are highlighted. Selected under-expressed genes in DN T cells relative to SP T cells are also highlighted. (B) Validation of expression of selected genes by real-time PCR. Expression level of each gene was normalized relative to expression of 18 s rRNA in the same cell subset. X-axis shows -fold change in expression of indicated genes in DN T cells relative to SP T cells. (C) Validation of specific expression of sdc1 by flow cytometry. Splenocytes were isolated from 16-week-old C3H-gld/gld mice, stained with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-sdc1 or with APC-TCRβ, PerCP-CD4, FITC-CD8α, and PE-B220 mAbs and analyzed by FACS. Dot plot: TCR⁺ cells were gated followed by specific gating of DN (R5), CD4⁺ (R3), and CD8⁺ (R4) subsets. Histograms: Overlays show relative expression of TCR, B220, and sdc1 by gated CD4, CD8, and DN subsets.

(A) Apoptosis of DN T cells in the gut epithelium is Fas-independent. IEL from 12-14-week-old C3H-gld and wt mice were stained for TCR, CD4/CD8, and annexin V binding. The graph show percentages of annexin V⁺, TCR-gated DN or SP in small (SI) and large intestine (LI) and of gld/gld and wt mice. Results are presented as mean±SEM (n = 4). There is no significant difference in apoptosis of DN T cells or SP T cells between wt and gld mice; p>0.05). (B) Apoptosis of peripheral DN T cells is Fas-dependent. Lymph node and spleen cells were stained for TCR, CD4/CD8, and annexin V binding. The frequencies of annexin V⁺ DN T cells were determined as above. The graph shows apoptosis of gated DN and SP T cells in lymph nodes (LN) and spleens of gld/gld and wt mice. The results are presented as average±SEM (n = 6 per group). Apoptosis of peripheral DN T cells is significantly higher (***, p<0.0001) than that of SP T cells in wt mice but in the absence of functional FasL in gld mice is comparable to that of SP T cells. (C) Wild-type C3H mice were injected i.p. with anti-FasL (0.5 mg) or control saline twice within a 24-h period. Lymph node and/or spleen cells from mice in each group were stained for TCR, CD4+CD8, and annexin V and analyzed by FACS. TCR+ cells were gated and analyzed for annexin V binding by SP (CD4⁺/CD8⁺) and DN (CD4⁻/CD8⁻) subsets. Quadrants were set using samples stained with TCR, CD4/CD8 but not annexin V. The data are pooled from two independent experiments (n = 4); numbers indicate mean±SEM of annexin V⁺ DN T cells. The graph shows mean±SEM of apoptosis. Apoptosis of DN T cells was significantly reduced by FasL blockade; p<0.0376. Apoptosis of SP T cells, which is significantly lower than of DN T cells, was further reduced by FasL blockade from 23±2 to 16±0.2; p<0.042. (D) DN T cells that accumulate in the periphery of gld mice are sensitive to FasL-induced apoptosis in the absence of concomitant TCR stimulation. DN and CD4 T cells were isolated from lymph nodes of gld mice and assessed for purity as described in (each >95% pure, data not shown). Levels of Fas expression by DN and CD4 T cells isolated from C3H-gld/gld mice were determined (middle histogram). Left histogram shows isotype control staining. Right histogram shows apoptosis of DN (80%) and CD4 (19%) T cells incubated overnight with soluble FasL in the presence of a crosslinker without TCR activation. Background staining of DN T cells incubated with the enhancer was less than 20%. Results from one of two independent experiments are shown. (E) Higher rate of BrdU⁺ DN T cell accumulation in the periphery of gld mice. C3H-gld/gld mice were injected i.p. with 2 mg of BrdU and then maintained on BrdU (0.8 mg/ml) in drinking water for 21 days. Rates of BrdU labeling and apoptosis of DN and SP T cells were monitored in pooled peripheral blood from mice in the same group (n = 3 per group). The best-fit lines represent the relative rate of accumulation of different subsets.