Fig 2: Apoptotic T cells and AE-specific CD8+ T cells accumulate in the CNS of mice with EAE.A, B C57BL/6 mice (n = 6) were immunized with 100 µg MOG35-55 and 400 µg heat-killed M. tuberculosis in IFA at day 0, and received 200 ng PTX i.v. at days 0 and 2. After 14 days, when all the mice had developed EAE, inguinal lymph nodes (LN) and brains and spinal cords (CNS) were collected, and flow cytometry analysis was performed. A Representative staining of active Caspase 3 in gated live single CD3+ CD4+ T cells (left) and of CD44 versus CD40L in gated apoptotic cells (right). B Cumulative analysis in 6 mice. Bars represent means ± SEM. **P < 0.01, by Mann–Whitney test. C, D Mice were immunized as above (n = 5) and sacrificed between days 28 and 48 after EAE induction. As control, not immunized (CTRL) mice were used. Lymphocytes from spleen (SPL) and central nervous system (CNS) were challenged in vitro with MOG35-55 or with AE-peptide pools and IFN-γ ELISpot was performed. The sum of IFN-γ spots, per 106 cells, against all AE-peptide pools was calculated. C Cumulative analysis of IFN-γ production against MOG35-55 (left) or AE-peptides (right) in MOG35-55-immunized (n = 5) or control (n = 4) mice. Bars represent means ± SEM. *P < 0.05, by Mann–Whitney test. D Spearman correlation between IFN-γ response to the indicated peptides and the cumulative disease score. ns, not significant. The data are from a single experiment representative of three independent experiments.

Fig 3: 6PGD blockade induce mitochondrial ROS and expression of antioxidant enzymes(A) Naive CD8+ T cells from 6PGD-/- and 6PGDfl/fl mice were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), and expression of CD62L and CD44 was examined by flow cytometry.(B–L) Naive CD8+ stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), mitochondrial ROS was assessed using MitoSOX Red (B), and lipid peroxidation capacity was assessed using BODIPY 581/591 C11 (C) and flow cytometry. (D–L) The indicated enzymes of ROS metabolism pathway were assessed by real-time PCR (D–K), and their quantitative changes in 6PGDfl/fl and 6PGD-/- T cells during activation were also depicted by the heatmap (L). Results are representative of three independent experiments with n = 3 per experiment. Gsr, glutathione reductase; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; Prdx2, peroxiredoxin 2; SOD2: superoxide dismutase 2; Txn1, thioredoxin 1; Txnrd1, thioredoxin reductase 1. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 4: FACS-sorted CTC populations derived from primary breast tumors.(a) Breast cancer gene expression array profiling of FACS-enriched CD45-/EpCAM-negative/CD24−/CD44+/uPAR+/−/int β1+/− CTC subsets derived from BCBM and no BCBM patients. mRNAs were amplified by REPLI-g WTA single cell kit (Qiagen) followed by real-time PCR analysis. Ct values and fold expression were calculated by online RT2 PCR profiler array data analyses software version 3.5 (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) (Qiagen). Heat map and clustergram analyses were generated by online software Treeview and Cluster (Eisen lab, University of California, Berkeley). BCBM, Breast Cancer Brain Metastasis; (b) STR DNA fingerprinting of FACS-sorted CTC subsets derived from BCBM patients have unique profiles over cell lines available to NCI databases.

Fig 5: The effect of CCR2 and CX3CR1 deficiency on monocyte and CD8+ T cell activation status in the blood of L31 mice. (A) Representative flow cytometry dot plot and quantification analysis showed a decrease in the number of CD115+CD11b+ monocytes in the blood of L31/CX3CR1KO mice and an almost abolishment of CD115+CD11b+ monocytes in L31/CCR2KO mice. (B) Compared to L31 mice, the frequency and number of CD8+ T cells were reduced in the blood of L31/CX3CR1KO mice, but not affected in L31/CCR2KO mice (C) The majority of the reduced CD8+ T cells in L31/CX3CR1KO mice were of the activated subset (CD44+CD43+). (D) The frequency and number of CD8+ T cells with effector memory phenotype (CD44+CD62L-) were significantly reduced in the blood of L31/CX3CR1KO mice compared to L31 and L31/CCR2KO mice. Disease was induced by PSNL and experiments done 30 days post PSNL. All quantitative analyses are shown as number of cells per µl blood. n = 5-8/group; student’s t test; *p < 0.05; ***p < 0.001.