Fig 1.
Experimental strategy used to evaluate the first T/DC interactions in the spleen.
The panel shows the kinetics of injection, the number and color labeling of each cell type, and the time points when experimental data was collected. Lineage- BM cells (0.5×106) from male or female Rag2-/- mice expressing GPF under chicken beta-actin promoter were injected i.v. in sub-lethally irradiated 400Rad (lethal dose 1300Rad) Rag2-/- hosts. Three days later, recipients were adoptively transferred with 1.5×106 monoclonal TCR-Tg T cells specific for the male antigen: Cell Trace Violet labeled Marilyn CD4+ T cells and DSRed+ CD8+ T cells.
Fig 2.
Characterization of BM derived cells in the spleen of mice injected with Rag2-/-GFP+ BM.
Upper panels: Rag2-/- female mice were irradiated (400Rad) and injected i.v. with 0.5×106 male Rag2-/- GFP+ BM cells. Results show the phenotype and frequencies of GFP+ cells in the spleen, 3.5 days after BM transfer; Middle panels: the same populations in a control Rag2-/- female mouse, studied simultaneously. Results are from one mouse representative of 4 mice studied in two independent experiments. The gate to identify CD11c+ and MHC class II expression was established in non-labeled cells. The MFI of MHC class II expression by CD11c+ cells for reconstituted mice was 9131±4055 (n = 4), and for Rag2-/- mice 10299±4547 (n = 3) (not statistically significant). Lower panels: the numbers of the different DC populations in reconstituted and control mice. Symbols show individual mice and bars the mean±SD of all the mice studied. Please note: Although the numbers of DCs were much lower in BM reconstituted/T cell injected animals than in non irradiated mice, the number of T cells injected was also proportionally much lower than that present in an intact spleen.
Fig 3.
T cells migration velocities in the intact spleen.
Rag2-/- female mice were irradiated (400Rad) and injected i.v. with 0.5x106 male (A) or female (B) Rag2-/- GFP+ BM cells. 3.5 days later, 1.5x106 of monoclonal male-specific TCR-Tg T cells were injected i.v.: CellTrace Violet labeled CD4+ T cells (blue), and DSRed CD8+ T cells (red). Each dot corresponds to an individual cell. Black dots show motile and grey dots represent arrested T cells (velocity <2 μm min-1). The red lines indicate the mean velocity values of motile T cells. (A) T cell motility was evaluated from 15 min to 4 hours after T cell transfer. A total of 198 CD4+ and 86 CD8+ T cell tracks ≥ 600 seconds were analyzed in three independent experiments. For CD4+ T cells the average track duration was 36.5 min, the average track length = 69.9 μm the average track displacement length = 15.2 μm. For CD8+ T cells the average track duration was 33.6 min, the average track length = 59.3 μm the average track displacement length = 12.4 μm. The velocity of CD4+ and CD8+ T cells was not statistically different (Student t-test). (B) Mice were studied 24h after T cell transfer in three independent experiments. Cell motility was evaluated as described above. For 100 CD4+ T cells the average track duration was 17.59 min, the average track length = 60.71μm the average track displacement length = 17.92 μm. For 125 CD8+ T cells the average track duration was 20.87 min, the average track length = 44.54 μm the average track displacement length = 13.93 μm.
Fig 4.
The formation of ternary T/Ag+DC complexes.
Rag2-/- female mice were irradiated (400Rad), reconstituted with Ag+ (male) Rag2-/-GFP+ BM cells and 3.5 days later received Mo TCR Tg CD4+ (blue) and CD8+ (red) T cells as described in Fig 1. TPLSM imaging of the intact spleen was performed from 4–7 hours after T cell transfer. Results show the two different types of complexes and are from two independent experiments out of the three performed. (A-F): 1st type: snap-shots of the TPLSM imaging from S5 Video; (A) initial cell locations; (B-C) the tracks of the different cells: (B) from 0 until 19min; (C) from 34 until 54min; (D-F) cellular interactions at the indicated time points: between a CD8+ and a CD4+ T cell (magenta); between CD8+ and the Ag+DC (yellow); between CD4+ and the Ag+DCs (cyan). (G-I): 2nd type: snap-shots of the TPLSM imaging from S6 Video. (G-I) cellular interactions between CD8+ and a CD4+ T cells at the indicated time points, represented by the same colors as in (D-F). Arrows indicate CD4+/CD8+ T cell contact zones. Scale bars in both experiments correspond to 10 μm.
Fig 5.
Cognate T/DC interactions induce DC activation and their migration to the spleen white pulp.
Rag2-/- female mice were irradiated (400Rad), and injected i.v. with female Ag- (A, C) or male Ag+ (B) Rag2-/-GFP+ BM cells. Three days later mice were injected with 1.5x106 male antigen-specific Mo TCR-Tg CD4+ (blue) and CD8+ (red) T cells (A, B); or 5,000 CFU of Listeria monocytogenes (LM) (C). PFA fixed spleens were cryo-sectioned and studied by confocal microscope 24 hours after T cell injection (A, B); or 5 days after LM infection (C). The spleen stroma was labeled with Alexa Fluor 635 phalloidin to better visualize the white pulp (grey). Arrows in (A) indicate the localization of DCs; the MZ in (C) is depicted by a red dashed line. Images are from a representative experiment out of five (A, B) and three (C) independent experiments. Scale bars correspond to 100 μm.
Fig 6.
Evaluation of cellular interactions in T/Ag+DC complexes.
Rag2-/- female mice were irradiated (400Rad), injected i.v. with male Rag2-/-GFP+ BM (green) and three days later with male specific Mo TCR Tg: CD8+ (red) and CD4+ (blue) T cells, as described in Fig 1. 24 hours after T cell transfer spleens were cryo-sectioned and analyzed using confocal microscopy to identify the types of T/DC complexes. Left panels: The DC was first identified using a low magnification and the green channel alone. A number was attributed to each DC. Attached T cells were then visualized. The square delineates where T cells were present, selected for magnification (scale bar-5 μm). Right panels: The region of the DC where T cells were present was magnified (scale bar 1 μm). From left to right: The contacts between different cells: CD4+/CD8+: Magenta; CD8+/Ag+DCs: Yellow; CD4+/Ag+DCs: Cyan. The two upper panels show examples of complexes harboring CD4+ and CD8+ T cells interacting directly and also both interacting with the Ag+DCs. The two lower panels exemplify complexes where CD4+ and CD8+ interact directly, but only the CD8+ interacts with the Ag+DCs.
Table 1.
Cellular composition and interactions in T/Ag+DC complexes.
Fig 7.
Expression of MHC class II by CD8+ T cells.
Rag2-/- female mice were irradiated (400Rad), injected i.v. with either Ag+ (male) BM or Ag- (female) BM from Rag2-/- donors, and male-specific Mo TCR-Tg CD4+ and CD8+ T cells as described in Fig 1. Results are from one representative experiment out of five performed and show: (A) The expression of the MHC class IIb by CD8+ T cells at different days after T cell transfer in mice injected with male (Ag+) Rag2-/- GFP+ BM (Upper graphs) or female (Ag-) Rag2-/- GFP+ BM (Lower graphs). (B) The specificity of MHC class II labeling: from left to right: expression of the MHC IIb in B cells, naïve and primed CD8+ T cells 4 days after T cell transfer; expression of the Balb/c class IId in primed CD8+ T cells, 4 days after T cell transfer. (C) The distribution of MHC class II in B cells (left panels) and CD44+CD8+ T cells (middle and right panels). B lymphocytes were labeled with anti-IgD (red) and MHC class II Abs (green). The nuclei were stained with DAPI (blue). Male specific DSRed CD44+CD8+T cells were sorted from the spleen of female mice injected with male Rag2-/- GFP+ BM and T cells 4 days after T cell transfer. Cells were labeled with anti-MHC class IIb or anti-MHC IId (green), nuclei stained with DAPI (blue), and studied by confocal microscopy. Results show examples of MHC class IIb expression by two CD8+ lymphocytes (red). All CD8+ T cells showed similar patterns of MHC class II labeling, and none expressed MHC class IId (not shown). Scale bars correspond to 5 μm.
Fig 8.
CD44+CD8+ T cells present the specific peptide and induce the division of naïve CD4+ T cells.
Rag2-/- female mice were irradiated (400Rad) and injected i.v. with either Ag+ (male) BM or Ag- (female) Rag2-/- GFP+ BM. Three days later they were injected i.v. with male-specific Mo TCR-Tg CD4+ and CD8+ T lymphocytes, as described in Fig 1. At day 4 after T cell transfer, CD8+ T cells were double-sorted from spleen cells suspensions pooled from 5–6 mice/group, and cultured in the presence of double-sorted CellTrace Violet labeled naïve CD4+ T cells from Marilyn mice and (when mentioned) the optimal concentration (1μg/ml) of Marilyn (MP) or the class I HY peptide. Results show CellTrace Violet dilution, as indicator of cell division in four days cultures. (A) Total spleen cells from Marilyn mice; (B) Double-sorted CD4+ T cells from Marilyn mice cultured alone; (C) Double-sorted CD4+ T cells from Marilyn mice cultured in the presence of double-sorted naïve CD8+ T cells recovered from mice injected with Ag- (female) Rag2-/- GFP+ BM; (D) Double-sorted CD4+ T cells from Marilyn mice cultured in the presence of double-sorted CD44+CD8+ T cells recovered from mice injected with Ag+ (male) Rag2-/- GFP+ BM and 1μg/ml of the class I HY peptide recognized by the CD8+ TCR-Tg cells; (E) Double-sorted naïve CD4+ T cells cultured in the presence double-sorted CD44+CD8+ T cells from mice injected with male Rag2-/- GFP+ BM and the MP.
Fig 9.
The role of CD8+ T cells in promoting CD4+ activation.
Rag2-/- female mice were irradiated (400Rad) and injected i.v. with Ag+ (male) Rag2-/-GFP+ BM cells. Three days later they received 1.5x106 male-specific Mo TCR-Tg naïve Marilyn CD4+ T cells alone, or together with 1.5x106 male specific Mo TCR-Tg CD8+ T cells. CD4+ T cells were studied three days after T cell transfer. Upper histograms: Cell size (left histograms) and CD25 expression (right histograms) of naïve CD4+ T cells (grey), CD4+ T cells injected alone (black) or together with CD8+ T cells (red). Middle dot plots: CXCR5 expression by male-specific Vβ6+ (CD4+) T cells injected alone (left) or together with male-specific CD8+ T cells (right). Numbers indicate the percentage of CXCR5 positive cells in the total spleen population. Lower dot plots: CXCR5 expression by CD4+ T cells gated on CD3+CD4+Vβ6+CD25+ from the middle dot plots. The gates defining positive cells were established in the same population not labeled with anti-CXCR5 Abs. Result are from one mouse representative of the four studied in two different experiments.
Table 2.
The role of CD8+ T cells in CD4+ responses.