Volume 11, Issue 2, Pages 173-181 (August 1999) The Peptide Ligands Mediating Positive Selection in the Thymus Control T Cell Survival and Homeostatic Proliferation in the Periphery  Bettina Ernst, Dong-Sup Lee, Jennifer M Chang, Jonathan Sprent, Charles D Surh  Immunity  Volume 11, Issue 2, Pages 173-181 (August 1999) DOI: 10.1016/S1074-7613(00)80092-8

Figure 1 Expansion of Donor T Cells upon Transfer into T-Depleted Syngeneic Hosts (A) Small doses (2 × 106/mouse) of whole B6.PL (Thy1.1+) LN cells labeled with CFSE (Experimental Procedures) were i.v. injected into the indicated hosts (all Thy1.1− B6); 8 days later, LN cells from the injected mice were double stained for Thy1.1 and CD4. CFSE levels on gated donor CD4+ and CD8+ cells are shown. To facilitate detection, the unirradiated hosts in group I received a higher dose of LN cells (2 × 107). Mice were irradiated 2 days prior to donor cell injection. For depletion of Thy1.2+ cells, adult thymectomized mice were injected with anti-Thy1.2 mAb (50 μl of ascites fluid/day) for 3 days starting 1 week prior to the donor cell injection. The recoveries of total numbers of donor cells in pooled LN and spleen relative to the numbers initially injected are shown in the lower graph. The data are means of 2–3 hosts analyzed individually and are representative of two separate experiments. (B) Small doses (2 × 106) of CFSE-labeled whole B6.PL LN cells were injected into irradiated (600 cGy) B6 mice either alone or together with the indicated numbers of B6 whole LN cells or purified T cells subsets. Mice were analyzed 8 days later for donor T cells as described in (A). Similar results were obtained in two other experiments. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)

Figure 2 Capacity of Naive T Cells to Proliferate in T-Deficient Syngeneic Hosts A mixture of FACS-sorted CD4+ and CD8+ B6.Ly5.1 LN cells and FACS-sorted CD44− CD4+ and CD44− CD8+ B6.PL (Thy1.1) LN cells were CFSE-labeled and transferred into irradiated (600 cGy) B6 hosts; 9 days later host LN cells were triple stained for Thy1.1, Ly 5.1, and CD4. To detect CD44 expression after transfer, unlabeled aliquots of the cell mixture were injected into separate groups of irradiated (600 cGy) B6 hosts and analyzed at day 9 by staining host LN cells for Thy1.1, Ly5.1, CD4, and CD44. CFSE and CD44 intensities are shown on gated Thy1.1+ versus Ly5.1+ CD4+ and CD8+ donor cells. As controls, nonirradiated B6 mice were injected with CFSE-labeled or unlabeled sorted whole B6.Ly5.1 CD4+ and CD8+ LN cells and analyzed 9 days later; data on gated CD4+ and CD8+ donor cells are shown. One other experiment showed similar results. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)

Figure 3 Capacity of Transgenic T Cells to Proliferate in Irradiated Syngeneic Hosts Small doses (1–2 × 106/mouse) of CFSE-labeled TCR transgenic CD4+ or CD8+ cells were transferred into nonirradiated versus irradiated (600 cGy) syngeneic hosts and analyzed 7–14 days later. Donor 2C and P14 CD8+ cells from a B6 background were sorted for CD44− 1B2hi and CD44− Vα2hi cells, respectively, and injected into B6.PL hosts. Donor DO11 CD4+ cells from a BALB/c.TCRα− background were transferred into BALB/c hosts, and OT-II CD4+ cells and anti-HY CD8+ cells from a B6 and B10 background, respectively, were transferred into B6.PL hosts. To detect donor cells in host LN, TCR clonotype+ (1B2+) 2C cells were detected by staining for 1B2, CD8, and Thy1.2; P14 cells were detected by staining for Vα2, Vβ8, and Thy1.2; TCR clonotype+ (KJ1-26+) DO11 donor cells were detected by staining for KJ1-26 and CD4 expression; OT-II cells were detected by staining for Vα2, Vβ5, and Thy1.2; and anti-HY cells were detected by staining for T3.70, CD8, and Thy1.2. The data show CFSE levels on gated donor cells 7 days post transfer for 2C, P14, and anti-HY, and 14 days post transfer for DO11 and OT-II. Findings are representative of at least two experiments. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)

Figure 4 B6 T Cell Proliferation in Irradiated B6 versus B6.H2-A− Hosts Small doses (2 × 106/mouse) of CFSE-labeled purified B6.PL CD4+ and CD8+ cells were i.v. injected into the indicated hosts; 7 days later, the host LN cells were double stained for Thy1.1 and CD4. To facilitate detection, unirradiated hosts received a larger dose of 1 × 107 donor T cells. The donor cells consisted of 61% CD4+ cells and 39% CD8+ cells. The B6.H2-A− hosts used were backcrossed 5–6 times to B6. At the top, CFSE levels on gated donor CD4+ and CD8+ cells are shown. The data on the far right show the ratio of donor CD4+ cells (Thy1.1+ CD4+) to donor CD8+ (Thy1.1+ CD4−) cells recovered from host LN as calculated from the adjacent dot plots. The percent of recoveries of the donor cells (calculated as in Figure 1) is shown in the lower graph; the data are means of 2–3 hosts analyzed individually and are representative of two separate experiments. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)

Figure 5 Capacity of B6 versus H2-M− CD4+ Cells to Proliferate in T-Deficient H2-M− Hosts (A) Small doses (2 × 106/mouse) of CFSE-labeled or unlabeled purified donor B6.Ly5.1 CD4+ cells were i.v. injected into groups of irradiated (600 cGy) H2-M− mice and H2-M+ littermates; the phenotype of the injected cells is shown at the top. The H2-M− host mice, which originated from a (B6 × 129)F2 (H2b) background, were backcrossed six times to B6. Recipients injected with CFSE-labeled cells were analyzed 8 days later by staining LN cells for Ly5.1 and CD4; CFSE levels on gated donor cells are shown in the left column. For CD44 expression, hosts injected with unlabeled cells were analyzed 1, 8, and 16 days later by staining for Ly5.1, CD4, and CD44; data for CD44 expression on gated donor CD4+ cells and Ly5.1 versus CD4 expression on all LN cells on day 8 are shown in the middle. The graph (far right) shows the total numbers of donor cells recovered in pooled LN plus spleen from the H2-M− and H2-M+ hosts on days 1, 8, and 16. (B) CFSE-labeled or unlabeled mixtures of purified B6.Ly5.1 (2.3 × 106) and H2-M− (Ly5.2) CD4+ (1.7 × 106) cells (both Thy1.2+) were i.v. injected into a group of irradiated (600 cGy) H2-M−.Thy1.1 hosts. H2-M−.Thy1.1 mice were obtained by backcrossing H2-M− mice in a (B6 × 129)F2 background twice to B6.PL. To prevent possible rejection of donor cells, we used adult thymectomized hosts pretreated with anti-CD8 and anti-NK1.1 mAbs for 3 days starting 1 week prior to donor cell injection. Hosts injected with unlabeled cells were analyzed on 1, 7, 15, and 35 days later by staining LN and spleen cells for Thy1.2, Ly5.1, and CD44. The data show donor Thy1.2 versus Ly5.1 expression as dot plots on pooled LN cells on days 1 and 15 (top right); CD44 expression on the donor cells at these same time points is shown in the left side. The data on the far right show the ratio of the two donor cell populations recovered from host LN. The graph (bottom right) shows the total numbers of the two donor cell populations recovered from pooled host LN plus spleen at all time points. Recipients of the CFSE-labeled cell mixture were analyzed on day 15 by staining LN cells for Thy1.2 and Ly5.1; CFSE levels on the two donor cell populations are shown (bottom row). All of the data are representative of or means of 2–3 mice analyzed individually; a separate experiment showed similar results. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)

Figure 6 Differential Capacity of DO11 and OT-II Transgenic T Cells to Proliferate and to Survive in H2-M− Hosts (A) LN cells from BALB/c.DO11 and B6.OT-II transgenic mice backcrossed to either a B6 or an H2-M− (H2b) background were double stained for TCR clonotype (KJ1-26+) versus CD4 for DO11 and for Vβ5 versus CD4 for OT-II T cells. For B6.OT-II mice, >99% of Vβ5+ cells were Vα2+; for H2-M−.OT-II mice, by contrast, <0.1% of Vβ5+ cells were Vα2+. (B) Small doses (2 × 106) of CFSE-labeled or unlabeled cells comprising a mixture of purified CD4+ cells from B6.OT-II and H2-M−.DO11 mice were injected into a group of irradiated (600 cGy) H2-M−.Thy1.1 mice; hosts were treated (as in Figure 5B) to prevent rejection of donor cells. We calculated that each host received 0.6 × 106 OT-II, 0.5 × 106 H2-M−.DO11, and 0.9 × 106 nontransgenic CD4+ cells. Separate staining showed that for OT-II cells, >99% of Vβ5+ cells were Vα2+. Recipients of unlabeled cells were analyzed 1, 8, 15, and 35 days later by staining LN and spleen cells for Thy1.2, CD4, Vβ5, and Vα2 for OT-II cells and for Thy1.2, CD4, and KJ1-26 for DO11 cells. Levels of CD44 on gated transgenic T cells at days 1 and 8 are shown; CD44 expression was detected by staining for Thy1.2, CD4, Vβ5, and CD44 for OT-II cells and for Thy1.2, CD4, KJ1-26, and CD44 for DO11 cells. The graphs at the bottom show the total numbers of the two transgenic T cell populations recovered from pooled LN plus spleen at the times shown. Hosts injected with CFSE-labeled donor cells were analyzed on day 15 by staining for Thy1.2, CD4, and Vβ5 for OT-II cells and for Thy1.2, CD4, and KJ1-26 for DO11 cells; the data show CFSE levels on gated OT-II and DO11 cells (middle row). Data are representative of or means of 2–3 mice analyzed individually. One other experiment showed similar results. Immunity 1999 11, 173-181DOI: (10.1016/S1074-7613(00)80092-8)