Figure 6.

Naïve CD8⁺ lymphocytes vigorously proliferate and differentiate in the mediastinal LNs of influenza infected mice in the absence of ICAM-1 and ICAM-2 and independently of DC ICAM-1. (A) The total numbers of CD45⁺CD8⁺ T cells in the resting MedLNs of both WT and ICAM-1/2^-/- mice analyzed by flow cytometry. (B) Experimental design and timeline protocol for OT-I splenocyte homing to resting MedLNs of WT and ICAM-1/2^-/- mice. Naïve CD45.1 OT-I (2x10⁶) were preincubated with α4 mAb (α4 block) or isotype control (iso) and then I.V. transferred into WT and ICAM-1/2^-/- mice. An additional dose of α4 blocking mAb or an isotype control mAb was I.V. administered 4 hours later. MedLNs were harvested 18 hours after the initial cell transfer and the accumulation of naïve OT-I (CD45.1⁺CD8⁺CD62L⁺CD44^-) T cells was determined by flow cytometry. Results shown (right panel) are the mean +- SEM. (C) Experimental design and timeline of accumulation of CD45.1 OT-I CD8⁺ T cells in the MedLNs of resting or PR8-SIINFEKL infected WT and ICAM-1/2^-/- mice. The OT-I totals in the MedLNs of resting or infected WT or ICAM-1/2^-/- mice are shown (right panel). Where indicated, OT-I T cells were preincubated with α4 mAb (α4 block) and recipient mice were treated with an additional dose of α4 blocking mAb 4 hours later as in (B). I.N. PBS administration was performed in control experiments. (D) Experimental design and timeline of the proliferation and differentiation of transferred OT-I spenocytes into IFN-γ producing OT-I effector cells in the MedLNs of PR8-SIINFEKL infected WT or ICAM-1/2^-/- mice. A total of 2x10⁶ CFSE-labeled CD45.1 OT-I cells were I.V. transferred into both mice groups. After 18 hours, mice were I.N. infected with a sublethal dose of PR8-SIINFEKL. (E) Four days post infection MedLNs were harvested and the numbers of effector (CFSE-labeled CD45.1⁺CD8⁺CD62L^-CD44⁺) OT-I T cells were determined by flow cytometry. Where indicated, OT-I T cells were treated with α4 mAb (α4 block) as in (C). (F) Proliferation histograms of the CFSE-labeled CD45.1⁺CD8⁺CD62L^-CD44⁺ OT-I effector T cells recovered from WT or ICAM-1/2^-/- MedLNs post infection described in (E) or under steady state (PBS). Inset: The percentage of effector OT-I CD8⁺ T cells that divided at least once and their division index determined four days post PR8-SIINFEKL infection. (G) The number of OT-I effector cells described in (E) with high IFN-γ content. (H) Experimental design and timeline of the proliferation and differentiation in the MedLNs, of OT-I CD8⁺ T cells 7 days post PR8-SIINFEKL infection of WT or ICAM-1/2^-/- mice. The numbers of OT-I CD8⁺ effector T cells and of high IFN-γ producing T cells harvested 7 days post infection in the MedLN are shown in (I, J), respectively. (K) Experimental design and timeline of the proliferation and differentiation in the MedLNs, of OT-I CD8⁺ T cells 4 days post PR8-SIINFEKL infection of WT (ICAM-1^fl/fl) or DC^ΔICAM-1 mice. The numbers of OT-I CD8⁺ effector T cells (L) and of high IFN-γ producing T cells (M). Statistical significance was determined by two-tailed, unpaired Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. The numbers of each experimental group are indicated in the graphs. The error bars indicate the SEM of each measurement.