Deficiency in CD103⁺ RDC impaired anti-IAV CD8⁺ Teff accumulation in the lungs, but not DLN. (A – D) Batf3^−/− and B6 mice were infected with IAV and specific CD8⁺ T cells in the lungs (A and B) and DLN (C and D) were enumerated over time after tetramer staining. Rep. flow analyses (left panels in A and C) and combined NP- and PA-specific tetramer⁺ CD8⁺ T total cell numbers (right panels in A and C) were depicted. (B and D) IFNγ-secreting CD8⁺ T cells in the infected lung (B) and DLN (D) at d7 p.i. were identified after restimulation with IAV-infected BMDC for 5 hrs in vitro (n = 6 - 8 mice/genotype). (E and F) Langerin-DTR mice were depleted of CD103⁺ RDC by i.n. administration of DTx (or control PBS) followed by IAV infection. At d7 p.i specific CD8⁺ T cells in the inflamed lung (E) and the responding DLN (F) were enumerated as shown in A and C (n = 7 - 9). See also .

Egress and initial activation of CD8⁺ T cells in the DLN of Batf3^−/− mice. (A) Tet⁺ CD8 T cells in the circulation of infected Batf3^−/− and B6 mice were enumerated over time. A rep. FACS plot evaluated at d11 p.i. is shown (n = 8 – 10). (B and C) Congenic mice that had received CFSE labeled OT1 cells (Thy1.1) were infected with IAV-OT1. Division (CFSE dilution) (B) and accumulation of divided (CFSE^low) (C) OT1 cells in the DLN were measured at d3.5 p.i. (n > 5). (D – F) At d3.5 p.i., divided OT1 cells in the DLN were examined for surface marker and Granzyme B expression directly ex vivo, and IFNγ expression after ex vivo cognate peptide restimulation (D), T-bet (E) and Blimp-1 (F) protein expression by flow cytometry (n = 4 - 6). (G) Gene expression profiles of endogenous CD8 T cells activated in the WT (blue) or Batf3^−/− (red) mice. At d5 p.i., activated (CD44^hi) and naïve (CD44^lo/−) CD8⁺ T cells were sorted from the DLN of infected mice. T-bet, Blimp-1, Granzyme B and IFNγ gene expression was evaluated in total mRNA. Data in G represent at least two ind. expts. of cells from pools of 3 - 5 mice prior to sorting. See also .

CD8⁺ T cells differentiated in the absence of CD103⁺ RDC exhibit central memory-like T cell properties. (A - C) At > d60 p.i., circulating specific CD8⁺ T cells in the blood of recovered mice were analyzed for their surface expression of CD62L (A), CCR7 (B) and IL-7RαC - D and E) Frequency of Tet⁺ CD8⁺ T cells in the circulation over time (D), and in the DLN (left panel in E) and lung (right panel in E) at d65 p.i. (F) The frequency of CD62L^hi or CCR7^hi CD8⁺ T cells was measured in the DLN of recovered WT (blue) and Batf3^−/− (red) mice at d65 p.i. (n = 5 - 8). See also .

Trafficking and recall response of CD8⁺ T cells primed by CD103⁺ and CD11b^hi RDC subsets and costimulatory molecule expression. (A - D) CFSE-labeled naive specific Cl4 cells were stimulated in vitro with DLN-derived CD103⁺ or CD11b^hi RDC isolated at d3 p.i. of PR/8 IAV infected mice (from n = 15 – 20 donors/sorting). After d4, the CFSE^low cells were sorted, and equivalent cell numbers were individually transferred into Thy-mismatched recipient mice. For trafficking analyses, activated Cl4 cells (1 × 10⁴/mouse) were infused into d5 p.i. B/Lee IAV-infected mice and enumerated in the lungs and DLN of the recipients 24 hr p.t. (A and B). To further analyze the trafficking of the Cl4 cells following IAV challenge, in vitro activated Cl4 cells were transferred into uninfected recipient mice and maintained in recipients for 7 days prior to infection with A/PR8 IAV. At d8 p.i., responding Cl4 cells in the lungs and DLN of the infected recipients were evaluated (C and D). Data represent at least two ind. expts. with similar results (n = 4 – 6/group). (E) Expression level of markers by the RDC subsets following migration to the DLN is represented as the ratio of MFI of CD103⁺ RDC relative to the MFI of CD11b^hi RDC. (F) CD24 mRNA in total RNA from the sorted CD103⁺ RDC and CD11b^hi RDC isolated from the pooled DLN (from n = 15 – 20 mice/sorting) was analyzed at d3 p.i. Data represent three independent experiments. See also .

Impact of CD24 blockade on anti-IAV CD8⁺ T cell responses in vivo. (A) To monitor the effect of CD24 blockade on early events during T cell activation in vivo, CFSE-labeled OT1 cells were transferred into B6 WT mice, infected with PR8-OT1 IAV 24 hrs. p.t. These infected recipients were infused i.p. with αCD24 mAb or IgG mAb at both d1 and d3 p.i. (B) Cell division (left panel), the fraction (%) and total numbers of divided OT1 cells (right panels) were enumerated (n = 4 – 5 recipients). (C - E) At d7 p.i., the frequency and total numbers of IAV-responding lung and DLN CD8⁺ T cells was determined by IFNγ-secretion after infected BMDC restimulation ex vivo for 5 hrs (C) and direct ex vivo analysis of tet⁺ (NP and PA) CD8⁺ T cells in the lungs (D) and DLN (E). Data in C - E represent at least three ind. exps. (n = 4 – 5 mice/group). (F) Requirement of CD24 expressed RDC subsets for full expansion of naïve CD8 T cells. CFSE-labeled naïve Cl4 cells were co-cultured with sorted CD11b^hi or CD103⁺ RDC from d3 p.i DLN (n = 15 – 20 mice/sorting) in the presence (filled in red) or absence (white lines) of αCD24 mAb. At d4, the extent of cell division (left panels) and total divided Cl4 cells (right panel) were examined. Data represent at least three ind. expts. See also .

Elevated CD24 expression by DC enhances CD8⁺ T cell response and promotes lung homing. (A) Effect of in vitro CD24 blockade on αCD3-CD28 Ab-stimulated proliferation of dye labeled Cl4 cells or polyclonal CD8⁺ T cells (data not shown) as determined by CFSE dilution at d3 of culture. Data is a rep. of three ind. expts. (B and C) IAV-infected BMDC were treated with blocking CD24 mAb prior to transfer into naïve mice. At d7 post infected BMDC transfer, the frequency and total number of tet⁺ CD8⁺ T cells (B) and IFNγ secreting CD8⁺ T cells following ex vivo peptide re-stimulation (C) in the spleens were determined. (D) BMDC over-expressing CD24 by retrovirus transduction (pCD24; E – F and control vector BMDC) were pulsed with OT1 peptide prior to co-culture with naïve OT1 cells (Thy1.1) for 4 days. Activated OT1 cells (5 × 10⁴/mouse) were then transferred into d5 p.i. WT IAV-infected mice (Thy1.2). Accumulation of OT1 cells in the lungs and DLN was evaluated 24 hr later. Data in B - D represent at least three ind. Exps (n = 4 – 5 mice/group). See also .

CD24 controls naïve CD8⁺ T cell fate decision via RAGE engagement. (A - C) RDC subsets isolated from the lung (A) and DLN (B), respectively, at d3 p.i. were stained for cell-bound HMGB1. Shown are rep. flow cytometric plots (left panels in A) and MFI (right panels in A and B; n = 4 – 5 mice/group). (C) Migrant RDCs in the DLN at d3 p.i. were stained simultaneously with control and mAbs specific for HMGB1 and CD24. Data in C are representative of three independent experiments. (D) CFSE-labeled OT1 cells were transferred into naïve WT mice, which were subsequently infected with PR/8-OT1 IAV. αHMGB1 Ab was administered i.n. at d1 and d3, p.i., respectively. OT1 cell proliferation in the DLN was examined at d4 p.i. (n = 4 – 6 mice/group). (E) Naïve Cl4 cells were co-cultured for 4 days with DLN derived CD103⁺ RDC in the presence or absence of αRAGE Ab. Activated Cl4 cells (5 × 10⁴/mouse) were transferred into d6 p.i., B/Lee infected mice and Cl4 cell accumulation in the lungs and DLN was evaluated 24 hr later (n = 4 – 5 mice/group). Cl4 cells activated by CD11b^hi RDC were used as controls. Data in D and E represent at least two independent experiments. (F) Mixed BM chimeric mice between WT and Ager^−/− marrows (1:1) were generated and infected with PR/8. IAV-specific CD45 congenic memory CD8⁺ T cells were identified in the lungs and DLN (left panels) and enumerated (right panels) at d60 p.i. Data represent at least two ind. expts. (n = 4 – 6/expt). See also .