Antigen persistence and the control of local T cell memory by migrant respiratory dendritic cells after acute virus infection

Abstract

Acute viral infections induce robust adaptive immune responses resulting in virus clearance. Recent evidence suggests that there may be depots of viral antigen that persist in draining lymph nodes (DLNs) after virus clearance and could, therefore, affect the adaptive immune response and memory T cell formation. The nature of these residual antigen depots, the mechanism of antigen persistence, and the impact of the persistent antigen on memory T cells remain ill defined. Using a mouse model of influenza virus infection of the respiratory tract, we identified respiratory dendritic cells (RDCs) as essential for both sampling and presenting residual viral antigen. RDCs in the previously infected lung capture residual viral antigen deposited in an irradiation-resistant cell type. RDCs then transport the viral antigen to the LNs draining the site of infection, where they present the antigen to T cells. Lastly, we document preferential localization of memory T cells to the DLNs after virus clearance as a consequence of presentation of residual viral antigen by the migrant RDC.

Figure 1.

Viral antigen persistence in the influenza-infected lungs. (A) Kinetic analyses of infectious virus recovery from the influenza (A/PR/8)-infected lungs. Bronchoalveolar lavages were collected at the indicated days p.i. and assayed for infectious virus titers using MDCK cells. The horizontal line indicates the limit of detection. Viral titers are represented as mean TCID₅₀ (median tissue culture infective dose) ± SEM. (B) Duration of antigen presentation in the regional LN draining the lung. Previously, influenza-infected mice received i.v. CFSE-labeled HA-specific TCR Tg CD8⁺ (Cl-4) or CD4⁺ (TS1) T cells at the indicated days p.i. Proliferation in vivo was monitored by CFSE dilution 4 d later in the draining MLN. Antigen specificity was measured by transferring the reporter Tg T cells into mice that were previously infected with an antigenically irrelevant type A influenza virus (X31). Data are representative of at least five independent experiments. (C–E) Viral antigen persistence in the infected lung. Total RNA was extracted from MLN or lungs at the indicated days p.i. and subjected to RT-PCR analyses to detect NP RNA (with two rounds of PCR; C) or unspliced and spliced variants of NS gene (one round of PCR; E). RNA from RSV-infected lungs or an influenza-infected mouse lung epithelial cell line (MLE) was included as a control for primer specificity. Lung sections were stained for viral NP at day 5 (d5; top) or day 30 (bottom) p.i. and visualized by immunofluorescence microscopy (20×, D). Left, light fields; middle, anti-NP immunostaining; right, inverted view of the anti-NP immunostaining. TA, terminal airway. Data are representative of at least four independent experiments. Arrows indicate NP-positive cells. Bar, 200 µm.

Figure 2.

Characterization of RDC subsets in the lung after i.n. influenza virus infection. (A) CD103⁺ RDC (I), CD11b^hi RDC (II), and MoRDC (III) in the uninfected and infected lungs are identified among CD45⁺CD11c ⁺ lung cells by flow cytometry at the indicated days p.i. Data are representative of analyses from >20 individual mice performed at each time point. (B) Surface marker expression on subsets of RDC. Lung-residing DC subsets (CD103⁺ RDC, I; CD11b^hi RDC, II; and MoRDC, III) were examined at day 20 p.i. for surface expression of costimulatory molecules as indicated (open histograms). Staining with isotype-matched control antibodies was included (filled histograms). Data are representative of results from >10 mice analyzed in three independent experiments.

Figure 3.

Migrant RDC in the regional DLN of the previously infected mice support antigen-specific T cell proliferation. (A) Flow cytometry–based categorization of RDC subpopulations in the MLN of previously infected mice. The CD11c⁺ cells found in the MLN are divided into three major subsets: pDC (B220⁺ MHC II^lo, I), CD8α⁺ LNDC (CD8α⁺ MHC II^int, II), and migrant RDC (CD8α⁻B220⁻MHC II^hi, III). The migrant RDCs are further divided into two populations based on CD103 marker expression: CD103⁺ (CD103⁺ RDC) and CD103⁻ (CD11b^hi RDC) cells. Data are representative of more than five mice analyzed in five independent experiments. (B) In vivo uptake by RDC of soluble protein delivered to the respiratory tract before RDC migration to the DLN. Mice (day 20 p.i.) were given i.n. fluorescent dye–labeled OVA (OVA-DQ) or unlabeled OVA proteins. Migrant DCs that had taken up the soluble OVA protein in the respiratory tract were identified in the MLN 1 d later. Data are representative of four mice in two independent experiments. (C) Phenotypic features of DC subsets in the MLN of influenza-infected mice (day 20 p.i.). Levels of costimulatory molecule expression on CD8α⁺ LNDC, CD103⁺ RDC, and CD11b^hi RDC were determined at either day 3 (gray) or day 20 (open) after infection. Black histograms represent isotype-matched control Ab stainings. Data are representative of two mice in two independent experiments. (D) Migrant RDC isolated from MLN can stimulate proliferation of naive Tg CD8⁺ T cells directly ex vivo. The indicated individual DC subsets were separated by cell sorting of MLN cell suspensions from pooled LN of previously infected mice (day 20 p.i., n = 15–20 mice/experiment) and then co-cultured with CFSE-labeled HA-specific naive TCR Tg CD8⁺ T cells for 4 d in cultures. Sorted DC preparations were incubated with the preprocessed synthetic cognate peptide (HA_533-541) before co-culture with the responding T cells as a positive control. Data are representative of three independent experiments.

Figure 4.

Residual antigen presentation to CD8⁺ T cells in vivo in the MLN depends on migrant RDC. (A and B) Selective ablation of CD103⁺ and CD11b^hi RDC from the previously infected lungs resulted in near absence of the corresponding populations in the lung-draining MLN. CD11c-DTR mice recovering from influenza virus (day 20 p.i.) were given DTx i.n., and depletion of RDC in the lung was analyzed 24 h later (percentage of DC among CD11c⁺SiglecF⁻ cells: 15.2 ± 3.7 vs. 1.5 ± 0.7 for CD103⁺, P < 0.012, and 47.5 ± 6.4 vs. 17.6 ± 5.3 for CD11b^hi RDC, P < 0.006, for PBS-treated vs. DTx-treated mouse panels, respectively; mean ± SEM; A) or in the DLN at 72 h (percentage of DC among total LN cells: 0.09 ± 0.01 vs. 0.03 ± 0.006 for CD103⁺, P < 0.003, and 0.12 ± 0.02 vs. 0.04 ± 0.001 for CD11b^hi RDC, P < 0.008, for PBS-treated vs. DTx-treated mouse panels, respectively; mean ± SEM; B) after DTx treatments. Data in A and B is representative of at least three independent experiments with two animals per experiments. (C and D) The ability of T cells to proliferate in vivo in the lung-draining MLN was abrogated with the depletion of migrant RDC. PR/8-infected CD11c-DTR mice (day 20 p.i.) were treated with DTx (or PBS). CFSE-labeled HA-specific CL-4 CD8⁺ Tg T cells were then transferred i.v. into the previously infected treated mice 3 d later. The ability of the reporter T cells to proliferate in the MLN was examined 4 d after the adoptive transfer and depicted as the percentage of divided cells (mean ± SEM; P = 0.0003; C). Transferred T cells in the DLN of migrant RDC-depleted mice proliferate in response to i.v. influenza virus administration (D). Data in C and D are representative of two to four individual recipient mice analyzed in two separate experiments.

Figure 5.

The depot of viral antigen in the lung transported by RDC is radio resistant. (A) Schematic depiction of irradiation and BM transplantation. Wild-type mice recovering from acute influenza virus infection (∼3 wk after infection) underwent total body irradiation with a lethal dose and then were reconstituted with BM cells prepared from naive (uninfected) CD11c-DTR Tg mice. Mice were analyzed >4 wk after BM transplantation. (B) Antigen presentation persists in the DLN of the irradiated and BM reconstituted mice. Irradiated/reconstituted and control unirradiated mice received either CFSE-labeled Cl-4 (CD8⁺) or TS1 (CD4⁺) T cells by i.v. at day 50 p.i. T cell proliferation (CFSE dilution) in the DLN was measured 4 d later. X31-infected mice were included as a control for antigen specificity of the T cell response. (C) Engraftment of CD11c-DTR–derived RDC in the previously infected non-Tg mice. Reconstituted mice were treated i.n. with DTx (or PBS) route at >4 wk after BM reconstitution. Depletion of two major RDC subsets (CD103⁺ and CD11b^hi) from the lungs was determined 24 h after DTx treatment. Percentages (mean ± SEM) of DC among CD11c⁺MHC II^hi cells were 13.2 ± 2.5 versus 0.6 ± 0.08 for CD103⁺ (P < 0.003) and 42 ± 5.5 versus 8.2 ± 1.1 for CD11b^hi RDC (P < 0.001) for PBS-treated versus DTx-treated mouse panels, respectively. Data in B and C is representative of two independent experiments using two mice per experiment. (D) BM donor RDC were required for the presentation of residual vial antigen in the DLN of the previously infected mice. RDC-depleted mice, as described in C, were infused i.v. with CFSE-labeled reporter CD8⁺ or CD4⁺ Tg T cells 3 d after DTx (right) or PBS (left) treatments, and T cell proliferation in vivo was examined 4 d after transfer. Percentages (mean ± SEM) of divided cells (CFSE^low) were 25.2 ± 4.2 versus 5.1 ± 0.9 for CD8 T cells (P < 0.004) and 65.1 ± 3.5 versus 15.3 ± 2.6 for CD4 T cells (P = 0.0001) for PBS-treated versus DTx-treated mice, respectively. (E) CD8⁺ T cells were capable of proliferating in the MLN when antigen was delivered via the i.v. route. Mice treated as in D received i.v. infectious A/PR/8 virus at the time of T cell transfer. Data in D and E are representative of three independent experiments using one to two mice per experiment.

Figure 6.

Enrichment of memory T cell numbers in the DLN requires the continuous recruitment of antigen-bearing migrant RDC. (A) Enrichment of antigen-specific memory T cells in the LN draining the initial site of virus replication. Single cell suspensions prepared from lung–draining (MLN) or axillary and inguinal LN (pLN) of mice previously infected with A/PR/8 at the indicated time points were co-cultured with A/PR/8-infected BMDC, as APC and IFN-γ-secreting T cells were detected by intracellular cytokine staining. Data for each day analyzed are representative of four independent experiments using one mouse per experiment. (B–D) Lung-resident CD11c^hi DCs are required for maintaining the elevated number of antigen-specific polyclonal T cells in the lung DLN. (B) Schematic depiction of the time course of ablation of RDC from previously infected DTx-treated CD11c-DTR mice and reconstitution of the RDC-depleted lungs with DC. CD11c-DTR mice (day 30 p.i.) were given DTx i.n. The DTx-treated DTR mice then received either DC or alveolar macrophages (AM) i.n. 6 h after DTx treatment. Antigen-specific memory T cells were enumerated 4 d later. (C and D) Absolute numbers of IFN-γ–secreting polyclonal CD8⁺ (C) or CD4⁺ (D) T cells in the DLN upon restimulation with infected BMDC in vitro for 5 h as described in A. Data in C and D represent mean ± SEM values from four to seven individual experiments each using one animal per experiment.