Dendritic cell migration limits the duration of CD8+ T-cell priming to peripheral viral antigen

Abstract

CD8(+) T cells (T(CD8(+))) play a crucial role in immunity to viruses. Antiviral T(CD8(+)) are initially activated by recognition of major histocompatibility complex (MHC) class I-peptide complexes on the surface of professional antigen-presenting cells (pAPC). Migration of pAPC from the site of infection to secondary lymphoid organs is likely required during a natural infection. Migrating pAPC can be directly infected with virus or may internalize antigen derived from virus-infected cells. The use of experimental virus infections to assess the requirement for pAPC migration in initiation of T(CD8(+)) responses has proven difficult to interpret because injected virus can readily drain to secondary lymphoid organs without the need for cell-mediated transport. To overcome this ambiguity, we examined the generation of antigen-specific T(CD8(+)) after immunization with recombinant adenoviruses that express antigen driven by skin-specific or ubiquitous promoters. We show that the induction of T(CD8(+)) in response to tissue-targeted antigen is less efficient than the response to ubiquitously expressed antigen and that the resulting T(CD8(+)) fail to clear all target cells pulsed with the antigenic peptide. This failure to prime a fully functional T(CD8(+)) response results from a reduced period of priming to peripherally expressed antigen versus ubiquitously expressed antigen and correlated with a brief burst of pAPC migration from the skin, a requirement for induction of the response to peripheral antigen. These results indicate that a reduced duration of pAPC migration after virus infection likely reduces the amplitude of the T(CD8(+)) response, allowing persistence of the peripheral virus.

FIG. 1.

T_CD8⁺ responses after i.v. immunization with AdCMVNP or AdK14NP. Mice were immunized i.v. with AdCMVNP (□) or AdK14NP (▪) (A) or additionally with AdSPCNP or rVACV-NT60 NP (B). (A) Production of IFN-γ in response to NP_366-374 peptide by in vitro restimulated splenocytes removed 30 days after immunization. Error bars are too small to observe. There was no statistically significant difference between the response observed in mice infected with either AdK14NP or AdSPCNP compared to uninfected mice. (B) Expansion of adoptively transferred F5 T_CD8⁺ (CD8⁺ V_β11⁺) 4 days after immunization. **, P < 0.01.

FIG. 2.

Proliferation and effector activity of T_CD8⁺ after i.d. immunization with AdCMVNP or Adk14NP. Mice were immunized i.d. in the ear pinnae with AdSPCNP (A and D), AdK14NP (B and E), or AdCMVNP (C and F). Proliferation of adoptively transferred F5 T_CD8⁺ in the draining cervical lymph node was assessed 3 days postinfection by monitoring CFDA-SE dilution (A to C). The data shown are representative, but the statistics shown are derived from four repeat experiments. (D to K) Cytotoxic activity was assessed 3 days (D to G) or 30 days (H to K) postinfection by examining clearance of cells pulsed with either the OVA_257-264 peptide (labeled with 0.5 μM CFDA-SE, lower peak) or with the NT60 NP_366-374 peptide (labeled with 5 μM CFDA-SE, upper peak) from the spleen 18 h after injecting target cells. Similar results were obtained when splenocytes were taken 4 h after the injection of labeled cells. *, P < 0.05; **, P < 0.01. The data in panels D to F and H to J are representative, and cumulative data from three independent experiments are shown with statistical analyses in panels G and K.

FIG. 3.

Localization of T_CD8⁺ after i.d. immunization with AdCMVNP or AdK14NP. CFDA-SE-labeled (A) or unlabeled (B and C) F5 T_CD8⁺ were adoptively transferred into mice that were immunized 24 h later with rAd as shown. Spleens and lymph nodes were removed 3 days (A), 4 days (B) or 30 days (C) after immunization, and F5 T_CD8⁺ (CD8⁺, CD45.1⁺) proliferation (A), quantified by dilution of CFDA-SE, or the numbers of cells (B and C) were measured by flow cytometry. (A) Representative plots are shown, but the statistics from three independent experiments are included. The numbers of proliferating cells in both spleen and node were different in AdCMVNP- and AdK14NP-infected mice (P < 0.01). Panels B and C: **, P < 0.01; *, P < 0.05.

FIG. 4.

Time course and efficacy of T_CD8⁺ activation after i.d. infection with AdCMVNP or AdK14NP. Mice were immunized i.d. and tested for either the ability to stimulate proliferation of adoptively transferred F5 T_CD8⁺ via CFDA-SE dilution (A and B) or the effector activity via clearance of specific peptide-pulsed CFDA-SE^HI labeled targets (C) on day 3 (A) or days 2, 3, and 4 (B and C) postinfection. (A) The site of infection was removed at the times shown postinfection. (D) The sensitivity of effector F5 T_CD8⁺ was assessed by measuring the clearance of cells pulsed with various amounts of NP_366-374 peptide 6 days after i.d. immunization. The data in panel C are expressed as a percentage of the maximal killing of NP_366-374 peptide-pulsed CFDA-SE^HI cells compared to levels after immunization with AdSPCNP. *, P < 0.05; **, P < 0.01.

FIG. 5.

Duration of antigen presentation. (A) Mice were immunized with viruses as shown, and enriched populations of DC were isolated from the draining lymph node at 24 h (VACV) or 48 h (rAd) postinfection. DC were incubated with CFDA-SE-labeled F5 T_CD8⁺ for 60 h, and proliferation of the T_CD8⁺ was measured. In four separate experiments, we observed no proliferation after immunization with AdCMVNP. (B) CFDA-SE-labeled F5 T_CD8⁺ were adoptively transferred into recipients at the days shown after i.d. infection. The proliferation of F5.SJL cells was assessed 3 days after adoptive transfer. The data shown are representative of three independent experiments, but the statistics of pooled data for AdK14NP and AdSPCNP stimulated proliferation are shown. Proliferation in response to AdK14NP was significantly different to that in response to AdSPCNP on 0 and 1 day postinfection (P < 0.01) but was not statistically different on days 3 through 7. Proliferation in response to AdCMVNP infection was statistically different (P < 0.01) from that observed after infection with either Adk14NP or AdSPCNP at all time points examined.

FIG. 6.

DC Migration following rAd infection. (A) The ears of mice were immunized and then painted with FITC at the time periods shown postinfection with rAd. The percentage of FITC⁺ CD11c⁺ DC in the draining lymph node was quantified 18 h later. (B) Similar to panel A, but unimmunized mice were painted with FITC, and the numbers of FITC⁺ CD11c⁺ DC were quantified at the times shown postpainting.