Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation

<div><p>Naïve anti-viral CD8⁺ T cells (T_CD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I <i>in vivo</i> can increase the number of responding T_CD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve T_CD8+ with similar efficiencies <i>in vitro</i>. We also provided evidence that these same cell-types presented antigen <i>in vivo</i>, as they form contacts with antigen-specific T_CD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.</p></div

Antigen specific T cells relocate to the LN periphery where they interact with infected pAPC expressing antigen <i>in vivo</i>.

<p>(A and B) Localization of OT-I T_CD8+ following ECTV infection. Naïve OT-I T_CD8+ were labeled with cell tracker CMTMR dye (red) and adoptively transferred. Mice were injected with NP-S-EGFP or NP-EGFP i.d., and 12 h.p.i., D-LN were harvested and analyzed by fluorescence microscopy. (C, E, G, I) Mice were injected with NP-S-EGFP or NP-EGFP i.d., and 24 h.p.i. D-LN were harvested and stained for B220⁺ B cells, CD169⁺ macrophages, CD11c⁺ DC and CD103⁺ DC, and analyzed by fluorescence microscopy. (D, F, H, J) High power view of interaction of naïve OT-I T_CD8+ and ECTV-infected pAPC. The insets (D, F, H, and J) show 2-dimensional projections of one plane of the 3-dimensional datasets. Each image is representative of 3 experiments, with a minimum of 4 infected nodes per experiment.</p

DC subsets are equally efficient in direct antigen presentation.

<p>(A) Mice were injected with ECTV, D-LN harvested and cells stained with antibodies to identify non-NK, non-B, non T cell, GFP⁺ CD11c⁺ DC subsets as: pDC (B220⁺CD11b^-), CD8α (B220^-CD8α ⁺CD11b^-), CD11b⁺ (B220^-CD11b⁺CD8α ^-). Nos. represent % of cells in 3 representative experiments using 3 mice per condition. (B and C) Mice were injected with NP-S-EGFP or NP-EGFP, D-LN harvested and stained as described in (A), with the addition anti- K^b-SIINFEKL. Quantification of K^b-SIINFEKL expression and efficiency was determined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004941#ppat.1004941.g002" target="_blank">Fig 2C</a>. Data are pooled from 3 experiments using 3 mice per condition. (D) Mice were injected with NP-EGFP or NP-S-EGFP, then D-LN cells were FACS sorted for EGFP⁺ or EGFP^- pDC, CD8α ⁺, or CD11b⁺ DC, as above. Each population was co-cultured with OT-I T_CD8+ that were then analyzed for proliferation as above. Data are pooled from 3 experiments, using 15 mice per condition to obtain sufficient cells. (E) As in (A), except for addition of anti-CD80 and anti-CD86 antibodies. Data are pooled from 3 experiments. All graphs show (mean ± standard error), P values *p<0.05, **p<0.01, ***p<0.001, NS (not significant) using Student’s unpaired t-test.</p

Treatment with TLR agonists <i>in vivo</i> inhibits viral infectivity but does not enhance direct antigen presentation.

<p>(A) Mice were injected i.v. with LPS, and 12 hr later splenocytes stained to identify DC, and examine expression of MHC class II, CD40, CD80, and CD86. Representative of 3 experiments, using 3 mice per condition. (B) CFDA-SE-labeled OTI T_CD8+ were adoptively transferred into mice that were then treated with LPS i.v. and 12 hours later injected i.p. with β₂m^-/- cells infected with NP-EGFP or NP-S-EGFP as above. Three days later, OTI T_CD8+ cell proliferation was determined by CFDA-SE dye dilution. Nos. represent % of cells representative of 3 experiments, using 3 mice per condition. (C) Mice were injected with LPS as above, and 12 h later, the mice were infected i.v with NP-S-EGFP. Twelve h.p.i., splenocytes were stained for DC subsets. Graphs depict ECTV-infection of DC (top panel, left) or DC subsets (top panel, right), and direct presentation by DC (bottom panel, left) or DC subsets (bottom panel, right). Data are pooled from 3 experiments, using 3 mice per condition (mean ± standard error). (D) Splenocytes were harvested and treated with LPS for 12 h, then infected with NP-S-EGFP (MOI = 10). Twelve h.p.i., cells were stained as described in (C). Graphs depict ECTV-infection of DC (top panel, left) or DC subsets (top panel, right), and direct presentation by DC (bottom panel, left) or DC subsets (bottom panel, right). Data are pooled from 3 experiments, using 3 mice per condition (mean ± standard error). (E) Splenocytes were treated with LPS for 12 h, then infected with NP-S-EGFP at the MOI indicated. Graphs depict infection of DC (top panel) and direct presentation by DC (bottom panel). Data are representative of two independent experiments (mean ± standard dev). P values *p<0.05, **p<0.01, ***p<0.001, NS (not significant). Student’s unpaired t-test.</p

EGFP⁺ cells are infected by ECTV and directly present antigen in a TAP dependent manner.

<p>(A and B) Expression of EGFP 12 h.p.i with NP-EGFP i.d. or vehicle. D-LN were analyzed by flow cytometry (12 h.p.i) (A) or by fluorescence microscopy (6 h.p.i) (B) (Representative of 5 experiments). (C) C57BL/6.SJL cells were infected with ECTV wt or NP-S-EGFP <i>in vitro</i>, treated with UV-C and psoralen and injected i.v. into C57BL/6 mice. Positive control C57BL/6 mice were injected i.v. with ECTV NP-S-EGFP. Twelve hours later, spleens were harvested and recipient cells were analyzed for EGFP expression by flow cytometry (Representative of 3 experiments. Nos. are % of cells). (D) Expression of K^b-SIINFEKL complexes by splenocytes 24 hr after immunization with NP-S-EGFP or NP-EGFP i.v. analyzed by flow cytometry (Representative of >10 experiments (n = 3 mice per condition per experiment) and nos. represent % of cells). (E) TAP1^-/- or C57BL/6 mice were injected with NP-S-EGFP, as described in (D) (Representative of 3 experiments and nos. represent % of cells).</p