Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization

Abstract

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

Figure 1

Number of N418⁺ DC recruited to the draining LNs after gene gun immunization. Groups of C57BL/6 mice (n = 3–6) received five abdominal deliveries of gold beads coated with different plasmids. 24 h after bombardment, nucleated LN cells from draining inguinal nodes of immunized or naive mice were isolated using the collagenase method and pooled. FcγII and III receptors were blocked, and cells were stained with N418 followed by PE-goat anti–hamster antibodies (adsorbed on mouse and rat Ig). The number of N418⁺ cells was monitored by FACS^® (>10⁵ total cells/sample) and calculated per one draining LN (DLN) based on the number of nucleated cells in the pooled LNs and the number of LNs in each group. (A) Three different experiments (Exp.) comparing DC number in inguinal LNs between naive mice and mice bombarded with pCMV/β-gal. (B) Comparison of DC number in inguinal LNs among naive mice and mice bombarded either with gold beads alone or with gold beads coated with pCMV/β-gal or control plasmids (Mock 1, Mock 2). Mock 1, pCDNA3, which contains the ampicillin resistance gene. Mock 2, Wrg7077, which contains the kanamycin resistance gene. Similar results were obtained in another experiment.

Figure 2

Number of directly transfected (β-gal–positive) cells in draining LNs. Groups of C57BL/6 mice (n = 3) received nonoverlapping abdominal deliveries (one, two, or five shots) of gold beads coated with pCMV/β-gal. 24 h after bombardment, draining LNs were harvested, pooled, and sectioned for analysis (A), or nucleated cells from the LNs were pooled and plated, 10⁶/well, into 24-well plates (B and C). Frozen sections and 24-well plates were stained for β-gal activity. To assure accurate enumeration of β-gal–positive cells, we (a) screened between 100 and 200 alternate serial sections (10 μm cuts) per each experimental group to evaluate a tissue mass equal to one to two LNs (A) and (b) screened wells containing cells from a total of two to three pooled LNs (B). No cells showing strong uniform staining were observed in draining LNs from unimmunized mice or mice bombarded with a control plasmid. The results in A and B/C are from two different experiments. Similar results were obtained in another experiment.

Figure 3

Analysis of APCs in draining LNs presenting the class I–restricted epitope. Groups of C57BL/6 mice (n = 4–5) received five abdominal deliveries of gold beads coated with different plasmids. 24 h after bombardment, inguinal LN cells were harvested using the collagenase method and pooled. (A) APCs and control APCs are from mice immunized with pCMV/β-gal– and mock plasmid–coated particles, respectively. APCs (10⁶) were depleted of either macrophages, B cells, DEC205⁺ cells, or class II⁺ cells using specific mAbs and magnetic beads as described in Materials and Methods. Cells then were incubated with 10⁵ T cells specific for a K^b-restricted β-gal epitope for 18 h in U-bottomed 96-well plates. IFN-γ levels in the supernatants were measured using an ELISA kit, and results are presented as OD ± SE. (B) APCs are from mice immunized with pCMV/β-gal–coated gold beads (β-gal) or from mice immunized with pCMV/β-gal and T4TM (hCD4 plasmid)–cocoated gold beads (β-gal+hCD4). APCs (experiment [Exp.] 1, DC-enriched LN cells, 10⁵; experiment 2, LN cells, 10⁶) were either mock-depleted (i.e., treated only with magnetic beads) or depleted of cells expressing cell surface hCD4 protein as described in Materials and Methods (hCD4 dep). Cells were then incubated with T cells, and IFN-γ levels were measured as described above. Background OD, obtained from T cells without APCs (0.15– 0.19), was subtracted from the results in B. Bars, ±SE. Experiments were repeated three times with similar results.

Figure 4

CTL responses induced by gene gun delivery of NPo and B7.2 plasmids. Groups of BALB/c mice were immunized once by gene gun with a total of six shots, with gold particles either coated with the colinear construct NPo/B7.2 or cocoated with NPo and B7.2 constructs. Another set of mice received overlapping shots of gold separately coated with NPo or B7.2 as described in Materials and Methods. 4 wk after immunization, spleens were harvested and restimulated as described in Materials and Methods. Influenza virus–infected mice were used as positive controls. Lysis of P815 cells not pulsed with peptide was <5%. Each point corresponds to the mean ± SE obtained from two mice in each group. The experiment was repeated twice with similar results.