DNA as an adjuvant: capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen

Abstract

How strong adjuvants such as complete Freund's adjuvant (CFA) promote T cell priming to protein antigens in vivo is still unclear. Since the unmethylated CpG motifs in DNA of bacteria and other nonvertebrates are stimulatory for B cells and antigen-presenting cells, the strong adjuvanticity of CFA could be attributed, at least in part, to the presence of dead bacteria, i.e., a source of stimulatory DNA. In support of this possibility, evidence is presented that insect DNA in mineral oil has even stronger adjuvant activity than CFA by a number of parameters. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs mimic the effects of insect DNA and, even in soluble form, ODNs markedly potentiate clonal expansion of T cell receptor transgenic T cells responding to specific peptide.

Figure 1

Adjuvant function of insect DNA for T cell responses to FγG. Normal B6 mice were immunized in the hind limbs with FγG (25 μg/limb for A, top and B, 5 μg/limb for A, bottom) mixed with CFA, IFA, DNA, or IFA and DNA; for DNA, insect DNA was injected at 50 μg/limb. Cell suspensions were prepared from the DrLN 9 d later. For A, top and B, unseparated LN cells were cultured at 2.5 × 10⁵ cells/well ± FγG (20 μg/well, 100 μg/ml). For A, bottom, purified CD4⁺ cells were prepared from DrLN cells and cultured at 10⁵/well in the presence of T-depleted spleen cells (4 × 10⁵/well) ± FγG (10 μg/ml). To measure [³H]TdR incorporation (A), cultures were pulsed with 1 μCi/well of [³H]TdR on day 3 and harvested for radioactive counting on day 4. For IFN-γ production (B), supernatants were tested on day 3. The data show means of triplicate cultures; SDs were within 10–20% of the means.

Figure 2

Adjuvant function of ODNs for T cell responses to FγG. Mice were immunized to FγG (5 μg/limb) as described for Fig. 1, using CpG and ZpG ODNs (25 μg/limb) instead of DNA; ODNs were either mixed with IFA (A; B, left; and C, right) or suspended in saline (A; B, right; and C, left). Antiserum was collected by tail bleeding on day 9 and tested at 1:100 or 1:1,000 dilution (C). For Ab production, some mice received CpG ODNs in the front limbs and FγG in the hind limbs (FγG + CpG*). For in vitro responses (A and B), DrLN cells were removed on day 9, depleted of B cells, and cultured with graded doses of FγG for 3 d (IFN-γ production) or 4 d ([³H]TdR incorporation). The data show means of triplicate cultures. Two other experiments gave similar findings.

Figure 3

Capacity of ODNs to augment clonal expansion of 2C transgenic CD8⁺ cells responding to specific peptide. Groups of B6 mice were injected intravenously with 2 × 10⁷ 2C lymphoid cells and then injected subcutaneously in the hind limbs with specific peptide (25 μg/limb) ± soluble CpG or ZpG ODNs (25 μg/limb). At 3, 4, or 5 d after immunization, the mice received a single intraperitoneal injection of 1 mg BrdU. Lymphoid organs were removed from the mice 4 h later and cell suspensions were stained for CD8 and 1B2 expression and then for BrdU incorporation followed by FACS^® analysis. The data show mean values (2–3 mice/group) for total numbers of 1B2⁺ CD8⁺ cells (A) and BrdU⁺ 1B2⁺ CD8⁺ cells (B) in the DrLN, spleen, and MLN. Essentially identical results were seen in a second experiment.