Introduction to immunostimulatory DNA sequences.

Abstract

Complete Freund’s adjuvant (CFA) consisting of mycobacterial extract in oil immersion was initially characterized 60 years ago [7] as an immune adjuvant. Although it contains many immunologically active substances, it has recently been shown that much of the immunostimulatory ability of CFA is attributable to the bacterial DNA fraction (discussed by S. Yamamoto et al. in this volume). Tokunaga and coworkers discovered that DNA purified from Bacillus Calmette-Guerin (BCG) induced an innate antitumor activity that appeared to be mediated through its ability to activate NK cells and induce production of interferons (IFNs) α/β/γ [26]. A variety of synthetic 45-mer oligodeoxynucleotides (ODNs) corresponding to various coding regions of three BCG proteins were synthesized. The ODNs that induced IFN secretion and augmented NK cell activity included CpG dinucleotides within a palindromic hexamer (also known as CpG motifs), e.g., 5’-GACGTC-3’, 5’-AGCGCT-3’ and 5’-AACGTT-3’ [27]. Furthermore, only bacterial and not vertebrate DNA produced the NK-immunostimulatory effect. This initial observation demonstrated for the first time that DNA carries information about its originating organism in addition to its genetic blueprints (discussed by D. Pisetsky in this volume). A consensus motif of rrCGyy (where r = purine and y = pyrimidine) has been proposed [12]; however, this CpG motif is only a loose rule: there are sequences that are stimulatory that do not follow the motif and sequences that would be included within the consensus that are not stimulatory. Recent work proposes that the optimal human immunostimulatory sequences are 5’-rTCGyy-3’ or a shortened version, 5’-TCG-3’ (discussed by H. Liang et al. in this volume). However, we have found that the sequence 5’-AACGTTCG-3’ has pronounced stimulatory ability in both mouse and human systems. Some DNA sequences without CpG have stimulatory ability, which is why we prefer the functionally defined name immunostimulatory DNA sequence (ISS) to the structurally defined name CpG motif. Table 1 gives an annotated list of the sequences that have been demonstrated to have potent immunostimulatory activity to date.