SummaryAllergen specific CD4+ T cell clones generated from allergic individuals have been shown to produce increased levels of the cytokine interleukin 4 (IL-4), compared to allergen specific clones generated from nonallergic individuals. This difference between CD4+ T cells from allergic and nonallergic individuals with regard to cytokine production in response to allergen is thought to be responsible for the development of allergic disease with increased IgE synthesis in atopic individuals. We examined the production ofIL-4 in subjects with allergic rhinitis and in allergic individuals treated with allergen immunotherapy, a treatment which involves the subcutaneous administration of increasing doses of allergen and which is highly effective and beneficial for individuals with severe allergic rhinitis. We demonstrated that the quantity of IL-4 produced by allergen specific memory CD4+ T cells from allergic individuals could be considerably reduced by in vivo treatment with allergen (allergen immunotherapy) . Immunotherapy reduced IL-4 production by allergen specific CD4+ T cells to levels observed with T cells from nonallergic subjects, or to levels induced with nonallergic antigens such as tetanus toxoid . In most cases the levels of IL-4 produced were inversely related to the length of time on immunotherapy. These observations indicate that immunotherapy accomplishes its clinical effects by reducing IL-4 synthesis in allergen specific CD4+ T cells . In addition, these observations indicate that the cytokine profiles of memory CD4+ T cells can indeed be altered by in vivo therapies . Thus, the cytokine profiles of memory CD4+ T cells are mutable, and are not fixed as had been suggested by studies of murine CD4+ memory T cells. Finally, treatment of allergic diseases with allergen immunotherapy may be a model for other diseases which may require therapies that alter inappropriate cytokine profiles of memory CD4+ T cells .

Recent reports have identified two major classes of CpG motif-containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)-alpha, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)-derived IFN-alpha but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN-gamma/IFN-alpha from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN-alpha. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG-containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN-alpha/IFN-gamma induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.

There are two principle subsets of dendritic cells (DCs); CD11c+CD123− myeloid DCs (MDCs) and CD11c−CD123+ plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin−HLA-DR+CD11c+CD123− MDCs and Lin−HLA-DR+CD11c−CD123+ PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-α secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-α release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-γ production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.

IntroductionImmunostimulatory sequences (ISS) are short oligonucleotides (ODNs) that mimic the innate immune response to microbial DNA. 1 ISS contain one or more cytosine-phosphate-guanine (CpG) dinucleotide-containing motifs with unmethylated cytosine residues and are recognized by Toll-like receptor-9 (TLR-9), one of a family of receptors prominent in innate responses to microbial pathogens. 2,3 TLR-9 expression is not widely distributed, and ISS-responsive cells in human peripheral blood mononuclear cells (PBMCs) are limited to B cells and plasmacytoid dendritic cells (PDCs). ISS activate B cells to proliferate, secrete interleukin (IL)-6, and differentiate to plasma cells. 4 PDCs respond to ISS by secreting type I interferons, tumor necrosis factor ␣ (TNF-␣), and, upon signaling through CD40, IL-12. 2,5 In both cell types, ISS are potent enhancers of antigen-presenting cell (APC) function and induce key costimulatory molecules, such as CD40, CD80, and CD86. 6 In mixed cell populations, such as human PBMCs, the direct response to ISS initiates a cascade of secondary responses, including activation of macrophages and natural killer (NK) cells and the induction of interferon ␥ (IFN-␥) and a wide range of other cytokines and chemokines characteristic of inflammatory responses. [6][7][8][9][10] At least 3 classes of ISS can be distinguished on the basis of both structure and function. Uniformly modified phosphorothioate (PS) ISS, called CpG-B, strongly activate B cells but are weak stimulators of IFN-␣ from human PBMCs. Phosphodiester (PO)-linked sequences flanked by PS-linked poly-G ends, called CpG-A ISS, in contrast, are potent inducers of IFN-␣ and IFN-␥ but are weak activators of B cells. 11 Recently, a third class of ISS has been defined, called CpG-C. CpG-C ISS retain distinctive properties of both CpG-A and -B ISS with respect to IFN-␣ production and B-cell activation. 6 Preliminary data have shown that ISS affect PDC survival, maturation, and cytokine production, but the differential properties of these ISS on PDCs are otherwise undefined. 5,6,12 The efficient activation of APCs and induction of IL-12, IFN-␣, and IFN-␥ explains the potent ability of ISS to act as a strong T-helper cell 1 (Th)1-polarizing adjuvant. 13 Besides inducing Th1 responses, administration of ISS mixed or covalently linked to antigens can inhibit Th2 responses 14-17 and stimulate CD8 T-cell responses. 7 These activities have stimulated much interest in the clinical use of ISS as a vaccine adjuvant and in the treatment of allergy, asthma, cancer, and infectious diseases, and clinical trials are currently being conducted in all of these disease areas.IL-10 is a potent anti-inflammatory cytokine that can act as a feedback regulator of the inflammatory response to many microbial stimuli. 18 IL-10 can be produced by a number of different cell types and can inhibit both Th1 and Th2 responses by affecting APC function and dendritic cell (DC) maturation. 18-20 IL-10 inhibits activation and induces death of PDCs in vitro and can reduce PDC...

Alveolar macrophages, resident phagocytic cells in the lung that derive from peripheral blood monocytes, are paradoxically ineffective in presenting antigen to T cells. We found that antigen presentation by alveolar macrophages could be restored by the addition of anti-CD28 mAb to cultures of T cells and macrophages, indicating that costimulation by alveolar macrophages via the CD28 pathway was defective. In addition, we found that alveolar macrophages activated with IFN-y failed to express B7-1 or B7-2 antigens, which normally ligate CD28 on T cells and provide a costimulatory signal required for the activation of T cells. These observations are the first to demonstrate the inability of a "professional" antigen-presenting cell type to effectively express the costimulatory molecules B7-1 and B7-2. Inasmuch as immune reactions within the lung are inevitably associated with inflammatory injury to pulmonary tissue, these observations suggest that reduced expression of B7-1 and B7-2 by alveolar macrophages may be advantageous, as a critical mechanism involved in the induction of peripheral tolerance to the abundance of antigens to which mucosal tissues are continuously exposed. (J. Clin. Invest. 1995Invest. . 95:1415Invest. -1421