Cloning of IL-12, a Critical Advance in Th1 and Th17 Discovery

Cytokines, soluble factors with immune-modulating properties, were first reported in the late 1950s and 1960s, with the discovery of IFN-α and then lymphotoxin. This Pillars of Immunology was article published in 1991 by Giorgio Trinchieri, Bice Perussia, and colleagues (1) describing the identification of the genes for IL-12 subunits p35 and p40, and it confirms that they encode the NK and T cell–activating cytokine NK cell stimulatory factor (NKSF), now known as IL-12.

NKSF was first identified 2 y earlier by the same research group as a secreted soluble factor produced by the feeder B cell line RPMI-8866, capable of inducing IFN-γ production, cytotoxicity, and proliferation in human peripheral blood leukocytes (2). NKSF was found to be a heterodimeric protein made up of a 35-kDa subunit disulfide linked to a 40-kDa subunit. The Pillars of Immunology study identified amino acid sequences from each of the subunits, made oligonucleotide probes based on those sequences, and screened against phage libraries to identify the genes (1). The genes included sequences known to be typical of cytokines, including signal peptides and cleavage sites needed for secretion and repetitive ATTTA destabilization sequences. By expressing plasmids encoding cDNAs for each subunit individually or together in cos-1 cells, the authors confirmed the production of secreted proteins that together formed a p70 heterodimer. This recombinant factor recapitulated activities of purified NKSF when applied to PBLs, and both subunits were required to be expressed in the same cell line, suggesting formation of the cytokine before secretion. These elegantly executed and controlled experiments thus identified the first heterodimeric cytokine with biologically distinct subunits.

As every immunology student now knows, IL-12 is the major Th1 skewing factor, and rIL-12 is routinely used for in vitro Th1 cell differentiation. In 1984, Trinchieri et al. (3) found that IL-2 was able to induce IFN-γ production from NK cells, but not from T cells. The Th1–Th2 paradigm was described 2 y later (4). However, in vitro differentiation of Th1 cells was not successful until the cloning of IL-12 (in 1991), with subsequent use of rIL-12 to demonstrate the induction of IFN-γ from human and mouse resting PBLs (5). The cloning and characterization of the heterodimeric IL-12R followed soon after. rIL-12 also facilitated elucidation of the critical roles of STAT4 and T-bet in Th1 differentiation (6–8). Additional studies determined macrophages and dendritic cells to be the major sources of IL-12 production, suggesting that IL-12 bridges the gap between the innate response and adaptive polarization (9, 10).

In 1996, p40 knockout mice were generated (11), allowing in vivo studies that confirmed the critical importance of IL-12 for IFN-γ–mediated immunity to intracellular pathogens (12, 13). Shortly thereafter, case studies of human patients with IL-12 deficiency were described with increased susceptibility to infections, such as M. tuberculosis, S. pneumoniae, and S. enterica, and significantly impaired IFN-γ production (14–16), further solidifying the role of IL-12 in driving the Th1 response in both mice and humans.

Mice deficient in p40 were presumed to be IL-12 knockouts. However, Wolf et al. (1) presciently noted the possibility of additional partners for p40. In 2002, comparison of IL-12p35– and IL-12p40–deficient mice revealed that deletion of IL-12p40 protected against experimental autoimmune encephalomyelitis, whereas deletion of IL-12p35 did not (17). IL-23, a close relative of IL-12, had recently been identified as a heterodimer consisting of IL-12p40 and IL-23p19 (18). In 2003, Cua et al. (19) published a paradigm-shifting study that identified IL-23 as the critical IL-12 family member in experimental autoimmune encephalomyelitis, leading to a slew of investigations re-evaluating the role of IL-12 and Th1 cells in autoimmune and infectious diseases. Ultimately, these studies led to the discovery of the Th17 lineage of CD4⁺ Th cells more than two decades after the initial description of Th1 and Th2 cells (20).

Discovery and characterization of cytokines and other secreted proteins was expedited by advances in cDNA cloning and subsequent transfection into prokaryotic or eukaryotic cells for recombinant protein production. Insulin was the first Food and Drug Administration–approved recombinant protein in 1984, and since then >130 recombinant proteins have been approved for clinical use in humans. In this regard, rIL-12 was evaluated as a possible cancer treatment because of its IFN-γ–inducing activity. However, early clinical trials found that IL-12 caused systemic toxicity (21–23). Targeting IL-12p40 with mAbs for autoimmunity saw initial clinical success, specifically the use of ustekinumab in psoriasis, and with the realization that IL-23 was the key target, the next generation of mAbs targeted the IL-23 p19 subunit with improved efficacy (24). Hence pioneering work, including this study by Wolf et al. (1), provided key biological tools for the next 30 y of studying type 1 and type 17 immunology, as well as a number of new therapeutic approaches.