After the lecture, an elderly lady came up and told the scientist that he had it all wrong. “The world is really a flat plate supported on the back of a giant tortoise.” The scientist asked “And what is the turtle standing on?” To which the lady triumphantly replied: “You’re very clever, young man, but it’s no use—it’s turtles all the way down.”—Stephen Hawking, A Brief History of Time (1988)

When The Journal of Immunology approached me to write a commentary on the 1990 Science paper “Homology of Cytokine Synthesis Inhibitory Factor (IL-10) to the Epstein-Barr Virus Gene BCRF1” (1), I asked the editors this one question: “Why?”

Only not as it first sounds. I meant “why” in a very good way, as in which of the many elegant features of this gem happened to be their basis for its selection as a Pillars of Immunology article—because I could think of so many. It is, after all, on the cloning of IL-10, founding member of a terrifically important cytokine family (2, 3) that includes IL-19, IL-20, IL-22, IL-24, IL-26, and the related IL-28A, IL-28B, and IL-29. It is, arguably, the first molecular example of viral immunoevasion through cytokine mimicry. It is, in my opinion, about as artistic a fusion of molecular and cellular immunology as I have seen. Specifically, it fused a technically demanding molecular problem with the subtle insight of assaying a factor’s indirect action on T cells, not its direct effect on APCs, to clone a cDNA with this factor’s activity. I think it likely that this paper’s subtlety is lost on most readers today, and I would guess the same was true in 1990.

Given the power of today’s technologies, mostly nonexistent in the late 1980s, it is easy to miss the beauty of this paper and overlook its impact. For these reasons, some background may help set the stage. In 1980, the entrepreneur chemist Dr. Alejandro Zaffaroni from Syntex founded the DNAX Research Institute in the heart of Silicon Valley and hired a mixture of molecular biologists and cellular immunologists. DNAX soon became a truly extraordinary place. By the late 1980s, DNAX had already made several major accomplishments, including the cloning of several human cytokine genes (4, 5).

Relevant to this story, Dr. Kevin W. Moore, the paper’s first author, had studied at the California Institute of Technology and trained in molecular immunology with Dr. Leroy Hood in the regulation of RNA splicing and cloning of MHC genes. He moved from Pasadena, CA, straight to DNAX in Palo Alto, CA, and spent much of the 1980s cloning several Fc receptors for IgG (6) and receptors for IgE (7). Doing the work with Kevin was a fresh postdoctoral fellow, Dr. Paulo Vieira, who had recently moved to DNAX from the University of Cologne, where he had trained with Dr. Klaus Rajewsky.

Dr. Tim Mosmann’s pathway to DNAX included training in microbiology at the University of British Columbia and a few years at the Department of Immunology at the University of Alberta, focusing on various aspects of the control of Ab responses. By 1982, he had moved to DNAX, where he began an interest in establishing T cell clones, and in 1986 along with Dr. Robert L. Coffman, he published the watershed paper in T cell biology describing the existence of CD4+ T cell subsets and defining type 1 and type 2 Th cells (8).

In examining the behaviors of Th1 and Th2 cells, Mosmann realized that the cytokines produced by one had inhibitory actions on the other. In one case, Th1 cells produced a known cytokine, IFN-γ, that inhibited Th2 proliferation (9, 10). Symmetrically, Mosmann discovered that Th2 clones secreted a product that diminished IFN-γ production by Th1 cells (11). In recognizing this, he discovered a central principle allowing the stable divergence of T cell subsets through systems of mutual repression. Whereas IFN-γ controlled proliferation of Th2 cells, he called the Th2-derived factor “cytokine synthesis inhibitory factor” (CSIF). Mosmann recognized an additional complexity. The previous year, working with David Fiorentino, one of the present study’s coauthors, Mosmann had published that the action of CSIF on Th1 cytokine synthesis seemed to be indirect and was most pronounced only when splenic APCs were present (11). From today’s vantage point, it is easily understood that IL-10–mediated repression of costimulation or IL-12 production by macrophages or dendritic cells might lead to decreased IFN-γ production by Th1 cells. However, in 1990, the roles of B7 and CD28 were unknown, and the factor called “NK cell stimulatory factor” (later IL-12) had only been identified by Dr. Giorgio Trinchieri (12) the previous year and would not be cloned for another (13). Only the next year, Fiorentino would work with Dr. Anne O’Garra to show that CSIF, by then called IL-10, acted on macrophages, and not B cells, in the splenic APC population to suppress Th1 cytokine production (14, 15), but at the time, it was only clear that the actions of CSIF were complex and indirect, casting a shadow over the cloning strategy. Indeed, one might say the authors were cloning in the dark.

The approach was beautifully simple, but technically demanding. On Moore and Vieira’s side, individual cDNAs from Dr. Charles Janeway’s famous Th2 clone D10 (16) were pooled and transfected into COS7 cells to produce supernatants. These supernatants were tested on Mosmann and Fiorentino’s side for inhibition of IFN-γ production by the Th1 clone HDK-1. Now this is much trickier than it sounds, and there are many ways this could have gone wrong, such as if the cytokine required two subunits, and so on. Furthermore, it was recognized that TGF-β could act similarly to CSIF, so conceivably this screen could have pulled out copies of this or any other nonspecific inhibitory factor. Fortunately, the CSIF activity was acid sensitive (11), in contrast to that of TGF-β, so it was at least possible to distinguish these activities experimentally.

When a clone isolated from this process was sequenced, no homology to known cytokines was found, but one open reading frame from the EBV genome was identified that was clearly related. However, at first it was not so clear which way things had evolved. Had mice picked up a viral fragment in the genome left over from some ancient infection, as is the case in endogenous retrovirus proteins encoding IgE receptors (7), or had the virus stolen the mammalian gene to use for some purpose of its own?

Here is where the brilliance of this study shines through. At that time, viral immunoevasion was a recognized process, but knowledge of this process was mechanistically restricted to blockade of Ag presentation by viral proteins (17, 18). However, it was known that “IFN-γ inhibits the … outgrowth of EBV-transformed B cells in vitro” (1). Considering the possibility that the mammalian IL-10 gene had been taken in by the virus, the authors developed the idea that “EBV has exploited the biological activity of the product of a captured cytokine gene to manipulate the immune response …” (1). Then, they carefully articulated the proposal that “Conceivably, mimicry of host cytokines could be a defense mechanism also utilized by other pathogens or parasites” (1). When I asked Dr. Moore about the study’s priority as the first example of viral immunoevasion through cytokine mimicry, he modestly pointed to a contemporary study in the very same volume of Science describing soluble “cytokine receptors” encoded by Pox virus related to the TNF receptor (19). But typically, he took pleasure in adding that this study’s first author was also a DNAX alumnus.

Subsequent studies have only slightly altered Moore and Mosmann’s initial interpretation (2, 20), suggesting that viral IL-10 may be a weak agonist at the IL-10 receptor and may convert an otherwise harmful pathogen to a mostly benign viral parasite by reducing collateral tissue damage. Vieira went on the next year with Moore to clone human IL-10 by homology to murine IL-10 (21), and Fiorentino worked with O’Garra’s laboratory at DNAX to sort out the nature of the indirect action of IL-10 on APCs (14, 15). It is regrettable that IL-10 is not a cytokine that has achieved status as a drug. Because it was identified before the days of industrial “biologics,” time was lost in developing suitable forms for in vivo use, and now, as patent-related protection nears expiration, development as a drug seems doubtful.

A final thought about the long-term impact of this study was suggested by some of its authors. By 1990, immune suppression as a mechanism had come into some doubt (22). With the discovery of IL-10 as a new suppressive cytokine, these authors helped to provide a solid framework for negative feedback mechanisms in T cell biology. Importantly, these authors had close relationships with Drs. Robert Coffman and Fiona Powrie at DNAX, who led a quiet counterrevolution in the area that critically helped restore some confidence in cellular suppressive mechanisms (23, 24). So, besides being an example of an entirely novel category of viral immunoevasion, this study helped set up a new viewpoint for thinking about immunosuppression as well. I would add, “It’s Pillars all the way down.”

Abbreviation used in this article:

CSIF

cytokine synthesis inhibitory factor.

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The author has no financial conflicts of interest.