From IL-10 to IL-12: how pathogens and their products stimulate APCs to induce TH1 developmentAnne O’Garra & Kenneth M Murphy

The authors recount their discovery of how pathogen-induced interleukin 12 production leads to TH1 T cell polarization. Simultaneously they discovered the suppressive cytokine interleukin 10 inhibits antigen-presenting cells, thus regulating development of TH1 cells.

Successful eradication of a pathogen requires that the right type of protective

immune response be elicited. Furthermore, the response must be regulated to prevent damage to the host. By the mid-1970s, it became clear that heterogeneity of CD4+ T cells can determine whether humoral or cell-mediated immune responses are elicited in response to antigen, and many studies had demonstrated that such responses are often mutually exclusive1. In 1986, seminal work by Bob Coffman and Tim Mosmann at the DNAX Research Institute showed that mouse CD4+ T helper cell clones isolated from chronically immunized or infected mice could be subcategorized according to the patterns of cytokines they secreted2, which offered some explanation for the reciproc-ity of cell-mediated and humoral immune responses3. T helper type 1 (TH1) clones were characterized by their hallmark cytokine interferon-γ (IFN-γ) and were shown to be important for cell-mediated immune responses by their activation of macrophages

and the killing of intracellular pathogens. In contrast, TH2 cells produced interleukin 4 (IL-4) and IL-5, cytokines that they showed were involved in antibody responses3. By this stage, the following two key questions arose. First, could TH1 and TH2 cells differentiate from a naive CD4+ T cell and, if so, how? Second, how is cytokine production by these effector cells regulated to avoid immunopa-thology? Here we will focus on how our vari-ous lines of research at the DNAX Research Institute and the Department of Pathology of Washington University in St. Louis School of Medicine led us to join forces in the early 1990s to answer these questions.

IL-10 acts on macrophages and DCs (Anne)IL-10 was first described by Tim Mosmann, David Fiorentino and Martha Bond at DNAX as a cytokine synthesis–inhibitory factor produced by TH2 cells that inhibited the pro-duction of cytokines by TH1 cells4 and was suggested to explain part of the apparent reciprocal regulation of cell-mediated (TH1) and humoral (TH2) immune responses3. However, when Kevin Moore and Paulo Vieira screened cDNA libraries from TH2 clones supplied by Mosmann’s group to determine the gene encoding the cytokine synthesis–inhibitory factor activity5,6, it became clear that IL-10 had additional activities and was produced by cells other than TH2 cells7.

I was intrigued with the discovery of TH1 and TH2 cells by Tim and Bob, having started working on T cell–derived cytokines and B cell

responses when I was a postdoctoral fellow at the National Institute for Medical Research in Mill Hill, London. I obtained a PhD for studies of bacterial adhesion there, and then decided to do a postdoctoral fellowship in immunology, which offered perfect systems for investigating cell differentiation, one of my passions. At the National Institute for Medical Research, I then studied the function of cytok-ines in B cell growth and differentiation in the lab of Gerry Klaus and much enjoyed the collaborative spirit of the institute. I joined DNAX in 1987 as a postdoctoral fellow with Maureen Howard to continue some of this work, but then was given the opportunity to start my independent line of research with a small group of my own. My question was as follows. Do different antigen-presenting cells (APCs) produce particular factors that determine the development of TH1 and TH2 responses? Because my background was in studying B cells, I had started analyzing B cell populations for their production of various known cytokines or activities that we could test on many cell lines that would then allow the purification and/or cloning of the mole-cule that had the activity. We identified a mast cell activity distinct from the activities of IL-3 and IL-4, which turned out to be attributable to IL-10. I remember the excitement after discussing this with Paulo Vieira (Fig. 1) and Kevin Moore at the time, when we discovered that the B cell lines I was working on expressed IL-10 mRNA, whose abundance correlated with the mast cell activity we had identified

Anne O’Garra is with the Division of

Immunoregulation, The Medical Research

Council National Institute for Medical Research,

London, UK. Kenneth M. Murphy is in the

Department of Pathology and Centre for

Immunology, Howard Hughes Medical Institute,

Washington University in St. Louis School of

Medicine, St. Louis, Missouri, USA.

e-mail: aogarra@nimr.mrc.ac.uk or

kmurphy@wustl.edu

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in the various B cell lines8. We showed that IL-10 is also produced by normal peritoneal B cells after stimulation with lipopolysaccha-ride9. IL-10 was confirmed to be pleiotropic, as shown earlier for many other cytokines10, and to have many different activities in cells of the immune system7.

Our finding, that IL-10 is produced by B cells, led us to address the mechanism by which cytokine production by TH1 cells is inhibited by this cytokine, particularly as B cells could perfectly stimulate the production of IFN-γ by TH1 cell clones together with anti-gen. David Fiorentino, after Tim Mosmann left DNAX (Fig. 1), had joined Kevin Moore’s laboratory to continue his research assistant job before going to graduate-medical school, joined me in addressing this question, with a lot of input from Paulo. To do so, we purified macrophages and B cells and used them to present antigen to TH1 cell clones specific for various antigens. We did these assays in the presence or absence of IL-10 (or IL-10-specific antibodies) and measured the effects on IFN-γ production by the TH1 clone. This work led to our findings that IL-10 inhibits macrophages but not B cells from stimulating TH1 cells to produce IFN-γ11. Later, Steven Macatonia (Fig. 1) joined my lab, after his extensive work on dendritic cells with Stella Knight, and showed that IL-10 also inhibits dendritic cells from stimulating IFN-γ production by

TH1 clones12. We then continued this work further to identify potential mechanisms whereby IL-10 inhibits macrophage APC activity needed to stimulate TH1 cells and found that IL-10 profoundly inhibits cytok-ines produced by macrophages in response to lipopolysaccharide and thus its effects on TH1 cells are indirect13.

Many studies followed that confirmed IL-10 as a broadly suppressive cytokine7,14. At this early stage, I started thinking about how these data impinged on the original question of how TH1 and TH2 cells differentiate from a naive CD4+ T cell. We started looking for systems to address these questions. I attended a Keystone conference on tolerance at that time and was taken by the fact that many researchers were using antigen-specific T cell antigen receptor (TCR)–transgenic mice for tolerance studies but none were using such mice to address the question of how TH1 and TH2 cells may dif-ferentiate. The aim was to test different APCs stimulated in different ways (with cytokines or microbial products) for their ability to induce naive CD4+ T cells to differentiate into either TH1 or TH2 cells. I searched around for mice expressing major histocompatibility complex class II–restricted TCR–transgenic CD4+ T cells specific for soluble peptide antigens and found the name Kenneth (Ken) Murphy (Fig. 1); he had just published a paper on mechanisms of tolerance by induction of

CD4+ T cell apoptosis using such a mouse15. I rang him up and there started our collabora-tion. We both had small groups and decided to join forces to address this question of TH1 and TH2 differentiation. By that time, Suzy Swain’s and Bill Paul’s groups had published some findings showing that the addition of IL-4 to CD4+ T cells stimulated in the absence of APCs but with antibody to CD3 or con-canavalin A resulted in IL-4-producing TH2 cells after restimulation16,17.

Factors acting on APCs (Ken)After finishing my degrees at John Hopkins School of Medicine, I did a residency in pathology at Washington University under a new chairman, Emil Unanue. After my clini-cal training, Emil advised me to do a fellow-ship with Dennis Loh, an immunologist and Howard Hughes Medical Institute investiga-tor who was particularly strong in molecular biology. Dennis was one of the earliest to make TCR-transgenic mice to examine T cell devel-opment. His student Bill Sha had made the 2C mouse line and had made important find-ings about positive and negative selection. But the peptide for 2C was still unknown and the precise function of this peptide was still unex-plored. A hybridoma made by Pippa Marrack and John Kappler, called DO11.10, offered the advantage of a known peptide as well as a clonotype-specific antibody that could be used to help track transgenic T cells. Following Bill Sha’s exact strategy, I made the DO11.10 TCR–transgenic line and initially used it to test the consequences of T cell encounter with cognate peptide during their development15. After Emil offered me a position to stay in St. Louis, it seemed appropriate to look into areas that would not conflict with Dennis’s interests in the thymus. Luckily, Casey Weaver, a friend and fellow pathology resident, had exposed me to the work of Mosmann and Coffman on helper T cells2,3, and I thought that DO11.10 could provide an ‘in vitro prim-ing’ model that could be useful in exploring this problem. At about the same time, two other fortunate things happened. Chyi-Song Hsieh (Fig. 1) joined my new lab, already hav-ing extensive experience with T cells from his undergraduate experiences with Frank Fitch, and he began ‘banging on’ the TH1-TH2 problem. Also, I got a phone call from Anne O’Garra (Fig. 1), who had similar interests. Anne and I talked by phone and, after a visit to DNAX, we cemented a collaboration of thoughts and reagents that resulted in many intense and fun interactions through the years to follow. Using DO11.10 cells, Chyi showed that IL-4 directs development toward the TH2 phenotype18, which was in accordance

930 volume 10 number 9 september 2009 nature immunology

Figure 1 Players in the story. Top row, Anne O’Garra, Steven Macatonia and Kenneth Murphy. Bottom row, Chyi-Song Hsieh, Paulo Vieira and David Fiorentino.

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with findings of Swain and Paul16,17. We also showed that splenic APCs can direct develop-ment toward the TH1 phenotype when endog-enous IL-10 is neutralized18. The splenic APCs mediating these effects were probably macrophages or dendritic cells and not B cells, as IL-10 was unable to affect TH phenotype development when Chyi used B cells as the APC18, consistent with Anne’s earlier results with TH1 clones11. These results suggested that early regulation of IL-4 and IL-10 in a devel-oping immune response and the identity of the initiating APCs are critical in determining the TH phenotype of the developing T cells. However, these results did not resolve the issue of how TH1-TH2 development is controlled in the animal, as antibodies were unlikely to be the answer. It is important here to note ideas elaborated by Charlie Janeway at this time that it is microbial products themselves that act as ‘nature’s adjuvant’ to induce ‘sig-nal 2’ (now called ‘costimulation’19); we began thinking along similar lines. Chyi started to intentionally contaminate his cultures with various microbial products, a sort of crazy idea at the time. However, using heat-killed Listeria monocytogenes provided by Emil, Chyi discovered that very robust induction of TH1 development occurred when splenic APCs were used but not when the APCs were the TA3 B cell hybridoma. However, the addi-tion of flow cytometry–sorted macrophages to cultures using TA3 cells as APCs restored the ability of listeria to induce TH1 devel-opment20. Although the presence of IFN-γ was necessary for listerial induction of TH1 development, IFN-γ alone was insufficient to induce TH1 development. During this time, Chyi went from St. Louis to DNAX for several months in Anne’s lab to expedite our stud-ies and further cement our collaboration. Our findings showed that listeria may act by inducing soluble factors from macrophages that would induce a TH1 phenotype. Having ruled out several known factors produced by listeria-activated macrophages, which could not replace the effect of the activated mac-rophages, we determined that this process may involve an as-yet-unknown cytokine. By now, this work indicated that Charlie’s ideas about microbial products were not limited to just the initiation of adaptive immunity but also included the possibility that they serve a critical function in directing the differentia-tion of helper T cells. The next question was how they did this.

Inducing IL-12 in APCs (Anne and Ken)After much labor in ruling out other cytokines, we tested the ability of IL-12 to replace liste-ria-stimulated macrophages in inducing TH1

cell development. IL-12 had been defined by Giorgio Trinchieri et al. as a natural killer cell–stimulatory factor for IFN-γ expression21,22 and thus was a promising candidate. We then found that listeria or lipopolysaccharide induced TH1 development in vitro through the production of IL-12 by macrophages23,24. Moreover, inhibition of macrophage produc-tion of IL-12 seemed to explain the ability of IL-10 to suppress TH1 development and the production of IFN-γ by TH1 clones23,25. As dendritic cells are APCs that are unique in their potency in stimulating the proliferation of primary antigen-specific responses in vitro and in vivo, we were interested in determin-ing whether they too could, in the right con-ditions, produce IL-12 and thus induce TH1 cell development. Steven Macatonia, who had joined Anne’s lab to determine potential effects of IL-10 on dendritic cell function, then demonstrated that dendritic cells can produce IL-12, which is enhanced in the pres-ence of activated CD4+ T cells and thus can direct the development of TH1 cells26. Mouse in vivo immune responses to L. monocytogenes are of the appropriate TH1 phenotype.

Although our work predated the discovery by Jules Hoffman of the function of Toll-like receptors in innate immunity27 and the iden-tification of their vertebrate homologs28–30, work by Theresa Murphy recognized that the pathway leading to IL-12 induction by microbial products involved activation of the transcription factor NF-κB pathway in the APCs31. Therefore, we postulated at that time that this regulatory pathway may have evolved to enable cells of the innate immune system, through interactions with microbial pathogens, to direct development of specific immunity toward the appropriate TH pheno-type.

IL-12 and IL-10 in TH differentiationKen’s lab went on to show that IL-12 sig-naled through the IL-12 receptor to activate the transcription factor STAT4 to direct TH1 development32. Susanne Szabo, Kenneth’s stu-dent, then described a previously unknown mechanism that governs stable commitment to TH2 differentiation by extinction of IL-12 signaling33. Using flow cytometry to detect single cells producing IFN-γ and IL-4, Anne’s lab continued cellular studies and showed that polarized TH1 and TH2 populations assessed by immunoassay are heterogeneous and are reversible at a population level34.

These systems allowed numerous further studies by many labs defining signaling path-ways and transcription factors involved in both TH1 and TH2 differentiation and other cytokines, such as IL-18, important for TH1

responses35,36. Furthermore, IL-10, now known to be produced by many cells of the immune system, by its inhibition of both macrophage and dendritic cell function has broad anti-inflammatory properties37, serv-ing as a feedback regulator of not only a TH1-type response but also, in some circum-stances, TH2-type responses38. However, it would seem, as we originally hypothesized, that pathogens and their products stimulate dendritic cells and macrophages of the innate immune response to direct development of specific immunity toward the appropriate TH phenotype and that this is tightly controlled by IL-10 (refs. 14,35,37). These fundamental rules seem to apply to the broad number of different TH cell subsets that have now been defined on the basis of their different cytokine profiles.

ACKNOWLEDGMENTSWe thank other research assistants, PhD students, postdoctoral fellows and collaborators who have worked with us through the years and contributed to our work and apologize that we have not been able to include all here. We also thank our colleagues at DNAX and in the Department of Pathology at Washington University for their support and interaction. We thank P. Vieira for comments and reading of the manuscript.

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