Interleukin 4 is arguably one of the best-studied and most influential cytokines of the immune system. It was originally described in 1982 as a B cell stimulatory factor present in the supernatants of the phorbol ester-stimulated T cell tumor line EL-4 by Maureen Howard and Bill Paul in the Laboratory of Immunology at the National Institute of Allergy and Infections Diseases (NIAID) (1). However, it is now known that this cytokine regulates a myriad of immune functions including Ig isotype switching, class II MHC expression by B cells, and the differentiation fate of certain T cell subsets (2). In 1986 the gene encoding mouse IL-4 was cloned from Con A-activated Th cells by two laboratories and the race was on to identify the range of cells that express this molecule and determine how it is regulated (3, 4). Surprisingly, it was discovered that few if any of the conventional, nontransformed T cell lines derived in the Laboratory of Immunology at NIAID at that time produced IL-4. This observation was among the first evidence that there might be some selectivity in the cytokines expressed by Th cells. Indeed, not long afterward, Mossman and Coffman published their seminal paper showing dichotomy in CD4+ Th cell subsets based on cytokine-producing potential and showed that there is a reciprocal expression pattern of IL-4 and IFN-γ in Th2 and Th1 cells, respectively (5). So why were the majority of clones derived in vitro of the Th1 phenotype? As we know now, there are very strict requirements for IL-4 expression by T cells. In this month’s Pillars of Immunology article, “Generation of Interleukin 4 (IL-4)-producing Cells In Vivo and In Vitro: IL-2 and IL-4 Are Required for In Vitro Generation of IL-4-producing Cells,” Graham Le Gros, Shlomo Ben-Sasson, Robert Seder, Fred Finkelman and Bill Paul present the first evidence that IL-4 is absolutely required for its own robust production by T cells (6).

Their studies were prompted by two earlier observations. First, IL-4 promotes B cell expression of IgG1 and IgE in vitro (7, 8, 9). Second, administration of anti-IgD to mice results in high production of these Ab isotypes in vivo (10). Le Gros et al. reasoned that anti-IgD treatment may act to influence Ig isotype switching by inducing high levels of IL-4 (6). In line with their prediction, T cells isolated from the lymph nodes of mice injected with anti-IgD produced substantial amounts of IL-4 relative to those from untreated mice when cultured with anti-CD3 and IL-2. It is notable that their in vivo model system was paradoxical at the time, yet it was nevertheless very useful. How anti-IgD, a reagent that engages a BCR, could activate T cells was completely unclear. Fred Finkelman’s group subsequently demonstrated that “foreign Ags” imbedded in the injected anti-IgD Abs were taken up by the B cell, processed, and presented to T cells in concert with MHC class II molecules (11). This preferential B cell presentation of Ag in the absence of dendritic cell-derived IL-12 is thought to cause a default to the Th2 pathway.

The authors made a number of other significant observations. Their data were the first to show that IL-4 must be present during the initial activation of a naive T cell and that it “primes” that cell for subsequent IL-4 expression upon challenge by promoting a specific differentiation program in the naive precursor cell. The formulation of these ideas is particularly impressive because markers of T cell activation had not been identified and the ability to define naive and activated cells was limited to measurements of relative density after separation on a Percoll gradient. Analysis of T cells from anti-IgD-treated mice revealed that appreciable amounts of IL-4 are produced only by cells of medium and low density and not by the high-density population, cells that we now realize largely represent the “activated” and “naive” T cells, respectively. Because T cells from primed donors can produce more IL-4 than those from naive animals, Le Gros and colleagues next examined the requirements for IL-4 production in naive cells in vitro. To do this, they cocultured high-density, lymph node-derived T cells from untreated mice with anti-CD3 and IL-2 with or without anti-IL-4 or anti-IL-2. Their results established the following: 1) that appreciable amounts of IL-4 were not made by high-density (naive) cells in a primary culture, but IL-2 was easily detected; and 2) that culturing anti-CD3-stimulated, high-density cells for 2–5 days in the presence of IL-2 and IL-4 followed by a secondary activation with anti-CD3 alone resulted in IL-4 production. The robust production of IL-4 in secondary cultures could be explained by preferential proliferation of a cell subset already capable of producing IL-4. However, the authors argued correctly that the more likely scenario is that IL-4-producing cells selectively develop from a naive precursor. Their argument was based on the rarity of high density IL-4-producing cells in naive animals as well as kinetic considerations, because culturing the anti-CD3-stimulated, high-density cells from naive donors for as little as 2 days in the presence of IL-4 (and IL-2) was likely not enough time for sufficient proliferation but would allow the initiation of a differentiation program. In their discussion, they also asked the inevitable question: if IL-4 is needed for its own production by naive T cells, what is the source of the initial priming IL-4 in vivo? Although several IL-4-producing cells are candidates, including naive T cells themselves, this question still has not been satisfactorily answered.

In retrospect, there are several remarkable aspects of these studies that qualify this work as a “pillar of immunology”. Using only an amazingly simple coculture system and an IL-4-dependent cell line to assay cytokine production, and capitalizing on the ability to make key connections between in vitro and in vivo observations with respect to IL-4’s link to isotype switching, a paradigm was conceived that has not changed much over the years. With the experimental tools available to today’s scientists, including the ability to track the fate of a single naive CD4+ T cell precursor, we now know how IL-4, acting through STAT6 and GATA3, can determine Th2 cell fate (12). The idea that the particular cytokine microenvironment during initial Ag encounter dictates whether a cell will produce IL-4 has much wider applicability to all CD4+ T cell subsets. IL-4, as well as IL-12, TGFβ, or TGFβ plus IL-6, can promote one of at least four alternative differentiation pathways (13). These studies provide proof that extraordinary scientific insight, as shown by Le Gros, Paul, and colleagues, can go a long way to advance our understanding of biology, even in the absence of advanced technology.

I thank Dr. Graham Le Gros for clearly explaining to me how anti-IgD might induce T cell IL-4 production in vivo. I also thank Alison Christy and Dr. Kavitha Rao for helpful comments.

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