One of the most important and intensively studied properties of naive CD4+ T cells is their capacity to differentiate into cells with unique functions, with the capacity to produce distinctive sets of cytokines that are protective against different pathogens and that mediate distinct immunopathologic conditions. At least four such cell types exist: Th1, Th2, Th17, and induced Tregs (iTregs) (1).
That clearly differentiated types of CD4+ T cells exist was first definitively established by Tim Mosmann and Bob Coffman in 1986 (2) in their landmark study of CD4+ T cell clones. They showed that most clones could either be characterized as Th1 cells, unique producers of IFN-γ, or as Th2 cells, for which IL-4 was the signature cytokine. At about the same time, Kim Bottomly and her colleagues were reporting that CD4+ T cell clones had functional differences and those that were the best helper cells produced IL-4 (3).
Once the Th1/Th2 paradigm had been demonstrated for CD4+ T cell clones, it was observed that infection could lead to immune responses in which Th1 or Th2 CD4+ T cells would dominate. Among the most influential early reports are those from Richard Locksley and his colleagues (4). They reported that the previously demonstrated strain difference in the capacity of mice to resolve infections with Leishmania major could be attributed to differences in the dominant types of CD4+ T cell responses mounted by the infected animals, with those that resolved the infection making an IFN-γ (Th1)-dominated response and those in which the infection was progressive making an IL-4 (Th2)-dominated response.
These results implied that naive CD4+ T cells had the capacity to develop into different phenotypic types of CD4+ T cells with different functional properties and that these functions were critically important in immune responses to pathogens. An appropriate response, such as one to L. major dominated by IFN-γ, would be protective, but an inappropriate one (an IL-4-dominated response to this pathogen) would be ineffective or could even result in a more serious infection than might have occurred if only the innate immune response had functioned.
This work was followed by a flood of publications indicating the existence of Th1 and Th2 cells in many settings and implicating them in both humans and animals in distinct types of protective responses, with Th1 cells playing the lead role in intracellular infections and Th2 cells in helminth infections and in Ig class switching to IgE (5).
These observations indicated that understanding the molecular basis of CD4+ T cell differentiation to each of the potential differentiated states could be extremely important. The first progress in this direction was made in determining how naive CD4+ T cells could develop into IL-4-producing Th2 cells. In 1990, both our research group (6) and that of Susan Swain (7) showed that acquisition of the Th2 phenotype in vitro occurred in ∼3 days when naive CD4+ T cells were stimulated by polyclonal activators in the presence of IL-4, establishing what would eventually become a general rule, i.e., that a principal product of the differentiated cells was a critical inducer (1). The initial Th2 differentiation work was extended in an important way by Ken Murphy’s and Anne O'Garra’s groups (8) and by Bob Seder, Barbara Fazekas de St. Groth, Mark Davis, and myself (9) in 1992 when both groups showed that CD4+ T cells from TCR transgenic mice required IL-4 to develop into Th2 cells when stimulated with their cognate Ag.
While progress in understanding the Th2 differentiation process was quite rapidly made, the Th1 differentiation problem was more difficult and its solution is the subject of the Pillars of Immunology article discussed here. Both Ken Murphy’s group and Anne O'Garra’s were deeply interested in this problem and they collaborated in the work described in a landmark Science paper (10). Hsieh and Macatonia and their colleagues observed that when CD4+ T cells from BALB/c mice transgenic for genes encoding a TCR specific for an OVA peptide were cultured with OVA and with a B cell hybridoma as an APC without the addition of “biasing” cytokines, they mainly developed into IL-4 producers. This is in keeping with the known propensity of BALB/c cells to develop a Th2 response. When IL-4 was neutralized in these experiments, the CD4+ T cell response was dominated by IFN-γ, confirming that IL-4 was essential for Th2 differentiation. The authors must have reasoned that agents that induced Th1 responses in vivo would be good candidates to play a similar role in vitro. The addition of heat-killed Listeria monocytogenes had no effect on the cytokine profile under the culture conditions described above, but when a source of macrophages was added, the heat-killed L. monocytogenes caused a robust IFN-γ response without any IL-4 production. To be certain that the macrophages were not adding to the APC activity of the culture, the authors employed allogeneic macrophages, which were unable to present OVA to the responding T cells.
Hsieh and colleagues then went on to test whether soluble factors could replace the heat-killed L. monocytogenes, particularly in the absence of added macrophages. Of the molecules they tested, including IL-1, IL-6, TNF-α, TGFβ, and IL-12, only the latter was effective, and anti-IL-12 Ab blocked the macrophage-dependent action of heat-killed L. monocytogenes, implying that IL-12 was the active principle and that stimulation with heat-killed L. monocytogenes induced IL-12 production by the macrophages.
IL-12 had been discovered in 1989 by Trinchieri and colleagues (11) and by Gately and associates (12) and was originally designated NK cell stimulatory factor (NKSF). In the original description of NKSF it was shown that it could induce IFN-γ production from human PBL populations, anticipating its important role in the differentiation of naive CD4+ T cells to become Th1 cells.
Further study of Th1 differentiation has shown that IFN-γ plays an important role in this process and that IFN-γ, rather than IL-12, is the major inducer of T-bet (13), the master regulator of Th1 differentiation (14). In addition, an important role of IFN-γ is to up-regulate the IL-12 receptor and thus to enhance the IL-12 signaling that, in turn, is important in IFN-γ production (15). Indeed, STAT4 knockout mice, which are unresponsive to IL-12, have a major impairment in in vivo Th1 responses, strikingly validating the importance of IL-12 in this process (16).
The 1993 Science paper by Hsieh, Macatonia, Tripp, Wolf, O'Garra and Murphy is a landmark in the progress made in understanding the mechanisms of differentiation of naive CD4+ T cells. The lessons learned from the study of this pathway and that of the pathway of Th2 differentiation have instructed our knowledge of all aspects of CD4+ T cell differentiation and have had an enormous influence on our understanding of how CD4+ T cells mount effective responses against distinct pathogens and how abnormalities in these cells lead to an array of immunopathologic conditions, including tissue-specific and generalized autoimmune disorders and asthma and allergic disorders.
This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.