The survival of lymphocytes is tightly regulated at each stage of their lifespan. Although this may seem obvious to us today, prior to the early 1990s, the focus of most studies of T lymphocyte activation emphasized the regulation of cellular proliferation, with studies of cell death focused on apoptosis during development (1). The paper of Boise et al. (2), highlighted in this Pillars of Immunology article, provided two important new insights that brought to the forefront the concept that T cell costimulation provides critical survival signals to T cells. The first important insight was that costimulation through CD28 induces intrinsic survival signals in T cells, independently of the exogenous proliferative/survival signals induced by IL-2. Secondly, Boise et al. provided evidence that the intrinsic survival signal induced by CD28 was distinct from the homeostatic survival mechanisms in place in resting T cells.

The two signal model of lymphocyte activation, as conceptualized for T lymphocytes by Lafferty (3), posited that “both antigen and an inductive stimulus are required for T cell activation and neither factor alone induces detectable T cell activation.” It was already recognized in the 1970s that this inductive process required a metabolically active accessory cell. This concept was revisited again in the 1980s by Jenkins and Schwartz (4) and by Quill and Schwartz (5), who showed that provision of signal 1 without signal 2 (using either fixed APCs or purified MHC class II in planar membranes) led to clonal inactivation of murine T cell clones. These findings invigorated the search for the key molecule(s) that could provide signal 2. However, it was an independent line of research on the characterization of human T cell surface molecules using monoclonal Abs that led to the identification of CD28 as a key receptor for signal 2 for naive T cell activation. Costimulation of T cells through the TCR and CD28 greatly increased human T cell proliferation and IL-2 production (reviewed in Ref. 6). These findings were soon followed by the identification of the ligands for CD28, now known as B7.1 and B7.2 (711), as well as a second, inhibitory, B7 binding receptor, CTLA-4 (12). Although not all immune responses were found to be CD28 dependent (13), studies showing that CD28–B7 interactions could costimulate T cells and prevent T cell clonal anergy, as well as the dramatic inhibitory effects of B7 blockade by CTLA-4-Ig in vivo, cemented the role of CD28 as a key receptor for signal 2 for T lymphocyte activation (1419).

At the time of the studies of Boise et al. (2), it had been established that CD28 engagement could induce high level IL-2 production by increasing both the transcription of the IL-2 gene (20) and the stability of its message (21). There was also evidence that signals through the TCR in the absence of accessory cells could induce apoptosis of mature T cells and that this could be prevented by addition of IL-2 (2224). In contrast, mitogen-activated lymphocytes were resistant to irradiation-induced apoptosis (25), leading Boise et al. to suspect a role for costimulation in prevention of T cell death. At the same time, data were emerging suggesting that there were intrinsic mechanisms that regulated lymphocyte survival. The Bcl-2 gene had been identified through its aberrant expression in B cell lymphomas with the t(14;18) translocation (reviewed in Refs. 26 and 27). Bcl-2 overexpression allowed the survival of lymphocytes upon growth factor withdrawal and had recently been shown to be critical for lymphoid homeostasis (27, 28). Importantly, Boise et al. (2) had just cloned a homolog of Bcl-2, Bcl-xL, whose overexpression could replace Bcl-2 in sustaining survival of IL-3–dependent cells upon cytokine withdrawal (29). These findings led Boise et al. (2) to hypothesize that the costimulatory signal induced by CD28 might induce intrinsic survival signals in activated T cells.

To test this concept, the authors first demonstrated that activated T cells were more resistant than resting lymphocytes to cell death induced by gamma irradiation. The advantage of this approach was that irradiation could induce cell death independently of surface death receptors whose expression could change with activation. Initial experiments showed that T cells activated by plate-bound anti-CD3 were more resistant to cell death than cells left untreated, independently of the inclusion of anti-CD28 in the stimulation. However, if they washed the cells to remove accumulated cytokines in the supernatant, “the anti-CD3 only” cultures were more radiation sensitive, whereas the anti-CD3 plus anti-CD28–treated cultures remained resistant to irradiation-induced death. Addition of IL-2 restored the resistance of the anti-CD3–treated cells, confirming that IL-2 could provide an extrinsic survival signal to activated T cells, but also demonstrating that costimulation reduced the requirement for IL-2 in T cell survival. However, at this stage they could not rule out that the anti-CD28 treatment was simply increasing the amount of IL-2 produced, rendering the washout experiment less effective.

Boise et al. (2) next went on to examine the induction of Bcl-2 and Bcl-xL by anti-CD3 and anti-CD28. When examined at the protein level, Bcl-2 levels were constant in resting versus activated T cells, whereas Bcl-xL protein was mildly induced by anti-CD3 and strongly induced by anti-CD3 plus anti-CD28. Although Bcl-2 message increases with T cell activation, it is not regulated by CD28 and the mRNA appears to increase to maintain steady state levels of Bcl-2 protein as the cells divide. Moreover, CD28 costimulation, but not stimulation through other surface molecules, such as LFA-1 or CD2 or addition of exogenous IL-2, could augment Bcl-xL expression. Addition of CTLA-4-Ig to the cultures resulted in the complete abrogation of Bcl-xL induction by anti-CD3. Thus, the low level induction of Bcl-xL by anti-CD3 alone may have been due to the presence of B7 on the activated T cells. The peak induction of Bcl-xL expression by CD28 was coincident with the peak resistance to exogenous death signals induced by FAS cross-linking. Moreover, the overexpression of Bcl-xL prevented apoptosis induced by FAS, anti-CD3, or cytokine withdrawal. Together, these experiments showed that in addition to IL-2–induced extrinsic survival signaling, CD28–B7 interaction induces an intrinsic survival pathway, leading to the accumulation of Bcl-xL in the cell and protection from intrinsic or extrinsic cell death signals. A key finding of the study was that it defined the distinct roles of Bcl-2 and Bcl-xL in regulation of survival of quiescent versus expanding lymphocyte populations.

In the years since this landmark paper was published, the signaling pathways leading to Bcl-xL induction and T cell survival in response to CD28 costimulation have been further fleshed out and the studies extended in vivo (3032). The importance of mitochondrial integrity and mechanisms of cell death regulation by the Bcl-2 family have been extensively studied (33, 34) and a previously unexpected role for CD28 in the control of regulatory T cell development and homeostasis was also revealed (35). The concept of costimulation leading to T cell survival via Bcl-xL has also been extended to members of the TNFR family that can sustain effector and memory T cell survival at the later stages of T cell activation (36, 37). The paper of Boise et al. (2) represented a turning point in the way we think about costimulation of T lymphocytes by showing us that costimulation influences not only proliferation and cytokine production by T cells but also their survival. Subsequently, Thompson’s group (38, 39) further broadened the concept of T cell costimulation by demonstrating that CD28-mediated costimulation is also important for increasing glucose uptake to provide the metabolic energy needed for T cell expansion. Thus the current paradigm for costimulation of T cell responses is that the Ag receptor provides the specificity for T cell activation, whereas costimulation is an essential driver of T cell expansion through effects on proliferation, survival, and energy metabolism. Many aspects of these signaling pathways are still under active investigation and no doubt further insights await.

I thank Laura M. Snell for critical reading of the manuscript.

Disclosures The author has no financial conflicts of interest.

T.H.W. holds the Sanofi Pasteur Chair in Human Immunology at the University of Toronto.

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