Lawrence H. Boise

CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL

T cell activation through the TCR can result in either cell proliferation or cell death. The role of costimulatory receptors in regulating T cell survival has not been defined. Here, we present data demonstrating that CD28 costimulation enhances the in vitro survival of activated T cells. One mechanism for this enhancement is the ability of CD28 costimulation to augment the production of IL-2, which acts as an extrinsic survival factor for T cells. In addition, CD28 costimulation augments the intrinsic ability of T cells to resist apoptosis. Although CD28 signal transduction had no effect on Bcl-2 expression, CD28 costimulation was found to augment the expression of Bcl-XL substantially. Transfection experiments demonstrated that this level of Bcl-XL could prevent T cell death in response to TCR cross-linking, Fas cross-linking, or IL-2 withdrawal. These data suggest that an important role of CD28 costimulation is to augment T cell survival during antigen activation.

Bcl-2 and Bcl-2-related proteins in apoptosis regulation.

In this review we have discussed the importance of Bcl-2 and related proteins in the regulation of apoptotic cell death in mammalian systems. It is clear that Bcl-2 plays a critical role in controlling many forms of PCD. Bcl-2 seems to have particular significance in lymphocyte development and the function of the immune system. We have also discussed the increasing size of the newly identified Bcl-2 family. There are a number of Bcl-2 homologues in human, murine, avian, nematode, and viral systems. The evolutionary conservation of the function of the Bcl-2 homologues, reinforces the importance of PCD in all complex organisms. Some of these bcl-2-like genes function as agonists and others as antagonists. Despite the seemingly universal importance of Bcl-2, it is unable to prevent PCD in all systems. In addition, we have described a role for other Bcl-2 family members in systems in which Bcl-2 is ineffective and supplied a potential rationale for the large number of genes involved in the regulation of PCD. Identification and functional analysis of the Bcl-2 family members reveals the complex nature of cell death regulation. As we begin to appreciate the significance of PCD in the control of development and homeostasis, its regulation at the molecular level is becoming better understood. Bcl-2 has long been the only known intracellular regulator of the PCD pathway(s), although its ability to prevent apoptosis is not universal. We now know that bcl-2 is only one member of an evolutionary conserved family of genes which display different patterns of expression as well as function. At least two family members, Bcl-xs and Bax, act in opposition to Bcl-2. The discovery of these new family members, including those with Bcl-2-like function and antagonists, should help clear up the discrepancies seen in Bcl-2s ability to protect cells from PCD. In doing so, we will be able to further define the pathways associated with cell death signaling. The study of these family members, as well as the non-related genes of the PCD pathways (ced-3, ced-4, ice) should lead us to understanding of how cells of multicellular organisms make decisions to die.

Impaired Induction of the Apoptosis-Protective Protein Bcl-xL in Activated PBMC from Asymptomatic HIV-Infected Individuals

Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = −0.695, P = 0.005). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.

Introduction of the cell survival gene bcl-xL improves the viability of CTLL-2 cells without affecting their IL-2 proliferative response Implications for the development of bioassays

A standard method for the quantitation of cytokines is to perform a bioassay in which aliquots of samples are compared to known concentrations of a cytokine in supporting the proliferation of a cytokine-dependent cell line. In most instances however, these cell lines are dependent on the cytokine not only for proliferation but also for survival. For example, a cell line that is commonly utilized for interleukin-2 (IL-2) bioassays is the IL-2-dependent line, CTLL-2. CTLL-2 cells will die rapidly by apoptosis if withdrawn from IL-2, thus these cells can be difficult to maintain in culture for extended periods. Overexpression of the anti-apoptotic protein Bcl-x(L) can enhance CTLL-2 survival in the absence of IL-2. However, while overexpression of Bcl-x(L) can prevent CTLL-2 cells from dying in the absence of IL-2, overexpression of Bcl-x(L) does not impair the ability of CTLL-2 cells to be used for proliferation-based IL-2 bioassays. Thus the bcl-x(L)-transfected CTLL-2 cells are equivalent to the parental cell line for determination of IL-2 levels in a culture supernatant, yet are easier to maintain in culture. Introduction of Bcl-x(L) or Bcl-2 into other factor-dependent cell lines may also simplify their maintenance without significantly affecting their utility in bioassays.