David A. Hildeman

Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim.

At the end of the T cell response, the majority of the activated T cells die. We activated Vbeta8(+) T cells with staphylococcal enterotoxin B (SEB) in vivo and monitored the expansion and deletion of Vbeta8(+) T cells. We found that, in response to SEB, activated T cells died in vivo in the absence of Fas or TNF-R signaling but not when they overexpressed human Bcl-2. We also found that Vbeta8(+) T cells from Bim-deficient mice are resistant to SEB-induced deletion. While Bim levels did not change, endogenous Bcl-2 levels within Vbeta8(+) T cells decrease following SEB injection. Thus, the death of superantigen-stimulated T cells in vivo is mediated by Bim and may be modulated by a decrease in Bcl-2.

Reactive Oxygen Species Regulate Activation-Induced T Cell Apoptosis

Reactive oxygen species (ROS) mediate apoptosis in a number of cell types. We studied the role that ROS play in activated T cell apoptosis by activating T cells in vivo and then culturing them for a short time. Activated T cells died independently of Fas and TNF alpha. Their death was characterized by rapid loss of mitochondrial transmembrane potential (delta psi(m)), caspase-dependent DNA fragmentation, and superoxide generation. A superoxide dismutase mimetic, Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), protected T cells from superoxide generation, caspase-dependent DNA loss, loss of delta psi(m), and cell death. These results indicate that ROS can regulate signals involved in caspase activation and apoptosis and may contribute to peripheral T cell deletion.

Molecular mechanisms of activated T cell death in vivo.

The culmination of the immune response involves the death of the majority of the activated antigen-specific T lymphocytes. The death of these cells is important to prevent autoimmunity, to decrease the metabolic cost to the organism and to ensure T cell homeostasis. This review will focus on the mechanisms that, in animals, control the death of these activated cells. At least two separate types of cell death can occur (activation-induced cell death and activated T cell autonomous death) via death receptors such as Fas or the Bcl-2 related protein Bim, respectively. Finally, adjuvants that enable T cell survival may operate via NF-kappaB and Bcl-3 rather than cytokines.

Homeostasis of alpha beta TCR+ T cells.

Cytokines contribute to T cell homeostasis at all stages of T cell existence. However, the particular cytokine involved varies as T cells progress from a naïve through an activated to a memory state. In many cases the important cytokines are members of the interleukin 2 subfamily of the short-chain type I cytokines. A case is made for the idea that the evolutionary divergence of the short-chain family allowed for concurrent divergence in leukocytes.

Functional Regulatory T Cells Accumulate in Aged Hosts and Promote Chronic Infectious Disease Reactivation

Declines in immune function are well described in the elderly and are considered to contribute significantly to the disease burden in this population. Regulatory T cells (Tregs), a CD4+ T cell subset usually characterized by high CD25 expression, control the intensity of immune responses both in rodents and humans. However, because CD25 expression does not define all Tregs, especially in aged hosts, we characterized Tregs by the expression of FOXP3, a transcription factor crucial for Treg differentiation and function. The proportion of FOXP3+CD4+ Tregs increased in the blood of the elderly and the lymphoid tissues of aged mice. The expression of functional markers, such as CTLA-4 and GITR, was either preserved or increased on FOXP3+ Tregs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral Tregs from elderly humans improves effector T cell responses in most subjects. Importantly, Tregs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young Tregs. The increased proportion of Tregs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old Tregs efficiently suppressed the production of IFN-γ by effector T cells. Finally, in vivo depletion of Tregs in old mice attenuated disease severity. Accumulation of functional Tregs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of Treg numbers and/or activity may be envisioned to enhance the control of infectious diseases in this fragile population.

Nonredundant Roles for B Cell-Derived IL-10 in Immune Counter-Regulation

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8+ T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.

IL-7 Promotes T Cell Viability, Trafficking, and Functionality and Improves Survival in Sepsis

Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-γ production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-γ– and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.

Control of Bcl-2 expression by reactive oxygen species

Reactive oxygen species (ROS) mediate apoptosis in many different cell types. We have previously shown that the antioxidant Mn(III) tetrakis(5,10,15,20-benzoic acid)porphyrin (MnTBAP) decreased intracellular ROS and prevented the apoptosis of activated T cells in vitro. To determine the mechanism(s) by which MnTBAP afforded such protection, we used Affymetrix (Santa Clara, CA) gene arrays to compare gene expression in T cells activated with staphylococcal enterotoxin B in vivo then cultured with or without MnTBAP. This analysis showed that the antioxidant increased the expression of Bcl-2, an antiapoptotic molecule whose levels are normally decreased by T cell activation. Culture with MnTBAP revealed a tight inverse correlation between the levels of Bcl-2 and ROS within T cells. In vivo, production of ROS in activated T cells occurred before Bcl-2 down-regulation. Furthermore, MnTBAPs ability to prevent death required the expression of Bcl-2 in most T cells. Finally, neither ROS production nor the effects on Bcl-2 expression required Bim, the Bcl-2 antagonist that mediates the death of activated T cells in vivo. Taken together, our results suggest that ROS sensitize T cells to apoptosis by decreasing expression of Bcl-2.

Bim/Bcl-2 balance is critical for maintaining naive and memory T cell homeostasis

We examined the role of the antiapoptotic molecule Bcl-2 in combating the proapoptotic molecule Bim in control of naive and memory T cell homeostasis using Bcl-2−/− mice that were additionally deficient in one or both alleles of Bim. Naive T cells were significantly decreased in Bim+/−Bcl-2−/− mice, but were largely restored in Bim−/−Bcl-2−/− mice. Similarly, a synthetic Bcl-2 inhibitor killed wild-type, but not Bim−/−, T cells. Further, T cells from Bim+/−Bcl-2−/− mice died rapidly ex vivo and were refractory to cytokine-driven survival in vitro. In vivo, naive CD8+ T cells required Bcl-2 to combat Bim to maintain peripheral survival, whereas naive CD4+ T cells did not. In contrast, Bim+/−Bcl-2−/− mice generated relatively normal numbers of memory T cells after lymphocytic choriomeningitis virus infection. Accumulation of memory T cells in Bim+/−Bcl-2−/− mice was likely caused by their increased proliferative renewal because of the lymphopenic environment of the mice. Collectively, these data demonstrate a critical role for a balance between Bim and Bcl-2 in controlling homeostasis of naive and memory T cells.

Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3 + regulatory T cells

Foxp3+ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4+ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2–dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.