Ameeta Kelekar

BH3-only proteins of the Bcl-2 family of apoptosis regulators are sensors of cellular stress. They are activated in response to a wide range of stress stimuli and, once activated, commit cells to apoptosis via the intrinsic pathway, which involves mitochondria. The BH3-only protein subfamily shares a single conserved domain, the proapoptotic BH3 domain, with members of the large Bcl-2 family. BH3-only proteins utilize their BH3 domains to interact either with prosurvival binding partners to inhibit their function or with multidomain proapoptotic Bcl-2 family members to activate them. These interactions cause permeabilization of the mitochondrial outer membrane and lead to apoptosis. As promoters of apoptotic cell death, BH3-only proteins help eliminate damaged and potentially harmful cells to maintain tissue homeostasis. They also play key roles in preventing cancer and in enhancing the response to anticancer therapies. While apoptotic pathways activated by BH3-only proteins are highly conserved, the stress stimuli, signaling pathways, and the underlying mechanisms that activate the proapoptotic function of these proteins are not. In fact, BH3-only proteins are known to be regulated by a variety of mechanisms, both transcriptional and posttranslational, a majority of which involve phosphorylation. Focusing on a selection of well-studied and unique members of the BH3-only subclass of Bcl-2 proteins, this chapter describes how phosphorylation controls the canonical and noncanonical functions of these proteins through regulation of their structure, localization, activation, protein-protein interactions, and binding specificity, and discusses the outcomes of the regulation in terms of cell fate.

Naive or memory T cells reprogram their metabolism upon antigenic stimulation. They increase their glucose uptake, relying on aerobic glycolysis for generating biomass while switching to glutamine to fuel energy production. Here we have identified a requirement for human Bcl-2 family, Noxa, in the metabolic switch to glutamine dependence in activated CD8+ T cells, that is independent of its canonical role in apoptosis at the end of the immune response. Using an in vitro co-stimulation model, we demonstrate that Noxa is induced in CD8+ T cells and remains elevated during the proliferative and differentiation phases of the response and through the onset of apoptosis. Noxa protein induction requires glutamine, is mediated via mTOR, and is independent of glutaminolysis. Glutamine, in turn, requires Noxa to facilitate its conversion to glutamate. CD8+ T cells lacking Noxa showed reduced levels of intracellular glutamate but no impairment of mitochondrial or effector function, and decreased dependence on glutamine for both respiration and growth during the proliferative phase. NOXA knockout CD8⁺ T cells also displayed significantly higher viability in the apoptotic phase of the immune response. CD8+ T cells from a human NOXA gene-replacement mouse responded normally to in vitro stimulation and in vivo acute infection. However, human Noxa-expressing murine CD8+ T cells displayed a distinctly proliferative gene signature in their transcriptome following activation, supporting an early growth-promoting role for this BH3-only protein. Our studies suggest that knocking out NOXA in human CD8+ T cells to increase their lifespan as well as their ability to survive and function in glutamine-poor microenvironments could be a promising immunotherapeutic strategy.

Supplementary Table from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Supplementary Figure from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Supplementary Figure from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Supplementary Table from P2RX7 Enhances Tumor Control by CD8+ T Cells in Adoptive Cell Therapy

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.