Tullia Lindsten

The Combined Functions of Proapoptotic Bcl-2 Family Members Bak and Bax Are Essential for Normal Development of Multiple Tissues

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.

Growth Factor Regulation of Autophagy and Cell Survival in the Absence of Apoptosis

In animals, cells are dependent on extracellular signals to prevent apoptosis. However, using growth factor-dependent cells from Bax/Bak-deficient mice, we demonstrate that apoptosis is not essential to limit cell autonomous survival. Following growth factor withdrawal, Bax-/-Bak-/- cells activate autophagy, undergo progressive atrophy, and ultimately succumb to death. These effects result from loss of the ability to take up sufficient nutrients to maintain cellular bioenergetics. Despite abundant extracellular nutrients, growth factor-deprived cells maintain ATP production from catabolism of intracellular substrates through autophagy. Autophagy is essential for maintaining cell survival following growth factor withdrawal and can sustain viability for several weeks. During this time, cells respond to growth factor readdition by rapid restoration of the ability to take up and metabolize glucose and by subsequent recovery of their original size and proliferative potential. Thus, growth factor signal transduction is required to direct the utilization of sufficient exogenous nutrients to maintain cell viability.

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.

Effector and memory CD8 + T cell fate coupled by T-bet and eomesodermin

Two seemingly unrelated hallmarks of memory CD8+ T cells are cytokine-driven proliferative renewal after pathogen clearance and a latent effector program in anticipation of rechallenge. Memory CD8+ T cells and natural killer cells share cytotoxic potential and dependence on the growth factor interleukin 15. We now show that mice with compound mutations of the genes encoding the transcription factors T-bet and eomesodermin were nearly devoid of several lineages dependent on interleukin 15, including memory CD8+ T cells and mature natural killer cells, and that their cells had defective cytotoxic effector programming. Moreover, T-bet and eomesodermin were responsible for inducing enhanced expression of CD122, the receptor specifying interleukin 15 responsiveness. Therefore, these key transcription factors link the long-term renewal of memory CD8+ T cells to their characteristic effector potency.*Note: In the version of this article initially published online, the third sentence of the abstract was incorrect. The correct sentence is as follows: “We now show that mice with compound mutations of the genes encoding the transcription factors T-bet and eomesodermin were nearly devoid of several lineages dependent on interleukin 15, including memory CD8+ T cells and mature natural killer cells, and that their cells had defective cytotoxic effector programming.” The error has been corrected for the HTML and print versions of the article. Additionally, in the print version of this article and the version initially published online, some labels for Tbx21 in Figure 7b are incorrect. This correction has been appended to the PDF version.

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax − / − bak − / − cells. In bax − / − bak − / − cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.

T-cell proliferation involving the CD28 pathway is associated with cyclosporine-resistant interleukin 2 gene expression.

CD28 is a homodimeric glycoprotein expressed on the surface of a major subset of human T cells that has recently been identified as a member of the immunoglobulin supergene family. The binding of monoclonal antibodies to the CD28 antigen on purified T cells does not result in proliferation; however, previous studies have shown that the combination of CD28 stimulation and protein kinase C activation by phorbol myristate acetate (PMA) results in T-cell proliferation that is independent of both accessory cells and activation of the T-cell receptor-CD3 complex. In the present study, effects of stimulation by anti-CD28 on cell cycle progression and on the interleukin 2 (IL-2) and IL-2 receptor system have been investigated on primary cultures of purified peripheral-blood CD28+ T cells. There was no measurable effect on cell size or on DNA synthesis after stimulation of resting (G0) cells by CD28 alone. After 3 h of activation of T cells by PMA alone, a slight (8%) increase in cell volume occurred that did not progress to DNA synthesis. In contrast, T-cell stimulation by CD28 in combination with PMA resulted in a progressive increase in cell volume in approximately 100% of cells at 12 to 14 h after stimulation. Northern blot (RNA blot) analysis revealed that CD28 stimulation alone failed to cause expression of the alpha chain of the IL-2 receptor or of IL-2 mRNA, and in accord with previous studies, stimulation by PMA alone resulted in the accumulation of IL-2 receptor transcripts but no detectable IL-2 mRNA. In contrast, T-cell stimulation by the combination of CD28 and PMA resulted in the appearance of IL-2 transcripts and enhanced expression of IL-2 receptor mRNA. Functional studies revealed that the proliferation induced by CD28 and PMA stimulation was entirely resistant to cyclosporine, in contrast to T-cell activation induced by the CD3-T-cell receptor complex. Cyclosporine was found not to affect the accumulation of IL-2 mRNA after CD28 plus PMA stimulation, although there was no detectable IL-2 mRNA after stimulation by CD3 in the presence of the drug. Furthermore, stimulation by CD28 in combination with immobilized CD3 antibodies caused a striking enhancement of IL-2 mRNA expression that was, in part, resistant to the effects of cyclosporine. These studies indicate that the CD28 molecule synergizes with protein kinase C activation to induce IL-2 gene expression and demonstrate that stimulation by the CD28 pathway can cause vigorous T-cell proliferation even in the presence of cyclosporine and that cyclosporine does not prevent transcription of 16-2 mRNA, as has been suggested previously. Moreover, these findings suggest that a potential role for the CD28 molecule in vivo may be to augment IL-2 production after stimulation of the CD3-T-cell receptor molecular complex and thereby to amplify an antigen-specific immune response. Finally, these results provide further evidence that the CD28 molecule triggers T-cell proliferation in a manner that differs biochemically from CD3-T-cell receptor-induced proliferation.

Characterization of XIAP-Deficient Mice

ABSTRACT The inhibitor of apoptosis protein (IAP) family consists of a number of evolutionarily conserved proteins that function to inhibit programmed cell death. X-linked IAP (XIAP) was cloned due to its sequence homology with other family members and has previously been shown to prevent apoptosis by binding to active caspases 3, 7, and 9 in vitro. XIAP transcripts can be found in a variety of tissues, and the protein levels are regulated both transcriptionally and posttranscriptionally. To better understand the function of XIAP in normal cells, we generated mice deficient in XIAP through homologous gene targeting. The resulting mice were viable, and histopathological analysis did not reveal any differences between XIAP-deficient and wild-type mice. We were unable to detect any defects in induction of caspase-dependent or -independent apoptosis in cells from the gene-targeted mice. One change was observed in cells derived from XIAP-deficient mice: the levels of c-IAP1 and c-IAP2 protein were increased. This suggests that there exists a compensatory mechanism that leads to upregulation of other family members when XIAP expression is lost. The changes in c-IAP1 and c-IAP2 expression may provide functional compensation for loss of XIAP during development or in the induction of apoptosis.

The Transcription Factors T-bet and Eomes Control Key Checkpoints of Natural Killer Cell Maturation

Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.

Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation.

Production of a red blood cells hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.

Anomalous Type 17 Response to Viral Infection by CD8+ T Cells Lacking T-bet and Eomesodermin

When intracellular pathogens invade mammalian hosts, naïve CD8+ T cells differentiate into cytotoxic killers, which lyse infected target cells and secrete cytokines that activate intracellular microbicides. We show that CD8+ T cells deficient in the transcription factors T-bet and eomesodermin (Eomes) fail to differentiate into functional killers required for defense against lymphocytic choriomeningitis virus. Instead, virus-specific CD8+ T cells lacking both T-bet and Eomes differentiate into an interleukin-17–secreting lineage, reminiscent of the helper T cell fate that has been implicated in autoimmunity and extracellular microbial defense. Upon viral infection, mice with T cells lacking both T-bet and Eomes develop a CD8+ T cell–dependent, progressive inflammatory and wasting syndrome characterized by multi-organ infiltration of neutrophils. T-bet and Eomes, thus, ensure that CD8+ T cells adopt an appropriate course of intracellular rather than extracellular destruction.