Interleukin-2 Withdrawal Research Articles

Differential cytokine withdrawal-induced death sensitivity of effector T cells derived from distinct human CD8+ memory subsets

CD8+ central memory (CM) and effector memory (EM) T-cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T-cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal-induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells displayed more mitochondrial instability and greater caspase activity. Indeed, we found that heightened CWID sensitivity in EM-derived effectors coincided with higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to ‘imprinted’ differences in BIM protein regulation, preserved by CD8+ CM and EM progeny, which govern their relative sensitivity to CWID. In addition, we detected a burst of autophagy after IL-2 withdrawal, which was better maintained in CM-derived T cells. Both subsets showed increased, equivalent CWID sensitivity upon treatment with autophagy inhibitors, suggesting sustained autophagy could preferentially protect CM-derived T cells from apoptosis. These findings offer new insight into how CM CD8+ T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based in part on decreased CWID sensitivity.

Differential apoptosis sensitivity of human effector CD8+ T cells derived from central versus effector memory subsets in response to IL-2 withdrawal

Abstract CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells display more mitochondrial instability and greater basal caspase activation. Indeed, we found that heightened CWID sensitivity in EM-derived effectors is linked to higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to “imprinted” differences in BIM protein regulation preserved between CD8+ CM and EM progeny that govern their relative sensitivity to CWID. These findings offer new insight into why CM-derived T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based on differential apoptosis sensitivity.

Prevention of Interleukin-2 Withdrawal-Induced Apoptosis in Lymphocytes Retrovirally Cotransduced With Genes Encoding an Antitumor T-cell Receptor and an Antiapoptotic Protein

Adoptive cell transfer using autologous tumor infiltrating lymphocytes or lymphocytes transduced with antitumor T-cell receptor (TCR) is an effective therapy for patients with metastatic melanoma. A limiting factor in the effectiveness of this treatment is the apoptosis of the transferred cells when Interleukin-2 (IL-2) administration is withdrawn. In an attempt to improve persistence of the transferred lymphocytes, we cotransduced human peripheral blood lymphocytes with retroviruses encoding Bcl-2 or Bcl-xL, antiapoptotic genes of the BCL2 family, and the MART-1 melanoma tumor antigen-specific TCR, DMF5. Lymphocytes were cotransduced with 38% to 64% cotransduction efficiency, and exhibited a marked delay in apoptosis after IL-2 withdrawal. Cotransduction with Bcl-2 or Bcl-xL did not affect cytokine secretion or lytic ability of the DMF5-transduced lymphocytes. After 5 days of IL-2 withdrawal, cotransduced lymphocytes produced similar levels of IFN-γ per cell as DMF5-alone transduced lymphocytes in response to tumor cells. Cotransduction did not alter the phenotype of lymphocytes with respect to a panel of T-cell differentiation markers. In a mouse model of melanoma, adoptively transferred T cells transduced with Bcl-2 persisted better in vivo at the site of tumor, 13 and 21 days after adoptive transfer (P=0.0064 and 0.041, respectively), with evidence of enrichment of the Bcl-2-transduced population over time (P<0.0001). Thus, by coexpressing Bcl-2 or Bcl-xL with a tumor-specific TCR, we have engineered a lymphocyte that resists apoptosis owing to IL-2 withdrawal without altering its tumor-specific function or phenotype, and thus may show improved antitumor effectiveness in vivo after cell transfer.

CD25high T Cells with a Prolonged Survival Inhibit Development of Diabetes

The goal of this study is to examine a novel hypothesis that the progression of diabetes is partially due to the weakened survival of CD25high T cells, and prolonging survival of CD25high T cells inhibits the development of diabetes. Since CD28 co-stimulation is essential for the survival of CD4+CD25high T cells, we determined whether CD28-upregulated translationally controlled tumor protein (TCTP) prolongs the survival of CD4+CD25high regulatory T cells (Tregs) by a transgenic approach. The TCTP transgene prevents Tregs from undergoing apoptosis induced by interleukin-2 withdrawal-, dexamethasone-, cyclophosphamide-, and anti-Fas treatment in vitro. In addition, transgenic Tregs express higher levels of FOXP3 than wild-type counterparts and maintain suppressive activity, suggesting that TCTP promotes Tregs escape from thymic negative selection, and that prolonged survival does not attenuate Treg suppression. Moreover, TCTP transgenic Tregs inhibit the development of autoimmune diabetes due to increased survival of suppressive Tregs and decreased expression of pancreatic TNF-alpha. Promoting the survival of CD25high T cells leads to prolonged survival of Tregs but not activated CD25+ non-Treg T cells. Thus, we propose a new model of "two phase survival" for Tregs. Our results suggest that modulation of Treg survival can be developed as a new therapy for autoimmune diseases.

Expression of TCTP antisense in CD25 high regulatory T cells aggravates cuff-injured vascular inflammation

This study examines our hypothesis that translationally controlled tumor protein (TCTP) expression in CD4+ CD25 high regulatory T cells (Tregs) is critical for the interleukin-2 (IL-2) withdrawal-triggered apoptosis pathway in Tregs, and modulation of Treg apoptosis pathway affects development of vascular inflammation. To test this hypothesis, we established a Tregs-specific TCTP antisense transgenic mouse model. Lower TCTP expression in Tregs than in CD4+ CD25− T cells is associated with the higher susceptibility of Tregs to apoptosis induced by IL-2 withdrawal. Overexpression of TCTP antisense in Tregs leads to decreased positive selection of CD25 high thymic Tregs and reduced survival of peripheral Tregs, which is correlated to our previous report that TCTP antisense knocks-down TCTP protein expression and promotes apoptosis. In addition, TCTP antisense transgene confers higher susceptibility of Tregs to apoptosis induced by IL-2 withdrawal than wild-type Tregs, which can be suppressed by exogenous supply of IL-2, suggesting that IL-2 promotes Treg survival at least partially due to promoting TCTP expression. Finally, decreased expression of TCTP in Tregs aggravates experimental vascular inflammation, presumably due to increased Treg apoptosis and failure of decreased Tregs in suppressing inflammatory cells and immune cells. These results suggest that the modulation of Tregs apoptosis/survival may be used as a new therapeutic approach for inflammatory cardiovascular diseases.

Higher expression of Bax in regulatory T cells increases vascular inflammation

This study is to examine our hypothesis that CD4+CD25(high)Foxp3+ regulatory T cells (Tregs) have an interleukin-2 (IL-2) withdrawal-triggered apoptosis pathway, and modulation of Treg apoptosis pathway affects development of vascular inflammation. We found that pro-apoptotic protein Bax upregulation in Tregs is induced by IL-2 withdrawal. Treg apoptosis induced by IL-2 withdrawal is inhibited by a Bax inhibitor, suggesting that highly expressed Bax is functional. To define the role of upregulated Bax in Treg apoptosis, we established a Tregs-specific Bax transgenic mouse model. Enforced expression of Bax in Tregs promotes Treg apoptosis triggered by IL-2 withdrawal and other apoptosis stimuli, suggesting pro-apoptotic role of highly expressed Bax in wild-type Tregs. Finally, higher expression of Bax in Tregs decreases the striking threshold of vascular inflammation due to the failure of suppression of inflammatory cells resulting from Treg apoptosis. These results have demonstrated the proof of principle that the modulation of Tregs apoptosis/survival could be used as a new therapeutic approach for inflammatory cardiovascular diseases.

Bcl-2 Overexpression Enhances Tumor-Specific T-Cell Survival

Although immunotherapy based on the adoptive transfer of tumor-specific T lymphocytes has been shown to result in dramatic clinical responses in some patients, the relatively low levels of engraftment and persistence of the adoptively transferred cells may limit these responses in many patients. In an attempt to develop strategies for prolonging the survival of adoptively transferred T cells, we have carried out studies in which T cells obtained from healthy donors as well as tumor-specific T cells were transduced with a retrovirus expressing the human Bcl-2 gene. Our results indicate that these transduced T cells overexpress Bcl-2, are resistant to death, and have a survival advantage following interleukin-2 withdrawal compared with control T cells transduced with a retrovirus expressing green fluorescent protein. Tumor-specific T cells overexpressing Bcl-2 maintained their ability to specifically recognize and respond to target cells. Furthermore, we show that adoptive immunotherapy of an established B16 tumor can be significantly enhanced by overexpressing Bcl-2 in melanoma-specific T-cell receptor transgenic T cells. Our data suggest that adoptive immunotherapy approaches to the treatment of cancer patients may be enhanced using Bcl-2-modified tumor-reactive T cells.

GILZ, a new target for the transcription factor FoxO3, protects T lymphocytes from interleukin-2 withdrawal–induced apoptosis

AbstractInterleukin-2 (IL-2) withdrawal is a physiologic process inducing cell death in activated T lymphocytes. Glucocorticoid-induced leucine zipper (GILZ) has recently been identified as a protein modulating T-cell receptor activation by repressing various signaling pathways. We report here that IL-2 deprivation leads to expression of GILZ in T lymphocytes. We then characterized the human gilz promoter and showed that FoxO3 (Forkhead box class O3) binding to the Forkhead responsive elements identified in the promoter is necessary for induction of gilz expression upon IL-2 withdrawal. To assess the functional consequences of this induction, we used 2 strategies, GILZ overexpression and GILZ silencing in murine IL-2–dependent CTLL-2 cells. GILZ overexpression protects CTLL-2 cells from IL-2 withdrawal–induced apoptosis, whereas cell death is accelerated in cells unable to express GILZ. Concomitantly, the expression of Bim is inhibited in GILZ-overexpressing cells and enhanced when GILZ expression is impaired. Furthermore, GILZ inhibits FoxO3 transcriptional activity that leads to inhibition of Bim expression but also to down-regulation of GILZ itself. Therefore, GILZ is a transiently expressed protein induced upon IL-2 withdrawal that protects T cells from the onset of apoptosis.

Promoting a Deadly Rise in Calcium

Devireddy and Green used expression profiling to uncover genes that were transcriptionally activated in response to interleukin 2 (IL-2) deprivation and identified a protein implicated in learning and memory as a novel proapoptotic factor in T lymphocytes. After termination of an immune response, the expanded population of T cells is eliminated through apoptosis. T cell apoptosis in response to IL-2 deprivation requires transcriptional activation; however, the details remain unclear. Devireddy and Green identified genes whose transcription was activated or inhibited after IL-2 withdrawal in an IL-2-dependent T cell line. Among the genes that were activated was RC3 (or neurogranin ), whose protein product is a calmodulin (CaM)-binding protein, previously considered to be neuron-specific. Other IL-2-dependent cells also upregulated RC3 following IL-2 deprivation; whereas cells dependent on other cytokines did not show this response after cytokine deprivation nor did apoptotic cells from an IL-2-independent T cell line. RC3 overexpression promoted apoptosis in T cell lines; mutational analysis indicated that this depended on an intact CaM-binding domain. Fluorescent imaging with the Ca 2+ -indicator fura-2 AM showed that both IL-2 deprivation and overexpression of RC3 led to similar increases in intracellular Ca 2+ concentration. Blocking this increase with a Ca 2+ chelator inhibited apoptosis. A second CaM-binding protein that was previously identified as a proapoptotic factor in various cell types was also up-regulated, leading the authors to propose a model whereby IL-2 withdrawal promotes apoptosis through multiple pathways involving CaM segregation and a consequent deadly rise in intracellular Ca 2+ . L. Devireddy, M. R. Green, Transcriptional program of apoptosis induction following interleukin 2 deprivation: Identification of RC3, a calcium/calmodulin binding protein, as a novel proapoptotic factor. Mol. Cell. Biol. 23 , 4532-4541 (2003). [Abstract] [Full Text]

P27kip1 regulates the cell cycle arrest and survival of activated T lymphocytes in response to interleukin-2 withdrawal.

The majority of activated T lymphocytes undergo cell death at the end of a primary immune response, while a minority survive as memory cells. The mechanisms that control the decision between these two fates are unknown. In the present study we examined the response of activated T cells to interleukin-2 (IL-2) withdrawal. Within hours, the percentage of T lymphocytes in cell cycle showed a steady decrease, while the percentage arrested in G1 increased proportionally. Deprivation of IL-2 resulted in upregulation of the cell cycle inhibitor p27kip1. Comparison with resting T-cell populations revealed that the highest expression of p27kip1 occurs in activated T cells undergoing cell cycle arrest following IL-2 withdrawal. T cells deficient in p27kip1 expression showed an impaired ability to undergo cell cycle arrest in response to IL-2 deprivation. Moreover, T cells deficient in p27kip1 showed significantly more apoptosis after IL-2 withdrawal. Collectively, this study demonstrates that p27kip1 regulates both the cell cycle arrest and the apoptosis of antigen-specific T lymphocytes.

Selective Modification of Antigen-Specific CD4 + T Cells by Retroviral-Mediated Gene Transfer and in Vitro Sensitization with Dendritic Cells

Adoptive therapy with antigen-specific T cells is a potential treatment against cancers and viral diseases. To establish a system to modify the genes of these cells to increase their effectiveness, we examined whether the combined use of retroviral vector, which only infects dividing cells, and in vitro sensitization of T cells with antigen-loaded dendritic cells (DCs) could selectively modify antigen-specific T cells with a bcl-2 gene. Human CD4 + T cells were used as target cells. Autologous DCs transfected with genes of hepatitis B virus (HBV) stimulated a specific T cell proliferation. Importantly, these proliferating T cells were selectively transduced by a bcl-2-retrovirus, and CD25 + T cells isolated from them contained higher levels of integrated provirus. To select bcl-2-transduced, activated T cells, cells were subjected to interleukin-2 (IL-2) withdrawal. In contrast to CD25 − and mock-infected CD25 + T cells, 70% of CD25 + T cells transduced with bcl-2-retrovirus survived IL-2 withdrawal. These surviving T cells were demonstrated to contain integrated bcl-2 provirus and exhibited HBV-specific proliferation and interferon-γ secretion. In addition, bcl-2 overexpression protected HBV-specific T cells from transforming growth factor (TGF)-β-induced cell death. These results demonstrate the feasibility of our strategy in the generation of genetically modified antigen-specific CD4 + T cells and show that bcl-2-transduced antigen-specific T cells survive IL-2 withdrawal and TGF-β-induced apoptosis.

Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy.

NK-92 is a highly cytotoxic natural killer (NK) tumor cell line that possesses properties that make it an excellent candidate for adoptive cellular immunotherapy. However, the cytotoxicity of NK cells is dependent on cytokines such as interleukin 2 (IL-2). Although NK-92 cells maintain cytotoxicity for a time after withdrawal of IL-2, clinical use will probably require prolonged treatment with fully activated cells to eliminate disease effectively. The ability to support cytotoxic cells with exogenously administered IL-2 is limited by associated toxicity. Therefore, we describe the transfection of the IL-2-dependent NK-92 cell line with human IL-2 (hIL-2) cDNA by particle-mediated gene transfer to create two IL-2-independent variants, NK-92MI and NK-92 CI, and describe their characterization and comparison with parental cells. Both variants were shown to contain, express, and synthesize the hIL-2 cDNA. IL-2 synthesis was higher in NK-92MI cells compared with NK-92CI cells, with no expression in parental cells. Functionally, the cytotoxicity of all three cell lines was similar and coincubation with IL-2-independent variants did not affect hematopoietic progenitor cells. NK-92MI and NK-92CI cells were more radiosensitive than NK-92 cells, with proliferation inhibited at lower radiation doses and increased morality and decreased cytotoxicity compared with parental cells. Data presented here show that we have created by particle-mediated gene transfer two IL-2-independent variants of NK-92 that are identical to parental cells in virtually all respects, including high cytotoxic activity. The nonviral transfection of these cells makes them suitable for clinical applications. These IL-2-independent cells should allow prolonged treatment with fully active natural killer cells without the need for exogenous IL-2 support.

Acceleration of apoptotic cell death after the cleavage of Bcl-XL protein by caspase-3-like proteases.

Interleukin-2 (IL-2)-dependent T cell clone CTLL-2 underwent apoptosis by deprivation of IL-2 from culture medium. The decrease in the anti-apoptotic Bcl-XL protein level was observed during apoptosis after IL-2 withdrawal. We found that Bcl-XL protein was cleaved to produce two 18 kDa fragments during CTLL-2 cell apoptosis. When the activation of caspases was suppressed by overexpressing human Bcl-2 protein or by the addition of caspase inhibitors, cleavage of Bcl-XL protein was suppressed in vivo. Bcl-XL protein cleavage by incubation with apoptosed CTLL-2 cell lysate was suppressed by the caspase-3/CPP32-specific tetrapeptide inhibitor in vitro. Therefore, caspase-3/CPP32-like proteases were activated and involved in the cleavage of Bcl-XL protein during CTLL-2 cell apoptosis. We found that Bcl-XL protein was cleaved by caspase-3/CPP32 at two sites in the loop domain (i.e., HLAD61/S and SSLD76/A). The transfection of the carboxy-terminal 18 kDa Bcl-XL fragment increased the sensitivity to apoptosis. These results indicate that caspase-3/CPP32-like proteases cleaved anti-apoptotic Bcl-XL protein and resulted in accelerated apoptotic cell death.

Exposure of Phosphatidylethanolamine on the Surface of Apoptotic Cells

In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells. The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells. The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells. The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a fluorescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V. This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.

Autologous bone marrow purging by in situ IL-2 activation of endogenous killer cells.

The major cause of treatment failure in hematologic malignancies is the development of resistance to chemotherapeutic agents and the presence of clonogenic tumor cells in remission bone marrow. In the present study, we tested the optimal conditions for activation by interleukin-2 (IL-2) of endogenous bone marrow effector cells to develop cytotoxicity against solid tumor and leukemia cells and the effects of IL-2 treatment on eradication of malignant cells from the marrow product while preserving hematopoiesis. Normal donor bone marrow was contaminated with 10% A549 lung cancer cells, MCF-7 breast cancer, or EM2 leukemia cells, and then combined with peripheral blood-derived lymphokine-activated killer (LAK) cells which had been incubated in the presence of IL-2, 6000 IU/ml, for 5 days. Eradication (A549 and MCF-7) or >90% reduction in tumor cell number (EM2) was achieved upon exposure of the marrow to LAK cells but not to control, non-IL-2-exposed PBMC. The non-cross-resistance of chemotherapy and cell-mediated cytotoxicity were tested by using chemoresistant target cells for LAK cytotoxicity assays. Ara-C-resistant K562 cells were sensitive to LAK cytotoxicity, similar to sensitive controls. Similar effects were seen in daunomycin-resistant HL60 leukemia cells and VP-16-resistant K562 cells. The daunomycin-resistant K562 cells were also sensitive to LAK cell killing similar to sensitive controls. Activation of bone marrow mononuclear cells (BMMC) with IL-2, 6000 IU/ml for 24 h, significantly enhanced cytotoxicity against the NK-resistant, LAK-sensitive Daudi cell line. This procedure did not result in significant loss of total BMMC or hematopoietic colony-forming units. We then studied the possibility that the effector cell activity of the IL-2-activated BMMC which is diminished after removal of the IL-2 at 24 h could be recovered upon re-exposure of the marrow to IL-2. We found that the optimum conditions for preserving cytotoxicity were prolonged continuous exposures to IL-2 but that re-exposure of the 24-h IL-2-activated BMMC to IL-2 after a 1- or 2-week IL-2 withdrawal was associated with full recovery of cytotoxicity. These experiments also demonstrated that the overall recovery of viable cells from the BMMC was over 100%, possibly due to a proliferative effect of the IL-2 exposure on BMMC. These results demonstrate that the use of IL-2-activated marrow in patients with acute leukemia in remission undergoing autologous bone marrow transplantation (aBMT) may overcome the obstacles of clonogenic, drug-resistant minimal residual disease. The effector cell activity and hematopoietic capacity of the activated bone marrow product can also be maintained even if optimal clinical care requires an interruption in the in vivo exposure to IL-2. This therapeutic approach is currently being tested in acute leukemia patients undergoing IL-2-activated aBMT followed by prolonged IL-2 treatment before and after hematologic reconstitution.

16S Mitochondrial Ribosomal RNA Degradation Is Associated with Apoptosis

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 μmol H 2O 2 per 10 7 cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis. © 1997 Elsevier Science Inc.

Detection of apoptosis by flow cytometry of cells simultaneously stained for intracellular pH (carboxy SNARF-1) and membrane permeability (Hoechst 33342).

Intracellular acidification appears to be a frequent and possibly universal occurrence during apoptosis. We have used carboxy SNARF-1 to measure intracellular pH by flow cytometry. We report here that the addition of Hoechst 33342 concurrently with carboxy SNARF-1 provides a clearer discrimination of the apoptotic population. Apoptotic cells accumulate Hoechst 33342 more rapidly than do viable nonapoptotic cells. The cells with greater Hoechst 33342 fluorescence are the same cells as those with decreased intracellular pH; these cells also have decreased volume. The different parameters in this analysis are presented for three models of apoptosis: human myeloblastoid ML-1 cells incubated with etoposide, murine cytotoxic T cells following withdrawal of interleukin-2, and Chinese hamster ovary cells incubated with staurosporine. In most circumstances, carboxy SNARF-1 provides excellent resolution of viable and apoptotic cells. However, the addition of Hoechst 33342 is particularly valuable when carboxy SNARF-1 alone can not fully discriminate the two cell populations. This situation occurs in Chinese hamster ovary cells, which undergo a smaller degree of acidification than do the other cell models. This situation also occurs when the extracellular pH is experimentally reduced to investigate the mechanism of pH dysregulation; the apoptotic cells appear to retain the ability to regulate intracellular pH at low pH, although they are defective in proton export at neutral pH.

The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation.

Apoptosis following interleukin-2 withdrawal from T cells: evidence for a regulatory role of CD18 (beta 2-integrin) molecules.

Following a successful immune response against invading microorganisms, the majority of activated T cells is eliminated, while a minor fraction survives as memory T cells. A decline in T lymphocyte growth factors such as interleukin-2 (IL-2) appears to play a role in the elimination of previously activated T cells. Thus, removal of IL-2 from proliferating T cells not only induces growth arrest, but triggers a massive cell death due to apoptosis. While the apoptotic response involves a series of well-described events, it remains less clear how apoptosis is regulated following IL-2 withdrawal. Here, we provide evidence that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following removal of IL-2 from previously activated, antigen specific CD4+ T cell lines. Thus, CD18 mAb inhibited the apoptotic response to IL-2 deprivation, whereas mAb against other adhesion molecules (CD28, CD29, CD49d, CD80, CD86) did not. Secondly, IL-2 withdrawal resulted in a retarded apoptotic response in LFA-1 (CD11a/CD18) negative T cells obtained from a leukocyte adhesion deficiency (LAD) patient, as compared to LFA-1 positive T cell lines. Thirdly, co-culture of LFA-1 positive- and negative-T cells at different ratios induced apoptotic responses that were higher than expected, had the two lymphocyte populations not been interacting and significantly higher than that seen in pure LFA-1 negative T cells. Supernatants from LFA-1 positive T cell cultures undergoing apoptosis did not induce an enhanced apoptotic responses in LFA-1 negative T cells, and, reversely, culture supernatants from LFA-1 negative T cells did not rescue LFA-1 positive cells from undergoing apoptosis. The apoptotic response was partly blocked by IL-15, a newly identified T cell growth factor. Taken together, these findings suggest that CD18 molecules (beta 2-integrins) play a regulatory role in the apoptotic response following cytokine withdrawal, and that the regulation is mediated, at least partly, through T-T cell interactions. Thus, apoptotic death following IL-2 deprivation appears to be under "social" control by surrounding T cells.

Apoptosis in an Interleukin-2-dependent Cytotoxic T Lymphocyte Cell Line Is Associated with Intracellular Acidification: ROLE OF THE Na+/H+-ANTIPORT

Apoptosis is a form of cell death associated with DNA fragmentation and chromatin condensation. We recently established that intracellular acidification occurred during apoptosis following cytotoxic insult. The current studies were designed to determine whether intracellular acidification was more generally associated with apoptosis, specifically in a model of growth factor withdrawal. Upon withdrawal of interleukin-2, CTLL-2 cells accumulated in the G1 phase of the cell cycle and started to fragment their DNA around 12 h concurrent with both decreased pH and increased Ca2. Chelation of Ca2+ did not inhibit DNA digestion, whereas incubation with a calcium ionophore prevented both acidification and DNA digestion. Hence, acidification rather than increased Ca2+ was associated with apoptosis. The acidified cells represented a discrete population up to 0.7 pH units below normal. The extent of acidification depended upon the extracellular pH; above pH 6.3, intracellular pH was significantly below extracellular pH, whereas below pH 6.3, the cells still regulated their pH. Inhibition of the Na+/H(+)-antiport prevented the apoptotic cells from regulating their intracellular pH under these acidic conditions. These intracellular pH under these acidic conditions. These results demonstrate that apoptotic cells retain a functional antiport but that its set-point has changed. Many survival factors are known to phosphorylate and activate the antiport, hence apoptosis is likely to be associated with dephosphorylation. Although acidification always occurred during apoptosis, maintaining intracellular pH above 7.2 did not prevent apoptosis, suggesting that an acid pH is not essential for apoptosis. We hypothesize that other critical regulators of apoptosis must be subject to dephosphorylation.