Rapid induction of apoptosis in CD8+ HIV-1 envelope-specific murine CTLs by short exposure to antigenic peptide

During primary viral infection, in vivo exposure to high doses of virus causes a loss of Ag-specific CD8(+) T cells. This phenomenon, termed clonal exhaustion, and other mechanisms by which CTLs are deleted are poorly understood. Here we show evidence for a novel form of cell death in which recently stimulated CD8(+) HIV-1 envelope gp160-specific murine CTLs become apoptotic in vitro after brief exposure to free antigenic peptide (P18-I10). Peak apoptosis occurred within 3 h of treatment with peptide, and the level of apoptosis was dependent on both the time after initial stimulation with target cells and the number of targets. Using T cell-specific H-2D(d)/P18-I10 tetramers, we observed that the apoptosis was induced by such complexes. Induction of apoptosis was blocked by cyclosporin A, a caspase 3 inhibitor, and a mitogen-activated protein kinase inhibitor, but not by Abs to either Fas ligand or to TNF-alpha. Thus, these observations suggest the existence of a Fas- or TNF-alpha-independent pathway initiated by TCR signaling that is involved in the rapid induction of CTL apoptosis. Such a pathway may prove important in the mechanism by which virus-specific CTLs are deleted in the presence of high viral burdens.     

Pyp3 PTPase acts as a mitotic inducer in fission yeast.

The p34cdc2 M-phase kinase is regulated by inhibitory phosphorylation of Tyr15, largely through the actions of the p107wee1 tyrosine kinase and p80cdc25 protein tyrosine phosphatase (PTPase). In this study we demonstrate that a second PTPase, encoded by pyp3, also contributes to tyrosyl dephosphorylation of p34cdc2. Pyp3 was identified as a high copy suppressor of a cdc25- mutation. The pyp3 gene encodes a 33 kDa PTPase that is more closely related to human PTP1B and fission yeast pyp1 and pyp2 PTPases than to cdc25. Pyp3 does not share an essential overlapping function with pyp1 or pyp2. We demonstrate that disruption of pyp3 causes a mitotic delay that is greatly exacerbated in cells that are partially defective for cdc25 function and that pyp3 function is essential in cdc25-disruption wee1- strains. Pyp3 PTPase effectively dephosphorylates and activates the p34cdc2 kinase in vitro. We conclude that the pyp3 PTPase acts cooperatively with p80cdc25 to dephosphorylate Tyr15 of p34cdc2.     

The redox-sensing quinone reductase Lot6p acts as an inducer of yeast apoptosis

Mammalian NAD(P)H:quinone oxidoreductases such as human NQO1 act as inducers of apoptosis. Quinone reductases generated interest over the last decade due to their proposed function in the oxidative stress response. Furthermore, human NQO1 was reported to regulate p53 stability and p53-dependent apoptosis through regulation of cellular oxidation–reduction events. In this study, we have used low concentrations of hydrogen peroxide (0.4 and 0.6 mM) to induce apoptosis-like cell death in wild type, an LOT6 overexpressing and a Δ lot6 yeast strain to monitor cell survival. Using this approach, we demonstrate that yeast quinone reductase Lot6p, an orthologue of mammalian quinone reductases, plays a pivotal role in apoptosis-like cell death in Saccharomyces cerevisiae . Overexpression of LOT6 results in enhanced cell death, as shown by an investigation of the morphological hallmarks of apoptosis-like fragmentation of DNA and externalization of phosphatidylserine, whereas the deletion strain displays a deficiency in apoptosis-like cell death as compared with the wild type. Thus, we propose that Lot6p is directly involved in the control of the apoptosis-like cell death in yeast.     

Induction of TCR Vbeta-specific CD8+ CTLs by TCR Vbeta-derived peptides bound to HLA-E

Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vbeta families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vbeta peptides and whether the HLA-E/Vbeta-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vbeta1 and Vbeta2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vbeta1 (C1R-E/V1) or Vbeta2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vbeta peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vbeta-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vbeta1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vbeta-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vbeta peptides can be presented by HLA-E to further induce Vbeta-specific CD8+ CTLs.     

IL-12 acts selectively on CD8 alpha- dendritic cells to enhance presentation of a tumor peptide in vivo.

Previous work has shown that a significant proportion of murine splenic dendritic cells (DC) express a high affinity receptor for IL-12, thus accounting for the adjuvanticity of the cytokine when DBA/2 mice are transferred with syngeneic DC exposed in vitro to rIL-12 and an otherwise poorly immunogenic tumor peptide. In DBA/2 mice, splenic DC consist of 90-95% CD8- and 5-10% CD8+ cells. To detect any difference in IL-12 responsiveness among phenotypically distinct DC subtypes, enriched CD8- (>99% pure) and CD8+ ( approximately 80% pure) populations of DC from DBA/2 spleens were assayed for APC function in vivo following exposure to rIL-12 and tumor peptide in vitro. Unlike unfractionated DC, the CD8- fraction was capable of effective presentation of the peptide even when the cells had not been pretreated with IL-12 before peptide pulsing. The addition of as few as 3% CD8+ cells during pulsing blocked in vivo priming by the CD8- fraction. However, pretreatment of CD8- DC with IL-12 before cell mixing and peptide pulsing ablated the inhibitory effect of the CD8+ fraction. CD8-, but not CD8+, DC showed significant message expression for the beta 1 and beta 2 subunits of the IL-12 receptor. These data suggest that a minority population of CD8+ DC, which appeared to secrete IL-10 in vitro, negatively regulates the induction of T cell reactivity by peptide-loaded CD8- DC in DBA/2 mice. However, the CD8- fraction can be primed by IL-12 to overcome the inhibitory effect of the CD8+ subtype.     

CD4+CD25+ regulatory T cells attenuate the phosphatidylinositol 3-kinase/Akt pathway in antigen-primed immature CD8+ CTLs during functional maturation

This study was designed to determine the role of CD25(+)CD4(+) regulatory T (Tr) cells in CTL maturation and effector functions using a murine CTL line and in vitro MLC. Tr cells inhibited CTL functional maturation, but had no effect on CTL effector functions. In CD4(+) responder T cell-depleted MLC supplemented with IL-2, Tr cells suppressed mature CTL generation only when added within the first 2 days of culture. Tr cells down-regulated levels of active Akt, but not STAT5 or ZAP70 in Ag-primed immature CTLs. Down-regulation of active Akt was accompanied by a reduction in CTL cell size and IL-2Ralpha expression. In Tr cell-depleted MLC, CTLs were generated that exhibited high levels of nonspecific cytotoxicity. Our in vitro findings suggest that Tr cells regulate functional CTL maturation to generate optimal Ag-specific immune responses through the control of the PI3K/Akt pathway.     

Induction of obliterative airway disease in murine tracheal allografts by CD8+ CTLs recognizing a single minor histocompatibility antigen

The role of minor histocompatibility Ag (mHAg)-specific CD8+ CTLs in the pathogenesis of chronic lung allograft rejection (bronchiolitis obliterans syndrome) remains to be elucidated. Thus, the goal of this study was to define the role of a single mHAg mismatch at the polymorphic H13 allele in the development of obliterative airway disease (OAD) after murine heterotopic tracheal transplantation. The H13a and H13b alleles encode for the SSVVGVWYL (SVL9) and SSVIGVWYL (SIL9) mHAgs, respectively, presented in the context of the H2Db MHC class I molecule. Toward this, C56BL/10SnJ (H13a) tracheal allografts were transplanted into congenic B10.CE-H13b Aw(30NX)/Sn (H13b) recipients. The allografts were harvested at 30, 60, and 90 days after transplantation, and OAD lesions (epithelial damage, cellular infiltration, and luminal fibrosis) were confirmed histologically. Selected groups of mice were immunized (s.c.) or tolerized (i.v.) with the SVL9 peptide before transplantation. This single mHAg mismatch induced the development of OAD within 90 days. SVL9 immunization significantly accelerated the kinetics of the OAD lesions. In contrast, SVL9 tolerization completely abrogated the development of OAD. This was correlated with a complete abrogation of H13a-specific CD8+ CTL responses with a significant reduction in the frequency of IFN-gamma-producing CTLs and the activation of TGF-beta-producing CD8+ T cells. In conclusion, a single mHAg mismatch can induce the development of OAD. These data also suggest that mHAg-reactive CD8+ CTLs may play an important role in the pathogenesis of chronic lung allograft rejection in humans.     

A viral long terminal repeat expressed in CD4+CD8+ precursors is downregulated in mature peripheral CD4-CD8+ or CD4+CD8- T cells.

The long terminal repeat from a thymotropic mouse mammary tumor virus variant, DMBA-LV, was used to drive the expression of two reporter genes, murine c-myc and human CD4, in transgenic mice. Expression was observed specifically in thymic immature cells. Expression of c-myc in these cells induced oligoclonal CD4+ CD8+ T-cell thymomas. Expression of human CD4 was restricted to thymic progenitor CD4- CD8- and CD4+ CD8+ T cells and was shut off in mature CD4+ CD8- and CD4- CD8+ T cells, known to be derived from the progenitor double-positive T cells. These results suggest the existence of similar and common factors in CD4+ CD8- and CD4- CD8+ T cells and support a model of differentiation of CD4+ CD8+ T cells through common signal(s) involved in turning off the expression of the CD4 or CD8 gene.     

Generation of CD8 (T8) cytotoxic cells has a preferential requirement for CD4+2H4- inducer cells

CD8 (T8) cells are capable of both suppression and cytotoxicity. However, we have found that the activation of CD8 cytotoxic cells has a preferential requirement for a different CD4 (T4) subset from that previously reported for the activation of CD8 suppressor cells. We have recently characterized two monoclonal antibodies which subdivide CD4 cells into inducers of help for antibody production (CD4+ 4B4+) and inducers of CD8 mediated suppression (CD4+2H4+). We now report that CD4+4B4+2H4- cells also preferentially induce CD8-mediated cytotoxicity. Human peripheral blood T cells were fractionated into CD8, CD4, CD4+2H4+, and CD4+2H4- populations by both the adherence to antibody-coated plates and the fluorescence-activated cell sorter. The cells were cultured 6 days with irradiated allogeneic non-T cells and a cytotoxicity assay was then performed using cryopreserved non-T cells as targets. It was found that the combination of CD4+2H4- cells and CD8 cells resulted in greater cytotoxicity than either CD4 + CD8, or CD4+2H4+ + CD8. The combination of CD4+2H4+ cells with CD8 cells resulted in minimal cytotoxicity, which was similar to that generated by CD8 cells alone. These results were confirmed using anti-4B4 to positively select the reciprocal CD4 subset. Furthermore, the cytotoxicity induced by CD4+2H4- cells was alloantigen specific and Class I major histocompatibility complex restricted. As both CD4+2H4+ and CD4+2H4- cells proliferate equally well to alloantigen and produce similar levels of interleukin 2 (IL-2), it is likely that the generation of CD8 cytotoxic cells requires a signal in addition to IL-2.     

The marine cytotoxin portimine is a potent and selective inducer of apoptosis

Portimine is a recently discovered member of a class of marine micro-algal toxins called cyclic imines. In dramatic contrast to related compounds in this toxin class, portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells. In this study we show that portimine kills human Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs), with LC₅₀ values of 6 and 2.5 nM respectively. Treated cells displayed rapid caspase activation and phosphatidylserine exposure, indicative of apoptotic cell death. Jurkat cells overexpressing the anti-apoptotic protein Bcl-2 or Bax/Bak knockout MEFs were completely protected from portimine. This protection was apparent even at high concentrations of portimine, with no evidence of necrotic cell death, indicating that portimine is a selective chemical inducer of apoptosis. Treatment of the Bcl-2-overexpressing cells with both portimine and the Bcl-2 inhibitor ABT-737 proved a powerful combination, causing >90?% death. We conclude that portimine is one of the most potent naturally derived inducers of apoptosis to be discovered, and it displays strong selectivity for the induction of apoptotic pathways.     

CD8 functions as an inhibitory ligand in mediating the immunoregulatory activity of CD8+ cells.

Antisense and sense transfection technologies were employed in cellular coculture systems to investigate the physiologic requirements for CD8-dependent immunoregulation. Our data indicate that cells bearing genetically engineered CD8 molecules incorporating a glycoinositolphospholipid membrane anchor, as well as fixed cells bearing natural CD8 molecules, retain specific, CD8-dependent immunoregulatory activity. These findings together support the novel concept that CD8, a molecule traditionally thought of as a receptor, can function as an inhibitory ligand. CD8-dependent inhibition was shown to induce nonresponsiveness, persisting for at least 24 h, in Ag-specific responders. Moreover, only cells undergoing primary, but not secondary, antigenic stimulation through their TCR were found to be susceptible to CD8-dependent inhibition. Both CD4+ and CD8+ responding T cells were inhibited by CD8+ modulatory cells. These functional analyses of inhibitory and responder cells in CD8-dependent inhibition lay the groundwork for developing artificial CD8-based immunomodulatory peptides and deciphering CD8's role in natural immunoregulation.     

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR-NF-kappaB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.     

CD4+CD25+ and CD4+CD25- T cells act respectively as inducer and effector T suppressor cells in superantigen-induced tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4(+) TCRBV8(+) T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-gamma secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4(+) T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4(+)CD25(+) splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4(+)CD25(-) T regulatory cells prevented SEB-induced death independently of CD4(+)CD25(+) T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25(+) cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4(+)CD25(+) T regulatory cells are required for the induction of SAg-induced tolerance, CD4(+)CD25(-) T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.     

Concomitant helper response rescues otherwise low avidity CD8+ memory CTLs to become efficient effectors in vivo

This report seeks a means of maximizing memory CD8 T cell responses to peptide immunization. Delivery of the CD8 peptide epitope by stress protein, heat shock protein (hsp)70, results in excellent immunogenicity at the acute phase but memory responses were poor both in terms of the number of responding cells as well as their functional avidity. We demonstrate for the first time that hsp70 can also be used as a vehicle to achieve CD4 T cell responses to loaded peptide epitopes and that coimmunization with hsp70 loaded with both CD8 and CD4 peptide epitopes may increase memory up to 3-fold. Furthermore, CD8+ T cell memory responses were of higher avidity measured both by in vitro cytotoxicity assays and a new methodology that measures the avidity of CTL activity in vivo in mice. Our results emphasize that peptide immunization remains a viable approach to induce long-term CD8+ T cell function, providing steps are taken to assure appropriate stimulation of Th cell responses.     

IL-21 induces apoptosis of antigen-specific CD8+ T lymphocytes

IL-21, a member of the common gamma-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common gamma-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.     

A peptide epitope derived from the cancer testis antigen HOM-MEL-40/SSX2 capable of inducing CD4+ and CD8+ T-cell as well as B-cell responses

Background

Antigen-derived HLA class I-restricted peptides can generate specific CD8⁺ T-cell responses in vivo and are therefore often used as vaccines for patients with cancer. However, only occasional objective clinical responses have been reported suggesting the necessity of CD4⁺ T-cell help and possibly antibodies for the induction of an effective anti-tumor immunity in vivo. The SSX2 gene encodes the cancer testis antigen (CTA) HOM-MEL-40/SSX2, which is frequently expressed in a wide spectrum of cancers. Both humoral and cellular immune responses against SSX2 have been described making SSX2 an attractive candidate for vaccine trials.

Methods

SYFPEITHI algorithm was used to predict five pentadecamer peptides with a high binding probability for six selected HLA-DRB1 subtypes (*0101, *0301, *0401, *0701, *1101, *1501) which are prevalent in the Caucasian population.

Results

Using peripheral blood cells of 13 cancer patients and 5 healthy controls, the HOM-MEL-40/SSX2-derived peptide p101-111 was identified as an epitope with dual immunogenicity for both CD4⁺ helper and cytotoxic CD8⁺ T cells. This epitope also reacted with anti-SSX2 antibodies in the serum of a patient with breast cancer. Most remarkably, SSX2/p101-111 simultaneously induced specific CD8, CD4, and antibody responses in vitro.

Conclusions

p101-111 is the first CTA-derived peptide which induces CD4⁺, CD8⁺, and B-cell responses in vitro. This triple-immunogenic peptide represents an attractive vaccine candidate for the induction of effective anti-tumor immunity.     

Phosphorylation and down-regulation of CD4 and CD8 in human CTLs and mouse L cells

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4⁺ or CD8⁺ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4⁺ or CD8⁺ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4⁺ and CD8⁺ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4⁺ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8⁺ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.     

Isolation of primary HIV-1 that target CD8+ T lymphocytes using CD8 as a receptor

HIV-1 use CD4 receptors to infect their primary targets, CD4+ cells, whereas CD8+ cells have a protective role against HIV-1. We recently isolated HIV-1-producing CD8+ clones from two AIDS patients. Here we show that although HIV-1 produced by CD8+ cells maintained the ability to infect CD4+ cells, these viruses were able to infect CD8+ cells independent of CD4. Evidence indicates that these viruses used CD8 as a receptor to infect CD8+ cells. First, expression of CD8 was downmodulated after infection. Second, anti-CD8 antibodies blocked viral entry and replication in CD8+ cells. Finally, resistant cells became susceptible after expression of CD8. Although these viruses used CXCR4 to enter CD4+ cells, it seems that infection of CD8+ cells was independent of CXCR4 or CCR5 co-receptors. Novel changes were observed in envelope sequences of CD8-tropic viruses. These results provide initial evidence that HIV-1 can mutate to infect CD8+ cells using CD8 as a receptor.     

Salicylic acid glucoside acts as a slow inducer of oxidative burst in tobacco suspension culture

Salicylic acid beta-glucoside (SAG) is a storage form of a defense signal against pathogens, releasing free salicylic acid (SA), to meet the requirements in plants. Since excess SA induces locally restricted cell death following oxidative burst and Ca2+ influx in plants, the effects of SAG on cell viability, Ca2+ influx, and generation of superoxide (O2*-) were examined in suspension-cultured tobacco BY-2 cells expressing aequorin. Among SA-related chemicals tested, only SAG induced the slow and long-lasting O2*- generation, although SAG was less active in acute O2*- generation, Ca2+ influx and induction of cell death. The prolonging action of SAG is likely due to gradual release of SA and the data suggested that a peroxidase-dependent reaction is involved. Notably, pretreatment with low-dose SA (50 micromu) enhanced the response to SAG by 2.5-fold. There are four possible secondary messengers in early SA signaling detectable in the BY-2 culture, namely O2*-, H2O2, Ca2+ and protein kinase (PK). If these messengers are involved in the low-dose SA-dependent priming for SAG response, they should be inducible by low-dose SA. Among the four SA-inducible signaling events, PK activation was excluded from the low-dose SA action since a much higher SA dose (> 0.4 mmu) was required for PK activation.