Effects of extracellular pH and hypoxia on the function and development of antigen-specific cytotoxic T lymphocytes.

The major effector cells for cellular adaptive immunity are CD8(+) cytotoxic T lymphocytes (CTLs), which can recognize and kill virus-infected cells and tumor cells. Although CTLs exhibit strong cytolytic activity against target cells in vitro, a number of studies have demonstrated that their function is often impaired within tumors. Nevertheless, CTLs can regain their cytotoxic ability after escaping from the tumor environment, suggesting that the milieu created by tumors may affect the function of CTLs. As for the tumor environment, the patho-physiological situation present in vivo has been shown to differ from in vitro experimental conditions. In particular, low pH and hypoxia are the most important microenvironmental factors within growing tumors. In the present study, to determine the effect of these factors on CTL function in vivo, we examined the cytolytic activity of CTLs against their targets using murine CTL lines and the induction of these cells from memory cells under low pH or hypoxic conditions using antigen-primed spleen cells. The results indicated that both cytotoxic activity and the induction of functional CTLs were markedly inhibited under low pH. In contrast, in hypoxic conditions, although cytotoxic activity was almost unchanged, the induction of CTLs in vitro showed a slight enhancement, which was completely abrogated in low pH conditions. Therefore, antigen-specific CTL functions may be more vulnerable to low pH than to the oxygen concentration in vivo. The findings shown here provide new therapeutic approaches for controlling tumor growth by retaining CTL cytotoxicity through the maintenance of higher pH conditions.

Effects of Dendritic Cell Subset Manipulation on Airway Allergy in a Mouse Model.

BACKGROUND: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR). METHODS: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA. RESULTS: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion. CONCLUSIONS: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo.

Inactivation of tumor-specific CD8⁺ CTLs by tumor-infiltrating tolerogenic dendritic cells.

Cancer immunosurveillance failure is largely attributed to the insufficient activation of tumor-specific class I major histocompatibility complex (MHC) molecule (MHC-I)-restricted CD8⁺ cytotoxic T lymphocytes (CTLs). DEC-205⁺ dendritic cells (DCs), having the ability to cross-present, can present captured tumor antigens on MHC-I alongside costimulatory molecules, inducing the priming and activation of tumor-specific CD8⁺ CTLs. It has been suggested that reduced levels of costimulatory molecules on DCs may be a cause of impaired CTL induction and that some tumors may induce the downregulation of costimulatory molecules on tolerogenic DCs. To examine such possibilities, we established two distinct types of murine hepatoma cell lines, named Hepa1-6-1 and Hepa1-6-2 (derived from Hepa1-6 cells), and confirmed that they display similar antigenicities, as well as identical surface expression of MHC-I. We found that Hepa1-6-1 had the ability to grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and did not prime CD8⁺ CTLs. In contrast, Hepa1-6-2 cells, which display reduced levels of adhesion molecules, such as Intercellular Adhesion Molecule 1 (ICAM-1), failed to grow in vivo and efficiently primed CTLs. Moreover, Hepa1-6-1-derived factors, such as transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP), converted CD11c(high) MHC-II(high) DEC-205⁺ DC subsets into tolerogenic cells, displaying downregulated costimulatory molecules and having impaired cross-presenting capacities. These immunosuppressive tolerogenic DCs appeared to inhibit the induction of tumor-specific CD8⁺ CTLs and suppress their cytotoxic functions within the tumor. Together, the findings presented here provide a new method of cancer immunotherapy using the selective suppression, depletion or alteration of immunosuppressive tolerogenic DCs within tumors.

Induction of rapid apoptosis for class I MHC molecule-restricted CD8(+) HIV-1 gp160-specific murine activated CTLs by free antigenic peptide in vivo.

We have previously reported that the cytotoxic activity of murine CD8(+) CTLs specific for HIV-1 gp160 envelope protein was markedly inhibited in vitro by brief exposure to a free epitope peptide P18-I10 (aa: RGPGRAFVTI) using the epitope-specific CTL line (LINE-IIIB) or a clone (RT-1). We have also shown that recently stimulated P18-I10-specific murine CTLs rapidly fell into apoptosis in vitro after brief exposure to the free epitope peptide. In the present study, we examined whether similar inactivation or apoptosis of recently stimulated CTLs occurred in vivo by exposure to the free epitope peptide using TCR transgenic (Tg-RT-1) mice expressing TCRαß genes of CTL clone RT-1. When the Tg mice were inoculated with recombinant vaccinia virus expressing HIV-1-IIIB gp160 genes followed by injection of P18-I10 epitope peptide, apparent reduction in the number of CTLs determined by flow cytometry using H-2D(d)/P18-I10 pentamer was observed within a few hours after the injection. Most of the H-2D(d)/P18-I10 pentamer-stained cells were positive for Annexin V and apoptosis was confirmed by microscopic analyses. Moreover, when mice were pretreated with immunosuppressive agents, such as cyclosporin A and tacrolimus (FK506), induction of apoptosis by P18-I10 was significantly inhibited and CTL cytotoxicity was maintained. These results suggest that the rapid loss of virus-specific CD8(+) CTLs might occur in vivo through apoptosis in the early stages of viral infection when activated CTLs may encounter viral epitope(s) released from virus-infected cells attacked by CTLs and we can prevent the loss by pretreatment with immunosuppressive agents.

Induction of apoptosis-resistant and TGF-ß-insensitive murine CD8(+) cytotoxic T lymphocytes specific for HIV-1 gp160.

Although TGF-ß and IL-6 would turn CD8(+) T cells to differentiate into non-cytotoxic state, these treated cells were converted to cytolytic phenotypes after re-exposure to their antigenic epitope in vitro. Here, using spleen cells from TCR transgenic mice expressing TCRαß genes of clone RT1 recognizing an epitope peptide (P18-I10: RGPGRAFVTI) of HIV-1 gp160, we generated CD8(+) cytotoxic T lymphocytes (CTLs) activated by re-exposure to P18-I10 after primarily cultured with TGF-ß and IL-6 in vitro to examine their effector function. The CTLs, having strong cytotoxic activity in vitro, were not only resistant to Fas-FasL mediated apoptosis, but also insensitive to the suppression of their cytotoxicity by re-exposure to TGF-ß in vitro. Moreover, adoptive transfer experiments indicated that the CTLs are capable of eliminating recombinant vaccinia virus expressing HIV-1 gp160 in vivo. Taken together, our data suggest that TGF-ß and IL-6 may play pivotal roles in inducing apoptosis-resistant and TGF-ß-insensitive CTLs in vitro.

Production of autoantibodies by murine B-1a cells stimulated with Helicobacter pylori urease through toll-like receptor 2 signaling.

Helicobacter pylori infection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purified H. pylori urease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purified H. pylori urease or H. pylori coated onto plates (referred to hereafter as plate-coated H. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted from H. pylori but was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coated H. pylori stimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matched H. pylori did not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pylori urease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pylori urease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coated H. pylori or H. pylori urease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface of H. pylori will directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders.

One-step simple assay to determine antigen-specific cytotoxic activities by single-color flow cytometry.

Assays for cytotoxicity of CTLs in vivo using a fluorescent-based dye, 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE), have been established and widely used. On the basis of this experience, we applied it to in vitro assay system and established a simpe, highly sensitive flow cytometric assay for CTL activity. In our assay, specific activities of CTLs could be detected by a reduction in sensitive target cell numbers on single-color histogram plot analysis. By using this assay, we could determine the changes in cytotoxic activity by single amino acid substitution within an epitope peptide. Adherent cells were also used as target cells in this assay by treatment with excess EDTA and trypsin reagents after incubation with effector CTLs. Furthermore, when fluorescent calibration beads were used as a control, we could determine the cytotoxicity of CTLs against tumor cells. The results obtained from our assay were almost consistent with those from the conventional ( 51)Cr-release assay.Because our assay uses only a stable non-radioactive reagent, CFSE, this assay is safe, inexpensive and extremely easy. These results indicated that this new assay (FACS-CTL assay) would be sufficiently acceptable alternative to classical (51)Cr-release assay.

Suppression of an already established tumor growing through activated mucosal CTLs induced by oral administration of tumor antigen with cholera toxin.

Priming of CTLs at mucosal sites, where various tumors are originated, seems critical for controlling tumors. In the present study, the effect of the oral administration of OVA plus adjuvant cholera toxin (CT) on the induction of Ag-specific mucosal CTLs as well as their effect on tumor regression was investigated. Although OVA-specific TCRs expressing lymphocytes requiring in vitro restimulation to gain specific cytotoxicity could be detected by OVA peptide-bearing tetramers in both freshly isolated intraepithelial lymphocytes and spleen cells when OVA was orally administered CT, those showing direct cytotoxic activity without requiring in vitro restimulation were dominantly observed in intraepithelial lymphocytes. The magnitude of such direct cytotoxicity at mucosal sites was drastically enhanced after the second oral administration of OVA with intact whole CT but not with its subcomponent, an A subunit (CTA) or a B subunit (CTB). When OVA plus CT were orally administrated to C57BL/6 mice bearing OVA-expressing syngeneic tumor cells, E.G7-OVA, in either gastric tissue or the dermis, tumor growth was significantly suppressed after the second oral treatment; however, s.c. or i.p. injection of OVA plus CT did not show any remarkable suppression. Those mucosal OVA-specific CTLs having direct cytotoxicity expressed CD8alphabeta but not CD8alphaalpha, suggesting that they originated from thymus-educated cells. Moreover, the infiltration of such OVA-specific CD8(+) CTLs was observed in suppressed tumor tissues. These results indicate that the growth of ongoing tumor cells can be suppressed by activated CD8alphabeta CTLs with tumor-specific cytotoxicity via an orally administered tumor Ag with a suitable mucosal adjuvant.

Molecular analysis of TCR and peptide/MHC interaction using P18-I10-derived peptides with a single D-amino acid substitution.

For the structural analysis of T-cell receptor (TCR) and peptide/MHC interaction, a series of peptides with a single amino acid substitution by a corresponding D-amino acid, having the same weight, size, and charge, within P18-I10 (aa318-327: RGPGRAFVTI), an immunodominant epitope of HIV-1 IIIB envelope glycoprotein, restricted by the H-2Dd class I MHC molecule, has been synthesized. Using those peptides, we have observed that the replacement at positions 324F, 325V, 326T, and 327I with each corresponding D-amino acid induced marked reduction of the potency to sensitize targets for P18-I10-specific murine CD8+ cytotoxic T lymphocytes (CTLs), LINE-IIIB, recognition. To analyze further the role of amino acid at position 325, the most critical site for determining epitope specificity, we have developed a CTL line [LINE-IIIB(325D)] and its offspring clones specific for the epitope I-10(325v) having a D-valine (v) at position 325. Taking advantage of two distinct sets of CD8+ CTLs restricted by the same Dd, three-dimensional structural analysis on TCR and peptide/MHC complexes by molecular modeling was performed, which indicates that the critical amino acids within the TCRs for interacting with 325V or 325v appear to belong to the complementarity-determining region 1 but not to the complementarity-determining region 3 of Vbeta chain.

Resistance to viral infection by intraepithelial lymphocytes in HIV-1 P18-I10-specific T-cell receptor transgenic mice.

For the analysis of mucosal immunity to HIV-1, we have recently established a line of transgenic (Tg) mice expressing the TCRalpha and TCRbeta genes of the murine CTL clone RT1 specific for P18-I10 (RGPGRAFVTI), an immunodominant gp160 envelope-derived epitope of IIIB isolate, restricted by the H-2D(d) MHC-I molecule. Here we examine those cells bearing specific TCR among the intraepithelial lymphocytes (IELs), with flow cytometric analysis using H-2D(d)/P18-I10 tetramers. We observed three distinct CD3(+), tetramer positive populations among the IELs: extra-thymic CD8alphabeta(+), alphabetaTCR T-cells; CD8 alphaalpha+, gammadeltaTCR T-cells; and thymus-derived CD8alphabeta+, alphabetaTCR T-cells. Challenge of these Tg mice with P18-I10 encoded by a vaccinia virus vector, either intrarectally (i.r.) or intraperitoneally (i.p.), revealed that the intraepithelial compartment seems to be a major site for prevention of the spread of viral infection. Such immunity appears due to the thymus-derived, CD8alphabeta+ antigen-specific CTLs together with CD8alphaalpha+ gammadelta cells, which regulate virus spread. This model system for studying CTL based immunity at mucosal sites should prove helpful in developing rational approaches for HIV control.

Polyriboinosinic polyribocytidylic acid [poly(I:C)]/TLR3 signaling allows class I processing of exogenous protein and induction of HIV-specific CD8+ cytotoxic T lymphocytes.

In the case of viral infection, various viral proteins and genetic components are disseminated in the body. The former viral proteins may be captured by immature dendritic cells (DC) and the latter genetic components may stimulate the antigen-loading DC to maturate via specific Toll-like receptors (TLR), leading to the establishment of virus-specific cellular immunity; in particular, cytotoxic T lymphocytes (CTL) that control intracellular virions. Polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural genetic product from a variety of viruses during replication, has recently been identified as one of the critical stimuli for TLR3. Based on these observations, we speculated that stimulation of TLR3 with poly(I:C) might drive the direction of acquired/adaptive immunity to the cellular arm. Indeed, when BALB/c mice were immunized with purified recombinant HIV-1 envelope gp120 or influenza hemagglutinin (HA) protein together with poly(I:C), epitope-specific CD8(+) class I MHC molecule-restricted CTL were primed from naive CD8(+) T cells in vivo. In contrast, when the same proteins were immunized with lipopolysaccharide, a stimulant of TLR4, specific CTL were not primed at all. Moreover, we show here that immature DC could present processed antigen from captured purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that we are able to manipulate the direction of acquired/adaptive effector immune responses using an appropriate stimuli and the findings presented in this paper will offer a new therapeutic strategy using poly(I:C) administration for priming antigen-specific CD8(+) CTL with purified viral protein in vivo.

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances.

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8(+) cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D(b) class I MHC-restricted antigens and CD1d-restricted substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4(-)CD8(-) and CD8(+) NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4(-)CD8(-), but not CD8(+) NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes.

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.

Induction of CD4+ murine natural killer T-like cells by immunization with syngeneic thymoma expressing embryonic alpha-fetoprotein.

In embryo, before the establishment of acquired immunity, a variety of embryonic antigens like alpha-fetoprotein (AFP) are produced and secreted in the sera, which rapidly disappear after the birth. Such embryonic antigens sometimes reappear from various tumor cells and decrease in the case of remission, indicating embryonic antigens may alert immune system to control tumors. In the present study, to examine the evoked immune responses against the tumors expressing embryonic antigen, we administered AFP-gene-transfected EL4 cells into syngeneic C57BL/6 mice and established a killer line against the tumor cells. To our surprise, the killer line was CD4+ NK1.1+, natural killer T (NKT)-like cells and eliminated not only AFP-expressing EL4 but YAC-1 cells. Moreover, the established line uniformly expressed Vbeta11 and secreted IL-4, IL-10, IL-13, and IFN-gamma. In vivo inoculation of the line markedly reduced the tumor growth in SCID mice, suggesting novelty of the NKT-like line for tumor surveillance.

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.

Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system.

The CDR3 regions of T cell receptor (TCR)-alpha and -beta chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-alpha and -beta to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-alpha or -beta chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-beta or -alpha chains. Surprisingly, one-third of such TCR-beta containing random CDR3 beta from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-alpha. A similar dominant function of TCR-alpha in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus-specific TCR. Subsequently, we generated TCR-alpha and/or -beta transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-alpha Tg generated CTLs with heterogeneous TCR-beta, whereas TCR-beta Tg-induced CTLs bearing a single TCR-alpha. These findings of Ag recognition with minimum involvement of CDR3 beta expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-alpha chain in determining the preimmune repertoire of Ag-specific TCR.