Formation of supramolecular activation clusters on fresh ex vivo CD8+ T cells after engagement of the T cell antigen receptor and CD8 by antigen-presenting cells.

Upon productive interaction of CD4 T cells with antigen-presenting cells (APCs), receptors and intracellular proteins translocate and form spatially segregated supramolecular activation clusters (SMACs). It is not known whether SMACs are required for CD8 T cell activation. CD8 T cells, unlike CD4 T cells, can be activated by a single peptide-MHC molecule, or by purified monovalent recombinant peptide-MHC molecules. We studied, by three-dimensional digital microscopy, cell conjugates of fresh ex vivo CD8 T cells (obtained from OT-1 mice, which are transgenic for T cell antigen receptor reactive with the complex of H-2K(b) and the ovalbumin octapeptide SIINFEKL) and peptide-pulsed APCs. Remarkably, even in T cell:APC conjugates that were formed in the presence of the lowest concentration of peptide that was sufficient to elicit T cell proliferation and IFN-gamma production; the theta isoform of protein kinase C was clustered in a central SMAC, and lymphocyte function-associated antigen 1 and talin were clustered in the peripheral SMAC. Conjugation of T cells to APCs that were pulsed with concentrations of peptide smaller than that required to activate T cells was greatly reduced, and SMACs were not formed at all. APCs expressing mutant H-2K(b) (Lys(227)) molecules that do not bind CD8 were unable to form stable conjugates with these T cells, even at high peptide concentrations. Thus, although CD8 and CD4 T cells may display different sensitivity to the concentration and oligomerization of surface receptors, SMACs are formed and seem to be required functionally in both cell types. However, unlike CD4 T cells, which can form SMACs without CD4, CD8 T cells from OT-1 transgenic mice depend on their coreceptor, CD8, for the proper formation of SMACs.

Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL).

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-alpha and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-alpha and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-alpha- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.

Immunization with f-Met peptides induces immune reactivity against Mycobacterium tuberculosis.

OBJECTIVE: To determine whether synthetic peptides containing an amino terminal formyl-methionine residue and corresponding to the sequence of several proteins produced by Mycobacterium tuberculosis, would elicit an immune response in mice. DESIGN: Peptides corresponding to the amino termini of 8 M. tuberculosis proteins and initiating with formyl methionine residues were synthesized. The ability of these peptides to bind to the mouse non-classical MHC class I molecule H-2M3a was determined by flow microfluorimetry. These peptides were used to pulse dendritic cells that were then injected into normal mice. These mice were subsequently challenged with aerosolized M. tuberculosis and, 30 days later, the number of viable bacteria in the lungs was determined. RESULTS: Four of the 8 synthetic peptides bound to H-2M3a and stabilized its expression on the cell surface. Injection of mice with dendritic cells pulsed with H-2M3a binding peptides elicited non-MHC restricted cytotoxic T lymphocytes that killed peptide pulsed target cells and macrophages infected with M. tuberculosis. Immunization of mice with syngeneic dendritic cells pulsed in vitro with 2 of these peptides led to retardation of the growth of M. tuberculosis following aerosol challenge. CONCLUSION: Peptides that bind to non-polymorphic class I molecules can elicit immune reactivity directed towards M. tuberculosis.

Intravenous cytokine gene delivery by lipid-DNA complexes controls the growth of established lung metastases.

Local expression of cytokine genes by ex vivo transfection or intratumoral gene delivery can control the growth of cutaneous tumors. However, control of tumor metastases by conventional nonviral gene therapy approaches is more difficult. Intravenous injection of lipid-DNA complexes containing noncoding plasmid DNA can significantly inhibit the growth of early metastatic lung tumors. Therefore, we hypothesized that delivery of a cytokine gene by lipid-plasmid DNA complexes could induce even greater antitumor activity in mice with established lung metastases. The effectiveness of treatment with lipid-DNA complexes containing the IL-2 or IL-12 gene was compared with the effectiveness of treatment with complexes containing noncoding (empty vector) DNA. Treatment effects were evaluated in mice with either early (day 3) or late (day 6) established lung tumors. Lung tumor burdens and local intrapulmonary immune responses were assessed. Treatment with either noncoding plasmid DNA or with the IL-2 or IL-12 gene significantly inhibited the growth of early tumors. However, only treatment with the IL-2 or IL-12 gene induced a significant reduction in lung tumor burden in mice with more advanced metastases. Furthermore, the reduction in tumor burden was substantially greater than that achieved by treatment with recombinant cytokines. Treatment with the IL-2 or IL-12 gene was accompanied by increased numbers of NK cells and CD8+ T cells within lung tissues, increased cytotoxic activity, and increased local production of IFN-gamma by lung tissues, compared with treatment with noncoding DNA. Thus, cytokine gene delivery to the lungs by means of intravenously administered lipid-DNA complexes may be an effective method of controlling lung tumor metastases.

Systemic and local interferon gamma gene delivery to the lungs for treatment of allergen-induced airway hyperresponsiveness in mice.

Allergen-induced airway hyperresponsiveness, an animal model of asthma in humans, may respond to immunotherapy with Th1 cytokines. For example, local administration of recombinant IL-12 or IFN-gamma, or intratracheal delivery of the genes for these cytokines, has been shown to reduce the severity of allergen-induced airway hyperresponsiveness (AHR) in rodent models. We reasoned that systemic cytokine gene delivery to the lungs by intravenous injection of lipid-DNA complexes might also be an effective approach to treatment of allergen-induced AHR. Therefore, the effects of either systemic or local pulmonary IFN-gamma gene delivery were evaluated in mice with allergen-induced AHR. The effects of treatment on AHR, airway eosinophilia and cytokine production, and serum IgE concentrations were evaluated in mice that were first sensitized to ovalbumin and then subjected to aerosol ovalbumin challenge. Intravenous IFN-gamma gene delivery significantly inhibited development of AHR and airway eosinophilia and decreased serum IgE levels, compared with control mice or mice treated with noncoding DNA. Intratracheal IFN-gamma gene delivery also significantly inhibited AHR and airway eosinophilia, but did not affect serum IgE levels. Treatment with recombinant IFN-gamma was much less effective than IFN-gamma gene delivery by either route. We conclude that either systemic or local pulmonary delivery of a Th1 cytokine gene such as IFN-gamma may be an effective approach for treatment of allergen-induced asthma.

Lipid-DNA complexes induce potent activation of innate immune responses and antitumor activity when administered intravenously.

Cationic lipid-DNA complexes (CLDC) are reported to be safe and effective for systemic gene delivery, particularly to the lungs. However, we observed that i.v. injection of CLDC induced immunologic effects not previously reported. We found that even very low doses of CLDC administered i.v. induced marked systemic immune activation. This response included strong up-regulation of CD69 expression on multiple cell types and systemic release of high levels of Th1 cytokines, from both lung and spleen mononuclear cells. CLDC were much more potent immune activators on a per weight basis than either LPS or poly(I:C). The remarkable potency of CLDC appeared to result from enhancement of the immune stimulatory properties of DNA, since cationic lipids alone were without immune stimulatory activity. Systemic treatment with CLDC controlled tumor growth and significantly prolonged survival times in mice with metastatic pulmonary tumors. NK cells accumulated to high levels in the lungs of CLDC-treated mice, were functionally activated, and released high levels of IFN-gamma. The antitumor activity induced by CLDC injection was dependent on both NK cells and IFN-gamma. Thus, DNA complexed to cationic liposomes becomes highly immunostimulatory and capable of inducing strong antitumor activity when administered systemically.

Antigen secreted from noncytosolic Listeria monocytogenes is processed by the classical MHC class I processing pathway.

Intracellular bacteria can reside in a vacuolar compartment, or they can escape the vacuole and become free living in the cytoplasm. The presentation of Ag by class I MHC molecules has been defined primarily for Ag present in the cytoplasm. It was therefore thought that Ags from bacteria that remain in a vacuole would not be presented by MHC class I molecules. Although some studies have provided data to support this idea, it is not necessarily true for all intracellular bacteria. For example, we have previously demonstrated that an epitope from the p60 protein secreted by LLO- Listeria monocytogenes, which does not reside in the cytoplasm, can be presented by MHC class I molecules to a T cell clone specific for the epitope, p60217-225. We have further examined the route by which Ag secreted by LLO- L. monocytogenes is presented by MHC class I molecules. Using pharmacological inhibitors, we demonstrate that MHC class I presentation of the p60 epitope derived from by LLO- L. monocytogenes requires phagolysosome fusion and processing by the proteasome. Lysosomal cathepsins, however, are not required for processing of the p60 epitope. Similarly, processing of the AttM epitope, secreted by LLO- L. monocytogenes and presented by H2-M3, also requires phagolysosome fusion and cleavage by the proteasome. Thus, p60 and AttM secreted by LLO- L. monocytogenes are processed via the classical class I pathway for presentation by MHC class I molecules.

Polymerase chain reaction for the detection of Toxoplasma gondii within aqueous humor of experimentally-inoculated cats.

The purpose of this study was to determine the temporal appearance of T. gondii in aqueous humor of cats orally inoculated with T. gondii using polymerase chain reaction (PCR) for the detection of the B1 gene. Serum and aqueous humor were collected from five SPF cats prior to oral inoculation with T. gondii and days 7, 14, 21, 28, 42, 84, 140, 147, 154, 161, 168, and 182 after inoculation. Cats were inoculated orally with T. gondii tissue cysts on day 0 and day 140. T. gondii-specific IgM and IgG were measured in serum and aqueous humor from the cats at each sample data. T. gondii B1 gene PCR was performed on all the aqueous humor samples and the amplified DNA was detected by Southern blotting. Chorioretinitis developed in three out of the five cats, but anterior uveitis was not detected. All cats developed T. gondii-specific IgG titers in serum, and had T. gondii-specific IgG C-values > 1 in both eyes at varying times during the study. T. gondii was detected by PCR and Southern blotting in aqueous humor in both eyes of all cats at times varying from days 14-84 after primary inoculation and days 14-42 after challenge inoculation.

In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma.

In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.

Alloreactive T cells that do not require TCR and CD8 coengagement are present in naive mice and contribute to graft rejection.

Class I alloreactive CTL populations have been defined as either CD8 dependent or CD8 independent, based upon their ability to kill target cells in the presence of Ab to CD8. The CD8-dependent population uses CD8 in a coreceptor role with the TCR, and mutations in the class I molecule that destroy the CD8 binding site abrogate CTL killing, even if the target cell expresses other allelic forms of class I molecules with an intact binding site for CD8. The CD8-independent population apparently does not require CD8, as Ab to CD8 has no effect on the ability of these cells to kill appropriate target cells. We have isolated a third population of CTL that is inhibited by the addition of CD8 Ab yet can kill target cells that express the alloantigenic molecule incapable of binding CD8, provided that the target cells also express non antigenic class I molecules that contain an intact binding site for CD8. We refer to these cells as CD8 bystander-dependent CTL. Many (10 of 12) of these CTL were able to kill H-2Kb-expressing transfectants of T2 cells, consistent with the idea that they recognize a peptide-independent determinant that may be expressed at a high density on the cell surface. These CD8 bystander-dependent CTL are only readily detectable in vitro when spleen cells from mice primed in vivo with a skin graft are used.

Cytokine-induced survival of activated T cells in vitro and in vivo.

Many antigen-specific T cells die after exposure to antigen in animals. These cells also die if they are isolated from animals shortly after activation and cultured. Various cytokines were tested for their ability to interfere with this in vitro death. Surprisingly, tumor necrosis factor alpha and other inflammatory cytokines did not prevent the in vitro death of activated T cells, even though these cytokines do prevent activated T cell death in animals. Therefore, the inflammatory cytokines probably act on T cells in vivo via an intermediary factor. Four cytokines, interleukin (IL)-2, IL-4, IL-7, and IL-15, did prevent activated T cell death in vitro, with IL-4 and IL-15 more effective than IL-2 or IL-7. These cytokines share a component of their receptors, the common gamma chain, gammac. Therefore, their collective ability to protect activated T cells from death may be mediated by signals involving gammac. To assess their activity in vivo, two of the cytokines, IL-2 and IL-4, were expressed in animals at local sites of superantigen responses. Both cytokines increased the numbers of T cells found at the local sites 14 days later. Interleukin 4 was more effective than IL-2, even though IL-2 stimulates T cell proliferation better than IL-4. This result suggested that IL-4 and related cytokines can promote T cell survival in vivo as well as in vitro. The ability of these cytokines to prevent the death of activated T cells may be important at certain stages of immune responses in animals.

Mechanisms of CD8beta-mediated T cell response enhancement: interaction with MHC class I/beta2-microglobulin and functional coupling to TCR/CD3.

CD8beta expression results in enhanced IL-2 production and/or altered specificity in allogeneic MHC class I-restricted T cell hybridomas. Expression of chimeric CD8beta-alpha molecules (extracellular CD8beta, transmembrane and cytoplasmic CD8alpha) also results in enhancement of T hybridoma responses to alloantigen, suggesting that at least part of CD8beta's ability to influence responses similar to those of mature CD8+ T cells is mediated by its extracellular domain. Current data suggest that CD8beta-mediated response enhancement proceeds through mechanisms similar to those mediated by CD8alpha, i.e., interacting with MHC class I and stabilizing CD8-associated Lck activity. In this study we present evidence that the extracellular portion of CD8beta is capable of independent interaction with MHC class I/beta2m dimers in the absence of CD8alpha. In addition, CD8beta may enhance interaction with MHC class I/beta2m when associated with CD8alpha. We also present evidence from T hybridoma responses suggesting that the extracellular portion of CD8beta is uniquely capable of efficient interaction with the TCR/CD3 complex and may couple the TCR/CD3 complex to other surface components capable of enhancing TCR-mediated signals. This represents the first evidence that a critical coreceptor function can be preferentially associated with the CD8beta subunit.

Expression of bacterial superantigen genes in mice induces localized mononuclear cell inflammatory responses.

Bacterial superantigens are potent T cell activators, and superantigen proteins have been injected into mice and other animals to study T cell responses in vivo. When superantigen proteins are injected, however, the T cell stimulatory effects cannot be confined to specific tissues. Therefore, to target superantigen expression to specific tissues, we used gene transfer techniques to express bacterial superantigen genes in mammalian cells in vitro and in tissues in vivo. Murine, human, and canine cells transfected with superantigen genes in vitro all produced superantigen proteins both intracellularly and extracellularly, as assessed by bioassay, immunocytochemistry, and antigen ELISA. Superantigens produced by transfected eukaryotic cells retained their biologic specificity for T cell receptor binding. Intramuscular injection of superantigen plasmid DNA in vivo induced an intense intramuscular mononuclear cell infiltrate, an effect that could not be reproduced by intramuscular injection of superantigen protein. Intradermal and intravenous injection of superantigen DNA induced cutaneous and intrapulmonary mononuclear cell inflammatory responses, respectively. Thus, superantigen genes can be expressed by mammalian cells in vivo. Superantigen gene therapy represents a novel method of targeting localized T cell inflammatory reactions, with potential application to treatment of cancer and certain infectious diseases.

Peptide-independent recognition by alloreactive cytotoxic T lymphocytes (CTL).

We have isolated several H-2K(b)-alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2K(b). These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2K(b) molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2K(b) on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2(b)) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2K(bm8) molecule. These findings suggested that the clones recognized determinants on H-2K(b) that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2K(b) produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2K(b) molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2-incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent.

Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines.

Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with noninvasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57Bl/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.

Polymerase chain reaction for the detection of Toxoplasma gondii in aqueous humor of cats.

OBJECTIVES: To develop Toxoplasma gondii B1 gene polymerase chain reaction (PCR) for use with aqueous humor of cats, and to report PCR and antibody detection results in naturally exposed cats with and without uveitis. SAMPLE POPULATION: Serum and aqueous humor samples from client-owned, healthy cats (n = 23) and client-owned cats with uveitis (n = 43). PROCEDURE: T gondii-specific IgM and IgG were measured in serum and aqueous humor from all cats. The Goldman-Witmer coefficient for ocular antibody production was calculated for cats positive for T gondii-specific IgM or IgG in aqueous humor. Aqueous humor from all cats was assessed by the B1 gene PCR. RESULTS: T gondii was detected in aqueous humor by PCR from 2 of 23 (8.7%) healthy cats and 8 of 43 (18.6%) cats with uveitis. T gondii-specific IgM in either serum or aqueous humor was detected in 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor. All cats with uveitis and T gondii in aqueous humor had anterior segment disease. In 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor, ocular production of T gondii antibodies was not detected. T gondii was not detected in aqueous humor from 14 of 17 (82.4%) cats with ocular production of T gondii-specific antibody. CONCLUSIONS: The presence of T gondii in aqueous humor may correlate to clinical disease in some, but not all, cats. CLINICAL RELEVANCE: T gondii-specific aqueous humor antibody tests and PCR should be used together to aid in the diagnosis of ocular toxoplasmosis in cats.

Glu227-->Lys substitution in the acidic loop of major histocompatibility complex class I alpha 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions.

Cytotoxic T lymphocyte (CTL) activation requires specific T cell receptor (TCR)-class I major histocompatibility complex (MHC) antigen complex interactions as well as the participation of coreceptor or accessory molecules on the surface of CTL. CD8 can serve as a coreceptor in that it binds to the same MHC class I molecules as the TCR to facilitate efficient TCR signaling. In addition, CD8 can be "activated" by TCR stimulation to bind to class I molecules with high avidity, including class I not recognized by the TCR as antigenic complexes (non-antigen [Ag] class I), to augment CTL responses and thus serve an accessory molecule function. A Glu/Asp227-->Lys substitution in the class I alpha 3 domain acidic loop abrogates lysis of target cells expressing these mutant molecules by alloreactive CD8-dependent CTL. Lack of response is attributed to the destruction of the CD8 binding site in the alpha 3 domain which is likely to disrupt CD8 coreceptor function. The relative importance of the class I alpha 3 domain acidic loop Glu227 in coreceptor as opposed to accessory functions of CD8 is unclear. To address this issue, we examined CTL adhesion and degranulation in response to immobilized class I-peptide complexes formed in vitro from antigenic peptides and purified class I molecules containing wild-type or Glu227-->Lys substituted alpha 3 domains. The alpha 3 domain mutant class I-peptide complexes were bound by CTL and triggered degranulation, however to much lower levels than wild-type class I-peptide complexes. In further experiments, it is directly demonstrated that the alpha 3 domain mutant class I molecules, which lack the Glu227 CD8 binding site, still serve as TCR-activated, avidity-enhanced CD8 accessory ligands. However, mutant class I peptide Ag complexes failed to effectively serve as CD8 coreceptor ligands to initiate TCR-dependent signals required to induce avidity-enhanced CD8 binding to coimmobilized non-Ag class I molecules. Thus the Glu227-->Lys mutation effectively distinguishes CD8 coreceptor and avidity-enhanced CD8 accessory functions.

E1A oncogene expression in target cells induces cytolytic susceptibility at a post-recognition stage in the interaction with killer lymphocytes.

E1A oncogene expression increases the susceptibility of cells from several species to lysis by natural killer lymphocytes (NK cells). We asked whether this E1A-induced cellular phenotypic conversion is specific for NK cell recognition interactions with target cells or whether it results from an E1A effect that is mediated independently of recognition. E1A-positive and E1A-negative cell pairs were compared for cytolytic susceptibility to other types of killer cells that use recognition mechanisms different from those of NK cells. E1A-positive, NK-susceptible target cells were also preferentially lysed by cytotoxic T lymphocytes (CTL) that recognize only foreign MHC molecules, lymphokine-activated T cells that lack recognition specificity, and CTL whose conventional recognition mechanisms were bypassed by lectin treatment of target cells. E1A expression increased cellular susceptibility to both major mechanisms of killer cell lysis-perforin/granzyme lysis and Fas-dependent lysis. Furthermore, anti-Fas antibody lysed E1A-positive, but not E1A-negative, cells expressing comparable levels of cell surface Fas antigen. These results indicate that a major mechanism by which E1A induces cellular susceptibility to lysis involves a stage in the interaction of killer cells with their targets that follows and is independent of cell surface recognition.

Peptide epitopes from noncytosolic Listeria monocytogenes can be presented by major histocompatibility complex class I molecules.

Listeria monocytogenes is an intracellular pathogen which escapes the phagosome and resides in the cytosol of the host cell. Using Listeria innocua and a mutant strain of L. monocytogenes (listeriolysin O negative), which do not enter the cytosol of the host cell, we demonstrate class I presentation of an epitope of p60, a protein secreted by L. monocytogenes, to a class I-restricted CD8+ cytotoxic T lymphocyte clone.

Requirement for CD8+ T cells in the development of airway hyperresponsiveness in a marine model of airway sensitization.

To study the role of CD8+ T cells in allergic sensitization, we examined the effects of in vivo depletion of CD8+ T cells prior to sensitization on IgE production, immediate type cutaneous hypersensitivity and development of altered airway responsiveness. BALB/c mice were thymectomized and treated with anti-CD8 antibody resulting in depletion of CD8+ T cells (<1%) in spleen and lymphoid tissues. In these mice, sensitization to ovalbumin (OVA) via the airways still resulted in IgE anti-OVA responses and immediate cutaneous reactions to OVA, but the animals were unable to develop airway hyperresponsiveness, eosinophil infiltration of the lung parenchyma, or IL-5 production in the local lymph nodes of the airway. Transfer of CD8+ T cells from naive animals during sensitization (on day 8 of the 10-d protocol) fully restored the ability to develop airway hyperresponsiveness and this was accompanied by IL-5 production and eosinophil accumulation in the lung. These data indicate a critical role for CD8+ T cells in the production of IL-5 and the development of altered airway responsiveness after antigen sensitization through the airways.