Mechanism of glucocorticoid action on murine natural killer cell activity.

With the use of an in vitro model system, the mode of action of glucocorticoids on murine natural killer (NK) cell-mediated cytotoxicity of tumor cells was investigated. Of the steroids tested, only the glucocorticoids notably suppressed NK activity. Glucocorticoids were not toxic to the NK effector cell since inhibitors of protein synthesis protected NK activity from the suppressive action of glucocorticoids. Glucocorticoid-treated C57BL/6J spleen cells, although suppressed in NK activity, were unable to suppress the NK activity of normal syngeneic spleen cell cultures. Similarly, the supernatants of glucocorticoid-treated cultures were also unable to suppress normal NK activity. Thus a role for suppressor cell activity or soluble suppressive factors was excluded. Results of analyses of the NK activity of Percoll-fractionated glucocorticoid-treated C3H/HeN (nu/nu) spleen cells at the single-cell level demonstrated that NK effector cells could efficiently bind to YAC-1 lymphoma cells but were incapable of inducing cytolysis. Moreover, the production of NK cytotoxicity factor(s) in tumor cell-stimulated nude mouse spleen cell cultures was severely depressed after glucocorticoid treatment. The results of these studies suggest that glucocorticoids suppress murine NK activity by acting directly on the NK effector cells, possibly by inhibiting the formation or release of specific effector molecules that are cytotoxic to NK-sensitive tumor cells.

Direct suppression of natural killer activity in human peripheral blood leukocyte cultures by glucocorticoids and its modulation by interferon.

In vitro treatment of human peripheral blood leukocytes for 18 to 24 hr with physiological concentrations of glucocorticoids resulted in a marked decrease (up to 90%) in natural killer (NK) activity. The effect on NK activity was both dose and time dependent and was specific for glucocorticoids. Glucocorticoids had no effect when added directly to the 4-hr 51Cr release cytotoxicity assay, nor did they alter the susceptibility of K562 cells to NK-mediated cytolysis. Glucocorticoid-induced inhibition occurred in Percoll-fractionated peripheral blood leukocytes enriched for NK activity. Viabilities of steroid-treated and untreated cultures were similar. Mixing experiments failed to demonstrate the involvement of suppressor activity in the inhibition. Purified cloned human leukocyte interferon subtype A and inducers of interferon enhanced NK activity in the presence of glucocorticoid, although the levels of enhancement were lower than those produced by these agents in the absence of the steroid. Thus, glucocorticoids appear to suppress human NK activity by interacting directly with the NK effector cell, and our results obtained with physiological concentrations of these steroids suggest that they may play an important role in regulating NK activity in vivo. Additionally, these findings suggest a possible means for overriding this immunosuppressive side effect of glucocorticoid therapy by simultaneous treatment with interferon.

Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients.

The functional characteristics of CD8+ T cells specific for melanoma antigens (MAs) have often been defined after in vitro culture using nonprofessional antigen-presenting cells. We have examined CD8+ T-cell immunity to MAs and a viral antigen (influenza) in uncultured T cells of healthy donors and melanoma patients using autologous, mature, monocyte-derived dendritic cells (DCs) pulsed with peptide antigens and viral vectors. Antigen-specific IFN-gamma-producing T cells reactive with HLA-A*0201-restricted peptides from four melanoma antigens (MelanA/MART-1, MAGE-3, tyrosinase, and gp100) were detected only at low frequencies (<30 per 2 x 10(5) peripheral blood mononuclear cells for each of the MAs) from HLA-A2.1-positive healthy donors (n = 12) and patients with stages III/IV melanoma (n = 8). Detection of MA-specific, but not influenza matrix peptide (Flu-MP)-specific, T cells required a high concentration (10 microg/ml) of the peptide in this assay. Furthermore, these T cells did not recognize endogenously processed antigen on tumor cell lines or cells infected with viral vectors capable of expressing MAs. The use of autologous, mature DCs led to a significant increase in the number of Flu-MP, but not MA-specific, T cells in 16-h ELISPOT assays for both melanoma patients and healthy donors. In 1-week cocultures with DCs pulsed with 10 microg/ml peptide, MelanA/MART-1-specific T cells did not readily proliferate or differentiate into lytic effectors, in contrast to strong influenza-specific lytic responses. Therefore, despite distinct memory responses to influenza antigens, melanoma patients and healthy controls have a paucity of MA-reactive memory T cells, failing to rapidly generate IFN-gamma-secreting lytic effectors in short-term assays, even when stimulated by DCs.

A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals.

OBJECTIVES: HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay. METHODS: Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase. RESULTS: In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays. CONCLUSIONS: Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.

Induction of immune responses to HIV-1 by canarypox virus (ALVAC) HIV-1 and gp120 SF-2 recombinant vaccines in uninfected volunteers. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVE: To determine the ability of live attenuated canarypox virus expressing HIV antigens to induce CD8+ cytotoxic T-cell responses and to prime for neutralizing antibody responses to boosting with purified recombinant gp120 subunit vaccine. DESIGN: A prospective, double-blind, randomized, immunogenicity and safety study was conducted in healthy adults at low risk for acquiring HIV infection and who were seronegative for HIV. METHODS: CD8+ cytotoxic T-cells directed against Env or Gag expressing target cells were measured after live recombinant canarypox-HIV-1 vaccine priming (vaccine given at days 0, 7, 14 and 21). Neutralizing antibodies were measured after subunit boosting (vaccine given at days 28 and 84). RESULTS: CD8+ CTL were induced in 64% of volunteers by the live recombinant canarypox-HIV-1 vaccine. All volunteers who received two doses of subunit vaccine after live recombinant canarypox priming developed neutralizing antibodies directed against laboratory strains of HIV-1 and seven out of eight volunteers tested developed neutralizing antibodies to the primary isolate, BZ167, but to none of eight other primary isolates. Unprimed controls had low or absent neutralizing antibodies after two doses of subunit vaccine. CONCLUSIONS: The live canarypox vector was safe, stimulated cytotoxic T-cells and primed for a vigorous neutralizing antibody response upon boosting with subunit gp120 vaccine. This vaccine combination should be evaluated further for inducing protection against HIV infection.

Dietary nucleotide effects upon immune function in infants.

Nucleotide (NT) nitrogen, a component of nonprotein nitrogen, accounts for approximately 0.1% to 0.15% of the total nitrogen content of human milk. The results of studies in animals indicate that dietary NTs may be required for maintenance of normal immune function. Thirty-seven healthy term infants were either breast-fed (n = 9) or fed SMA formula supplemented with 33 mg of NTs per liter (n = 13, NT+) or standard SMA formula (n = 15; NT-). At 2 months of age, natural killer cell percent cytotoxicity was significantly higher in the breast-fed and NT+ groups compared with the NT- group (41.7 +/- 4.7, 32.2 +/- 3.4, 21.7 +/- 2.2%, respectively). Interleukin-2 production by stimulated mononuclear cells was higher in the NT+ compared with the NT- group at 2 months of age (0.90 +/- 0.28 U/mL, 0.27 +/- 0.11 U/mL, respectively); neither formula-fed group differed significantly from the breast-fed group. Rate of growth and incidence and severity of infections did not differ significantly among dietary groups. Nucleotides may be a component of human milk that contributes to the enhanced immunity of the breast-fed infant.

Dietary nucleotide effects upon murine natural killer cell activity and macrophage activation.

Weanling mice fed chow or chow plus water supplemented with 3.5 mg of nucleotides per 100 ml of water for 6 weeks exhibited increased natural killer cell (NK) activity and lower macrophage activation compared to mice fed chow plus nonsupplemented water. In a dose-response study, NK activity, macrophage activation and spleen weight (as a percentage of body weight) were higher in mice fed up to 0.035% w/w nucleotides, however macrophage activation was decreased by feeding over 0.35% w/w compared to those receiving basal purified diet (BPD). Nucleotides in human milk may affect the immune response in breast-fed infants.

Induction of cytotoxic T lymphocytes by recombinant canarypox (ALVAC) and attenuated vaccinia (NYVAC) viruses expressing the HIV-1 envelope glycoprotein.

Successful immunization against many viruses, including retroviruses such as HIV-1, is thought to depend upon the roles of both antibody and cytotoxic T-lymphocyte responses. With safety a major concern, we developed two poxvirus recombinants expressing the envelope glycoprotein of HIV-1 IIIB. Canarypox (ALVaC), which is not known to replicate in mammalian cells, and a highly attenuated vaccinia (NYVAC) virus deleted of 18 open reading frames associated with virulence and host range were used as vectors. Upon inoculation into BALB/c mice, both the ALVAC and NYVAC recombinants were capable of inducing antibody responses to HIV gp120 and provoking remarkable levels of primary and memory Thy1.2+, CD4-, CD8+ cytotoxic T-lymphocyte responses to the hypervariable V3 loop of the HIV-1 envelope glycoprotein.

Safety and immunogenicity of recombinants based on the genetically-engineered vaccinia strain, NYVAC.

NYVAC-based recombinants expressing pertinent immunogens from equine influenza virus (EIV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and human immunodeficiency virus (HIV) were used to evaluate the safety and immunogenicity of this vector. Administration of either NYVAC recombinants or parenteral virus to mice, horses and swine was well tolerated with no notable local or systemic reactivities. Further, despite a highly attenuated phenotype, NYVAC was found to function effectively as an immunization vehicle capable of eliciting both humoral and cell-mediated immune responses.

Dimethyl sulfoxide-induced inhibition of the immunosuppressive activity of cultured Friend erythroleukemia cells.

Two Friend leukemia virus-induced tumor cell lines lost their immunosuppressive properties in vitro when treated with dimethyl sulfoxide (DMSO), a known cellular differentiation agent. Incubation of the cell lines, GM 979 and GM 86, with DMSO for 4 days or longer, inhibited their ability to suppress the antibody forming capacity of normal murine spleen cells immunized in vitro with sheep red blood cells. Suppression of the inhibitory capability of the tumor cell lines by DMSO was time dependent. Three days incubation caused only slight, if any, inhibition, while a shorter period of treatment had no effect. Inhibition of the immunosuppressive properties of the tumor cell lines was not due to a decrease in tumor cell viability. The development of previously reported metabolic alterations in the treated cells, such as increased hemoglobin synthesis and other physicochemical alterations, paralleled cellular differentiation, and loss of immunosuppressive properties.

Mature dendritic cells infected with canarypox virus elicit strong anti-human immunodeficiency virus CD8+ and CD4+ T-cell responses from chronically infected individuals.

Recombinant canarypox virus vectors containing human immunodeficiency virus type 1 (HIV-1) sequences are promising vaccine candidates, as they replicate poorly in human cells. However, when delivered intramuscularly the vaccines have induced inconsistent and in some cases transient antigen-specific cytotoxic T-cell (CTL) responses in seronegative volunteers. An attractive way to enhance these responses would be to target canarypox virus to professional antigen-presenting cells such as dendritic cells (DCs). We studied (i) the interaction between canarypox virus and DCs and (ii) the T-cell responses induced by DCs infected with canarypox virus vectors containing HIV-1 genes. Mature and not immature DCs resisted the cytopathic effects of canarypox virus and elicited strong effector CD8+ T-cell responses from chronically infected HIV+ individuals, e.g., cytolysis, and secretion of gamma interferon (IFN-gamma) and beta-chemokines. Furthermore, canarypox virus-infected DCs were >30-fold more efficient than monocytes and induced responses that were comparable to those induced by vaccinia virus vectors or peptides. Addition of exogenous cytokines was not necessary to elicit CD8+ effector cells, although the presence of CD4+ T cells was required for their expansion and maintenance. Most strikingly, canarypox virus-infected DCs were directly able to stimulate HIV-specific, IFN-gamma-secreting CD4 helper responses from bulk as well as purified CD4+ T cells. Therefore, these results suggest that targeting canarypox virus vectors to mature DCs could potentially elicit both anti-HIV CD8+ and CD4+ helper responses in vivo.

Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion.

Vaccinia virus employs multiple mechanisms to evade the immune system, yet is highly immunogenic. We studied the interaction between vaccinia and human dendritic cells (DCs), potent APCs. DCs develop from precursor cells in two stages: an immature stage in which Ag uptake and processing occur, and a mature stage in which there is up-regulation of costimulatory and HLA molecules and efficient T cell activation. Vaccinia virus undergoes an abortive replication in both stages of DCs and induces apoptotic cell death. Furthermore, maturation of immature DCs and consequently T cell activation are inhibited. Obstruction of DC maturation may constitute a novel mechanism by which vaccinia attempts to evade the immune response.

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells.

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.

Cytotoxic T lymphocyte (CTL) responses to human cytomegalovirus pp65, IE1-Exon4, gB, pp150, and pp28 in healthy individuals: reevaluation of prevalence of IE1-specific CTLs.

The prevalence of human cytomegalovirus (HCMV) pp65-, pp150-, IE1-exon4-, gB- and pp28-specific cytotoxic T lymphocyte (CTL) responses was compared among 34 healthy individuals, grouped by neutralizing antibody titers. Moderately and highly seropositive donors showed predominantly pp65- and IE1-exon4-specific CTL responses (92% and 76% of the donors, respectively), with similar precursor frequencies in the 2 donors tested. In addition, highly seropositive and a few moderately seropositive donors showed CTL responses to gB and pp150 (33% and 30% of the donors, respectively). No individual recognized pp28 as a target in the CTL assay. Phenotypic analysis revealed a mixed effector population of CD4+ and CD8+ (1 donor) or only CD8+ cells for pp65-specific effectors (2 donors). IE1-exon4- and pp150-specific effectors were CD8+ (2 donors and 1 donor, respectively), whereas gB-specific CTLs were CD4+ (1 donor). These data may help to design a cellular immunity-based vaccine effective against HCMV diseases.

Strong human immunodeficiency virus (HIV)-specific CD4+ T cell responses in a cohort of chronically infected patients are associated with interruptions in anti-HIV chemotherapy.

Virus-specific CD4+ T-helper cell function is important in controlling human immunodeficiency virus (HIV) infection but is impaired in patients with progressive HIV disease. It has been reported that after highly active antiretroviral therapy (HAART), HIV-specific lymphoproliferative responses remain absent, whereas responses to non-HIV microbial antigens are restored. However, in analyzing immune responses in a cohort of chronically infected adults on HAART, we observed strong HIV-specific CD4+ T cell responses of Th-1 phenotype in 11 of 22 patients. The magnitude and frequency of HIV-specific lymphoproliferative responses was strongly associated with previous interruptions in HAART (P=.001). In contrast, the magnitude of CD8+ T cell responses to HIV Gag, Pol, Env, and Nef was similar in patients who had and those who had not interrupted HAART. We conclude that (1) a significant proportion of chronically HIV-infected patients on HAART can generate strong HIV-specific CD4+ and CD8+ T cell immunity and (2) transient interruptions in antiviral treatment may prime or boost HIV-specific CD4+ T-helper responses.

Replication-defective canarypox (ALVAC) vectors effectively activate anti-human immunodeficiency virus-1 cytotoxic T lymphocytes present in infected patients: implications for antigen-specific immunotherapy.

In the attempt to develop immunotherapeutic strategies for acquired immunodeficiency syndrome capable of activating effector cells in an antigen-specific manner while maintaining the broadest possible T-cell repertoire, we evaluated two canarypox (ALVAC)-based vectors for their capacity to induce ex vivo activation/expansion of human immunodeficiency virus (HIV)-specific CD8+ cytotoxic lymphocyte precursors (CTLp) obtained from HIV-1-infected donors. These two vectors, vCP205 encoding HIV-1 gp120 + TM (28 amino acid transmembrane anchor sequence) in addition to Gag/protease and vCP300 encoding gp120 + Gag/protease as well as Nef and Pol CTL determinants, are pancytotropic but replication incompetent in mammalian cells. Bulk peripheral blood mononuclear cells (PBMCs) or enriched CD8+ T cells were stimulated for 10 days with autologous ALVAC-infected PBMCs in the presence of different cytokine combinations (interleukin-2 [IL-2], IL-4, IL-7, and IL-12). Activation by ALVAC constructs was highly antigen-specific, because vCP205 elicited only Env and Gag CTL, whereas vCP300 elicited broader reactivities against Env, Gag, Pol, and Nef determinants. The ALVAC activation of CTLp was IL-2 dependent and enhanced by the addition of IL-7, whereas IL-4 and IL-12 failed to augment cytotoxic reactivities elicited by these constructs. The expansion of enriched CD8+ T cells after activation with vCP300 was higher in patients with CD4 counts greater than 400 cells/microL. Two rounds of in vitro stimulation (IVS) with vCP300 resulted in nearly an eightfold expansion of CD8+ lymphocytes over a 25-day period. After the second IVS, an average 3.2-fold increase among the different antigen-specific CTL frequencies was achieved. These studies clearly show that HIV-recombinant ALVAC vectors represent powerful polyvalent antigenic stimuli for activation and expansion of the CD8 lymphocyte response that occurs as a result of HIV infection.

A canarypox vector-expressing cytomegalovirus (CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects.

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8(+). Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.