Alloantigenic stimulation bypasses CD28-B7 costimulatory blockade by an interleukin-2-dependent mechanism.

Allogeneic leukocytes have been used as biological adjuvants for T cell-specific responses to tumor and recall antigens, but the mechanisms underlying this effect have not been fully understood. The present study investigates whether alloantigen stimulation of human T cells would bypass an in vitro T cell costimulatory dysfunction induced by CTLA4Ig blockage of CD28-B7 interaction. Here, we demonstrate that costimulation with intact allogeneic leukocytes plus viral antigen circumvented the inhibition of this costimulatory pathway via interleukin-2 (IL-2) production, resulting in the generation of influenza-specific cytotoxic T lymphocytes (CTL). The alloantigen-induced help for influenza-specific CTL generation did not require cell-to-cell contact between responding and allogeneic stimulator cells. These results suggest that alloantigens can be used to bypass defects in the CD28-B7 costimulatory pathway and, therefore, may contribute to understanding the mechanisms of alloantigen-induced restoration of T cell-mediated immunity.

Comparison of in vitro immunostimulatory potential of live and inactivated influenza viruses.

Live influenza viruses, heat-inactivated virus, and a trivalent formalin-inactivated influenza vaccine were analyzed for their in vitro stimulatory properties on immune cells from healthy donors. Lymphocyte proliferation induced by each influenza antigen was comparable. Influenza vaccine stimulated significantly lower production of interferon-gamma (IFN-gamma) compared with live and heat inactivated viruses, whereas both vaccine and heat-inactivated influenza induced lower levels of IFN-alpha compared with live virus. Furthermore, only live virus generated influenza-specific cytotoxic T lymphocyte (CTL) activity. A significant increase in monocyte expression of CD80, CD86, CD40, and human leukocyte antigen-DR (HLA-DR) was also induced by live influenza virus. Our results suggest that immunization with live influenza vaccines might induce immune responses that would not be induced by conventional inactivated vaccines, including CTL generation, antiviral IFN-gamma and IFN-alpha cytokine production, and increased antigen presentation and costimulatory capacity on antigen presenting cells (APC).

Anthracycline antibiotic blockade of SV40 T antigen helicase action.

We previously showed that anthracycline antibiotics potently block SV40 large T antigen helicase; in the present study, we describe the kinetics and the structure-activity characteristics of this process. The concentration vs effect data for helicase blockade were fitted by the Hill equation to yield nearly parallel log-concentration effect curves for a series of active anthracycline antibiotics. The effective concentration for 50% helicase blockade (EC50) values ranged from 0.34 microM for daunorubicin to 40.8 microM for 3'-deaminodaunorubicin. Clinically inactive 3'-N-acyl anthracyclines produced no blockade. The Hill constants for the blockade ranged from 1.1 to 1.6 for the entire series of active anthracyclines, indicating no positive cooperativity and suggesting that a single molecule of bound drug is sufficient to block helicase action. The EC50 values for several clinically effective anthracyclines showed a relationship to the average DNA binding constants for these drugs, and Lineweaver-Burk analysis of the blockade kinetics indicated non-competitive inhibition. The kinetics of the blockade indicated that the anthracycline, DNA, and helicase form a ternary complex that is irreversible under the reaction conditions. This mechanism may be central to the cytotoxic and anti-cancer activities of anthracycline antibiotics and may be useful in understanding the enzymatic mechanism of DNA helicase action.

Antihelicase action of CI-958, a new drug for prostate cancer.

CI-958, a new DNA-intercalating drug derived from a series of substituted 2H-[1] benzothiopyrano[4,3,2-cd]indazoles, is being tested in clinical trails because of its curative properties against murine solid tumor models and because it has demonstrated activity in a pilot phase II study of patients with hormone-refractory prostate cancer. However, the mechanism of anticancer action of CI-958 has not been established. Because CI-958 binds to DNA and DNA helicases are profoundly affected by DNA-binding drugs, we examined the effects of CI-958 on human DNA helicase action. DNA helicase activity was measured by strand dissociation of double-stranded (ds) DNA with a gel electrophoresis assay, and ATPase activities were determined on thin-layer chromatography by measurement of the conversion of ATP to ADP. For human helicase blockade, CI-958 is slightly more potent than doxorubicin (EC50 values 0.17 and 0.26 microM, respectively). We observed no difference in helicase-blockade EC50 values recorded for three helicase substrates containing A-T rich, G-C rich, and both types of oligonucleotide sequences. The effects of CI-958 helicase blockade and DNA-dependent ATPase activities were similar for the two reactions. The kinetics of the blockade by CI-958 of the human DNA helicase indicates that it involves a reversible ternary complex of helicase-drug-dsDNA. CI-958 produces potent blockade of human DNA helicases with no apparent strong DNA sequence-binding preference. Similar potency against helicase strand dissociation and DNA-dependent ATPase suggests that the mechanism against these reactions is the same. The blockade of DNA helicases by CI-958 may be central in its mechanism of action as an anticancer drug.

Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses.

The present study analyzes the role of CD28-B7-mediated costimulation during in vitro human peripheral blood memory T cell activation by influenza A virus. Inhibition studies using the B7-binding fusion protein CTLA4Ig and antibodies against CD80 and CD86 demonstrate that CTLA4Ig and anti-CD86 inhibited influenza-specific T cell proliferation, interleukin (IL)-2 and interferon (IFN)-gamma production, and generation of influenza-specific CD8+ CTL. The production of IL-10 and IL-18, which are known to modulate T cell immune responses, were not affected by blocking the CD28-B7 costimulatory pathway. Inhibition of diverse influenza-specific T cell functions could be reversed by the addition of exogenous IL-2 or IL-12 but not by the addition of IFN-gamma or IL-18. Although IL-2 is known to overcome CD28-B7 costimulatory requirements, this is the first report showing that exogenous IL-12 is able to bypass CD28-B7 costimulatory blockade induced by CTLA4Ig in vitro. The induction of IFN-gamma production with the recently described IFN-gamma inducing cytokine IL-18 was not detected. In conclusion, these results demonstrate that CD86 represents a major costimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens. These observations may have important implications for the development of immunotherapeutic strategies in diverse immunodeficiency diseases as well as in tumor immunotherapy.

Differential incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation.

Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4(+) T cells long before a quantitative decline in circulating CD4(+) T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4(+) T cells remains unclear. Both direct effects of cytopathic infection of CD4(+) cells and indirect effects in which uninfected "bystander" cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.

Influenza virus-stimulated generation of anti-human immunodeficiency virus (HIV) activity after influenza vaccination in HIV-infected individuals and healthy control subjects.

Influenza virus stimulation of leukocytes induces factors that suppress human immunodeficiency virus (HIV). The effect of influenza vaccination on influenza-induced anti-HIV activity was investigated. Influenza vaccine was administered to 25 control subjects and 20 HIV-infected patients. Antiviral activity, cytokine production, and influenza antibodies were assessed before and 2 and 6 weeks after vaccination. Immunization induced a statistically significant increase in antiviral activity in control subjects but not in HIV patients, although the number of patients who generated this activity increased. Pre- and postvaccination levels of anti-HIV activity were significantly lower in HIV patients. Vaccination of control subjects and HIV patients induced increases in production of interleukin-2 and interferon (IFN)-gamma, but not of IFN-alpha. Virus load and CD4 cell counts were not significantly altered. This study demonstrates impairment of antiviral activity in HIV patients, in addition to deficiencies in antibody responses and cytokine production. In summary, influenza vaccination can induce an increase in multiple immunologic components that remained impaired in HIV patients.

Human herpesvirus 8 cellular immune responses in homosexual men.

Little is known about cellular immunity to human herpesvirus 8 (HHV-8), the virus associated with Kaposi's sarcoma (KS). T cell proliferative responses to purified HHV-8 were measured in homosexual men, a group with elevated HHV-8 seroprevalence and high risk of KS. None of 20 blood donor controls had T cell responses to HHV-8. Among human immunodeficiency virus (HIV)-negative homosexual men, 8 (42%) of 19 HHV-8 seropositive men responded as did 4 (16%) of 25 HHV-8 seronegative men. Among HIV-positive homosexual men, however, none of 21 HHV-8 seropositives had T cell responses to HHV-8, even though most responded to common recall antigens, and 10 had >/=400 CD4 cells/mm3. The results suggest that HHV-8 T cell proliferative responses are common in HIV-negative homosexual men and that HIV infection may be associated with diminished HHV-8 cellular immunity, possibly before there is substantial depletion of CD4 cells. If correct, this could explain why KS occurs relatively early in HIV infection/AIDS.