George J. Cianciolo

Immunosuppressive activity of the retroviral envelope protein P 15E and its possible relationship to neoplasia

Type C retroviral infections can cause profound immunosuppression as well as neoplasms. The retroviral envelope protein p15E has both immunosuppressive and anti-inflammatory activities which may contribute to the pathogenicity of retroviruses. Murine and human neoplastic cells, not infected with retroviruses, have recently been found to contain p15E like antigens. In this article Ralph Snyderman and George Cianciolo discuss the potential relationship between p15E production, immunosuppression and neoplasia.

Immunosuppressive retroviral peptides: immunopathological implications for immunosuppressive influences of retroviral infections.

Studies of the effects of retroviruses on the immune system, which date back through thirty years of investigations, are reviewed. In the earliest published studies in the 1960s, it was demonstrated that mice infected with oncogenic viruses were immunosuppressed. Since then, numerous articles have been published describing profound immunodeficiencies observed in vivo in humans infected with human immunodeficiency virus and in animals such as cats infected with the feline immunodeficiency virus. In vitro investigations have shown that inactivated retroviruses or transmembrane envelope protein pl5E as well as a synthetic 17‐amino acid peptide (CKS‐17) impressively conserved within the transmembrane envelope protein of several animal or human retroviruses are highly immunosuppressive. More recently, dysfunction of cytokines produced by CKS‐17 at both a cellular and molecular level have been found to mimic influences observed in vivo in patients infected with the human immunodeficiency virus. CKS‐17 has also been shown to induce cAMP in vitro. The significance of these observations to understanding the immunological disturbances observed in malignancy, cytokine biosynthesis, and modulations of immune functions through cAMP is discussed. J. Leukoc. Biol. 61:654–666; 1997.

Inhibition of endothelial activation: a new way to treat cerebral malaria?

Background Malaria is still a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria (CM), remains incompletely understood. However tumor necrosis factor (TNF) is thought to play a key role in the development of this neurological syndrome, as well as lymphotoxin α (LT). Methods and Findings Using an in vitro model of CM based on human brain–derived endothelial cells (HBEC-5i), we demonstrate the anti-inflammatory effect of LMP-420, a 2-NH2-6-Cl-9-[(5-dihydroxyboryl)-pentyl] purine that is a transcriptional inhibitor of TNF. When added before or concomitantly to TNF, LMP-420 inhibits endothelial cell (EC) activation, i.e., the up-regulation of both ICAM-1 and VCAM-1 on HBEC-5i surfaces. Subsequently, LMP-420 abolishes the cytoadherence of ICAM-1-specific Plasmodium falciparum–parasitized red blood cells on these EC. Identical but weaker effects are observed when LMP-420 is added with LT. LMP-420 also causes a dramatic reduction of HBEC-5i vesiculation induced by TNF or LT stimulation, as assessed by microparticle release. Conclusion These data provide evidence for a strong in vitro anti-inflammatory effect of LMP-420 and suggest that targeting host cell pathogenic mechanisms might provide a new therapeutic approach to improving the outcome of CM patients.

Inverse Correlation of Maturity and Antibacterial Activity in Human Dendritic Cells

Dendritic cells (DCs) are a key part of host defense against microbial pathogens, being part of the innate immune system, but also instructing the adaptive T cell response. This study was designed to evaluate whether human DCs directly contribute to innate immunity by killing intracellular bacteria, using tuberculosis as a model. DCs were detected in bronchoalveolar lavage samples indicating that DCs are available for immediate interaction with Mycobacterium tuberculosis (M. Tb) after inhalation of the pathogen. The phenotype of DC in bronchoalveolar lavage closely resembles monocyte-derived immature DC (iDC) according to the expression of CD1a, CD83, and CCR7. The antimicrobial activity of iDC against intracellular M. Tb inversely correlated with TNF-α-release and was enhanced by treatment with anti-TNF-α Abs. Differentiation of iDC into mature DC by addition of TNF-α or activation via Toll-like receptors further reduced killing of M. Tb. The antibacterial activity against intracellular M. Tb of all DCs was significantly lower than alveolar macrophages. Therefore, the maintenance of a pool of DCs at the site of disease activity in tuberculosis, and the maturation of these DC by TNF-α provides a mechanism by which M. Tb escapes the innate immune system.

Human retrovirus-related synthetic peptides inhibit T lymphocyte proliferation

Immunosuppression frequently accompanies infections with the human retroviruses HTLV-1 and HIV. Previous studies have shown that UV-inactivated and detergent-disrupted preparations of either virus can produce immune dysfunction in vitro although the active component of such preparations has not yet been identified. We now report that synthetic peptides corresponding to a conserved sequence within the transmembrane envelope proteins of both HTLV-1 (CS-1) and HIV (CS-3) are capable of suppressing the in vitro proliferative responses of human T lymphocytes to mitogenic or allogeneic stimulation. CS-1 inhibits proliferation in response to stimulation by anti-CD3 antibody by up to 57% a concentration of 3 nmol per culture well. CS-3 inhibits by up to 49% at a similar concentration. CS-1 and CS-3 inhibit two-way mixed leukocyte culture reactions by 81 and 88%, respectively, at the 3 nmol per well concentration. The inhibition by such human retrovirus-related peptides appears to be T cell-specific in that B cell proliferation in response to stimulation with anti-IgG is not affected by the CS-3 peptide. The release of such immunosuppressive peptides may thus play a role in the pathogenesis of human exogenous retroviral disease such as AIDS.

Effects of CKS-17, a synthetic retroviral envelope peptide, on cell-mediated immunity in vivo: Immunosuppression, immunogenicity, and relation to immunosuppressive tumor products

SummaryCKS-17 is a heptadecapeptide corresponding to a region highly conserved in retroviral transmembrane proteins such as p15E. Because a relationship had previously been determined between p15E and immunosuppressive tumor cell products, we examined the effect of CKS-17, control peptides and conjugates thereof on the expression of cell-mediated immunity (delayed-type hypersensitivity, DTH) in mice. Conjugates of CKS-17 inhibited DTH reactions to sheep erythrocytes in the feet of mice. The degree of inhibition was dose-dependent. Unconjugated CKS-17 had almost no effect, and control peptide conjugates had no inhibitory effect. Immunization of mice with CKS-17 conjugates, but not with control conjugates, rendered them resistant to the depression of DTH reactions, not only by CKS-17 conjugates, but also by products of cultured tumor cells. CKS-17 conjugates, but not control conjugates, also depressed the cellular inflammatory reactions induced in mouse footpads by concanavalin A (ConA) and immunized mice against the depression of ConA reactions by products of cultured tumor cells. Injections of globulin from sera of mice immunized with CKS-17 conjugates conferred upon normal recipients resistance to the depression of footpad reactions to ConA by products of cultured tumor cells. Globulin from sera of normal mice or control immunized mice did not confer such resistance. Thus conjugates of a synthetic peptide not only mimic the immunosuppressive effects of tumor products in vivo, but can also immunize mice against those effects.

A Synthetic Peptide Homologous to Retroviral Transmembrane Envelope Proteins Depresses Protein Kinase C Mediated Lymphocyte Proliferation and Directly Inactivated Protein Kinase C: A Potential Mechanism for Immunosuppression

CKS‐17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS‐17‐BSA). To define its site of action, we determined if CKS‐17 conjugated to human serum albumin (CKS‐17‐HSA) could block the direct activation of lymphocytes by phorbol‐12‐myristate‐13‐acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiCs). CKS‐17‐HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose‐dependent manner with up to 88% inhibition occurring with 15μM CKS‐17‐HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 μM CKS‐17‐HSA. Based on these findings we investigated the effect of CKS‐17‐HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS‐17‐HSA caused a dose‐dependent enzyme inhibition with a concentration giving half‐maximal inhibition (IC50) of ca.3 μM and >95% inhibition at 15 μM CKS‐17‐HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS‐17‐HSA did not inhibit the activity of adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP)‐dependent protein kinase (PK‐A) nor the calcium and phospholipid‐independent form of PKC (PK‐M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA‐stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS‐17‐HSA may be due to the direct inactivation of PKC.

Covalent complexes of antigen and α2-macroglobulin: evidence for dramatically-increased immunogenicity

A safe, effective, more potent adjuvant than currently available would be beneficial in developing new therapeutics and diagnostic reagents. We report here a technique for the rapid, efficient incorporation of non-proteolytic antigens into alpha(2)-macroglobulin (alpha(2)M; tradename, SynerVax), allowing us to covalently couple much larger subunit antigens to alpha(2)M than previously possible. Our goal was to determine if incorporation of HB, the monomeric form of Hepatitis B virus (HBV) surface antigen (HBsAg), into alpha(2)M would result in increased immune reactivity. Earlier attempts to immunize animals using HB did not generate significant levels of antibodies. Using HB complexes prepared with alpha(2)M we now report dramatically-increased immunogenicity of HB in BALB/c mice. Combining these soluble complexes with a depot-generating agent (alum), titers>1:1,000,000 are obtained with a single injection. This novel adjuvant technology should provide a valuable tool for the development of either prophylactic and therapeutic vaccines, or monoclonal antibodies against hitherto poorly-immunogenic subunit antigens.

SP-A Preserves Airway Homeostasis During Mycoplasma pneumoniae Infection in Mice

The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A−/− mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A−/− mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-α. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-α response.

Increased immunogenicity of HIV envelope subunit complexed with α2-macroglobulin when combined with monophosphoryl lipid A and GM-CSF

Critical to the success of HIV-1 subunit vaccines is the development of strategies to augment vaccine immunogenicity. Successful adjuvants must not only improve immunogenicity above current adjuvant levels, but must also decrease the dose of immunogen required for optimal immunogenicity. We have evaluated activated alpha2-macroglobulin (alpha2M*) and a squalene-based stable emulsion containing monophosphoryl lipid A (MPL-SE) with granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvants to enhance the immunogencity of candidate HIV immunogens. Balb/c mice were subcutaneously immunized on days 0, 14 and 28 with 100-0.1 microg of HIV-1 envelope gp120 C4-V3 immunogens from either HIV IIIB (C4-V3(IIIB)) or SHIV 89.6P (C4-V3(89.6P)). Immunogens were tested covalently coupled to alpha2M*, formulated with MPL-SE/GM-CSF, or as a combination of both. Using CFA/IFA, only 50 and 100 microg, but not lower doses of C4-V3(IIIB) peptides, induced antibody responses. In contrast, peak antibody responses were detected in mice immunized with 10 microg of C4-V3 peptide coupled to alpha2M* (alpha2M*-peptide). Similar to CFA/IFA, MPL-SE/GM-CSF induced optimal antibody responses at 50 and 100 microg of C4-V3 immunogen. However, the combination of MPL-SE/GM-CSF with alpha2M*-C4-V3 peptide decreased the dose of C4-V3 required for optimal response to 5 microg for C4-V3(IIIB), and to 0.1 microg for C4-V3(89.6P). Taken together, HIV envelope gp120 C4-V3 peptides covalently complexed with alpha2M* and formulated with MPL-SE/GM-CSF resulted in a subunit HIV immunogen capable of inducing anti-HIV envelope antibody responses at doses up to 100-fold less than those needed with CFA/IFA or MPL-SE/GM-CSF alone.