Biomedical subjects
Publications and source records attributed to C Lapham.
Brucella abortus is an intracellular pathogen that causes disease in cattle and in humans. The response against B. abortus involves the whole gamut of the immune system, from innate to adaptive immunity resulting from stimulation of antigen-presenting cells, NK cells, CD4(+) and CD8(+) T cells, and B cells.
The effect of interferon gamma (IFN-gamma) and interleukin 6 (IL-6) on infection of macrophages with human immunodeficiency virus type 1 (HIV-1) was investigated. By using a polymerase chain reaction-based viral entry assay and viral infectivity assay, it was demonstrated that IL-6 and IFN-gamma augmented susceptibility of monocyte-derived macrophages (MDMs) to infection with T-cell tropic CXCR4-utilizing (X4) HIV-1 strains. Consistent with this finding, IFN-gamma and IL-6 augmented fusion of MDMs with T-tropic envelope-expressing cells. The enhanced fusion of cytokine-treated MDMs with T-tropic envelopes was inhibited by the CXCR4 ligand, SDF-1, and by T22 peptide. IFN-gamma and IL-6 did not affect expression of surface CXCR4 or SDF-1-induced Ca(++) flux in MDMs. In contrast to the effect of IFN-gamma on the infection of MDMs with X4 strains, IFN-gamma inhibited viral entry and productive infection of MDMs with macrophage-tropic (M-tropic) HIV-1. Consistent with this finding, IFN-gamma induced a decrease in fusion with M-tropic envelopes that correlated with a modest reduction in surface CCR5 and CD4 on MDMs. It was further demonstrated that macrophage inflammatory protein (MIP)-1alpha and MIP-beta secreted by cytokine-treated MDMs augmented their fusion with T-tropic-expressing cells and inhibited their fusion with M-tropic envelope-expressing cells. These data indicate that proinflammatory cytokines, which are produced during opportunistic infections or sexually transmitted diseases, may predispose macrophages to infection with X4 strains that, in turn, could accelerate disease progression.
CD4-specific monoclonal antibodies (CG1, CG7, and CG8), which bind with a 5- to 10-fold higher avidity to preformed CD4-gp120 complexes than to CD4, were previously shown to recognize newly identified conformational epitopes in the D1-CDR3 region of CD4. In the current study, these and other complex-enhanced MAbs were tested in three separate assays of HIV-1 coreceptor (CXCR4/CCR5) recruitment. In these assays, the CD4-specific MAbs CG1, -7, and -8 stabilized the association of coreceptor, gp120, and CD4 in trimolecular complexes. In contrast, the gp120-specific, complex-enhanced MAbs 48d and 17b were inhibitory. These data suggest that conformational changes in the CDR3 region of CD4-D1, induced by gp120 binding, may be involved in coreceptor association and thus play a positive role in the HIV-1 cell fusion process.
We have previously shown that immunization of mice with human immunodeficiency virus (HIV)-derived proteins or peptides conjugated to inactivated Brucella abortus induces the secretion of virus-neutralizing antibodies, predominantly of the immunoglobulin G2a (IgG2a) isotype. In addition, B. abortus activates human CD4+ and CD8+ cells to secrete gamma interferon. Since these are both characteristics of a Th1-type immune response, which is associated with the development of cell-mediated immunity, it was important to determine if B. abortus conjugates would also act as a carrier to induce a cytotoxic T-lymphocyte (CTL) response. To test this hypothesis, we conjugated an 18-amino-acid peptide from the V3 loop of the MN strain of HIV-1 gp120 that contains both B- and cytotoxic T-cell epitopes to B. abortus (B. abortus-MN 18-mer). A 10-amino-acid fragment of this peptide has been shown to be the minimal CTL determinant presented by murine H-2Dd. It was found that two in vivo immunizations with 10(8) organisms of B. abortus-MN 18-mer followed by in vitro stimulation with peptide induced a virus-specific CTL response. Conjugation to B. abortus was required for in vivo priming, since there was no induction of memory CTLs when B. abortus was only mixed with peptide. Targets pulsed with peptide as well as those infected with a vaccinia virus encoding HIV gp160 were killed, demonstrating recognition of naturally processed envelope. Also, major histocompatibility complex-incompatible L cells which were infected with vaccinia viruses that encoded H-2Dd, but not H-2Kd, and pulsed with peptide were lysed. This demonstrated the appropriate major histocompatibility complex class I restriction. Treatment of the mice with anti-L3T4 prior to immunization caused a severe depletion of CD4+ lymphocytes, yet it did not decrease the CTL priming. Thus, inactivated B. abortus can induce non-CD4+ cells to produce the cytokines required for CTL induction. We conclude that B. abortus stimulates a cellular as well as a humoral immune response, even in the relative absence of CD4+ helper cells. It may be a particularly useful vaccine carrier in HIV-1-infected individuals or others with impaired CD4+ T-cell function.
LMP2 and LMP7 are proteins encoded by MHC genes that are tightly linked to the genes encoding TAP, the transporter that conveys peptides from the cytosol to the endoplasmic reticulum for assembly with MHC class I molecules. LMP2 and LMP7 are subunits of a subset of proteasomes, large molecular assemblies with multi-proteolytic activities believed to degrade damaged and unwanted cellular proteins. Like TAP and class I molecules themselves, expression of LMP genes is enhanced after exposure of cells to IFN-gamma. These findings implicate LMP2 and LMP7 in the cytosolic production of antigenic peptides. Doubts have been cast, however, on the role of LMP2 and LMP7 in Ag processing, because cells lacking these proteins possess class I molecules that contain peptides quantitatively and qualitatively indistinguishable from the peptides bound to class I molecules derived from normal cells. In this paper we show that cells lacking LMP2 and LMP7 present seven TAP-dependent determinants derived from viral proteins. For two determinants, the kinetics of presentation are shown to be similar for LMP-expressing and -nonexpressing cells. We also demonstrate biochemically that peptide is not limiting in the assembly of class I molecules in LMP-nonexpressing cells. These findings provide additional evidence that LMP2 and LMP7 are not required for efficient Ag presentation, and suggest that these proteins have either a more specialized role in the production of class I-associated peptides, or are not involved in the processing of proteins for association with class I molecules.
It is well established that tumor-specific CD8+ T cells have the capacity to prevent and cure malignancies in animals under experimental conditions. This has raised expectations that it will prove possible to achieve similar successes with human cancers. CD8+ T cells recognize peptides of 8-10 residues derived from cytosolic proteins that are bound to the class I molecules of the major histocompatibility complex. To most effectively manipulate the T-cell response to tumor cells, it is essential to understand the means by which the peptide-class I complex is created in cells. An overview of this process is provided with an emphasis toward the recent findings made by our laboratory.
Products prepared from broth extracts of beta-hemolytic Group A streptococci activate human natural killer (NK) cells. The active moiety is likely a protein since the enhancing capability is destroyed by the proteolytic enzyme pronase, although not by trypsin. The enhancement in NK cytotoxicity is due at least in part to lymphokines, since normal peripheral blood mononuclear cells and T cells, upon incubation with streptococcal products (SP), release supernatant factors which augment NK activity. These cell culture supernatants contain interferons (IFN) as well as low levels of interleukin-2 (IL-2). Treatment of supernatants with anti-IFN antibodies has variable effects, depending on the donor cells used to produce the factors. In most cases, anti-IFN-gamma totally abrogates enhancement. Treatment of supernatants with antibodies to IFN-alpha modestly decreases enhancement of most donor cells; however, IFN-alpha appears not to be a major factor in SP-activated lymphokines. Pretreating effector cells with a monoclonal antibody directed against the IL-2 receptor (anti-Tac) usually reduces the supernatant effect. The combination of anti-Tac and anti-IFN-gamma totally nullifies enhancement. Thus T lymphocytes stimulated with streptococcal products augment NK activity at least in part by producing IFN-gamma and a factor whose activity is reduced by the interaction of the IL-2 receptor with anti-Tac.
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