C. David Pauza

Vaccination with Tat toxoid attenuates disease in simian/HIV-challenged macaques

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo. We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-alpha, and chemokine receptor expression (CXCR4 and CCR5) on CD4(+) T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

Human γδ T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement

Natural killer (NK) cells are innate effector lymphocytes that control the growth of major histocompatibility complex class I negative tumors. We show here that γδ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell-mediated killing of tumors that are usually resistant to NK cytolysis. The induction of cytotoxicity toward these resistant tumors requires priming of NK cells by immobilized human immunoglobulin G1 and costimulation through CD137L expressed on activated γδ T lymphocytes. This costimulation increases NKG2D expression on the NK-cell surface, which is directly responsible for tumor cell lysis. Moreover, culturing peripheral blood mononuclear cells with zoledronic acid, a γδ T lymphocyte activating agent, enhances NK-cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors. Our data reveal a novel function of human γδ T lymphocytes in the regulation of NK cell-mediated cytotoxicity and provide rationale for the use of strategies to manipulate the CD137 pathway to augment innate antitumor immunity.

Monocytes Treated with Human Immunodeficiency Virus Tat Kill Uninfected CD4+ Cells by a Tumor Necrosis Factor-Related Apoptosis-Induced Ligand-Mediated Mechanism

ABSTRACT The human immunodeficiency virus (HIV) Tat protein has a critical role in viral transcription, but this study focuses on its additional role as an extracellular effector of lymphocyte cell death. It is well known that Tat induces tumor necrosis factor-related apoptosis-induced ligand (TRAIL) in peripheral blood mononuclear cells (PBMC), and we show that the majority of TRAIL is produced by the monocyte subset of PBMC. Human monocytes and U937 monoblastoid cells did not take up soluble HIV Tat-86, as T cells did, yet produced more TRAIL than did T cells. TRAIL secretion was induced by Tat and by a cysteine-rich peptide of Tat but not by sulfhydryl-modified Tat toxoid. Although there was only a slight increase in cell surface expression of TRAIL on monocytes, sufficient TRAIL was secreted to be toxic for T cells. The cytotoxicity of Tat-stimulated monocyte medium could be blocked by a TRAIL-neutralizing antibody. T cells treated with Tat did not secrete enough TRAIL to mediate cell death in our assay. Remarkably, uninfected T cells are more susceptible to TRAIL than are HIV-infected T cells. The production of TRAIL by Tat-stimulated monocytes provides a mechanism by which HIV infection can destroy uninfected bystander cells.

Isopentenyl Pyrophosphate–Activated CD56+ γδ T Lymphocytes Display Potent Antitumor Activity toward Human Squamous Cell Carcinoma

Purpose: The expression of CD56, a natural killer cell–associated molecule, on αβ T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of γδ T cells. However, antitumor effector functions of CD56+ γδ T cells are poorly characterized. Experimental Design: To investigate the potential effector role of CD56+ γδ T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2–expanded γδ T cells from peripheral blood mononuclear cells of healthy donors. Results: Thirty to 70% of expanded γδ T cells express CD56 on their surface. Interestingly, although both CD56+ and CD56− γδ T cells express comparable levels of receptors involved in the regulation of γδ T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56+ γδ T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56+ γδ T lymphocytes expressed higher levels of CD107a compared with CD56− controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-γδ T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56+ γδ T cells involves the perforin-granzyme pathway and is mainly γδ T-cell receptor/NKG2D dependent. Importantly, CD56-expressing γδ T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. Conclusions: Our data indicate that CD56+ γδ T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

rhesus Macaques That Become Systemically Infected With Pathogenic Shiv 89.6-pd After Intravenous, Rectal, or Vaginal Inoculation and Fail to Make an Antiviral Antibody Response Rapidly Develop Aids

A new simian-human immunodeficiency virus (SHIV) stock (SHIV 89.6-PD), derived from plasma of a rhesus macaque used for in vivo serial passage of virulence-attenuated SHIV 89.6, produces systemic infection after intravenous, intravaginal, or intrarectal inoculation of rhesus macaques. Infection with this virus results in high levels of viral antigen in plasma, a precipitous decline in CD4+ T-cell counts, and a disease syndrome that is characteristic of AIDS. Rapid progression to disease was associated with failure to seroconvert to viral antigens, whereas longer survival was associated with production of antiviral antibodies. In intravenously inoculated animals, peak antigenemia occurred at 7 days postinjection (PI) and severe CD4+ depletion occurred at 14 days PI. In mucosally infected animals, peak antigenemia occurred at 14 days PI and severe CD4+ depletion was not evident until 21 days PI. The 1-week delay in both viral antigenemia and CD4+ T-cell decline in mucosally infected animals is consistent with the hypothesis that, following vaginal inoculation, virus dissemination proceeds in a stepwise manner from the mucosal surface to the draining lymph nodes and subsequently to the bloodstream. This animal model can be used to test the ability of HIV-1 envelope-based vaccines to prevent infection or disease after challenge by the three major routes of HIV transmission.

Gamma Interferon Secretion by Human Vγ2Vδ2 T Cells after Stimulation with Antibody against the T-Cell Receptor plus the Toll-Like Receptor 2 Agonist Pam3Cys

ABSTRACT Circulating Vγ2Vδ2 T-cell populations in healthy human beings are poised for rapid responses to bacterial or viral pathogens. We asked whether Vγ2Vδ2 T cells use the Toll-like receptor (TLR) family to recognize pathogen-associated molecular pattern molecules and to regulate cell functions. Analysis of expanded Vγ2Vδ2 T-cell lines showed the abundant presence of TLR2 mRNA, implying that these receptors are important for cell differentiation or function. However, multiple efforts to detect TLR2 protein on the cell surface or in cytoplasmic compartments gave inconsistent results. Functional assays confirmed that human Vγ2Vδ2 T cells could respond to the TLR2 agonist (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride (Pam3Cys), but the response required coincident stimulation through the γδ T-cell receptor (TCR). Dually stimulated cells produced higher levels of cytoplasmic or cell-free gamma interferon and showed increased expression of the lysosome-associated membrane protein CD107a on the cell surface. A functional TLR2 that requires coincident TCR stimulation may increase the initial potency of Vγ2Vδ2 T-cell responses at the site of infection and promote the rapid development of subsequent acquired antipathogen immunity.

Whole-body positron emission tomography in patients with HIV-1 infection.

Positron emission tomography with fluorine-18-deoxyglucose (FDG-PET) detects active lymphoid tissues during HIV-1 infection in man. We used FDG-PET to study anatomical correlates of HIV-1 infection in man. Whole-body FDG-PET images from 15 patients with HIV-1 showed distinct lymphoid tissue activation in the head and neck during acute disease, a generalised pattern of peripheral lymph-node activation at mid-stages, and involvement of abdominal lymph nodes during late disease. Unexpectedly, HIV-1 progression was evident by distinct anatomical correlates, suggesting that lymphoid tissues are engaged in a predictable sequence. Understanding the anatomy of HIV-1 infection could encourage use of surgical or radiological interventions to supplement chemotherapy.

Early Blood Profiles of Virus Infection in a Monkey Model for Lassa Fever

ABSTRACT Acute arenavirus disease in primates, like Lassa hemorrhagic fever in humans, begins with flu-like symptoms and leads to death approximately 2 weeks after infection. Our goal was to identify molecular changes in blood that are related to disease progression. Rhesus macaques (Macaca mulatta) infected intravenously with a lethal dose of lymphocytic choriomeningitis virus (LCMV) provide a model for Lassa virus infection of humans. Blood samples taken before and during the course of infection were used to monitor gene expression changes that paralleled disease onset. Changes in blood showed major disruptions in eicosanoid, immune response, and hormone response pathways. Approximately 12% of host genes alter their expression after LCMV infection, and a subset of these genes can discriminate between virulent and nonvirulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and will be valuable candidates for future validation as biomarkers for arenavirus disease.

In vitro stimulation with a non-peptidic alkylphosphate expands cells expressing Vγ2-Jγ1.2/Vδ2 T-cell receptors

The majority of peripheral blood γδ T cells in healthy adult humans express the Vγ2/Vδ2 T‐cell receptor (TCR) and generate TCR‐mediated, major histocompatibility complex (MHC)‐unrestricted proliferative responses to low molecular weight alkylphosphates. Vγ2/Vδ2 populations after antigen proliferation maintained diversity in the CDR3s of Vγ2 mRNA, indicating that the response was polyclonal or oligoclonal, and were enriched for Vγ2 TCR chains containing the Jγ1.2 segment. Alkylphosphate stimulation further skewed an already biased peripheral blood γδ T‐cell population and increased the abundance of Vγ2‐Jγ1.2/Vδ2 T cell receptors, suggesting similarities between the alkylphosphate response and peripheral selection mechanisms shaping this repertoire in human beings.

A Neonatal Fc Receptor-Targeted Mucosal Vaccine Strategy Effectively Induces HIV-1 Antigen-Specific Immunity to Genital Infection

ABSTRACT Strategies to prevent the sexual transmission of HIV include vaccines that elicit durable, protective mucosal immune responses. A key to effective mucosal immunity is the capacity for antigens administered locally to cross epithelial barriers. Given the role of neonatal Fc receptor (FcRn) in transferring IgG across polarized epithelial cells which line mucosal surfaces, FcRn might be useful for delivering HIV vaccine antigens across mucosal epithelial barriers to the underlying antigen-presenting cells. Chimeric proteins composed of HIV Gag (p24) fused to the Fc region of IgG (Gag-Fc) bind efficiently to airway mucosa and are transported across this epithelial surface. Mice immunized intranasally with Gag-Fc plus CpG adjuvant developed local and systemic immunity, including durable B and T cell memory. Gag-specific immunity was sufficiently potent to protect against an intravaginal challenge with recombinant vaccinia virus expressing the HIV Gag protein. Intranasal administration of a Gag-Fc/CpG vaccine protected at a distal mucosal site. Our data suggest that targeting of FcRn with chimeric immunogens may be an important strategy for mucosal immunization and should be considered a new approach for preventive HIV vaccines.