Customizing Maxpar Direct Immune Profiling Assay with Additional Surface Marker and Intracellular Cytokine Staining Workflows for Expanded Mass Cytometry Panels.

Mass cytometry, or cytometry by time-of-flight (the basis for Fluidigm CyTOF technology), is a system for single-cell detection using antibodies tagged with metal probes. Without the need for compensation, the highly parametric Helios™ mass cytometer has a detection range of 135 distinct mass channels (75-209 Da). Optimized for mass cytometry, the Maxpar Direct™ Immune Profiling Assay™ is a dry, metal-tagged antibody cocktail for immunophenotyping 37 immune cell populations found in human peripheral blood in a single tube.

Elevated glycolysis imparts functional ability to CD8 T cells in HIV infection.

The mechanisms inducing exhaustion of HIV-specific CD8 T cells are not fully understood. Metabolic programming directly influences T-cell differentiation, effector function, and memory. We evaluated metabolic profiles of ex vivo CD8 T cells in HIV-infected individuals.

HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals.

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4 T cells).

Effect of IL-7 Therapy on Phospho-Ribosomal Protein S6 and TRAF1 Expression in HIV-Specific CD8 T Cells in Patients Receiving Antiretroviral Therapy.

IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB-mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice.

Pharmacologic HIV-1 Nef blockade promotes CD8 T cell-mediated elimination of latently HIV-1-infected cells in vitro.

Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef-mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1-infected CD4 T cells protects them from immune-mediated clearance.

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials.

Tim-3 is a Marker of Plasmacytoid Dendritic Cell Dysfunction during HIV Infection and Is Associated with the Recruitment of IRF7 and p85 into Lysosomes and with the Submembrane Displacement of TLR9.

In chronic diseases, such as HIV infection, plasmacytoid dendritic cells (pDCs) are rendered dysfunctional, as measured by their decreased capacity to produce IFN-α. In this study, we identified elevated levels of T cell Ig and mucin-domain containing molecule-3 (Tim-3)-expressing pDCs in the blood of HIV-infected donors. The frequency of Tim-3-expressing pDCs correlated inversely with CD4 T cell counts and positively with HIV viral loads.

HIV-Specific Granzyme B-Secreting but Not Gamma Interferon-Secreting T Cells Are Associated with Reduced Viral Reservoirs in Early HIV Infection.

A major barrier to a human immunodeficiency virus type 1 (HIV-1) infection cure is the establishment of a viral reservoir in spite of combined antiretroviral therapy (cART). It is unclear how HIV-specific cytotoxic T lymphocytes (CTLs) influence the size of the reservoir in early HIV infection. Twenty-eight subjects with early HIV infection were recruited to receive cART and followed for 48 weeks.

Self-assembling peptide for co-delivery of HIV-1 CD8+ T cells epitope and Toll-like receptor 7/8 agonists R848 to induce maturation of monocyte derived dendritic cell and augment polyfunctional cytotoxic T lymphocyte (CTL) response.

Peptide based vaccine that incorporates one or several highly conserved CD8+ T cells epitopes to induce potent cytotoxic T lymphocyte (CTL) response is desirable for some infectious diseases, such as HIV-1 (human immunodeficiency virus-1), and cancers. However, the CD8+ T cells epitope is often weakly immunogenic, and thus requires a specific adjuvant or delivery system to enhance the efficiency. Here we investigated the use of self-assembling peptide EAK16-II based platform to achieve the co-delivery of CD8+ T cells epitope and TLR7/8 agonists (R848 or R837) for augmenting DCs maturation and HIV-1 specific CTL response.

Early initiation of combination antiretroviral therapy in HIV-1-infected newborns can achieve sustained virologic suppression with low frequency of CD4+ T cells carrying HIV in peripheral blood.

Background: A human immunodeficiency virus type 1 (HIV-1)-infected infant started on combination antiretroviral therapy (cART) at 30 hours of life was recently reported to have no detectable plasma viremia after discontinuing cART. The current study investigated the impact of early cART initiation on measures of HIV-1 reservoir size in HIV-1-infected children with sustained virologic suppression.

Methods: Children born to HIV-1-infected mothers and started on cART within 72 hours of birth at 3 Canadian centers were assessed.

T cell Ig and mucin domain-containing protein 3 is recruited to the immune synapse, disrupts stable synapse formation, and associates with receptor phosphatases.

CD8(+) CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-γ) to aid this response.

LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events.

HERV-K-specific T cells eliminate diverse HIV-1/2 and SIV primary isolates.

The genetic diversity of HIV-1 represents a major challenge in vaccine development. In this study, we establish a rationale for eliminating HIV-1-infected cells by targeting cellular immune responses against stable human endogenous retroviral (HERV) antigens. HERV DNA sequences in the human genome represent the remnants of ancient infectious retroviruses.

Tim-3 negatively regulates cytotoxicity in exhausted CD8+ T cells in HIV infection.

Cytotoxic CD8(+) T cells (CTLs) contain virus infections through the release of granules containing both perforin and granzymes. T cell 'exhaustion' is a hallmark of chronic persistent viral infections including HIV. The inhibitory regulatory molecule, T cell Immunoglobulin and Mucin domain containing 3 (Tim-3) is induced on HIV-specific T cells in chronic progressive infection.

HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation.

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway.

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α.

Human endogenous retrovirus K(HML-2) Gag- and Env-specific T-cell responses are infrequently detected in HIV-1-infected subjects using standard peptide matrix-based screening.

T-cell responses to human endogenous retrovirus (HERV) K(HML-2) Gag and Env were mapped in HIV-1-infected subjects using 15 mer peptides. Small peptide pools and high concentrations were used to maximize sensitivity. In the 23 subjects studied, only three bona fide HERV-K(HML-2)-specific responses were detected.

A centrifugation-free high-throughput protein purification system using in-line microfluidization.

The majority of current high-throughput protein purification protocols include rate-limiting centrifugation steps. A column and nozzle assembly was developed that can be used in-line with microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols.

Human immunodeficiency virus type 1 escapes from interleukin-2-producing CD4+ T-cell responses without high-frequency fixation of mutations.

The presence of interleukin-2 (IL-2)-producing human immunodeficiency virus type 1 (HIV-1)-specific CD4(+) T-cell responses has been associated with the immunological control of HIV-1 replication; however, the causal relationship between these factors remains unclear. Here we show that IL-2-producing HIV-1-specific CD4(+) T cells can be cloned from acutely HIV-1-infected individuals. Despite the early presence of these cells, each of the individuals in the present study exhibited progressive disease, with one individual showing rapid progression.