Dendritic Cells Reveal a Broad Range of MHC Class I Epitopes for HIV-1 in Persons with Suppressed Viral Load on Antiretroviral Therapy

et al. (2010) Dendritic Cells Reveal a Broad Range of MHC Class I Epitopes for HIV-1 in Persons with Suppressed Viral Load on Antiretroviral Therapy. PLoS ONE 5(9): e12936. doi:10.1371/journal.pone.0012936 Dendritic Cells Reveal a Broad Range of MHC Class I Epitopes for HIV-1 in Persons with Suppressed Viral Load on Antiretroviral Therapy Xiao-Li Huang 0 Zheng Fan 0 LuAnn Borowski 0 Robbie B. Mailliard 0 Morgane Rolland 0 James I. 0 Mullins 0 Richard D. Day 0 Charles R. Rinaldo 0 Derya Unutmaz, New York University, United States of America 0 1 Department of Infectious Diseases and Microbiology, Graduate School of Public Health and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Microbiology, University of Washington, Seattle, Washington, United States of America, 3 Department of Biostatistics, Graduate School of Public Health and School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 4 Department of Pathology, Graduate School of Public Health and School of Medicine, University of Pittsburgh , Pittsburgh, Pennsylvania , United States of America Background: HIV-1 remains sequestered during antiretroviral therapy (ART) and can resume high-level replication upon cessation of ART or development of drug resistance. Reactivity of memory CD8+ T lymphocytes to HIV-1 could potentially inhibit this residual viral replication, but is largely muted by ART in relation to suppression of viral antigen burden. Dendritic cells (DC) are important for MHC class I processing and presentation of peptide epitopes to memory CD8+ T cells, and could potentially be targeted to activate memory CD8+ T cells to a broad array of HIV-1 epitopes during ART. Principal Findings: We show for the first time that HIV-1 peptide-loaded, CD40L-matured DC from HIV-1 infected persons on ART induce IFN gamma production by CD8+ T cells specific for a much broader range and magnitude of Gag and Nef epitopes than do peptides without DC. The DC also reveal novel, MHC class I restricted, Gag and Nef epitopes that are able to induce polyfunctional T cells producing various combinations of IFN gamma, interleukin 2, tumor necrosis factor alpha, macrophage inhibitory protein 1 beta and the cytotoxic de-granulation molecule CD107a. Significance: There is an underlying, broad antigenic spectrum of anti-HIV-1, memory CD8+ T cell reactivity in persons on ART that is revealed by DC. This supports the use of DC-based immunotherapy for HIV-1 infection. - Funding: This work was supported by the National Institutes of Allergy and Infectious Diseases grants P01 AI-055794, U01 AI-35041 and R37 AI-41870. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The breadth of CD8+ T cell reactivity specific for HIV-1 antigens is considered a key factor in host control of HIV-1 infection [1]. Production of interferon c (IFNc) by memory CD8+ T cells that are specific for a broad array of HIV-1 epitopes, especially those within the Gag protein, is associated with slower HIV-1 disease progression [2,3]. Control of HIV-1 infection has also been linked to polyfunctional reactivity of memory CD8+ T cells, i.e., T cells that produce more than one immune mediator in response to HIV-1 antigens [4], particularly Gag [5,6,7,8]. This has led to the concept that effective prophylactic and immunotherapeutic vaccines for HIV-1 will need to induce a broad, HIV-1 antigenic spectrum of CD8+ T cell reactivity. Induction of broad and robust T cell reactivity could be particularly important in immunotherapy of HIV-1 infection during antiretroviral therapy (ART) [9]. However, virus-suppressive ART results in a contraction of anti-HIV-1, CD8+ memory T cell function related to the lower HIV-1 antigenic burden [10,11,12,13]. Based on recent evidence that dendritic cells (DC) are important for activation of memory CD8+ T cell reactivity to influenza A virus, herpes simplex virus type 1 and human cytomegalovirus [14,15,16,17,18], we hypothesized that DC could enhance the breadth of T cell responses to HIV-1, particularly in persons on ART. In the present study, we therefore analyzed the breadth of memory, recall CD8+ T cell responses in vitro from HIV-1 infected subjects on ART to DC loaded with HIV-1 peptides. Our results show that HIV-1 peptide-loaded, mature DC induced IFNc production to a much broader range of HIV-1 Gag and Nef epitopes than did peptides without DC. The MHC class I restricted Gag and Nef epitopes included novel ones that could activate polyfunctional T cells producing various combinations of IFNc interleukin 2 (IL-2), TNFa, macrophage inhibitory protein 1b (MIP-1b) and the cytotoxic de-granulation molecule CD107a. This indicates that there is a broader and more robust array of memory CD8+ T cells specific for HIV-1 antigens circulating in persons on ART than has previously been appreciated, and supports use of DC-based immune therapies. Study subjects This research was part of the Pittsburgh Multicenter AIDS Cohort Study (MACS), an investigation of the natural history of HIV infection, and was approved by the University of Pittsburgh Institutional Review Board. 7 HIV-1 seropositive homosexual men on ART were randomly selected for study from the Pittsburgh, PA, portion of the MACS (Table S1). Four HIV-1 seronegative persons were included as controls. All study subjects gave written informed consent. DC cultures To obtain immature DC, CD14+ monocytes were positively selected from peripheral blood mononuclear cells (PBMC) using antiCD14 monoclonal antibody (mAb)-coated magnetic microbeads (StemCell Technologies, Vancouver, Canada) to a purity of .96%, cultured for 5 to 6 days in AIM V medium (GIBCO, Grand Island, NY) containing 1000 U/ml of recombinant IL-4 (R & D Systems, Minneapolis, Minn.) and 1000 U/ml of recombinant granulocytemonocyte colony stimulating factor (GM-CSF) (Amgen, Seattle, WA). Fresh IL-4 and GM-CSF were added every other day. The DC were treated with maturation factor CD40L (0.5 mg/ml; Amgen or Alexis, San Diego, CA) for 40 h to induce DC maturation. The number of viable DC was determined by typical morphology in trypan blue dye-stained preparations. The maturation status of the DC was determined by flow cytometry as the percent positive and mean fluorescent intensity of expression of MHC class II (HLADR), MHC class I (HLA ABC), CD80, CD86 and CD83. Viable DC displayed a characteristic DC morphology and cell surface marker expression and responded to stimulation with CD40L. Synthetic peptides A library of HIV-1 peptides (consecutive 15mers overlapping by 11 amino acids) spanning the consensus B HIV-1 proteome was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. These were used as singlets or in pools of con (...truncated)