Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Platelet-activating factor causes ventilation-perfusion mismatch in humans

We hypothesized that platelet-activating factor (PAF), a potent inflammatory mediator, could induce gas exchange abnormalities in normal humans. To this end, the effect of aerosolized PAF (2 mg/ml solution; 24 micrograms) on ventilation-perfusion (VA/Q) relationships, hemodynamics, and resistance of the respiratory system was studied in 14 healthy, nonatopic, and nonsmoking individuals (23 +/- 1 [SEM]yr) before and at 2, 4, 6, 8, 15, and 45 min after inhalation, and compared to that of inhaled lyso-PAF in 10 other healthy individuals (24 +/- 2 yr). PAF induced, compared to lyso-PAF, immediate leukopenia (P < 0.001) followed by a rebound leukocytosis (P < 0.002), increased minute ventilation (P < 0.05) and resistance of the respiratory system (P < 0.01), and decreased systemic arterial pressure (P < 0.05). Similarly, compared to lyso-PAF, PaO2 showed a trend to fall (by 12.2 +/- 4.3 mmHg, mean +/- SEM maximum change from baseline), and arterial-alveolar O2 gradient increased (by 16.7 +/- 4.3 mmHg) (P < 0.02) after PAF, because of VA/Q mismatch: the dispersion of pulmonary blood flow and that of ventilation increased by 0.45 +/- 0.1 (P < 0.01) and 0.29 +/- 0.1 (P < 0.04), respectively. We conclude that in normal subjects, inhaled PAF results in considerable immediate VA/Q inequality and gas exchange impairment. These results reinforce the notion that PAF may play a major role as a mediator of inflammation in the human lung.

Platelet-activating factor causes ventilation-perfusion mismatch in humans

We hypothesized that platelet-activating factor (PAF), a potent inflammatory mediator, could induce gas exchange abnormalities in normal humans. To this end, the effect of aerosolized PAF (2 mg/ml solution; 24 micrograms) on ventilation-perfusion (VA/Q) relationships, hemodynamics, and resistance of the respiratory system was studied in 14 healthy, nonatopic, and nonsmoking individuals (23 +/- 1 [SEM]yr) before and at 2, 4, 6, 8, 15, and 45 min after inhalation, and compared to that of inhaled lyso-PAF in 10 other healthy individuals (24 +/- 2 yr). PAF induced, compared to lyso-PAF, immediate leukopenia (P < 0.001) followed by a rebound leukocytosis (P < 0.002), increased minute ventilation (P < 0.05) and resistance of the respiratory system (P < 0.01), and decreased systemic arterial pressure (P < 0.05). Similarly, compared to lyso-PAF, PaO2 showed a trend to fall (by 12.2 +/- 4.3 mmHg, mean +/- SEM maximum change from baseline), and arterial-alveolar O2 gradient increased (by 16.7 +/- 4.3 mmHg) (P < 0.02) after PAF, because of VA/Q mismatch: the dispersion of pulmonary blood flow and that of ventilation increased by 0.45 +/- 0.1 (P < 0.01) and 0.29 +/- 0.1 (P < 0.04), respectively. We conclude that in normal subjects, inhaled PAF results in considerable immediate VA/Q inequality and gas exchange impairment. These results reinforce the notion that PAF may play a major role as a mediator of inflammation in the human lung.