Increased program cell death-1 expression on T lymphocytes of patients with progressive multifocal leukoencephalopathy

Abstract

The cellullar immune response is important in the containment of progressive multifocal leukoencephalopathy (PML). We examined program cell death-1 (PD-1) expression, a marker of cellular immune exhaustion, on T lymphocytes in PML. PD-1 expression was elevated on total CD4(+) and CD8(+) T cells (medians 36% and 24%) in PML patients compared with healthy control subjects (medians 14% and 18%; P = 0.0015 and P = 0.033). In PML patients, JC virus (JCV)-specific CD8(+) cytotoxic T lymphocytes expressed PD-1 more frequently than total CD8 T lymphocytes (means 39% and 78%, P = 0.0004). Blocking the PD-1 receptor increased JCV-specific T-cell immune response in a subgroup of PML patients.

Figure 1. Percentage of PD-1⁺ CD4⁺ and CD8⁺ T-cells in PML patients and controls

Blocking PD-1 receptors increased JCV-specific CTL response to JCV VP1_-p36 restricted by HLA A*0201 in a PML early patient but not in a PML survivor. Seven million PBMC were incubated with blocking antibody, anti-human CD279 (PD-1) (clone EH12.2H7) at 10ug/ml half-hour prior to addition of HLA-_A*0201-restricted nonamer epitope peptides of the JCV VP1 protein, VP1_p36-SITEVECFL or VP1_p100-ILMWEAVTL, at final concentrations of 2.5 μg/ml and 1 μg/ml, respectively. IL-2 was added 72 hours later; and every 2 days thereafter, half the medium was replaced. PD-1 blocking antibody was replenished on day 7. Intracellular staining assay was performed after 11 days in culture. HIV+/ PML E: HIV-positive PML early patient who was within 6 months of PML diagnosis; HIV+/PML S: HIV-positive PML survivor who was at least 1 year after PML diagnosis. Shaded histograms indicate IgG isotype control. Numbers on upper right quadrant indicate percentage of cells.

Figure 2. JCV-specific T-cell response can be augmented by blocking PD-1 in some patients

While PML patients with and without HIV have significantly elevated median PD-1 expression on CD4+ T-cells (A) and CD8+ T-cells (B) compared to healthy controls, their PD-1 expression levels on T-cells are similar to HIV-positive patients without PML. PD-1 staining was performed with anti-human CD279 (PD-1) (clone EH12.2H7, Biolegend) at 25ug/ml, and IgG₁(clone MOPC-21, Biolegend) was used as isotype control at 10ug/ml as per manufacturer’s recommendation. JCV-specific CD8+ T-cells had significant elevated levels of PD-1 expression when compared to total CD8+ T-cells in PML patients with and without HIV(C). This difference did not reach significance in the HIV-positive study group. Seven million PBMC of three HIV-positive patients and six patients with PML were stimulated with HLA-_A*0201-restricted nonamer epitope peptides of the JCV VP1 protein, VP1_p36-SITEVECFL or VP1_p100-ILMWEAVTL, at final concentrations of 2.5 μg/ml and 1 μg/ml, respectively. Cells were cultured in RPMI 1640-12% FCS in a well of a 12-well plate. After 72 h, an equal volume of medium containing 50U of recombinant interleukin-2 (IL-2) was added to each well, and every 2 days thereafter, half of the medium was replaced. Cells were then stained with JCV VP1_p36 and _-p100 tetramers on days 10-11 of culture. Horizontal line illustrates median of each group and Mann Whitney U test was used to compare groups in A) and B). Bar graphs express means and standard deviations of means and paired t-test was used to compare groups in C).