Increased programmed death-1 expression on CD4+ T cells in cutaneous T-cell lymphoma: implications for immune suppression

Abstract

Objectives: To investigate the expression profile of programmed death-1 (PD-1) on T cells derived from patients with cutaneous T-cell lymphoma (CTCL), analyze a potential mechanism responsible for upregulation of PD-1, and assess the correlation between blockade of its signaling pathway and improvement in immunological function.

Design: Translation research study.

Setting: University medical center.

Participants: Patients with Sézary syndrome, patients with mycosis fungoides, and healthy volunteers.

Main outcome measures: Programmed death-1 expression on T cells by flow cytometry and interferon γ (IFN-γ) production by enzyme-linked immunosorbent assay.

Results: We report significantly increased PD-1 expression on CD4(+) T cells from patients with Sézary syndrome compared with CD4(+) T cells from patients with mycosis fungoides and healthy volunteers. Both CD26(-) and CD26(+) populations of CD4(+) T cells demonstrated increased expression of PD-1, which was upregulated by the engagement of the T-cell receptor with anti-CD3/CD28 antibodies. In addition, blockade of the signaling pathway with blocking antibodies to PD-1 or its ligand PD-L1 led to an increase in the capacity to produce IFN-γ among some patients. Finally, longitudinal studies of 1 patient revealed a progressive decrease in PD-1 expression on CD4(+) T cells with improvement of clinical disease.

Conclusion: Our data imply that increased PD-1 expression in Sézary syndrome may play a role in attenuating the immune response and provide further insight into the immunosuppressive nature of CD4(+) T cells in Sézary syndrome and suggest another potential means of targeted therapy for these patients.

Figure 1

Increased expression of programmed death-1 (PD-1) on CD4⁺ T cells from patients with Sézary syndrome (SS). The peripheral blood mononuclear cells from patients with SS (n = 7), patients with mycosis fungoides (MF) (n = 4), and healthy donors (HDs) (n = 4) were stained with anti-CD4, anti–PD-1, and anti-CD26, and were analyzed by flow cytometry. The percentage of CD4⁺ PD-1⁺ is demonstrated according to patient population and CD26 status. Error bars indicate the standard deviation of uncertainty.

Figure 2

Activation of CD4⁺CD26⁻ T cells by anti-CD3/CD28 antibodies varies between patients with a low and high tumor burden in circulation. The peripheral blood mononuclear cells were isolated from either a patient with a low tumor burden (A) or a high tumor burden (B) and were cultured with medium alone or with anti-CD3/CD28. After 48 hours of stimulation, cells were harvested, stained with antibodies, and analyzed by flow cytometry. In both A and B, the top panels demonstrate cells cultured with medium alone, and the lower panels demonstrate cells cultured with anti-CD3/CD28 antibodies. Panels on the left reveal the expression of CD26 on CD4⁺ T cells, the center panels reveal the expression of programmed death-1 (PD-1) on the CD4⁺CD26⁻ population, and panels on the right reveal the expression of PD-1 on the CD4⁺CD26⁺ population. The numbers in the quadrants represent the percentages of cells that are double positive for each condition, and the numbers in parentheses represent the mean fluorescent intensity for PD-1 expression. Results shown are representative of a total of 12 patients tested. APC indicates allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; and PerCP, peridinin chlorophyll protein complex (all from BD Pharmingen, San Jose, California).

Figure 3

Blocking the pathway of programmed death-1 (PD-1) and its ligand PD-L1 results in increased interferon γ (IFN-γ) production by patients’ peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28. The PBMCs of patients with Sézary syndrome were cultured for 72 hours with either medium or anti-CD3/CD28 alone, and with the following: murine IgG (mIgG), anti–PD-1, or anti–PD-L1. Subsequently, culture supernatants were collected and tested for the presence of IFN-γ. There was no detectable level of IFN-γ in samples treated with medium only (data not shown).