Functional capacity of natural killer cells in HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

Abstract

Background: Natural killer (NK) cells are part of the innate immune system and provide surveillance against viruses and cancers. The ability of NK cells to kill virus-infected cells depends on the balance between the effects of inhibitory and activating NK cell receptors. This study aimed to investigate the phenotypic profile and the functional capacity of NK cells in the context of HTLV-1 infection.

Methods: This cross-sectional study sequentially recruited HTLV-1 infected individuals with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and asymptomatic HTLV-1 (AS) from the Integrated and Multidisciplinary HTLV Center in Salvador, Brazil. Blood samples from healthy blood donors served as controls. NK cell surface receptors (NKG2D, KIR2DL2/KIR2DL3, NKp30, NKG2A, NKp46, TIM-3 and PD-1), intracellular cytolytic (Granzyme B, perforin) and functional markers (CD107a for degranulation, IFN-γ) were assayed by flow cytometry in the presence or absence of standard K562 target cells. In addition, cytotoxicity assays were performed in the presence or absence of anti-NKp30.

Results: The frequency of NKp30⁺ NK cells was significantly decreased in HAM/TSP patients [58%, Interquartile Range (IQR) 30-61] compared to controls (73%, IQR 54-79, p = 0.04). The production of cytolytic (perforin, granzyme B) and functional markers (CD107a and IFN-γ) was higher in unstimulated NK cells from HAM/TSP and AS patients compared to controls. By contrast, stimulation with K562 target cells did not alter the frequency of CD107a⁺ NK cells in HAM/TSP subjects compared to the other groups. Blockage of the NKp30 receptor was shown to decrease cytotoxic activity (CD107a) and IFN-γ expression only in asymptomatic HTLV-1-infected individuals.

Conclusions: NK cells from individuals with a diagnosis of HAM/TSP present decreased expression of the activating receptor NKp30, in addition to elevated degranulation activity that remained unaffected after blocking the NKp30 receptor.

Keywords: CD107; HAM/TSP; HTLV-1; NK cells; NKp30; Natural cytotoxicity receptor.

Fig. 1

Phenotypic characteristics of NK cells from individuals with HTLV-1 associated myelopathy /Tropical spastic paraparesis (HAM/TSP), asymptomatic HTLV-1-infected individuals (AS) and uninfected controls (CTR). (A) Lymphocyte gate. (B) Selected NK cells (CD3⁻CD56⁺). (C) Expression of CD56. Activating/inhibitory receptors (D-H) and exhaustion markers (I-J) from one representative individual from each group, out of 17 independent experiments performed. Pink histograms represent specific–stained population and blue histograms represent background fluorescence observed with matched isotype control

Fig. 2

Analysis of cell-surface receptors. HAM/TSP: HTLV-1 associated myelopathy /Tropical spastic paraparesis, n = 12; AS: asymptomatic HTLV-1 carriers, n = 18. CTR: uninfected controls, n = 15. Activating receptors: a NKp30, (b) NKp46 and (d) NKG2D. Inhibitory receptors: c KIR2DL2/KIR2DL3 and (e) NKG2A exhaustion markers: f-h TIM-3 and PD-1. Data are expressed as medians and interquartile interval range. Differences were considered significant when P ≤ 0.05. Kruskal-Wallis test, followed by Dunn’s post-test

Fig. 3

Cytotoxic activity of NK cells from HTLV-1 infected individuals against K562 target cells. HAM/TSP: HTLV-1 associated myelopathy/Tropical spastic paraparesis, n = 6; AS: asymptomatic individuals, n = 6; CTR: uninfected controls, n = 6. Peripheral blood mononuclear cells (PBMC) were cultured in presence or absence of K562 cells (1:1) for 6 h. a expression of IFN-γ, (b) expression of perforin, (c) expression of granzyme B, (d) expression of CD107a. Data are expressed as median and interquartile interval range. Differences considered significant when P ≤ 0.05. Kruskal-Wallis test, followed by Dunn’s post-test

Fig. 4

Representative flow plots for cytokine staining and cytotoxic molecules of NK cells. Lymphocyte gate (A). Selected NK cells (CD3⁻CD56⁺) (B). Representative flow plots showing expression of IFN-γ (C), (CD107a (D), granzyme B (E) and perforin (F) by NK cells from one representative individual with HAM/TSP, an asymptomatic HTLV-1-infected individual (AS) and one uninfected control (CTR), cultured in the presence of K562 for 6 h. Pink dot plots represent specific–stained population and blue dot plots represent background fluorescence observed with matched isotype control. Results reflect one representative experiment out of six independent experiments performed

Fig. 5

Cytotoxic activity of NK cells from HTLV-1 infected individuals against K562 target in the presence or absence of anti-NKp30 monoclonal antibody. Peripheral blood mononuclear cells (PBMC) were cultured in the presence of K562 cells (1:1), and in the presence or absence of NKp30 monoclonal antibody (4 μg/ml), for 6 h. a Expression of CD107a, (b) expression of IFN-γ, (c) expression of granzyme B. In controls, cytotoxic activity (CD107a) was reduced by 36% in the presence of anti-NKp30 antibody. Data are expressed as medians and interquartile interval range. HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP), n = 6; AS: asymptomatic individuals, n = 6. Differences were considered significant when P ≤ 0.05, Wilcoxon-test. *p = 0.01