Immunoprofiling of fresh HAM/TSP blood samples shows altered innate cell responsiveness

Abstract

The Human T-cell Leukemia Virus-1 (HTLV-1)-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a devastating neurodegenerative disease with no effective treatment, which affects an increasing number of people in Brazil. Immune cells from the adaptive compartment are involved in disease manifestation but whether innate cell functions participate in disease occurrence has not been evaluated. In this study, we analyzed innate cell responses at steady state and after blood cell stimulation using an agonist of the toll-like receptor (TLR)7/8-signaling pathway in blood samples from HTLV-1-infected volunteers, including asymptomatic carriers and HAM/TSP patients. We observed a lower response of IFNα+ DCs and monocytes in HAM/TSP compared to asymptomatic carriers, as a potential consequence of corticosteroid treatments. In contrast, a higher frequency of monocytes producing MIP-1α and pDC producing IL-12 was detected in HAM/TSP blood samples, together with higher IFNγ responsiveness of NK cells, suggesting an increased sensitivity to inflammatory response in HAM/TSP patients compared to asymptomatic carriers. This sustained inflammatory responsiveness could be linked or be at the origin of the neuroinflammatory status in HAM/TSP patients. Therefore, the mechanism underlying this dysregulations could shed light onto the origins of HAM/TSP disease.

Fig 1. PVL in PBMCs does not correlate with the clinical status in HTLV-1 infected subjects.

(A) Proviral load (PVL) of the HTLV-1 asymptomatic carriers (AC) and HAM/TSP patients. (B) Graph representing motor scores (IPEC and Osame) and PVL in HAM/TSP patients. (C) Correlation between PVL of HTLV-1-infected individuals and the age at the time of the analysis.

Fig 2. Dendritic cells of HAM/TSP patients present impaired response to stimulation.

(A) Frequency of dendritic cell subsets (cDC1, cDC2 and pDC) among PBMCs in the clinical groups at steady state. HD: healthy donor; AC: asymptomatic carriers; and HAM/TSP: HTLV-1 associated myelopathy/Tropical spastic paraparesis (B) tSNE analysis of DC subsets in innate cell populations at steady state and after TLR7-stimulation with R848. (C) Frequency of the cells producing IFNα, IL-12, MIP-1α and TNFα at steady state and after R848 treatment in the different DC subsets. Statistical significance was determined using one-way ANOVA followed by Turkey post-hoc test. * p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001. (D) Pie-chart of the Boolean analysis, using the data from all samples of each clinical groups, for the cytokine production in DC subsets at steady state (SS) and after TLR7 stimulation (R848).

Fig 3. Monocytes from HAM/TSP patients present greater IL-12 and MIP-1α response to stimulation.

(A) Gating strategy for identification of the three monocyte subpopulations using CD16 and CD3-14-15-19 antibodies. (B) Frequency of innate subsets in the clinical groups evaluated in monocytes subsets (classical monocytes, intermediate monocytes and non-classical monocytes). (C) tSNE analysis of monocyte subset frequencies in whole blood samples at steady state and after TLR7-stimulation with R848. (D) Frequency of the cells producing IFNα, IL-12, MIP-1α and TNFα at steady state and after R848 treatment in the different monocyte subsets. HD: healthy donor; AC: asymptomatic carriers; and HAM/TSP: HTLV-1 associated myelopathy/Tropical spastic paraparesis. Statistical significance was determined using one-way ANOVA followed by Turkey post-hoc test. * p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001. (D) Pie-chart of the Boolean analysis for the cytokine production in monocyte subsets at steady state (SS) and after TLR7 stimulation (R848).

Fig 4. NK cells from HAM/TSP present greater response to IFNγ after stimulation.

(A) Violing-plot representation of the cell frequency of NK subpopulations in the 3 clinical groups. (B) t-SNE clustering of the NK cells reveals different subpopulations and (C) the histogram represents TNFα expression of the identified population (Pop1). (D) Bar-plot representing the frequency of cytokine producing cells by the gated NK subpopulations at steady state and upon R848 stimulation. (E) Boolean analysis of the multiple-cytokine production by the NK subtypes at steady state (NS) and after TLR7/8 stimulation (R848). (F) Correlation between the frequency of IFNγ⁺ NK CD16⁺ and the frequency of IL-12⁺ classical monocytes (left) or pDC (right) after stimulation.