cd8 t cells  (Sony Biotechnology)

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: HTLV-1–Infected CD4 + T Cells Drive Spontaneous Lymphoproliferation and Virus-Specific CD8 + Cytotoxic T Lymphocyte Expansion in HAM/TSP

doi: 10.1212/NXI.0000000000200581

Figure Lengend Snippet: (A) Time course of SP. PBMCs from patients with HAM/TSP and healthy controls (HCs) were cultured without IL-2, either in the absence (left panel) or presence (center panel) of PHA. The upper and lower rows show PBMCs from a patient with HAM/TSP and an HC, respectively, on day 6. Red and blue areas indicate proliferating or nonproliferating cells, respectively. The right panel illustrates the time-dependent proliferation of PBMCs from patients with HAM/TSP (upper panel) and HCs (lower panel) without stimulation. Subsequent analyses of SP were primarily performed on day 6, corresponding to the exponential phase of proliferation. (B) Gating strategy for cell subsets in flow cytometry. Proliferating (red area) and nonproliferating (blue area) cells were classified based on surface markers. αβ T cells: TCRαβ+CD3 + , γδ T cells: TCRγδ+CD3 + , NK cells: CD56 + CD16 + , NKT cells: CD56 + CD3 + , B cells: CD19 + . HTLV-1–infected cells: CADM1+CD4 + CD3 + , and HTLV-1–specific CTLs: HTLV-1 Tax 301–309 tetramer+CD8 + CD3 + . (C) Comparison of cell subsets between preculture and proliferating cells. Changes in cell populations from day 0 to day 6 were analyzed in 10 patients with HLA-A*24:02-positive HAM/TSP. Bar graphs show the proportions of each cell subset (left panel). CD8 + T cells exhibited marked proliferation (second panel), with the proportion of HTLV-1 Tax 301–309-tetramer + cells among CD8 + T cells increasing from an average of 3.81%–17.67% after SP (third panel). Although the proportion of CD4 + T cells among CD3 + cells decreased (fourth panel), the percentage of CADM1+CD4 + cells within the CD4 + T-cell population increased from an average of 13.14%–38.15% (right panel). *Significant differences: p < 0.05, p < 0.01 (Wilcoxon signed-rank test). CTL = cytotoxic T lymphocyte; HAM = human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy; HTLV-1 = human T-cell leukemia virus type 1; TSP = tropical spastic paraparesis; NK cells = natural killer cells; NKT cells = natural killer T cells; SP = spontaneous lymphoproliferation.

Article Snippet: PBMCs from 10 patients with HLA-A*24:02–positive HAM/TSP and 6 ACs were cultured for 6 days, after which CD8 + T cells were sorted using a Cell Sorter SH800 (Sony, Tokyo, Japan).

Techniques: Cell Culture, Flow Cytometry, Infection, Comparison, Virus

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: HTLV-1–Infected CD4 + T Cells Drive Spontaneous Lymphoproliferation and Virus-Specific CD8 + Cytotoxic T Lymphocyte Expansion in HAM/TSP

doi: 10.1212/NXI.0000000000200581

Figure Lengend Snippet: (A) HTLV-1–specific CTLs during SP in patients with HLA-A*24:02-positive HAM/TSP were identified by intracellular IFN-γ production. PBMCs were cultured without exogenous stimulation, and CD8 + T cells were isolated by cell sorting on day 6, followed by a 3-day resting period in culture. These resting CD8 + T cells were then cocultured for 6–8 hours with antigen-presenting cells pulsed with HTLV-1 peptides, including a Tax peptide mixture, an HBZ peptide mixture, and a predicted Gag/Env epitope mixture. After peptide stimulation, proliferating CD8 + T cells were gated, and IFN-γ+ cells were detected. The Tax 301–309 peptide and no-peptide control were used as positive and negative controls, respectively. Representative IFN-γ–producing CD8 + T cells in response to each peptide stimulation are shown for a patient with HAM/TSP. (B) Results from 4 patients with HLA-A*24:02-positive HAM/TSP are summarized after subtracting background responses determined using the no-peptide control for each antigen-specific condition. There were no significant differences among groups by the Kruskal-Wallis test. (C) HTLV-1–infected cells within the proliferating population (red area) were identified either by intracellular staining for HTLV-1 Tax protein using the Lt-4 antibody or by surface detection of CADM1 (a marker for HTLV-1–infected cells). For intracellular Tax detection, brefeldin A was added to the PBMCs on day 6, followed by overnight culture. After gating on CD4 + T cells within the proliferating population, Tax-expressing cells were detected on day 7. In a representative case, 71.14% of proliferating CD4 + T cells were Tax positive (third panel). CADM1-positive cells constituted 38.83% of all CD4 + T cells, with CADM1 expression predominantly observed in the CD4-high subset (fourth panel). Results from 10 patients with HAM/TSP are shown (right panel). Intracellular staining for Tax protein detected a mean of 61.84 ± 10.04% positive cells, demonstrating higher sensitivity compared with surface CADM1 detection, which yielded a mean of 9.42 ± 10.70%. Bars and vertical lines represent the mean and SD, respectively. (D) Changes in HTLV-1 PVL before and after SP in CD4 + and CD8 + T-cell populations. PBMCs were labeled with CellTrace Far Red, and half of the cells were sorted into CD4 + and CD8 + T cells for DNA extraction on day 0. The remaining PBMCs were cultured for 7 days without exogenous stimulation. On day 7, cultured PBMCs were first sorted into proliferating and nonproliferating subsets, followed by a second round of sorting to separate CD4 + and CD8 + T cells. PVLs were measured in each subset. Data from 3 cases are shown. On day 7, red and blue dots represent proliferating and nonproliferating cells, respectively. Dots indicate the mean, and vertical lines represent the SD. CTL = cytotoxic T lymphocyte; HTLV-1 = human T-cell leukemia virus type 1; SP = spontaneous lymphoproliferation.

Article Snippet: PBMCs from 10 patients with HLA-A*24:02–positive HAM/TSP and 6 ACs were cultured for 6 days, after which CD8 + T cells were sorted using a Cell Sorter SH800 (Sony, Tokyo, Japan).

Techniques: Cell Culture, Isolation, FACS, Control, Infection, Staining, Marker, Expressing, Labeling, DNA Extraction, Virus

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: HTLV-1–Infected CD4 + T Cells Drive Spontaneous Lymphoproliferation and Virus-Specific CD8 + Cytotoxic T Lymphocyte Expansion in HAM/TSP

doi: 10.1212/NXI.0000000000200581

Figure Lengend Snippet: (A) Whole PBMCs from patients with HAM/TSP were cultured, and the percentages of proliferating cells in CD4 + and CD8 + T-cell populations were measured on day 6. As shown in the figure, both populations exhibited proliferation, with 45.47% for CD8 + T cells undergoing proliferation (right panel). (B) On day 0, CD4 + and CD8 + T cells were isolated using a cell sorter and cultured independently for 6 days. CD4 + T cells proliferated (34.95%, left panel), whereas CD8 + T cells showed minimal proliferation (2.93%, right panel). (C) On day 0, CD4 + and CD8 + T cells were first separated and then remixed at a 1:1 ratio, followed by culture for 6 days. CD8 + T-cell proliferation was restored to 36.60% (right panel). (D) Results from 3 cases are summarized in the graph. CD8 + T cells do not proliferate in the absence of CD4 + T cells (right panel). HAM = human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy; PBMC = peripheral blood mononuclear cell; SP = spontaneous lymphoproliferation; TSP = tropical spastic paraparesis.

Article Snippet: PBMCs from 10 patients with HLA-A*24:02–positive HAM/TSP and 6 ACs were cultured for 6 days, after which CD8 + T cells were sorted using a Cell Sorter SH800 (Sony, Tokyo, Japan).

Techniques: Cell Culture, Isolation, Virus

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: HTLV-1–Infected CD4 + T Cells Drive Spontaneous Lymphoproliferation and Virus-Specific CD8 + Cytotoxic T Lymphocyte Expansion in HAM/TSP

doi: 10.1212/NXI.0000000000200581

Figure Lengend Snippet: Comparison between preculture cells (day0) and proliferating cells (day6 SP) is shown for CD3 + , CD4 + , and CD8 + T-cell population (n = 7). (A) T-cell differentiation status was assessed based on surface expression of CCR7 and CD45RA. The classification is as follows: naïve T cells (CCR7+CD45RA+), central memory T cells (TCM: CCR7+CD45RA-), effector memory T cells (TEM: CCR7-CD45RA-), and effector memory T cells reexpressing CD45RA (TEMRA: CCR7-CD45RA+). In CD8 + T cells during SP, TEMRA cells markedly decreased, whereas TCM and TEM cells increased. (B) 93.74 ± 8.47% of the proliferating CD4 + T cells expressed CXCR3, indicative of a Th1-like phenotype. Most proliferating CD8 + T cells were also CXCR3-positive (99.08 ± 2.02%) (upper panels). CD25, the α-chain of the IL-2 receptor and a marker of T cell activation, was upregulated after SP: 85.66% ± 13.88% in CD3 + cells, 89.53% ± 13.51% in CD4 + T cells, and 85.41% ± 13.63% in CD8 + T cells (middle panels). In SP cells, 94.46% ± 3.75% of CD3 + cells, 93.17% ± 3.95% of CD4 + T cells, and 96.94% ± 3.17% of CD8 + T cells coexpressed PD-1 and Tim-3, markers indicative of T-cell exhaustion (lower panels). (C) PBMCs from patients with HAM/TSP (n = 10) and HTLV-1–uninfected HCs (n = 6) were cultured without IL-2 or mitogenic stimulation for 6 days, and cytokine and chemokine levels in the supernatant were measured. Bars indicate median values. * p < 0.05, ** p < 0.01, *** p < 0.001 (Mann-Whitney U test). PBMC = peripheral blood mononuclear cell; SP = spontaneous lymphoproliferation.

Article Snippet: PBMCs from 10 patients with HLA-A*24:02–positive HAM/TSP and 6 ACs were cultured for 6 days, after which CD8 + T cells were sorted using a Cell Sorter SH800 (Sony, Tokyo, Japan).

Techniques: Comparison, Cell Differentiation, Expressing, Marker, Activation Assay, Cell Culture, MANN-WHITNEY