Figure 1.
CD161−CD56+ CD8 T cell subset in normal PBMC.
(A) Existence of CD161−CD56+ CD8 T cell subset in normal PBMC. Flow cytometric analysis of cell surface expression of CD3, CD8, CD161 and CD56 on PBMC from healthy donors. The left plot was gated on lymphocytes and the right plot on CD8 T cells. Plots are representative of PBMC from 22 healthy donors. (B) Percentage of CD161−CD56+ CD8 T cells in total CD8 T cells. Each dot represents one individual (n = 22). Horizontal bars indicate the mean. Cell subset gating was as shown in part (A) right plot.
Figure 2.
Distinct cell surface antigen expression profile of CD161−CD56+ CD8 T cells.
PBMC isolated from healthy donors (n = 5) were stained and analyzed as described in Materials and Methods. Plots shown are from one representative individual. Conventional CD8 T cells (CD161−CD56−) are compared to CD8 Treg (CD161−CD56+) and Control (non-stained lymphocytes).
Figure 3.
Cell surface antigens expressed by CD161−CD56+ CD8 T cells.
PBMC isolated from healthy donors were stained and analyzed as described in Materials and Methods. Intensity of cell surface expression of various markers shown in each graph is compared for different CD3+CD8+ subsets: CD8 Treg (CD161−CD56+); conventional CD8 T cells (CD161−CD56−); and CD8 NKT cells (CD161+CD56+). Mean and standard deviation are shown (n = 5).
Figure 4.
CD161−CD56+ CD8 Treg are CD8αβ+Vα24− cells with abundant cytoplasm.
(A) Morphology of CD161−CD56+ CD8 Treg. PBMC were stained as described in Materials and Methods. CD56pos (CD3+CD8+CD161−CD56+) CD8 Treg and CD56neg (CD3+CD8+CD161−CD56−) CD8 Tcon were purified by cell sorting. Isolated CD56pos and CD56neg cells were stained with Wright-Giemsa stain and visualized using a bright-field microscope. Photos are from one representative experiment of six. (B) Cell surface expression analysis of CD8β and Vα24 on CD56pos and CD56neg cells. CD56pos (CD8 Treg) and CD56neg (CD8 Tcon) were purified by fluorescence-activated cell sorting. Both subsets were CD161 negative. Sorted cells were analyzed for expressions of CD8β and Vα24 as described in Materials and Methods. Data are from one of four representative healthy donors.
Figure 5.
Cell surface phenotype of PHA-activated CD8 Treg.
(A) Representative histograms for cell surface antigen expression on PHA activated CD8 Treg (CD56pos) and CD8 Tcon (CD56neg). CD56pos and CD56neg cells isolated from PBMC were stimulated with PHA and cultured in the presence of feeder cells for 8 days before being analyzed for cell surface marker expression as described in Materials and Methods. Data shown are from one of 5 representative healthy donors. (B) Activated CD56pos cells maintained high expression of CD56 and CD94 and lower expression of CD62L and CD27. CD8 Treg (CD56pos) and CD8 Tcon (CD56neg) cells isolated from PBMC were stimulated with PHA and cultured in the presence of feeder cells for 9 to 16 days before being analyzed for intensity of expression of CD56, CD62L, CD27 and CD94 cell surface antigens. p-value was calculated with unpaired two-tailed Student’s t-test. Mean and standard deviation are shown (n = 5).
Figure 6.
Activated CD56pos cells induce cell death in TCR-activated allogeneic and autologous CD4 T cells.
(A) Suppression of activated allogeneic clonal CD4 T cells by CD8 Treg (CD56pos) cells. CD56pos and CD56neg cells isolated from healthy donors were stimulated with PHA and expanded for 7–9 days. Suppression assays were performed as described in Materials and Methods. (CD56pos, αCD3), the suppressive activity of CD8 Treg towards CD3-activated S2B5 cells; (CD56neg, αCD3), the suppressive activity of CD8 Tcon towards CD3-activated S2B5 cells; (CD56pos), the suppressive activity of CD8 Treg towards non-activated S2B5 cells; (CD56neg), the suppressive activity of CD8 Tcon towards non-activated S2B5 cells. *p<0.05. Results are pooled from three healthy donors (n = 3). p values were calculated with unpaired two-tailed Student’s t-test compared to (CD56neg, αCD3). Mean and standard deviation are shown. (B) Activated CD8 Treg induce lysis of TCR-activated autologous polyclonal CD4 T cells. Isolated CD8 Treg (CD56pos) and CD8 Tcon (CD56neg) were stimulated with PHA and expanded for 8–15 days. Isolated autologous polyclonal CD4 T cells were stimulated with PHA and expanded for 8 days. Suppression assays were performed as described in Materials and Methods. (CD56pos, αCD3), the suppressive activity of CD8 Treg towards pre-activated autologous CD4 T cells; (CD56neg, αCD3), the suppressive activity of CD8 Tcon towards pre-activated autologous CD4 T cells; (CD56pos), the suppressive activity of CD8 Treg towards non-activated autologous CD4 T cells; (CD56neg), the suppressive activity of CD8 Tcon towards non-activated autologous CD4 T cells. *p<0.05. Results are pooled from two independent sorting experiments. P values were calculated with unpaired two-tailed Student’s t-test compared to (CD56neg, αCD3). Mean and standard deviation are shown. (C) Activated CD8 Treg induce lysis of TCR-activated allogeneic polyclonal CD4 T cells. Isolated CD56pos and CD56neg cells were stimulated with PHA and expanded for 8–15 days. Isolated allogeneic polyclonal CD4 T cells were stimulated with PHA and expanded for 8–10 days. Suppression assays were performed as described in Materials and Methods. (CD56pos, αCD3), the suppressive activity of CD8 Treg towards pre-activated allogeneic polyclonal CD4 T cells; (CD56neg, αCD3), the suppressive activity of CD8 Tcon towards pre-activated allogeneic polyclonal CD4 T cells; (CD56pos, IgE), the suppressive activity of CD8 Treg towards isotype control mouse IgE-treated allogeneic polyclonal CD4 T cells; (CD56neg, IgE) the suppressive activity of CD8 Tcon towards isotype control mouse IgE-treated allogeneic polyclonal CD4 T cells. *p<0.05. Results are pooled from six healthy donors (n = 6). p-values were calculated with unpaired two-tailed Student’s t-test compared to (CD56neg, αCD3). Mean and standard deviation are shown.