Figure 1.
Expression of CD45RO on PBMC of pigtailed macaques.
Flow cytometry was performed on freshly isolated PBMC. A. CD45RO was detected by indirect immunofluorescence on PBMC from two uninfected pigtailed macaques and for comparison, a human sample. Cells were gated on singlets and lymphocytes by light scatter. Murine IgG2a isotype controls are shown in blue. Data represent 1 of 3 independent experiments. B–D. Direct immunofluorescent staining of PBMC with a panel of Abs to CD3, CD4, CD8, and CD45RO. Cells were gated on lymphocytes by light scatter, then on CD3+. Panel B shows fluorescence minus one (FMO) control used to determine the CD45RO gate (left) and UCHL1 stained sample (right) in a SHIV-infected macaque (representative of 2 independent experiments). In panel C, we examined CD4 and CD8 on T-cells gated for CD45RO expression (one of 21 macaques so tested). The proportion of CD4 or CD8 PBMCs expressing CD45RO in 21 SHIV-infected macaques is shown in panel D.
Figure 2.
Binding of UCHL1 to lymphocytes from different macaque species.
Cryopreserved PBMC from human, and uninfected pigtailed, rhesus and cynomolgus macaques were stained with commercially prepared UCHL1 conjugates with PE, APC, or FITC according to the manufacturer’s recommendation, or by indirect immunofluorescence using 10 µg/ml of unconjugated UCHL1 (red histograms). Directly conjugated isotype controls were run for human and pigtailed macaques (blue histograms), and unstained controls for rhesus and cynomolgus. Data representative of n=2 independent replicate experiments.
Figure 3.
Binding of directly conjugated UCHL1 to lymphocytes from rhesus macaques.
Direct immunofluorescence was performed on freshly isolated PBMC from four SHIV-infected and one uninfected rhesus macaques. UCHL1 conjugated to PE, APC, PerCP Cy5.5, or FITC and Ab to CD3, CD4 and CD8 were used. Fluors used in each panel were adjusted to accommodate the fluor on UCHL1. Cells were gated on singlets and lymphocytes by light scatter, and then on CD3+ CD4+ cells. FMO controls are shown in blue.
Figure 4.
Cryopreserved PBMC from an uninfected pigtailed macaque and a human were stained with panels of Abs to memory markers (CD45RO, CD45RA, CD95, CD28 or CD45RO, CD45RA, CCR7, CCR5). Cells were gated on singlets and lymphocytes by light scatter, and then gated on single-positive CD45RO or CD45RA. Human cells are displayed in the top row and macaque cells in the bottom row. Left: CD45RO and CD45RA expression on lymphocytes, showing gates used to distinguish CD45RO+ and CD45RA+ cells in center and right panels. Center: CD95 and CD28 expression on CD45RO and CD45RA single-positive cells. Right: CCR7 and CCR5 expression CD45RO and CD45RA single-positive cells. Data are from a single experiment.
Figure 5.
Relative avidity of UCHL1 for human and pigtailed macaque CD45RO.
A. Indirect immunofluorescence and flow cytometry were performed on human and 2 sets of pigtailed macaque (JV12 and JV18) PBMC. Equal numbers of thawed, rested PBMC were incubated with serial dilutions of UCHL1 before staining with an excess of Alexa-488 conjugated anti-mouse IgG. Acquired cells were gated on singlets and lymphocytes by light scatter. A gate for UCHL1 positive cells was then drawn. Data presented in red are the mean fluorescent intensity of the positive cells. Staining of cells by IgG2a isotype controls are shown in blue. Data representative of n=3 experiments. B. Human and pigtailed macaque (JV12 and JV18) PBMC were stained with biotinylated UCHL1 and streptavidin-PE in a direct or indirect staining method. For the direct method, biotinylated mAb was incubated with streptavidin-PE for twenty minutes before adding the multimeric complex (to a final concentration of 20µg/ml Ab and 2µg/ml streptavidin-PE) to the cells. For the indirect method, cells were first incubated with 20µg/ml of biotinylated mAb for thirty minutes, washed thoroughly and then incubated with 2µg/ml of streptavidin-PE. Acquired cells were gated on singlets and lymphocytes by light scatter. Direct immunofluorescence with multimeric UCHL1 is shown in red. Indirect immunofluorescence with monomeric UCHL1 is shown in blue. Autofluorescence of unstained PBMC is shown in grey. Data representative of a single experiment.
Figure 6.
CD45RO on T-cells isolated from tissue.
Lymphocytes were isolated from tissue explants taken at the time of necropsy from one uninfected (M04004) and three aviremic SHIV-infected pigtailed macaques. Lymphocytes were pooled from the mesenteric and bronchial lymph nodes (internal LN) and from the axillary and inguinal lymph nodes (peripheral LN). Cells were then stained with Abs to CD3, CD4, CD8, and CD45RO and analyzed by flow cytometry. Acquired cells were gated on lymphocytes by light scatter, and then on CD3+ cells. Stained cells are shown in red. Unstained controls are shown in blue. Data representative of n=5-6 macaques.
Figure 7.
CD45RO expression on T-cells in the gut.
Lymphocytes were isolated from GALT of colon and duodenum of a SHIV-infected pigtailed macaque at time of necropsy and stained with Abs to CD3, CD4, CD8, and CD45RO. Cells were analyzed by FACS. Lymphocytes were gated by light scatter, and then on CD3+ cells. Cells were further gated on CD4 and CD8. Expression of CD45RO is shown in red. Unstained cells are shown in blue. Data representative of n=5 macaques.
Figure 8.
Expression of memory markers on CD4+ lymphocytes from different tissues.
Cryopreserved lymphocytes from GALT of colon and duodenum, and PBMC from an uninfected pigtailed macaque and a human subject were stained with panels of Abs (either CD4, CD45RO, CD95, CD28 or CD4, CD45RO, CCR7, CCR5). Lymphocytes from the colon and duodenum were also live/dead stained. Acquired cells were gated on singlets, live cells and then lymphocytes before gating on CD4 and CD45RO. CD45RO+ and CD45RO- subsets of CD4+ cells were analyzed for CD95 and CD28, or CCR7 and CCR5. Left: CD4 and CD45RO expression. Center: CD95 and CD28 expression on CD4+ CD45RO- and CD4+ CD45RO+ cells. Right: CCR7 and CCR5 expression of CD4+ CD45RO- and CD4+ CD45RO+ cells.
Figure 9.
Mitogen stimulation and CD45RO expression.
Cryopreserved PBMC from human, and uninfected pigtailed (JV12 and JV18) and rhesus (HE91) macaques were stained with UCHL1-multimers or were stimulated with PHA for 48 hours, and then maintained in medium containing IL-2. The resulting blast cells were stained with UCHL1-multimers on days 7 and 14. Cells were analyzed by flow cytometry. Acquired cells were gated on singlets and lymphocytes by light scatter. UCHL1-multimer staining is shown in red. Isotype controls (biotinylated IgG2a-streptavidin-PE multimers) are shown in blue.
Figure 10.
In vivo administration of UCHL1 immunotoxin or mAb.
Groups of three aviremic SHIV-infected macaques each received escalating doses of UCHL1-ricin A chain immunotoxin (panels A and C) or UCHL1 mAb (panels B and D). A. Flow cytometry was performed prior to (blue histograms) and 1hr following (red) administration of the third dose of UCHL1-ricin A chain immunotoxin. Cells were stained with antibody specific for mouse IgG (top), ricin A chain (middle), or CD45RO (bottom). B. Flow cytometry was performed prior to and one hour following the first administration of UCHL1 mAb. Cells were stained with antibody specific for mouse IgG (top) or CD45RO (bottom). C. Percent of CD3+ cells that express CD45RO was followed with time using flow cytometry. Day 0 indicates the start of immunotoxin administration (vertical arrows). D. The effects of administering UCHL1 mAb were followed over time. Day 0 indicates the start of mAb administration (vertical arrows). Left: percent of CD3+ cells that express CD45RO, measured by flow cytometry. Middle: percent of cells detected with fluorescent anti-mouse IgG, measured by flow cytometry. Right: macaque anti-UCHL1 antibody measured by ELISA.