(A) Purified human CD3⁺ T cells were stained with antibodies to CD3, CD4 and CD7 before or after treatment with scFvCD7Cys. (B) CD7 expression was assessed at indicated times after preincubation with scFvCD7Cys. (C) siRNA was incubated with scFvCD7-9R or unconjugated scFvCD7Cys at the indicated molar ratios for 15 min and electrophoresed on 1% agarose gels. The position of the non-bound siRNA is indicated. (D) Purified human CD3⁺ T cells (upper panels), CD19⁺ B cells (bottom panel), and differentiated CD14⁺ monocyte-derived macrophages (bottom panel) were treated with FITC-labeled siRNA alone (grey, filled histograms) or siRNA mixed with the indicated reagents (black, open histograms). (E) PHA-activated PBMC were treated with anti-huCD4 siRNA complexed to scFvCD7-9R. CD4 and CD8 expression levels on CD3⁺ T cells were monitored 60 h later (black histograms). Grey filled histograms depict control PBMC treated similarly with scFvCD7-9R/siLuc.

(A) Protocol for scFvCD7-9R/siRNA administration and immunological and virological monitoring of Hu-PBL mice infected with HIV_BaL. (B-D) Hu-PBL mice were treated iv with siCCR5 or control siLuc 14 days after reconstitution. Two days later, the mice were ip infected with HIV_BaL and subsequently either mock-treated (n=2) or treated with a combination of siCCR5/Vif/Tat (test, n=4) or siLuc (control, n=4) complexed to scFvCD7-9R as indicated in (A) and CD3/CD4/CD8 T cell levels were monitored by flow cytometry. Representative dot plots from one test and one control mouse are shown in (B) and cumulative data in (C). Quadrants at each time point were drawn in comparison with corresponding isotype controls. Numbers in (B) represent CD4⁺ or CD8⁺ percentages as a proportion of total CD3⁺ T cells. Error bars indicate standard deviations. (D) Serum p24 levels were measured by ELISA at the indicated times after viral challenge. Horizontal lines indicate median values. (E, F) Hu-PBL mice were treated with siLuc (control) or siCCR5 or siCCR5/Vif/Tat combination (siTRIPLE) complexed to scFvCD7-9R as in (A) and CD4⁺ T cell ratios and plasma p24 levels tested as above.

(A) Peripheral blood from Hu-HSC mice was examined for the presence of human CD4 and CD8 T cells 12 weeks after reconstitution. (B, C) Hu-HSC mice were iv-injected twice, 16 h apart, with siCD4 (test) or control siLuc complexed to scFvCD7-9R and peripheral blood T cells were tested for CD4 and CD8 expression before and 3 days after treatment. Representative dot plots from one mouse in each group are shown in (B) and cumulative data from 3 mice in (C). Numbers indicated in (B) represent the percentage of total CD3⁺ T cells. In (C) the reduction in surface CD4 or CD8 levels was calculated as a percentage of the initial expression level before siRNA injection. (D) Splenocytes isolated from Hu-HSC mice 1 day after a single injection with scFvCD7-9R/siLuc (control) or siCCR5 (test) were examined for CCR5 mRNA levels by qPCR using β-actin mRNA levels for normalization. (E) Splenocytes in (D) were PHA-stimulated and infected with HIV_BaL at a moi of 3 and p24 antigen levels in culture supernatants were assayed in triplicate by ELISA at the indicated time points. Error bars indicate standard deviation. (F) Hu-HSC mice were either mock-treated (n=3), treated iv with siLuc (Control, n=3) or siVif/Tat (Test, n=4) complexed to scFvCD7-9R 22 weeks after reconstitution. 18 h later, the control and test animals were ip infected with HIV_BaL and further treated with scFv/siRNA every 4–5 days. Viral copy numbers in plasma measured by the Amplicor test (upper panel) and CD4⁺CD3⁺ T cell percentages monitored by flow cytometry (lower panel) at various times are shown. The grey dotted line in the upper panel represents the limit of detection of the Amplicor test. CD4 T cell ratios were calculated as a ratio of the entire CD3 population (CD4⁺CD3⁺:CD3⁺) and mean ratios (horizontal grey bars) at 40 d post challenge is shown. Individual animals in each group are represented by distinct symbols.

NOD/SCIDIL2rγ^−/− mice reconstituted with human PBMC were injected iv with siLuc (control) or siCD4 (test) complexed to scFvCD7-9R twice, 16 h apart and human CD3⁺ T cells in the peripheral blood, spleen and liver were analyzed for CD4 and CD8 expression 60 h later. Representative dot plots from one mouse (A), and cumulative data from 3 mice (B), are shown. Asterisks indicate significant and “ns” indicates no significant differences between test and control groups. P < 0.05. (C) PBMC isolated from groups of Hu-PBL mice were PHA-stimulated and infected with HIV_IIIB. Culture supernatants collected on day 10 after infection were tested for p24 antigen levels in triplicate by ELISA. (D) Mice were treated with siRNA 20 days after reconstitution as in (A) three times at 16 h intervals and CD4 and CD8 expression in peripheral blood T cells were determined on days 3, 6 and 9 after the last injection. Error bars indicate standard deviation.

(A) Protocol for siRNA/scFvCD7-9R administration and immunological and virological monitoring. (B, C) Mice transplanted with PBMC from a HIV-seropositive donor were treated iv with scFvCD7-9R complexed to either siLuc (control) or siCCR5/Vif/Tat (test) as indicated in (A) and CD4 T cell levels were monitored by flow cytometry. Representative dot plots from one mouse in each group are shown in (B) and cumulative data from 4 mice in (C). Numbers indicated in (B) represent CD4⁺ percentages as a proportion of total CD3⁺ T cells. (D) Viral copy numbers in plasma were measured by the Amplicor test on day 17 after reconstitution with donor PBMC. Error bars represent standard deviation.

(A) scFvCD7-9R/siLuc-treated or mock-treated PBMC stimulated with PHA were stained with Annexin-V on 4 consecutive days of culture. 24 h staurosporine-treated cultures served as positive control. (B) PBMC treated with scFvCD7-9R/siLuc were stimulated with PHA or antiCD3/CD28 beads for 3 days and pulsed with ³H-thymidine for 18 h. Fold stimulation was calculated by dividing the counts incorporated in the presence of to those in the absence of stimulating agent. (C) Purified human CD4⁺ T cells were stimulated with anti-CD3 mAb in the absence (no treatment) or presence of Pam₃CSK₄ (TLR2 ligand), Poly I:C (TLR3), LPS (TLR4), Flagellin (TLR5), CLO97 (TLR8/9) or anti-CD3/CD28 Dynabeads. IFN-γ was quantified by ELISA in 48 h culture supernatants. Error bars indicate standard deviation of triplicate cultures. (D) Expression profiles of miRNA in CD3⁺ T cells purified from Hu-HSC mice treated thrice with scFvCD7-9R/siCCR5 are depicted. Expression level was normalized to that of small non-coding RNA U6B. Mean of triplicate runs with two animals each ± SD is shown. (E) CD3⁺ T cells purified from Hu-HSC mice treated as in (D) were examined for c-Myb protein levels either immediately or after 48 h in culture with PHA. The numbers below represent the ratios of band intensities of c-Myb normalized to that of β-actin. Error bars in all cases represent standard deviation.