PubMed · 15603449
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John R Mascola. 2004. Neutralizing antibody quantification by flow cytometry.. https://doi.org/10.1016/s0091-679x(04)75030-x
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A combination gene therapy strategy using an ASPsi-gag antisense RNA (targeted against the packaging signal and the gag-coding region) and a multimeric hammerhead ribozyme Rz1-9 (targeted against nine sites within the env-coding region) or Rz1-14 (targeted against 14 sites within the 5' leader and the pro-, pol-, vif- and env-coding regions) was assessed for inhibiting HIV-1 replication. A murine stem cell virus (MSCV)-based MGIN vector was used to express Rz1-9, Rz1-14, ASPsi-gag, Rz1-9ASPsi-gag, or Rz1-14ASPsi-gag RNA in a CD4+ T lymphoid cell line. Stable transductants were shown to express similar levels of interfering RNA. HIV-1 replication was inhibited in cells expressing Rz1-9 and Rz1-14. Little inhibition of HIV-1 replication was observed in cells expressing ASPsi-gag RNA. Thus, the multimeric hammerhead ribozymes inhibit HIV-1 replication better than the antisense RNA. Inhibition of HIV-1 replication in cells expressing Rz1-9ASPsi-gag or Rz1-14ASPsi-gag RNA was worse than that obtained with the multimeric ribozymes alone. This result suggests that co-expression of antisense RNA decreases the anti-HIV potential of ribozymes. The multimeric ribozymes and the antisense RNA were designed to target different sites within the HIV-1 RNA. They are not expected to interact with each other. Neither are they expected to compete with each other for binding to the HIV-1 RNA. Instead, the antisense RNA binding to its (1553 nt-long) target site may have resulted in a decreased ribozyme turn over. Furthermore, since the antisense RNA/HIV-1 RNA hybrids are degraded by the cells, the co-expressed antisense RNA may have led to ribozyme degradation.
In the present study, we analyzed the involvement of the BCR-ABL protein in the induction of antigen-specific CTL in order to develop an immunotherapeutic approach in patients with chronic myelogenous leukemia (CML). To accomplish this, we generated dendritic cells (DC) in vitro and electroporated them with various sources of RNA harboring the chimeric bcr-abl transcript. These genetically engineered DCs were used as antigen-presenting cells for the induction of CTLs. By applying this approach, we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl-positive K-562 cells or CML blasts lysed DCs transfected with the corresponding RNA, but failed to recognize epitopes derived from the chimeric BCR-ABL fusion protein in (51)Cr-release assays. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia, indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. In patients with CML in complete cytogenetic remission during IFN-alpha treatment, we detected some reactivity of CD8(+) T cells against BCR-ABL in IFN-gamma ELISPOT assays, which was weaker as compared with proteinase 3 (PR3)- or prame-directed responses, suggesting that the BCR-ABL protein is less immunogenic as compared with other CML-derived antigens.
High-dose cyclophosphamide (Cy) and G-CSF are widely used to mobilize hemopoietic stem cells for treating patients with high-dose chemotherapy and autologous stem cell transplantation (ASCT). Because lymphocyte count in the graft collected after Cy-G-CSF treatment is an independent survival factor after ASCT for patients with multiple myeloma, our purpose was to study how Cy-G-CSF treatment affects the phenotype and function of T cells in patients with multiple myeloma. Cy induced a 3-fold decrease of T cell counts with a slow and partial T cell recovery of one-third at the time of hemopoietic stem cell collection. Cy-G-CSF treatment did not affect the relative ratios of central memory, effector memory, and late effector CD4+ or CD8+ T cells, but a decrease in the percentage of naive CD4+ cells was observed. The percentages of CD25+ cells increased 2- to 3-fold in CD4+ and CD8+ T cells, the former including both activated CD25low and CD25high cells. CD4+CD25high cells were regulatory T cells (Treg) that expressed high levels of FOXP3, CTLA-4, and GITR and displayed in vitro suppressive properties. The recovery of Treg absolute counts after Cy-G-CSF treatment was higher than the recovery of other lymphocyte subpopulations. In conclusion, Cy-G-CSF treatment induces a severe T cell count decrease without deleting Treg, which are potent inhibitors of antitumor response. The present data encourage novel therapeutic strategies to improve T cell recovery following ASCT while limiting Treg expansion.