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Inhibition of CD4 expression by antisense oligonucleotides in PMA-treated lymphocytes
Manuel Rabanal1, Angels Franch, Véronique Noé
1Department of Physiology, Division IV, Faculty of Pharmacy, University of Barcelona, Spain.
Abstract:
To decrease CD4 expression on T helper (Th) lymphocyte surface, antisense oligonucleotides (AS-ODNs), delivered by the cationic liposome DOTAP, were assayed in vitro on rat spleen lymphocytes. Four 21-mer ODNs (AS-CD4-1, AS-CD4-2, AS-CD4-3, and AS-CD4-4) directed against the translation start region of the cd4 gene were designed. AS-CD4-1 was phosphorothioate (PS)-modified in each base, and the other three were PS-modified at both ends and in the internal pyrimidine residues. Four ODN controls (fully PS-modified ODN-A and partially modified ODN-B, ODN-C, and ODN-D) were also assayed. CD4 resynthesis was stimulated by treatment with phorbol 12-myristate 13-acetate (PMA) at the same time as the incubations with the ODN. After 24 hours of treatment, CD4 expression was measured by immunofluorescence staining and flow cytometry. CD4 reexpression in rat PMA-treated lymphocytes was counteracted by 40% by means of AS-CD4-2 and AS-CD4-4 treatments. On the other hand, AS-CD4-3 produced only 20% inhibition, similar to that produced by ODN-B, and AS-CD4-1 did not have any significant effect compared with control ODNs. Both AS-CD4-2 and AS-CD4-4 decreased CD4 mRNA, as determined by RT-PCR, and in addition, they did not affect the expression of other surface lymphocyte molecules. Inhibition of surface CD4 expression remained at least 72 hours. The addition of both AS-ODNs did not further increase the effect obtained separately by each AS-ODN. Treatment of rat PMA-lymphocytes with two concentrations of AS-CD4-2 and AS-CD4-4 added 24 hours apart did not further decrease CD4 expression. In summary, AS-CD4-2 and AS-CD4-4 could constitute a good strategy to inhibit CD4 expression on Th lymphocytes and modulate their function.
Insights
Antisense oligonucleotides (AS-ODNs) effectively reduced CD4 expression on T helper lymphocytes. These AS-ODNs offer a promising strategy for modulating T helper cell function.
Area of Science:
- Immunology
- Molecular Biology
- Oligonucleotide Therapeutics
Background:
- T helper (Th) lymphocytes play a crucial role in immune responses.
- CD4 is a key surface marker for Th lymphocytes.
- Modulating CD4 expression is a potential therapeutic strategy.
Purpose of the Study:
- To investigate the efficacy of antisense oligonucleotides (AS-ODNs) in decreasing CD4 expression on rat T helper lymphocytes.
- To evaluate the impact of AS-ODN modifications and delivery methods on CD4 inhibition.
Main Methods:
- In vitro assay on rat spleen lymphocytes using cationic liposome DOTAP delivery.
- Design of four 21-mer AS-ODNs targeting the cd4 gene translation start region.
- Assessment of CD4 expression via immunofluorescence and flow cytometry after phorbol 12-myristate 13-acetate (PMA) stimulation.
- Analysis of CD4 mRNA levels using RT-PCR.
Main Results:
- AS-CD4-2 and AS-CD4-4 AS-ODNs achieved approximately 40% inhibition of CD4 reexpression in PMA-treated lymphocytes.
- These effective AS-ODNs reduced CD4 mRNA levels without affecting other lymphocyte surface markers.
- CD4 expression inhibition persisted for at least 72 hours.
Conclusions:
- AS-CD4-2 and AS-CD4-4 demonstrate significant potential for inhibiting CD4 expression on Th lymphocytes.
- These AS-ODNs represent a viable strategy for modulating Th lymphocyte function.
- Further research may explore their therapeutic applications in immune modulation.