Gene expression profile of human T cells following a single stimulation of peripheral blood mononuclear cells with anti-CD3 antibodies.

BACKGROUND: Anti-CD3 immunotherapy was initially approved for clinical use for renal transplantation rejection prevention. Subsequently, new generations of anti-CD3 antibodies have entered clinical trials for a broader spectrum of therapeutic applications, including cancer and autoimmune diseases. Despite their extensive use, little is known about the exact mechanism of these molecules, except that they are able to activate T cells, inducing an overall immunoregulatory and tolerogenic behavior. To better understand the effects of anti-CD3 antibodies on human T cells, PBMCs were stimulated, and then, we performed RNA-seq assays of enriched T cells to assess changes in their gene expression profiles. In this study, three different anti-CD3 antibodies were used for the stimulation: two recombinant antibody fragments, namely, a humanized and a chimeric FvFc molecule, and the prototype mouse mAb OKT3. RESULTS: Gene Ontology categories and individual immunoregulatory markers were compared, suggesting a similarity in modulated gene sets, mainly those for immunoregulatory and inflammatory terms. Upregulation of interleukin receptors, such as IL2RA, IL1R, IL12RB2, IL18R1, IL21R and IL23R, and of inhibitory molecules, such as FOXP3, CTLA4, TNFRSF18, LAG3 and PDCD1, were also observed, suggesting an inhibitory and exhausted phenotype. CONCLUSIONS: We used a deep transcriptome sequencing method for comparing three anti-CD3 antibodies in terms of Gene Ontology enrichment and immunological marker expression. The present data showed that both recombinant antibodies induced a compatible expression profile, suggesting that they might be candidates for a closer evaluation with respect to their therapeutic value. Moreover, the proposed methodology is amenable to be more generally applied for molecular comparison of cell receptor dependent antibody therapy.

MicroRNA expression profiles in human CD3+ T cells following stimulation with anti-human CD3 antibodies.

BACKGROUND: Anti-CD3 therapy can induce immunosuppression by several non mutually exclusive mechanisms that have been proposed to explain the therapeutic effect the administration anti-CD3 mAb, but its immunoregulatory mechanism is still not completely clear. In T cells, microRNAs (miRNAs) regulate several pathways, including those associated with immune tolerance. Here, we report changes in miRNA expression in T cells following treatment with anti-human CD3 antibodies. Peripheral blood mononuclear cells were cultured in the presence of the monoclonal antibody OKT3 or a recombinant fragment of humanized anti-CD3. Following these treatments, the expression profiles of 31 miRNA species were assessed in T cells using TaqMan arrays. RESULTS: Eight of the tested miRNAs (miR-155, miR-21, miR-146a, miR-210, miR-17, miR-590-5p, miR-106b and miR-301a) were statistically significantly up- or down-regulated relative to untreated cells. CONCLUSIONS: Stimulation of T cells with anti-human CD3 antibodies alters miRNA expression patterns, including of miRNA species associated with immune regulatory pathways.

Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody.

Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.