ODN 2216 Uptake Induced TLR9 Signaling Mediates Phenotypic Changes in CD4+ T Cells

Background: Oligodeoxynucleotides (ODNs) are established TLR9 ligands; however, their functional responses in CD4+ T cells are believed to be independent of TLR9 and MyD88. Methods: We studied ligand-receptor interactions for ODN 2216 and TLR9 in human CD4+ T cells using confocal microscopy and flow cytometry. We assessed the consequences of this interaction in terms of TLR9 signaling and its effects on cell proliferation and cytokine expression using real time RT-PCR and flow cytometry. Findings: We demonstrated that endocytosed ODN 2216 physically interacts with intracellular TLR9 inside lysosomes in CD4+ T cells. The uptake of ODN 2216, controlled by TLR9 signaling molecules, resulted in an increase in the expression of TLR9 signaling molecules. Further, we observed that downstream signaling molecules controlled the expression of TLR9 in CD4+ T cells in a feedback dependent manner. Next, the uptake of ODN 2216 resulted in TLR9 signaling dependent but MyD88 independent increase in expression of TGF-I². Finally, ODN 2216 treated CD4+ T cells showed an anti-inflammatory phenotype that was quite similar to Th3 type of regulatory T cells. These Th3 like cells were able to suppress the proliferation of untreated CD4+ T cells. Interpretation: Collectively, our results demonstrate a direct and interdependent relationship between ODN 2216 uptake and TLR9 signaling in CD4+ T cells. Our findings thus, pave the way for future research to explore direct modulation of adaptive immune cells, using innate immune ligands, to subvert exaggerated inflammatory responses. Funding Statement: Centre of Excellence, Advance Eye Centre, PGIMER funded by Department of Biotechnology (DBT), Government of India, New Delhi for support (Grant No. BT/01/CEIB/11/02) (awarded to AG and NS) and Indian Council of Medical Research for providing fellowship to RS (3/1/3/JRF-2012/HRD-87). This study was supported in part by the National Institutes of Health/Fogarty International Centre training grant D43 TW009588 (awarded to SL), and the 1R01NS097147 grant (awarded to PJ). Declaration of Interests: Authors declare no conflict of interest. Ethics Approval Statement: The Institutional ethics committee duly approved the study (No’s PGI/IEC/2011/532- 533/24/11/11 and NK/1385/PhD/2484/07/09/17). All subjects gave written informed consent in accordance with the Declaration of Helsinki