Introduction: The presence and relative abundance of specific CD4+ T cell populations are highly relevant for immunologic responses and allergic reactions. The activation and differentiation of naïve T cells into T helper (Th) cells are precisely controlled processes. Besides transcriptional regulation, mechanisms influencing the translational efficiency and stability of RNA can affect protein biosynthesis and therefore cell phenotype and function. Short open reading frames (sORF) encoding small peptides have been implicated to impact diverse cellular functions. Our aim is to determine the effect of translational regulation on T cell differentiation and activation and thereby contribute to elucidate the regulatory network of T cell differentiation beyond the role of transcriptional regulation.

Methods: We are using ribosome profiling and RNA sequencing of Th cell populations during differentiation and shortly after activation to detect so far unknown translation events and estimate translation efficiencies of RNAs. The sequencing reads resulting from these samples are mapped against the human reference genome including annotations of non-protein coding regions.

Results: We have been able to successfully generate ribosome profiling libraries of Th cell populations. Ribosome footprints can be detected in known coding sequences while they are absent in untranslated 3` regions of RNA. Identified sORFs will be further verified and analyzed for potential immunomodulatory capacities. Furthermore, we observed that standard pipelines intended for RNA sequencing should be used with care to analyze ribosome profiling data.

Conclusion: The procedure presented here promises to provide valuable insights into the translatome of T cell differentiation. Identifying differentially regulated peptides or other mechanisms could potentially provide a suitable target for the treatment of allergic diseases.