Rltpr Is A Central Scaffold Protein Regulating Human Tcr Co-Signaling And Cytoskeletal Dynamics

T cells are central players in adaptive immunity and T cell receptor (TCR) signaling and co-signaling govern immune defense, immune homeostasis and immune surveillance. Upon antigen encounter, the TCR becomes engaged and in concert with context-sensitive co-receptors, such as CD28, T cell activation, differentiation and function are achieved. Whereas TCR-dependent signaling has been subject to intense investigations, knowledge about CD28-dependent signal cascades is rather incomplete. In murine T cells, Rltpr was discovered to play an important role in CD28 co-signaling. We identified four patients from two consanguineous families presenting with generalized EBV-associated smooth muscle cell tumors and susceptibility to recurrent viral, bacterial, and fungal infections. All patients had homozygote loss-of-function mutations in RLTPR (c.489insG and c.871+1Gu003eT respectively) and did not express RLTPR protein. Primary RLTPR-mutated T cells were characterized by deficiency to signal via CD28. In addition, the cells showed disturbed cytoskeletal microtubule network and defective migratory pattern with increased spontaneous motility in 2D and 3D, but reduced migratory persistence. To determine the protein interactome of human RLTPR, we designed CRISPR/Cas9 constructs and targeted the RLTPR locus in Jurkat cells. We derived five RLTPR-defective Jurkat clones, as determined by Western blot analysis and Sanger sequencing studies. Next, these clones were engineered via retroviral vectors to re-express a GFP-tagged human RLTPR protein. Jurkat T-cell clones expressing RLTPR with N-terminal-GFP, RLTPR with C-terminal-GFP or GFP only were stimulated with anti-CD3/CD28 or left unstimulated and lysed after 15 minutes. Upon pull-down using GFP-Trap, the RLTPR interactome was comprehensively investigated by nano liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzed using Maxquant. Confirmation by immunoprecipitation and western blotting was performed for selected proteins. In seven independent experiments, 903 different proteins were detected in total, 192 proteins repeatedly in at least half of the RLTPR-GFP samples. Quantitative analysis and statistical evaluation yielded 26 proteins significantly enriched in the RLTPR-GFP compared to the control samples (permutation-based q-values The interactome of RLTPR in genetically modified Jurkat cells primarily included cytoskeletal proteins such as capping proteins and (co)chaperones. In addition, RLTPR also interacted with a metabolic enzyme involved in nucleotide synthesis and with vesicle transport proteins involved in the initiation of TCR signaling. Known TCR or CD28 signaling molecules were not detected indicating an indirect contribution of RLTPR in these pathways. In summary, studying rare patients with EBV-associated smooth-muscle cell tumors has allowed us to define RLTPR-deficiency as a novel human primary immunodeficiency disorder and to define RLTPR as a central scaffolding molecule for CD28-mediated TCR co-signaling, cytoskeletal dynamics and metabolic signaling. Disclosures No relevant conflicts of interest to declare.