Dinkel, Brittney A.

Abstract T cell cytokine production was recently shown to depend not only on the T cell antigen receptor (TCR), but also on the chemokine receptor CXCR4. Our lab recently published that stimulation of the TCR results in the TCR associating with and transactivating CXCR4, which, in turn, activates a PREX-1/Rac-1 pathway that stabilizes cytokine mRNA. This pathway therefore normally significantly increases IL-2, IL-4, and IL-10 production by activated T cells. Here, we further characterize the molecular mechanisms responsible for the first step of this pathway: TCR-mediated TCR-CXCR4 complex formation. First, we used FRET and proximity ligation assay (PLA) to define the required structural motifs of CXCR4, showing that this pathway requires the CXCR4 cytoplasmic tail domain and phosphorylation of CXCR4-S339. Second, we showed that TCR-induced TCR-CXCR4 complex formation requires TCR-induced activity of Src family and ZAP-70 tyrosine kinases, as well as one or more Ser/Thre kinases. Third, we found that the G protein-coupled receptor kinase-2 (GRK2) is required both for TCR-induced phosphorylation of CXCR4-S339 and for the formation of TCR-CXCR4 complexes. Finally, we found that GRK2 is required for robust IL-2, IL-4, and IL-10 cytokine secretion by normal human T cells. Together these results identify a novel role for TCR-induced GRK-2 in inducing the TCR-CXCR4 complex formation that subsequently signals to increase TCR-mediated cytokine production. Targeting GRK2 or other mechanisms required for TCR-induced TCR-CXCR4 formation may therefore be therapeutically useful for limiting production of T cell cytokines in humans, for example, during graft versus host disease or immunotherapy.

Abstract The rapidly changing nature of immunology makes it a challenging course for both undergraduate students and faculty. A teaching approach that incorporates essential immunological techniques and an introduction to the primary literature helps undergraduates navigate the discipline and remain current. Case studies are an effective active learning approach that provide real world examples to engage undergraduates and an opportunity for hands on problem solving, data analysis, and critical thinking as students must consider outcomes and consequences. Although case studies that explore clinical connections already exist for undergraduates, they do not include key techniques or provide experience working with the primary literature. We have developed several case studies to help undergraduate immunology students understand and analyze data involving: flow cytometry, CRISPR/Cas, CAR T cells, and ELISAs. The case studies 1) introduce students to techniques and methods they may not have access to in their laboratory courses due to the lack of appropriate instruments or the cost of reagents, 2) build on difficult concepts covered in the course, and 3) incorporate figures from papers that challenge students to apply what they have learned. Assessment of student learning includes in-class quizzes and small group responses to data analysis questions, the use of "clickers"/personal response system, and related exam questions. Students self-report that the case studies are a highly effective teaching method that allow them to both reinforce their understanding of the course material and apply it to practical problems.

Abstract T cell cytokine production has been shown to be dependent not only on T cell antigen receptor (TCR), but also the chemokine receptor CXCR4. Evidence in the literature suggests that, in addition to stimulating migration, CXCR4 might also more directly regulate T cell immune activation. Recent studies from our lab have revealed that TCR stimulation in the absence of SDF-1 induces association of TCR and CXCR4, and that this is inhibited by pretreating cells with the specific CXCR4 antagonist AMD3100. Cytokine production, particularly cytokines IL-2, IL-4, and IL-10, by TCR-stimulated cells was inhibited by using either AMD3100 or CXCR4 siRNA to prevent TCR-CXCR4 formation. Interestingly, TCR-CXCR4 was not required for activation of many TCR-stimulated signaling pathways or for cytokine gene transcription. Instead, TCR-CXCR4 formation during TCR-mediated T cell immune activation was required for the post-transcriptional stabilization of cytokine mRNAs through a Rac-1 mediated pathway. Our results suggest that targeting the TCR-CXCR4 complex formation or downstream signaling pathways may provide therapeutic targets to regulate cytokine production.

Key Points T-cell activation induces TCR transactivation of CXCR4 to stabilize cytokine mRNA transcripts via a PREX1-Rac1–signaling pathway. Inhibition of the TCR-CXCR4–signaling pathway impairs TCR-dependent and TCR-independent cytokine secretion by CTCL cells.