High School student Mount Lebanon High School Pittsburgh, Pennsylvania, United States
Introduction: Autoimmune diseases are a leading cause of death, especially in women, but little is known about which antigens trigger disease activation. Many autoimmune diseases begin when T cell receptors (TCRs) on CD4+ cells bind to class II peptide-major histocombatibility complexes (pMHCs) and activate, releasing cytokines that direct other immune cells to kill the pMHC-expressing cell. Determining cognate peptides of autoimmune TCRs will enable future studies to develop new antigen-specific treatments for autoimmune disorders. TCR-pMHC cognate pairs can be determined using Signaling and Antigen-presenting Bifunctional Receptors (SABRs), which present known peptides to CD4+ cells on an MHC-like structure. When recognized by a TCR, intracellular signaling in the SABR using GFP and CD69 is induced, which can be detected using flow cytometry. Initial experiments used Jurkat cells to express SABRs; however, since immortalized cells have different properties than primary cells, future experiments must express SABRs on primary CD4+ cells, which are much less readily available than Jurkat cells. Therefore, additional research is needed to determine the minimum quantity of TCR and SABR-expressing CD4+ cells necessary to achieve detectable CD69 and GFP signaling. By co-incubating varying concentrations of TCR and SABR-expressing cells and measuring CD69 expression, SABR efficacy in primary CD4+ cells can be determined.
Materials and
Methods: 293T packaging cells were transfected with DNA for BDC2.5 TCR and HIP SABR (a known cognate pair) using 8.9 and vsv-g packaging plasmids. The resulting virus was collected and purified. Primary CD4+ cells were extracted from non-obese diabetic (NOD) mice spleens using negative selection with EasySep Mouse CD4+ Isolation Kit and plated. Separate plates were transduced with BDC2.5 TCR and HIP SABR virus. The transduced cells were diluted to specific concentrations using a serial dilution and co-incubated at varying ratios. Flow cytometry was used to measure CD69 expression, gating on lymphocytes, live cells, and CD69-expressing cells, and subdividing into CD69 on BDC2.5 TCR stained with cell trace violet (CTV) and CD69 on 2.5 HIP SABR stained with cell trace far red (CTFR).
Results, Conclusions, and Discussions:
Results: CD69 expression on all live lymphocytes increased as the number of TCRs and SABRs increased, with a highest expression of 69.70% with 50,000 TCRs and 50,000 SABRs. Expression decreased with cell count to 3.19% at 0 TCRs and 0 SABRs. CD69 expression on CTFR-stained SABRs varied greatly between 100.00% with 10 SABRs and 50,000 TCRs and 6.64% with 50,000 SABRs and 10 TCRs. Conditions were optimized at 1,000 TCRs coincubated with 1,000 SABRs, with CD69 expression of 51.00% and a total cell count of 2,000. CD69 expression on CTV-stained TCRs also decreased with cell count from a high of 100.00% with 10,000 SABRs and 100 TCRs to a low of about 1-2% with less than 10 SABRs.
Conclusions: Low CD69 expression levels in live lymphocytes at 0 SABRs and 0 TCRs establishes a level of background CD69 expression between 0 and 3.5%. Any results in this range should be discarded as background. CD69 expression levels on BDC2.5 TCR indicates that TCRs were being activated by the antigen on the SABR; verifying SABR efficacy. CD69 levels on SABRs show that SABR activation can be detected in CD4+ cells with only 1,000 SABR and 1,000 TCR-expressing cells. Future experiments will use these data when coincubating SABR and TCR-expressing primary CD4+ cells to screen autoimmune TCRs for their cognate epitopes.. Identifying cognate epitopes of autoimmune TCRs will build understanding of autoimmunity triggers and may lead to the development of treatments or cures for these diseases that will benefit thousands.
Acknowledgements (Optional): Hillman Cancer Center Academy - David Boone, Joseph Ayoob, Steven Jones, Tullia Bruno, Ellen Scott Joglekar Lab - Alok Joglekar, Sanya Arshad
References (Optional): Joglekar, Alok V., et al. “T Cell Antigen Discovery via Signaling and Antigen-Presenting Bifunctional Receptors.” Nature News, Nature Publishing Group, 28 Jan. 2019, www.nature.com/articles/s41592-018-0304-8.
Walsh, S J, and L M Rau. “Autoimmune Diseases: A Leading Cause of Death among Young and Middle-Aged Women in the United States.” American Journal of Public Health, U.S. National Library of Medicine, Sept. 2000, www.ncbi.nlm.nih.gov/pmc/articles/PMC1447637/.
