I second Jun on this! Thanks, Soo Hyun. if you are interested in participating, don't hesitate to submit a proposal (link) :-)

As discussed, now that everyone has a better sense of that the immgenT program is about, we would be grateful if everyone could provide a somewhat detailed experimental plan, using the ProposedSamples spreadsheet attached. This isn’t in any way final, but will help to start putting thoughts into place. And serve as a basis for scoping, discussion of the experimental format, and coordination between labs. For clarification, each “experiment” here basically amounts to one run on the 10X instrument, and is to include 50,000 cells from the final sort. These can be composed of anywhere between 1 and 10 “samples”; the number of cells in each sample can vary (as long as the total is 50,000 per experiment). Don’t be shy at this stage :-)

Thanks, Dan! Yes, profiling thymocytes is undoubtedly within the project's scope, i.e., especially analyzing the types of T cells and states that emerge during differentiation. Thymus-specific states that don't make it to the periphery would be equally interesting. TCR repertoire is a tricky one considering its size. The technology isn't powered to answer broad repertoire analysis. Maybe we could consider experimental models with a limited range or models testing different T cell paths?

Hello Dr. Haley, thanks for reaching out! Your technology would nicely complement the workflow and bring valuable insights to the TCR repertoire analysis. Would you have time to discuss this further? Would you mind emailing me at davidpzATfas.harvard.edu?

Thanks for the suggestion! Certainly a fascinating topic. What would be the most relevant tissues and timepoint to compare virgin and pregnant mice? Any clonal expansion we could identify with the TCR sequencing? As a first experiment, we could sequence ~50,000 cells across ten conditions. Looking forward to describing pregnancy-specific T cells states!

Indeed, the TCR is a central molecule to understanding T cells, so TCR analysis is paramount to the project. Across all our samples, we are sequencing the TCRab in parallel to the RNA and surface protein expression (CITE-seq). It will track clones across tissues, mice, and conditions. But this will provide data for cells of unknown specificity. Profiling T cells of known antigen specificity will nicely complement this analysis and is therefore well within the project's scope. It will help understand the range of states that T cells with the same TCR can adopt.

Where is the list?

To second this point, since the goal is to understand the "natural states" that T cells can adopt in various environmental conditions, profiling KO T cells are out of scope per se. But if a genetically engineered mouse serves as a disease model, particularly if the mutation is extrinsic to T cells, then it is within the scope of the ImmGen T project.

Great suggestion!

Excellent point. Thanks for the suggestion. Peritoneal cavity T cells at baseline will be profiled soon. Including infectious and cancer models will certainly be valuable. If you know a lab working on these models, please inform them about ImmGenT.

In collaboration with BioLegend, we have optimized a panel of antibodies for profiling T cells. Of 128 antibodies, 110 are against antigens expressed in T cells, 9 are markers used to identify non-T cells, 9 are isotype controls.