A molecular signal recorder for mapping T cell differentiation in tumors

Introduction:: T cells circulate throughout the body to survey surrounding environments, counter immune threats, and store information about past pathogenic experiences. To bolster appropriate immune responses, T cells react appropriately to a milieu of environmental signals by differentiating into various T cell states, each with unique immune functions. In cancer contexts, T cells differentiate into dysfunctional states upon prolonged exposure to tumor microenvironments, resulting in reduced anti-tumor activity. To enable rational design of immunotherapies which counteract this process, it is critical to understand the mechanisms by which tumor signals drive dysfunctional T cell differentiation. Currently, we have rich information about T cell differentiation states and their phenotypes due to technologies such as snapshot RNA-seq which allow us to elucidate such differentiated states in T cell populations. However, it is still largely unclear how these states arise as a result of environmental exposure experienced by T cells, primarily due to a lack of technologies capable of non-invasively tracking T cell states upon signal contact over time.


Materials and Methods:: Given the gaps in the field, we are developing a DNA-based molecular recorder enabling T cells to irreversibly log environmental experience themselves over long time scales. This recorder tracks contact with a tumor cell via a synthetic receptor (SNIPR) which binds a model tumor-specific antigen (hCD19). When the SNIPR is bound, expression of a multi-sgRNA cassette is induced, an RNA processing protein (Csy4) frees functional sgRNAs from the larger cassette, and permanent CRISPR/Cas9 genome editing of a synthetic scratch pad is activated. The accumulation of genome edits over time encodes information about signal duration or magnitude. Coupling sequencing of the genomic scratch pad with single cell RNA sequencing (scRNA-seq) to identify T cell states, we can begin linking T cell differentiation to cancer signal exposure and construct T cell state trajectories that arise from various signal dynamics.


Results, Conclusions, and Discussions:: We have validated SNIPR recognition of hCD19 and subsequent activation of a fluorescent reporter in an in vivo mouse tumor model. Moreover, we have demonstrated successful knockout of a fluorescent reporter via an inducible, Csy4-processed sgRNA as well as scratch pad editing via an sgRNA processed out of a larger cassette in primary mouse T cells. Moving forward, we will genetically wire the sensory and recording arms of the T cell signal recording circuit together to track signal contact over time. T cell state information will be gathered via scRNA-seq and mapped to corresponding genomically-encoded signal experience data. Through this work, we will begin to decode T cell state transitions in response to tumor signaling environments.


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