As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment.
Transgelin-2 is a small 22-kDa actin-binding protein implicated in actin dynamics, which stabilizes actin structures and participates in actin-associated signaling pathways. Much curiosity regarding transgelin-2 has centered around its dysregulation in tumor development and associated diseases. However, recent studies have shed new light on the functions of transgelin-2, the only transgelin family member present in leukocytes, in the context of various immune responses. In this review, we outlined the biochemical properties of transgelin-2 and its physiological functions in T cells, B cells, and macrophages. Transgelin-2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse. Transgelin-2 in B cells also participates in the stabilization of T cell-B cell conjugates. While transgelin-2 is expressed at trace levels in macrophages, its expression is highly upregulated upon lipopolysaccharide stimulation and plays an essential role in macrophage phagocytosis. Since transgelin-2 increases T cell adhesion to target cells via boosting the "inside-out" costimulatory activation of leukocyte function-associated antigen 1, transgelin-2 could be a suitable candidate to potentiate the antitumor response of cytotoxic T cells by compensating for the lack of costimulation in tumor microenvironment. We discussed the feasibility of using native or engineered transgelin-2 as a synergistic molecule in cell-based immunotherapies, without inducing off-target disturbance in actin dynamics in other cells.
CD40 signaling in conventional type 1 dendritic cells (cDC1s) is required for CD8 T cell-mediated tumor rejection, but the underlying mechanisms are incompletely understood. Here, we identified CD40-induced genes in cDC1 and examined their contributions to anti-tumor immunity. cDC1-specific inactivation of CD70 and COX-2, and global CD27 inactivation, only partially impaired tumor rejection or tumor-specific CD8 T-cell expansion. Loss of 4-1BB, alone or in Cd27−/− mice, did not further impair anti-tumor immunity. However, cDC1-specific CD40 inactivation reduced cDC1 mitochondrial transmembrane potential and increased apoptosis in tumor-draining lymph nodes, reducing migratory cDC1 numbers in vivo. Similar impairments occurred during in vitro antigen presentation, which were reversed by re-expression of Bcl-xL. Thus, CD40 signaling in cDC1 not only induces co-stimulatory ligands for CD8 T cells, but also induces Bcl2l1 that sustains cDC1 survival during priming of anti-tumor responses.
This work was supported by grants from the NIH (R01AI150297, R01CA248919, and R21AI164142 to K.M.M., and F30CA247262 to R.W.).
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