Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.

T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.

Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However, the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic, whereas other responses, such as the ability to overcome tumor immunosuppression, are absent. Thus, the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles, biased T cell differentiation, and local delivery of non-native therapeutic payloads, such as antibodies, in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases.

The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation.

Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.

Domain-Specific and Stage-Intrinsic Changes in Tcrb Conformation during Thymocyte Development.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vß gene segments (Trbvs) and their DßJß targets. Formation of a functional VßDßJß join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DßJß segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DßJß segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VßDßJß join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DßJß targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.

The microRNA biogenesis machinery modulates lineage commitment during αß T cell development.

Differentiation of CD4(+) helper and CD8(+) cytotoxic αß T cells from CD4(+)CD8(+) thymocytes involves upregulation of lineage-specifying transcription factors and transcriptional silencing of CD8 or CD4 coreceptors, respectively, in MHC class II or I (MHCII or I)-restricted thymocytes. In this study, we demonstrate that inactivation of the Dicer RNA endonuclease in murine thymocytes impairs initiation of Cd4 and Cd8 silencing, leading to development of positively selected MHCI- and MHCII-restricted mature CD4(+)CD8(+) thymocytes. Expression of the antiapoptotic BCL2 protein or inactivation of the p53 proapoptotic protein rescues these thymocytes from apoptosis, increasing their frequency and permitting accumulation of CD4(+)CD8(+) αß T cells in the periphery. Dicer-deficient MHCI-restricted αß T cells fail to normally silence Cd4 and display impaired induction of the CD8 lineage-specifying transcription factor Runx3, whereas Dicer-deficient MHCII-restricted αß T cells show impaired Cd8 silencing and impaired induction of the CD4 lineage-specifying transcription factor Thpok. Finally, we show that the Drosha RNA endonuclease, which functions upstream of Dicer in microRNA biogenesis, also regulates Cd4 and Cd8 silencing. Our data demonstrate a previously dismissed function for the microRNA biogenesis machinery in regulating expression of lineage-specifying transcription factors and silencing of Cd4 and Cd8 during αß T cell differentiation.

Requirement for dicer in survival of proliferating thymocytes experiencing DNA double-strand breaks.

The Dicer nuclease generates small RNAs that regulate diverse biological processes through posttranscriptional gene repression and epigenetic silencing of transcription and recombination. Dicer-deficient cells exhibit impaired differentiation, activity, proliferation, and survival. Dicer inactivation in developing mouse lymphocytes impairs their proliferation and survival and alters Ag receptor gene repertoires for largely undefined reasons. To elucidate functions of Dicer in lymphocyte development and Ag receptor locus transcription and recombination, we analyzed mice with conditional Dicer deletion in thymocytes containing unrearranged or prerearranged TCRß loci. Expression of either a preassembled functional TCRß gene (Vß1(NT)) or the prosurvival BCL2 protein inhibited death and partially rescued proliferative expansion of Dicer-deficient thymocytes. Notably, combined expression of Vß1(NT) and BCL2 completely rescued proliferative expansion of Dicer-deficient thymocytes and revealed that Dicer promotes survival of cells attempting TCRß recombination. Finally, inclusion of an endogenous preassembled DJß complex that enhances Vß recombination increased death and impaired proliferative expansion of Dicer-deficient thymocytes. These data demonstrate a critical role for Dicer in promoting survival of thymocytes experiencing DNA double-strand breaks (DSBs) during TCRß recombination. Because DSBs are common and ubiquitous in cells, our findings indicate that impaired cellular survival in response to DSBs should be considered when interpreting Dicer-deficient phenotypes.

CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells.

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize programmed cell death protein 1 [PD-1]) have both opened new avenues for cancer treatment, but the clinical potential of combined disruption of inhibitory checkpoints and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient anti-CD19 CAR T cells. Pdcd1 (PD-1) disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies.

Molecular Analysis of Mouse T Cell Receptor α and ß Gene Rearrangements.

PCR on genomic DNA isolated from lymphocyte populations is an invaluable technique to analyze T cell receptor (TCR) α and ß gene rearrangements. Although this approach is powerful, it also has limitations that must be accounted for in experimental design and data interpretation. Here, we provide background required for understanding these limitations, and then outline standard PCR methods that can be used for analysis of TCRα and ß gene rearrangements in mice.

Synthetic biology approaches to engineer T cells.

There is rapidly growing interest in learning how to engineer immune cells, such as T lymphocytes, because of the potential of these engineered cells to be used for therapeutic applications such as the recognition and killing of cancer cells. At the same time, our knowhow and capability to logically engineer cellular behavior is growing rapidly with the development of synthetic biology. Here we describe how synthetic biology approaches are being used to rationally alter the behavior of T cells to optimize them for therapeutic functions. We also describe future developments that will be important in order to construct safe and precise T cell therapeutics.