Engineering T cell specificity and function at multiple loci can generate more effective cellular therapies, but current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here we develop a one-step process to enrich unlabeled cells containing knock-ins at multiple target loci using a family of repair templates named synthetic exon expression disruptors (SEEDs). SEEDs associate transgene integration with the disruption of a paired target endogenous surface protein while preserving target expression in nonmodified and partially edited cells to enable their removal (SEED-Selection). We design SEEDs to modify three critical loci encoding T cell specificity, coreceptor expression and major histocompatibility complex expression. The results demonstrate up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). This method is compatible with existing clinical manufacturing workflows and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

Multiplexed reprogramming of T cell specificity and function can generate powerful next-generation cellular therapies. However, current manufacturing methods produce heterogenous mixtures of partially engineered cells. Here, we develop a one-step process to enrich for unlabeled cells with knock-ins at multiple target loci using a family of repair templates named Synthetic Exon/Expression Disruptors (SEEDs). SEED engineering associates transgene integration with the disruption of a paired endogenous surface protein, allowing non-modified and partially edited cells to be immunomagnetically depleted (SEED-Selection). We design SEEDs to fully reprogram three critical loci encoding T cell specificity, co-receptor expression, and MHC expression, with up to 98% purity after selection for individual modifications and up to 90% purity for six simultaneous edits (three knock-ins and three knockouts). These methods are simple, compatible with existing clinical manufacturing workflows, and can be readily adapted to other loci to facilitate production of complex gene-edited cell therapies.

This review critically examines the evolving landscape of chimeric antigen receptor (CAR) T-cell therapy in treating solid tumors, with a particular focus on the metabolic challenges within the tumor microenvironment. CAR T-cell therapy has demonstrated remarkable success in hematologic malignancies, yet its efficacy in solid tumors remains limited. A significant barrier is the hostile milieu of the tumor microenvironment, which impairs CAR T-cell survival and function. This review delves into the metabolic adaptations of cancer cells and their impact on immune cells, highlighting the competition for nutrients and the accumulation of immunosuppressive metabolites. It also explores emerging strategies to enhance CAR T-cell metabolic fitness and persistence, including genetic engineering and metabolic reprogramming. An integrated approach, combining metabolic interventions with CAR T-cell therapy, has the potential to overcome these constraints and improve therapeutic outcomes in solid tumors.

Background and Significance: CD25 (IL2RA) has been extensively studied as IL2-receptor chains expressed on T- and NK-cells. However, CD25 is sharply upregulated upon BCR-signaling. Mature B-cell lymphoma subtypes (CLL, MCL, DLBCL) that depend on oncogenic BCR-signaling inactivate CD25 by proteolytic cleavage and aggressiveness of disease is associated with low expression CD25 surface levels and high serum levels of cleaved CD25. Results: Here, we discovered a mechanistic framework of dynamic BCR-feedback control and its dependency on coordinated activity of PKCδ and CD25. To study the role of CD25 in a model of aggressive B-cell lymphoma, we propagated splenic B-cells from CD25-fl/fl mice and transduced them with the CARD11-L232LI oncogene. Induction of Cre-activity in this model resulted in rapid loss of CD25 surface expression and dramatic acceleration of proliferation of CD25-/- B-cell lymphoma cells. Conditional ablation of CD25 during normal B-cell development in vivo (Cd25-fl/fl x Mb1-Cre) resulted in the development of B-cell autoimmunity with pervasive autoantibody production, development of spontaneous germinal centers. RNA-seq and mass spectrometry-based global phosphoproteomic studies revealed that acute ablation of CD25 in CARD11-L232LI B-cell lymphoma resulted in prominent activation of NF-kB gene expression programs, hyperphosphorylation of multiple substrates in the BCR-signaling pathway, including SYK, Src-family kinases, BLNK, NF-kB-components and PKCδ. Conversely, CD25-deletion caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Imbalances of control of BCR-signalling following CD25-deletion were confirmed by Western blot, namely hyperphosphorylation of SYK BLNK, ERK, NFKBIA and PKCδ as well as loss of SHP1- and SHIP1-phosphorylation. In addition to Cre-mediated deletion of CD25 in murine B-cell lymphoma cells, we confirmed these results by CRISPR-mediated deletion of CD25 in patient-derived mantle cell lymphoma xenografts (PDX). Mechanistic studies revealed that oncogenic BCR signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail (S268, T271). Genetic deletion of PKCδ and mutations of CD25 S268/T271 residues demonstrated that PKCδ-mediated CD25-phosphorylation was critical for CD25-dependent feedback control of oncogenic BCR signaling. Our genetic and interactome studies revealed that BCR-signaling induced PKCδ-mediated phosphorylation and recruitment of CD25 to the BCR. Instead of transducing IL2-signals, CD25 directed ITIM-mediated SHP1-activation towards BCR-signaling molecules, within range of SHP1-mediated dephosphorylation. In proximity of the BCR, CD25 attracted ITIM-receptor nanoclusters, resulting in local concentration and activation of SHP1 for negative feedback control of BCR-signaling. Defective BCR-feedback control in human CD25-/- germinal center B-cells could be restored by a novel bispecific antibody to direct ITIM-dependent SHP1 activation towards BCR-proximal signaling molecules. BCR-dependent B-cell lymphomas frequently harbor deleterious mutations of PKCδ and CD25 is typically inactivated by proteolytic cleavage. To investigate the interplay between PKCδ and CD25, we introduced genetic CD25 knockin alleles using HDR templates encoding GFP and wildtype CD25 or CD25-S268A/T271V (AV) mutations in primary human germinal center (GC) B-cells cultured on YK6 follicular dendritic cells. Interestingly, CD25-AV mutant knockin GC B-cells failed to terminate BCR-signaling and showed autonomous Ca2+ oscillations. In addition, CD25-AV knockin GC B-cells expressed increased levels of the activation markers CD69, CD80 and CD86 and exhibited constitutive activation of the NF-kB pathway. In coculture experiments determining how CD25 regulates competitive fitness, CD25-AV knockin GC B-cells rapidly outcompeted their CD25-wildtype counterparts. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, our findings highlight the previously unrecognized role of PKCδ and CD25 in assembling inhibitory phosphatases to control BCR signaling. Also in the context of oncogenic BCR-signaling in B-cell lymphoma, CD25 and PKCδ cooperate as negative regulators. These findings are consistent with frequent inactivation of CD25 by proteolytic cleavage as well as PKCδ-mutations in B-cell lymphomas.

Peyer’s patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFβ-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA + germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell–deficient mice had reduced PP cDC2s and IgA + cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell–guided positioning of GPR35 + cDC2s in the PP SED supports induction of intestinal IgA responses.

Initiation of B-cell receptor (BCR) 1 signaling, and subsequent antigen-encounter in germinal centers 2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) 4 and lymphoma 5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells 6-9 , the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ 10 - 12 , SHIP1 13,14 and SHP1 14, 15,16 , conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268 ). Genetic rescue experiments identified CD25-S 268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25 S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+ -oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent ‘all-or-none’ B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.

Successful initiation of the B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers are both marked by sharp increases of CD25 surface-expression. Likewise, oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma induces CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. We discovered that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells feedback regulates BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCD, SHIP1 and SHP1, conditional CD25-deletion suppressed early B-cell development but induced hyperactivation of antigen-experienced B-cells and autoimmunity. Additionally, B-cell-specific CD25 deletion resulted in formation of spontaneous germinal centers and expansion of autoreactive B cells. Biochemical and interactome studies revealed that BCR-signaling induced PKCd-dependent CD25-phosphorylation of the CD25 cytoplasmic tail (S268) to recruit inhibitory phosphatases to the BCR complex. Upon CD25-deletion, SHIP1 and SHP1 were no longer recruited and activated to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to proliferation and autoantibody production in antigen-experienced B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to prevent chronic BCR signaling and safeguard B cell tolerance.

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000’s of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei. Teaser Sentence CRISPR-Cas9 mediates nuclear transport and integration of nanostructured genes in human primary cells

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches. Combinations of single-stranded DNA repair templates and small molecules markedly enhance genome editing.

Mechanisms of local kinase recruitment to amplify signaling in lipid rafts have been extensively studied. However, mechanisms of phosphatase membrane-recruitment to terminate local signaling activity remained elusive. Here we identified CD25 (IL2Rα) as a central phosphatase membrane-shuttle and feedback regulator of B-cell receptor (BCR)-signaling in activated and transformed B-cells. This was unexpected because CD25 is known as one of three chains of the IL2-receptor on T- and NK-cells. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells recruits an inhibitory phosphatase complex for feedback control of BCR-signaling and its oncogenic mimics. Reminiscent of genetic phosphatase-ablation, conditional CD25-deletion profoundly depleted early B-cell development followed by dramatic expansion and hyperactivation of mature B-cell subsets and autoimmunity owing to imbalances of BCR-signaling. However, defects in CD25 deficient mice were not replicated in mice that express CD25 but lack expression of the IL2 cytokine, demonstrating IL2-independent functions of CD25 in B-cells. In six clinical cohorts, high expression levels of CD25 predicted poor outcomes for patients with pre-germinal center (GC), in contrast to favorable outcomes for patients with post-GC B-cell malignancies. Consistent with opposite clinical outcome-annotation, genetic CD25-deletion in B-cell precursor leukemia induced rapid cell death but accelerated proliferation in post-GC B-cell lymphomas. Mechanistically, BCR-engagement or oncogenic BCR-signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail at S268. Genetic deletion, in vitro kinase, and interactome studies revealed that PKCδ-dependent CD25-S268 phosphorylation triggered the PKCδ-scaffold RACK1 to recruit inhibitory phosphatases (SHP1, SHIP1) for membrane-translocation and feedback control of BCR-signaling. Deletion of CD25 in human B-cells induced autonomous BCR-signaling and Ca2+-oscillations: In the absence of CD25, inhibitory phosphatases were no longer recruited to the cell membrane to control duration and strength of BCR-signaling. Genetic rescue experiments identified membrane-tethered RACK1 and CD25-S268 tail-phosphorylation as central structural elements of this previously unrecognized phosphatase membrane-shuttle. As a result, CRISPR-mediated knock-in of CD25-S268A mutant that abrogates the formation of inhibitory phosphatase complexes caused accelerated proliferation with hyperactivation of BCR-signaling in primary human tonsillar germinal center B-cells. In conclusion, PKCδ-mediated phosphorylation of CD25 assembles RACK1-dependent inhibitory phosphatase complexes to enable feedback control of BCR-signaling to prevent autoimmunity and malignant transformation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce “CAR Pooling,” a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell–activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)–specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Introduction: CD25, also known as interleukin-2 receptor α chain (IL-2Rα), forms a heterotrimer with IL-2Rβ and common γ chain, and mediates IL-2 signaling. IL-2 signaling is essential for T- and NK-cell survival and proliferation. Additionally, CD25 is required for development of thymic regulatory T cells (Treg) and their suppressive function. Interestingly, CD25 is upregulated in a variety of lymphoid and myeloid malignancies, which do not depend on IL-2 signaling, suggesting IL-2-independent roles of CD25. Here, we seek to investigate whether CD25 has previously unrecognized functions in T cells and T-cell lymphomas. Results: Our previous study in B cells has discovered the cytoplasmic tail of CD25 as an essential structural element for site-specific recruitment of inhibitory phosphatases. CD25-mediated shuttling of inhibitory phosphatases balanced strength of oncogenic B-cell receptor (BCR)-signaling and was essential for cell-survival in B-cell malignancies. Interestingly, we found that CD25 expressed on B cells was monomeric and showed dynamic recruitment to BCR or its oncogenic mimics within lipid rafts. Studying gene expression data across different hematopoietic lineages, we found CD25 was strongly induced in T cell receptor (TCR)-activated T cells, as well as in T-cell malignancies that harbor oncogenic TCR-mimics, such as PTCL. Our mechanistic studies discovered that the serine residue 268 (S268) in the cytoplasmic tail of CD25 was a principal substrate of protein kinase C (PKC), and that S268 phosphorylation by PKC was critical for cell surface expression of CD25. The proximity-based labeling assays (Bio-ID) revealed that CD25-S268 but not its phospho-dead mutant recruits PKC and its adapter RACK1, which scaffold the inhibitory phosphatases SHIP1 and SHP1 for site-specific activation in lipid rafts. CRISPR-mediated knock-in of CD25-S268A that abrogates the formation of inhibitory phosphatase complexes in primary human T cells enabled us to specifically interrogate the functional relevance of CD25-S268 in the regulation of TCR signaling. By using single stranded homology-directed repair templates (ssHDRT) that contain a Cas9 shuttling sequence, we were able to achieve 70% knock-in efficiency in primary human T cells (Figure 1). Signaling studies with these cells revealed that S268A mutation significantly augmented TCR signaling strength. Additionally, S268A mutation resulted in acute inactivation of inhibitory phosphatases including SHP1. Consistent with these results, CD25 shuttling to TCR complex was promptly induced upon TCR stimulation and was mitigated by S268 mutation. Furthermore, immunophenotyping showed that loss of S268 phosphorylation resulted in increased expression of T-cell activation markers such as CD44 and CD69. Based on the ex vivo results, we generated a transgenic mouse model with germline knock-in of CD25-S268A, which would allow us to study S268-specific roles in CD25-mediated feedback regulation of TCR and oncogenic signaling in vivo. Conclusions: The role of CD25 in mediating IL-2 signaling has been extensively studied. However, increasing evidence suggests that CD25 plays a more sophisticated role in TCR signaling and T-lymphomagenesis. Here we discovered a previously unrecognized function of CD25 as a feedback regulator of TCR signaling. TCR activation or oncogenic TCR-mimics induce CD25-mediated recruitment of inhibitory phosphatases in lipid rafts to mitigate TCR signaling or curb excessive oncogenic signaling. Figure 1. CRISPR-mediated knock-out of CD25 alone, or followed by knock-in of CD25-S268-GFP or CD25-A268-GFP, in primary human T cells. The proportion of CD25-expressing GFP+ cells are analyzed by flow cytometry on day 4 and day 30 post CRISPR. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 10 9 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

Ag stimulation (signal 1) triggers B cell activation and proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). However, failure to receive signal 2 within a defined window of time results in an abortive round of proliferation, followed by anergy or apoptosis. Although the molecular basis of T cell help has been extensively dissected, the mechanisms that restrain Ag-stimulated B cells, and enforce dependence upon co-stimulation, are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell receptor (BCR) stimulation, yet little is known about their function in humoral immune responses. Here we use germline and conditional loss-of-function mouse models to show that Nr4a1 and Nr4a3 play partially redundant roles to restrain both the survival and proliferation of B cells that receive signal 1 in the absence of co-stimulatory signals, and do so in part by repressing expression of BATF and consequently c-MYC. Correspondingly, Ab responses to TI-2 immunogens are enhanced in the absence of Nr4a1, but are unaltered in response to immunogens that incorporate co-stimulatory signals. Unexpectedly, we also identify a role for the NR4A family in restraining B cell access to T cell help by repressing expression of the T cell chemokines CCL3/4, as well as CD86 and ICAM1, and show that this is relevant under conditions of competition for limiting T cell help. Our studies collectively reveal a novel negative feedback loop mediated by the NR4A family that increases B cell dependence upon T cell help and restrains strongly Ag-activated B cell clones from monopolizing limiting amounts of T cell help. We speculate that this imposes B cell tolerance and dampens immunodominance to facilitate preservation of clonal diversity during an immune response.

It has long been appreciated that highly autoreactive BCRs are actively removed from the developing B cell repertoire by Ag-dependent receptor editing and deletion. However, there is persistent debate about whether mild autoreactivity is simply tolerated or positively selected into the mature B cell repertoire as well as at what stage, to what extent, under what conditions, and into which compartments this occurs. In this study, we describe two minor, trackable populations of B cells in B1-8i Ig transgenic mice that express the VH186.2 H chain and recognize a common foreign Ag (the hapten 4-hydroxy-3-nitrophenylacetyl) but differ in L chain expression. We use the Nur77-eGFP reporter of BCR signaling to define their reactivity toward endogenous Ags. The less autoreactive of these two populations is strongly counterselected during the development of mature B1a, follicular, and marginal zone B cells. By genetically manipulating the strength of BCR signal transduction via the titration of surface CD45 expression, we demonstrate that this B cell population is not negatively selected but instead displays characteristics of impaired positive selection. We demonstrate that mild self-reactivity improves the developmental fitness of B cell clones in the context of a diverse population of B cells, and positive selection by endogenous Ags shapes the mature B cell repertoire.