Ag Receptor Research Articles

Involvement of the HCK and FGR src-family kinases in FCRL4-mediated immune regulation.

FCRL4 is an immunoregulatory receptor expressed by a subpopulation of memory B cells. These tissue-based cells express increased levels of the src-family kinases HCK and FGR. In this study, we investigate the roles of these src-family kinases in FCRL4-mediated immunoregulation of B cells in the context of previously unrecognized palmitoylation of the receptor. We observed enhanced phosphorylation of FCRL4 on tyrosine residues in the presence of the HCK p59 or FGR. This phosphorylation was markedly reduced in assays using a palmitoylation-defective mutant of FCRL4. In reporter gene studies, we observe that FCRL4 expression enhances CpG-mediated activation of NF-κB signaling. Surprisingly, using a reporter gene linked to activation of the MAPK substrate Elk-1 in response to Ag receptor ligation, we find that FCRL4 has inhibitory activity in cells coexpressing FGR but an activating function in cells coexpressing HCK p59. We provide evidence that in primary memory B cells, expression of FCRL4 leads to increased expression of IL-10 in the presence of FGR or HCK p59 in response to CpG, but increased levels of IFN-γ only in the context of coexpression of FGR. Our study supports the specific requirement of HCK p59 and FGR src-family kinases for FCRL4-mediated immunomodulatory activity and indicates that palmitoylation serves as an additional level of regulatory control of FCRL4.

Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.

Transcription-dependent generation of a specialized chromatin structure at the TCRβ locus.

V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dβ-Jβ-Cβ gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCβ clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. We propose that the entire DJCβ regions behave as transcription "initiation" platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Dβ and Jβ gene segments.

DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy.

NK cells are emerging as new effectors for immunotherapy of cancer. In particular, the genetic engraftment of chimeric Ag receptors (CARs) in NK cells is a promising strategy to redirect NK cells to otherwise NK cell-resistant tumor cells. On the basis of DNAX-activation protein 12 (DAP12), a signaling adaptor molecule involved in signal transduction of activating NK cell receptors, we generated a new type of CAR targeting the prostate stem cell Ag (PSCA). We demonstrate in this article that this CAR, designated anti-PSCA-DAP12, consisting of DAP12 fused to the anti-PSCA single-chain Ab fragment scFv(AM1) confers improved cytotoxicity to the NK cell line YTS against PSCA-positive tumor cells when compared with a CAR containing the CD3ζ signaling chain. Further analyses revealed phosphorylation of the DAP12-associated ZAP-70 kinase and IFN-γ release of CAR-engineered cells after contact with PSCA-positive target cells. YTS cells modified with DAP12 alone or with a CAR bearing a phosphorylation-defective ITAM were not activated. Notably, infused YTS cells armed with anti-PSCA-DAP12 caused delayed tumor xenograft growth and resulted in complete tumor eradication in a significant fraction of treated mice. The feasibility of the DAP12-based CAR was further tested in human primary NK cells and confers specific cytotoxicity against KIR/HLA-matched PSCA-positive tumor cells, which was further enhanced by KIR-HLA mismatches. We conclude that NK cells engineered with DAP12-based CARs are a promising tool for adoptive tumor immunotherapy.

Cutting edge: An in vivo reporter reveals active B cell receptor signaling in the germinal center.

Long-lasting Ab responses rely on the germinal center (GC), where B cells bearing high-affinity Ag receptors are selected from a randomly mutated pool to populate the memory and plasma cell compartments. Signaling downstream of the BCR is dampened in GC B cells, raising the possibility that Ag presentation and competition for T cell help, rather than Ag-dependent signaling per se, drive these critical selection events. In this study we use an in vivo reporter of BCR signaling, Nur77-eGFP, to demonstrate that although BCR signaling is reduced among GC B cells, a small population of cells exhibiting GC light zone phenotype (site of Ag and follicular helper T cell encounter) express much higher levels of GFP. We show that these cells exhibit somatic hypermutation, gene expression characteristic of signaling and selection, and undergo BCR signaling in vivo.

Beyond consolidation: auto-SCT and immunotherapy for plasma cell myeloma.

Autologous hematopoietic cell transplantation (auto-HCT) is the standard consolidation therapy for plasma cell myeloma patients following induction therapy. Auto-HCT improves disease-free survival (DFS), but is generally not curative. The allogeneic HCT experience demonstrated that T-cell immunotherapy can confer long-term DFS. Preclinical and clinical data indicate that myeloma-associated Ags elicit humoral and cellular immune responses (IRs) in myeloma patients. These findings strongly suggest that the immunotherapeutic strategies, including immune checkpoint inhibitors, therapeutic cancer vaccines and adoptive cellular therapies, are promising avenues of clinical research that may be most applicable in the minimal residual disease state following auto-HCT. These strategies are designed to prime or augment antimyeloma IRs and promote a 'host-vs-myeloma' effect that may result in durable DFS. Innovative clinical trials investigating immune checkpoint inhibitors and cancer vaccines have demonstrated that robust immunity against myeloma-associated Ags can be elicited in the setting of auto-HCT. A diverse array of immunotherapeutic strategies have entered clinical trials in myeloma, including PD-1/PD-L1 inhibitors, DC/myeloma cell fusion vaccines and adoptive chimeric Ag receptor T-cell therapy, and further investigation of combinations of immunologic and pharmaceutical agents are expected in the near future. In this review, we will discuss the preclinical data supporting immunotherapy in auto-HCT for myeloma, clinical investigation of these strategies and the future prospects of immunotherapy in pursuit of the goal of curative therapy.

Target antigen density governs the efficacy of anti-CD20-CD28-CD3 ζ chimeric antigen receptor-modified effector CD8+ T cells.

The effectiveness of chimeric Ag receptor (CAR)-transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR-T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR-T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR-T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

Rpl22 Loss Impairs the Development of B Lymphocytes by Activating a p53-Dependent Checkpoint.

Although ribosomal proteins facilitate the ribosome’s core function of translation, emerging evidence suggests that some ribosomal proteins are also capable of performing tissue-restricted functions either from within specialized ribosomes or from outside of the ribosome. In particular, we have previously demonstrated that germline ablation of the gene encoding ribosomal protein Rpl22 causes a selective and p53-dependent arrest of ab T cell progenitors at the b-selection checkpoint. We have now identified a crucial role for Rpl22 during early B cell development. Germline ablation of Rpl22 results in a reduction in the absolute number of B-lineage progenitors in the bone marrow beginning at the pro–B cell stage. Although Rpl22-deficient pro–B cells are hyporesponsive to IL-7, a key cytokine required for early B cell development, the arrest of B cell development does not result from disrupted IL-7 signaling. Instead, p53 induction appears to be responsible for the developmental defects, as Rpl22 deficiency causes increased expression of p53 and activation of downstream p53 target genes, and p53 deficiency rescues the defect in B cell development in Rpl22-deficient mice. Interestingly, the requirement for Rpl22 in the B cell lineage appears to be developmentally restricted, because Rpl22-deficient splenic B cells proliferate normally in response to Ag receptor and Toll receptor stimuli and undergo normal class-switch recombination. These results indicate that Rpl22 performs a critical, developmentally restricted role in supporting early B cell development by preventing p53 induction.

The molecular bases of δ/αβ T cell-mediated antigen recognition.

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.

Indirect CD4+ T-cell-mediated elimination of MHC II(NEG) tumor cells is spatially restricted and fails to prevent escape of antigen-negative cells.

Tumor-specific Th1 cells can activate tumor-infiltrating macrophages that eliminate MHC class II negative (MHC II(NEG)) tumor cells. Activated M1-like macrophages lack antigen (Ag) receptors, and are presumably unable to discriminate and thus kill both Ag-positive (Ag(POS)) and Ag-negative (Ag(NEG)) tumor cells (bystander killing). The lack of specificity of macrophage-mediated cytotoxicity might be of clinical importance as it could provide a means of avoiding tumor escape. Here, we have tested this idea using mixed populations of Ag(POS) and Ag(NEG) tumor cells in a TCR-transgenic model in which CD4(+) T cells recognize a secreted tumor-specific antigen. Surprisingly, while Ag(POS) tumor cells were recognized and rejected, Ag(NEG) cells grew unimpeded and formed tumors. We further demonstrated that macrophage-mediated cytotoxicity was spatially restricted to areas dominated by Ag(POS) tumor cells, sparing Ag(NEG) tumor cells in the vicinity. As a consequence, macrophage tumoricidal activity did not confer bystander killing in vivo. The present results offer novel insight into the mechanisms of indirect Th1-mediated elimination of MHC II(NEG) tumor cells.

Antigen-dependent versus -independent activation of invariant NKT cells during infection.

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation.

Effect of CDR3 sequences and distal V gene residues in regulating TCR-MHC contacts and ligand specificity.

The mature T cell repertoire has the ability to orchestrate immunity to a wide range of potential pathogen challenges. This ability stems from thymic development producing individual T cell clonotypes that express TCRs with unique patterns of Ag reactivity. The Ag specificity of TCRs is created from the combinatorial pairing of one of a set of germline encoded TCR Vα and Vβ gene segments with randomly created CDR3 sequences. How the amalgamation of germline encoded and randomly created TCR sequences results in Ag receptors with unique patterns of ligand specificity is not fully understood. Using cellular, biophysical, and structural analyses, we show that CDR3α residues can modulate the geometry in which TCRs bind peptide-MHC (pMHC), governing whether and how germline encoded TCR Vα and Vβ residues interact with MHC. In addition, a CDR1α residue that is positioned distal to the TCR-pMHC binding interface is shown to contribute to the peptide specificity of T cells. These findings demonstrate that the specificity of individual T cell clonotypes arises not only from TCR residues that create direct contacts with the pMHC, but also from a collection of indirect effects that modulate how TCR residues are used to bind pMHC.

IL-2: the first effective immunotherapy for human cancer.

The ability of IL-2 to expand T cells with maintenance of functional activity has been translated into the first reproducible effective human cancer immunotherapies. The administration of IL-2 can lead to durable, complete, and apparently curative regressions in patients with metastatic melanoma and renal cancer. The growth of large numbers of tumor-infiltrating lymphocytes with in vitro anti-cancer activity in IL-2 has led to the development of cell transfer therapies that are highly effective in patients with melanoma. The genetic modification of T cells with genes encoding αβ TCRs or chimeric Ag receptors and the administration of these cells after expansion in IL-2 have extended effective cell transfer therapy to other cancer types.

Specific Recognition of Mycobacterial Protein and Peptide Antigens by γδ T Cell Subsets following Infection with VirulentMycobacterium bovis

Promoting effective immunity to Mycobacterium bovis infection is a challenge that is of interest to the fields of human and animal medicine alike. We report that γδ T cells from virulent M. bovis-infected cattle respond specifically and directly to complex, protein, and nonprotein mycobacterial Ags. Importantly, to our knowledge, we demonstrate for the first time that bovine γδ T cells specifically recognize peptide Ags derived from the mycobacterial protein complex ESAT6:CFP10 and that this recognition requires direct contact with APCs and signaling through the T cell Ag receptor but is independent of MHC class I or II. Furthermore, we show that M. bovis infection in cattle induces robust IL-17A protein responses. Interestingly, in contrast to results from mice, bovine CD4 T cells, and not γδ T cells, are the predominant source of this critical proinflammatory mediator. Bovine γδ T cells are divided into subsets based upon their expression of Workshop Cluster 1 (WC1), and we demonstrate that the M. bovis-specific γδ T cell response is composed of a heterogeneous mix of WC1-expressing populations, with the serologically defined WC1.1(+) and WC1.2(+) subsets responding in vitro to mycobacterial Ags and accumulating in the lesions of M. bovis-infected animals. The results described in this article enhance our understanding of γδ T cell biology and, because virulent M. bovis infection of cattle represents an excellent model of tuberculosis in humans, contribute to our overall understanding of the role of γδ T cells in the mycobacterial-specific immune response.

Targeted Chromatin Profiling Reveals Novel Enhancers in Ig H and Ig L Chain Loci

The assembly and expression of mouse Ag receptor genes are controlled by a collection of cis-acting regulatory elements, including transcriptional promoters and enhancers. Although many powerful enhancers have been identified for Ig (Ig) and TCR (Tcr) loci, it remained unclear whether additional regulatory elements remain undiscovered. In this study, we use chromatin profiling of pro-B cells to define 38 epigenetic states in mouse Ag receptor loci, each of which reflects a distinct regulatory potential. One of these chromatin states corresponds to known transcriptional enhancers and identifies a new set of candidate elements in all three Ig loci. Four of the candidates were subjected to functional assays, and all four exhibit enhancer activity in B but not in T lineage cells. The new regulatory elements identified by focused chromatin profiling most likely have important functions in the creation, refinement, and expression of Ig repertoires.

T Cells Expressing Cars Directed Against HLA-0201 –Rmf WT-1 Peptide Complex Can Effectively Eradicate WT1+ A0201+ Tumor Cells in-Vitro

Adoptive transfer of ag specific T-cells can effectively eradicate viral infections and tumors. T-cells can be targeted to specific ags expressed on tumor cells by genetic modification to express chimeric ag receptors (CARs); which are fusion proteins incorporating an ag recognition moiety (ab) and T-cell activation domains. Human T-cells genetically modified to express a CD19-targeted CAR have been demonstrated to successfully eradicate CD19+ B cell malignancies in patients with refractory disease. The CARs developed thus far have been directed against native cell-surface antigens, such as CD19, that are recognized through antibody-derived antigen-binding motifs, independent of antigen processing or MHC-restricted presentation. However, tumor associated intracellular self ags such as WT-1 require processing following which antigenic epitopes are presented on the tumor cell surface in complex with HLA class-I or class-II. A strategy for targeting T-cells to specific peptide epitopes of such self ags expressed on tumor cells involves genetically introducing TCR mimic CARs containing a scFv ab directed against the specific peptide-MHC complexes. CARs containing a Fab directed against an HLA-A0201-NY-ESO-1 epitope have been generated, but functional data is lacking. We generated a TCR-like CAR containing a scFv specific for an HLA/peptide complex; the WT-1 RMFPNAPYL peptide presented by HLA-A0201 (A2-RMF CAR). The original A2-RMF ScFv, isolated using phage display technology, had a low binding affinity (Kd= 2.3 x 10-7 M). T-cells were transduced to express the A2-RMF CAR using MSCV retroviral vector. However, CAR transduced T-cells bearing the low affinity A2-RMF ScFv did not demonstrate any binding to the A2-RMF tetramers, and did not demonstrate any in-vitro cytotoxic activity against A2+/WT-1+ tumor cell targets. We subsequently developed an affinity matured ScFv (Kd= 3.08 x 10-9 M) to determine if ScFv binding affinity correlated with functional activity. T-cells transduced to express CARs bearing the affinity matured A2-RMF ScFv demonstrated strong binding to the A2-RMF tetramer in all transduced cells. The affinity matured A2-RMF CAR modified T-cells were able to kill A2+/WT-1+ solid tumor or B cell leukemia cell lines (29% -40% specific lysis) at E:T ratios of 50:1 and 20:1, in comparison to 1-4% specific lysis for HLA A2-/WT-1+ or A2+/WT-1- control tumor cell lines. These data demonstrate that a TCR-mimic CAR bearing scFv against an A2-RMF complex, can bind to A2-RMF tetramers and effectively lyse tumor cell targets co-expressing HLA A2 and WT1 in an HLA restricted and antigen specific manner. The results also suggest that the binding affinity of the antibody is important for effective functional activity of CAR modified T-cells directed against epitopes of self tumor associated antigens.

EndoU is a novel regulator of AICD during peripheral B cell selection

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

Coupled IL-2–Dependent Extracellular Feedbacks Govern Two Distinct Consecutive Phases of CD4 T Cell Activation

T cells integrate cell-specific Ag receptor signaling with shared signals mediated by secreted cytokines, which often involve regulatory feedback loops. IL-2 signaling, for example, reduces the synthesis of IL-2 and increases the synthesis of IL-2Rα-chain, whereas both genes require TCR signaling for their activation. The ways by which T cells dynamically integrate these private and public signals during activation are not well understood. We combined robotics, multiparameter flow cytometry, and real-time quantitative PCR to analyze T cell activation at high temporal resolution over several days. Two distinct temporal phases of T cell activation were evident. First, Ag-dependent signals activated low IL-2Rα and high IL-2 production, independent of IL-2 signaling. Subsequently, secreted IL-2 acted as a shared resource driving high IL-2Rα expression, reduced IL-2 synthesis, and cell proliferation. This transition was independent of continued TCR signaling. Our data allowed the determination of the parameters of the IL-2-mediated extracellular positive and negative feedback circuits and demonstrated that the two loops are coupled and become activated at a similar level of IL-2 signaling. We propose that temporal separation of private and shared signals allows T cells to first integrate Ag-specific responses and subsequently share information leading to collective decision making.

Preclinical in vivo modeling of cytokine release syndrome induced by ErbB-retargeted human T cells: identifying a window of therapeutic opportunity?

The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αβ (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4(+) T cells exhibit antitumor activity against several ErbB(+) cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB(+) cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4(+) T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4(+) T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.

Ebf1 and c-Myb Repress Rag Transcription Downstream of Stat5 during Early B Cell Development

The temporal control of RAG (Rag) expression in developing lymphocytes prevents DNA breaks during periods of proliferation that could threaten genomic integrity. In developing B cells, the IL-7R and precursor B cell Ag receptor (pre-BCR) synergize to induce proliferation and the repression of Rag at the protein and mRNA levels for a brief period following successful Ig H chain gene rearrangement. Whereas the mechanism of RAG2 protein downregulation is well defined, little is known about the pathways and transcription factors that mediate transcriptional repression of Rag. Using Abelson murine leukemia virus-transformed B cells to model this stage of development, we identified early B cell factor 1 (Ebf1) as a strong repressor of Rag transcription. Short hairpin RNA-mediated knockdown of either Ebf1 or its downstream target c-Myb was sufficient to induce Rag transcription in these highly proliferative cells. Ebf1 and c-Myb antagonize Rag transcription by negatively regulating the binding of Foxo1 to the Rag locus. Ebf1 accomplishes this through both direct negative regulation of Foxo1 expression and direct positive regulation of Gfi1b expression. Ebf1 expression is driven by the IL-7R downstream effector Stat5, providing a link between the negative regulation of Rag transcription by IL-7 and a novel repressive pathway involving Ebf1 and c-Myb.