Patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) are typically characterized by the presence of autoantibodies and an IFN-signature. The strength of the ...IFN-signature positively correlates with disease severity, suggesting that type I IFNs are active players in these diseases. BAFF is a cytokine critical for development and proper selection of B cells, and the targeting of BAFF has emerged as a successful treatment strategy of SLE. Previous reports have suggested that BAFF expression is directly induced by type I IFNs, but the precise mechanism for this remains unknown. In this article, we demonstrate that BAFF is a bona fide ISG and that IFN regulatory factors (IRFs) control the expression of BAFF. We identify IRF1 and IRF2 as positive regulators of BAFF transcription and IRF4 and IRF8 as potent repressors; in addition, we have mapped the precise binding site for these factors in the BAFF promoter. IFN-β injections induced BAFF expression mainly in neutrophils and monocytes, and BAFF expression in neutrophils from pSS patients strongly correlated with the strength of the IFN-signature. In summary, we show that BAFF expression is directly induced by type I IFNs via IRF1 and IRF2, whereas IRF4 and IRF8 are negative regulators of BAFF expression. These data suggest that type I IFN blockade in SLE and pSS patients will lead to downregulation of BAFF and a consequential reduction of autoreactive B cell clones and autoantibodies.
BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While ...remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.
Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular ...mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin IL 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway.
Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of ...their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Despite the high remission rates achieved using T cells bearing a chimeric antigen receptor (CAR) against hematogical malignancies, there is still a considerable proportion of patients who eventually ...experience tumor relapse. Clinical studies have established that mechanisms of treatment failure include the down-regulation of target antigen expression and the limited persistence of effective CAR T cells. We hypothesized that dual targeting mediated by a CAR and a chimeric costimulatory receptor (CCR) could simultaneously enhance T cell cytotoxicity and improve durability. Concomitant high-affinity engagement of a CD38-binding CCR enhanced the cytotoxicity of BCMA-CAR and CD19-CAR T cells by increasing their functional binding avidity. In comparison to second-generation BCMA-CAR or CD19-CAR T cells, double-targeted CAR + CD38-CCR T cells exhibited increased sensitivity to recognize and lyse tumor variants of multiple myeloma and acute lymphoblastic leukemia with low antigen density in vitro. In addition, complimentary costimulation by 4-1BB and CD28 endodomains provided by the CAR and CCR combination conferred increased cytokine secretion and expansion and improved persistence in vivo. The cumulatively improved properties of CAR + CCR T cells enabled the in vivo eradication of antigen-low tumor clones, which were otherwise resistant to treatment with conventional CAR T cells. Therefore, multiplexing targeting and costimulation through the combination of a CAR and a CCR is a powerful strategy to improve the clinical outcomes of CAR T cells by enhancing cytotoxic efficacy and persistence, thus preventing relapses of tumor clones with low target antigen density.
Melanoma tumors are highly metastatic partly due to the ability of melanoma cells to transition between invasive and proliferative states. However, the mechanisms underlying this plasticity are still ...not fully understood. To identify new epigenetic regulators of melanoma plasticity, we combined data mining, tumor models, proximity proteomics, and CUT&RUN sequencing. We focus on the druggable family of bromodomain epigenetic readers and identify TRIM28 as a new regulator of melanoma plasticity. We find that TRIM28 promotes the expression of pro‐invasive genes and that TRIM28 controls the balance between invasiveness and growth of melanoma cells. We demonstrate that TRIM28 acts via the transcription factor JUNB that directly regulates the expression of pro‐invasive and pro‐growth genes. Mechanistically, TRIM28 controls the expression of JUNB by negatively regulating its transcriptional elongation by RNA polymerase II. In conclusion, our results demonstrate that a TRIM28–JUNB axis controls the balance between invasiveness and growth in melanoma tumors and suggest that the bromodomain protein TRIM28 could be targeted to reduce tumor spread.
Synopsis
TRIM28 controls JUNB transcription that in turn induces RAS signature genes and suppresses YAP1 signature genes. TRIM28 depletion therefore leads to higher JUNB expression followed by increased melanoma growth and decreased melanoma invasiveness.
TRIM28 is highly expressed in aggressive melanoma tumors.
TRIM28 negatively regulates the transcriptional elongation of JUNB.
JUNB expression induces a switch from melanoma invasiveness to melanoma growth.
TRIM28 controls JUNB transcription that in turn induces RAS signature genes and suppresses YAP1 signature genes. TRIM28 depletion therefore leads to higher JUNB expression followed by increased melanoma growth and decreased melanoma invasiveness.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Tripartite-motif 21 (TRIM21) is an E3 ubiquitin ligase that regulates innate immune responses by ubiquitinating IFN regulatory factors (IRFs). TRIM21 is mainly found in hematopoietic cells in which ...its expression is induced by IFNs during viral. infections and in systemic autoimmune diseases such as systemic lupus erythematosus and Sjögren's syndrome. However, the exact molecular mechanism by which the expression of the Trim21 gene is regulated is unknown. In this study, we demonstrate that IFNs induce Trim21 expression in immune cells via IRFs and that IFN-α and IFN-β are the most potent inducers of Trim21. A functional IFN-stimulated response element but no conserved IFN-γ-activated site was detected in the promoter of Trim21. IRF1 and IRF2 strongly induced Trim21 expression in an IFN-stimulated response element-dependent manner, whereas IRF4 and IRF8 strongly repressed the IRF1-mediated induction of Trim21. Consistent with this observation, baseline expression of Trim21 was elevated in Irf4(-/-) cells. TRIM21, IRF1, and IRF2 expression was increased in PBMCs from patients with Sjögren's syndrome compared with healthy controls. In contrast, IRF4 and IRF8 expression was not increased in PBMCs from patients. The IFN-γ-mediated induction of Trim21 was completely abolished by inhibiting protein synthesis with cycloheximide, and Trim21 expression could not be induced by IFN-γ in Irf1(-/-) cells, demonstrating that IFN-γ induces Trim21 indirectly via IRF1 and not directly via STAT1 activation. Our data demonstrate that multiple IRFs tightly regulate expression of Trim21 in immune cells, suggesting that a well-controlled expression of the E3 ligase TRIM21 is important for regulation of immune responses.
Summary
TRIM21 is an interferon‐stimulated E3 ligase that controls the activity of pattern‐recognition signaling via ubiquitination of interferon regulatory factors and DDX41. Previous studies on the ...role of TRIM21 in innate immune responses have yielded contradictory results, suggesting that the role of TRIM21 is cell specific. Here, we report that bone‐marrow‐derived macrophages (BMDMs) generated from Trim21−/− mice have reduced expression of mature macrophage markers. Reflecting their reduced differentiation in response to macrophage colony‐stimulating factor (M‐CSF), Trim21−/− BMDMs had decreased expression of M‐CSF signature genes. Although Trim21−/− BMDMs responded normally to Toll‐like receptor 9 (TLR9) activation, they produced lower levels of pro‐inflammatory cytokines in response to the TLR2 agonist PAM3CSK4. In line with this, the response to infection with the Bacillus Calmette–Guérin strain of Mycobacterium bovis was also diminished in Trim21−/− BMDMs. Our results indicate that TRIM21 controls responses to TLR2 agonists.
Trim21‐deficient bone marrow‐derived macrophages had a reduced response to the Toll‐like receptor 2 agonists PAM3CSK4 and Bacillus Calmette–Guérin. This was reflected by a reduced production of pro‐inflammatory cytokines and iNOS.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Type I interferons (IFNs) are potent inducers of the first-line defense against pathogens. Theiractivity leads to the up- and downregulation of a large number of genes with various effects on ...theimmune system, including direct effects on the pathogens. Due to the strong response evoked byIFN signaling, the IFN pathway is tightly regulated by IFN stimulated genes to avoid detrimentaleffects of long-term exposure. If the IFN signaling pathway is not regulated properly, or for otherreasons constantly activated, it can lead to interferonopathies and autoimmune diseases such assystemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS). Indeed, many therapeuticstargeting the IFN pathway are currently in clinical trials. In contrast, type I IFNs are used to treatcertain types of cancer, virus infections and multiple sclerosis. The complex role of type I IFNsignaling in disease is not well understood and need further characterization for better therapeuticinventions.The aim of this thesis was to identify genes that are regulated by type I IFNs and investigate theirrole in the immune system. To do this, we quantified the expression of interferon-regulated genesin sorted immune cells from patients with primary SS, and from individuals treated with IFNβ.Using global gene expression analysis on PBMCs as well as qPCR on sorted cells, we could identifyseveral genes that were differentially regulated by type I IFNs in different immune cell populations.Among them were TRIM21 that was upregulated in T cells and B cells, BAFF that was upregulatedin T cells, monocytes and neutrophils and miR-150-5p that was selectively downregulated inmonocytes. The downregulated expression of miR-150-5p consequently led to an increasedexpression of its target c-Myb. In addition, monocytes from patients with SLE displayed an increasein c-Myb target genes. When investigating the regulation of BAFF and TRIM21, we found thatboth were regulated by members of the interferon regulatory factor (IRF) family. Specifically, bothwere upregulated by IRF1 and IRF2, and downregulated by IRF4 and IRF8. To further investigatethe role of TRIM21 in the immune system, we generated Trim21-/- mice. These mice were prone togranulocyte infiltrations and developed symptoms of autoimmune disease after being triggered bymetal ear tags. We found that TRIM21 negatively regulates the immune response by ubiquitinatingIRFs, and that naïve Trim21-/- mice control the development of eosinophils in the bone marrow.
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