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Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9 silencing for the improvement of adoptive T cell therapy. MicroRNA (miRNA)-mediated EBAG9 downregulation in transplanted cytolytic CD8+ T cells (CTLs) from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably fewer EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9 silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose.

Supplemental Movie S2. Follicular motility of B cells. The Movie complements Figure 4J.

Supplementary Movie S1. Follicular motility of Emu-Tcl1 leukemia cells. The Movie complements Figure 4J.

Figure S1. Gating strategy to characterize and quantitate CD19+CD5+ leukemia Emu-Tcl1 cell frequencies and homeostatic chemokine receptor expression on splenic, LN, PB, and BM derived CD19+CD5+ gated Emu-Tcl1 tumor cells. This Figure complements Figure 1. Figure S2. Surface marker expression of murine Emu-Tcl1 leukemia cells is shown. Human primary B-CLL cells share characteristic expression of CXCR5. This Figure complements Figure 1. Figure S3. Enrichment plots of seven proliferation-related signatures are presented. Short term transfer of CXCR5+/+Emu-Tcl1 or CXCR5-/-Emu-Tcl1 tumor cells shows upregulation of the gene encoding the cell-cycle inhibitor p21 in CXCR5-deficient leukemia cells. Growth and activation delay of CXCR5-/- Emu-Tcl1 tumor cells can be reversed by direct contacts with stromal cells in vitro. This Figure complements Figure 2. Figure S4. (A) shows that CCR7-deficiency on leukemia cells does not affect their migratory behavior and complements Figure 3. (B) Ebi2-/-Emu-Tcl1 leukemia cells enter B cell follicles and the GC light zone area in a pattern comparable to Ebi2++Emu-Tcl1 leukemia cells, as presented in Figure 4, and (C) tumor growth of Ebi2-/-Emu-Tcl1 mice is unaltered compared to Wt Emu-Tcl1 mice. Figure S5. Human primary B-CLLs exhibit a characteristic FDC staining. This Figure complements Figure 4. Figure S6. Pertussis toxin (PTX) treatment induces apoptosis in Emu-Tcl leukemia cells co-cultured with FDC-HK stroma cells. This Figure complements Figure 4. Figure S7. Intravital application of anti-CD21/CD35 Fab fragments stains specifically GC light zone localized FDCs. These stainings complement Figure 5. Figure S8. Challenge of Rag2-/- mice with Emu-Tcl1 leukemia B cells induces FDC networks and a tumor cell-promoting cytokine and chemokine secretion. This Figure accompanies Figure 6. Figure S9. Abrogation of LTbetaR-signaling leads to disappearance of characteristic stromal elements within the B cell follicle; however, CD31+ blood vessels are not affected. In accordance with Figure 7, Ltalpha -/- Emu-Tcl1 mice exhibit substantially delayed leukemia growth and progression.

Lack of hypoxia and tip cell signature

Neutrophil localization in human SMZL and benign spleen tissues.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Tumor-induced down- and upregulations of genes in the stroma.

Top GO and GSEA pathways

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma–induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis.Significance:In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.

Legends for Supplementary Figures S1-S6.

Supplementary Material+Methods, Legends, References

Recruitment of tumor-associated macrophages and neutrophils (TAM and TAN) to solid tumors contributes to immunosuppression in the tumor microenvironment; however, their contributions to lymphoid neoplasms are less clear. In human chronic lymphocytic leukemia (CLL), tumor B cells lodge in lymph nodes where interactions with the microenvironment occur. Tumor cell homing stimulates proliferation, such that engagement of the B-cell receptor is important for malignant progression. In the Eμ-Tcl1 murine model of CLL, we identified gene expression signatures indicative of a skewed polarization in the phenotype of monocytes and neutrophils. Selective ablation of either of these cell populations in mice delayed leukemia growth. Despite tumor infiltration of these immune cells, a systemic inflammation was not detected. Notably, in progressive CLL, splenic neutrophils were observed to differentiate toward a B-cell helper phenotype, a process promoted by the induction of leukemia-associated IL10 and TGFβ. Our results suggest that targeting aberrant neutrophil differentiation and restoring myeloid cell homeostasis could limit the formation of survival niches for CLL cells. Cancer Res; 76(18); 5253–65. ©2016 AACR.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPβ-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPβ protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPβ as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

Chimeric antigen receptor (CAR) T cell therapy that involves genetic engineering a patient's own immune cells with antigen-specific receptors has shown remarkable efficacy in blood cancer treatment. Numerous clinical studies with CAR T cells targeting the blood cell surface protein CD19 led to the FDA 's first approval of a genetically engineered cell therapy. The process of generating potent CAR T cells involves several carefully performed manufacturing steps. Here, we describe the generation of redirected engineered human CAR T cells for preclinical studies starting with the CAR design, retroviral gene transfer, detection of CAR expression, and expansion of transduced T cells.

Dendritic cell (DC) maturation is a prerequisite for the induction of adaptive immune responses against pathogens and cancer. Transcription factor (TF) networks control differential aspects of early DC progenitor versus late-stage DC cell fate decisions. Here, we identified the TF C/EBPβ as a key regulator for DC maturation and immunogenic functionality under homeostatic and lymphoma-transformed conditions. Upon cell-specific deletion of C/EBPβ in CD11c(+)MHCII(hi) DCs, gene expression profiles of splenic C/EBPβ(−/−) DCs showed a down-regulation of E2F cell cycle target genes and associated proliferation signaling pathways, whereas maturation signatures were enriched. Total splenic DC cell numbers were modestly increased but differentiation into cDC1 and cDC2 subsets were unaltered. The splenic CD11c(+)MHCII(hi)CD64(+) DC compartment was also increased, suggesting that C/EBPβ deficiency favors the expansion of monocytic-derived DCs. Expression of C/EBPβ could be mimicked in LAP/LAP* isoform knockin DCs, whereas the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a negative regulator of DC maturation, C/EBPβ(−/−) bone marrow-derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, lymphoma-exposed DCs exhibited an up-regulation of the E2F transcriptional pathways and an impaired maturation. Pharmacological blockade of C/EBPβ/mTOR signaling in human DCs abrogated their protumorigenic function in primary B cell lymphoma cocultures. Thus, C/EBPβ plays a unique role in DC maturation and immunostimulatory functionality and emerges as a key factor of the tumor microenvironment that promotes lymphomagenesis.

The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B-cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12-CXCR4 as the leading signaling axis for B-ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient-derived xenograft (PDX)-ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2-like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4-mediated functions using an anti-CXCR4 antibody treatment completely abolished testicular infiltration of PDX-ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.

Insight into processes that determine CD8(+) T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often employ high avidity CD8(+) T cells in adoptive transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon non-inflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8(+) T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8(+) memory commitment was analyzed in Ebag9-deficient mice that exhibit an enhanced tumor cell lysis. This property endowed Ebag9(-/-) mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9(-/-) mice, an expanded memory population was obtained for anti-HY and anti-SV40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8(+) T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9(+/+) and Ebag9(-/-) T cells. In Ebag9(-/-) cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation, along with anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8(+) memory lineage.

Simple Summary Efficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future. Abstract Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). Subsequently, we evaluated different pharmacological interventions to improve cell viability and relative knock-in rates in T cells. While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

The IκB kinase (IKK)‐NF‐κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double‐strand breaks elicit two subsequent phases of NF‐κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA‐sequencing reveals that the first‐phase controls anti‐apoptotic gene expression, while the second drives expression of senescence‐associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM‐PARP1‐TRAF6‐IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re‐expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF‐κB family member p65/RelA, in part mediated by GSK3β, results in transcriptional silencing of NFKBIA and IKK‐independent, constitutive activation of NF‐κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF‐κB activation with important implications for genotoxic cancer treatment., DNA damage induces two kinetically and functionally distinct phases of NF‐κB activation, the first being anti‐apoptotic and the non‐canonical second wave inducing the senescence‐associated secretory phenotype (SASP).

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma–induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTβ receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis. Significance: In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTβR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.

The IκB kinase (IKK) - NF-κB pathway is activated as part of the DNA damage response and controls both resistance to apoptosis and inflammation. How these different functions are achieved remained unknown. We demonstrate here that DNA double strand breaks elicit two subsequent phases of NF-κB activationin vivoandin vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The first, rapidly activated phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of IκBα and is terminated through IκBα (NFKBIA) re-expression. The second phase is activated days later in senescent cells but is independent of IKK and the proteasome. An altered phosphorylation status of p65, in part driven by GSK3β, results in transcriptional silencing ofNFKBIAand IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.

The IκB kinase (IKK)-NF-κB signaling pathway plays a multifaceted role in inflammatory bowel disease (IBD): on the one hand, it protects from apoptosis; on the other, it activates transcription of numerous inflammatory cytokines and chemokines. Although several murine models of IBD rely on disruption of IKK-NF-κB signaling, these involve either knockouts of a single family member of NF-κB or of upstream kinases that are known to have additional, NF-κB-independent, functions. This has made the distinct contribution of NF-κB to homeostasis in intestinal epithelium cells difficult to assess. To examine the role of constitutive NF-κB activation in intestinal epithelial cells, we generated a mouse model with a tissue-specific knockout of the direct inhibitor of NF-κB, Nfkbia/IκBα. We demonstrate that constitutive activation of NF-κB in intestinal epithelial cells induces several hallmarks of IBD including increased apoptosis, mucosal inflammation in both the small intestine and the colon, crypt hyperplasia, and depletion of Paneth cells, concomitant with aberrant Wnt signaling. To determine which NF-κB-driven phenotypes are cell-intrinsic, and which are extrinsic and thus require the immune compartment, we established a long-term organoid culture. Constitutive NF-κB promoted stem-cell proliferation, mis-localization of Paneth cells, and sensitization of intestinal epithelial cells to apoptosis in a cell-intrinsic manner. Increased number of stem cells was accompanied by a net increase in Wnt activity in organoids. Because aberrant Wnt signaling is associated with increased risk of cancer in IBD patients and because NFKBIA has recently emerged as a risk locus for IBD, our findings have critical implications for the clinic. In a context of constitutive NF-κB, our findings imply that general anti-inflammatory or immunosuppressive therapies should be supplemented with direct targeting of NF-κB within the epithelial compartment in order to attenuate apoptosis, inflammation, and hyperproliferation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Despite progress in exploiting therapeutically the genetic vulnerabilities of leukemia and lymphoma, the outcome is often extended progression-free survival but not tumor eradication. Lymphomagenesis is not a tumor-autonomous process, and the onset and progression of tumors requires reciprocal crosstalk with the tumor microenvironment (TME). Minimal residual disease and immunosurveillance are also regulated by factors in the TME. Here, we dissect the stromal compartment in lymphoid organs, focusing on conditions that are prevalent in an autochthonous environment for lymphoid neoplasia. Identification of tumor-promoting factors is instrumental in the selection of therapeutic targets that are part of the immune system. Targeting the TME is an appealing treatment option for lymphoma because this compartment is subject to low selective pressure for mutations.

How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.

Autologous T cells genetically modified with a chimeric antigen receptor (CAR) redirected at CD19 have potent activity in the treatment of B cell leukemia and B cell non-Hodgkin’s lymphoma (B-NHL). Immunotherapies to treat multiple myeloma (MM) targeted the B cell maturation antigen (BCMA), which is expressed in most cases of MM. We developed a humanized CAR with specificity for BCMA based on our previously generated anti-BCMA monoclonal antibody. The targeting single-chain variable fragment (scFv) domain exhibited a binding affinity in the low nanomolar range, conferring T cells with high functional avidity. Redirecting T cells by this CAR allowed us to explore BCMA as an alternative target for mature B-NHLs. We validated BCMA expression in diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia. BCMA CAR T cells triggered target cell lysis with an activation threshold in the range of 100 BCMA molecules, which allowed for an efficient eradication of B-NHL cells in vitro and in vivo. Our data corroborate BCMA is a suitable target in B cell tumors beyond MM, providing a novel therapeutic option for patients where BCMA is expressed at low abundance or where anti-CD19 immunotherapies have failed due to antigen loss.

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Microenvironmental regulation in lymphoid tissues is essential for the development of chronic lymphocytic leukemia. We identified cellular and molecular factors provided by the splenic marginal zone (MZ), which alter the migratory and adhesive behavior of leukemic cells. We used the Cxcr5-/-Eµ-Tcl1 leukemia mouse model, in which tumor cells are excluded from B cell follicles and instead accumulate within the MZ. Genes involved in MZ B cell development and genes encoding for adhesion molecules were upregulated in MZ-localized Cxcr5-/-Eµ-Tcl1 cells. Likewise, surface expression of the adhesion and homing molecules, CD49d/VLA-4 and CXCR7, and of NOTCH2 was increased. In vitro, exposing Eµ-Tcl1 cells or human CLL cells to niche-specific stimuli, like B cell receptor- or Toll-like receptor ligands, caused surface expression of these molecules characteristic for a follicular or MZ-like microenvironment, respectively. In vivo, inhibition of VLA-4-mediated adhesion and CXCL13-mediated follicular homing displaced leukemic cells not only from the follicle, but also from the MZ and reduced leukemia progression. We conclude that MZ-specific factors shape the phenotype of leukemic cells and facilitate their niche-specific retention. This strong microenvironmental influence gains pathogenic significance independent from tumor-specific genetic aberrations.

In human chronic lymphocytic leukemia (CLL) pathogenesis, B-cell antigen receptor signaling seems important for leukemia B-cell ontogeny, whereas the microenvironment influences B-cell activation, tumor cell lodging, and provision of antigenic stimuli. Using the murine Eμ-Tcl1 CLL model, we demonstrate that CXCR5-controlled access to follicular dendritic cells confers proliferative stimuli to leukemia B cells. Intravital imaging revealed a marginal zone B cell–like leukemia cell trafficking route. Murine and human CLL cells reciprocally stimulated resident mesenchymal stromal cells through lymphotoxin–β-receptor activation, resulting in CXCL13 secretion and stromal compartment remodeling. Inhibition of lymphotoxin/lymphotoxin–β-receptor signaling or of CXCR5 signaling retards leukemia progression. Thus, CXCR5 activity links tumor cell homing, shaping a survival niche, and access to localized proliferation stimuli. Significance: CLL and other indolent lymphoma are not curable and usually relapse after treatment, a process in which the tumor microenvironment plays a pivotal role. We dissect the consecutive steps of CXCR5-dependent tumor cell lodging and LTβR-dependent stroma–leukemia cell interaction; moreover, we provide therapeutic solutions to interfere with this reciprocal tumor–stroma cross-talk. Cancer Discov; 4(12); 1448–65. ©2014 AACR. See related commentary by López-Guerra et al., p. 1374 This article is highlighted in the In This Issue feature, p. 1355

Infection with helminths and exposure to antigens induce a strong type 2 immune response resulting in the secretion of the cytokines IL-4 and IL-13 by CD4(+) T cells and several innate cell types. IL-4 and IL-13 promote class switch recombination to IgG1 and IgE while their role for germinal center (GC) formation is poorly understood. We found a dramatic reduction in the numbers of GC B cells when investigating different type 2 immune responses in IL-4/IL-13-deficient mice. IL-4/IL-13 from T cells located outside B-cell follicles was sufficient for GC formation. We further revealed that IL-4/IL-13 acts directly on B cells for the formation of a robust GC response. The frequency of apoptotic GC B cells was not altered in the absence of IL-4/IL-13 and proliferation was even enhanced. However, deficiency of signal transducer and activator of transcription 6 signaling in B cells resulted in failure to downregulate the chemotactic receptor Gpr183 (Ebi2) and downregulation of this receptor has been shown to be essential for proper GC B-cell differentiation. Thus, T-cell-derived extrafollicular IL-4/IL-13 and signal transducer and activator of transcription 6-regulated genes in B cells play a critical role for orchestration of the GC response in type 2 immunity.

Aims The homeostatic chemokines, CCL19 and CCL21 and their receptor CCR7, have recently been linked to atherogenesis. We investigated the expression of CCL19/CCL21/CCR7 in carotid atherosclerosis as well as the ability of these chemokines to modulate lipid accumulation in macrophages and vascular smooth muscle cell (SMC) phenotype. Methods and results Our major findings were: (i) patients with carotid atherosclerosis ( n = 158) had increased plasma levels of CCL21, but not of CCL19, compared with controls ( n = 20), with particularly high levels in symptomatic ( n = 99) when compared with asymptomatic ( n = 59) disease. (ii) Carotid plaques showed markedly increased mRNA levels of CCL21 and CCL19 in symptomatic ( n = 14) when compared with asymptomatic ( n = 7) patients, with CCR7 localized to macrophages and vascular SMC (immunohistochemistry). (iii) In vitro , CCL21, but not CCL19, increased the binding of modified LDL and promoted lipid accumulation in THP-1 macrophages. (iv) CCL19, but not CCL21, increased proliferation and release and activity of matrix metalloproteinase (MMP) 1 in vascular SMC. (v) The differential effects of CCL19 and CCL21 in macrophages and SMC seem to be attributable to divergent signalling pathways, with CCL19-mediated activation of AKT in SMC- and CCL21-mediated activation of extracellular signal-regulated kinase 1/2 in macrophages. Conclusion CCL19 and CCL21 are up-regulated in carotid atherosclerosis. The ability of CCL21 to promote lipid accumulation in macrophages and of CCL19 to induce proliferation and MMP-1 expression in vascular SMC could contribute to their pro-atherogenic potential.

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor ROR{gamma}t and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7-/-Ror{gamma}t-/-, succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7-/-Ror{gamma}t-/- mice.

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates development of germinal center derived lymphomas in mice. In both human and murine lymphomas CREBBP loss of function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses including class II MHC. Mechanistically, CREBBP regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we find bind extensively to MHC class II loci. HDAC3 loss of function rescued repression of these enhancers and corresponding genes including MHC class II, and more profoundly suppress CREBPP mutant lymphomas in vitro and in vivo. Hence CREBBP loss of function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3 targeted therapy as a precision approach for CREBBP mutant lymphomas.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.