Search

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.

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.