The aged tumor microenvironment limits T cell control of cancer.

The etiology and effect of age-related immune dysfunction in cancer is not completely understood. Here we show that limited priming of CD8+ T cells in the aged tumor microenvironment (TME) outweighs cell-intrinsic defects in limiting tumor control. Increased tumor growth in aging is associated with reduced CD8+ T cell infiltration and function. Transfer of T cells from young mice does not restore tumor control in aged mice owing to rapid induction of T cell dysfunction. Cell-extrinsic signals in the aged TME drive a tumor-infiltrating age-associated dysfunctional (TTAD) cell state that is functionally, transcriptionally and epigenetically distinct from canonical T cell exhaustion. Altered natural killer cell-dendritic cell-CD8+ T cell cross-talk in aged tumors impairs T cell priming by conventional type 1 dendritic cells and promotes TTAD cell formation. Aged mice are thereby unable to benefit from therapeutic tumor vaccination. Critically, myeloid-targeted therapy to reinvigorate conventional type 1 dendritic cells can improve tumor control and restore CD8+ T cell immunity in aging.

CD19-targeting CAR T cells protect from ANCA-induced acute kidney injury.

OBJECTIVES: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are life-threatening systemic autoimmune diseases manifesting in the kidneys as necrotizing crescentic glomerulonephritis (NCGN). ANCA antigens are myeloperoxidase (MPO) or proteinase 3. Current treatments include steroids, cytotoxic drugs and B cell-depleting antibodies. The use of chimeric antigen receptor (CAR) T cells in autoimmune diseases is a promising new therapeutic approach. We tested the hypothesis that CAR T cells targeting CD19 deplete B cells, including MPO-ANCA-producing B cells, thereby protecting from ANCA-induced NCGN. METHODS: We tested this hypothesis in a preclinical MPO-AAV mouse model. NCGN was established by immunisation of MPO-/- mice with murine MPO, followed by irradiation and transplantation with haematopoietic cells from wild-type mice alone or together with either CD19-targeting CAR T cells or control CAR T cells. RESULTS: CD19 CAR T cells efficiently migrated to and persisted in bone marrow, spleen, peripheral blood and kidneys for up to 8 weeks. CD19 CAR T cells, but not control CAR T cells, depleted B cells and plasmablasts, enhanced the MPO-ANCA decline, and most importantly protected from NCGN. CONCLUSION: Our proof-of-principle study may encourage further exploration of CAR T cells as a treatment for ANCA-vasculitis patients with the goal of drug-free remission.

Analyses of murine lymph node endothelial cell subsets using single-cell RNA sequencing and spectral flow cytometry.

Blood endothelial cells (BECs) in lymph nodes are distinct stromal cells with a transcriptional profile allowing fast and specific adaptation to the functional requirements. Here, we describe a step-by-step protocol for the enzymatic digestion of lymph nodes, the enrichment of stromal cells, the sorting of BECs, and the processing of BEC-related data for modern analysis approaches as spectral flow cytometry and single-cell RNA sequencing (scRNA-seq). For complete details on the use and execution of this protocol, please refer to Menzel et al. (2021).

Lymphocyte access to lymphoma is impaired by high endothelial venule regression.

Blood endothelial cells display remarkable plasticity depending on the demands of a malignant microenvironment. While studies in solid tumors focus on their role in metabolic adaptations, formation of high endothelial venules (HEVs) in lymph nodes extends their role to the organization of immune cell interactions. As a response to lymphoma growth, blood vessel density increases; however, the fate of HEVs remains elusive. Here, we report that lymphoma causes severe HEV regression in mouse models that phenocopies aggressive human B cell lymphomas. HEV dedifferentiation occurrs as a consequence of a disrupted lymph-carrying conduit system. Mechanosensitive fibroblastic reticular cells then deregulate CCL21 migration paths, followed by deterioration of dendritic cell proximity to HEVs. Loss of this crosstalk deprives HEVs of lymphotoxin-ß-receptor (LTßR) signaling, which is indispensable for their differentiation and lymphocyte transmigration. Collectively, this study reveals a remodeling cascade of the lymph node microenvironment that is detrimental for immune cell trafficking in lymphoma.

CXCR5 CAR-T cells simultaneously target B cell non-Hodgkin's lymphoma and tumor-supportive follicular T helper cells.

CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME.

Lymphoma Angiogenesis Is Orchestrated by Noncanonical Signaling 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 LTß 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, LTßR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/80/6/1316/F1.large.jpg.