Work

733 Macrophage-dendritic cell-T-cell tetrads orchestrate antitumor immunity and response to checkpoint blockade

Year: 2026

Type: conference-abstract

Abstract: Background Immune checkpoint inhibitors (ICIs) induce durable responses in only a subset of patients with solid tumors, highlighting the need to define the multicellular interactions that coordinate effective antitumor immunity. We sought to identify the spatial immune architecture underlying successful responses to checkpoint blockade Therapy.Methods Multiplexed imaging (COMET in murine tumors and CODEX in human tumors), spatial transcriptomics (Xenium and Visium HD), flow cytometry, ex vivo multicellular coculture assays, and in vivo tumor models were integrated to characterize immune organization following anti-CTLA-4 or dual checkpoint blockade. Mechanistic studies incorporated adoptive macrophage transfer, ICOS- and ICAM1-knockout mice, and cDC1-specific ICOSL conditional knockout mice.Results We identified a previously unrecognized multicellular immune structure composed of macrophages, cDC1s, CD4 + T cells, and CD8+ T cells, which we term tetrads. Tetrads formed early following checkpoint blockade and were significantly enriched compared with triads in both mouse and human tumors (figure 1). Spatial analyses demonstrated that tetrads are enriched for ICOS+ Th1-like CD4+ T cells, ICOSLhigh cDC1s, activated CD8+ T cells, and IFNγ-responsive macrophages, consistent with a coordinated immune niche supporting productive antitumor immunity. Functional studies showed that macrophage ICAM1 promotes tetrad formation, while genetic disruption of the ICOS–ICOSL axis, including cDC1-specific ICOSL deletion, reduced tetrad abundance and impaired antitumor immunity, supporting a model in which ICOSL–ICOS signaling helps sustain tetrads during effective immune responses. Importantly, tetrads, but not dyads or triads, were associated with clinical response in patients with bladder cancer treated with neoadjuvant dual checkpoint blockade.Conclusions Tetrads represent a fundamental multicellular immune unit that coordinates productive antitumor immunity. Our findings support a model in which macrophage ICAM1 promotes tetrad assembly, whereas ICOSL–ICOS signaling helps sustain these structures and their immune function, establishing tetrads as a mechanistic framework for effective T-cell activation and immune checkpoint blockade efficacy ( figure 2).Ethics Approval For details on the clinical trial, see Gao et al 2020. Briefly, Patients with high-risk, cisplatin-ineligible urothelial carcinoma (UC) were enrolled onto a pilot, single-arm neoadjuvant clinical trial evaluating combined PD-L1 and CTLA-4 blockade (MD Anderson Cancer Center protocol 2016-0033; NCT02812420). The study was approved by the MDACC Institutional Review Board, and all participants provided written informed consent. The primary endpoint was safety and tolerability of durvalumab plus tremelimumab in the neoadjuvant setting, assessed at each clinical visit according to NCI CTCAE v4.03. Secondary endpoints included immunological and molecular responses to therapy and pathological responses at cystectomy. Patients in this cohort received durvalumab (1,500 mg) plus tremelimumab (75 mg) on weeks 1 and 5. Radical cystectomy with bilateral pelvic lymph-node dissection was performed between weeks 9 and 11. Peripheral blood and tumor samples were collected pre- and post-treatment under a correlative laboratory protocol (MDACC PA13-0291).Abstract 733 Figure 1Identification of immune tetrads induced by anti-CTLA-4 and dual checkpoint inhibition therapies in murine tumors. (A) Schematics illustrating the sequential immunofluorescence and RNAscope pipeline analysis for major cell phenotyping and tetrad identification in mouse B16F10 tumors collected on day 12 (n = 59 total biological replicates for entire study for B16F10 tumor models); (B) Representative COMET images highlighting tetrads (white circles). Tetrads are composed of CD8+ T-cells (red). CD4+ T-cells (blue cyan), cDC1 (green) and macrophages (purple). Nuclear staining (DAPI) is highlighted in dark blue. (C–D) Tetrad quantification representing absolute numbers per ROI (C), and numbers normalized to the total number of cells per each ROI (D). Data are pooled from 3 independent experiments (n = 8–16 biological replicates per group). Significance is calculated by One-way ANOVA with Tukey’s multiple comparisons test. (*p < 0.05, ** p < 0.01). Values are mean ± SEM. (E–F) Triad and tetrad comparison in mouse B16F10 tumors; (E) Schematics illustrating differences between triad and tetrad quantification (mutually exclusive); (F) Number of triads and tetrads per ROI (color-coded per treatment group). Data are pooled from 3 independent experiments (n = 48 biological replicates). ( **** p < 0.0001, unpaired T-test). Values are mean ± SEMAbstract 733 Figure 2Proposed model of tetrad formation and function during anti-tumor immunity. Immune checkpoint blockade, particularly anti-CTLA-4, promotes IFNy production within the tumor microenvironment, inducing activation of macrophages characterized by increased ICAM-1, MHC-II, and CXCL9/10 expression. Activated ICAM-1+ macrophages promote the assembly of a multicellular immune structure, termed the tetrad, consisting of a macrophage, cDC1, CD4+ T cell, and CD8+ T cell. Within tetrads, cDC1s provide antigen presentation to both CD4+ and CD8+ T cells, while ICOSL- ICOS interactions are proposed to sustain tetrad integrity and productive immune communication. Concurrently, macrophage ICAM-1 engages LFA-1 on T cells to stabilize cellular interactions and facilitate optimal immune activation. Tetrads support the generation of highly functional CD8+ T cells with enhanced cytotoxic activity (increased IFNγ, granzyme B, and perforin) and reduced expression of dysfunction-associated markers (TOX, PD-1, and TIM-3), resulting in improved responses to immune checkpoint blockade and durable tumor rejection

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0733