AbstractNatural killer (NK) cell function within tissues extends beyond exerting cytotoxicity, encompassing a range of functions that are just starting to become fully elucidated. In the context of human placentation, NK cells play a key role in enabling initial placentation, which is associated with the acquisition of tolerance-like properties. If and to which extent NK cells maintain these tolerance-like properties over the course of human pregnancy is still poorly understood. We asked if NK cells isolated from the decidual-placental interface of full-term human pregnancies are able to exert effector function. We observed a significant and striking lack in the ability of NK cells isolated from the decidual-placental interface (DPI) to produce interferon-g (IFN-γ) in response to the activating cytokines interleukin (IL)-12, IL-15, and IL-18. In contrast, NK cells from the decidua retained their responsiveness to cytokine-mediated activation. Notably, CD103+CD69+ tissue-resident NK cells were present in both DPI and decidua, yet exhibited distinct effector function from one another. Using high-parameter flow cytometry and single-cell sequencing, we found that this functional discrepancy was not directly predictable based on their cell surface phenotype or cell transcript. Together, our findings reveal the presence of distinct functional resident NK cell populations in 2 anatomically adjacent tissues at healthy full-term pregnancies.

UNLABELLED: Neoadjuvant immunotherapy improves outcomes in esophageal squamous cell carcinoma (ESCC), yet ∼70% of patients fail to respond. Pretreatment biopsies and plasma provide critical opportunities for biomarker discovery. In this study, we performed plasma proteomic profiling and identified soluble glycoprotein nonmetastatic melanoma protein B (sGPNMB) as the most elevated circulating protein in nonresponders. Mechanistically, tumor cell-derived sGPNMB suppressed CD8+ T-cell receptor signaling via the SDC4-CD148 axis to induce functional exhaustion, with secretion being required for its immunosuppressive activity. Cancer-associated fibroblast-epithelial (CAF-Epi) niches promoted SOX2 upregulation in tumor cells, transcriptionally activating GPNMB expression. In humanized patient-derived xenograft models, circulating GPNMB levels predicted response to PD-1 blockade, and GPNMB inhibition synergized with therapy. Across retrospective cohorts and a prospective clinical trial, a multimodal model combining plasma GPNMB levels, CAF-Epi niche detection, and clinical-pathologic features achieved robust predictive accuracy for immunotherapy response and survival. These findings establish a spatial-circulating biomarker framework for precision ESCC immunotherapy. SIGNIFICANCE: Tumor-derived soluble GPNMB, transcriptionally activated by SOX2 within CAF-Epi niches, drives CD8+ T-cell exhaustion and resistance to PD-1 blockade in ESCC. Integrating circulating GPNMB levels with CAF-Epi niche features and clinical-pathologic factors, we develop and validate a clinically scalable multimodal model for predicting immunotherapy response.

BACKGROUND: Chimeric antigen receptor (CAR) T cells are highly potent therapies approved by the U.S. Food and Drug Administration (FDA) for several hematological malignancies. However, efficacy remains variable due to resistance, antigen modulation, systemic toxicities, and relapse. Developing improved CAR T cell therapies relies on preclinical models that accurately predict clinical outcomes. For this purpose, immortalized tumor cell lines, such as the Burkitt's lymphoma line Raji, are commonly used. METHODS: CD19-targeting CAR (CAR19) and non-transduced (NT) human T cells were co-cultured with target cell lines in vitro to assess T cell cytotoxicity, cytokine production, activation, and proliferation. Major histocompatibility complex (MHC) class I and II blocking antibodies and TCR knockout (KO) T cells were used to validate T cell receptor interactions. In vivo, Raji wild-type (wt) or CD19 KO tumor-bearing mice were treated with CAR19 or NT T cells, and survival, tumor burden, serum cytokines, and T cell phenotype in tissues were analyzed. Statistical analyses using either paired/paired ratio/unpaired two-tailed Student's t tests, two-way analysis of variance, and log-rank (Mantel-Cox) tests were performed using GraphPad Prism 9. RESULTS: We report that T cells generated from multiple different donors exhibit not only strong CAR-mediated cytotoxicity against CD19-expressing Raji WT cells but also, unexpectedly, CAR-independent reactivity against Raji CD19 KO cells. This reactivity was observed both in vitro and in vivo, and was associated with pro-inflammatory cytokine secretion and upregulation of T cell activation markers, consistent with endogenous T cell receptor (TCR) engagement. NT T cells displayed similar activation, cytotoxic, and cytokine responses against Raji cells. These effects failed to occur using other B cell leukemia/lymphoma or solid tumor cell lines in vitro. Both CD4+ and CD8+ T cell populations contributed to this alloreactive response, which could be attenuated by blockade of MHC class I or II, highlighting a TCR:MHC-dependent mechanism. CONCLUSIONS: These findings demonstrate that certain tumor lines can elicit strong allogeneic T cell responses independent of CAR specificity, revealing an important limitation of commonly used preclinical models and highlighting the potential for false-positive assessments of CAR T cell efficacy. Furthermore, these results highlight the importance of validating tumor models for alloreactivity prior to use in preclinical testing.