Regulatory T cells (Treg) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations, which does not provide an accurate picture of a physiologically relevant response. To overcome this limitation, we here develop 'single cell suppression profiling of human Tregs' (scSPOT). scSPOT uses a 52-marker CyTOF panel, a cell division detection algorithm, and a whole PBMC system to assess the effect of Tregs on all other cell types simultaneously. In this head-to-head comparison, we find Tregs having the clearest suppressive effects on effector memory CD8 T cells through partial division arrest, cell cycle inhibition, and effector molecule downregulation. Additionally, scSPOT identifies a Treg phenotypic split previously observed in viral infection and propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is thus scalable, robust, widely applicable, and may be used to better understand Treg immunobiology and screen for therapeutic compounds.
© 2025. The Author(s).

Clinical efficacy of chimeric antigen receptor (CAR) T cells against pediatric osteosarcoma (OS) has been limited. One strategy to improve efficacy may be to drive chemokine-mediated homing of CAR T cells to tumors. We sought to determine the primary chemokines secreted by OS and evaluate the efficacy of B7-H3.CAR T cells expressing the cognate receptors.
We developed a pipeline to identify chemokines secreted by OS by correlating RNA-seq data with chemokine protein detected in media from fresh surgical specimens. We identified CXCR2 and CXCR6 as promising receptors for enhancing CAR T-cell homing against OS. We evaluated the homing kinetics and efficiency of CXCR2- and CXCR6.T cells and homing, cytokine production, and antitumor activity of CXCR2- and CXCR6.B7-H3.CAR T cells in vitro and in vivo.
T cells transgenically expressing CXCR2 or CXCR6 exhibited ligand-specific enhanced migration over T cells modified with nonfunctional control receptors. Differential homing kinetics were observed, with CXCR2.T-cell homing quickly and plateauing early, whereas CXCR6.T cells took longer to home but achieved a similar plateau. When expressed in B7-H3.CAR T cells, CXCR2- and CXCR6 modification conferred enhanced homing toward OS in vitro and in vivo. CXCR2- and CXCR6-B7-H3.CAR-treated mice experienced prolonged survival in a metastatic model compared with B7-H3.CAR T-cell-treated mice.
Our patient-based pipeline identified targets for chemokine receptor modification of CAR T cells targeting OS. CXCR2 and CXCR6 expression enhanced the homing and anti-OS activity of B7-H3.CAR T cells. These findings support clinical evaluation of CXCR-modified CAR T cells to improve adoptive cell therapy for patients with OS.
©2024 The Authors; Published by the American Association for Cancer Research.

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

CD4+ T cells are critical to the immune system and perform multiple functions; therefore, their identification and characterization are crucial to better understanding the immune system in both health and disease states. However, current methods rarely preserve their ex vivo phenotype, thus limiting our understanding of their in vivo functions. Here we introduce a flexible, rapid, and robust platform for ex vivo CD4+ T cell identification. By combining MHCII allele purification, allele-independent peptide loading, and multiplexed flow cytometry technologies, we can enable high-throughput personalized CD4+ T cell identification, immunophenotyping, and sorting. Using this platform in combination with single-cell sorting and multimodal analyses, we identified and characterized antigen-specific CD4+ T cells relevant to COVID-19 and cancer neoantigen immunotherapy. Overall, our platform can be used to detect and characterize CD4+ T cells across multiple diseases, with potential to guide CD4+ T cell epitope design for any disease-specific immunization strategy.
© 2023 The Authors.

Autologous T cells transduced to express a high affinity T-cell receptor specific to NY-ESO-1 (letetresgene autoleucel, lete-cel) show promise in the treatment of metastatic synovial sarcoma, with 50% overall response rate. The efficacy of lete-cel treatment in 45 synovial sarcoma patients (NCT01343043) has been previously reported, however, biomarkers predictive of response and resistance remain to be better defined. This post-hoc analysis identifies associations of response to lete-cel with lymphodepleting chemotherapy regimen (LDR), product attributes, cell expansion, cytokines, and tumor gene expression. Responders have higher IL-15 levels pre-infusion (p = 0.011) and receive a higher number of transduced effector memory (CD45RA- CCR7-) CD8 + cells per kg (p = 0.039). Post-infusion, responders have increased IFNγ, IL-6, and peak cell expansion (p < 0.01, p < 0.01, and p = 0.016, respectively). Analysis of tumor samples post-treatment illustrates lete-cel infiltration and a decrease in expression of macrophage genes, suggesting remodeling of the tumor microenvironment. Here we report potential predictive and pharmacodynamic markers of lete-cel response that may inform LDR, cell dose, and strategies to enhance anticancer efficacy.
© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.