Adipose tissue physiology and homeostasis depends on a healthy vascular network. Vascular malfunction is a hallmark of obesity, and vascular endothelial dysfunction, in particular, precipitates metabolic diseases, including obesity and type two diabetes. Although single-cell transcriptomics approaches have defined atlases of human white adipose tissue (WAT) cells, the associated adipose vascular cells remain relatively undefined. Specifically, there is limited information on their heterogeneity and function, and roles in metabolic disease. To address this gap, we created a single-cell transcriptome atlas of human subcutaneous adipose tissue (SAT), comprising nearly 70,000 vascular cells from 65 individuals. We identified eight adipose endothelial cell (AdEC) populations, comprising seven canonical subtypes and a previously undescribed, heterogeneous population we named sub-AdECs. Sub-AdECs exhibit gene signatures characteristic of multiple cell types, including mesenchymal, adipocytic, and immune cells, suggesting they possess diverse properties and identities. Furthermore, we compare the transcriptomes of vascular cells from individuals living with or without obesity and type two diabetes and find metabolic disease-associated inflammatory and fibrotic transcriptomic patterns. The atlas and accompanying analyses establish a solid foundation for future investigations into the biology of vascular cells within WAT and their contributions to metabolic diseases.

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy.
© 2024. The Author(s).

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current prediction algorithms for tumor neoantigens are not reliable and many tumor neoantigens are derive from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently need. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulating of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot (ELISPOT) assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxic of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8 + tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes (TILs) of patients with colorectal cancer (CRC), and tumor-specific TCRs can potentially be used for personalized TCR-Ts therapy.

M1- and M2-like macrophages infected with Mycobacterium tuberculosis (Mtb) have been found to differ in their capacity to elicit memory CD4+ T cell activation. Here, we present a protocol to quantify and isolate the subset of human memory CD4+ T cells activated in response to autologous monocyte-derived macrophages (MDMs) infected with virulent Mtb. We describe steps for CD14+ monocyte isolation, generating MDMs, culturing Mtb and infection of macrophages, and identifying activated CD4+ T cells by flow cytometry. For complete details on the use and execution of this protocol, please refer to Gail et al.1.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

T cell receptor (TCR)-engineered T cell therapy is a promising potential treatment for solid tumors, with preliminary efficacy demonstrated in clinical trials. However, obtaining clinically effective TCR molecules remains a major challenge. We have developed a strategy for cloning tumor-specific TCRs from long-term surviving patients who have responded to immunotherapy. Here, we report the identification of a TCR (10F04), which is human leukocyte antigen (HLA)-DRA/DRB1*09:01 restricted and human papillomavirus type 18 (HPV18) E784-98 specific, from a multiple antigens stimulating cellular therapy (MASCT) benefited metastatic cervical cancer patient. Upon transduction into human T cells, the 10F04 TCR demonstrated robust antitumor activity in both in vitro and in vivo models. Notably, the TCR effectively redirected both CD4+ and CD8+ T cells to specifically recognize tumor cells and induced multiple cytokine secretion along with durable antitumor activity and outstanding safety profiles. As a result, this TCR is currently being investigated in a phase I clinical trial for treating HPV18-positive cancers. This study provides an approach for developing safe and effective TCR-T therapies, while underscoring the potential of HLA class II-restricted TCR-T therapy as a cancer treatment.
© 2024. The Author(s).