Regulatory T cells (Tregs) maintain immune homeostasis in vivo. Similar to conventional T cells (Tconvs), Tregs are divided into naïve and memory cells. Tregs have unique metabolic properties, including enhanced oxidative phosphorylation. The lipidomic profiles of human Tregs have been studied previously; however, those of naïve and memory Tregs have not yet been consistently compared. Thus, in the present study, we used a combined transcriptomic and lipidomic analysis to assess the metabolic features of human naïve and memory Tregs upon activation. Using transcriptomic analysis, we identified distinct gene expression profiles in naïve and memory Tregs compared with those in Tconvs. Upon TCR stimulation, memory Tregs showed a lipidomic profile distinct from that of memory Tconvs, whereas the lipidomic profiles of naïve Tregs were similar to those of naïve Tconvs. Furthermore, upon TCR stimulation, memory Tregs expressed triglycerides (TGs) that were more enriched in monounsaturated fatty acids and polyunsaturated fatty acids (PUFAs) than those of memory Tconvs. However, memory Tconvs also had higher PUFA-TG levels than naïve Tconvs and Tregs after TCR stimulation, although their levels remained lower than those in memory Tregs. These findings suggest PUFA-TGs could be a marker of memory Tregs. In turn, higher frequency of memory cells within the Treg population may also contribute to the observed enrichment of PUFA-TGs in Tregs upon TCR activation. Our study demonstrated unique transcriptomic and lipidomic profiles and enhanced lipogenesis in memory Tregs upon TCR stimulation.

BACKGROUND: Cancer immunotherapies have significantly improved treatment efficacy and patient survival by exploiting antigen-specific T cells to eliminate cancer cells. However, current approaches for identifying and isolating antigen-specific T cells typically require prior knowledge of target antigens, limiting discovery, and reducing the ability to consistently detect rare tumor-reactive T cells. We therefore sought to develop an unbiased platform for the identification and enrichment of antigen-specific T cells using naturally processed and presented tumor antigens. METHODS: We developed ATTACH (Assessment of T cells Tethered to Antigen Class I Histocompatibility), a microfluidic platform that applies controlled shear stress and leverages tumor cells as a natural source of endogenous major histocompatibility complex (MHC)-peptide complexes to isolate antigen-specific T cells based on MHC/peptide binding avidity. ATTACH was evaluated in both human and mouse systems for its ability to enrich rare tumor-reactive T-cell populations and deplete bystander virus-specific T cells. RESULTS: ATTACH resulted in up to a 10-fold enrichment of antigen-specific T cells across both human and mouse systems, enabling the isolation of clonotypes present at frequencies as low as 0.1%. In addition to enriching rare tumor-reactive T cells, ATTACH efficiently depleted virus-specific bystander T cells. CONCLUSIONS: ATTACH provides a streamlined and unbiased approach for the rapid identification and isolation of antigen-specific T cells, and may facilitate the optimization of cellular therapies for the treatment of solid tumors.

The clinical efficacy of immune checkpoint inhibitors (ICIs) in colorectal cancer (CRC) remains limited. Modulation of the glucagon-like peptide-1 receptor (GLP-1R) may enhance T-cell-mediated antitumor responses. The present study aimed to evaluate the antitumor effects of the GLP-1R antagonist Exendin 9–39 (Exe-9) combined with anti-programmed cell death protein-1 (PD-1) treatment in preclinical CRC models. Using in vitro co-culture assays, ELISA and in vivo murine models, alongside immunohistochemical and molecular analyses of clinical samples, HT-29 and MC38-OVA colon cancer cell lines were co-cultured in vitro with activated T cells in the presence of Exe-9. In vivo, male BALB/c mice were injected with MC38 to establish a CRC model and nude mice were used to assess T-cell dependency. To evaluate this synergistic effect, BALB/c mice with CRC were treated with Exe-9, anti-PD-1 or a combination. Additionally, clinical CRC samples were analyzed to assess the association of GLP-1R expression with the immunotherapy response. Exe-9 significantly enhanced T-cell-mediated cytotoxicity in CRC cell lines and reduced tumor growth in immunocompetent CRC mice; however, this effect was not observed in nude mice. Furthermore, combination therapy with the GLP-1R antagonist and anti-PD-1 yielded an improved antitumor effect compared with either treatment alone, and high GLP-1R ex2pression in clinical samples correlated with poor ICI response. These findings suggest that GLP-1R antagonism potentiates T-cell-mediated antitumor immunity and may provide a promising adjunctive therapeutic strategy for patients with CRC when combined with ICIs in the future.