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.

As a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, butyrate possesses extensive immunomodulatory properties. Here, we uncover a novel mechanism wherein butyrate markedly upregulates the expression of the immune checkpoint PD-L1 across diverse tumor cell types. Mechanistically, epigenetic analyses revealed that butyrate drives PD-L1 transcriptional activation through dual synergistic pathways: increasing histone H3 acetylation (H3Ac) at the PD-L1 promoter via HDAC inhibition, and enhancing histone H3K4me3 modification by upregulating transglutaminase 2 (TGM2). Combining butyrate or high-fiber diets with anti-PD-L1 antibodies profoundly suppressed tumor growth and bolstered anti-tumor immunity in both subcutaneous and AOM-DSS-induced colorectal cancer (CRC) murine models. Ultimately, our results highlight butyrate and dietary fiber supplementation as promising sensitizing strategies to augment the efficacy of PD-L1 blockade in patients with low baseline PD-L1 expression.