In a recent study published in Nature, researchers developed a library of blood nutrient molecules and conducted screens to find nutritional components that influence anti-tumor immunity.
Background
Dietary nutrients are closely linked to human physiological processes, as they provide energy, biosynthesize building blocks and act as mediators.
However, the methods by which human nutrients circulate and influence certain physiological pathways are unclear, which warrants further investigation.
About the study
In the current study, researchers examined the impact of dietary transvaccenic acid (TVA) on effector cytotoxic T lymphocyte functions and antitumor immunity in the in vivo institutions.
The researchers examined the impact of different nutrients on human T lymphocyte cells using a blood nutrient molecule library-based screening technique.
All cell lines (Jurkat T, human Plat-E, B16-OVA mouse melanoma cancer cells, B16F10 mouse melanoma cancer cells, E0771 breast cancer cells and LLC1 Lewis lung carcinoma cells and MC38 mouse colorectal adenocarcinoma cells) were validated using genomic short tandem repeat (STR) profiling.
The initial screen was used to identify foods that promoted Jurkat T lymphocyte activation caused by a cluster of differentiation 23 (CD23) and CD28 antibodies. Screen 1b identified nutrients that reverse programmed death ligand 1 (PD-L1)-dependent depletion of Jurkat T cells stably expressing programmed cell death protein 1 (PD-1) produced by co-cultured human H596 lung cancer cells Expressing PD-L1.
The team performed magnetic bead purification to identify CRISPR-associated OT-I cells expressing protein 9 (Cas9) from the spleen and peripheral lymph nodes of Cas9-OT-I animals.
Commercial recipients of chimeric antigen receptor (CAR) T lymphocyte therapy provided serum samples. The researchers infected Jurkat T lymphocytes with a ready-made lentivirus expressing PD-1 and then selected 2.0 g/ml puromycin to obtain Jurkat T lymphocytes expressing PD-1.
Western blotting revealed the presence of PD-1. The study involved feeding Jurkat T lymphocytes with a nutritional library for two days, then stimulating them with anti-CD3 and anti-CD28 antibodies for 12 hours. Interleukin-2 (IL-2) levels were measured using enzyme-linked immunosorbent assays (ELISA).
In the second screen, Jurkat T lymphocytes were co-cultured with PD-1-expressing cells for 60 hours and then stimulated with anti-CD23 and anti-CD28 antibodies for 12 hours. Gpr43fl/fl, Gpr43−/−, and Cd8acre mouse animals were bred to produce Gpr43−/flCd8acre conditional knockout mice (Gpr43/flCd8acre) and develop the Gpr43/animal tumor model.
The team obtained cheek bleeds on days 3.0, 12 and 18 and performed flow cytometry to assess the effectiveness of CD8+ T cell reduction using antibodies directed against non-competing CD8 epitopes (BV711 anti-mouse CD8).
In vitro [13C] tracing of fatty acids, fatty acid oxidation assay in seahorses, measured calcium (Ca2+) level of cytotoxic (CD8+) T lymphocytes, clustered regularly spaced short palindromic repeats (CRISPR) editing of mouse OT-I cells, pull-down assay to to identify cross-linked proteins TVA complexes, co-culture assay with Blinatumomab and CAR-T cell expansion assay were performed. TVA levels were quantified by nuclear magnetic resonance (NMR) spectroscopy.
Results
TVA, a trans fatty acid component of breast milk, is obtained primarily from ruminant foods such as lamb, beef and dairy products. Humans and mice convert only 12% to 19% of dietary TVA into rumenic acid. TVA inhibited the immunoregulatory G protein-coupled receptor 43 (GPR43), a molecule stimulated by its SCFA ligands.
TVA activated the cyclic AMP (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) axis, improving cytotoxic T lymphocyte function, indicating that TVA from the diet, rather than from the gut microbiota-derived SCFAs from the host, a host-extrinsic reprogramming pathway for cytotoxic T lymphocytes.
TVA attenuated the activity of Gαi linkage GPR43 and increased cAMP levels, counteracting the influence of short-chain fatty acid molecules on cyclic AMP to improve the function of effector-type cytotoxic T lymphocytes. TVA promoted antitumor immunity through CD8+ T lymphocyte regulation, selectively enhancing stimulated cytotoxic T lymphocyte function.
The improvement of cytotoxic T lymphocyte function induced by TVA was regulated by the GPCR-CREB pathway, where positive feedback increased PKA and CREB expression at the gene level. TVA activity required CREB and could enhance cytotoxic T lymphocyte function and antitumor immunity in vivo. CREB inhibition antagonized the impact of dietary TVA on antitumor immunity.
TVA improved T cell-based treatments. For cytotoxic T lymphocytes, the GPR43-CREB pathway may be cell type specific. For example, TVA therapy enhanced the synthesis of interleukin-2 by helper T (CD4+) lymphocytes, but did not affect the formation of effector molecules such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) or the proliferation or apoptosis of helper T lymphocytes.
Therefore, the effects of dietary TVA on helper T lymphocytes were small compared with those on cytotoxic T lymphocytes, which may be due to the low GPR43 expression in helper T lymphocytes.
Conclusion
Overall, the study results showed that dietary trans-vaccenic acid increases effector cytotoxic T lymphocyte activity and antitumor immunity in the in vivo institutions.
Extraorganismal TVA reprogrammed CD8+ T lymphocytes through extrinsic regulation to inactivate GPR43, in contrast to gut microbe-derived intraorganismal SCFAs that act as GPR43 agonists.
The study results contribute to a better understanding of the molecular links between diet and human pathophysiology, with implications for future research into the function of circulating nutrients in health and disease.
Further research is needed to improve the understanding of GPR43's downstream effector pathways and elucidate the underlying processes.
