In a recent study published in Nature, researchers developed a blood nutrient molecule library and conducted screenings to find dietary components that impact anti-tumor immunity.

Study: Trans-vaccenic acid reprograms CD8⁺ T cells and anti-tumour immunity. Image Credit: Anusorn Nakdee/Shutterstock.com

Background

Dietary nutrients are intricately related to human physiological processes since they provide energy, biosynthesize building blocks, and act as mediatory substances.

However, the methods by which human nutrients circulate impact particular physiological pathways are unclear, warranting further research.

About the study

In the present study, researchers explored the impact of diet-derived trans-vaccenic acid (TVA) on effector cytotoxic T lymphocyte functions and anti-tumor immunity in the in vivo settings.

The researchers examined the impact of several nutrients on human T lymphocyte cells using a blood nutrition molecule library-based screening technique.

All cell lines (Jurkat T, human Plat-E, B16-OVA murine melanoma cancer cells, B16F10 murine melanoma cancer cells, E0771 breast cancer cells, and LLC1 Lewis lung carcinoma cells, and MC38 murine colorectal adenocarcinoma cells) were validated using genomic short tandem repeat (STR) profiling.

The first screen was used to identify foods promoting Jurkat T lymphocyte activation triggered by cluster of differentiation 23 (CD23) and CD28 antibodies. Screen 1b identified nutrients that reverse programmed death-ligand 1 (PD-L1)-dependent exhaustion of Jurkat T cells stably expressing programmed cell death protein 1 (PD-1) produced by co-cultured PD-L1-expressing human H596 lung cancer cells.  

The team performed magnetic bead purification to identify CRISPR-associated protein 9 (Cas9)-expressing OT-I cells from the spleen and peripheral lymph nodes of Cas9-OT-I animals.

Commercial chimeric antigen receptor (CAR) T lymphocyte therapy recipients provided serum samples. The researchers infected Jurkat T lymphocytes with a pre-made PD-1-expressing lentivirus and then chose 2.0 g/mL puromycin to yield PD-1-expressing Jurkat T lymphocytes.

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 murine 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 CD8+ T cell reduction effectiveness using antibodies targeting non-competing CD8 epitopes (BV711 anti-mouse CD8).

In vitro [13C] fatty acid tracing, seahorse fatty acid oxidation assay, measured calcium (Ca2+) level of cytotoxic (CD8+) T lymphocytes, clustered regularly interspaced short palindromic repeats (CRISPR) editing of mouse OT-I cells, pull-down assay to identify crosslinked protein-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 constituent of human milk, is derived predominantly from ruminant foods such as lamb, beef, and dairy. Humans and mice convert only 12% to 19% of diet-derived TVA into rumenic acid. TVA inhibited the immunoregulatory G protein-coupled receptor 43 (GPR43), a molecule stimulated by its SCFA ligands.

TVA triggered the cyclic AMP (cAMP)-protein kinase A (PKA)-cAMP-response element binding protein (CREB) axis, enhancing cytotoxic T lymphocyte function, indicating that dietary TVA, rather than intra-host gut microbiota-derived SCFAs, is a host-extrinsic reprogramming pathway for cytotoxic T lymphocytes.

TVA attenuated the activity of Gαi-coupling GPR43 and elevated cAMP levels, antagonizing the influence of short-chain fatty acid molecules on cyclic AMP to enhance the function of effector-type cytotoxic T lymphocytes. TVA promoted anti-tumor immunity through CD8+ T lymphocyte regulation, selectively enhancing stimulated cytotoxic T lymphocyte function.

The enhancement in cytotoxic T lymphocyte function induced by TVA was regulated by the GPCR-CREB pathway, with positive feedback augmenting PKA and CREB expression at the gene level. TVA activity required CREB and could enhance cytotoxic T lymphocyte function and anti-tumor immunity in vivo. CREB inhibition antagonized the impact of dietary TVA on anti-tumor immunity.

TVA improved T-cell-based treatments. For cytotoxic T lymphocytes, the GPR43-CREB pathway might be cell-type-specific. TVA therapy, for example, enhanced interleukin-2 synthesis by helper T (CD4+) lymphocytes but did not impact the generation 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 minor compared to those on cytotoxic T lymphocytes, which might be due to low GPR43 expression in helper T lymphocytes.

Conclusion

Overall, the study findings showed that dietary trans-vaccenic acid increases effector cytotoxic T lymphocyte activity and anti-tumor immunity in the in vivo settings.

Extraorganismal TVA, in contrast to gut microbe-derived intra-organismal SCFAs operating as GPR43 agonists, reprogramed CD8+ T lymphocytes through extrinsic regulation to inactivate GPR43.

The study findings contribute to a better understanding of the molecular linkages between nutrition and human pathophysiology, with implications for future research on the function of circulating nutrients in health and illness.

Further research is required to improve understanding of the downstream effector pathways of GPR43 and elucidate underlying processes.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.