Researchers devise a novel potential IBD treatment strategy that eliminates pathogenic T cells and encourages the proliferation of regulatory T cells

By Pooja Toshniwal Paharia Jan 31 2023 Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Science Immunology, researchers investigated the CAMCI (combination of cell activation and metabolic checkpoint inhibition) strategy as an immunotherapeutic option for inflammatory bowel disease (IDB).

Study: CD4+ T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis. Image Credit: Design_Cells/Shutterstock

Background

Studies have reported that microbiome-reactive CD4+ (cluster of differentiation 4+) memory T (TM) lymphocytes are produced during gut inflammation and infections in response to disease-causing CD4+ effector T (TE) lymphocytes, resulting in IBD chronicity. Contrary to TE lymphocytes, TM lymphocytes metabolize slowly unless stimulated by cognate antigenic molecules.

About the study

The present study investigated CAMCI application in IBD prevention and treatment.

A MEP1 (multiple flagellin T cell epitopes) construct was engineered as a template for activating transgenic (Tg) CD4+ (helper) T lymphocytes that can detect CBir1 flagellin, comprising three CBir1 p456–475 epitope repeats and one OVA p323–339 epitope. Additionally, the construct comprised the CBir1 p52–101 site of robust 3B3 mAb (monoclonal antibody) binding and one copy of the endogenously produced 2W1S epitope.

Dose-response assays were performed to assess MEPI presentation by APCs (antigen-presenting cells). Cellular activation was evaluated based on CD-25, -69 levels by MEP1-driven CBir1 T cell receptor (TCR) transgenic CD4+ T lymphocytes.C57BL/6 mice were used for helper T lymphocyte isolation. The presence or absence of TLR (toll-like receptor)-4, 5 agonisms by MEP1 was evaluated based on the interleukin-6 (IL-6) expression by MEP1-activated APCs, and APCs stimulated by E. coli lipopolysaccharides or CBir1 flagellin.

Further, the team investigated the effects of MEP1-driven CD4+ T lymphocyte activation and mTOR (mammalian target of rapamycin) inhibition on CD4+ TE metabolism and CD4+ regulatory T (Treg) lymphocyte functions in the in vitro setting and in vivo setting. Next, the team evaluated the cumulative impact of antigen-targeted helper TE suppression and Tregactivation with mTOR suppression on gut inflammation intensity using adoptively transferred naïve CBir1 T cell receptor transgenic CD4+ T lymphocytes in RAG1−/− murine animals.

The murine animals were administered rapamycin daily intraperitoneally, followed by histopathological assessments. Further, CD4+ T lymphocytes were isolated from lipopolysaccharide, spleen, and mesenteric lymph nodes, from mice and cultured in vitro in MEP1 presence with or without rapamycin. To investigate the effects of concomitant mTOR suppression and TCR stimulation on antigen-specific CD4+ memory T lymphocyte development, the physiological processes of peripheral microbiome-reactive CD4+ TM responses were mimicked.

C57BL/6 mice, who received adoptive transfers of CBir1 T cell receptor transgenic CD45.1 CD4+ CD44-naïve cells, were immunized with the CBir1 flagellin, adjuvanted by CT (cholera toxin), intraperitoneally. Rapamycin was administered intraperitoneally post-immunization to evaluate its effects on memory CD4+ response development. Antigen-specific TFH (follicular helper T) lymphocyte differentiation was assessed based on programmed cell death protein-1 (PD-1) and C-X-C chemokine receptor type 5 (CXCR-5) expression. Serological anti-CBir1 immunoglobulin G titers were determined using ELISA (enzyme-linked immunosorbent assays), and flow cytometry analysis was performed. Ribonucleic acid (RNA) sequencing, gene expression analysis, and antigen-specific T lymphocyte assays were also performed.

The team investigated whether metformin, an adenosine monophosphate-activated protein kinase (AMPK) pathway activator, with or without rapamycin could robustly inhibit CD4+ TM cells. They also investigated if the CAMCI approach could be used in humans and ablate disease-causing microbiome-reactive CD4+ T lymphocytes in circulation. For the investigation, PMBCs (peripheral blood mononuclear cells) obtained from Crohn’s disease patients were stimulated with Lachnospiraceae flagellin antigens such as CBir1, A4 Fla3, FlaX, and 14-2 Fla1. Bacterial colonization in the murine animals was verified by polymerase chain reaction (PCR) analysis.

Results

The findings showed that the CAMCI strategy, targeting the AMPK pathway and mTOR complex (mTORC), led to energy and cellular death but increased Treg generation. The parenteral use of CAMCI in combination with a MEP1-comprising synthetic peptide and metabolic suppression effectively inhibited CD4+ T lymphocyte-driven colitis development. Microbiome-targeted CD4+ T lymphocytes, particularly TE cells, were 10.0-fold lower within the lamina propria of the intestines.

Further, the CAMCI approach prevented the formation of antigen-targeted TMlymphocytes at the first antigenic encounter and ablated existing TM lymphocytes upon stimulation in the murine animals, resulting in altered transcriptomes within the remaining helper T lymphocytes post-ablation. Microbiome flagellin-targeted helper T lymphocytes obtained from Crohn’s disease patients were ablated similarly post-CAMCI application in vitro, and 50% of antigen-targeted T lymphocytes underwent cellular death.

Microbiome-targeted helper T lymphocyte stimulation and simultaneous metabolic checkpoint suppression effectively eliminated pathogenic TM lymphocytes and induced Treg lymphocytes that provide bystander and antigen-targeted inhibition and were successful in colitis prevention in CBir1 T cell receptor transgenic CD4+ T lymphocyte adoptive transfer modeling. Microbiome flagellin-targeted helper T lymphocytes isolated from Crohn’s disease patients were depleted similarly in vitro by CAMCI therapy. The findings indicated the potential for clinical use of the CAMCI strategy for IBD prevention and/or treatment.

Conclusion

Overall, the study findings showed that parenteral stimulation of microbiome-targeted helper T lymphocytes with simultaneous metabolic checkpoint inhibition could effectively ablate disease-causing TM lymphocytes and induce Treg lymphocytes for bystander and antigen-targeted suppression. The findings indicated that the CAMCI approach could be used for IBD prevention and/or treatment.