Abstract
Background and AimsLymphocyte activation gene [LAG]-3 is an immune checkpoint and its expression identifies recently activated lymphocytes that may contribute to inflammation. We investigated the role of LAG-3 by analysing its expression and function in immune cells from blood and tissue of patients with ulcerative colitis [UC].MethodsThe phenotypic properties of LAG-3+ T cells were determined by flow cytometry, qRT-PCR and single-cell RNA-sequencing. LAG-3+ cells were quantified and correlated with disease activity. The functional effects of LAG-3+ cells were tested using a depleting anti-LAG-3 monoclonal antibody [mAb] in a mixed lymphocyte reaction [MLR].ResultsLAG-3+ cells in the blood were negligible. LAG-3+ lymphocytes were markedly increased in inflamed mucosal tissue and both frequencies of LAG-3+ T cells and transcript levels of LAG3 correlated with endoscopic severity. LAG-3 expression was predominantly on effector memory T cells, and single-cell RNA-sequencing revealed LAG3 expression in activated and cytokine-producing T cell subsets. Foxp3+CD25hi Tregs also expressed LAG-3, although most mucosal Tregs were LAG-3−. Mucosal LAG-3+ cells produced mainly interferon γ [IFNγ] and interleukin-17A. LAG-3+ cell numbers decreased in patients who responded to biologics, and remained elevated in non-responders. Treatment with a depleting anti-LAG-3 mAb led to a reduction in proliferation and IFNγ production in an MLR.ConclusionsLAG-3+ cells are increased in the inflamed mucosa, predominantly on effector memory T cells with an activated phenotype and their cell numbers positively correlate with disease activity. Depleting LAG-3 eliminates activated proliferating T cells, and hence LAG-3 could be a therapeutic target in UC.
Highlights
Ulcerative colitis [ulcerative colitis (UC)] is a chronic mucosal inflammatory condition in which T lymphocytes play a critical role.[1,2] The importance of T cells is demonstrated both by murine T cell transfer models of experimental colitis,[3] and by the clinical efficacy of drugs targeting T cell trafficking in UC.[4,5,6] Selective depletion of newly activated T cells, with sparing of resting lymphocytes, could represent a promising therapeutic strategy in this context
LAG3+ cell numbers decreased in patients who responded to biologics, and remained elevated in nonresponders.Treatment with a depleting anti-lymphocyte activation-3 (LAG-3) mAb led to a reduction in proliferation and IFNγ production in an mixed lymphocyte reaction (MLR)
Flow cytometry plots for LAG-3 staining on CD3+ T cells from the uninflamed and inflamed lamina propria mononuclear cells (LPMCs) and corresponding peripheral blood mononuclear cells (PBMCs) are shown in Figure 1A and the fluorescence minus one [FMO] for LAG-3 is shown in Supplementary Figure 2A
Summary
Ulcerative colitis [UC] is a chronic mucosal inflammatory condition in which T lymphocytes play a critical role.[1,2] The importance of T cells is demonstrated both by murine T cell transfer models of experimental colitis,[3] and by the clinical efficacy of drugs targeting T cell trafficking in UC.[4,5,6] Selective depletion of newly activated T cells, with sparing of resting lymphocytes, could represent a promising therapeutic strategy in this context. We investigated the role of LAG-3 by analysing its expression and function in immune cells from blood and tissue of patients with ulcerative colitis [UC]. LAG-3 in Ulcerative Colitis activity.The functional effects of LAG-3+ cells were tested using a depleting anti-LAG-3 monoclonal antibody [mAb] in a mixed lymphocyte reaction [MLR]. LAG-3 expression was predominantly on effector memory T cells, and single-cell RNA-sequencing revealed LAG3 expression in activated and cytokineproducing T cell subsets. LAG3+ cell numbers decreased in patients who responded to biologics, and remained elevated in nonresponders.Treatment with a depleting anti-LAG-3 mAb led to a reduction in proliferation and IFNγ production in an MLR. Conclusions: LAG-3+ cells are increased in the inflamed mucosa, predominantly on effector memory T cells with an activated phenotype and their cell numbers positively correlate with disease activity. Depleting LAG-3 eliminates activated proliferating T cells, and LAG-3 could be a therapeutic target in UC
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