HLA-Restriction of Human Treg Cells Is Not Required for Therapeutic Efficacy of Low-Dose IL-2 in Humanized Mice

Abstract

Regulatory T (T_reg) cells are essential to maintain immune homeostasis in the intestine and T_reg cell dysfunction is associated with several inflammatory and autoimmune disorders including inflammatory bowel disease (IBD). Efforts using low-dose (LD) interleukin-2 (IL-2) to expand autologous T_reg cells show therapeutic efficacy for several inflammatory conditions. Whether LD IL-2 is an effective strategy for treating patients with IBD is unknown. Recently, we demonstrated that LD IL-2 was protective against experimental colitis in immune humanized mice in which human CD4⁺ T cells were restricted to human leukocyte antigen (HLA). Whether HLA restriction is required for human T_reg cells to ameliorate colitis following LD IL-2 therapy has not been demonstrated. Here, we show that treatment with LD IL-2 reduced 2,4,6-trinitrobenzensulfonic acid (TNBS) colitis severity in NOD.Prkdc^scidIl2rg^-/- (NSG) mice reconstituted with human CD34⁺ hematopoietic stem cells. These data demonstrate the utility of standard immune humanized NSG mice as a pre-clinical model system to evaluate therapeutics targeting human T_reg cells to treat IBD.

Keywords: IBD; IL-2; Tregs; colitis; humanized.

Figure 1

Low-dose (LD) interleukin-2 (IL-2)-mediated protection against trinitrobenzensulfonic acid (TNBS)-induced colitis is independent of T_reg cell human leukocyte antigen (HLA) restriction. (A) Schematic of LD IL-2/TNBS protocol. (B) Percent survival of NOD.Prkdc^scidIl2rg^-/- (NSG) human immune system (HIS) mice post-TNBS rectal administration (vehicle n = 11; PBS n = 28; LD IL-2 n = 30). (C) Percent initial body weight following induction of TNBS-induced colitis in NSG HIS mice treated with or without LD IL-2 (vehicle n = 11; PBS n = 28; LD IL-2 n = 30). (D) Colon length 3 days following vehicle or TNBS enema. Each dot represents an individual animal (vehicle n = 11; PBS n = 22; LD IL-2 n = 23). (E) Representative H&E-stained distal colon section at 10X magnification (left) and histology score (right) where each circle represents an individual animal (vehicle, n = 11; PBS n = 22; LD IL-2, n = 23). Scale bar = 200 µm. *P < 0.05, ***P < 0.001. Image generated using BioRender.

Figure 2

T_reg cell expansion in peripheral compartments following low-dose (LD) interleukin-2 (IL-2). (A) Representative flow cytometry dot plots of human T_reg cells of CD4⁺ in blood and spleen (left) with quantified data (right). (B) Quantification of FOXP3⁺ cells via immunohistochemistry in the colonic lamina propria. (C) Correlation between T_reg cell number and histology score in colon (R^{2 =} 0.38, P < 0.001). Data were pooled from three independent experiments and results were expressed as mean ± SEM. **P < 0.01, ***P < 0.001; n.s., not significant.

Figure 3

Alteration in the lymphocyte population in blood and spleen of trinitrobenzensulfonic acid (TNBS)-induced colitis in humanized NOD.Prkdc^scidIl2rg^-/- (NSG) mice treated with low-dose (LD) interleukin-2 (IL-2). (A, B) Representative flow cytometry dot plots and quantified data of human CD4⁺ and CD8⁺ T cells subsets in blood and spleen where each circle represents an individual mouse. (C) Representative flow cytometry dot plots and quantified frequencies of splenic and blood natural killer (NK) cell subsets 3 days post-rectal challenge in NSG human immune system (HIS) mice (n ≥ 7). Data were pooled from three independent experiments and histograms depict the mean ± SEM. *P < 0.05, ***P < 0.001.

Figure 4

Interleukin-2 (IL-12) production is reduced in mice treated with low-dose (LD) interleukin-2 (IL-2). Concentration of human cytokines in colon tissue of NOD.Prkdc^scidIl2rg^-/- (NSG) human immune system (HIS) mice 3 days post-rectal challenge with trinitrobenzensulfonic acid (TNBS) or vehicle as a control determined by Luminex (n ≥ 11 per group). Histograms are the mean concentration pooled from three independent experiments ± SEM. *P < 0.05, **P < 0.01.