Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease

Abstract

Background and aim: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown.

Methods: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background.

Results: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4β7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa.

Conclusions: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

Keywords: IBD; IBD BASIC RESEARCH; IBD CLINICAL; IMMUNOTHERAPY; INTESTINAL T CELLS.

Figure 1

Expansion, phenotype and potency of in vitro expanded T_regs. (A) Cumulative fold expansion of T_reg lines at days 12 and 24 of culture, grouped according to CD4⁺CD25^hiCD127^loCD45RA⁺ or CD4⁺CD25^hiCD127^loCD45RA⁻ precursors; n=13 each, bar: median. (B) Representative FACS plots gated on live events showing CD25 and FOXP3 expression at D24. (C) Proportion of T_regs with a CD4⁺CD25^hiCD127^lo T_reg phenotype at D24. (D) Representative plots from a proliferation assay, illustrating dose-dependent suppression of T_con proliferation by CD45RA⁺ T_regs. Proliferation CTV-labelled autologous CD4⁺CD25⁻ T_cons alone (filled) or with T_regs at various T_con:T_reg ratios (bold line) is shown. (E) D24 T_reg-mediated suppression of T_con proliferation. Cumulative data showing mean±SEM suppression seen at each T_con:T_reg ratio. Pooled data from 29 T_reg lines. Comparisons between suppression seen in study conditions and mean non-specific suppression seen in ‘2X’ control condition (dotted line) are shown. *p<0.05,***p<0.001 and ****p<0.0001. T_regs, thymus-derived regulatory T cells; FACS, fluorescence-activated cell sorting; T_cons, conventional CD4⁺CD25^lo/int T cells; CTV, Cell Trace Violet; NS, not significant.

Figure 2

D24 CD45RA⁺ T_regs are resistant to IL-17 induction. (A) Relative expression of IL17A, RORC and AHR in D24 CD45RA⁺ and CD45RA⁻ T_regs, relative to GAPDH; n=16, bar at median. (B) D24 T_reg IL-17, IFN-γ and TNF secretion in 24 h culture supernatants; n=20, bar at median. (C) IL-17 detected by ELISA from 5-day culture supernatants of D24 T_regs cultured in the absence of rapamycin but with supplemental IL-2 alone, a cocktail of IL-2, IL-1, IL-6 and TGF-β or a cocktail of IL-2, IL-21, IL-23 and TGF-β. n=17, bar at median. (D) % FOXP3 TSDR demethylation; n=15, bar at median. *p<0.05,**p<0.01,***p<0.001. T_regs, thymus-derived regulatory T cells; IL, interleukin; IFN, interferon; TNF, tumour necrosis factor; TGF, transforming growth factor; TSDR, T_reg-specific demethylated region; NS, not significant.

Figure 3

D24 CD45RA⁺ T_regs express gut and lymphoid homing receptors and home to inflamed human LP in a C.B-17 severe combined immunodeficiency (SCID) mouse human intestinal XG model. (A) Representative FACS plots illustrating gut and lymphoid homing receptor expression on D24 CD45RA⁺ T_regs (bold line). Gates were drawn on the basis of fully stained CD4⁺ lymphocytes (filled) and fluorochrome minus one (FMO) controls. (B) Dot plots showing expression of intestinal and lymphoid homing receptors in D24 CD45RA⁺ and CD45RA⁻ T_regs. n=17; *p<0.05. (C) Design of the XG mouse experiment. (D) Left panel: mature XGs (circled) are visible subcutaneously on the dorsum of the mouse. Right panel: dorsal skin has been removed in an anaesthetised mouse to reveal the mucus-filled XG in situ (right panel). Microscopic images of the XG are shown in online supplementary figure S4A. (E) FACS plots showing live human CD45⁺CD3⁺CD4⁺ events in single cell suspensions prepared from murine spleen, non-inflamed and inflamed XGs, 24 h after intravenous phosphate buffered saline (PBS) (left panels) or adoptive transfer of T_regs (right panels). The absolute numbers of CD3⁺CD4⁺ events in the XG human CD45⁺ gates are highlighted. The gating strategy is illustrated in online supplementary figure S4B. (F) Immunofluorescence staining of XG cryosections with antihuman CD3 (red), antihuman CD45 (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). (E and F) Representative of two independent experiments. T_regs, thymus-derived regulatory T cells; LP, lamina propria; XG, xenograft; FACS, fluorescence-activated cell sorting; EPEC, enteropathogenic Escherichia coli; NS, not significant.

Figure 3

D24 CD45RA⁺ T_regs express gut and lymphoid homing receptors and home to inflamed human LP in a C.B-17 severe combined immunodeficiency (SCID) mouse human intestinal XG model. (A) Representative FACS plots illustrating gut and lymphoid homing receptor expression on D24 CD45RA⁺ T_regs (bold line). Gates were drawn on the basis of fully stained CD4⁺ lymphocytes (filled) and fluorochrome minus one (FMO) controls. (B) Dot plots showing expression of intestinal and lymphoid homing receptors in D24 CD45RA⁺ and CD45RA⁻ T_regs. n=17; *p<0.05. (C) Design of the XG mouse experiment. (D) Left panel: mature XGs (circled) are visible subcutaneously on the dorsum of the mouse. Right panel: dorsal skin has been removed in an anaesthetised mouse to reveal the mucus-filled XG in situ (right panel). Microscopic images of the XG are shown in online supplementary figure S4A. (E) FACS plots showing live human CD45⁺CD3⁺CD4⁺ events in single cell suspensions prepared from murine spleen, non-inflamed and inflamed XGs, 24 h after intravenous phosphate buffered saline (PBS) (left panels) or adoptive transfer of T_regs (right panels). The absolute numbers of CD3⁺CD4⁺ events in the XG human CD45⁺ gates are highlighted. The gating strategy is illustrated in online supplementary figure S4B. (F) Immunofluorescence staining of XG cryosections with antihuman CD3 (red), antihuman CD45 (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). (E and F) Representative of two independent experiments. T_regs, thymus-derived regulatory T cells; LP, lamina propria; XG, xenograft; FACS, fluorescence-activated cell sorting; EPEC, enteropathogenic Escherichia coli; NS, not significant.

Figure 4

In vitro expanded CD45RA⁺ T_regs suppress CD3⁺ T cell responses from inflamed Crohn's MLN and LP. (A) Suppression of proliferation of a single lot of freeze-thawed, allogeneic T_cons by freshly isolated PB CD4⁺CD25^hiCD127^loCD45RA⁺ T_regs, or D24 CD45RA⁺ T_regs that were expanded in vitro from these freshly isolated precursors. Pooled data from three sets of freshly isolated PB T_regs and subsequently expanded T_reg populations. Data points are mean±SEM. (B) Fresh ileal resection specimen opened longitudinally to show ileal stricture (marked ‘S’) and proximal inflamed, haemorrhagic mucosa with deep ulceration (arrows). Scale bar: 2 cm. (C) Representative microscopic image from this resection showing mucosal distortion, ulceration (marked ‘U’) and transmural inflammation, including lymphoid aggregates (arrows). 12.5× H&E. Scale bar: 2 mm. (D) Representative FACS plots gated on live CD3⁺ events, showing proliferation of MLN T_cons cultured alone (top left panel) or with T_regs at a 1:1 MLNMC:T_reg ratio (bottom left panel). Pooled data showing T_reg-mediated suppression of MLN CD3⁺ proliferation (right panel, n=5). Box and whisker plot shows median, IQR and range. (E) Representative FACS plots gated on live MLN CD3⁺ events showing CD154 expression on MLN T_cons cultured alone (top left panel) or with T_regs at a 1:1 MLNMC:T_reg ratio (bottom left panel). Pooled data showing T_reg-mediated suppression of CD154 expression in live MLN CD3⁺ cells (right panel, n=5). (F) Representative FACS plots gated on live LP CD3⁺ events showing CD154 expression on LP T_cons cultured alone (top left panel) or with T_regs at a 1:1 LPMC:T_reg ratio (bottom left panel). Pooled data showing T_reg-mediated suppression of CD154 expression in live LP CD3⁺ cells (right panel, n=5). (D–F) Dotted line shows non-specific suppression from ‘2X control’. Comparisons between observed suppression and non-specific suppression (†p<0.05, ††p<0.01, †††p<0.001, ††††p<0.0001) and observed suppression and no suppression (zero, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001) are shown. T_regs, thymus-derived regulatory T cells; MLN, Mesenteric lymph node; LP, lamina propria; T_cons, conventional CD4⁺CD25^lo/int T cells; PB, peripheral blood; FACS, fluorescence-activated cell sorting; MLNMC, MLN mononuclear cell; LPMC, LP mononuclear cell; CTV, Cell Trace Violet.