IL23R-Specific CAR Tregs for the Treatment of Crohn's Disease

Abstract

Background and aims: Regulatory T cells (Tregs) are key regulators in maintaining tissue homeostasis. Disrupted immune homeostasis is associated with Crohn's disease (CD) pathogenesis. Thus, Treg therapy represents a promising long-acting treatment to restore immune balance in the diseased intestine. Chimeric antigen receptor (CAR) T-cell therapy has revolutionized cancer treatment. This innovative approach also provides the opportunity to improve therapy for CD. By targeting a disease-relevant protein, interleukin-23 receptor (IL23R), we engineered Tregs expressing IL23R-CAR for treating active CD.

Methods: Intestinal IL23R expression from active CD was verified by immunohistochemical analysis. Phenotypic and functional characteristics of IL23R-CAR Tregs were assessed using in vitro assays and their migration capacity was monitored in a xenograft tumor model. Transcriptomic and proteomic analyses were performed to associate molecular profiles with IL23R-CAR Treg activation against colon biopsy-derived cells from active CD patients.

Results: Our study showed that IL23R-CAR displayed negligible tonic signaling and a strong signal-to-noise ratio. IL23R-CAR Tregs maintained regulatory phenotype during in vitro expansion, even when chronically exposed to proinflammatory cytokines and target antigen. IL23R engagement on IL23R-CAR Tregs triggered CAR-specific activation and significantly enhanced their suppressive activity. Also, IL23R-CAR Tregs migrated to IL23R-expressing tissue in humanized mice. Finally, IL23R-CAR Tregs elicited a specific activation against colon biopsy-derived cells from active CD, suggesting an efficient CAR engagement in active CD. Molecular profiling of CD patient biopsies also revealed transcriptomic and proteomic patterns associated with IL23R-CAR activation.

Conclusions: Overall, our results demonstrate that IL23R-CAR Tregs represent a promising therapy for active CD.

Keywords: CAR Treg; Crohn’s disease; IBD; IL23R; cell therapy.

Graphical Abstract

Figure 1

IL23R expression in intestinal biopsies from CD patients. (A) Representative human IL23R or isotype (mouse IgG2b) staining and (B) combined score (intensity score × percentage score) of IL23R in the colon laminar propria of healthy or CD subjects. The IL23R score reflects the percentage of IL23R positive cells graded as follows: 0, none; 1, 1%-25%; 2, 26%-50%; 3, 51%-75%; 4, 76%-100%. The IL23R intensity score is determined by immunostaining intensity rated as follows: 0, none; 1, weak; 2, moderate; 3, intense. Scale bar is 200 µm. (C) IL23R or isotype staining of representative colon or ileum section from 2 CD subjects. (D) IL23R intensity score and percentage of IL23R⁺ cells in lamina propria. Pool of 14 samples from 10 moderate-severe CD subjects.

Figure 2

IL23R-CAR Tregs maintain regulatory phenotype during expansion and after antigen stimulation. (A) Schematic diagram of the IL23R-CAR and the control IL23R-ΔCAR which lacks a signaling domain. (B) Naïve Tregs were left UT or transduced with a nonsignaling truncated IL23R-CAR (IL23R-ΔCAR), IL23R-CAR, or GFP-tagged IL23R-CAR (IL23R-CAR_GFP). After 12 days of expansion, cells were analyzed for CAR expression. (C) Percentage of CD69 expression on UT Tregs or CAR Tregs after overnight activation with culture media (NS), anti-CD3/CD28 beads, or IL23R coated beads. Two-way ANOVA with Tukey’s multiple comparisons test was performed. (D) Phenotype and (E) TSDR demethylation of IL23R-CAR Tregs after 12 days of expansion. (F) Schematic diagram of IL23R-CAR Treg chronic stimulation with IL23R-coated beads at days 15 and 22 in the presence of IL-2 alone or IL-2 combined with proinflammatory cytokines (IL-6, IL-1β, and TNFα) replenished every 2 days. (G) Percentage of intracellular IL-17A produced by IL23R-CAR Tregs at day 26. (H) Percentage and intensity of FOXP3 expression in IL23R-CAR Tregs after chronic stimulation in the presence of proinflammatory cytokines. Abbreviations: PMA/iono, stimulated with PMA and ionomycin; Unstim, not treated; UT, untransduced.

Figure 3

IL23R-CAR Tregs exhibit IL23R-dependent suppressive activity in vitro. (A) Suppression of polyclonally stimulated responder T-cell (Tconv) proliferation by untransduced (UT) Tregs, IL23R-ΔCAR-Tregs, or IL23R-CAR Tregs in coculture or transwell culture. Tregs were prestimulated with culture media (NS), anti-CD3/CD28 beads, or IL23R-coated beads. Data are shown as mean ± SD (n = 7). Two-way ANOVA with Dunnett’s multiple comparisons test was performed. *p < 0.05, **p < 0.01, ***p < 0.001. (B, C) IL23R-CAR Tregs inhibit the maturation of monocyte-derived dendritic cells (DC). Autologous immature DCs were treated with LPS to induce maturation (DC alone) or cocultured with prestimulated IL-23R-CAR Tregs in the presence of LPS (CAR-Treg DC). After 3-day coculture, DC phenotype was analyzed by (B) surface expression of CD80, CD86, CD40, and HLA-DR, (C) percentage of immature DC. One-way ANOVA with Tukey’s multiple comparisons test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

IL23R-CAR Tregs migrate into human IL23R-expressing tissues. (A) Schematic diagram of the luciferase-expressing lentiviral constructs. (B) Three weeks after engraftment of IL23R⁻ and IL23R⁺ tumors in both orientations, luciferase-expressing human ΔNGFR-Tregs, IL23R-ΔCAR Tregs, or IL23R-CAR Tregs were injected intravenously. After 6 days, luciferase expression was monitored in mice by quantification of the total bioluminescence signal (photons/sec) in IL23R⁻ or IL23R⁺ tumors. (C) Data are presented as box and whisker plots. Each dot represents an individual tumor (n = 10 per group, 2-way ANOVA with Sidak’s multiple comparisons test, ****p < 0.0001; ns, not significant). (D) As a control, the size of the tumors was measured in all the conditions (no statistical differences).

Figure 5

Colon biopsy-derived cells from CD patients induce IL23R-CAR Treg activation. (A) Schematic diagram of activation assay. Created with BioRender.com. (B) Percentage of CD69 expressing IL23R-ΔCAR_GFP or IL23R-CAR_GFP Tregs after overnight stimulation with culture media (NS), anti-CD3/CD28 beads, or IL23R-coated beads. (C, D) Percentage of activated (CD69 or GARP expressing) IL23R-ΔCAR_GFP or IL23R-CAR_GFP Tregs after overnight stimulation with culture media (NS) or colon biopsy-derived cells from (C) severe or (D) mild-moderate CD patients. Biopsies from less or more inflamed areas were collected from the same patients. Two-way ANOVA with Dunnett’s multiple comparisons test was performed.

Figure 6

Transcriptomic and proteomic patterns associated with CD biopsy-induced IL23R-specific Treg activation. (A) Transcriptomic profiling (RNA-Seq) was performed on CD colon biopsy samples. The CD69-fold change between biopsy-induced activation and basal activation (NS) of IL23R-CAR Tregs was used as a surrogate outcome variable in the correlation analysis. (A) Correlation analysis between WGCNA modules and biopsy-induced IL23R-specific CAR activation was performed (N = 11). Enrichment analysis of genes within positively correlated modules was performed using EnrichR. Top 20 ranked Gene Ontology (GO) terms according to adjusted p value are displayed here. Log_padj, log 10 of the adjusted p value; Overlap_n, number of enriched genes in a GO term; gene ratio, percentage of enriched genes in the given GO term. (B) Correlation analysis between plasma IL-23 concentration and biopsy-induced IL23R-specific CAR activation (N = 11). A line is used to approximate the relationship between the 2 variables. (C) Plasma protein screening using proximity extension assay. Left, correlation analysis between normalized protein expressions (NPX) and patient biopsy-induced IL23R-specific CAR activation (N = 11). The dotted line represents the FDR threshold of 0.05. Right, volcano plot of the Group strong (N = 4) versus Group weak/no (N = 7) IL23R-specific CAR activation. The dotted line represents the uncorrected significance threshold of 0.05. The x-axis depicts the NPX difference between the groups for each protein measured.