Differences in Peripheral and Tissue Immune Cell Populations Following Haematopoietic Stem Cell Transplantation in Crohn's Disease Patients

Abstract

Background and aims: Recent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn's disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available.

Methods: We followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison.

Results: Severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content.

Conclusions: Peripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα.

Keywords: Crohn’s disease; anti-TNFα; autologous haematopoietic stem cell transplantation.

Figure 1.

Transcriptional blood signatures following HSCT reveal changes in T and B cells. [A,C] Heatmap representation of microarray expression of top genes regulated at week 13 [A] or week 26 [C] compared to week 0. Each row shows one individual probe and each column an experimental sample. High expression levels are shown in red and low expression levels in green. An unsupervised hierarchical cluster method, using a Pearson distance and average linkage method, was applied for each gene classification. [B,D] Polar graphs showing the top functions identified by ingenuity pathways analysis [IPA] for the genes significantly regulated at week 13 [B] and week 26 [D]. Results are shown graphically as a negative logarithm of the probability score [the most statistically significant pathways have the highest value in the graph]. [E] Absolute numbers (per µL) of blood CD3⁺, CD4⁺ and CD8⁺ cells [mean ± SEM] at baseline [W0] and after haematopoietic stem cell transplantation [HSCT] in Crohn’s disease patients [n = 18]]. [F] Absolute numbers of blood CD19⁺ [mean ± SEM] at baseline [W0] and after HSCT in CD patients [n = 18]. *p < 0.05, **p < 0.01, ***p < 0.001; significant by Wilcoxon signed-rank paired test.

Figure 2.

Differential recovery of blood naive and memory cells after autologous haematopoietic stem cell transplantation [HSCT] in Crohn’s disease patients. [A] Dot plots representing naïve [CD45RA⁺] and memory/activated [CD45RO⁺] compartments within CD4⁺ T cells at baseline [W0] and after HSCT [W13, W26 and W52]. Data are from one representative patient. Below, the mean ± SEM is represented for all patients included at all time points [n = 18]. [B] Dot plots representing naïve [CD27^–] and memory [CD27⁺] CD19⁺ B-cell subsets at baseline [W0] and after HSCT [W13, W26 and W52]. Data are from one representative patient. The mean ± SEM for the naïve and memory B-cell absolute numbers is represented [n = 18]. *p < 0.05, **p < 0.01, ***p < 0.001; significant by Wilcoxon signed-rank paired test.

Figure 3.

Blood transcriptional analysis of selected T- and B-cell-expressed genes following autologous haematopoietic stem cell transplantation [HSCT]. Relative mRNA expression [mean ± SEM] of CCR7 and PTK7 [A], IL7R, CD40LG, and CD28 [B], and CD79A and IGHD [C] in whole blood of ten healthy non-IBD controls and 18 patients with Crohn’s disease [CD] undergoing HSCT at baseline [W0] and after HSCT [W13, W26, W52]. Gene expression in CD patients is shown for remitters and non-remitters separately. Remission to HSCT is defined as drug-free endoscopic remission at W52. *p < 0.05, **p < 0.01, ***p < 0.001; significant by Wilcoxon signed-rank paired test.

Figure 4.

IgG antibody levels in serum remain unchanged after autologous haematopoietic stem cell transplantation [HSCT] in Crohn’s disease patients. [A] Serum IgG, IgA and IgM concentrations [g/L; mean ± SEM] at baseline [W0] and after HSCT [W13, W26 and W52] in Crohn’s disease patients [n = 18]. Dotted line represents the serological protection level. *p < 0.05, ***p < 0.001, ****p < 0.0001; significant by Wilcoxon signed-rank paired test. [B] Anti-tetanus toxoid [TT] antibodies [U.I./mL] detected by ELISA in patient serum at baseline [W0] and after HSCT [W13, W26 and W52]. Dotted line represents the serological protection level. [C] Serum anti-ASCA IgG antibodies at baseline [W0] and after HSCT [W13, W26 and W52]. Values represent extinctions at an optical density [O.D.] at 620 nm obtained by ELISA. The dotted line represents the threshold established for seroreactivity [mean O.D.] in a group of healthy controls.

Figure 5.

Transcriptional intestinal signatures following HSCT reveal significant changes after HSCT. [A] Volcano plot representation of transcriptional changes in biopsies from patients in remission at week 52 compared to week 0. The log₂[fold change] is shown along the x-axis and the −log₁₀[corrected p-value] along the y-axis. Differentially expressed genes that reach significance [considered as corrected p < 0.05 and |FC|>1.5] are shown in colour [downregulated genes at week 52 in green and upregulated genes in red]. [B] Heatmap representation of RNAseq expression of the top 100 genes significantly regulated at week 52 [that were also regulated at week 26] compared to week 0. Each row shows one gene and each column an experimental sample. High expression levels are shown in red and low expression levels in green. An unsupervised hierarchical cluster method, using a Pearson distance and average linkage method, was applied for each gene and sample classification. [C] Polar graph showing the top canonical pathways identified by IPA significantly regulated in remission at week 52 compared to week 0. Results are shown graphically as a negative logarithm of the probability score [the most statistically significant pathways have the highest value in the graph]. [D] Bar plot showing the activation z-score of the top upstream regulators at week 52 in patients in endoscopic remission compared to week 0 found by IPA.

Figure 6.

HSCT exclusive remission-induced gene signature is related to down-regulation of T-cell canonical pathways. [A] Venn diagram showing the number of genes that are differentially expressed in CD-remitting colonic mucosa at week 52 after HSCT or week 46 after anti-TNF treatment compared to their respective baseline expression values. Genes are classified based on their significance: FDR < 0.05 or p < 0.05. About 40% of the genes [882 transcripts] within the HSCT-induced signature were not changed by anti-TNFα. [B] Polar plot representing the top six pathways significantly regulated in the HSCT-induced signature that is not regulated by anti-TNF. The negative logarithm of the probability score [the most statistically significant pathways have the highest value in the graph] for each pathway is represented.

Figure 7.

Immune cell deconvolution analysis reveals unique changes in intestinal cellular composition in patients responding to HSCT treatment compared to anti-TNFα. [A] Cell deconvolution analysis [CIBERSORT] of biopsy samples from healthy non-IBD controls [n = 19] and CD patients before and after treatment with anti-TNFα [n = 22, 13 responders] and HSCT [n = 14, 8 responders]. The figure shows the results for naïve B cells [A], resting memory T cells [B], M1 macrophages [C] and neutrophils [D]. Information on colon biopsy samples from responders to HSCT at week 106 [2 years following HSCT; n = 7] is also included in this analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; significant by Mann–Whitney–Wilcoxon test.

Figure 8.

T-cell-related gene expression by RNAseq before and after treatment with anti-TNFα and HSCT. Gene expression was determined in intestinal biopsies by RNAseq in CD patients treated with anti-TNFα therapy or autologous HSCT. The expression values are normalized and represent log-transformed counts for each gene. Dotted lines show the SEM for each gene from the control samples in each cohort. Expression at different time points for endoscopic remitters and non-remitters is shown for T-cell-related genes CD3E, CD28 and CD40LG [A]; tissue-resident T cells [T_RM] CD69, ITGEA and ITGA1 [B], activated effector T cells S1PR1, IL2RA and CTLA4 [C], and neutrophils or the macrophage genes CXCL9, CXCL10 and CD16B [B]. Asterisks mark the statistically significant differences in each group for each treatment compared to baseline, by Mann–Whitney–Wilcoxon test; *p > 0.05.