PMC9459022_01
Clinical characteristics in the patient with both ICA and CD. (A) Timeline of the clinical events and treatment strategies. (B) Abdominal CT images of the congenitally asplenic patient (left) and the control (right). (C) Morphological examination of peripheral blood smear of the congenitally asplenic patient showed the acanthocytes and Howell-Jolly bodies within red blood cells (arrows). (D) Colonoscopy demonstrating multiple longitudinal deep ulcers with mucosal oedema and cobblestone appearance in the hepatic flexure (top panel) and the sigmoid colon (bottom panel) of the asplenic CD patient. (E) Hematoxylin-eosin staining of the colonoscopic biopsy tissue from the asplenic CD patient showing the infiltration of lymphocytes, plasma cells and neutrophils in the lamina propria of the mucosa, discontinuous severe crypt distortion (top panel) as well as the ulcer (middle panel) and crypt abscess (bottom panel, arrow) formation. (F) The histograms demonstrating the elevated WBC (white blood cell count), PLT (platelet count), LYMPH (absolute number of lymphocytes), LYMPH% (percentage of lymphocytes), MONO (absolute number of monocytes) and MONO% (percentage of monocytes) of the asplenic CD patient in remission compared to remission controls (n=32) with normal spleens randomly selected from our center's CD cohort. (G) Line graph showing the difference in disease progression between the asplenic patient and the control after the first CyTOF analysis (HBI score used to evaluate disease activity of CD).
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During the remission stage of CD, when the Harvey Bradshaw Index (HBI) score was 3, the noticeable increase in the absolute numbers of peripheral blood leucocytes, monocytes, lymphocytes and platelets were observed in this asplenic patient compared to other CD patients in remission with normal spleens, as were the percentages of monocytes and lymphocytes ( Figure 1F ). To facilitate comparison, another CD patient with a normal spleen who had the same sex (male), disease phenotype (Montreal classification, A2L3B1) and medication history (prednisone, thalidomide, mesalazine and infliximab), as well as very similar age (34-year-old), medical history (9 years of CD) and clinical manifestations, was selected as the optimal control for the more in-depth study, namely cytometry by time of flight (CyTOF) ( Figure 1B ). It was found in amazement that the disease severity of this asplenic patient altered significantly during the following 1 year compared with the control ( Figure 1G ). The timeline of this case is showed in  Figure 1A .
PMC9459022_02
Clinical characteristics in the patient with both ICA and CD. (A) Timeline of the clinical events and treatment strategies. (B) Abdominal CT images of the congenitally asplenic patient (left) and the control (right). (C) Morphological examination of peripheral blood smear of the congenitally asplenic patient showed the acanthocytes and Howell-Jolly bodies within red blood cells (arrows). (D) Colonoscopy demonstrating multiple longitudinal deep ulcers with mucosal oedema and cobblestone appearance in the hepatic flexure (top panel) and the sigmoid colon (bottom panel) of the asplenic CD patient. (E) Hematoxylin-eosin staining of the colonoscopic biopsy tissue from the asplenic CD patient showing the infiltration of lymphocytes, plasma cells and neutrophils in the lamina propria of the mucosa, discontinuous severe crypt distortion (top panel) as well as the ulcer (middle panel) and crypt abscess (bottom panel, arrow) formation. (F) The histograms demonstrating the elevated WBC (white blood cell count), PLT (platelet count), LYMPH (absolute number of lymphocytes), LYMPH% (percentage of lymphocytes), MONO (absolute number of monocytes) and MONO% (percentage of monocytes) of the asplenic CD patient in remission compared to remission controls (n=32) with normal spleens randomly selected from our center's CD cohort. (G) Line graph showing the difference in disease progression between the asplenic patient and the control after the first CyTOF analysis (HBI score used to evaluate disease activity of CD).
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During the remission stage of CD, when the Harvey Bradshaw Index (HBI) score was 3, the noticeable increase in the absolute numbers of peripheral blood leucocytes, monocytes, lymphocytes and platelets were observed in this asplenic patient compared to other CD patients in remission with normal spleens, as were the percentages of monocytes and lymphocytes ( Figure 1F ). To facilitate comparison, another CD patient with a normal spleen who had the same sex (male), disease phenotype (Montreal classification, A2L3B1) and medication history (prednisone, thalidomide, mesalazine and infliximab), as well as very similar age (34-year-old), medical history (9 years of CD) and clinical manifestations, was selected as the optimal control for the more in-depth study, namely cytometry by time of flight (CyTOF) ( Figure 1B ). It was found in amazement that the disease severity of this asplenic patient altered significantly during the following 1 year compared with the control ( Figure 1G ). The timeline of this case is showed in  Figure 1A .
Single-cell mapping of peripheral blood mononuclear cells showed the significant differences of immune cell subsets among the control patient in remission and the asplenic patient in remission or active stage of CD. (A) Mass cytometric analysis and mapping of PBMC from the control patient in remission (left), the asplenic patient in remission (medial) and the asplenic patient in active stage of CD (right) using Cytometry by Time Of Flight (CyTOF) mass spectroscopy, t-distributed stochastic neighbor embedding (t-SNE) and clustering algorithms. (Upper panel: mapping of PBMC into 37 clusters based on differences in cell surface marker expression; lower panel: depicting differences in the distribution of cell subsets in PBMC based on 37 clusters.) (B) Bar graphs showing the difference in frequency of each cluster (left panel) or cell subset (right panel) across different samples. (C) Annotation of the immune cell subsets based on different clusters. (D) Differences in the frequency of each subset of CD4+ T cells and CD8+ T cells among the different samples. (Tem, effector memory T cells; Tcm, central memory T cells; Teff, effector T cells).
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The PhenoGraph clustering algorithm was then applied to all samples from the control and asplenic patient in remission, as well as the asplenic patient in active stage for comparative analysis ( Figures 2 ,  3 ). These alterations were more pronounced during the active stage of CD ( Figures 2A, B ;  Figures 3A, B ). As for the adaptive immune system, in the T lymphocyte population, elevated CD8+ T cells, predominantly CD8+ effector T cells (cluster 21, 22, 23), were identified in the asplenic patient in remission compared with the control, and reached its highest enrichment in the active period ( Figures 2A, B, D ). Meanwhile, the imbalance in T-cell homeostasis became more severe during the active CD ( Figures 2A, B ). Meanwhile, plasmablasts (cluster 34) also showed a tendency to be elevated in the asplenic patient ( Figures 2A, B ). Patients with an active or remitting CD and healthy individuals were selected as controls ( Figure 4 ;  Supplementary Figure 2 ).
High-dimensional analysis revealing the differences in the expression of specific surface markers on PBMC samples from the control patient and congenitally asplenic patient with remission CD, as well as the congenitally asplenic patient in the active stage of CD. (A) The t-distributed stochastic neighbor embedding plots colored by expression of CD38, CCR4, CCR6 and CD94 of each group. (B) The histograms displaying differences in the expression of surface markers (CD38, CCR4, CCR6 and CD94) across patient samples. (C) Heatmap representing the marker expression profiles of the 12 cell populations in PBMC. The heatmap colors represent the average expression of a given marker for each cell population. (D) Boxplot showing the difference in the expression of CCR4 (top) and CCR6 (bottom) in colon tissues from healthy controls, remission IBD and active IBD patients, with differences assessed by the GEO2R bioinformatics tool. (Raw data from GEO database, GSE59071; ns, not significant; *P<0.05; ***P<0.001).
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The PhenoGraph clustering algorithm was then applied to all samples from the control and asplenic patient in remission, as well as the asplenic patient in active stage for comparative analysis ( Figures 2 ,  3 ). These alterations were more pronounced during the active stage of CD ( Figures 2A, B ;  Figures 3A, B ). Meanwhile, compared with the control, down-regulated expression of the inhibitory receptor CD94 in clusters 18 and 19, which represent the terminally differentiated CD57+ NK cells, was noticeable in the asplenic patient during disease remission, which became more obvious as the disease progressed ( Figures 3A, C ). More interestingly, CD4+ T cells from the asplenic patient showed higher expression of CCR4 and CCR6, and their expression levels increased progressively with disease severity, which was not observed on CD8+ T cells ( Figures 3A-C ). Figures 3A, B ). Furthermore, compared with the remission phase, the asplenic patient with active CD showed a dramatic reduction in circulating naive B cells accompanied by the highest expression of CCR6. Similarly, memory B cells (cluster 33) reduced in the asplenic patient, but the expression of CCR6 was upregulated, which was more pronounced in the active stage ( Figure 3B ).
Flow cytometry analysis demonstrating the differences in proportions of Th1, Th17 and Treg cells from PBMC and the Th17/Treg ratio among healthy controls, remission controls, active controls and the congenitally asplenic patient with active CD. (A) Representative FACS plots showing the percentage of CD3+CD4+IFN-gamma+ (Th1) cells (top panel: gated on CD3+ cells), CD3+CD4+IL-17A+ (Th17) cells (middle panel: gated on CD3+ cells) and CD3+CD4+CD25+Foxp3+ (Treg) cells (bottom panel: gated on CD3+CD4+ cells) of each group [asplenia with active CD (n=1), controls with active CD (n=3), controls with remission CD (n=6), healthy controls (n=5)]. (B) The histograms showing the difference in the percentage of Th1, Th17 and Treg cells in CD4+ T cell, as well as the Th17/Treg ratio. Data were analyzed by One-way ANOVA, and all values are reported as means and SD. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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Patients with an active or remitting CD and healthy individuals were selected as controls ( Figure 4 ;  Supplementary Figure 2 ). Notably, despite the similar disease activity scores, Th1 and Th17 cells were significantly enriched in PBMC from the asplenic patient compared to other active CD patients, accompanied by a dramatic increase in the ratio of Th17/Treg cells ( Figures 4A, B ).