Fig 2: Expression of CEACAM1 associates with LT-HSC activity in expanded UCB cells. (A) Schematic representation of experimental strategy for in vivo studies. Data presented as mean ± SD for 2 independent biological replicates. (B) Long-term engraftment of cells from indicated populations in NSG mice 3, 12, and 24 weeks after transplantation. Each dot represents 1 mouse from 2 independent experiments. Black bars indicate median engraftment values. **P = .0018, ***P = .0003 (Mann-Whitney U test, 2-tailed). Cells were transplanted at cell doses reflecting population frequency at sort to compare the contribution of each subset to engraftment. (C) Lineage potential of indicated populations assessed for each engrafted mouse of the experiment presented in panel B. The percentage of human CD45+ myeloid (CD33+), B lymphoid (CD19+), T lymphoid (CD3+), and primitive (CD34+) cells for each condition is shown. Each dot represents 1 mouse. ***P = .0007 (CD19+), ***P = .0009 (CD33+), ***P = .0001 (CD34+), **P = .0035 (CD3+) (Mann-Whitney U test, 2-tailed). (D) LT-HSC frequency of CEACAM1+ and CEACAM1- populations sorted from CD34+CD45RA-CD90+EPCR+ITGA3+ cells. Estimated frequencies (red lines) presented as 1 per number of sorted cells and 95% CIs (gray boxes) are shown. P = .05 (Mann-Whitney U test, 1-tailed). The objective of these experiments was to determine if the LT-HSC frequency of the CEACAM1+ITGA3+ population is higher than that of the CEACAM1-ITGA3+ subset. The hypothesis assumed for the test is unidirectional, supporting the use of a 1-tailed test. (E) Human engraftment in secondary NSG mice 18 weeks after transplantation. Primary NSG mice transplanted with CEACAM1+ITGA3+ and CD90+EPCR+ parental cells were sacrificed 24 weeks after transplantation, and 80% of BM was collected and transplanted; n = 2 biological replicates.

Fig 3: SSc serum induced CEACAM6 expression on classical monocytes. CD14+16- monocytes were isolated from a healthy donor and were treated with serum from HCs (n=7) and patients with systemic sclerosis (n=14) for 15 h. The proportion of CEACAM-positive classical monocytes from patients with SSc who provided serum was also analyzed. (A) Dot plots show the correlation between the proportion of CEACAM+ monocytes from a healthy donor treated with SSc serum and that from patients with SSc obtained at the same time as the serum. Statistical analysis was performed using Spearman’s rank correlation test. (B) The frequency of serum-induced CEACAM+CD14+16- monocytes was compared among HCs (n=7), inactive SSc (n=7), and active SSc (n=7). Each analysis was performed in duplicates. The left figure shows a histogram of CEACAM. (C, D) Data show the frequency of CEACAM1, 3, 5, and 6+ monocytes treated with serum from HCs or patients with dcSSc. Gating of CEACAM6+ monocytes is shown in (C) Data are presented as mean ± standard deviation (SD). P values were calculated using an unpaired t-test. *P < 0.05, ***P < 0.001.

Fig 4: CEACAM-positive monocytes present with an inflammatory phenotype. The GO functional category analysis (A) and the KEGG pathway functional classification (B) of genes differentially expressed in CEACAM+CD14+16- monocytes and CEACAM-CD14+16- monocytes from a patient with dcSSc. DAVID v6.7 functional annotation bioinformatics microarray analysis software was used to obtain the GO biological process functional category and the KEGG pathway functional classification. Only GO and KEGG pathway terms for categories that showed statistically significant differences are shown (P-value = 0.01 in A or P-value = 0.05 in B). GO functional category analysis (C) and KEGG pathway functional classification (D) of genes differentially expressed in CEACAM6+CD14+16- monocytes and CEACAM1+CD14+16- monocytes. Only GO and KEGG pathway terms with P-value = 0.05(C) or P-value = 0.1(D) are shown. (E) ELISA assay for the secretion of TNF-a and IL-1ß by CEACAM+CD14+16- and CEACAM-CD14+16- monocytes treated with LPS (50 ng/mL) for 6h. Data are expressed as the mean ± SD, and P values were calculated using an unpaired t-test. (F) The intracellular expression of TNF-a was analyzed using CyTOF (n=5). Representative histogram (left) and the Mean Metal Intensity (MMI) (right) are shown. P-values were calculated using paired t-tests. *P < 0.05, ***P < 0.001.

Fig 5: Expression of each subtype of CEACAM on SSc monocytes. (A-D) Dot plots show the proportion of positivity of CEACAM1 (A), CEACAM3 (B), CEACAM5 (C), and CEACAM6 (D) on CD14+CD16- classical monocytes from HCs and patients with RA and SSc. P values were calculated using a Mann–Whitney U test. **P < 0.01, ***P < 0.001. ns, not significant.