Fig 2: Isolation and characterisation of non-myogenic mesenchymal cells.A Schematic representation of non-myogenic mesenchymal cell isolation protocol (panel created with Biorender.com). B Gating strategy for non-myogenic mesenchymal cell sorting from the CD56- cell fraction. C Representative histograms of specific markers in the CD56- cell fraction. D Heatmap showing the relative expression levels of positive markers. Original values are ln(x)-transformed. Rows are centred and scaled. Rows are clustered using Euclidean distance and Ward linkage. 9 rows, 13 columns. E PCA analysis by singular value decomposition of the same dataset showed in D. X and Y axis show principal component 1 (PC1) and principal component 2 (PC2). The percentage of the total variance explained by each PC is indicated. F Loadings (i.e. contributions) of each variable to PC2. G–J Relative expression, expressed as median fluorescence intensity (MFI) of CD201, CD140a, CD73 and CD90.

Fig 3: Survival and retention of peripheral blood lymphocytes in coculture of gingival fibroblast (GF) over time. Percentages of cells present in cocultures. (A) The proportion of CD14+ cells (percentage ± SEM) present in GF cocultures with peripheral blood mononuclear cells (PBMCs) (solid line) and monocytes (dotted line). (B) Geometric mean fluorescence intensity (gMFI) of CD14+ cells over time of GF cocultures with PBMCs (solid line) and monocytes (dotted line). No significant difference of CD14+ percentage (A) or gMFI (B) after 7 days was observed. Percentages ±SEM of (C) CD19+, (D) CD56+CD3-, (E) CD3+, (F) CD3+CD4+, (G) CD3+CD8+, (H) CD3+CD4+CD196+ cells present in GF cocultures with PBMCs (solid line) and peripheral blood lymphocytes (PBLs) (dotted line). No significant increase or decrease of percentage (D) CD56+CD3-, (E) CD3+, (F) CD3+CD4+ cells is shown over time in both cocultures of PBMCs and PBLs. (C) A significant decrease of CD19+ cells after 7 days is shown in PBL cocultures. Significant decrease of CD3+CD8+ cells is shown after 7 days in PBMC cocultures. A significant decrease of CD3+CD4+CD196+ cells after 14 days is shown in PBMC and PBL cocultures. Time point 0 represents monocultures of (A,B) monocytes, (C–H) PBMCs, and PBLs. All significant differences were tested after 7 days since time point 0 represents a characterization of one experiment. n = 10 GF donors, n = 2 buffy coats, *p < 0.05, **<0.01.

Fig 4: Peripheral blood lymphocytes cells survive in presence of gingival fibroblasts (GFs) after 21 days. Distribution of CD56+CD3-, CD3+, CD14+, and CD19+ as indicated by stack bars over time; (A) before coculturing (0 days), (B) after 7 days, and (C) after 21 days. The proportion of cells (in percentages ± SEM) in live populations of (A) monocultures and (B,C) cocultures are presented in the table below (A–C) per cell subtype for cocultures with GFs. (D–G) Specification of (C) at 21 days. (D) CD3+ T cells, (E) CD19+ B cells, (F) CD56+CD3- NK cells, (G) CD14+ monocytes. (D–F) For all peripheral blood lymphocytes (PBLs), significantly more events per microliters were detected in cocultures with peripheral blood mononuclear cells and PBLs. (H) Specification of CD3+ cell population at baseline levels where CD3+CD4+, CD3+CD8+, and CD3+CD4+CD196+ T cells are shown. (I) Specification of CD3+ cell populations after 21 days where CD3+CD4+, CD3+CD8+, and CD3+CD4+CD196+ T cells are shown. Tables below figures (H,I) represent proportion (percentages ± SEM) of cell populations per (H) monoculture and (I) coculture. Data are presented as events per microliter (A–C,H,I) ±SEM (D–G). n = 10 GF donors, n = 2 buffy coats, *p < 0.05, **<0.01.

Fig 5: Monocytes play a role in the retention of PBLs and is not affected by TLR agonists. Characterization of heterogeneous cell populations: (A) after 7, (B) after 14, and (C) after 21 days. The different culture conditions are presented on x-axes (A–C), with or without monocytes, and in the presence or absence of TLR agonists. Fewer cells retain over time (note differences in values at y-axes) and in PBL conditions (without monocytes). Specification of cells from (C) (at 21 days) are shown for (D) CD56+CD3- (NK cells), (E) CD14+ cells (monocytes), (F) CD19+ (B cells), and (G) CD3+ (T cells). No significant differences were found between cell compositions of control, TLR2, TLR4, and TLR2+TLR4 conditions (calculated using an ANOVA with multiple comparisons, Tukey post-hoc). Data are presented as events per microliter, n = 2 buffy coats. Significant differences between PBMC and PBL cultures were calculated (D–G) using paired t-tests. * p < 0.05, ** p < 0.01, *** p < 0.001.