TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

Abstract

Background: In addition to their multilineage potential, mesenchymal stem cells (MSCs) have a broad range of functions from tissue regeneration to immunomodulation. MSCs have the ability to modulate the immune response and change the progression of different inflammatory and autoimmune disorders. However, there are still many challenges to overcome before their widespread clinical administration including the mechanisms behind their immunoregulatory function. MSCs inhibit effector T cells and other immune cells, while inducing regulatory T cells (T regs), thus, reducing directly and indirectly the production of pro-inflammatory cytokines. TNF/TNFR signaling plays a dual role: while the interaction of TNFα with TNFR1 mediates pro-inflammatory effects and cell death, its interaction with TNFR2 mediates anti-inflammatory effects and cell survival. Many immunosuppressive cells like T regs, regulatory B cells (B regs), endothelial progenitor cells (EPCs), and myeloid-derived suppressor cells (MDSCs) express TNFR2, and this is directly related to their immunosuppression efficiency. In this article, we investigated the role of the TNFα/TNFR2 immune checkpoint signaling pathway in the immunomodulatory capacities of MSCs.

Methods: Co-cultures of MSCs from wild-type (WT) and TNFR2 knocked-out (TNFR2 KO) mice with T cells (WT and TNFα KO) were performed under various experimental conditions.

Results: We demonstrate that TNFR2 is a key regulatory molecule which is strongly involved in the immunomodulatory properties of MSCs. This includes their ability to suppress T cell proliferation, activation, and pro-inflammatory cytokine production, in addition to their capacity to induce active T regs.

Conclusions: Our results reveal for the first time the importance of the TNFα/TNFR2 axis as an active immune checkpoint regulating MSC immunological functions.

Keywords: Immune checkpoint; Immune regulation; Mesenchymal stem cells; Regulatory T cells; TNFα/TNFR2 signaling pathway; Tolerance induction.

Fig. 1

MSC WT and TNFR2 KO characterization. a MSCs WT showed normal spindle-shaped fibroblast-like appearance (passage 3) (× 4) while MSCs TNFR2 KO exhibited a more heterogeneous morphology (passage 3) (× 4). b Flow cytometry analyses of the surface expression of CD45, CD34, CD44, CD105, CD73, CD90, and SCA1 in MSCs WT and TNFR2 KO (passage 3). Both MSC populations were negative for CD45 and CD34 and positive for the rest of the markers studied. The dark gray histograms represent isotype controls. Data are representative of n = 6 in 3 independent experiments. c Osteogenic differentiation: both MSCs WT and TNFR KO (passage 3) were incubated in osteogenic differentiation medium for 17 days followed by Alizarin Red S staining (× 4). d Adipogenic differentiation: both MSCs WT and TNFR2 KO (passage 3) were incubated in adipogenic differentiation medium for 21 days followed by Oil Red O staining (× 4)

Fig. 2

MSCs’ suppressive effect is diminished in the absence of the TNFα/TNFR2 signaling pathway. Activated CFSE⁺ CD4⁺ or CD8⁺ effector T cells were co-cultured with MSCs WT or TNFR2 KO in different MSC/T cell ratios (n = 6). Proliferation of CD4⁺ T cells (a) and CD8⁺ T cells (b) was measured by flow cytometry. The first bar represents the unstimulated T cells alone (n = 6), the second bar represents the bead-stimulated T cells alone in RPMI (n = 8), and the third bar is for the stimulated T cells alone in 50% RPMI+50% MEMα (n = 6). All data are collected from 3 different experiments. c A flow cytometry representative of proliferation assay at a 1:4 MSC/T cell ratio. Non-stimulated T cells have a single peak represented in blue. Upon stimulation, T cells alone or in co-culture with MSCs start to proliferate, and one can see less intensity of CFSE fluorescence. Data are represented as mean value ± SEM. One-way ANOVA analysis was performed to generate P values. ns, non-significant; *P < .05, **P < .01, ***P < .001, ****P < .0001. b Anti-CD3 and anti-CD28 activation beads

Fig. 3

The expression of TNFR2 on MSCs is directly correlated to their higher immunomodulation of CD4⁺ T convs. Activated CD3⁺ effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected and different activation markers were studied. Cells were gated on CD4⁺Foxp3⁻ T conventional cells. For each marker, the strategy of gating is indicated on the left (y-axis) and below (x-axis) the figure. The x-axis represents each activation marker, and the y-axis represents the CD4 population. Left dot plots represent the Fluorescence Minus One (FMO) controls, and right dot plots represent the main samples. Each dot represents a measured value (n = 6) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. MFI values have been normalized with the T cells alone control group. One-way ANOVA analysis was performed to generate P values. ns, non-significant; *P < .05, **P < .01, ***P < .001, ****P < .0001

Fig. 4

The expression of TNFR2 on MSCs is directly correlated to their higher immunomodulation of CD8⁺ T convs. Activated CD3⁺ effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected and different activation markers were studied. Cells were gated on CD8⁺Foxp3⁻ T conventional cells. For each marker, the strategy of gating is indicated on the left (y-axis) and below (x-axis) the figure. The x-axis represents each activation marker, and the y-axis represents the CD8 population. Left dot plots represent the Fluorescence Minus One (FMO) controls, and right dot plots represent the main samples. Each dot represents a measured value (n = 6) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. MFI values have been normalized with the T cells alone control group. One-way ANOVA analysis was performed to generate P values. ns, non-significant; *P < .05, **P < .01, ***P < .001, ****P < .0001

Fig. 5

MSCs can modulate T cell capacity to produce pro or anti-inflammatory cytokines via the TNFα/TNFR2 signaling pathway. Effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected, activated with PMA/ionomycin, and then blocked with GolgiPlug (a protein transport inhibitor). Intracellular pro-inflammatory cytokine (a, b) and anti-inflammatory cytokine (c, d) production was determined in both CD4⁺ and CD8⁺ T cells. Cells were first gated on CD4⁺Foxp3⁻ or CD8⁺Foxp3⁻ T cells to precisely analyze the conventional T cell population. For each marker, the strategy of gating is indicated on the left (y-axis) and below (x-axis) the figure. The x-axis represents each cytokine, and the y-axis represents the CD4 or CD8 populations. Each dot represents a measured value (n = 12) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. One-way ANOVA analysis was performed to generate P values. ns, non-significant; **P < .01, ***P < .001

Fig. 6

Expression of TNFR2 by MSCs is directly correlated to their higher Foxp3 T reg induction capacity. Activated CD4⁺ or CD8⁺ effector T cells were co-cultured with MSCs WT or TNFR2 KO in different ratios (n = 9). After 3 days, the expression of Foxp3 among CD4⁺ T cells (a) and CD8⁺ T cells (b) was measured by flow cytometry. Cells were gated on CD4⁺CD25⁺Foxp3⁺or CD8⁺CD25⁺Foxp3⁺ T cells. The gating strategy is indicated on the left and under the figures. The first black bar represents the percentage of Foxp3-expressing cells among total effector T cells used in co-cultures at day 0 (n = 9) while the second bar represents the percentage of Foxp3-expressing T cells alone after 3 days of culture (n = 9). (c) CD4⁺ T cells collected from the spleen of WT or TNFα deficient mice were co-cultured with MSCs WT. Cells were gated on CD4⁺CD25⁺Foxp3⁺. Data are represented as the mean value ± SEM collected from 3 different experiments. One-way ANOVA or unpaired Student t test analysis was performed to generate P values; ***P < .001. D0, day 0

Fig. 7

The expression of TNFR2 on MSCs is leading to the induction of T regs with a more activated phenotype. The activation status of the T regs that were induced after the co-culture of CD4⁺ conventional T cells with MSCs WT or TNFR2 KO was evaluated. Cells were gated on CD4⁺Foxp3⁺T cells. For each marker, the strategy of gating is indicated on the left and under the figure. Each dot represents a measured value (n = 4) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. Unpaired Student t test analysis was performed to generate P values. ns, non-significant; **P < .01, ***P < .001. iTregs, induced T reg cells