Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells

Abstract

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8⁺ and CD4⁺ T cells. Instead, Tregs and CD3⁺ CD4^- CD8^- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.

Keywords: DN T cells; Immunomodulation; Stem Cells; Tregs; renal diseases.

Figure 1

TLR2-activated- Adult Renal Stem/Progenitor Cells (ARPCs) were able to decrease Peripheral blood mononuclear cell (PBMC) proliferation. (A) TLR2-activated-ARPCs were able to decrease the viable count of Con-A activated PBMCs in an independent set of 3 experiments with 3 ARPC clones and PBMC from 3 different healthy donors after both 5 days and 15 days of co-culture. Data from 15 days of co-culture were normalized to 5 days of co-culture in order to compare differences in viable counts. (B) BrdU assays showed that LTA-stimulated ARPCs decreased PBMCs’ proliferation rate both after the 5th and 15th day of co-culture. The histograms represent the mean ± SEM. (C) PBMCs’ morphology was examined under a light-inverted microscope. Con-A activated PBMCs’ morphology was observed after 5 and 15 days of culture in different conditions. Con-A activated PBMCs formed rounded cellular aggregate after 5 and 15 days of culture in response to the Con-A mitogen effect. When Con-A activated PBMC were co-cultured with ARPCs or LTA-activated ARPCs, a lower number of cellular aggregates was observed both after 5 and 15 days. PBMC images were acquired by an upright microscope with a 10× objective. (D) Comparative analysis of apoptosis in Con-A activated PBMCs revealed no significant increase in early and late apoptosis or necrosis when cells were co-cultured with ARPCs and LTA-activated ARPCs after 5 days of co-culture. After 15 days of co-culture, early and late apoptosis increased slightly. Results are representative of 3 independent experiments on 3 different cell lines.

Figure 2

Gating strategy for T cell analysis. PBMC isolated from the peripheral blood of healthy donors, were activated for 24 h with Concanavalin and were cultured alone (A) or co-cultured with ARPCs (B) or with LTA-activated ARPCs (C) for 5 or 15 days and analyzed by flow cytometry. The figure is representative of one experiment showing the gating strategy used to identify lymphocytes (left panel), CD3⁺ T cells and T cells subsets: CD4⁺ T cells, CD8⁺ T cells, DN T cells and Tregs.

Figure 3

ARPCs are able to modulate Tregs and DN T cells. After 5 and 15 days of co-culture with ARPC or LTA-activated ARPCs, no changes were observed in both CD4⁺ T cells and CD8⁺ T cell subsets. After 5 days of co-culture, LTA acti-vated-ARPCs were able to significantly decrease the % of Tregs and DN T cells in Con-A activated PBMC (A). After 15 days of co-culture, LTA activated-ARPCs significantly decreased the % of Tregs and increased the % of DN T cells in Con-A activated PBMC (B). Data represent the fold change of the PBMC percentage respect to PBMC Con-A (basal). Data are representative of four independent experiments (means ± SEM).

Figure 4

The immunomodulation effect was specific for ARPCs. No significant changes in the % of T cell subsets were observed when ConA activated PBMC were co-cultured with RPTECs for 5 (A) or 15 days (B). Data represent the fold change of the PBMC percentage with respect to PBMC Con-A. Data are representative of three independent experiments (means ± SEM).

Figure 5

Activated ARPCs secreted a specific pattern of cytokines. The Human Cytokine Array detects 36 human cytokines in cell culture supernatants. The arrows and numbers indicate the cytokine array dot blots. (A) Dot blot pixel intensity is graphed. (B) Data are representative of three independent experiments (means ± SEM).

Figure 6

A cocktail of cytokines mediated the immunomodulatory effect of ARPCs after 5 days. (A) Con-A activated PBMC were exposed to different crescent concentrations of PAI1, CXCL1, GM-CSF, and MCP1 for 5 days. Percentages of Tregs and DN T cells were analyzed using flow cytometry. PAI1 was able to significantly decrease the % of Tregs at 6 nM and 12 nM but significantly increased DN T cells at 6 nM. None of CXCL1, GM-CSF, and MCP1 were able to significantly modulate T cell subsets alone. (B) When Con-A activated PBMC were exposed to the 4 cytokines in mix for 5 days, an immunomodulatory effect was observed on Tregs and DN subsets, similar to the ARPCs co-culture effect. Data represent the fold change of PBMC percentage respect to PBMC Con-A (Basal). Data are representative of six independent experiments (means ± SEM).

Figure 7

A cocktail of cytokines mediated the immunomodulatory effect of ARPCs after 15 days. (A) Con-A activated PBMC were exposed to different crescent concentration of PAI1, CXCL1, GM-CSF and MCP1 for 15 days. None of PAI1, CXCL1, GM-CSF, and MCP1 alone were able to significantly modulate T cell subsets. (B) When Con-A activated PBMC were exposed to the 4 cytokines in a mix for 15 days, an immunomodulatory effect was observed on Treg and DN subsets, perfectly comparable to the ARPCs’ co-culture effect. Data represent the fold change of PBMC percentage respect to PBMC Con-A (Basal). Data are representative of six independent experiments (means ± SEM).

Figure 8

ARPCs can mediate immunomodulation and affect inflammatory state. “The physiological response to tissue damage can be divided into three phases: inflammatory, reparative, and remodeling. During this process, inflammatory status (defined as the types and concentrations of cytokines and cells of the immune system present) changes considerably: proinflammatory influences (red dashed line) are dominant in the inflammatory, infection-fighting phase and diminish in the reparative and remodeling phases that follow, which allows wound healing. In the context of the intensity of the immune response (right vertical axis), the inflammatory response (red dashed line) fluctuates during the wound-healing process. Such changes in inflammation substantially alter the effects of mesenchymal stem cells (MSC)-meditated immunomodulation, which results in a variable correlation between the intensity of inflammation and efficacy of MSC treatment (solid black line)”. Such changes in inflammation are also affected by ARPC-meditated immunomodulation. ARPCs cause DN T cell decrease at the initial phase, promoting inflammation, and DN T cell increase in the late inflammation stages, favoring the inflammation quenching. Adapted by permission from Springer Nature: Springer Nature, NATURE IMMUNOLOGY, Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications, Ying Wang, Xiaodong Chen, Wei Cao, and Yufang Shi, COPYRIGHT 2014.