Bone marrow-derived mesenchymal stromal cells harness purinergenic signaling to tolerize human Th1 cells in vivo

Abstract

The use of bone marrow-derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) mediated by human CD4(+) Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; furthermore, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression.

Keywords: Adenosine; Mesenchymal stromal cells; T cells; x-GVHD.

Figure 1. Bone Marrow Derived Mesenchymal Stromal Cells Can Treat Human X-GVHD In Mice

Female NSG mice were reconstituted with human Th1 cells (5 million) and human monocytes (3 million). Between day 20 and day 35 post-transplant, mice developed severe x-GVHD (A; left and middle panel). Within 24 hrs of GVHD onset, mice were treated three times with BMSC (2 million) and each injection was separated by four days. Photographs from a representative mouse from each group are shown (A; right panel). Summary of percent weight loss, from three experiments, in control and treated cohorts (B). Cumulative kaplain-meier survival curve from three experiments (C). For weight loss, P value was determined using a two-way anova test. For survival curve, P values were determined using log-rank test. Each experiment had n=5 animals per cohort, experiments were performed in triplicate with 3 different BMSC clinical products. *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 2. BMSC Treatment Decreased Human Th1 cell Engraftment, Effector Cytokine Production With A Concomitant Reduction In FoxP3⁺ Tregs

Female NSG mice were reconstituted with human Th1 cells (5 million) and Monocytes (3 million). Within 24 hrs of GVHD onset (between day 20 to day 35), the BMSC cohort was treated 3 times, each injection separated by four days. Post-treatment, the mice were euthanized and the lymphoid organs such as spleen were evaluated for human Th1 cell engraftment. Representative flow plot showing human CD45⁺ cells versus mouse CD45⁺ cells (A). Absolute numbers of human CD45⁺ Th1 cells were calculated from total splenocyte number (B). Representative flow cytometry plots of IFNγ and TNF-α expression determined in the recovered human Th1 cells by intracellular cytokine staining (C). Cumulative absolute numbers of IFNγ producing human Th1 cells (D) and TNF-α producing human Th1 cells in the spleen (E). Absolute number of FoxP3⁺ human Tregs determined by intracellular staining (F). Absolute numbers of human CD39+ Th1 cells in the spleen (G). Absolute numbers of cytokine producing cells, T-regs and CD39⁺ Th1 cells were obtained from the absolute numbers of hCD45⁺ Th1 cells in the spleen. Each experiment had n=5 animals per cohort, experiments were performed in triplicates with 3 different BMSC clinical products. The data shown are cumulative of all three experiments. *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 3. Th1 Cells Exposed To Adenosine Show Significant Reduction In CD39 Expression And Enhanced Apoptosis

Human CD4⁺ T cells were expanded in Th1 conditions for 6 days. Th1 cells were washed in media and then dispensed into 24 well tissue culture plates at a concentration of 1 million cells per well in 1 ml of media. Adenosine was added at a concentration of 100μM. Effect of adenosine on Th1 cell CD39 frequency and protein expression (A). Summary of CD39 frequency (B; left panel) and CD39 protein expression (B; right panel) in three different normal donors. Apoptosis of human Th1 cells in the presence of adenosine at 2hrs (C upper panels) and 24 hrs (C lower panels). Summary of Annexin V+ for three different normal donors at 2 hrs (D; left panel) and at 24 hrs (D; right panel). N=5 animals per cohort, experiments were repeated 3 times with 3 different BMSC clinical products. P values were obtained by performing a student t- test where *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 4. BMSC Exposure Enhances Purine Metabolism In Human Th1 Cells In Vitro

Human Th1 cells from 3 different donors were co-cultured with clinical grade BMSC from 3 different donors in the presence of ATP (100μM) for 2 hrs at a 1:1 ratio. Some culture conditions included the inhibitor for CD73 (Adenosine 5’-(α,β-methylene) diphosphate sodium; 100μM). The supernatants were harvested and adenosine measured by GC-MS/MS._Representative GC-MS/MS graphs (A) and cumulative adenosine production in the supernatant from three experiments (B). The co-culture was repeated in a phosphate free reaction buffer and the amount of phosphate released in the supernatant was measured using Malachite Green Phosphate detection assay (C). P values were obtained by performing a student t- test where *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 5. In Vivo Administration Of Human BMSC Results In Enhanced Human Exosome Presence In The Serum Of Murine Recipients With x-GVHD

Female NSG mice that had severe x-GVHD were treated with BMSC as previously described. Serum 24 hrs post BMSC infusion, was collected from control and treated mice. Exosomes isolated from the serum were visualized by electron microscopy (A). Flow cytometric analysis of enriched human exosomes (B). Functional phosphate production of exosomes from AMP was detected using the malachite green phosphate assay (C). Experiments were performed in duplicates with BMSC from 3 different donors. P values were obtained by performing a student t- test where *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 6. Inhibiting A2aR In Human Th1 Cells Abrogates The Effect Of BMSC In Vivo

Female NSG mice were reconstituted with human Th1 cells (5 million) and Monocytes (3 million). Within 24hrs of GVHD onset (between day 20 to day 35), the BMSC cohort was treated 3 times with one injection of BMSC every four days. Some cohorts were treated by I.P. injections with the drug ZM241385 (1.5mg/kg/day) every day for the duration of BMSC treatment. Post-treatment, mice were euthanized and their spleen was evaluated for human Th1 cell engraftment (A). Human Th1 cells from the spleen were stimulated ex-vivo for 4 hrs with PMA/ionomycin along with GolgiStop and GolgiPlug for the last 2 hrs. Cells were then stained for Intracellular cytokine expression. Representative flow cytometry plots for intracellular IFNγ and TNF-α staining (B). Cumulative absolute numbers of IFNγ-producing human Th1 cells (C) and TNF-α-producing human Th1 cells in the spleen (D). Cumulative histological findings in the lung and liver of treated animals (E). Data was pooled from two different experiments using two different BMSC donors. P values were obtained by performing a one way anova where *=P<0.05, **=P<0.01 and ***=P<0.001.

Figure 7. Adenosine Generating Functional Exosomes Are Present In Patient Samples Post BMSC Treatment

Plasma from patients who underwent BMSC treatment was collected at different time points. Exosomes were isolated and characterized by flow cytometry for the expression of CD73 (A). Functional phosphate production of exosomes from AMP was detected using the malachite green phosphate assay (B). P values were obtained by performing a paired student t- test *=P<0.05, **=P<0.01, ***=P<0.001 and ns=not significant