Effect of Timing and Complement Receptor Antagonism on Intragraft Recruitment and Protolerogenic Effects of Mesenchymal Stromal Cells in Murine Kidney Transplantation

Abstract

Background: Mesenchymal stromal cells (MSCs) have protolerogenic effects in renal transplantation, but they induce long-term regulatory T cells (Treg)-dependent graft acceptance only when infused before transplantation. When given posttransplant, MSCs home to the graft where they promote engraftment syndrome and do not induce Treg. Unfortunately, pretransplant MSC administration is unfeasible in deceased-donor kidney transplantation.

Methods: To make MSCs a therapeutic option also for deceased organ recipients, we tested whether MSC infusion at the time of transplant (day 0) or posttransplant (day 2) together with inhibition of complement receptors prevents engraftment syndrome and allows their homing to secondary lymphoid organs for promoting tolerance. We analyzed intragraft and splenic MSC localization, graft survival, and alloimmune response in mice recipients of kidney allografts and syngeneic MSCs given on day 0 or on posttransplant day 2. C3a receptor (C3aR) or C5a receptor (C5aR) antagonists were administered to mice in combination with the cells or were used together to treat MSCs before infusion.

Results: Syngeneic MSCs given at day 0 homed to the spleen increased Treg numbers and induced long-term graft acceptance. Posttransplant MSC infusion, combined with a short course of C3aR or C5aR antagonist or administration of MSCs pretreated with C3aR and C5aR antagonists, prevented intragraft recruitment of MSCs and graft inflammation, inhibited antidonor T-cell reactivity, but failed to induce Treg, resulting in mild prolongation of graft survival.

Conclusions: These data support testing the safety/efficacy profile of administering MSCs on the day of transplant in deceased-donor transplant recipients and indicate that complement is crucial for MSC recruitment into the kidney allograft.

Figure 1.. C3aR and C5aR expression on MSCs.

Representative overlay histograms of flow cytometric analysis of C3aR and C5aR expression on BL6 bone marrow–derived MSCs (A). Mean percentages of positive MSC are reported in the figure (B). Median fluorescence intensity (MFI) over isotype (after subtracting MFI of isotype antibody) for C3aR and C5aR expression on MSC. (Data are mean ± SD of n = 4 MSC preparations.)

Figure 2.. MSCs homing to the kidneys and spleen of recipient mice and impact on early graft function and inflammation.

Number (mean ± SD) of PKH26⁺ MSCs in kidney grafts (A) and spleens (B) from mice given MSCs the day of transplantation (2 hrs after surgery) or MSCs 2 days after transplantation in combination with either C3aR-A or C5aR-A or preexposed to C3aR-A and C5aR-A and euthanized 24 hrs after cell infusion, (n = 3 mice per group). Representative images of a PKH26⁺ MSC in the kidney (C) and in the spleen (D), original magnification 600X. Linear regression analysis of MSC numbers in the kidney grafts (cells/mm²) versus the MSCs in the spleen (cells/mm²) from the above mice and including naïve untransplanted mice (n = 3) given MSCs (MSCs in the kidney: 3.6 ± 1.4 cells/mm²; MSCs in the spleen 375 ± 58 cells/mm²) or mice given MSC preexposed to vehicle for C3aR-A and C5aR-A (see Figure S2) (E). Representative immunohistochemistry images of neutrophils (F–G) and complement C3 (J) staining in kidney graft tissues from mice given MSCs alone on day 2 (F–J) or together with C5aR-A (G) (original magnification 400X). Number of graft infiltrating neutrophils (cells/mm²) (H), intragraft complement C3b deposition in kidney allografts (semiquantitative scores on peritubular capillaries and glomeruli) (I) and kidney graft function (as ratio between BUN levels 24 hrs after versus before MSC infusion) (K) from mice given peri-transplant MSCs or given MSCs 2 days after transplantation in combination with either C3aR-A or C5aR-A or preexposed to C3aR-A and C5aR-A. Data are mean ± SD; *P < 0.05 versus MSC day 2; °P < 0.05 versus MSC day 0. BUN: blood urea nitrogen, PTC: peritubular capillaries.

Figure 3.. Kidney allograft survival in mice given MSC infusion alone or combined with C3aR and C5aR antagonists.

BALB/c kidney allograft survival (A) and function (B, as BUN levels) in BL6 mice given syngeneic BM-MSC infusion (5 × 10⁵; i.v.) on the day of transplantation (MSCs day 0, n = 7) or 2 days posttransplant either alone (MSCs day2, n = 7) or combined with C3aR-A (3 injections of 15 mg/kg; MSCs day2 + C3aR-A, n = 7) or C5aR-A (3 injections of 0.5 mg/kg; MSCs day2 + C5aR-A, n = 8) or given MSC preexposed in vitro to C3aR-A/C5aR-A (n = 3). Mice left untreated (no treatment, n = 7) or given only C3aR-A and C5aR-A treatment (C3aR-A or C5aR-A, n = 6) were considered as control groups. *P < 0.05 versus C3aR-A or C5aR-A alone control group, ^§P < 0.05 versus MSCs day 2.

Figure 4.. Ex vivo antidonor alloreactivity, Treg expansion, and suppressive function in MSC-infused mice.

Frequency of BALB/c reactive IFNγ-producing splenocytes (A), percentage of CD25⁺Foxp3⁺ cells among splenic CD4⁺ T cells (B), percentage of Helios negative Tregs (D), and ratios between Treg and CD44^highCD62L⁻ effector memory (T_EM) CD4⁺ and CD8⁺ T cells (F) from BL6 mice given syngeneic BM-derived MSCs after the day of transplantation (MSCs day 0) or from BL6 recipients given MSCs 2 days posttransplant either alone (MSCs day 2, none) or with C3aR-A or C5aR-A treatment. All mice were transplanted with a BALB/c kidney allograft at day 0 and were sacrificed 4 days after transplant. Transplanted mice not given an MSC infusion (no MSCs) or given C3aRA and C5aRA (MSC + RAs) and sacrificed 4 days posttransplant or naïve untransplanted BL6 mice (n = 6) were considered controls. (C) Representative dot plots of Foxp3⁺CD25⁺ on CD4⁺ T cells and representative histograms of Helios expression by Tregs (E) from kidney transplant mice given MSCs at day 0 or MSC at day 2 in combination with C3aR-A, respectively. (G) Frequency of IFNγ-producing CD4⁺CD25⁻ T cells isolated from naïve untransplanted BL6 mice alone (first white column on the left) or in the presence of CD4⁺CD25⁺ T cells (at ratios of 2:10 CD4⁺CD25⁺ T cells:CD4⁺CD25⁻ T cells) isolated from mice given syngeneic BM-derived MSCs the day of transplantation (MSCs day 0) or from BL6 recipients given MSCs 2 days posttransplant, either alone (MSCs day 2, none) or with C3aR-A or C5aR-A treatment and euthanized 4 days after transplant and from mice of control groups. Percentages in spot images in panel A represent the area covered by spots. Data are mean ± SD; §P < 0.05 versus CD4⁺CD25⁻ T cells alone, °P < 0.05 compared with all the other groups, *P < 0.05 versus MSCs day 0.