Adoptive Transfer of Treg Cells Combined with Mesenchymal Stem Cells Facilitates Repopulation of Endogenous Treg Cells in a Murine Acute GVHD Model

<div><p>Therapeutic effects of combined cell therapy with mesenchymal stem cells (MSCs) and regulatory T cells (Treg cells) have recently been studied in acute graft-versus-host-disease (aGVHD) models. However, the underlying, seemingly synergistic mechanism behind combined cell therapy has not been determined. We investigated the origin of Foxp3⁺ Treg cells and interleukin 17 (IL-17⁺) cells in recipients following allogeneic bone marrow transplantation (allo-BMT) to identify the immunological effects of combined cell therapy. Treg cells were generated from eGFP-expressing C57BL/6 mice (Treg^egfp cells) to distinguish the transferred Treg cells; recipients were then examined at different time points after BMT. Systemic infusion of MSCs and Treg cells improved survival and GVHD scores, effectively downregulating pro-inflammatory Th×and Th17 cells. These therapeutic effects of combined cell therapy resulted in an increased Foxp3⁺ Treg cell population. Compared to single cell therapy, adoptively transferred Treg^egfp cells only showed prolonged survival in the combined cell therapy group on day 21 after allogeneic BMT. In addition, Foxp3⁺ Treg cells, generated endogenously from recipients, significantly increased. Significantly higher levels of Treg^egfp cells were also detected in aGVHD target organs in the combined cell therapy group compared to the Treg cells group. Thus, our data indicate that MSCs may induce the long-term survival of transferred Treg cells, particularly in aGVHD target organs, and may increase the repopulation of endogenous Treg cells in recipients after BMT. Together, these results support the potential of combined cell therapy using MSCs and Treg cells for preventing aGVHD.</p></div

eGFP detection in aGVHD target organs.

<p>(A) Immunofluorescence staining with confocal examination illustrates eGFP⁺ cells in recipients. eGFP⁺ cells were counted in the liver and small intestine (right). (B–C) Expression of eGFP mRNA was examined by real-time PCR in recipient spleen and small intestine on day 7 after BMT (<i>n</i> = 6). Data are shown as the mean ± SEM. The results are representative of three independent experiments. <i>P</i> values were determined using Student’s <i>t</i>-test. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Improvement in aGVHD severity with donor-derived MSCs and Treg cells.

<p>Recipient mice (BALB/c, H-2^d) were irradiated with 800 cGy and injected intravenously (i.v.) with 5×10⁶ BMCs and 5×10⁶ spleen cells from donor mice (C57BL/6, H-2^b) and donor-derived MSCs (1×10⁶) and C57BL/6 background eGFP mice-derived Treg cells (2×10⁶) on days 0 and 4, and mice were evaluated at different time points after BMT (<i>n</i> = 10). (A) Increase in survival rate in aGVHD mice model after co-administration of MSCs and Treg cells in allogeneic transplantation. (B–C) The degree of clinical aGVHD was assessed weekly using a scoring system that summed changes in five clinical parameters: weight loss, posture, activity, fur texture, and skin integrity. (D) Histological scores were evaluated in aGVHD target organs: skin (200×), liver (200×), and small intestine (200×). Data are shown as the mean ± SEM. The results are representative of three independent experiments. <i>P</i> values were determined using Student’s <i>t</i>-test. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Characterization of donor-derived MSCs and Treg cells.

<p>(A) Culture-expanded MSCs were distinguishable from hematopoietic cells by being positive for Sca-1, CD44, and CD29, but were negative for the cell surface markers c-kit, CD11b, CD34, CD106, CD45, and CD31. (B) The purity of Treg cells was greater than 96% by flow cytometry. Treg cells were analyzed by FACS for intracellular Foxp3, CTLA-4, PD-1, GITR, ICAM-1, and ICOS, and surface expression of the indicated cellular markers CD44, CD62L, and CD103 in the gated CD4⁺ T cell population.</p

Reduction of IL-17 after co-administration of donor-derived MSCs and Treg cells.

<p>Recipient mice were evaluated at different time points after BMT. (A) The percentage of CD4⁺ IL-17⁺ cells and CD4⁺ IL-6⁺ cells were identified by flow cytometry at 12 days after BMT (<i>n</i> = 6). (B) Expression of p-STAT3 was significantly downregulated in the combined cell therapy group compared to the single groups, as determined by Western blotting. (C) RORγ-t mRNA levels were significantly decreased in the combined cell therapy group versus the Treg cells single group using real-time PCR. The percentage of IL-17⁺ cells detected in the (D) spleen and (E) blood by flow cytometry at 7, 14, and 21 days after BMT. CD4⁺ IL-17⁺ eGFP⁺ cells (white bar) were detected as a small proportion of CD4⁺ IL-17⁺cells. Data are shown as means ± SEM. The results are representative of three independent experiments. <i>P</i> values were determined by Student’s <i>t</i>-test. * <i>p</i> < 0.05.</p

Identification of the origin of CD4⁺ Foxp3⁺ Treg cells that increased in recipients using <i>ex vivo</i>-expanded Treg cells from eGFP⁺ (H-2^b) mice.

<p>Recipients (BALB/c, H-2^d) were administered donor-derived MSCs (1×10⁶) and eGFP+ Treg cells (2×10⁶) on days 0 and 4, and were sacrificed on days 7, 14, and 21 after BMT (<i>n</i> = 6). (A) The gated CD4⁺ CD25⁺ populations of H-2K^b+ Foxp3⁺ cells were examined at 12 days after BMT by flow cytometry. (B–C) Expression of SOCS3 and Foxp3 was significantly upregulated in the combined cell therapy group at 12 days after BMT by real-time PCR. Animals in the GVHD and MSCs groups all died within 20 days after BMT. CD4⁺ Foxp3⁺ Treg cells were measured in recipients’ (D) spleen and (E) blood by flow cytometry at days 7, 14, and 21 after BMT. H-2b cells indicate endogenous donor-origin CD4⁺ Foxp3⁺ Treg cells (black bar), and eGFP⁺ cells indicate adoptive transferred Treg cells (white bar). Host-origin (H-2d) cells were not detected. Data are shown as means ± SEM. The results are representative of three independent experiments. <i>P</i> values were determined using a Student’s <i>t</i>-test. * <i>p</i> < 0.05, ** <i>p</i> < 0.01.</p

Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation

<div><p>Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.</p></div

At weeks 2, 4, 8, and 12 after BMT, leukocytes isolated from mesenterial lymph nodes were stained with anti-TCRβ,-CD4,-CD8,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>(A) The numbers in the dot plot show the percentage of B6-originated TCRβ⁺ T cells among the total leukocytes. (B) The bars show the ratio and absolute numbers of B6-orginated TCRβ⁺ T cells (■) and Balb/c-originated TCRβ⁺ T cells (□). (C) The bars show the ratio and absolute numbers of B6(Balb/c)-originated TCRβ⁺ CD4⁺ and TCRβ⁺ CD8⁺ T cells. Data are shown as the mean ± SD. p values compared to donor-derived cells two weeks after BMT. ***, p ≤ 0.0001 or p ≤ 0.001; **, p ≤ 0.01; and *, p ≤ 0.05.</p

At one week after BMT, leukocytes isolated from the indicated organs were stained with anti-TCRβ,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4).

<p>(A) The numbers in the dot plot show the percentage of B6-originated cells among the total leukocytes. (B) The bars show the ratio of B6- (■) and Balb/c-originated cells (□). Data are shown as the mean ± SD.</p

At weeks 2, 4, 8, and 12 after BMT, leukocytes isolated from the thymus (A) and liver (B) were stained with αGalCer-loaded CD1d dimer or anti-TCRβ,-K^b, and-D^d monoclonal antibodies (<i>n</i> = 4 at each time point).

<p>The bars show the ratio and absolute numbers of B6-orginated NKT cells (■) and Balb/c-originated NKT cells (□). Data are shown as the mean ± SD. p values compared to donor-derived cells one week after BMT. *, p ≤ 0.05.</p