Adipose-tissue derived mesenchymal stem cells protect against bone marrow GvHD

Graft-versus-host disease (GvHD) is a common complication of allogeneic hematopoietic stem cell transplant (allo-HSCT) which is used for the treatment of many hematological malignancies. First- and second-line treatment options, and their associated outcomes are shown in Table 1.

Table 1. Treatment options for acute GvHD (aGvHD)

aGvHD, acute graft-versus-host disease; ATG, antithymocyte globulin; EBMT, European Society for Blood and Bone Marrow Transplantation; European Leukemia Net; JAK, Janus kinase; MSCs, mesenchymal cells
First-line treatment1,2	Recommended second-line treatment 1,2
First-line treatment for aGvHD is corticosteroids; 60–70% of patients respond to this treatment Systemic treatment ≥ grade II: 2 mg/kg/day methylprednisolone or 2-2.5 mg/kg/day prednisone equivalent	·       Alemtuzumab ·       Alpha1-antitrypsin ·       Basiliximab ·       Cellular therapies, such as MSCs, and regulatory T-cells ·       Daclizumab ·       Extracorporeal photopheresis ·       Fecal microbiota transplantation ·       JAK inhibitors ·       Methotrexate ·       Mycophenolate mofetil ·       Pentostatin ·       Rabbit ATG ·       Sirolimus ·       Vedolizumab  EBMT ELN recommendations for the treatment of aGvHD
Those who do not respond have a 10–30% chance of long-term survival	Due to the higher risk of infectious complications, immunosuppression-mediated toxicity and incomplete remission of GvHD, these patients still have poor prognoses

aGvHD mainly targets the skin, liver and gastrointestinal (GI) tract and is also associated with cytopenia and bone marrow suppression. This can cause life-threatening infections and therefore there is an unmet need to prevent this from occurring.

MSCs are multipotent cells with specific surface antigen expression and an ability to suppress immunological responses, support hematopoiesis and repair tissues. MSCs are found in many tissues including; bone marrow, peripheral blood, umbilical cord blood and adipose tissue. The potential of MSCs to prevent and treat GvHD is a topic of discussion in the field. A recent systematic literature review, covered by the GvHD Hub in July 2019, confirmed the efficacy of MSC treatment to improve complete response (CR) rates and overall survival for chronic GvHD (cGvHD) with a trend towards lower risk of aGVHD.³

Currently, there are no reports in the GvHD field focusing on bone marrow aplasia related with GvHD. Most murine aGvHD models use an irradiation regimen prior to transplant which destroys the bone marrow, making it difficult to study in current models. In order to study bone marrow aplasia related to GvHD, Yukiko Nishi and Akikazu Murakami from the University of the Ryukyus, Okinawa, JP, and colleagues, conducted a study using a murine model with a major histocompatibility complex (MHC)-homozygous donor to heterozygous-recipient, without irradiation or pretreatment with chemotherapy. Their aim was to study the prophylactic potential of adipose-derived-MSCs (AD-MSCs) in GvHD, with a focus on bone marrow aplasia related to aGvHD.

AD-MSCs can be collected via a minimally invasive liposuction procedure, offering a unique advantage over other MSCs. AD-MSCs are also equipotent with a higher immunoregulatory capacity compared to bone marrow-derived MSCs. This model correlates to transfusion-related GvHD, which is different to clinically-occurring GvHD, but allows analysis of AD-MSCs on bone marrow aplasia caused by GvHD.

Study design

• Induction of GvHD:
  • Single cell suspension spleen cells (1 x 10⁸) obtained from C57BL/6 (B6-Ly-5.1, CD45.1) donor mice
  • Recipient mice, CB6F1 (CD45.2), were intravenously (IV) injected with the splenocytes via the tail vein without receiving irradiation or chemotherapy
• GvHD was assessed by:
  • Flow cytometry analysis of peripheral blood cells (T-cell subsets) on day 14
  • Histopathologic analysis of target organs
  • GvHD score to assess severity of GvHD
    • Criteria: percentage of weight loss, posture, activity, fur texture and skin integrity (score 0–2 per criteria, to a maximum of 10)
• Five groups were used (Table 2)
  • The control group were age- and sex-matched F1 mice who received phosphate buffer saline (PBS) alone
  • A positive control group were prepared by administering CTLA 4-Ig
• Statistical significance was observed at p values < 0.05

Table 2. Treatment received by each of the CB6F1 subgroups

Group	1 x 108 CD57BL/6 splenocytes administered?	Additional administration
AD-MSC, adipose-derived mesenchymal stem cell; GvHD, graft-versus-host disease
GvHD	Yes	None
AD-MSC group 1	Yes	1 x 106 AD-MSCs via IV injection on days -1 to 13
AD-MSC group 2	Yes	1 x 106 AD-MSCs via IV injection on days -1 to 8
CTLA4-Ig	Yes	CTL4A-Ig*
Control	No	PBS on days -1 to 13

* 200 µg of CTLA 4-Ig IV at the day of GvHD induction and on day 1, and 100 µg administered as an intraperitoneal (IP) injection on days 2–4

Results

AD-MSC characterization

• Highly expressed markers for authentic mesenchymal stem cells
  • Sca-I, CD29, CD90 and CD105
• Did not express hematopoietic and endothelial markers (e.g. CD31 and CD45)
• Were multi-potent: differentiated into adipocytes, osteocytes and chondrocytes

AD-MSC protection against GvHD

• AD-MSC group 1 had a significantly (p< 0.001) lower percentage of donor CD8⁺ and CD4⁺ T-cells compared to the GvHD group
• AD-MSC group 1 had a significantly (p< 0.001) lower GvHD score compared to the GvHD group
• The AD-MSC mice had a significantly (p< 0.01) lower percentage of activated T-cells (CD44^highCD62L^lowCD8⁺) compared to the GvHD group
  • Indicating AD-MSCs have a suppressive effect on activation and expansion of donor T-cells

AD-MSC protection of organ function

• Liver and bone marrow analysis on day 21:
  • AD-MSC group: infiltration of inflammatory cells was reduced dramatically and suppression was dependent on number of AD-MSCs administered
• Using ImageJ software, bone marrow aplasia was evaluated:
  • Number of bone marrow cells was higher in AD-MSC group 1 compared to the GvHD group
• Hematopoietic colony forming cell assay
  • Number of colony- forming cells from the bone marrow was significantly higher in AD-MSC group 1 compared to the GvHD group

Potential effect of AD-MSC on cell counts in peripheral blood

• White blood cell (WBC), platelet and neutrophil counts were significantly higher (p< 0.05) in AD-MSC group 1 compared to GvHD group

Limitations

• Mouse model was not representative of the clinical setting due to the lack of pre-conditioning chemotherapy that was necessary to evaluate the effect of AD-MSCs on bone marrow aplasia
• Compared to previous studies, high-doses of AD-MSCs were used
• Not possible to confirm that the observed benefit with AD-MSC on bone marrow and peripheral blood was not due to an indirect suppressive effect on global GvHD

Conclusion

This is the first study to report results that AD-MSCs are effective against bone marrow aplasia in aGvHD. Administering MHC-mismatched AD-MSCs inhibited the proliferation of donor CD4⁺ and CD8⁺ T-cells and increased peripheral blood cell counts indicating the bone marrow of the AD-MSC group contained more hematopoietic stem cells than the GvHD group.

The results support the rationale of using AD-MSCs for ameliorating bone marrow suppression and susceptibility to infections after allo-HSCT in humans.