Adoptive Therapy with Cord Blood Regulatory T Cells Can Treat Graft Vs Host Disease

Background

Adoptive therapy with regulatory T cells (Tregs) has already been established as a promising strategy for prevention of graft vs. host disease (GVHD) in clinical trials. Our group at MD Anderson Cancer Center has previously shown that a significantly lower dose of cord blood (CB) Tregs as compared to conventional T cells (Tcon) in the donor graft is able to prevent GVHD while preserving the graft vs. leukemia (GVL) effect. Therefore, we now examined the efficacy of using CB Tregs in the treatment of GVHD.

Method: Xenogenic GVHD mouse model was established using NOD/SCID/IL2Rgnull (NSG) mice were sublethally irradiated at 300 cGy followed by injection of 1x10⁷ peripheral blood (PB) mononuclear cells on day 0, as previously described. Ex vivo expanded CB Tregs were injected on day -1 (for prophylaxis) or at different days post PBMC injection for treatment. Mice were serially examined for appearance, weight, posture, GVHD score and survival. Serial peripheral blood sampling for flow cytometry and serum cytokine analysis. CB Tregs were also analyzed by flow cytometry. In order to understand the impact of the routine immunosuppressive agents on the function of CB Tregs, we incubated the CB Tregs in culture with cyclosporine (200ng/ml) or sirolimus (20 ng/ml) from day 8 to day 14. Cells were harvested on day 14 and analyzed by flow cytometry and CellTrace Violet suppression assay.

Result: A single dose of 1x10⁷ CB Tregs injected at day +7 did not result in a survival difference compared to the control arm (data not shown). Therefore, we froze multiple aliquots of expanded CB Tregs to be injected at different intervals post-transplant. Thawed CB Tregs showed stable phenotype of CD4⁺25⁺127^lo: 94.7%; intracellular Helios⁺: 98.5% and intracellular FOXP3⁺: 99.4% and were able to suppress 87% of the proliferating conventional T-cells (Tcons). In order to compare the efficacy of the CB Tregs for GVHD treatment, we set up 3 arms: i) Control: PBMC alone; ii) Prophylaxis: 1x10⁷ CB Tregs injected on day -1 and iii) Treatment: 1x10⁷ CB Tregs injected on day +4, +7, +18 and +25. The mice in the prophylaxis and treatment arm retained their weight as compared to the control arm (p<0.003) (Fig 1A) and showed significantly better overall survival (P=0.01) (Fig 1B), which correlated with the decrease in circulating inflammatory cytokines including TNFa (Fig 1C). Since the standard of care for acute GVHD still remains high dose steroids, we evaluated the effect of continued exposure to steroids (prednisone-100ug/ml) for a period of 96 hours on the viability of CB Tregs. When compared to CB Tcons, 90.3% CB Tregs remained alive and viable compared to 64.7% of Tcons (Fig 1D). No differences were observed in the intracellular expression of FOXP3 or Helios in the control vs. cyclosporine or sirolimus exposed cells (Fig 1E). Similarly, no significant impact was observed on their suppressor function (Fig 1F).

Conclusions: Multiple injections with CB Tregs can effectively treat GVHD. Combination therapy of CB Tregs with the commonly used GVHD treatments can be explored.

Figure 1

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Figure 1

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