Functional Comparison of Freshly Isolated and Ex-Vivo Expanded CD4+CD25+Foxp3+ Regulatory T Cells in Suppressing Murine Acute GvHD

Graft versus host disease (GVHD) is a major complication of hematopoietic stem cell transplantation resulting from donor T cell reactivity against host tissue antigens. CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) are known to be important in maintaining self tolerance and preventing autoimmunity. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown that donor Treg are able to suppress GVHD induced by donor allogeneic T cells and dramatically improve survival. Treg are rare and suppression of GVHD requires adequate numbers of Treg in relation to the number of conventional T cells (Tcon). To overcome this problem, expansion of Treg has been performed, however there has not been a head to head comparison of the function of expanded vs fresh Treg. Highly purified CD4⁺CD25⁺Foxp3⁺ T cells (>98% purity) were expanded using anti-CD3/anti-CD28 dynabeads and 1000 U/ml IL-2. Under these conditions, after five days Treg expanded up to 13 fold while maintaining high Foxp3 expression levels (85–90%). Longer expansion periods result in more T cell expansion but an overgrowth of Foxp3 negative T cells. In a mixed lymphocyte reaction assay, the ex-vivo expanded Treg efficiently suppressed the proliferation of alloreactive T cells. The expanded Treg were evaluated in an in vivo acute GVHD mouse model in direct comparison with freshly isolated Treg using a novel bioluminescent imaging assay that allowed for assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight gain, survival and GVHD score. Initial experiments show that, similar to freshly isolated Treg, the ex-vivo expanded Treg suppress GVHD symptoms and improve survival, although a greater number of expanded Treg were required comparable to freshly isolated Treg. The mean GVHD score for the Tcon alone group was 5.8±1.02. Fresh Treg added at 1:1 ratio decreased the GVHD score to 0.75±0.25 (p=0.0036). Ex-vivo expanded Treg demonstrated a dose-dependent decrease in GVHD score, although four times more expanded Treg were needed to obtain a similar reduction in GVHD score (0.50±0.5, p=0.0036). This observed difference in potency was not due to the ex-vivo expanded Treg being short-lived when infused in mice. Bioluminescence imaging of luciferase positive (luc⁺) cultured Treg showed the same in vivo persistence as freshly isolated Treg. The ability to expand ex-vivo generated Treg is greater than the difference in potency, making ex-vivo expanded Treg potentially a viable option for treatment of GVHD, however, increased ratios of Treg:Tcon are likely to be required.