Blockade of TNFα to Improve Human CD34+ Cell Repopulating Activity in Allogeneic Stem Cell Transplantation

Early release of TNFα after hematopoietic stem cell transplantation (HSCT) correlates with development of acute graft-vs.-host disease (GVHD). Here we tested the effect of TNFα and alloreactive T cells on early hematopoietic HSC genotype and function. Addition of TNFα (10 ng/ml) in liquid cultures with CD34+ cells for 6-72 h resulted in the downregulation of genes associated with stem cell activity, such as DNMT3A, DNMT3B, TET1, TET2, SOX2, NANOG, and OCT4, whereas no significant effect was observed on DNMT1 and GATA2 expression. These findings were reversed by using an anti-TNFα antibody. Similar gene downregulation was observed when CD34+ cells were co-cultured with alloreactive T cells CD34+ cells for 48-72 h, and this effect was partially prevented by rapamycin and an anti-TNFα antibody. CD34+ cells pre-incubated with TNFα for 48 h and transplanted in irradiated NOD-SCID ɤ^null (NSG) mice showed a reduced myeloid engraftment compared to control mice. By using a xenograft model recently developed in our lab, we co-transplanted CD34+ cells and allogeneic T lymphocytes at 1:0.1 ratio in one group that also received etanercept (TNFα inhibitor) at 100 μg intra-peritoneum (i.p.) on days -1,+1,+3,+5 post-HSCT, and in the control group. At 6 weeks post-transplant, mice that received etanercept had a significantly higher number of marrow huCD45+CD34+CD38- early stem cells (p = 0.03) and a reduced number of huCD45+CD3+ splenic T cells (p = 0.04) compared to controls. The repopulating activity of marrow cells from mice treated with etanercept vs. controls was tested in secondary transplants. Although the overall engraftment was similar in the two groups, CD34+ cells isolated from recipients of marrow from the etanercept group showed a significantly greater expression of stem cell-associated genes and a higher number of CD45+CD34+CD38- cells than in controls (p = 0.03). Our findings suggest that early TNFα increase post-transplant can affect long-term stem cell engraftment, and that blockade of TNFα early after transplant may limit a cytokine-mediated suppressive effect on repopulating stem cell function.