Monday September 14, 2020 - 23:30 to 00:15
Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) administration enriches for highly suppressive CD4+CD45RA-Foxp3hi cells in leukapheresis products of rhesus monkeys
Kazuki Sasaki1, Yu-Chao Wang1, Lien Lu1, Julia Hughes1, Veronica Vujevich1, Angus W. Thomson1, Mohamed B. Ezzelarab1.
1Department of Surgery, Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States
Introduction: In humans, CD4+CD45RA-Foxp3hi T cells comprise a regulatory T cell (Treg) subset with highly suppressive function. We have previously shown that granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) administration enhances the incidence of peripheral blood CD4+CD25hiFoxp3hi Treg in non-human primates. Here, we examined whether GM-CSF/G-CSF administration enhances the incidence of CD4+CD45RA-Foxp3hi Treg in leukapheresis products (LP) of rhesus monkeys.
Methods: Rhesus monkeys peripheral blood mononuclear cells (PBMC, n=3) were cultured with either GM-CSF (1ng/ml) or G-CSF (1ng/ml). Treg in PBMC were assessed based on their expression of CD45RA, CD25 and Foxp3. The percentages and phenotype of CD4+CD45RA-Foxp3hi Treg and CD4+CD45RA-Foxp3lo populations were evaluated. Naïve rhesus monkeys (n=6; 5-7kg) were treated sequentially with recombinant human GM-CSF (10 mg/kg/day for 4 days), then recombinant human G-CSF (10 mg/kg/day for 4 days), followed by leukapheresis. Similarly, Treg in PBMC, obtained before treatment, and in LP obtained after GM-CSF/GCSF administration were assessed. Additionally, LP CD4+CD45RA-CD25lo and CD4+CD45RA-CD25hi populations were flow-sorted (n=5) and assessed for their suppressive function.
Results: In vitro, G-CSF did not enhanced Treg percentages. Meanwhile, GM-CSF enhanced the percentages of both CD4+CD25hiFoxp3hi and CD4+CD45RA-Foxp3hi Treg populations. In vivo, while G-CSF administration increased lymphocyte numbers, no increase in Treg percentages was observed. On the other hand, GM-CSF administration augmented the percentages of both CD4+CD25hiFoxp3hi and CD4+CD45RA-Foxp3hi Treg populations in peripheral blood. After combined G-CSF/GM-CSF administration, the incidences of CD4+CD45RA-Foxp3hi Treg increased significantly in LP (p<0.01), compared to peripheral blood before treatment. Furthermore, the ratio of CD4+CD45RA-Foxp3hi to CD4+CD45RA-Foxp3lo cells increased significantly (p<0.05). In LP, The CD4+CD45RA-CD25hi population expressed significantly higher levels of Foxp3, CTLA4 and Helios, compared to the CD4+CD45RA-CD25lo population (p<0.05). In 5 animals, LP flow-sorted CD4+CD45RA-CD25hi cells exhibited higher suppressive capacity of T cell proliferation in response to anti-CD2/CD3/CD28 stimulation, compared to the CD4+CD45RA-CD25hi population.
Conclusions: G-CSF/GM-CSF administration enhances the incidences of highly suppressive CD4+CD45RA-Foxp3hi T cells in LP of mobilized rhesus monkeys. These observations further support the therapeutic potential of Treg-enriched LP from mobilized individuals.
NIH grant U19 AI131453..