Introduction: In humans, CD4⁺CD45RA^-Foxp3^hi 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⁺CD25^hiFoxp3^hi Treg in non-human primates. Here, we examined whether GM-CSF/G-CSF administration enhances the incidence of CD4⁺CD45RA^-Foxp3^hi 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^-Foxp3^hi Treg and CD4⁺CD45RA^-Foxp3^lo 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^-CD25^lo and CD4⁺CD45RA^-CD25^hi 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⁺CD25^hiFoxp3^hi and CD4⁺CD45RA^-Foxp3^hi 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⁺CD25^hiFoxp3^hi and CD4⁺CD45RA^-Foxp3^hi Treg populations in peripheral blood. After combined G-CSF/GM-CSF administration, the incidences of CD4⁺CD45RA^-Foxp3^hi Treg increased significantly in LP (p<0.01), compared to peripheral blood before treatment. Furthermore, the ratio of CD4⁺CD45RA^-Foxp3^hi to CD4⁺CD45RA^-Foxp3^lo cells increased significantly (p<0.05). In LP, The CD4⁺CD45RA^-CD25^hi population expressed significantly higher levels of Foxp3, CTLA4 and Helios, compared to the CD4⁺CD45RA^-CD25^lo population (p<0.05). In 5 animals, LP flow-sorted CD4⁺CD45RA^-CD25^hi cells exhibited higher suppressive capacity of T cell proliferation in response to anti-CD2/CD3/CD28 stimulation, compared to the CD4⁺CD45RA^-CD25^hi population.
Conclusions: G-CSF/GM-CSF administration enhances the incidences of highly suppressive CD4⁺CD45RA^-Foxp3^hi T cells in LP of mobilized rhesus monkeys. These observations further support the therapeutic potential of Treg-enriched LP from mobilized individuals.