*Purpose: Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) are known to mobilize immune cells to the peripheral blood. We evaluated regulatory T cells (Treg) percentages, phenotype and function in leukapheresis products obtained from G-CSF/GM-CSF-treated monkeys.

*Methods: Juvenile rhesus monkeys (n=6; 5-7kg) were treated 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. Absolute numbers of lymphocytes in the peripheral blood were evaluated before and after treatment. Leukapheresis products were evaluated for CD4⁺CD25^hiFoxp3^hi Treg percentages and absolute numbers. Pre-(peripheral blood) and post-(leukapheresis product) treatment CD4⁺CD25⁺ Treg were isolated, followed by polyclonal expansion up to 12 days only. Pre-treatment, post-treatment, and expanded Treg were evaluated for Treg-specific markers, chemokine receptors, transcription factors and suppressive function.

*Results: Following G-CSF/GM-CSF administration, peripheral blood lymphocyte numbers increased significantly (p<0.05). Notably, the percentage CD4⁺CD25^hiFoxp3^hi Treg increased significantly from 2.6% to 5% (p<0.01). In leukapheresis products, the mean absolute number of Treg was 26x10⁶ (4×10⁶/kg). No significant differences in the expression of Foxp3, CTLA4, CD27, and chemokine receptors (CCR4/CCR7/CXCR3) by Treg were observed after treatment. However, Helios expression was significantly increased (p<0.05). While no significant difference was observed in the expression of the transcription factors T-bet and RORγt by Treg, GATA3 expression was significantly decreased (p<0.05). After polyclonal stimulation, the expansion capacity of pre-treatment Treg was 4-26 fold on day 6 and 380-660 fold on day 12, while the expansion capacity of post-treatment Treg was 6-26 fold on day 6 and 311-314 fold on day 12. Both pre- and post-treatment expanded polyclonal Treg exhibited comparable phenotype and efficiently suppressed allogeneic T cell proliferation in response to αCD2/CD3/CD28 stimulation.

*Conclusions: n nonhuman primates, G-CSF/GM-CSF administration augments the percentages of peripheral blood Treg with preserved phenotypic signature, expansion capacity and suppressive function. Sufficient Treg numbers can be obtained from Treg-enriched leukapheresis products, circumventing long-term ex vivo expansion required for therapeutic Treg infusion.

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