Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

Prophylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro.

CD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4⁺/CD25^high/Foxp3⁺/Helios⁺, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators.

In vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4⁺CD25^highFoxp3⁺/Helios⁺ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28⁺/CD27⁺ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25^high Tregs.

Our results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients.

No relevant conflicts of interest to declare.