Facilitating Engraftment After MHC-Matched, Allogeneic BMT by IL-2 / Anti IL-2 Complex Treatment Requires Targeting CD25 On, and Activation in Situ of, Residual CD4 Tregs.
Authors: Shatry Alwi , Gorin Michael A , Chirinos Jackelin G , Levy Robert B. 
Published: October 2019

Abstract 66

We recently demonstrated that administration of IL-2 complexed to the anti IL-2 antibody JES6-A12 (IAC) induced stable chimerism and engraftment of donor HSCT (BBMT 15:785, 2009). Based on suppression of anti-donor MiHA-specific host T cells, it was concluded that IAC administration enhanced chimerism by suppression of HVG. We proposed that in situ manipulation of host Tregs was crucial to facilitating engraftment and establishing tolerance in this model and hypothesized that the enhanced chimerism induced by this strategy was a direct result of host Treg activation, expansion and function following engagement of the IL-2 receptor CD25. To directly test this hypothesis, B6 CD4−/− (H-2b, Ly9.1−) mice were infused with highly enriched CD4 cells from B6-WT or B6-IL-2Rβ−/− mice deficient in CD25 expression and 4 days later conditioned with 5.5 Gy TBI. One day later, these mice were transplanted with MHC-matched, MiHA-mismatched 4 × 106 BALB.B (H-2b, Ly9.1+) TCD-BM. At days +3 and +5, all recipients were administered IAC and subsequently assessed for peripheral donor chimerism. By 2 weeks post-HCT, untreated control mice had increased circulating levels of CD8TETRAMER+ T cells (representing specific host anti-donor H60 MiHA reactive T cells) vs. IAC-treated recipients. Three months post-HCT, CD4−/− recipients of WT but not IL-2Rβ−/− CD4 cells were chimeric as evidenced by high levels of circulating donor cells (60% vs. <1%). These findings demonstrate that IAC effects require host CD25+ Treg cells and we propose that facilitation of engraftment by this strategy was a direct result of Treg cell activation and expansion following engagement of IAC with CD25. To assess Treg activation, we examined these cells in our standard BALB.B à B6 HSCT model. Tregs isolated 7 days post-HCT from IAC-treated but not untreated recipients expressed readily demonstrable levels of pStat-5a expression (∼2X increase in IAC-treated vs. PBS controls). Moreover, culture of the former Tregs in the presence of rmIL-2 illustrated their heightened sensitivity to activation by this cytokine as virtually all Tregs from IAC-treated animals exhibited high levels of pStat-5a expression (3.2 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.6) compared to control mice (0.4 × 106 FoxP3+ P-Stat5a+ cells ± SE 0.5). Following this activation, increased numbers of host CD4+ FoxP3+ Tregs were readily identified in the PB and spleen of IAC vs. PBS-treated recipients. We next directly examined functional capacity of residual host Tregs exposed to IAC post-5.5Gy TBI and-transplant. Eight days post-HSCT, host Tregs (0 – 25 × 103/well) were isolated and highly enriched populations assessed for suppression of TCONV in anti-CD3 activation assays. Tregs from IAC-treated recipients efficiently mediated suppression at least equivalent to that by normal, untreated Tregs in these assays. Additionally, these IAC treated residual B6 Tregs also effectively inhibited allogeneic MLR responses by B6 CD4+ CD25− responder cells. Therefore, the capacity of host Tregs to respond to IAC activation signals and suppress T cell activation remained intact following conditioning and HSCT. We conclude that host Treg cell activation/expansion is central to the suppression of host effector cell responses to donor hematopoietic antigens leading to the inhibition of HVG following IAC administration resulting in enhanced engraftment.

Disclosures:

No relevant conflicts of interest to declare.