Murine CD4+CD25+ Treg cells derived from donor B6 mice were generated with autologous specificity (H-2Ab) or direct allospecificity for MHC Class II H-2Ad alloantigens using an expansion protocol, or indirect allospecificity for MHC Class I H-2Kd allopeptide presented by autologous-MHC H-2Ab using a retroviral TCR gene transduction method we have previously established [27]. Each Treg-cell line maintained equivalent levels of CD62L, CD25 and FoxP3 expression following in vitro expansion (Fig. 2A). The suppressive capacity and antigen specificity of each Treg-cell line was assessed by their ability to suppress

polyclonal or antigen-specific T-cell proliferation in vitro, which was greater than 90% suppression when applied at a ratio of 1:1 of Treg to Teff cells (Fig. 2B–H). Co-culture of Treg cells with autospecificity (auto-Treg), were able to potently suppress autologous B6 CD4+ T-cell responses to a polyclonal stimulus induced by autologous Lorlatinib in vivo B6 APC combined with a TCR stimulatory antibody (Fig. 2B). HDAC inhibitor The suppressive function of Treg cells with indirect allospecificity (indirect Treg cells) was assessed using CD4+ T cells with the same indirect allospecificity derived from TCR75 transgenic mice [32]. Co-culture of indirect Treg cells

with TCR75 was able to efficiently inhibit T-cell proliferation in response to indirect presentation of H-2Kd peptide by autologous B6 APCs (Fig. 2C), and also in response to stimulation with CB6F1 APCs, which constitutively present H-2Kd alloantigen via the indirect pathway (Fig. 2D). To study the suppressive function of Treg cells with direct specificity for H-2Ad (direct Treg cells), autologous B6 CD4+ T cells were stimulated with BALB/c and also CB6F1 APCs (Fig. 2E). As expected, Anacetrapib direct Treg cells were able to effectively suppress a proliferative response against both stimuli. The capacity of each Treg-cell line to mediate

linked suppression was also examined in vitro (Fig. 2F–G) using CD4+ responder T cells isolated from the OT-II TCR transgenic mouse, with specificity for ovalbumin peptide 323–339 (OVAp) presented by H-2Ab. As anticipated, while auto-Treg-cell mediated linked suppression of an OT-II T-cell response to B6 APCs pulsed with OVAp, direct Treg cells were unable to demonstrate any suppressive effect in the absence of their ligand (Fig. 2F), while indirect Treg cells were demonstrated potent dose-dependent suppression of OT-II proliferation only in the presence of H-2Kd peptide (Fig. 2G). Of particular importance, all Treg-cell lines maintained an equivalent capacity to suppress a polyclonal T-cell response in vitro (Fig. 2H). These results demonstrate that the Treg-cell lines were highly specific for their respective auto- or alloantigens, which also described their ability to effect linked suppression. Murine donor-derived Treg-cell lines (4 × 106 cells) were co-administered with donor CD8−CD25− B6 splenocytes (7 × 107) at the time of cGVHD induction.