Introduction: Allogeneic liver transplantation (LTx) outcomes have improved significantly, due in part to more efficacious immunosuppressive therapy (IST). However, pharmacologic IST utilization is accompanied by substantial rates of cardiovascular and renal damage, diabetes, infection, and therapy-related malignancies. Therefore, alternatives producing similarly low rates of acute and/or chronic graft rejection while minimizing toxicities of chronic pharmacologic IST are desirable. 

The potential of adoptive cell therapy with regulatory T cells (Treg) to promote transplant tolerance is under active exploration. However, the impact of specific transplant settings and protocols on Treg manufacturing is not well-delineated.
Here, we evaluated pre-and post-LTx peripheral blood mononuclear cells (PBMC), from baseline characteristics through ex vivo mixed lymphocyte reaction (MLR) using costimulatory blockade (CSB) (CSB-MLR) manufacturing conditions, to assess feasibility and then improve the outcome of CSB-MLR as a Treg production strategy for LTx.

Methods: We compared the use of PBMCs from patients with end-stage of liver failure before or after LTx to the use of PBMC from healthy controls (HC) to determine their suitability for Treg, defined by a CD4+CD25hiCD127loFoxp3+ phenotype, manufacture using ex vivo CSB with belatacept; 2nd generation CTLA4-Ig fusion protein, during an MLR. CSB-MLR supports Treg expansion using PBMC responders from patients and HC or HLA-mismatched healthy donor PBMC stimulators. Tregs were isolated from HC and post-LTx responder PBMC at baseline (unstimulated) or derived from primary CSB-MLRs. Treg-mediated suppression was assessed by adding purified CSB-Tregs to a fresh in vitro MLR.

Results: Treg frequency in CD4+ T-cell was significantly greater in pre- (median 4.47) and lower in post-LTx (median 2.29) samples than in HC (median 3.09). Despite post-LTx IST, alloproliferation of post-LTx PBMC responders in MLRs without CSB was very similar to HC (p = 7508). The addition of belatacept reduced alloproliferation in both HC and post-LTx responders with significantly greater inhibition using post-LTx (median 82.7%) than HC (median 71.0%) PBMC. Despite liver failure or IST, the capacity for Treg expansion during the manufacturing process was preserved (Figure 1, 2). These experiments did not identify performance issues or reasons to disqualify use of post-transplant PBMC, the favored protocol design. However, as Treg input correlated with output, significant CD4-lymphopenia in both pre- and post-transplant patients did limit Treg yield.

Conclusion: These studies provide support for use of post-LTx PBMC as a manufacturing substrate to generate Treg via CSB-MLR. This Treg expansion strategy works in a liver transplant context, this preclinical study illustrates how characterizing cellular input populations can both inform and respond to Treg manufacturing conditions and clinical trial design.