In Vivo Selection of Mycophenolate Mofetil-Resistant T Cells for Adoptive Immunotherapy.

Following allogeneic solid organ or hematopoietic stem cell transplantation, adoptive transfer of therapeutic T cells may be hindered by the requirement for immune suppressive drugs to prevent rejection or graft-versus-host disease. Mycophenolate mofetil (MMF) is a non-competitive inhibitor of inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), an inducible enzyme that generate guanine nucleotides for DNA and RNA synthesis in T cells. In this study, we have evaluated the potential for gene transfer to T cells of a mutated IMPDH2 that confers >2000-fold resistance to MMF (IMPDH2^R; T333I, S351Y).

Wild type IMPDH2^WT, IMPDH2^R and IMPDH2 with a catalytic site mutation (IMPDH2^CS; C331A) were cloned into SFG retroviral vectors as fusions to eGFP reporter sequences. Murine thymoma (BW 5147) or CD8 T cells were transduced with each vector and their phenotype and function evaluated in the presence or absence of mycophenolic acid (MPA), the active metabolite of MMF.

BW thymoma cells transduced with IMPDH2^R exhibited less apoptosis than cells transduced with IMPDH2^CS in response to MPA (ratio % Annexin V+ MPA: no MPA- IMPDH2^R = 1.2; IMPDH2^CS = 2.9, p=0.01). Cells transduced with IMPDH2^R were also able to overcome the G₁ cell-cycle arrest induced by MPA when compared to control IMPDH2^CS cells (Ratio %cells in S-G2/M phases MPA: no MPA- IMPDH2^R = 1.0; IMPDH2^CS = 0.3, p=0.03). This led to selective enrichment of IMPDH2^R transduced cells in the presence of MPA. At low dose MPA (450nM), IMPDH2^R transduced cells enriched compared to IMPDH2^CS but not IMPDH2^WT (ratio % GFP MPA: no MPA- IMPDH2^R = 1.6 vs IMPDH2^CS = 1.1, p=0.04 and IMPDH2^WT = 1.5 p= 0.14). However, at high dose (4500nM) IMPDH2^R exhibited enhanced enrichment (ratio % GFP MPA: no MPA- IMPDH2^R = 2.8 vs IMPDH2^CS = 1.0 p=0.03 and vs IMPDH2^WT= 1.5 p=0.03).

Gene transfer of IMPDH2^R into murine CD8 T cells also led to selective enrichment compared to controls in the presence of MPA when cultured with proliferation-inducing common gamma-chain cytokines (ratio MPA: no MPA IMPDH2^R = 4.4 vs. IMPDH2^CS = 1.10, p=0.02). Strong selection for IMPDH2^R-tranduced CD8 OT-1 T cells in the presence of MPA was also observed under conditions of antigen-induced proliferation (ratio IMPDH2^R= 7.1, control = 0.8, p=0.002).

To assess in vivo selection, sub-lethally irradiated (2Gy) B6.PL (Thy1.1) mice were injected with a 1:1 mix of OT1 TCR transgenic Thy1.2 CD8 T cells transduced with IMPDH2^R or IMPDH2^CS that could be differentiated by the congenic markers CD45.1 and CD45.2. Transferred cells were stimulated by s.c. injection with cognate peptide (SIINFEKL) in IFA and MMF (200mg/kg/day) was given by daily ip injection. As in vitro, IMPDH2^R-transduced OT1 cells were preferentially selected over IMPDH2^CS-transduced cells following MMF treatment (day 14 ratio IMPDH2^R to IMPDH^CSwas 19.3 vs 1.2 in the absence of MMF).

T cells transduced with IMPDH2^R are resistant to the anti-proliferative and apoptotic effects of MPA in vitro and demonstrate strong selection in vivo compared to controls at therapeutic levels of MMF. These data support the potential of conferring MMF resistance as a strategy to permit the survival of therapeutic T cells in immunosuppressed allograft recipients.

Stauss:Cell Medica: Scientific Advisor Other.