Belatacept

Fresh PBMCs were isolated from healthy donors under IRB approval using CPT tubes and incubated in 24-well flat-bottomed plates in culture medium (R10) consisting of 1640 RPMI medium supplemented with 10% heat-inactivated FBS (Mediatech, Herdon, VA), 1% L-glutamine (200mM), 1% penicillin/streptomycin (100×), 1% Hepes (1M), 1% 2-ME (5mM). Cells were processed unstimulated, stimulated with anti-CD3/CD28 Dynabeads (ThermoFisher) per manufacturer instructions, or stimulated with 3 μg/mL plate-bound functional grade anti-CD3 (OKT3; eBiosciences) with clinically therapeutic concentrations of belatacept (10 μg/mL; Bristol-Myers Squibb, NY) (34 , 35 (link)) for 3 days at 37°C and 5% CO₂, humidified atmosphere. Where indicated, cultures were treated with 10 μg/mL LPS- and azide-free agonistic αTIGIT (A15153G; BioLegend) or mouse IgG2a isotype control (MOPC-173; BioLegend). Where indicated, cultures were washed twice with media and restimulated with 50 ng/mL PMA (Sigma) and 1 μg/mL Ionomycin (Sigma) in the presence of GolgiStop (BD Biosciences) for 4 h at 37°C, 5% CO₂. Where indicated, conventional CD4⁺ or CD8⁺ T cells or CD25⁺ Treg were purified by MACS according to the manufacturer’s instructions (Miltenyi Biotech).

Fresh or frozen PBMC from healthy donors cells were cultured in 96 well flat-bottomed plates in RPMI supplemented with 10% human AB serum (Mediatech, VA) and 2.4 mM L-glutamine. Frozen PBMC were rested overnight before stimulation. Cells were stimulated with either 1 µg/mL (PBMC) or 2 µg/mL (lymph node T cells) functional grade anti-CD3 (OKT3; eBiosciences) in the presence of belatacept (100 µg/mL; Bristol-Myers Squibb, NY) or human IgG1-Fc control (BioXCell, Lebanon, NH), or with anti-CD3/CD28 Dynabeads (Invitrogen) in the presence of 10 µg/mL anti-CTLA-4 (BN13; BioXCell, Lebanon, NH) or mouse IgG1 (BioXCell, Lebanon, NH), as indicated. Cells were washed twice with media and restimulated with PMA/Iono for 4 h as described above. CD4⁺ T cell subsets were defined by the following gating strategy: CD45RA⁺ Th1, CD4⁺CD45RA⁺IFN-γ⁺; CD45RA⁻ Th1, CD4⁺CD45RA⁻ IFN-γ⁺; CD45RA⁻CCR6⁺ Th17, CD4⁺CD45RA⁻CCR6⁺IL-17⁺. The change in frequency of CD4+ populations was calculated as (% Cytokine⁺ Blockade/% Cytokine⁺ IgG)×100 of the indicated population.

Induction immunosuppression for all recipients consisted of a single dose of anti-thymocyte globulin (ATG) (10 mg/kg) (Genzyme, Cambridge, MA, USA) on Day −2, as well as Cobra Venom Factor (CVF) (100 ug/kg) (Complement Technology, Tyler, TX, USA) for complement factor (CH50) depletion on Day −1. Maintenance immunosuppression consisted of a continuous tacrolimus (FK-506) infusion (0.20 mg/kg/day; with target serum levels of 10–20 ng/ml) starting on Day −2 and a methylprednisone taper starting on Day 0. Additional maintenance immunosuppression was provided with belatacept for co-stimulation blockade (n = 3) (20 mg/kg on Days −1, 0, 4, 7, 14 and 21, Bristol-Myers Squibb, New York, NY, USA) or anti-CD40 monoclonal antibody (mAb) (n = 1) (20 mg/kg on Days 0 and 5 and 10 mg/kg on Day 7 and weekly thereafter, NHP Reagent Resource, Boston, MA, USA).

Immunosuppression was induced with anti-CD40 mAb (2C10R4, NIH NHP Reagent Resource), sirolimus (Rapamune®, Wyeth), and ATG (Thymoglobulin®, Genzyme) (Table 1). Anti-CD40 mAb (20∼50 mg/kg) was infused i.v. on days -4, 0, 4, 7, 10, 14 of the transplantation, weekly for three months, and biweekly thereafter. Sirolimus was orally administered daily to achieve stable trough levels (3∼8 ng/ml). ATG (5 mg/ml) was administered i.v. on days -3 and -1. Cobra venom factor (CVF) (100 U/kg, Quidel, San Diego, CA) was administered i.v. on day -1 of the transplant to deplete the complement. TNF-α neutralizing mAb, adalimumab (Humira^®, Abbott Laboratories, Queensborough, UK) was administered subcutaneously 2∼3 h before islet infusion at a dose of 5 mg/kg. Belatacept (20 mg/kg, Nulojix^®, Bristol-Myers Squibb Korea, Seoul, Korea) was i.v. administered at days -2, 0, 3, 7, and then weekly. Tacrolimus (Advagraf^®, Astellas Pharma Korea, Seoul, Korea) was orally administered daily from day -3 up to 56 (R92; 56, R015; 22, R087; 25, R008; 15, R131; 18) to achieve stable trough levels (3∼6 ng/ml). The target trough levels were sustained by adjusting dosages of tacrolimus (0.5-2.5 mg/kg/day) and sirolimus (0.08-0.3 mg/kg/day) after confirming serum concentration by liquid chromatography-tandem mass spectrometry.