HLA Class Ia and Ib Polyreactive Anti-HLA-E IgG2a Monoclonal Antibodies (TFL-006 and TFL-007) Suppress Anti-HLA IgG Production by CD19+ B Cells and Proliferation of CD4+ T Cells While Upregulating Tregs

Figure 4

The human hybridoma cell line HML16 was generated from the resting B cells by EBV transformation; then the clone was fused with the murine, nonproducing myeloma cell line P3X63-Ag8.653 (ATCC® CRL 1580™). HML16 produced high MFI allo-Abs against B0702, B8101, B6701, and B4201 and low MFI allo-Abs against B2708, B2705, B5501, B5601, and B8201. Figures are restricted to two of allo-HLA-I Abs, namely, B0702 (a and b) and B8101 (c and d). HML16 cells (cultured in RPMI-1640 + 20% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate + L-glutamine-pen-strep solution + 50 μM 2-ME) were seeded at 1000/100 μl/well in a Falcon 96-well flat-plate and divided into 3 treatment groups: medium control, mouse IgG control (100 and 50 μg/ml), and TFL-007a. Medium control was compared with treatment by IVIg preparations (GamaSTAN and Gamunex-C; three subgroups for each), or four subgroups for TFL-007a were established for different doses except medium control. Six or more repetitions were performed with each subgroup (sample size is shown in (b) and (d)). The three subgroups of GamaSTAN-IVIg were at dilutions 1/10 (15 mg/ml), 1/20 (7.5 mg/ml), and 1/40 (3.75 mg/ml); the subgroups of Gamunex-C were at 1/10 (10 mg/ml), 1/20 (5 mg/ml), and 1/40 (2.5 mg/ml); and those of mAb TFL-007a were at 1/10 (100 μg/ml), 1/20 (50 μg/ml), 1/40 (25 μg/ml), and 1/80 (12.5 μg/ml). Twenty μl of culture supernatant from each well was analyzed for allo-HLA Abs at hours 0 and 72. The anti-HLA-E mAb TFL-007a (stock 627 μg/ml) at different concentrations (62.7, 32.35, 16.17, and 8.9 μg/ml) but not IVIg preparations (GamaStan (15, 7.5, and 3.75 mg/ml) and Gammunex (10, 5 and 2.5 mg/ml)) inhibited the production of anti-HLA Abs against B0702 and B8102 produced by EBV-immortalized B cell line, HML-16. Mean and SD of the hexaplicate samples are presented. The paired sample two-tailed t-test was carried out. Two-tailed values are provided in the figure [49].

(a)

(b)

(c)

(d)