Expression of the ULBP Ligands for NKG2D by B-NHL Cells Plays An Important Role in Determining Their Susceptibility to Rituximab-Induced ADCC
Authors: Inagaki Atsushi , Ishida Takashi , Yano Hiroki , Mori Fumiko , Ito Asahi , Ri Masaki , Ding Jianmin , Kusumoto Shigeru , Komatsu Hirokazu , Iida Shinsuke , Ueda Ryuzo 
Published: April 2020

Abstract
Purpose: ADCC is a major antitumor mechanism for the action of therapeutic monoclonal antibodies (mAbs) such as rituximab, trastuzumab and cetuximab. Therefore, a better understanding of ADCC will allow the development of novel, more effective treatment strategies, and may help overcome the resistance which can develop against the effects of the therapeutic mAbs. However, the tumor-associated factors which determine susceptibility to rituximab-induced ADCC have not been identified. In the present study, we focused on this issue, especially focused on the molecules expressed by the tumor cells that interact with NK cells, such as NKG2D ligands, because of the importance of NK cells for rituximab-induced ADCC. The aim of this study was to identify tumor associated factors which determine susceptibility to rituximab-induced ADCC.
Experimental Design: 30 different CD20+ non-Hodgkin lymphoma (B-NHL) cell lines were phenotyped for characteristics of cell surface protein: expression levels of CD20, MHC class I, NKG2D ligands (ULBP1-3, MICA, and MICB), CD48 (2B4 ligands), HLA-G, cathepsin B, and complement inhibitors (CD46, CD55, and CD59), and the influence thereof on susceptibility to rituximab-induced ADCC was established.
Result: The degree of rituximab-induced ADCC were correlate with the expression levels of CD20 and ULBP1-3, and inversely correlate with the expression levels of MHC class I among 30 different CD20+ B-NHL cell lines. The importance of the ULBPs was confirmed using antibody blockade. In the presence of blocking mAb to ULBP1, 2 or 3, a decrease of rituximab-induced ADCCs against B-NHL cell lines were observed. In addition, the present study clearly identified the key mechanism of rituximab-induced ADCC as antibody-dependent target-specific cytotoxicity mediated by highly activated NK cells. Strong NK cells activation was due to the combination of Fc„dR stimulation via the Fc portion of rituximab, together with stimulation of activating NK cell receptors via their ligands expressed on the tumor cells, particularly ULBPs, which occurred in a robustly synergistic manner.
Conclusions: Tumor cell susceptibility to rituximab-induced ADCC was determined by three major tumor-associated factors:
the amount of the target molecule, CD20; the amount of the ligands for inhibitory killer Ig-like receptors, MHC class I; and the amounts of some of the NKG2D ligands, especially ULBP1-3.
This is the first report to show the importance of ULBPs on tumor cells for rituximab-induced ADCC. The ULBPs could be valuable diagnostic biological makers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs.
Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.