Page 77 - 2021_07-Haematologica-web

anti-RhD antibodies modulate NK cell activity
(FcR) CD32B (FcγRIIb).4 However, Fcγ receptors have not been shown to play a role in AMIS.8
Since the clinical effect of anti-RhD antibodies implies that they convey some immune suppression, we wondered whether these preparations affect immune cells other than B cells.
Here we concentrate on natural killer (NK) cells, which are innate lymphoid cells that play a significant role in elim- inating virus-infected and malignant cells.9 NK-cell activity is governed by a balance of signals from a vast array of acti- vating (e.g., CD16a, FcγRIIIa) and inhibitory receptors that are activated by self or foreign ligands. NK cells can also interact with dendritic cells (DC) and are able to kill DC in peripheral tissues, but this cytotoxic effect mostly affects immature DC (iDC).10 Notably, the killing of iDC by NK cells might attenuate adaptive immunity. 10 NK cells are also able to kill cells coated with antibody, a phenomenon known as antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated by the Fc fragment of antibod- ies which bind to CD16, the main FcR expressed on NK cells.11 Freshly isolated primary NK cells are mainly com- posed of a large, CD56dimCD16+ subpopulation (which expresses CD56 at intermediate levels and CD16 at high levels), and a much smaller, CD56brightCD16- subpopulation (which expresses CD56 at high levels and does not express CD16).12
Here we show that anti-RhD antibodies activate NK cells via binding of their Fc segment to CD16 in a glycosylation- dependent manner. We show that this activation occurs not only in vitro, but also in vivo, in patients who receive this treatment. We further show that the anti-RhD drug KamRho enhances killing of iDC by NK cells and discuss how this might lead to immune suppression.
Methods
Erythrocyte extraction and staining
We used commercial erythrocytes of defined phenotype (cat. 004310, Bio-Rad) or erythrocytes extracted from whole blood samples from healthy volunteers with a known RhD expression profile. For extraction of erythrocytes from whole blood samples, the samples were centrifuged and the supernatant was discarded. Erythrocytes were then washed three times with phosphate buffered saline (PBSx1). For flow cytometry analysis, erythrocytes were incubated at 37°C for 15 minutes with antibodies, washed and then incubated with a secondary antibody at room tempera- ture for 30 minutes. Details of other procedures related to erythro- cytes are described in the Online Supplementary Appendix.
Natural killer cell purification and CD107a degranulation assay
Primary NK cells were isolated from the peripheral blood of healthy human volunteers, and tested for purity as previously described.13 Unless stated otherwise, in all experiments we used NK cells which were activated as previously described.13
For the CD107a degranulation assay, primary NK cells were incubated with different polyclonal antibodies (described in the Online Supplementary Appendix) together with anti-CD107a and anti-CD56 antibodies. The NK and target cells were incubated at 37°C and 5% CO2 for 2 hours and analyzed by flow cytometry. In each well we used 50,000 NK cells and 0.5 μg of the relevant anti- body in a final volume of 100 μL. Degranulation of NK cells was assessed by calculating the percent of CD107a+ NK cells out of the total NK cells in a given well. The data was normalized to the
basal percent of CD107a+ NK cells (without addition of antibod- ies). The assay was performed in triplicate. For the mIgG2a block- ing experiment, NK cells were pre-incubated for 30 minutes on ice with 5 μg per well of the mouse IgG2a isotype control.
Ethics
The collection of patient samples was approved by the Institutional Review Board of The Hadassah Medical Center and informed con- sent was obtained from all participants (HMO-0091-18).
Human samples
Two peripheral blood samples were collected from RhD- women who received prophylactic anti-RhD treatment while hos- pitalized. After erythrocyte lysis, NK cell degranulation was quan- tified using flow cytometry as the percent of CD107a+ CD56+ CD3- cells out of CD56+CD3- cells. The assay was performed in triplicate.
Dendritic cell purification
Monocyte-derived DC were generated from CD14+ peripheral blood mononuclear cell (PBMC), which were isolated as described previously.14 Briefly, anti-CD14 magnetic beads were used to iso- late monocytes from PBMC according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA, USA). Monocytes were placed in wells at a concentration of 1.25×106 cells/1.5mL culture medium.
Cytotoxicity assays
The in vitro cytotoxic activity of NK cells was assessed in 5-hour 35S-release assays as previously described.13 DC were incubated for 48 hours with 20 ml of 35S- methionine prior to incubation with NK cells. K562, 721.221 or primary B cells were incubated for 24 hours with 4 ml of 35S- methionine, in methionine-free medium prior to incubation with NK cells. NK cells were pre-incubated for 2 hours at 37°C with different polyclonal antibodies (described in the Online Supplementary Appendix), at a concentration of 0.5 μg per well, diluted in RPMI medium. For the blocking experiment, NK cells were pre-incubated for 30 minutes on ice with 5 μg mIgG2a per well.
Results
Anti-RhD preparations induce natural killer cell degranulation
In order to investigate the effect of anti-RhD antibodies on NK cells, we used two common commercial prepara- tions of anti-RhD antibodies: Rhophylac and KamRho. Both are produced from human sera and used clinically. In order to verify that these preparations specifically bind the RhD antigen, we first stained erythrocytes with known RhD expression. As expected, RhD+ erythrocytes were rec- ognized by both anti-RhD products, in contrast to RhD- erythrocytes (Figure 1A). Next, we tested whether the anti- RhD preparations affect NK cell activation. We incubated activated human NK cells (which only contain CD56dimCD16+ cells, Online Supplementary Figure S1A) with anti-RhD antibodies and used flow cytometry to assess the level of CD107a, a marker of NK cell degranulation.15 Since anti-RhD preparations also contain non-specific human antibodies, we used intravenous immunoglobulin (IVIG) at the same concentration as the total antibody concentration of these preparations as a control. Neither of the IVIG prod- ucts (Intratect and Kiovig) significantly affected the degran- ulation level of NK cells (Figure 1B). Both of the anti-RhD
haematologica | 2021; 106(7)
1847