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S. Elias et al.
regulating adaptive immunity10, we tested whether anti- RhD antibodies affect this process.
We performed NK cytotoxicity assays, in which we meas- ured the killing of mature DC (mDC) activated by lipopolysaccharide (LPS) and of iDC by NK cells which were pre-incubated with KamRho, Rhophylac or IVIG. The phe- notype of iDC and mDC is presented in the Online Supplementary Figure S4A. At all effector to target ratios test- ed, pre-incubation with KamRho increased the killing of iDC by NK cells (Figure 6A). KamRho also increased the killing of mDC by NK cells, although the killing levels were significantly lower (Figure 6B), which is in line with previous reports.22-24 Pre-blocking of CD16 on NK cells with mouse IgG2a abolished the differences between KamRho and IVIG (Figure 6C). Interestingly, pre-incubation of NK cells with Rhophylac did not increase the killing of neither iDC nor mDC (Online Supplementary Figure S4B and C, respec- tively), although both drugs were able to induce degranula- tion of NK cells (Figure 1C).
Because anti-RhD antibody preparations contain mainly non-specific antibodies, we next verified that the increased killing effect is specifically due to anti-RhD antibodies. We repeated the cytotoxicity assay, this time using the bound antibody fractions we eluted from RhD- and RhD+ erythro- cytes incubated with KamRho (as shown in Figure 2 E to H). As expected, the bound fraction from RhD+ erythro- cytes (which contains the specific anti-RhD antibodies) increased NK-cell-mediated killing of iDC significantly while the bound fraction obtained from RhD- erythrocytes did not (Online Supplementary Figure S4D).
In order to check whether any other cells, particularly B cells, could also be more effciently killed in the presence of KamRho, we repeated the NK cytotoxicity assay with pri- mary B cells isolated from human donors, the B-cell line 721.221 or the myeloid-derived cell line K562, and observed no effect (Figure 6D).
Discussion
Anti-RhD Antibodies are widely used in clinical practice, mainly to prevent immunization against the RhD antigen. Surprisingly, although prophylactic administration of anti- RhD antibodies is a common, safe, and successful treat- ment (in terms of preventing HDFN), the mechanism of this therapy remains unclear. One of the common theories sug- gests that this treatment can cause AMIS, though there is no single definitive mechanism for this suppression.
One of the limitations of anti-RhD antibodies is that these products are manufactured from the sera of human subjects who are immunized against RhD. In light of the success of the preventive anti-RhD therapy, the number of immunized donors has decreased significantly.4 In order to overcome this drawback, in recent years several recombi- nant monoclonal antibodies against RhD have been gener- ated.25-27 Some studies have demonstrated clinical efficiency of monoclonal anti-RhD antibodies in preventing RhD immunization, and have displayed encouraging laboratory markers (e.g., clearance of erythrocytes or antibody-depen- dent cytotoxicity).26-28 Nonetheless, polyclonal RhD anti- bodies are still considered the standard of care, which emphasizes that the precise mechanism of this treatment has not yet been elucidated. Deciphering this mechanism would potentially assist in generating efficient recombinant substitutions which would replace serum-based products.
Here we explored the effect of two commercial anti-RhD antibody preparations on NK-cell activity. We showed that both products induce significant degranulation of NK cells, even in the absence of target cells, and that this is mediated specifically by anti-RhD antibodies. Importantly, we also observed an increase in NK-cell degranulation following prophylactic treatment with anti-RhD antibodies in human patients.
We demonstrated that this effect occurs via binding of the Fc segment of the antibodies to CD16 in a glycosyla- tion-dependent manner. It is possible that anti-RhD anti- bodies have unique glycosylations which are critical for their interaction with NK cells. This is consistent with a study which demonstrated that anti-RhD antibodies isolat- ed from the plasma of alloimmunized pregnant women are less fucosylated.29 These properties should by explored in further studies.
We demonstrated a significant binding of labeled anti- RhD antibodies to the CD56dim, but not to the CD56bright NK-cell population of freshly isolated NK cells. However, when we used the non-labeled anti-RhD antibodies, little staining was observed. This is probably due to limitations of staining human NK cells by unlabeled human antibodies which requires anti-human secondary antibody staining. Since NK cells are coated with human immunoglobulins,30,31 the non-specific background staining with secondary anti- human antibodies is high, seemingly masking any specific binding.
We also demonstrated the role of CD16 in the anti-RhD -mediated NK-cell degranulation in other experiments (e.g., degranulation with blocking of CD16 and with low and high-affinity CD16a-expressing donors). Blocking of CD16 abolished anti-RhD-mediated degranulation significantly, yet not completely. This incomplete blocking could result from inefficiency of the blocking or might indicate that additional NK-cell receptors are involved (even though we have not observed binding of anti-RhD antibodies to sever- al other NK-cell receptors).
We next found that the anti-RhD drug KamRho increases killing of iDC. KamRho also increased killing of mDC, but to a lesser extent. This is in line with previous reports of the sensitivity of iDC as compared to mDC to killing by NK cells, which is attributed to the increased levels of major histocompatibility complex (MHC) on mDC.22-24.
Surprisingly, although the drugs Rhophylac and KamRho had similar effects in most in vitro assays performed, Rhophylac did not increase NK killing of DC. One possibil- ity is that this difference is related to the presence of a dif- ferent stabilizer in each of these drugs: albumin in Rhopylac and glycine in KamRho (glycine is also used as stabilizer in the IVIG regent we used as a control).
Interestingly, increased NK-cell activation mediated by KamRho did not enhance killing of other cell types, includ- ing B cells. The reasons behind this observation are not completely understood. One possible explanation is that MHC class I proteins are involved. This option seems unlikely as K562 and 721.221 cells express little or no MHC class I, while primary B cells, iDC and to a greater extent mDC express high levels of MHC class I. Alternatively, it is possible that anti-RhD antibodies not only bind NK but can also bind DC through their Fab segment, therefore inducing ADCC. This possibility seems unlikely as the RhD antigen is expressed only on erythrocytes16. A final explanation might be that CD16 binds an unknown cellular ligand expressed on DC. Such a cellular ligand was previously
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haematologica | 2021; 106(7)