Recent clinical studies demonstrated that a bi-allelic polymorphism in the human FCGRIIIA gene critically determines the clinical outcome of rituximab therapy in certain hematologic malignancies and in autoimmune diseases. Thus, patients homozygous for the 158V allele of the FcγRIIIa receptor demonstrated significantly better response rates to rituximab therapy compared to homozygous 158F donors. However, confliciting data were reported for heterozygous patients - which account for approximately 45 % of the investigated population. Furthermore, most of the in vitro reports about functional effects of this polymorphism did not address the function of 158V/F heterozygotes. In all these studies, genomic DNA was used for allotyping of the FCGRIIIA 158V/F polymorphism. Thus, these studies were not set up to investigate potential allele- specific differences at mRNA or protein levels, which were demonstrated for several other NK cell- related genes. Therefore, we were interested to analyze receptor expression and function in 158V/F heterozygous donors in more detail.

First, we established FCGRIIIA allotyping at the mRNA level. Thus, isolated mRNA was reversely transcribed, PCR amplified and sequenced using BigDye terminator mix. Results from homozygous donors were consistent and homogeneous, whereas sequencing profiles from heterozygotes appeared heterogeneous. Some 158V/F heterozygotes demonstrated similar expression of both alleles, whereas sequencing profiles in others were almost similar to either homozygous 158V/V or homozygous 158F/F donors. Next, we analyzed the quantitative expression of the corresponding proteins by immunofluorescence experiments with antibodies 3G8 and MEM-154. While 3G8 binds similarly to both allelic forms of FcγRIIIa, MEM-154 preferentially recognizes the 158V allele, as confirmed by immunofluorescence studies with FcγRIIIa 158F or 158V transfected cell lines. These experiments again demonstrated that FcγRIIIA 158V/F heterozygous donors are a heterogeneous group - with 158V protein levels ranging from similar to 158F/F homozygotes to similar to 158V/V homozygous donors. Furthermore, we investigated whether 158V expression levels have functional implications in classical 51Cr- release assays against ARH-77 B cells. We used isolated NK cells as effector cells, and rituximab as sensitizing antibody. Effector cells were isolated from matched sets of FCγRIIIA allotyped donors to directly compare 158V/V or 158F/F homozygotes with 158V/F heterozygotes. As described by others, we observed statistically significant differences between 158V/V and 158F/F homozygous donors at suboptimal antibody concentrations. Interestingly, NK cells from 158F/V heterozygous donors, which appeared similar by sequencing profiles and by immunofluorescence studies to effector cells from 158V/V homozygotes, also demonstrated killing levels similar to those from 158V/V homozygous donors.

In conclusion, our data demonstrate that donors heterozygous for FcγRIIIA at position 158 are a heterogeneous population - potentially explaining controversial data published about 158F/V heterozygotes in clinical studies. In order to further assess the biological impact of our results, clinical trials may address whether FcγRIIIA 158V expression levels will correlate with clinical responses to rituximab therapy. These studies could help to predict which patients may optimally benefit from rituximab therapy.

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