puromycin selection is shown in Fig. 1A . Activation of the reporter cells is measured by 125 quantification of mouse IL-2 (mIL-2) secretion into the cell culture supernatant using an anti-126 IL-2 sandwich ELISA as described previously (Corrales-Aguilar et al., 2013) . In order to employ 127 the original assay, designed to measure ICs on adherent infected cells, for the detection of 128 soluble ICs we first determined the suspension of IgG achieved by pre-blocking a 96 well 129 There are only few commercially available antibody-antigen pairs that meet both the above 177 mentioned high grade purity requirements while also allowing for multimeric sIC formation. We 178 focused on three pairs of multivalent antigens and their respective mAbs that were available 179 in required amounts enabling large-scale titration experiments; trimeric rhTNFα:IgG1 infliximab 180 (TNFα:Ifx), dimeric rhVEGFA: IgG1 bevacizumab (VEGFA/Bvz) and dimeric rhIL-5: IgG1 181 mepolizumab (IL-5/Mpz). As lymphocytes express TNFα-receptors I and II while not 182 expressing receptors for IL-5 or VEGFA, we tested whether our mouse lymphocyte derived 183 BW5147 thymoma reporter cell line is sensitive to high concentrations of rhTNFα. Toxicity 184 testing revealed that even high concentrations up to 76.75 nM rhTNFα did not affect viability 185 of reporter cells (Fig. S1 ). Next, we measured the dose-dependent activation of human FcγRs 186 comparing immobilized IgG to soluble ICs using the FcγR reporter cell panel (Fig. 3) Next, to validate the measured differences in response to sICs vs. immobilized IC for primary 219 cells, we determined FcγRIIIA activation using primary human NK cells isolated from PBMCs 220 of healthy donors. Measuring a panel of activation markers and cytokine responses by flow 221 cytometry, we observed a differential activation pattern depending on ICs being soluble or 222 immobilized at equal molarity (Fig. 4A) . We chose IL-5/Mpz sICs as NK cells do not express 223 the IL-5 receptor. While MIP1-β responses were comparable between the two triggers, 224 degranulation (CD107a) and TNFα responses showed a trend towards lower activation by sICs 225 compared to immobilized IgG (Mpz). Strikingly, IFNγ responses were significantly weaker 226 when NK cells were incubated with sICs compared to immobilized IgG. In order to confirm this 227 hierarchy of responses and to enhance the overall low activation by Fcγ compared to the PMA 228 control, we changed the IC setup by generating reverse-orientation sICs consisting of human 229
FcγR-specific mAbs and goat-anti-mouse IgG F(ab)2 fragments. NK cell activation by reverse 230 sICs was compared to NK cell activation by immobilized FcγR specific mAbs (Fig. 4B) . Here, 231
we not only confirm our previous observations regarding MIP-1β and IFNγ, but we also confirm 232 significantly lower TNFα and CD107a responses towards soluble complexes compared to 233 immobilized mAbs. Importantly, these experiments validate that sICs readily activate primary 234 NK cells and induce immunological effector functions. As in 10% of the population NK cells 235 express FcγRIIC (Breunis et al., 2008; Metes et al., 1998) , we also tested if this receptor plays 236 a role in our measurements. Using the same three donors and an FcγRII specific mAb as 237 described above, we did not observe an FcγRII-mediated response. Accordingly, we conclude 238 that FcγRIIC expression did not play a role in our experiments (Fig. 4C) . Taken together, we 239
show that multivalent but not dimeric soluble immune complexes govern primary NK cell 240 response and FcγRIIIA/B activation ( Fig. 2A) . 241 complexed FcγRIII-specific mAb (reverse sICs), immobilized IgG of non-FcγRIII-specificity 250 (isotype control) or soluble F(ab)2 complexed isotype control (all at 1µg, 10 6 cells). Incubation 251
with PMA and Ionomycin served as a positive control. Incubation with medium alone served 252 as a negative control. C) As in B using an FcγRII-specific mAb. NK cells from the tested donors 253 in this study do not react to FcγRII activation. 254 255 256
We observed that the dimeric sIC CD20:Rtx completely failed to activate our reporter cells, 258 while potentially larger sICs based on multimeric antigens showed an efficient dose-dependent 259
FcγR activation. In order to determine whether the assay is able to respond to changes in sIC 260 size, we tested cross-titrated amounts of antibody (mAb, infliximab, Ifx) and antigen (Ag, 261 rhTNFα). To this end, the reporter cells were incubated with sIC of different mAb:Ag ratio by 262 fixing one parameter and titrating the other. According to the Heidelberger-Kendall precipitation 263 curve (Heidelberger and Kendall, 1929) describing sIC size as being dependent on the mAb:Ag 264 ratio, this should result in varying sIC sizes as an excess of either antigen or antibody results 265 in the formation of smaller complexes compared to the large molecular complexes formed at 266 around equal molarity. Changes in sIC size due to a varying mAb:Ag ratio were quantified 267 using asymmetrical flow-field flow fractionation (AF4) ( Fig. 5A and Table S1 ). precipitation curve with a functional read-out. A) infliximab (mAb) and rhTNFα (Ag) were 296 mixed at different ratios (17 µg total protein, calculated from monomer molarity) and analysed 297 via AF4. sIC size is maximal at a 1:3 ratio of mAb:Ag and reduced when either mAb or Ag are 298
given in excess. <M>w = mass-weighted mean of the molar mass distribution. Three 299 independent experiments. Error bars = SD. Data taken from Table S1. One complete run 300 analysis is shown in Fig. S2 . B) FcγR BW5147 reporter cell activation is sensitive to sIC size. 301 sICs of different size were generated by cross-titration according to the AF4 determination. 302
Reporter cells were incubated with fixed amounts of either mAb (infliximab, left) or Ag (rhTNFα, 303 right) and titrated amounts of antigen or antibody, respectively. X-Axis shows titration of either 304 antigen or antibody, respectively (TNFα calculated as monomer). IL-2 production of reporter 305 cells shows a peak for FcγRIIB/C and FcγRIIIA/B activation at an antibody:antigen ratio 306 between 1:2 and 1:4. FcγRs I and IIA show no activation towards sICs in line with previous 307
observations. Two independent experiments. Error bars = SD. C) Primary NK cells purified by 308 negative selection magnetic bead separation from three different donors were incubated with 309 cross-titrated sICs as in A. NK cells were measured for MIP-1β expression (% positivity). 310
Incubation with PMA and Ionomycin served as a positive control. Incubation with medium alone 311 served as a negative control. Measured in technical replicates. Error bars = SD. 312
In order to apply the assay to a clinically relevant condition we measured circulating sICs 314 present in the serum of SLE patients with variable disease activity. Sera from 4 healthy donors 315 and 25 SLE patients were investigated for FcγRIIIA/B and FcγRIIB/C activation. Reporter cells 316 produced IL-2 in response to patient sera in a dose-dependent manner (shown exemplarily for 317 one group of SLE patients in Fig. 6A) , which was not the case when sera from healthy controls 318 were tested. Consistent with the observation that FcγRI and FcγRIIA do not respond to 319 synthetic sICs, reporter cells expressing these receptors did also not respond to the tested 320 anti-dsDNA titers and C3d (p=0.0078 and p=0.0118, respectively). FcγRIIB/C on the other 331 hand showed no significant correlation with either biomarker. We assume this may be due to 332 the influence of IgG sialyation found to be reduced in active SLE (Vuckovic et al., 2015) . 333
Generally, de-sialylation of IgG leads to stronger binding by the activating receptors FcγRI, 334
FcγRIIA and FcγRIII while it reduces the binding affinity of the inhibitory FcγRIIB (Kaneko et 335 al., 2006 have already been generated using the here described method and were successfully used to immune function (Bruhns and Jonsson, 2015) . Incubation with synthetic sICs generated from 369 rhTNFα and mouse-anti-hTNFα IgG1 showed both of the mFcγR reporter cells to be 370 responsive to sICs (Fig. 7A) . Parental BW5147 cells expressing no FcγRs served as a negative 371
control. The sera of three mice per group were analysed and compared to sera from wildtype 372 C57BL/6 mice, which served as a negative control. We consistently detected mFcγR activation 373 by sera from K/BxN or NZB/WF1 compared to BL6 mice (Fig. 7B) . While the mFcγRIII 374 responses were generally high and similar between K/BxN and NZB/WF1 mice, we found a 375 lower and more variable mFcγRIV responsiveness. We assume this might be explained by 376 differences in mFcγR affinity to mouse IgG subclasses. While mFcRIII binds IgG1, IgG2a and 377
IgG2b with comparable affinity, IgG1 is not detected by mFcRIV (Bruhns and Jonsson, 2015) . 378
Further, we found that the response of mFcγRIV-expressing reporter cells shows no strict 379 dose-dependency. This effect was more pronounced for NZB/WF1 compared to K/BxN mice. 380
In addition, in this case we assume an influence of factors such as glycosylation patterns or 381 subclass composition, not further addressed here. Nevertheless, the assay enables the 382 reliable detection of sICs in sera of mice with immune-complex mediated diseases making it a 383 promising novel tool to monitor sICs as a biomarker of disease activity. 384 We provide for the first time a simultaneous functional and biophysical assessment of the 454 paradigmatic Heidelberger-Kendall precipitation curve (Heidelberger and Kendall, 1929) . 455
While previous work revealed that large and small sICs show differential impact on IL-6 456 production in PBMCs (Lux et al., 2013) , the dynamics of FcγR activation resulting from 457 constant changes in sIC size have not been explored in great detail yet. This was achieved in 458 this study by directly analyzing native molecules via AF4 (Fig. 5A, Fig. S2 , Table S1 ). Our data 459 reveal that sIC size is indeed governed by antibody:antigen ratios covering a wide range of 460 sizes up to several megadaltons. In the presence of increasing amounts of antibody or antigen 461 deviating from an optimal antibody:ratio, sIC size steadily decreases. 
We found that FcγRI is not activated by sICs in our assay. We assume that FcγRI activation 472 by sICs might require a native cellular environment given that a major function is the uptake of 473
ICs even in the absence of a signaling motif (Indik et al., 1994) . However, we find that FcγRI 474 ectodomains alone are not responsive to sICs indicating a different cross-linking threshold for 475
FcγRI possibly linked to it being the only high-affinity FcγR with three extracellular Ig domains 476 compared to the two domains found in other FcγRs. This reflects a general consideration 477 regarding the reporter system. While providing a robust and unified read-out using a scalable 478 cell-based approach, the assay is not able to reflect native immune cell functions governed by 479 cell specific signalling cascades. The major advancements of this reporter system include i) a 480 higher accuracy regarding FcγR activation compared to strict affinity measurement, ii) an sIC 481 size dependent quantification of FcγR responsiveness and iii) the identification of FcγR 482 activating sICs in autoimmunity but also infection. Finally, this scalable, sensitive and robust were not available and difficult to calculate as the exact stoichiometry is not known. An 583 extinction coefficient of 1.450 mL/(mg cm) was used for calculating the molar masses of all 584
ICs. Especially in the case of ICs rich in TNFα, the true coefficients should be lower, and the 585 molar masses of these complexes are overestimated by not more than 14 %. The determined 586 molar masses for TNFα-rich complexes are therefore biased but the observed variations in 587 molar mass for the different ICs remain valid. The mass-weighted mean of the distribution of 588 molar masses for each sample was calculated using the ASTRA 7 software package (Wyatt). Table S1. Analysis of the molar mass distribution of ICs from AF4 data. 852
For a given elution time, the AF4 profiles provide the concentration (UV) at which a given molar 853 mass (MALS) of a protein is present in the sample. The molar mass distribution of Ifx, TNFα 854
and their immune complexes (sICs) was obtained by plotting the cumulative frequency as a 855
function of molar mass. For a selected range of molar masses, a mass-weighted mean value 856 (<Mw>) was calculated. All detected molar masses were selected in the case of Ifx and TNFα 857 whereas only molar masses larger than the maximal molar mass found for Ifx were assigned 858
to sICs. The table shows the range of assigned molar masses and the calculated <Mw> for 859 each AF4 run (n = 3). 860 861