Mice used in this study were bred and maintained in local facilities with experiments approved through local ethics committees and performed according to Home Office guidelines.

Recipient mice were provided with acid water, pH 2.5 on day −7 until 14 days after bone marrow receipt. Recipients received 1.1 Gy radiation on days −1 and 0 using a MultiRad 350 X-ray Irradiator (Faxitron). Bone marrow was harvested from donor mice and 3–8×10⁶ cells injected intravenously (I.V.) into recipients. Systemic reconstitution was confirmed by flow cytometry 8–10 weeks after engraftment.

This model has been described previously.18 19 Briefly, 1×10⁷ cryopreserved Eµ-TCL-1 transgenic (Tg) or hCD20⁺ Eµ-TCL-1 Tg tumor splenocytes were injected intraperitoneally (I.P). into recipient mice. The presence of tumor was monitored in peripheral blood. Once tumor cells (CD19⁺CD5^mid) were detectable by flow cytometry, mice were treated. The white cell count was determined using a Coulter Z1 particle counter with red blood cells (RBC) lysed using ZAP-OGLOBIN II (both Beckman Coulter) or by flow cytomery using Precision Count beads (Biolegend).

All clinical antibodies were gifted from the Southampton General Hospital oncology pharmacy. Others were produced in-house. 18B12 and Rituximab isotype variants were cloned onto the appropriate IgG framework, produced in CHO cells and purified from culture supernatant with protein A. Purity was assessed by electrophoresis (Beckman EP; Beckman) and lack of aggregation confirmed by size exclusion (SEC) high performance liquid chromatography (HPLC). Unless otherwise stated, all antibodies were administered I.P. in 200 µL sterile PBS (Severn Biotech).

Flow cytometry was performed using the antibodies listed in online supplementary table 1. Anti-mFcγR has been reported previously.20 21 Following staining, RBC lysis buffer was added (AbD Serotec) and cells washed before analysis on a FACS Canto or FACS Calibur flow cytometer (BD Biosciences). Alexafluor 647 labeled MST-HN and H435A Abdegs for analyzing FcRn expression were a kind gift from Prof Sally Ward (University of Southampton) and used at 5 µg/mL with Fc block (2.4G2, 5 µg/mL) prior to extracellular staining.

The tibia and femur of mice were flushed with sterile complete RPMI (RPMI 1640 (Life Technologies), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin (all Life Technologies), 10% fetal calf serum (Sigma-Aldrich). Cells were plated in 6-well plates at 0.8×10⁶ cells/mL in complete RPMI +20% L929 conditioned media for 7–10 days.

IgG concentration was determined by ELISA with reference to a standard curve of the same antibody as follows: for hIgG, maxisorp plates (Thermo Scientific) were coated with 5 µg/mL goat antihuman Fc-specific polyclonal antibody (Sigma-Aldrich) and blocked with PBS+1% BSA before addition of serum for 1 hour and washing. Detection was with horseradish peroxidase (HRP) conjugated F(ab’)₂ goat anti-hFc specific antibody (Jackson Immunoresearch). Plates were incubated with o-phenylenediamine dihydrochloride (OPD) substrate and OD₄₉₅ measured using an Epoch microplate spectrophotometer (Biotek). For quantification of mIgG, plates were coated with rabbit anti-mIgG and detected with HRP- rabbit anti-mIgG (both Jackson Immunoresearch).

Purified IgG was heated to 64°C for 30 min. The aggregated fraction was purified on a superdex S200 column (GE Healthcare). Aggregation was confirmed by HPLC using a Zorbax GF-250 column (Agilent).

RNA was isolated and cDNA generated from SCID or NOD SCID bone marrow derived macrophages (BMDM) and the mFcγRII gene amplified using gene specific primers. Subsequently, the extracellular domain (residues 1–207) of mFcγRII were cloned with the addition of a 6xHis tag. The construct was transfected into MEXi −293E cells (IBA lifesciences) and FcγRII-His expressed according to the manufacturer’s protocol and protein purified using a HisTrap HP column on an AKTA prime system (Both GE biosciences) and purity confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).

Surface plasmon resonance (SPR) was performed using a Biacore T100 system upgraded to a T200 (GE Life Sciences). For mFcγRII isoforms, anti-His capture antibody was immobilized on a CM5 chip (GE life Sciences). Purified FcγRII-His (10 µg/mL) was flowed over the chip at 30 µL/min for capture. IgG was injected at 30 µL/min. For all other analysis, IgG was immobilized via amine coupling with a target of 2000 RU. Recombinant mFcγRII or mFcRn (R&D systems) was flowed over the immobilized IgG in HBS-EP +buffer (GE Life Sciences) at pH7.4 or pH6.0. Affinity constants were determined by analysis with Biacore Bioevaluation software assuming 1:1 binding.

mRNA was extracted from SCID or NOD SCID splenocytes and hepatocytes using an RNeasy Mini Kit (Qiagen) and cDNA generated using a Superscript III reverse transcription kit (Life Technologies). Quantitative PCR (qPCR) was performed using GoTaq qPCR master mix (Promega) using gene specific primers, with HPRT1 as a control. Ct values were normalized using HPRT1 values and the ΔΔCt method used to calculate fold change.

Lysates were produced from 5×10⁶ SCID or NOD SCID splenocytes and hepatocytes using RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Deoxycholate, 0.1% SDS, 50 mM Tris, pH 8) and run on a 12% Novex Nupage BIS-TRIS gel (Thermo Fisher) before transfer to a methylcellulose membrane (GE Lifesciences). Primary antibodies were antimouse FcRn and Lamin B with detection using an HRP-conjugated donkey anti-goat antibody (online supplementary table 1).

Liver tissue from BALB/c or NSG mice was embedded in OCT (CellPath) and frozen in isopentane on dry ice. 8 µm sections were cut and transferred to Superfrost plus slides (Thermo Scientific), air dried overnight and fixed in 100% acetone. Following blocking, primary antibodies against FcRn or FcγRII were added overnight before detection with Alexafluor 488-labeled secondary antibody (online supplementary table 1) for 45 min. Subsequently, primary antibodies against Clec4F or cytokeratin 8 were added for 2 hours before detection with AlexaFluor 549 or AlexaFluor 568 conjugated secondary antibodies (online supplementary table 1). Slides were mounted using Vectashield hardset with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories).

Images were collected using a CKX41 inverted microscope with a reflected fluorescence system equipped with a DP22 camera running CellSens software, using Plan Achromat 10×0.25 and 40×0.65 objective lenses (all from Olympus). Images were transferred to ImageJ (Fiji) or photoshop (Adobe) where background autofluorescence was removed, contrast stretched and brightness adjusted to maximize clarity, with all images treated equivalently.

To explore potential differences of recipient mouse strains on immunotherapy efficacy, SCID and NOD SCID mice bearing established hCD20⁺ Eµ-TCL-1 tumors were treated with rituximab. Although initial tumor clearance was comparable between strains, 14 days after mAb treatment there were significantly more tumor cells in the peripheral blood of NOD SCID compared with SCID mice (figure 1A,B). To determine if this was associated with rituximab’s type I nature,22 23 we repeated the experiment with the type II anti-hCD20 mAb, BHH2,18 and observed the same reduced efficacy in NOD SCID compared with SCID mice (figure 1B).

To understand this difference in efficacy, the concentration of injected hIgG in the plasma of mice following treatment was determined (figure 1C). This revealed that 7 days after mAb treatment there was significantly less (~10-fold) hIgG in the plasma of NOD SCID compared with SCID mice (16.7 vs 1.6 µg/mL) suggesting that rapid hIgG1 clearance in NOD SCID mice was responsible for the less prolonged tumor deletion.

To investigate whether the rapid clearance was related to the mAb, strain and/or tumor, an alternative hIgG1 mAb, cetuximab, was administered to non-tumor bearing SCID and NOD SCID mice. Cetuximab was also more rapidly cleared from NOD SCID compared with SCID mice with hIgG being undetectable in the plasma of NOD SCID mice by day 7 post-administration (figure 1D). Similar results were also observed with other hIgG1 mAb including trastuzumab (online supplementary figure 1), showing that the rapid clearance is directly related to the NOD SCID strain, independent of tumor and a common feature of therapeutically-relevant hIgG1 mAb. Importantly, the hIgG1 clearance in SCID and NOD mice was comparable to that of immune-competent BALB/c mice (online supplementary figure 2 and previously shown24), confirming fast hIgG1 clearance in NOD SCID mice, rather than slow clearance in SCID or NOD mice. Furthermore, the lack of a difference in SCID mice demonstrates that rapid hIgG clearance does not result from the absence of endogenous IgG or immune deficiency per se.

To determine if rapid mAb clearance in NOD SCID mice extended beyond hIgG1, isotype switch variants of rituximab were generated and administered to SCID or NOD SCID mice. Similar to hIgG1, mIgG2a also had a significantly faster mAb clearance in NOD SCID mice (figure 1D), being no longer detectable in the plasma by day 14. In contrast, hIgG2 and mIgG1 had similar clearance rates in both strains. These results demonstrate that faster mAb clearance in NOD SCID mice is isotype dependent.

We next assessed whether the rapid IgG clearance occurred in NOD and NSG strains. NOD mice had a normal hIgG1 clearance rate, akin to that seen in SCID and BALB/c (figure 2A). However, NSG mice displayed rapid clearance, comparable to that in NOD SCID mice (figure 2B). These data demonstrate that both NOD and SCID phenotypes are necessary to confer rapid IgG clearance. Moreover, the differences between isotypes in NOD SCID mice also occurred in the NSG strain, with hIgG1 and mIgG2a but not mIgG1 exhibiting rapid clearance (figure 2B).

Given that mIgG2a and hIgG1 have similar FcγR binding profiles (binding to all mFcγR, with substantial affinity for several activatory FcγR), we hypothesized that the rapid mAb clearance of hIgG1 and mIgG2a isotypes in NOD SCID mice was mediated by FcγR.25 26 This was investigated using a N279Q (NQ)-mutant of rituximab which lacks glycosylation at N297 and does not robustly engage mFcγR (without compromising interaction with FcRn).27 The NQ-mutant remained present in the plasma of NOD SCID mice at significantly higher concentrations at all time-points, supporting mFcγR involvement in the rapid hIgG1 clearance in NOD SCID mice (figure 2C). Moreover, the concentration of rituximab-NQ was comparable between SCID and NOD SCID mice at all time-points suggesting that abrogation of mFcγR binding restored normal mAb clearance rate.

Having established that the rapid hIgG1 clearance rate in NOD SCID mice was likely dependent on mFcγR, the relative expressions levels of these receptors in SCID and NOD SCID mice was investigated (figure 3A). While there were no statistically significant differences in expression levels (two-way analysis of variance p>0.05) trends toward differential expression were observed. mFcγRII expression was lower on both Ly6C^Hi and Ly6C^Lo monocytes in NOD SCID compared with SCID mice (figure 3B). Neutrophil and splenic macrophage FcγRIII expression was higher in SCID mice, with a similar expression profile for BMDM (online supplementary figure 3a). The expression of mFcγRI was not investigated as it is known to contain multiple polymorphisms in NOD SCID mice which prevent its detection using available antibodies.28 The subtle differences in activatory mFcγR expression detailed above appear to be compensatory with a similar overall expression of activatory mFcγR in each strain. In summary, only monocyte FcγRII was found to differ between SCID and NOD SCID mice; the relevance of this to mAb clearance rate remains to be determined.

To understand the contribution of specific mFcγR to rapid antibody clearance in NOD SCID mice, we made use of animals lacking different classes of mFcγR. In NOD SCID FcR γ-/- mice (which express no activatory FcγR at the cell surface29) there was no significant difference in the concentration of hIgG1 over time compared with NOD SCID mice (figure 4A), demonstrating that a lack of activatory mFcγR does not influence hIgG1 clearance. However, in NOD SCID mice deficient in the inhibitory mFcγRII, the concentrations of hIgG1 were significantly increased compared with wild-type NOD SCID mice retaining mFcγRII (figure 4B) and comparable with SCID mice. These results demonstrate that the rapid hIgG1 clearance in NOD SCID mice is dependent on mFcγRII. Moreover, this result suggests that the somewhat reduced FcγRII expression seen previously in NOD SCID mice is not responsible for the fast hIgG1 clearance rate.

A number of autoimmune strains including NOD express the ly17.1 form of mFcγRII while most other in-bred strains, including BALB/c, express the ly17.2 variant.30 These two polymorphic forms vary in four amino acids, three of which are located in the extracellular domain.30 The extracellular domain of FcγRII from SCID and NOD SCID mice was cloned and expressed; their relative affinity for IgG was then determined by SPR. Neither heat aggregated, pooled hIgG or individual isotypes of IgG displayed substantially different binding affinities to the ly17.1 and ly17.2 variants (table 1).

mFcγRII is expressed on both hematopoietic and non-hematopoietic cells.30 31 We, therefore, sought to determine which mFcγRII-expressing cells were responsible for the rapid clearance of hIgG1. Accordingly, NOD SCID mice were irradiated and reconstituted with bone marrow from NOD SCID FcγRII-/- mice (figure 4C,D). These mice, reconstituted with hematopoietic cells lacking mFcγRII displayed rapid clearance of hIgG1 and mIgG2a, indicating that mFcγRII on cells of the hematopoietic system were not responsible for the rapid mAb clearance (figure 4E). We next considered whether phagocytes, particularly tissue resident macrophages, might be responsible and so deleted them with clodronate liposomes. This approach effectively removed macrophages (online supplementary figure 3b) but only resulted in a small increase in circulating hIgG1 7 days after mAb administration, with no hIgG1 detectable by day 14 (figure 4F). This suggests that phagocytes in NOD SCID mice are not primarily responsible for the rapid hIgG1 clearance and that a non-hematopoietic cell type is responsible. Given their high expression of mFcγRII, the liver sinusoidal endothelial cells (LSEC) seem the most likely candidate.31 We confirmed high expression of mFcγRII on these cells by immunofluorescence of livers from both BALB/c and NOD SCID mice (online supplementary figure 4a and b). Moreover, we found hIgG detectable at substantially higher levels within the liver of NSG than SCID mice following administration of hIgG1 mAb confirming a role for the liver as a site of hIgG1 accumulation (online supplementary figure 4c).

As mFcγRII is not known to directly regulate mAb clearance, we next considered whether FcRn might be involved in the process of controlling clearance rate in the NOD SCID mouse. Importantly, FcRn in NOD SCID mice does not to contain any sequence variations compared with other strains and has normal binding to both human and mouse IgG.14 However, qPCR revealed significantly lower FcRn transcription in both spleen and liver of NOD SCID versus SCID mice (figure 5A). This result was confirmed at the protein level by western blotting (figure 5B) and flow cytometry using the MST-HN protein which maintains FcRn binding at both acidic and neutral pH (figure 5C).32 This latter approach demonstrated a lower expression of FcRn in Ly6C+monocytes from NSG compared with SCID mice (MFI 354 vs 3061, mean of n=4). Combined, these results demonstrate that there is a lower expression of FcRn in the tissues known to be important for IgG recycling (spleen and liver) of NOD SCID mice compared with SCID.33 mFcγRII has a pH dependent affinity for IgG isotypes and is expressed on the same cell types as FcRn.

While reduced FcRn could explain rapid IgG clearance, it does not provide an explanation for the isotype dependent nature of the effects seen, as all isotypes should be affected equally. In contrast, mFcγRII is known to display differential affinity for IgG isotypes (high for mIgG1, low for mIgG2a and hIgG1) and so we considered if mFcγRII specificity might be involved in regulating the clearance of the different isotypes. Using SPR and two different mAb of each isotype, we confirmed that at pH7.4 mFcγRII had ~10-fold higher affinity for mIgG1 (2.74×10^–7M) vs mIgG2a (1.18×10^–6M) and hIgG1 (3.02×10^–6) while the affinity for hIgG2 was lower still (7.65×10^–6M) (table 2 and figure 5D).

We next considered that mFcγRII might internalize cell-surface bound IgG and by virtue of its higher affinity, preferentially protect mIgG1 from degradation following internalization. To do this, it would need to remain bound to IgG in a low pH environment, akin to FcRn. We therefore repeated SPR analysis at pH6.0, and revealed that mFcγRII retained binding at low pH, with affinity for mIgG1, mIgG2a and hIgG1 ~100-fold higher than at pH7.4. Notably, the KD for mIgG1 binding mFcγRII was 2.77×10^–9M,>10-fold higher than for hIgG1 and mIgG2a. This suggests that mFcγRII is capable of binding IgG at an acidic pH with the potential to protect IgG from degradation being greatest for mIgG1. Using previously published affinity data for IgG binding to mFcRn, we calculated the ratio of mFcγRII:FcRn binding for different isotypes at pH6.0 (table 2 and figure 5D).34 hIgG2 exhibited a high mFcγRII:FcRn ratio, suggesting preferential binding for FcRn at an acidic pH. In comparison, hIgG1 had a ratio around 1 (indicating no overall preference) whereas mIgG1 had a low ratio, preferentially binding with a higher affinity to mFcγRII than to FcRn.

Having hypothesized that the differential interaction with FcRn and FcγRII may play a role in the recycling of IgG, and with the knowledge that the liver expresses 75% of the FcγRII in the mouse, we sought to determine the distribution of these two receptors within the liver.31 The majority of mFcγRII was expressed by LSEC as determined by their morphology (online supplementary figure 4). We did not see mFcγRII onClec4F ⁺ Kupffer cells or Cytokeratin 8 ⁺ hepatocytes. This is consistent with a previous study reporting 90% of the liver mFcγRII as being expressed by LSEC when assessing immunofluorescence by pixel intensity.31 We found FcRn to be widely expressed throughout the liver including on Kupffer cells and hepatocytes (online supplementary figure 4). This is consistent with a previous report showing FcRn mRNA in various cell types, additionally identifying LSEC as having the highest expression level.31 Together, these results suggest that LSEC may be the predominant cell type coexpressing FcγRII and FcRn.

Overall, these data provided the possibility that antibody clearance in NOD SCID strains is controlled through differential engagement of the various isotypes by mFcγRII and FcRn. However, these effects have not been reported previously in standard in-bred strains, or the single NOD and SCID strains, indicating that the proposed pathway, which is at least in part mediated by isotype-dependent binding of mFcγRII, is only revealed in the absence of endogenous IgG.

We, therefore, hypothesized that the addition of exogenous mIgG would restore normal mAb clearance rate in NOD SCID mice. Accordingly, SCID and NOD SCID mice were reconstituted with mIgG1 and mIgG2a to a level equivalent to that seen in the plasma of wild type BALB/c mice (online supplementary figure 5). Subsequently, the clearance of hIgG1 was investigated (figure 5E). Reconstitution with mIgG overcame the rapid clearance of hIgG1 in NOD SCID mice such that it became comparable to that observed in SCID mice. In contrast, mIgG addition did not significantly alter the clearance of hIgG in SCID mice. Additionally, reconstitution with mIgG substantially reduced the accumulation of hIgG in the liver (online supplementary figure 4c). Importantly, the expression of FcRn was not altered by reconstitution with mIgG (figure 5C). Finally, we sought to determine if overcoming rapid clearance of hIgG1 by mIgG reconstitution could improve therapy, using an Eμ-TCL1 tumor and a hIgG1 antibody targeting mouse CD20 (18B12). Using this second tumor model we found the differences in duration of therapy between SCID and NSG mice was maintained, with tumor growth recurring in NSG before SCID mice (figure 5F). We then compared the duration of tumor deletion in NSG mice versus NSG mice reconstituted with mIgG, using the same protocol as before. Reconstitution with mIgG was able to overcome the impaired therapy and restored comparable levels of tumor deletion and control to that observed in SCID mice both in terms of tumor percentage (figure 5F) and number of tumor cells (online supplementary figure 6). This demonstrates that reconstitution with mIgG is able to restore mAb efficacy in NSG mice.