Fcγ receptors (FcγRs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal FcγR engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the FcγR gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region.

Convincing evidence exists to support a role for skewed XCI in female subjects with AITD, which may, in part, explain the strong female preponderance observed in this disease.

Key Points• TGN1412-induced T-cell activation following highdensity preculture of PBMCs is a consequence of FcgRIIb upregulation on monocytes.• In vivo, cytokine release syndrome may be due to the close association of FcgRIIbbearing cells with T cells in lymphoid tissues.The anti-CD28 superagonist antibody TGN1412 caused life-threatening cytokine release syndrome (CRS) in healthy volunteers, which had not been predicted by preclinical testing. T cells in fresh peripheral blood mononuclear cells (PBMCs) do not respond to soluble TGN1412 but do respond following high-density (HD) preculture. We show for the first time that this response is dependent on crystallizable fragment gamma receptor IIb (FcgRIIb) expression on monocytes. This was unexpected because, unlike B cells, circulating monocytes express little or no FcgRIIb. However, FcgRIIb expression is logarithmically increased on monocytes during HD preculture, and this upregulation is necessary and sufficient to explain TGN1412 potency after HD preculture. B-cell FcgRIIb expression is unchanged by HD preculture, but B cells can support TGN1412-mediated T-cell proliferation when added at a frequency higher than that in PBMCs. Although lowdensity (LD) precultured PBMCs do not respond to TGN1412, T cells from LD preculture are fully responsive when cocultured with FcgRIIb-expressing monocytes from HD preculture, which shows that they are fully able to respond to TGN1412-mediated activation. Our novel findings demonstrate that cross-linking by FcgRIIb is critical for the superagonist activity of TGN1412 after HD preculture, and this may contribute to CRS in humans because of the close association of FcgRIIb-bearing cells with T cells in lymphoid tissues. (Blood. 2015;125(1):102-110)

SummaryThe advent of next-generation sequencing (NGS) and 'omic' technologies has revolutionized the field of genetics, and its implementation in health care has the potential to realize precision medicine. Primary immunodeficiencies (PID) are a group of rare diseases which have benefited from NGS, with a massive increase in causative genes identified in the past few years. Common variable immunodeficiency disorders (CVID) are a heterogeneous form of PID and the most common form of antibody failure in children and adults. While a monogenic cause of disease has been identified in a small subset of CVID patients, a genomewide association study and whole genome sequencing have found that, in the majority, a polygenic cause is likely. Other NGS technologies such as RNA sequencing and epigenetic studies have contributed further to our understanding of the contribution of altered gene expression in CVID pathogenesis. We believe that to unravel further the complexities of CVID, a multi-omic approach, combining DNA sequencing with gene expression, methylation, proteomic and metabolomics data, will be essential to identify novel disease-associated pathways and therapeutic targets.

Graves’ disease results from thyroid-stimulating Abs (TSAbs) activating the thyrotropin receptor (TSHR). How TSAbs arise from early precursor B cells has not been established. Genetic and environmental factors may contribute to pathogenesis, including the bacterium Yersinia enterocolitica. We developed two pathogenic monoclonal TSAbs from a single experimental mouse undergoing Graves’ disease, which shared the same H and L chain germline gene rearrangements and then diversified by numerous somatic hypermutations. To address the Ag specificity of the shared germline precursor of the monoclonal TSAbs, we prepared rFab germline, which showed negligible binding to TSHR, indicating importance of somatic hypermutation in acquiring TSAb activity. Using rFab chimeras, we demonstrate the dominant role of the H chain V region in TSHR recognition. The role of microbial Ags was tested with Y. enterocolitica proteins. The monoclonal TSAbs recognize 37-kDa envelope proteins, also recognized by rFab germline. MALDI-TOF identified the proteins as outer membrane porin (Omp) A and OmpC. Using recombinant OmpA, OmpC, and related OmpF, we demonstrate cross-reactivity of monoclonal TSAbs with the heterogeneous porins. Importantly, rFab germline binds recombinant OmpA, OmpC, and OmpF confirming reactivity with Y. enterocolitica. A human monoclonal TSAb, M22 with similar properties to murine TSAbs, also binds recombinant porins, showing cross-reactivity of a spontaneously arising pathogenic Ab with Y. enterocolitica. The data provide a mechanistic framework for molecular mimicry in Graves’ disease, where early precursor B cells are expanded by Y. enterocolitica porins to undergo somatic hypermutation to acquire a cross-reactive pathogenic response to TSHR.

Cancer immunotherapy has been revolutionised by the use monoclonal antibodies (mAb) that function through their interaction with Fc gamma receptors (FcγRs). The low-affinity FcγR genes are highly homologous, map to a complex locus at 1p23 and harbour single nucleotide polymorphisms (SNPs) and copy number variation (CNV) that can impact on receptor function and response to therapeutic mAbs. This complexity can hinder accurate characterisation of the locus. We therefore evaluated and optimised a suite of assays for the genomic analysis of the FcγR locus amenable to peripheral blood mononuclear cells and formalin-fixed paraffin-embedded (FFPE) material that can be employed in a high-throughput manner. Assessment of TaqMan genotyping for FCGR2A-131H/R, FCGR3A-158F/V and FCGR2B-232I/T SNPs demonstrated the need for additional methods to discriminate genotypes for the FCGR3A-158F/V and FCGR2B-232I/T SNPs due to sequence homology and CNV in the region. A multiplex ligation-dependent probe amplification assay provided high quality SNP and CNV data in PBMC cases, but there was greater data variability in FFPE material in a manner that was predicted by the BIOMED-2 multiplex PCR protocol. In conclusion, we have evaluated a suite of assays for the genomic analysis of the FcγR locus that are scalable for application in large clinical trials of mAb therapy. These assays will ultimately help establish the importance of FcγR genetics in predicting response to antibody therapeutics.

Monoclonal antibody (mAb) immunotherapy has transformed the treatment of allergy, autoimmunity, and cancer. The interaction of mAb with Fc gamma receptors (FcγR) is often critical for efficacy. The genes encoding the low-affinity FcγR have single nucleotide polymorphisms (SNPs) and copy number variation that can impact IgG Fc:FcγR interactions. Leukocyte-based in vitro assays remain one of the industry standards for determining mAb efficacy and predicting adverse responses in patients. Here we addressed the impact of FcγR genetics on immune cell responses in these assays and investigated the importance of assay format. FcγR genotyping of 271 healthy donors was performed using a Multiplex Ligation-Dependent Probe Amplification assay. Freeze-thawed/pre-cultured peripheral blood mononuclear cells (PBMCs) and whole blood samples from donors were stimulated with reagents spanning different mAb functional classes to evaluate the association of FcγR genotypes with T-cell proliferation and cytokine release. Using freeze-thawed/pre-cultured PBMCs, agonistic T-cell-targeting mAb induced T-cell proliferation and the highest levels of cytokine release, with lower but measurable responses from mAb which directly require FcγR-mediated cellular effects for function. Effects were consistent for individual donors over time, however, no significant associations with FcγR genotypes were observed using this assay format. In contrast, significantly elevated IFN-γ release was associated with the FCGR2A-131H/H genotype compared to FCGR2A-131R/R in whole blood stimulated with Campath (p ≤ 0.01) and IgG1 Fc hexamer (p ≤ 0.05). Donors homozygous for both the high affinity FCGR2A-131H and FCGR3A-158V alleles mounted stronger IFN-γ responses to Campath (p ≤ 0.05) and IgG1 Fc Hexamer (p ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14hi monocytes, CD56dim NK cells (p ≤ 0.05) and FcγRIIIa expression (p ≤ 0.05), in donor-matched freeze-thawed PBMC compared to whole blood samples, likely explaining the difference in association between FcγR genotype and mAb-mediated cytokine release in the different assay formats. These findings highlight the significant impact of FCGR2A and FCGR3A SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release in vitro. This knowledge can better inform on the utility of in vitro assays for the prediction of mAb therapy outcome in patients.

A non‐gall‐forming aphid, Macrosiphum euphorbiae, was reared within the galls of Dysaphis devecta and Aphis pomi which had been induced in apple seedlings. Similar aphids were also reared on ungalled seedling leaves of a similar age and also on mature leaves and in old galls. There is a weak positive linear relationship between lipid content and pre‐reproductive adult dry weight, but aphids living on young galled or ungalled plant tissue have 30% lipid compared to 11% for aphids living on old plant tissue. Adult aphids gradually decline in weight, losing between 24% and 38% of their pre‐reproductive biomass before death. The energy content of this loss subsidises reproduction. Reproduction of aphids feeding in old galls or on mature leaves was negligible, but on younger tissues average fecundity was 31, there being little difference between aphids living on galled and ungalled tissue. There is no simple relationship between pre‐reproductive adult embryo content and fecundity. The principal factor mediating honeydew production was aphid size and this factor overrides any variation caused by different feeding sites, Honeydew production averaged 0.43 mg during a life of 30 days. Aphids successfully completing their life cycle have a life‐time energy consumption of 30.58 joules of which 18% is lost as heat during respiration (R), 30% is contained in honeydew (F + U), 1% is contained in exuviae (Pe). Larval growth accounts for 17% (Pg), and adult reproduction for 34% (Pr). The P/C ratio for M. euphorbiae is 53% and is broadly comparable with aphids living on herbaceous plants. RÉSUMÉ Biologie et énergétique du puceron Macrosiphum euphorbiae vivant dans des galles des pucerons Dysaphis devecta et Aphis pomi Un puceron ne formant pas de galles, Macrosiphum euphorbiae, a été élevé dans des galles de Dysaphis devecta et Aphis pomi formées sur semis de pommiers. D'autres M. euphorbiae ont été élevés sur feuilles sans galles du měme ǎge provenant de semis identiques, sur feuilles mǔres et sur vieilles galles. Il y a une faible relation positive linéaire entre la teneur en lipides et le poids sec des adultes avant la reproduction, mais les pucerons élevés sur tissus végétaux jeunes avec ou sans galles contiennent 30% de lipides contre 11% pour ceux élevés sur tissus ǎgés. Le poids des pucerons adultes diminue progressivement, la perte avant la mort se situant entre 24 et 38% de la biomasse avant la reproduction. Le contenu énergétique de ces pertes alimente la reproduction. La reproduction de pucerons consommant les vieilles galles ou les feuilles mǔres était négligeable, mais sur jeunes tissus la fécondité moyenne était de 31 – avec peu de différences entre pucerons sur galles ou feuilles –. Il n'y a pas de corrélation simple entre le contenu en embryon des adultes avant la reproduction et la fécondité. Le principal paramètre conditionnant la production de miellat est la taille du puceron; ce paramètre surpasse toute variation provoquée par les divers lieux d'alimentation. La product...