Complement component C1q is an immunological rheostat that regulates Fc:Fc[Formula: see text]R interactions.

Though binding sites for the complement factor C1q and the canonical fragment crystallizable (Fc) gamma receptors (Fc[Formula: see text]Rs) on immunoglobulin G (IgG) molecules overlap, how C1q decoration of immune complexes (ICs) influences their ability to engage Fc[Formula: see text]Rs remains unknown. In this report, we use recombinant human Fc multimers as stable IC mimics to show that C1q engagement of ICs directly and transiently inhibits their interactions with Fc[Formula: see text]RIII (CD16) on human natural killer (NK) cells. This inhibition occurs by C1q engagement alone as well as in concert with other serum factors. Furthermore, the inhibition of Fc[Formula: see text]RIII engagement mediated by avid binding of C1q to ICs is directly associated with IC size and dependent on the concentrations of both C1q and Fc multimers present. Functionally, C1q-mediated Fc blockade limits the ability of NK cells to induce the upregulation of the cosignaling molecule, 4-1BB (CD137), and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Although C1q is traditionally viewed as a soluble effector molecule, we demonstrate that C1q may also take on the role of an "immunologic rheostat," buffering Fc[Formula: see text]R-mediated activation of immune cells by circulating ICs. These data define a novel role for C1q as a regulator of immune homeostasis and add to our growing understanding that complement factors mediate pleiotropic effects.

NK cell expression of Tim-3: First impressions matter.

Given the heightened interest in manipulation of co-signaling cascades for cancer immunotherapy, we sought to determine how/whether tumors decorated with therapeutic monoclonal antibodies (mAbs) impact the expression of co-signaling molecules on human NK cells. Stimulation of NK cells with aggregated IgG1 resulted in the upregulation of HAVCR2 - the gene encoding T-cell immunoglobulin and mucin-containing domain (Tim)-3 - known to be involved in the induction of peripheral T cell tolerance. This upregulation of HAVCR2 was recapitulated at the protein level, following NK cell stimulation by either mAb opsonized tumors, recombinant human IgG1 Fc multimer, and/or non-Fc stimuli e.g. IL-12/IL-18. The patterns of Tim-3 expression were temporally distinct from the FcR mediated induction of the co-signaling molecule, 4-1BB (CD137), with Tim-3 increases observed twenty minutes following exposure to Fc multimers and remaining at high levels for at least six hours, while increases in CD137 expression were first observed at the four-hour time point. Importantly, these Tim-3+ NK cells were functionally diverse, as evidenced by the fact that their ability to produce IFN-γ in response to an NK cell responsive tumor was strictly dependent upon the stimuli employed for Tim-3 induction. These data suggest that Tim-3 upregulation is the common end-result of NK cell activation by a variety of unique and overlapping stimuli and is not an independent marker of NK cell exhaustion. Furthermore, our observations potentially explain the diverse functionality attributed to Tim-3+ NK cells and should be considered prior to use of anti-Tim-3 inhibitory mAbs for cancer immunotherapy.

Impact of Detachment Methods on M2 Macrophage Phenotype and Function.

The methods of cell detachment influence phenotype and function of human macrophages cultured in vitro. However, comparative studies defining the influence of cell detachment techniques on secondary characterization of M1 or M2 polarized macrophages are largely absent from the literature. In this study we evaluated the impact of trypsin, accutase, EDTA, PBS, and cell scraping on: A. cell recovery, B. phenotype and C. function of in vitro polarized macrophages. Our data demonstrate that while exposure to trypsin or accutase yields highly efficient recovery of viable cells, such chemical cleavage results in loss of select M2 cell surface markers with correlative changes in cell function. In contrast, phenotype and function are maintained following detachment by EDTA on ice. Our data suggest that seemingly "trivial" changes in methodologies for macrophage detachment induce both variable and profound changes on cell phenotype and function which can dramatically impact the results of polarization experiments.

The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII(-/-) mice.

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc-FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR(-/-) strains. 2A-treated FcRγ(-/-) mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII(-/-) animals. These improved anti-tumor responses were associated with increased splenic CD8ß T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.

Tumour antigen targeted monoclonal antibodies incorporating a novel multimerisation domain significantly enhance antibody dependent cellular cytotoxicity against colon cancer.

Tumour antigen targeted antibodies (mAbs) can induce natural killer (NK) cells to kill tumours through antibody dependent cellular cytotoxicity (ADCC) upon engagement of NK cell expressed FcγRIIIa. FcγRIIIa polymorphisms partially dictate the potency of the ADCC response. The high affinity FcγRIIIa-158-valine (V) polymorphism is associated with more potent ADCC response than the low affinity FcγRIIIa-158-phenylalanine (F) polymorphism. Because approximately 45% of patients are homozygous for the FcγRIIIa-158-F polymorphism (FF genotype), their ability to mount ADCC is impaired. We investigated whether a novel mAb capable of binding multiple antigen specific targets and engaging multiple low affinity FcγRIIIa receptors could further enhance ADCC against colon cancer in vitro. Specifically, we generated a novel anti-epidermal growth factor receptor (EGFR) antibody (termed a stradobody) consisting of an unmodified Fab sequence and two Immunoglobulin G, subclass 1 (IgG1) Fc domains separated by an isoleucine zipper domain and the 12 amino-acid IgG2 hinge. The stradobody framework induced multimerisation and was associated with increased binding to the EGFR and FcγRIIIa. From a functional perspective, when compared to an unmodified anti-EGFR mAb with a sequence identical to cetuximab (a commercially available anti-EGFR mAb), stradobodies significantly enhanced ADCC. These effects were observed using both KRAS wild type HT29 and KRAS mutant SW480 colon cancer cells as targets, and by NK cells obtained from healthy donors and a cohort of patients with colon cancer. These data suggest that high avidity cross-linking of multiple tumour surface antigens and multiple NK cell associated FcγRIIIa molecules can enhance ADCC and partially overcome impaired ADCC by FF genotype individuals in vitro.

A high throughput method for enrichment of natural killer cells and lymphocytes and assessment of in vitro cytotoxicity.

In vitro assessment of lymphocyte and natural killer (NK) cell cytotoxicity typically employs density gradient centrifugation and magnetic cell separation to isolate effector cells, and chromium release to assess cytotoxicity. In order to improve the rapidity and scalability of in vitro cytotoxicity assessment, we evaluated the efficacy of a protocol utilizing tetrameric antibody complexes and SepMate™ isolation tubes to negatively select NK cells (TACs/Sep), and calcein-AM release to measure cytotoxicity. We compared the efficiency and accuracy of this protocol to a conventional approach employing density gradient centrifugation and magnetically labeled antibodies (DG/MACS) to isolate NK cells and chromium release to measure cytotoxicity. The TACs/Sep method significantly decreased the time required for NK cell isolation (1h vs. 4h), but resulted in higher red blood cell contamination. NK cell activation marker expression (including CD94, NKG2D, NKp30, NKp46, DNAM-1, 2B4, KIR2DL1/S1, KIR2DL2/L3, intracellular granzyme B, and perforin) was similar when comparing NK cells isolated by the TACs/Sep or DG/MACS methods, but the TACs/Sep method induced higher expression of CD16. In vitro cytotoxicity against HT29 colon cancer and K562 leukemia cells was not affected by the isolation method. Lastly, by combining the TACs/Sep NK cell isolation method with calcein-acetoxymethyl diacetylester (calcein-AM) release, the time required to assess in vitro cytotoxicity was reduced by 33% (4h) compared to protocols employing DG/MACS and chromium release. Altogether, these results provide the foundation for the development of a rapid, high throughput functional assay, and make it practical for the multiplexing of downstream applications, such as flow cytometric analysis and enzyme-linked immunosorbent assays (ELISAs).

A novel mechanism of thyroid hormone-dependent negative regulation by thyroid hormone receptor, nuclear receptor corepressor (NCoR), and GAGA-binding factor on the rat cD44 promoter.

CD44 is an adhesion molecule in the extracellular matrix that shows various functions, including tumor genesis and metastasis. A recent study showed that CD44 expression level was strongly correlated with the generation of papillary thyroid carcinomas, the most prevalent malignancy of the thyroid gland. We report here that CD44 is negatively regulated by thyroid hormone (T(3)) through a novel mechanism. We demonstrate that nuclear receptor corepressor (NCoR) enhances thyroid hormone receptor (TR)-mediated basal transactivation by a weak TR.DNA interaction in the absence of T(3), which is repressed by T(3) through a transient TR .DNA interaction. Initially, we identified that CD44 was negatively directly transcriptionally T(3) -responsive. Deletion and mutation analysis indicated that both a weak TR and a GAGA-binding factor (GAF) binding sites on the CD44 promoter were required for negative regulation by T(3). The weak TR.DNA interaction was further confirmed by electrophoretic gel mobility shift assay, chromatin immunoprecipitation, and transfection assays using a non-DNA-binding TRalpha1 mutant. More interestingly, NCoR acted as a co-activator to enhance TR-mediated basal transactivation in the absence of T(3). This effect was eliminated by removal of TR or NCoR binding. Most strikingly, T(3) induced a remarkable increase in TR.DNA binding at 40-60 min after T(3) exposure that rapidly returned to basal levels, suggesting a T(3)-induced remodeling of chromatin structure at the early stage of T(3) stimulation resulting in repression. Therefore, we propose a mechanism by which NCoR, GAF, and TR interact with the CD44 negative T(3)-responsive element to enhance basal transactivation, whereas T(3) induces the remodeling of chromatin structure for repression.

A novel cell type-specific mechanism for thyroid hormone-dependent negative regulation of the human type 1 deiodinase gene.

We have identified a cell type-specific, negative thyroid hormone-responsive element in the human type 1 iodothyronine deiodinase (hdio1) gene. This fragment, termed a JEG response element, bound tightly to a JEG-cell nuclear protein [JEG cell-specific transcription factor (JTF)] also present in placenta but not in COS-7, HeLa, or human embryonic kidney-293 cells. In JEG-3 cells, three copies of the JEG response element conferred a more than 40-fold transcriptional stimulation to the heterologous rat GH promoter which was further increased 2-fold by apo-thyroid hormone receptor (TR) and reduced 3-fold by T(3). Dimethyl sulfide footprinting showed overlapping contact sites for the high-affinity interaction of JTF and low-affinity binding of TR-retinoid X receptor. Expression of the same construct was unaffected by TR or T(3) in COS cells, indicating JTF was required for negative regulation by T(3)-TR. Mutations of the critical thyroid hormone responsive element binding P box amino acids EG to GS in TRalpha1 or TRbeta2 eliminated the apo-TR and T(3)-TR effects. These studies identify a novel mechanism for cell type-specific, promoter-independent negative regulation by T(3).