Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies.

Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity.

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox.

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

Targeting the high affinity receptor, FcγRI, in autoimmune disease, neuropathy, and cancer.

The Fc gamma receptor I (FcγRI or CD64) is the only human Fc receptor with a high affinity for monomeric IgG. It plays a crucial role in immunity, as it mediates cellular effector functions of antibodies including phagocytosis, antigen presentation, and cytokine production. FcγRI is constitutively saturated with monomeric IgG and this feeds the dogma that it has no role in immune responses. However, recent findings have implicated a role for FcγRI in various autoimmune disorders, neuropathies, and antibody therapy in tumor models. By a process known as 'inside-out' signaling, stimulation of myeloid cells with cytokines such as tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) enhances FcγRI binding to immune complexes (ICs), including antibody-opsonized pathogens or tumor cells. This review focuses on the current knowledge on interaction of FcγRI with IgG and ICs and the effect of inside-out signaling on FcγRI functioning. Additionally, this review will address potential clinical applications of targeting FcγRI, and the tools that can be used to overcome IC-mediated autoimmune diseases on the one hand, and to enhance antibody-based anti-cancer therapy on the other.

Elevated Mutational Age in Blood of Children Treated for Cancer Contributes to Therapy-Related Myeloid Neoplasms.

Childhood cancer survivors are confronted with various chronic health conditions like therapy-related malignancies. However, it is unclear how exposure to chemotherapy contributes to the mutation burden and clonal composition of healthy tissues early in life. Here, we studied mutation accumulation in hematopoietic stem and progenitor cells (HSPC) before and after cancer treatment of 24 children. Of these children, 19 developed therapy-related myeloid neoplasms (t-MN). Posttreatment HSPCs had an average mutation burden increase comparable to what treatment-naïve cells accumulate during 16 years of life, with excesses up to 80 years. In most children, these additional mutations were induced by clock-like processes, which are also active during healthy aging. Other patients harbored mutations that could be directly attributed to treatments like platinum-based drugs and thiopurines. Using phylogenetic inference, we demonstrate that most t-MN in children originate after the start of treatment and that leukemic clones become dominant during or directly after chemotherapy exposure. SIGNIFICANCE: Our study shows that chemotherapy increases the mutation burden of normal blood cells in cancer survivors. Only few drugs damage the DNA directly, whereas in most patients, chemotherapy-induced mutations are caused by processes similar to those present during normal aging. This article is highlighted in the In This Issue feature, p. 1825.

MutationalPatterns: the one stop shop for the analysis of mutational processes.

BACKGROUND: The collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data. RESULTS: Here, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights. CONCLUSIONS: This novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns .

Plasmodium early transcribed membrane proteins appear tailored to the host range of malaria parasites.

Early transcribed membrane proteins form a unique protein family in malaria parasites. These molecules are expressed during Plasmodium intracellular phases and inserted at the parasite parasitophorus vacuole membrane, which constitutes the host-parasite interface. Upregulated in infectious sporozoites 4 (UIS4) is an essential early transcribed membrane protein of liver stages of the murine malaria model parasite Plasmodium berghei. Despite its relevance for liver stage maturation, the molecular functions of UIS4 remain elusive, and UIS4 orthologs in human malaria parasites have not yet been identified. In order to characterise functional domains of UIS4, we generated P. berghei parasites carrying a carboxy-terminally truncated version of UIS4. We observed that uis4Δc parasites are severely impaired in liver stage development, similar to uis4(-) parasites, indicating an important role of the C-terminal domain for UIS4 function. To test whether members of the P. falciparum early transcribed membrane protein family are potential UIS4 orthologs, we selected candidates based on structural homology and parasitophorous vacuole membrane localization. We generated transgenic P. berghei parasites where UIS4 was replaced by Plasmodium falciparum ETRAMP8 or ETRAMP10.3. Both early transcribed membrane proteins were expressed in transgenic parasite lines, but liver stage maturation was impaired, indicating that the selected early transcribed membrane proteins failed to substitute the function of UIS4. As a control, we included the UIS4 ortholog from the murine parasite Plasmodium chaubaudi. We observed that PcUIS4 successfully restores UIS4 function in P. berghei. Together, these results suggest that Plasmodium parasites express tailor-made parasitophorous vacuole membrane proteins that might at least partially explain the narrow host range of malaria parasites.

Mutation signatures of pediatric acute myeloid leukemia and normal blood progenitors associated with differential patient outcomes.

Acquisition of oncogenic mutations with age is believed to be rate limiting for carcinogenesis. However, the incidence of leukemia in children is higher than in young adults. Here we compare somatic mutations across pediatric acute myeloid leukemia (pAML) patient-matched leukemic blasts and hematopoietic stem and progenitor cells (HSPCs), as well as HSPCs from age-matched healthy donors. HSPCs in the leukemic bone marrow have limited genetic relatedness and share few somatic mutations with the cell-of-origin of the malignant blasts, suggesting polyclonal hematopoiesis in pAML patients. Compared to normal HSPCs, a subset of pAML cases harbored more somatic mutations and a distinct composition of mutational process signatures. We hypothesize these cases might have arisen from a more committed progenitor. This subset had better outcomes than pAML cases with mutation burden comparable to age-matched healthy HSPCs. Our study provides insights into the etiology and patient stratification of pAML.

Antiviral treatment causes a unique mutational signature in cancers of transplantation recipients.

Genetic instability is a major concern for successful application of stem cells in regenerative medicine. However, the mutational consequences of the most applied stem cell therapy in humans, hematopoietic stem cell transplantation (HSCT), remain unknown. Here we characterized the mutation burden of hematopoietic stem and progenitor cells (HSPCs) of human HSCT recipients and their donors using whole-genome sequencing. We demonstrate that the majority of transplanted HSPCs did not display altered mutation accumulation. However, in some HSCT recipients, we identified multiple HSPCs with an increased mutation burden after transplantation. This increase could be attributed to a unique mutational signature caused by the antiviral drug ganciclovir. Using a machine learning approach, we detected this signature in cancer genomes of individuals who received HSCT or solid organ transplantation earlier in life. Antiviral treatment with nucleoside analogs can cause enhanced mutagenicity in transplant recipients, which may ultimately contribute to therapy-related carcinogenesis.

Biology and Clinical Applicability of Plasma Thymus and Activation-Regulated Chemokine (TARC) in Classical Hodgkin Lymphoma.

Thymus and activation-regulated chemokine (TARC) is produced by different cell types and is highly expressed in the thymus. It plays an important role in T cell development, trafficking and activation of mature T cells after binding to its receptor C-C chemokine receptor type 4 (CCR4) and consecutive signal transducer and activator of transcription 6 (STAT6) activation. Importantly, TARC is also produced by malignant Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). In cHL, HRS cells survive and proliferate due to the micro-environment consisting primarily of type 2 T helper (Th2) cells. TARC-mediated signaling initiates a positive feedback loop that is crucial for the interaction between HRS and T cells. The clinical applicability of TARC is diverse. It is useful as diagnostic biomarker in both children and adults with cHL and in other Th2-driven diseases. In adult cHL patients, TARC is also a biomarker for treatment response and prognosis. Finally, blocking TARC signaling and thus inhibiting pathological Th2 cell recruitment could be a therapeutic strategy in cHL. In this review, we summarize the biological functions of TARC and focus on its role in cHL pathogenesis and as a biomarker for cHL and other diseases. We conclude by giving an outlook on putative therapeutic applications of antagonists and inhibitors of TARC-mediated signaling.

Potent Fc Receptor Signaling by IgA Leads to Superior Killing of Cancer Cells by Neutrophils Compared to IgG.

Antibody therapy of cancer is increasingly used in the clinic and has improved patient's life expectancy. Except for immune checkpoint inhibition, the mode of action of many antibodies is to recognize overexpressed or specific tumor antigens and initiate either direct F(ab')2-mediated tumor cell killing, or Fc-mediated effects such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/P) after binding to activating Fc receptors. All antibodies used in the clinic are of the IgG isotype. The IgA isotype can, however, also elicit powerful anti-tumor responses through engagement of the activating Fc receptor for monomeric IgA (FcαRI). In addition to monocytes, macrophages and eosinophils as FcαRI expressing immune cells, neutrophils are especially vigorous in eliminating IgA opsonized tumor cells. However, with IgG as single agent it appears almost impossible to activate neutrophils efficiently, as we have visualized by live cell imaging of tumor cell killing. In this study, we investigated Fc receptor expression, binding and signaling to clarify why triggering of neutrophils by IgA is more efficient than by IgG. FcαRI expression on neutrophils is ~2 times and ~20 times lower than that of Fcγ receptors FcγRIIa and FcγRIIIb, but still, binding of neutrophils to IgA- or IgG-coated surfaces was similar. In addition, our data suggest that IgA-mediated binding of neutrophils is more stable compared to IgG. IgA engagement of neutrophils elicited stronger Fc receptor signaling than IgG as indicated by measuring the p-ERK signaling molecule. We propose that the higher stoichiometry of IgA to the FcαR/FcRγ-chain complex, activating four ITAMs (Immunoreceptor Tyrosine-based Activating Motifs) compared to a single ITAM for FcγRIIa, combined with a possible decoy role of the highly expressed FcγRIIIb, explains why IgA is much better than IgG at triggering tumor cell killing by neutrophils. We anticipate that harnessing the vast population of neutrophils by the use of IgA monoclonal antibodies can be a valuable addition to the growing arsenal of antibody-based therapeutics for cancer treatment.

Glutaminyl cyclase is an enzymatic modifier of the CD47- SIRPα axis and a target for cancer immunotherapy.

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.

Mechanisms of inside-out signaling of the high-affinity IgG receptor FcγRI.

Fc receptors (FcRs) are an important bridge between the innate and adaptive immune system. Fc gamma receptor I (FcγRI; CD64), the high-affinity receptor for immunoglobulin G (IgG), plays roles in inflammation, autoimmune responses, and immunotherapy. Stimulation of myeloid cells with cytokines, such as tumor necrosis factor-α ( TNFα) and interferon-γ ( IFNγ), increases the binding of FcγRI to immune complexes (ICs), such as antibody-opsonized pathogens or tumor cells, through a process known as "inside-out" signaling. Using super-resolution imaging, we found that stimulation of cells with IL-3 also enhanced the clustering of FcγRI both before and after exposure to ICs. This increased clustering was dependent on an intact actin cytoskeleton. We found that chemical inhibition of the activity of the phosphatase PP1 reduced FcγRI inside-out signaling, although the phosphorylation of FcγRI itself was unaffected. Furthermore, the antibody-dependent cytotoxic activity of human neutrophils toward CD20-expressing tumor cells was increased after stimulation with TNFα and IFNγ. These results suggest that nanoscale reorganization of FcγRI, stimulated by cytokine-induced, inside-out signaling, enhances FcγRI cellular effector functions.

FcαRI Dynamics Are Regulated by GSK-3 and PKCζ During Cytokine Mediated Inside-Out Signaling.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Single Nucleotide Polymorphisms of the High Affinity IgG Receptor FcγRI Reduce Immune Complex Binding and Downstream Effector Functions.

Binding of IgG Abs to FcγRs on immune cells induces FcγR cross-linking that leads to cellular effector functions, such as phagocytosis, Ab-dependent cellular cytotoxicity, and cytokine release. However, polymorphisms in low affinity FcγRs have been associated with altered avidity toward IgG, thereby substantially impacting clinical outcomes of multimodular therapy when targeting cancer or autoimmune diseases with mAbs as well as the frequency and severity of autoimmune diseases. In this context, we investigated the consequences of three nonsynonymous single nucleotide polymorphisms (SNPs) for the high affinity receptor for IgG, FcγRI. Only SNP V39I, located in the extracellular domain of FcγRI, reduces immune-complex binding of FcγRI whereas monomeric IgG binding is unaffected. This leads to reduced FcγRI effector functions, including Fc receptor γ-chain signaling and intracellular calcium mobilization. SNPs I301M and I338T, located in the transmembrane or intracellular domain, respectively, have no influence on monomeric IgG or immune complex binding, but FcRγ signaling is decreased for both SNPs, especially for I338T. We also found that the frequency of these SNPs in a cohort of healthy Dutch individuals is very low within the population. To our knowledge, this study addresses for the first time the biological consequences of SNPs in the high affinity FcγR, and reveals reduction in several FcγRI functions, which have the potential to alter efficacy of therapeutic Abs.

Fc receptor inside-out signaling and possible impact on antibody therapy.

Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR-mediated signaling and triggering of cellular effector functions. FcR activation is tightly regulated to prevent immune responses by non-antigen bound antibodies or in the absence of 'danger signals'. FcR activity may be modulated at the plasma membrane via cross-talk with integrins. In addition, cytokines at the site of infection/inflammation can increase FcR avidity, a process referred to as inside-out signaling. This regulatory mechanism has been described for FcγRI (CD64), FcγRIIa (CD32a), and FcαRI (CD89) and is also well-known for integrins. Key cellular events during inside-out signaling are (de)phosphorylation, clustering, cytoskeleton rearrangements, and conformational changes. The latter can be studied with antibodies that specifically recognize epitopes exposed by the active (high affinity) or inactive (low affinity) state of the FcR. These antibodies are important tools to investigate the role of FcR activation in disease settings. Research on FcR has gained momentum with the rise of monoclonal antibodies (mAb) entering the clinic for the treatment of cancer and other diseases. The clinical outcome of mAb therapy may be improved by increasing FcR avidity by cytokine stimulation.

Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing.

Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressing effector cells and therefore initiate different effector mechanisms in vivo compared with IgG. Here, we studied killing of tumor cells coexpressing EGFR and HER2 by the IgG mAbs cetuximab and trastuzumab and their IgA variants. In the presence of a heterogeneous population of effector cells (leukocytes), the combination of IgG and IgA mAbs to two different tumor targets (EGFR and HER2) led to enhanced cytotoxicity compared with each isotype alone. Combination of two IgGs or two IgAs or IgG and IgA against the same target did not enhance cytotoxicity. Increased cytotoxicity relied on the presence of both the peripheral blood mononuclear cell and the polymorphonuclear (PMN) fraction. Purified natural killer cells were only cytotoxic with IgG, whereas cytotoxicity induced by PMNs was strong with IgA and poor with IgG. Monocytes, which coexpress FcγRs and FcαRI, also displayed increased cytotoxicity by the combination of IgG and IgA in an overnight killing assay. Coinjection of cetuximab and IgA2-HER2 resulted in increased antitumor effects compared with either mAb alone in a xenograft model with A431-luc2-HER2 cells. Thus, the combination of IgG and IgA isotypes optimally mobilizes cellular effectors for cytotoxicity, representing a promising novel strategy to improve mAb therapy.

A functional CFTR assay using primary cystic fibrosis intestinal organoids.

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.