Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

c-Maf-dependent Treg cell control of intestinal TH17 cells and IgA establishes host-microbiota homeostasis.

Foxp3+ regulatory T cells (Treg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (TH17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORγt+ Treg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled Treg cell-derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal Treg cells. c-Maf deficiency in Treg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host-microbe symbiosis.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T. gondii or Citrobacter rodentium infection, but also maintained the homeostatic amount of proIL-18 in the ileum. IL-22, however, did not induce the processing to active IL-18, suggesting a two-step regulation of IL-18 in these cells. Although IL-18 exerted pathogenic functions during ileitis triggered by T. gondii, it was required for host defense against C. rodentium. Conversely, IL-18 was required for the expression of IL-22 in innate lymphoid cells (ILCs) upon T. gondii infection. Our results define IL-18 as an IL-22 target gene in epithelial cells and describe a complex mutual regulation of both cytokines during intestinal infection.

Transcription factor c-Maf mediates the TGF-ß-dependent suppression of IL-22 production in T(H)17 cells.

Interleukin 22 (IL-22), which is produced by cells of the T(H)17 subset of helper T cells and other leukocytes, not only enhances proinflammatory innate defense mechanisms in epithelial cells but also provides crucial protection to tissues from damage caused by inflammation and infection. In T(H)17 cells, transforming growth factor-ß (TGF-ß) regulates IL-22 and IL-17 differently. IL-6 alone induces T cells to produce only IL-22, whereas the combination of IL-6 and high concentrations of TGF-ß results in the production of IL-17 but not IL-22 by T cells. Here we identify the transcription factor c-Maf, which is induced by TGF-ß, as a downstream repressor of Il22. We found that c-Maf bound to the Il22 promoter and was both necessary and sufficient for the TGF-ß-dependent suppression of IL-22 production in T(H)17 cells.

Functions and regulation of T cell-derived interleukin-10.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine and functions as a negative regulator of immune responses to microbial antigens. IL-10 is particularly important in maintaining the intestinal microbe-immune homeostasis. Loss of IL-10 promotes the development of inflammatory bowel disease (IBD) as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions more generally to prevent excessive inflammation during the course of infection. Although IL-10 can be produced by virtually all cells of the innate and adaptive immune system, T cells constitute a non-redundant source for IL-10 in many cases. The various roles of T cell-derived IL-10 will be discussed in this review. Given that IL-10 is at the center of maintaining the delicate balance between effective immunity and tissue protection, it is not surprising that IL-10 expression is highly dynamic and tightly regulated. We summarize the environmental signals and molecular pathways that regulate IL-10 expression. While numerous studies have provided us with a deep understanding of IL-10 biology, the majority of findings have been made in murine models, prompting us to highlight gaps in our knowledge about T cell-derived IL-10 in the human system.

Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells.

T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-ß signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.

NRROS negatively regulates reactive oxygen species during host defence and autoimmunity.

Reactive oxygen species (ROS) produced by phagocytes are essential for host defence against bacterial and fungal infections. Individuals with defective ROS production machinery develop chronic granulomatous disease. Conversely, excessive ROS can cause collateral tissue damage during inflammatory processes and therefore needs to be tightly regulated. Here we describe a protein, we termed negative regulator of ROS (NRROS), which limits ROS generation by phagocytes during inflammatory responses. NRROS expression in phagocytes can be repressed by inflammatory signals. NRROS-deficient phagocytes produce increased ROS upon inflammatory challenges, and mice lacking NRROS in their phagocytes show enhanced bactericidal activity against Escherichia coli and Listeria monocytogenes. Conversely, these mice develop severe experimental autoimmune encephalomyelitis owing to oxidative tissue damage in the central nervous system. Mechanistically, NRROS is localized to the endoplasmic reticulum, where it directly interacts with nascent NOX2 (also known as gp91(phox) and encoded by Cybb) monomer, one of the membrane-bound subunits of the NADPH oxidase complex, and facilitates the degradation of NOX2 through the endoplasmic-reticulum-associated degradation pathway. Thus, NRROS provides a hitherto undefined mechanism for regulating ROS production--one that enables phagocytes to produce higher amounts of ROS, if required to control invading pathogens, while minimizing unwanted collateral tissue damage.

IL-22, not simply a Th17 cytokine.

Interleukin-22 (IL-22) has important functions in host defense at mucosal surfaces as well as in tissue repair. It is unique as a cytokine that is produced by immune cells, including T-helper (Th) cell subsets and innate lymphocytes, but acts only on non-hematopoietic stromal cells, in particular epithelial cells, keratinocytes, and hepatocytes. Although IL-22 is beneficial to the host in many infectious and inflammatory disorders, depending on the target tissue it can be pathogenic due to its inherent pro-inflammatory properties, which are further enhanced when IL-22 is released together with other pro-inflammatory cytokines, in particular IL-17. To avoid pathology, IL-22 and IL-17 production have to be controlled tightly and independently. While common factors such as signal transducer and activator of transcription 3 (STAT3) and retinoid orphan receptor γt (RORγt) drive the expression of both cytokines, other factors, such as c-Maf act specifically on IL-22 and enable the separate expression of either cytokine. Here, we discuss the production of IL-22 from various T-cell populations as well as protective versus pathogenic roles of IL-22. Finally, we focus on recent advances in our understanding of the molecular regulation of IL-22 in T cells.

Regulation of Interleukin-10 Expression.

Interleukin (IL)-10 is an essential anti-inflammatory cytokine that plays important roles as a negative regulator of immune responses to microbial antigens. Loss of IL-10 results in the spontaneous development of inflammatory bowel disease as a consequence of an excessive immune response to the gut microbiota. IL-10 also functions to prevent excessive inflammation during the course of infection. IL-10 can be produced in response to pro-inflammatory signals by virtually all immune cells, including T cells, B cells, macrophages, and dendritic cells. Given its function in maintaining the delicate balance between effective immunity and tissue protection, it is evident that IL-10 expression is highly dynamic and needs to be tightly regulated. The transcriptional regulation of IL-10 production in myeloid cells and T cells is the topic of this review. Drivers of IL-10 expression as well as their downstream signaling pathways and transcription factors will be discussed. We will examine in more detail how various signals in CD4+ T cells converge on common transcriptional circuits, which fine-tune IL-10 expression in a context-dependent manner.

Role of IL-22 in microbial host defense.

Interleukin (IL)-22 is a member of the IL-10 family of cytokines, which, besides IL-10, contains seven additional cytokines. Although the founding member IL-10 is an important immunoregulatory cytokine that represses both innate and adaptive immunity, the other family members preferentially target epithelial cells and enhance innate host defense mechanisms against various pathogens such as bacteria, yeast, and viruses. Based on their functions, the IL-10 family can be further divided into three subgroups, IL-10 itself, the IL-20 subfamily, and the IFNλ subfamily. IL-22 is the best-studied member of the IL-20 subfamily, and exemplifies the diverse biological effects of this subfamily. IL-22 elicits various innate immune responses from epithelial cells and is essential for host defense against several invading pathogens, including Citrobacter rodentium and Klebsiella pneumonia. IL-22 also protects tissue integrity and maintains the mucosal homeostasis. On the other hand, IL-22 is a proinflammatory cytokine with the capacity to amplify inflammatory responses, which might result in tissue damage, e.g., the IL-22-dependent necrosis of the small intestine during Toxoplasma gondii infection.

Regulation of epithelial immunity by IL-17 family cytokines.

Cutaneous and mucosal epithelial cells function as both a physical barrier and as immune sentinels against environmental challenges, such as microbial pathogens, allergens and stress. The crosstalk between epithelial cells and leukocytes is essential for orchestrating proper immune responses during host defense. Interleukin (IL)-17 family cytokines are important players in regulating innate epithelial immune responses. Although IL-17A and IL-17F promote antibacterial and antifungal responses, IL-17E is essential for defense against parasitic infections. Emerging data indicate that another member of this family, IL-17C, specifically regulates epithelial immunity. IL-17C production serves as an immediate defense mechanism by epithelial cells, utilizing an autocrine mechanism to promote antibacterial responses at barrier surfaces.

A real-time antibody-dependent cellular phagocytosis assay by live cell imaging.

Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated in vitro. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.

Preclinical and translational pharmacology of afucosylated anti-CCR8 antibody for depletion of tumour-infiltrating regulatory T cells.

BACKGROUND AND PURPOSE: RO7502175 is an afucosylated antibody designed to eliminate C-C motif chemokine receptor 8 (CCR8)+ Treg cells in the tumour microenvironment through enhanced antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL APPROACH: We report findings from preclinical studies characterizing pharmacology, pharmacokinetics (PK)/pharmacodynamics (PD) and safety profile of RO7502175 and discuss the translational PK/PD approach used to inform first-in-human (FiH) dosing strategy and clinical development in solid tumour indications. KEY RESULTS: RO7502175 demonstrated selective ADCC against human CCR8+ Treg cells from dissociated tumours in vitro. In cynomolgus monkeys, RO7502175 exhibited a biphasic concentration-time profile consistent with immunoglobulin G1 (IgG1) antibodies, reduced CCR8+ Treg cells in the blood, induced minimal and transient cytokine secretion, and was well tolerated with a no-observed-adverse-effect level (NOAEL) of 100 mg·kg-1. Moreover, RO7502175 caused minimal cytokine release from peripheral blood mononuclear cells (PBMCs) in vitro. A quantitative model was developed to capture surrogate anti-murine CCR8 antibody PK/PD and tumour dynamics in mice and RO7502175 PK/PD in cynomolgus monkeys. Subsequently, the model was used to project RO7502175 human PK and receptor occupancy (RO) in patients. Because traditional approaches resulted in a low FiH dose for this molecule, even with its superior preclinical safety profile, an integrated approach based on the totality of preclinical data and modelling insights was used for starting dose selection. CONCLUSION AND IMPLICATIONS: This work demonstrates a translational research strategy for collecting and utilizing relevant nonclinical data, developing a mechanistic PK/PD model and using a comprehensive approach to inform clinical study design for RO7502175.

Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

Argonaute3-SF3B3 complex controls pre-mRNA splicing to restrain type 2 immunity.

Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134Δ) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4+ T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis.

Activated B cells can regulate immunity and have been envisaged as a potential cell-based therapy for treating autoimmune diseases. However, activated human B cells can also propagate immune responses, and the effects resulting from their infusion into patients cannot be predicted. This led us to consider resting B cells, which in contrast are poorly immunogenic, as an alternative cellular platform for the suppression of unwanted immunity. Here, we report that resting B cells can be directly engineered with lentiviral vectors to express antigens in a remarkably simple, rapid, and effective way. Notably, this neither required nor induced activation of the B cells. With this approach we were able to produce reprogrammed resting B cells that inhibited antigen-specific CD4(+) T cells, CD8(+) T cells, and B cells upon adoptive transfer in mice. Furthermore, resting B cells engineered to ectopically express myelin oligodendrocyte glycoprotein antigen protected recipient mice from severe disability and demyelination in EAE, and even induced complete remission from disease in mice lacking functional natural Tregs, which otherwise developed chronic paralysis. In conclusion, our study introduces reprogrammed quiescent B cells as a novel tool for suppressing undesirable immunity.