The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes had not been available. Having now developed mice lacking the three ...immunoproteasome catalytic subunits, we found that the dendritic cells of these mice had defects in presenting several major histocompatibility complex (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes was markedly reduced in immunoproteasome-deficient animals compared with wild-type animals, whereas presentation of MHC class II peptides was unaffected. According to mass spectrometry, the repertoire of MHC class I-presented peptides was ∼50% different from that in wild-type mice, and these differences were sufficient to stimulate robust transplant rejection of wild-type cells in mutant mice. These results indicated that immunoproteasomes were more important in antigen presentation than previously thought.
Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic ...humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome–based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.
Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully ...human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.
The eotaxin chemokines have been implicated in allergen-induced eosinophil responses in the lung. However, the individual and combined contribution of each of the individual eotaxins is not well ...defined. We aimed to examine the consequences of genetically ablating eotaxin-1 or eotaxin-2 alone, eotaxin-1 and eotaxin-2 together, and CCR3. Mice carrying targeted deletions of these individual or combined genes were subjected to an OVA-induced experimental asthma model. Analysis of airway (luminal) eosinophilia revealed a dominant role for eotaxin-2 and a synergistic reduction in eotaxin-1/2 double-deficient (DKO) and CCR3-deficient mice. Examination of pulmonary tissue eosinophilia revealed a modest role for individually ablated eotaxin-1 or eotaxin-2. However, eotaxin-1/2 DKO mice had a marked decrease in tissue eosinophilia approaching the low levels seen in CCR3-deficient mice. Notably, the organized accumulation of eosinophils in the peribronchial and perivascular regions of allergen-challenged wild-type mice was lost in eotaxin-1/2 DKO and CCR3-deficient mice. Mechanistic analysis revealed distinct expression of eotaxin-2 in bronchoalveolar lavage fluid cells consistent with macrophages. Taken together, these results provide definitive evidence for a fundamental role of the eotaxin/CCR3 pathway in eosinophil recruitment in experimental asthma. These results imply that successful blockade of Ag-induced pulmonary eosinophilia will require antagonism of multiple CCR3 ligands.
Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including ...viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.
Background:
Inflammatory bowel disease (IBD) results from the chronic dysregulation of the mucosal immune system and the aberrant activation of both the innate and the adaptive immune responses. We ...used two complementary models of colonic inflammation to examine the roles of interleukin (IL)‐19 in colonic inflammation and thus its possible role in IBD.
Methods:
Using gene‐targeting, we generated IL‐19‐deficient mice. To study the activation of the innate immune response during colonic inflammation we characterized an innate immune‐mediated model of colitis induced by dextran sulfate sodium (DSS). DSS can induce not only acute colitis but also chronic colitis. In addition to the acute DSS‐induced colitis model, we used a chronic DSS‐induced colitis model that is associated with the activation of both Th1 and Th2 cytokines as well as innate immune response in the colon.
Results:
We show that IL‐19‐deficient mice are more susceptible to experimental acute colitis induced by DSS, and this increased susceptibility is correlated with the accumulation of macrophages and the increased production of IFN‐γ, IL‐1β, IL‐6, IL‐12, TNF‐α, and KC. Additionally, cytokine production in IL‐19‐deficient macrophages was enhanced on stimulation of lipopolysaccharide (LPS) through reduced phosphorylation of STAT1 and STAT3. Moreover, our results clearly demonstrate that IL‐19 is required for B‐cell infiltration during chronic DSS‐induced colitis, which may be mediated by IL‐13 and IL‐6.
Conclusions:
The finding that IL‐19 drives pathogenic innate immune responses in the colon suggests that the selective targeting of IL‐19 may be an effective therapeutic approach in the treatment of human IBD. Inflamm Bowel Dis 2009
The cytokine, interleukin (IL)-19, is a member of the IL-10 family that includes IL-20, IL-22, IL-24, and IL-26. Recent studies have shown that IL-19 is produced by keratinocytes, epithelial cells, ...macrophages, and B-cells. Little is known about the exact biological role of IL-19 in immunological regulation, although there is an increasing body of data demonstrating that IL-19 is associated with the development of Th2 responses and the pathogenesis of psoriasis. In this review, I shall attempt to discuss current knowledge about the role of IL-19 on macrophages and the potential role in inflammatory bowel disease.
BackgroundInterleukin-2 (IL-2) is a driver of T and NK cell proliferation and activation, and has produced remarkable clinical efficacy in a few cancer patients. However, its clinical use is limited ...by its narrow therapeutic index, and potentially by its preferential stimulation of immune suppressing Treg cells. TNRX-257, a novel multi-specific LAG3 antagonist with unique LAG3-conditional partial agonism of the IL2Rγ/β receptors, was developed using Tentarix’s propriety Tentacles™ platform, which is based on fully human, stabilized antibody VH domains. LAG3 expression is restricted to antigen-experienced and tumor-reactive immune cells with little expression on peripheral PBMC or immune cells in normal tissues. Thus, TNRX-257 is anticipated to drive the proliferation of this tumor-antigen positive pool of cells, while minimizing the dose limiting toxicity seen in the pre-clinical models and human clinical trials, as well as the bias towards Treg proliferation seen with WT IL-2.MethodsTNRX-257 was assessed for the affinity for human and cynomolgus LAG3, IL2Rγ and IL2Rβ affinities by Bio-Layer Interferometry, and by cytometry for binding and pSTAT5 induction on CD3/CD28 activated human and cynomolgus monkey PBMCs. To determine the pharmacokinetics and pharmacodynamics of TNRX-257, the molecule was dosed intravenously at 2 dose levels, 2.5mg/kg or 9mg/kg, to 2 cynomolgus monkeys each, and blood was harvested at multiple timepoints over 14 days for analysis. Pharmacokinetics was determined by ELISA, cytokine release by MSD assays, and Ki67 positivity of NK and T cells by cytometry.ResultsTNRX-257 was shown to have equivalent affinities for human and cynomolgus LAG3, IL2Rγ, IL2Rβ. The bioactivity of TNRX-257 on cynomolgus monkey PBMCs is highly similar to its human responses, retaining its unique IL2Rγ/β partial agonism and full antagonist activity for LAG3. At both dose levels, TNRX-257 was well tolerated and blood levels remained above the minimal concentration required for IL2Rγ/β agonism throughout the study period. TNRX-257 bioactivity was seen at both day 5 and day 7 post-dosing, as increases in the percentage of Ki67 positive NK cells, CD4 and CD8 T cells over the pre-dose level with the higher dose.ConclusionsThese data show that TNRX-257 has pharmacokinetics, bioactivity and a unique safety profile that support its clinical development for treating multiple cancer indications.Ethics ApprovalThis study was approved by Labcorp institution’s IACUC Board; study number 8486120
Resistin-like molecule (RELM) β is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function ...primarily remains an enigma.
We sought to elucidate the function of RELM-β in the gastrointestinal tract.
We generated RELM-β gene–targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation.
We show that RELM-β is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-β. Using acute colonic inflammatory models, we demonstrate that RELM-β has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-β regulates expression of type III regenerating gene (REG) (REG3β and γ), molecules known to influence nuclear factor κB signaling.
These data define a critical role for RELM-β in the maintenance of colonic barrier function and gastrointestinal innate immunity.
These findings identify RELM-β as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies.
IntroductionThe clinical benefit of the anti-CTLA-4 monoclonal antibody (mAb) ipilimumab has been well established but limited by immune-related adverse events, especially when ipilimumab is used in ...combination with anti-PD-(L)1 mAb therapy. To overcome these limitations, we have developed XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 mAb.MethodsXTX101 consists of an anti-human CTLA-4 mAb covalently linked to masking peptides that block the complementarity-determining regions, thereby minimizing the mAb binding to CTLA-4. The masking peptides are designed to be released by proteases that are typically dysregulated within the tumor microenvironment (TME), resulting in activation of XTX101 intratumorally. Mutations within the Fc region of XTX101 were included to enhance affinity for FcγRIII, which is expected to enhance potency through antibody-dependent cellular cytotoxicity.ResultsBiophysical, biochemical, and cell-based assays demonstrate that the function of XTX101 depends on proteolytic activation. In human CTLA-4 transgenic mice, XTX101 monotherapy demonstrated significant tumor growth inhibition (TGI) including complete responses, increased intratumoral CD8+T cells, and regulatory T cell depletion within the TME while maintaining minimal pharmacodynamic effects in the periphery. XTX101 in combination with anti-PD-1 mAb treatment resulted in significant TGI and was well tolerated in mice. XTX101 was activated in primary human tumors across a range of tumor types including melanoma, renal cell carcinoma, colon cancer and lung cancer in an ex vivo assay system.ConclusionsThese data demonstrate that XTX101 retains the full potency of an Fc-enhanced CTLA-4 antagonist within the TME while minimizing the activity in non-tumor tissue, supporting the further evaluation of XTX101 in clinical studies.
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