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The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUTMPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUTMPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUTneoepitopes with high affinity are underrepresented in patients with CALRMUTMPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUTMPN would not efficiently respond to a CALRMUTfragment cancer vaccine but would when immunized with a modified CALRMUTheteroclitic peptide vaccine approach. We found that heteroclitic CALRMUTpeptides specifically designed for the MHC-I alleles of patients with CALRMUTMPN efficiently elicited a CALRMUTcross-reactive CD8+T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUTnative peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+T cell response to the CALRMUTfragment upon immunization with a CALRMUTheteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALRMUTMPN.

We conducted a systematic evaluation of lung inflammation indued by repeated intranasal exposure (for 10 consecutive days) to a human aeroallergen, house dust mite (HDM) in BALB/c mice. Peak influx of neutrophils, monocytes/lymphocytes, and eosinophils was observed in bronchoalveolar lavage (BAL) on days 1, 7 and 11, respectively, and normalized to baseline by day 21. Peak elevations of Th2, myeloid‐derived cytokines/chemokines and serum IgE were seen both in BAL and lung tissue homogenates between days 7 and 11, and declined thereafter; however, IL‐33 levels remained elevated from day 7 to day 21. Airway hyperreactivity to inhaled methacholine was significantly increased by day 11 and decreased to baseline by day 21. The lung tissue showed perivascular and peribronchial cuffing, epithelial hypertrophy and hyperplasia and goblet cell formation in airways by day 11, and resolution by day 21. Levels of soluble collagen and tissue inhibitors of metalloproteinases (TIMP) also increased reflecting tissue remodeling in the lung. Microarray analysis demonstrated a significant time‐dependent up‐regulation of several genes including IL‐33, CLCA3, CCL17, CD4, CD10, CD27, IL‐13, Foxa3, IL‐4, IL‐10, and CD19, in BAL cells as well as the lung. Pre‐treatment of HDM challenged mice with CCL17 and IL‐13 antibodies reduced BAL cellularity, airway hyper‐responsiveness (AHR), and histopathological changes. Notably, anti‐IL‐13, but not anti‐CCL17 monoclonal antibodies (mAbs) reduced BAL neutrophilia while both mAbs attenuated eosinophilia. These results suggest that CCL17 has an overlapping, yet distinct profile versus IL‐13 in the HDM model of pulmonary inflammation and potential for CCL17‐based therapeutics in treating Th2 inflammation.

Veglia et al. characterize the heterogeneity of polymorphonuclear neutrophils (PMNs) in the tumor microenvironment, spleen, and peripheral blood. The identification of neutrophil populations with potent immune suppressive activity opens selective targeting opportunities., In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

RORγt and RORα are transcription factors of the RAR-related orphan nuclear receptor (ROR) family. They are expressed in Th17 cells and have been suggested to play a role in Th17 differentiation. Although RORγt signature genes have been characterized in mouse Th17 cells, detailed information on its transcriptional control in human Th17 cells is limited and even less is known about RORα signature genes which have not been reported in either human or mouse T cells. In this study, global gene expression of human CD4 T cells activated under Th17 skewing conditions was profiled by RNA sequencing. RORγt and RORα signature genes were identified in these Th17 cells treated with specific siRNAs to knock down RORγt or RORα expression. We have generated selective small molecule RORγt modulators and they were also utilized as pharmacological tools in RORγt signature gene identification. Our results showed that RORγt controlled the expression of a very selective number of genes in Th17 cells and most of them were regulated by RORα as well albeit a weaker influence. Key Th17 genes including IL-17A, IL-17F, IL-23R, CCL20 and CCR6 were shown to be regulated by both RORγt and RORα. Our results demonstrated an overlapping role of RORγt and RORα in human Th17 cell differentiation through regulation of a defined common set of Th17 genes. RORγt as a drug target for treatment of Th17 mediated autoimmune diseases such as psoriasis has been demonstrated recently in clinical trials. Our results suggest that RORα could be involved in same disease mechanisms and gene signatures identified in this report could be valuable biomarkers for tracking the pharmacodynamic effects of compounds that modulate RORγt or RORα activities in patients.

Background: Checkpoint blockade of PD-1/PDL-1 or CTLA-4 has demonstrated clinical benefit in a subset of cancer patients (pts). To further improve the outcome, approaches to deplete immunosuppressive Tregs are being developed. Emerging evidence supports that the TNF receptor superfamily member 18 (TNFRSF18 or GITR) is highly expressed on intratumoral (i.t.) Tregs with limited expression on effector T cells or circulating immune cells (1,2). This differential expression allows for development of monoclonal antibody (mAb) therapies that eliminate this population while sparing other immune subsets. Herein we characterize JNJ-64164711 (JNJ-711), a fully human anti-GITR mAb being developed to selectively deplete GITR+ Tregs and expand the population of pts that may respond to immunotherapy. Methods: Treg frequency and GITR expression on human cancer specimens was performed by IHC and multicolor flow cytometric analysis (FC). JNJ-711 was assessed for binding to GITR (human and cyno) and human Fcγ receptors, and inhibition of hGITRL binding to hGITR. The JNJ-711 binding epitope was mapped by hydrogen-deuterium exchange. JNJ-711 functional activity was determined by 1) an NF-kB reporter assay, 2) antibody dependent cellular cytotoxicity (ADCC) on GITR+ cell lines, and 3) JNJ-711 mediated Treg depletion in primary pts tumor samples. Results: GITR expression prevalence was evaluated by IHC in multiple solid tumors. Immune cell GITR expression was observed in head and neck (HN), esophageal (ESO), non-small cell lung carcinoma (NSCLC), colorectal (CRC) and prostate cancers. GITR was moderately expressed on HN and ESO tumor cells. GITR expression (by FC) was higher on tumor Tregs versus other T cell populations in the tumor and periphery across NSCLC, CRC, ovarian and renal cell carcinoma samples. GITR was differentially higher on i.t. effector Tregs (eTreg) whereas CCR4 was highest on CD4+ T cells in circulation. JNJ-711 binds to hGITR on cells with high affinity [KD= 180 pM], has a log greater affinity to FcγRIIIa [KD= 37 nM on 158V; KD= 180 nM on 158F], a slower dissociation rate than most GITR agonist (Ag) mAb tested [Koff= 5.47 x 10-4 s-1 for Fab], and binds to the CRD1 of GITR. Unlike other GITR Ag mAbs tested, JNJ-711 does not block GITR interaction with GITRL. JNJ-711 demonstrates higher ADCC mediated cell depletion than other GITR Ag mAbs in clinical development on 1) GITR+ hematologic tumor cell lines (HuT102, KMS-12-BM, RPMI 8226, JJN-3), 2) primary activated GITR+ T cells, and 3) in vitro expanded Tregs. Using dissociated primary pts samples, JNJ-711 selectively depleted i.t. eTregs. Conclusion: JNJ-64164711 selectively depletes hGITR+ cells including Tregs and has potential to enhance antitumor immune response. This antibody is being evaluated for clinical development. References: 1. Asma et al. Tumor Biol 2015;36:3727-34. 2. Pedroza-Gonzalez et al. OncoImmunology 2015;4:12, e1051297. Citation Format: Cam V. Holland, John Kehoe, Judith Hailey, Rupesh Nanjunda, Eilyn Lacy, Robin Ernst, Di Zhang, Sam Wu, Alexey Teplyakov, Weimin Li, Michelle Kinder, Edward W. Thompson, Patrick Wilkinson, Jackson Wong, Gerald Chu, Linda Okonkwo, John D. Alvarez, Anhco Nguyen, Iqbal S. Grewal, Moitreyee Chatterjee-Kishore, Mark Salvati, Marco Gottardis. Development of JNJ-64164711, a low fucose anti-GITR antibody for enhanced depletion of tumor regulatory T cells (Tregs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3813.

Diabetes is the leading cause of end stage renal disease (ESRD) in the United States, representing 44% of incident cases [1]. In this study, serum and peripheral blood collected from diabetic patients in five stages of chronic kidney disease (CKD), as defined by glomerular filtration rate (GFR), were compared to healthy (non-CKD) subjects.Serum samples were analyzed for 39 inflammatory or immune mediator protein levels and peripheral blood samples were analyzed for expression of 35 gene transcripts.In serum, MCP-1, FGF-2, VEGF, and EGF levels were elevated above controls at all stages of DN. Five mediator levels, GM-CSF, IL-1α, IL-1RA, IL-6, and MIP1β increased with disease progression until stage 4-5, at which point a decrease was observed paralleling a loss of functional renal mass that occurs in late stage CKD. Five mediator levels: GRO, IFNγ, MDC, Eotaxin, and G-CSF significantly differed from controls at one or more stages without apparent correlation with disease stage. Only a single mediator, sIL2RA, exhibited a linear increase with disease severity consistent with declining GFR. In peripheral blood, the transcript level of seven mediators, ICAM1, TNF-α, TGF-β, IL-8, IL17RA, IFNγ, and MYD88 were significantly elevated at all disease stages as compared to control.Statistically significant differences in protein and transcripts levels between diseased and control can be detected in serum and peripheral blood utilizing high content profiling. These changes occur as early as stage 1-2 before a significant decline in renal function.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2(Akita) mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11b(Cre)/TNF-α(Flox/Flox)). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-α(Flox/Flox) control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

Excessive deposition of type I collagen by activated fibroblasts is a hallmark of scarring and fibrotic pathologies. Quantitation of collagen I at the protein level is paramount to measure functionally relevant changes during pathological remodeling of the extracellular matrix. We describe two new cell-based assays to directly quantify the amount of collagen I incorporated into the extracellular matrix of primary human lung fibroblasts. Utilizing a monoclonal antibody specific to native human collagen I, we optimized conditions and parameters including incubation time, specificity and cell density to demonstrate dose-dependent induction of collagen I by transforming growth factor beta, as measured by in-cell enzyme linked immunosorbent assay. The results obtained by this assay were mimicked by an "In situ Quantitative Western Blot" on cultured cells using the same antibody. Results from these assays were comparable to those obtained with a commercial assay for collagen I N-propeptide, which is an index of collagen formation. These assays have been optimized for a 96-well format and provide a novel and useful approach for screening of anti-fibrotic agents in vitro. The assays described here also offer a significant improvement in throughput and specificity over conventional methods that primarily measure soluble collagen.

In 1957-58 and in March 1965, in the course of writing a book on E. M. Forster, I met with a number of Bloomsbury, and fringe Bloomsbury, figures. These encounters, here recorded from notes and memory, will perhaps provide a few footnotes to the Bloomsbury record. G. E. MOORE During an interview on Oct. 23, 1957, Forster reminded me that G. E. Moore was still alive and living in Cambridge and that I should try to see him. Forster himself, as I remember, arranged the meeting. I was invited for tea at 4:30 the following day. The prospect of such a meeting was a little daunting. Though I had read Principia Ethica and some Moore criticism in Paul Arthur Schilpp's important volume, I felt quite unready to talk philosophy with the great man. I hadn't understood much of what I had read in Principia Ethica, and much that I half understood I didn't think I agreed with. And that whole chapter on "The Ideal" - which had moved Keynes to see "a new heaven on a new earth" and Strachey to exclaim "The age of reason has come" (Keynes 82; Forster, Dickinson 110) - I found somewhere between unintelligible and plain silly. But who was I to hold such views when the whole world seemed agreed that Moore was a great man, if not a saint?(1) - and when his reputation as a "philosopher's philosopher" was undisputed? I took some heart from the fact that Forster could treat Moore lightly - even make puns on his work(2) - but I hardly had his rights in the matter. The house at 86 Chesterton Road was an old red-brick Victorian that had seen better days, one in a row paralleling the Cam at about a two-street remove. An air of postwar fatigue and shabbiness clung to the neighborhood, along with a stubborn respectability. Mrs. Moore, an energetic, rather bouncy woman of middle age, smiled a welcome and invited me in without ceremony. I was no sooner in the door, however, than she took me aside and whispered, conspiratorially, that she was to do all the talking. The doctor had forbidden Moore to talk philosophy because of his heart - he got too excited. I didn't know whether to be glad or disappointed. Mrs. Moore gave promise of being a lively and intelligent conversationalist, but she was hardly a proxy for the main exhibit. That exhibit shone splendidly forth, like a painter's model, in the center of the next room. There sat G. E. Moore in a straight-backed chair, posed in profile like Whistler's mother, staring fixedly before him, as if mesmerized by some pure and distant thought. It was a "beautiful" face - there is no other word for it - classical features, high brow, smooth white skin, silver hair, keen blue eyes. In some pantheon of ideal callings, he might serve, sculpted and labeled, to represent the philosopher, a picture of otherworldly contemplation fit to sit next to Rodin's The Thinker. One is tempted to hyperbole because there was something comic about the whole situation, especially when Mrs. Moore, after inviting me to sit down, settled into an armchair opposite and, smiling a little mischievously, lit up a pipe.(3) Throughout the interview, she puffed away happily as our words passed to and fro over the head of G. E. Moore, silent and immobile, gazing beatifically into space. I wasn't sure he even knew I was in the room. I remember only bits and pieces of what was said. We discussed Cambridge and Bloomsbury and my work, but Moore not at all, since Mrs. Moore declared herself unfit to talk philosophy, her husband's or any other. I remember her scolding me for not yet having read Keynes's "My Early Beliefs,"(4) which she called the best account she knew of Moore's influence on Bloomsbury (she always referred to him as "Moore," in the third person), and we gossiped a bit about Forster. I remember too the room we were sitting in, how dark and cluttered it was, and I wondered how the great "Apostle" of beauty could care so little about the aesthetics of his surroundings. The room had space and oaken comfort, but it seemed more like a warehouse filled with moveables than an area arranged after some principle of taste. …