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Multiplex immunofluorescence (MxIF) images provide detailed information of cell composition and spatial context for biomedical research. However, compromised data quality could lead to research biases. Comprehensive image quality checking (QC) is essential for reliable downstream analysis. As a reliable and specific staining of cell nuclei, 4′,6-diamidino-2-phenylindole (DAPI) signals were used as references for tissue localization and auto-focusing across MxIF staining–scanning–bleaching iterations and could potentially be reused for QC. To confirm the feasibility of using DAPI as QC reference, pixel-level DAPI values were extracted to calculate signal fluctuations and tissue content similarities in staining–scanning–bleaching iterations for identifying quality issues. Concordance between automatic quantification and human experts’ annotations were evaluated on a data set consisting of 348 fields of view (FOVs) with 45 immune and tumor cell markers. Cell distribution differences between subsets of QC-pass vs QC-failed FOVs were compared to investigate the downstream effects. Results showed that 87.3% FOVs with tissue damage and 73.4% of artifacts were identified. QC-failed FOVs showed elevated regional gathering in cellular feature space compared with the QC-pass FOVs. Our results supported that DAPI signals could be used as references for MxIF image QC, and low-quality FOVs identified by our method must be cautiously considered for downstream analyses.

Background Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. Methods Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression. Results GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. Conclusion These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

Through our involvement in KEYNOTE-059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti-VEGFR-2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/ paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST-defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression-free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5-fold (range 4–121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post-treatment versus pre-treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti-VEGFR-2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti-VEGF(R)/taxane are underway.

The concept of exploiting the immune system to treat cancer forms the basis of immuno-oncology. Since its birth in the late 1800s, immuno-oncology, or cancer immunotherapy, has come a long way. With better understanding of the complex relationship between tumor and the immune system, we have been able to explore and develop various modalities of anticancer therapies. In this review, we summarize the main strategies of immunotherapy that are available today: monoclonal antibodies, anticancer vaccines, cytokines, and adoptive T-cell therapy. We also highlight the unique set of adverse effects associated with modern immunotherapy and propose nonsteroidal immunomodulators and anticytokine antibodies as treatment options for toxicities. The future of immuno-oncology is discussed, including combination therapy, drug-antibody conjugates, epigenetic drugs, using nanoparticles for drug delivery, new antigen discovery, and developing biomarkers to assess treatment responses. A data search was conducted using PubMed and included studies published through November 1, 2017. Search terms used include cancer immunotherapy, pembrolizumab, ipilimumab, nivolumab, PD-1 inhibitors, PD-L1 inhibitors, checkpoint inhibitors, anticancer vaccines, TVEC, and adoptive cell therapy.

Epithelial tumors including melanoma often first metastasize to regional, sentinel lymph nodes (SLN). Thus, the presence of SLN metastases is a critical prognostic factor of survival. Prior to metastasis, accumulating evidence suggests the SLN is immunologically compromised; however, the process by which pre-metastatic niche formation occurs remains unknown. In this prospective study, freshly dissected, afferent lymphatic fluid was obtained during SLN biopsy in three patients with primary cutaneous melanoma. Lymphatic extracellular vesicles (L-EV) were visualized by transmission electron microscopy and proteomic cargo profiled by mass spectrometry. Flow cytometry assessed L-EV effects on autologous dendritic cell maturation in vitro. Immunogold electron microscopy and immunohistochemistry visualized expression of EV cargo within the primary tumor and SLN. Lymphatic extracellular vesicles from each afferent lymphatic channel demonstrated inhibition of autologous dendritic cell maturation. Proteomic profiling identified 81 peptides shared among the L-EV preparations including a signature of 18 immune-modulating proteins including previously established inhibitor of dendritic cell maturation, S100A9. Immunohistochemistry and immunogold electron microscopy confirmed S100A9 tracking along the lymphatic path, from keratinocytes in the primary tumor to sub-capsular macrophages in the SLN. Our findings suggest L-EV cargo may serve as early mediators of tumor-induced immune subversion in regional lymph nodes, by preceding malignant cells and trafficking within the lymphatic vasculature to harbor the first pre-metastatic niche.

Next‐generation sequencing has ushered in an era of individualized medicine enabling a patient's genome to be interrogated in search of variants implicated in disease. This has brought about diagnostic resolution for many patients suffering from previously undiagnosed genetic disorders. However, it has also created significant challenges in clinical interpretation, as many identified variants lack sufficient contextual information to allow clear association with disease and are classified as variants of uncertain significance (VUS). Here, we describe the sequential use of variant annotation, in silico protein modeling techniques, and in vitro functional studies to characterize a clinically reported VUS and reclassify it as a pathogenic variant. In this study, genetic panel testing in a 19‐month‐old boy with developmental delay, hypotonia, and abnormal brain MRI revealed a VUS, c.1126C>T, leading to p.R376W, in the glial fibrillary acidic protein (GFAP; OMIM:203450; NM_002055.4) which has previously been associated with Alexander disease (OMIM: 203450) 1. Alexander disease, a rare genetic disorder with more than 550 cases reported worldwide, is characterized as a progressive disorder of cerebral white matter 2. Alexander disease can present during infancy, childhood, or adulthood, with infantile onset being the most common presentation. The infantile form often presents with progressive psychomotor retardation, loss of developmental milestones, frontal bossing with megalencephaly, seizures, hyperreflexia, ataxia, and hydrocephalus, and frequently leads to death within the first decade 2. Juvenile and adult forms of Alexander disease often progress more slowly with ataxia, bulbar signs, and spasticity 3. The predominant genetic cause of Alexander disease was discovered after transgenic GFAP mice presented with astrocyte inclusions that pathologically mimicked the Rosenthal fibers in Alexander disease 4. The mouse model led to targeted GFAP sequencing in 11 cases of Alexander disease, 10 of which harbored a genetic variant. For cases with available parental samples, the variants were found to have arisen as de novo in the proband, and were absent in their larger cohort of control DNA samples 1. Since these initial studies, over 110 distinct pathogenic variants in GFAP have been identified 2. The vast majority of these variants are de novo missense heterozygous variants; however, there is some evidence supporting familial inheritance 5. Following the initial discovery of GFAP variants associated with Alexander disease, the functional characterization of newly identified variants was completed in mouse models 6. However, the vast majority of subsequently identified missense variants in GFAP were considered pathogenic without functional validation. Cellular‐based assays have been developed and used to examine the pathogenicity of variants in individuals with abnormal presentation or familial onset 3, 5. Results of these studies demonstrated that some variants elicit a wild‐type phenotype, suggesting they were benign in nature 3. These cases highlight the need for functional validation of GFAP variants associated with atypical Alexander disease. As our patient presented with developmental delay, hypotonia, and abnormal brain MRI, with no signs of regression, it could not confidently be concluded that the identified GFAP variant was associated with his disease phenotype. Because his phenotype may represent a subtle and atypical form of Alexander disease, we combined variant annotation using information on variant frequency in Alexander disease cases and publically available exome databases, protein studies via paralog modeling, and functional characterization utilizing a cellular model to determine the pathogenicity of p.R376W.

e15184 Background: Clinical responses to anti-PD1 immunotherapy in patients with metastatic melanoma (MM) remain challenging to predict. This clinical heterogeneity is also reflected in the tumor-immune microenvironment among patients and within a single tumor lesion. With the emergence of multiplex imaging platforms, defining complex phenotypes at single cell resolution has become possible. Here, we sought to objectively quantify paracrine tumor-immune interactions contributing to the variable clinical responses observed in patients receiving anti-PD1 therapy. Methods: Excisional lymph node (LN) biopsies were obtained from treatment-naïve patients with MM who underwent subsequent anti-PD1 therapy. A single 5µm section of LN tissue was used to assess a 42 analyte panel by multiplex immunofluorescence. From 30 LN samples, 418 fields of view (FOVs) were selected resulting in 14,360 high-resolution images of 4 anatomical subregions: tumor core, tumor-immune interface, tumor infiltrate and adjacent immune stroma. Following image processing, we developed an adaptive classification for tumor-centric cellular neighborhoods (TCCN) to identify and quantify critical paracrine interactions within the tumor-immune microenvironment. Results: Stratification based on responsiveness to anti-PD1 therapy resulted in 4 complete responders (CR) and 12 non-responders (NR) at 12-week follow-up. From 126 FOVs, we defined the cellular composition of 197,865 TCCN across patients based on clinical response and LN subregions. Overall, the percentage of TCCN devoid of any T cells, B cells or macrophages was significantly higher in NR compared to CR irrespective of subregion. However, other markers differentiated TCCN based on subregion. Specifically in CR, tumor core regions were enriched for CD8 T cells, while enrichment for B cells and endothelial cells was demonstrated at the tumor-immune interface. Strikingly, tumor infiltrate regions demonstrate robust immune reactivity with enrichment for M1 polarized macrophages, NK cells and B cells in CR compared to NR. Complete data from the 30 patient cohort across 418 FOVs will be presented. Conclusions: Taken together, this data suggests cellular composition of TCCN across subregions of the LN is dynamic within a single metastatic site. In this small cohort, we introduce a formalized stratification to quantify and classify critical paracrine interactions within the immune-tumor microenvironment with the potential to inform clinical responsiveness to therapy.

Glioblastoma (GBM) is the most common and lethal primary brain tumor, and novel therapeutic strategies that make a substantial impact on outcomes are sorely needed. Immunotherapies have shown great promise in the treatment of a number of cancers in recent year, and a concerted effort is being made to apply these treatment paradigms to GBM. However, many GBM patients exhibit profound immunosuppression, likely limiting the efficacy of such approaches. The mechanisms of this immunosuppression are poorly understood, but tumor-derived extracellular vesicles (EVs), may play a role. We demonstrate that GBM-derived EVs induce the development of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). We further demonstrate that these EV-induced monocytic cells are immunosuppressive, resulting in impaired T cell proliferation upon co-culture. We found that the immunosuppressive effects of tumor-derived EVs appear to be driven by the induction of these immunosuppressive monocytes, as EV treatment of T cells did not significantly impact T cell proliferation. Further, we sought to characterize the important of programmed death ligand 1 (PD-L1) in the induction of these immunosuppressive monocyte populations. We found PD-L1 to be expressed in the EVs from GBM cell lines, and that modulation in PD-L1 expression via either constitutive overexpression or shRNA-mediated knockdown resulted in concordant changes in expression in tumor-derived EVs. We demonstrate that PD-L1 is important for the induction of NCM but not for MDSCs. Taken together, these findings point to a significant role for tumor-derived EVs in the induction of immunosuppressive monocytes in GBM, and that these cells may be a driving force of systemic immunosuppression. PD-L1 is one factor expressed in EVs that has immunomodulatory properties, but additional EV cargo likely plays a major role in the induction of immunosuppressive cells.

Aiming to decipher immunological mechanisms of the autoimmune disorder alopecia areata (AA), we hypothesized that interleukin-6 (IL-6) might be associated with juvenile-onset AA, for which there is currently no experimental model. Upon intramuscular transgenesis to overexpress IL-6 in pregnant female C57BL/6 (B6) mice, we found that the offspring displayed an initial normal and complete juvenile hair growth cycle, but developed alopecia around postnatal day 18. This alopecia was patchy and reversible (non-scarring) and was associated with upregulation of Ulbp1 expression, the only mouse homolog of the human AA-associated ULBP3 gene. Alopecia was also associated with inflammatory infiltration of hair follicles by lymphocytes, including alpha-beta T cells, which contributed to surface hair loss. Despite these apparently shared traits with AA, lesions were dominated by follicular dystrophy that was atypical of human AA disease, sharing some traits consistent with B6 alopecia and dermatitis. Additionally, juvenile-onset alopecia was followed by complete, spontaneous recovery of surface hair, without recurrence of hair loss. Prolonging exposure to IL-6 prolonged the time to recovery, but once recovered, repeating high-dose IL-6 exposure de novo did not re-induce alopecia. These data suggest that although substantial molecular and cellular pathways may be shared, functionally similar alopecia disorders can occur via distinct pathological mechanisms.

The immune system has effectively evolved to protect the host against foreign invaders, including bacterial, viral, and parasitic infiltrates. Less clear has been the interaction and the protective effects the immune system mounts against its own infiltrates: cancer cells. Here we consider the dynamic interactions between cancer and the associated host immune response by highlighting the key players involved in engaging an effective antitumor immune response and the mechanisms responsible for enabling the evolution of cancer cells to escape immunosurveillance. By developing an appreciation for the dual function of the immune system in the setting of cancer biology, we also consider the clever strategies that have been employed to uncover tumor targets, including tumor-associated antigens and the mechanisms for enhancing or reengaging the immune system to mount an effective antitumor immune response. Finally, we incorporate these key findings into the context of immunotherapy, a rapidly evolving field aimed at combating tumor escape by enabling the host immune system to regain its tumor-eradicating functions. This review contains 5 figures, 9 tables and 60 references Key words: adoptive T cell therapy, checkpoint inhibitors, cytokine therapy, immunotherapy, neutralizing antibodies, tumor immunity, tumor microenvironment, vaccines

Evolution of melanoma from a primary tumor to widespread metastasis is crucially dependent on lymphatic spread. The mechanisms regulating the initial step in metastatic dissemination via regional lymph nodes remain largely unknown however evidence supporting the establishment of a pre-metastatic niche is evolving. We have previously described a dysfunctional immune profile including reduced expression of DC maturation markers in the first node draining from the primary tumor, the sentinel lymph node (SLN). Importantly, this phenotype is present prior to evidence of nodal metastasis. Herein, we evaluate melanoma-derived extracellular vesicles (EVs) as potential mediators of the pre-metastatic niche through cargo-specific polarization of dendritic cells (DCs). Matured in vitro in the presence of melanoma EVs, DCs show significantly impaired expression of co-stimulatory markers CD83 and CD86 compared to liposome treated DCs as well as decreased expression of Th1 polarizing cytokines Flt3L and IL15 and migration chemokines MIP1α and MIP1β. Profiling of melanoma EV cargo identified shared proteomic and RNA signatures including S100A8 and S100A9 protein cargo which in vitro compromised DC maturation similar to melanoma EVs. Early evidence demonstrates similar EVs can be isolated from human afferent lymphatic fluid ex vivo. Taken together, here we propose melanoma EV cargo as a mechanism by which DC maturation is compromised warranting further study to consider this as a potential mechanism enabled by the primary tumor to establish the pre-metastatic niche in tumor-draining SLNs of patients.