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Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low dendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg) in vitro and in vivo. Targeted depletion of this DC population in Mgl2DTR hosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell–mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection. Cancer Res; 77(15); 4158–70. ©2017 AACR.

Supplementary Figures S1-8. Fig. S1: CD11b+CD11c+MHC-II+ cells exhibit DC phenotype and accumulate at multiple metastatic sites. Fig. S2: PDAC cells produce large amounts of GM-CSF and stimulate Mo-DC differentiation. Fig. S3: Liver DC from tumor-bearing mice efficiently activate T cells in conventional assays. Fig. S4: Metastasis-associated DC stimulate nTreg proliferation while suppressing CD8 T cells through PD-1 ligand expression. Fig. S5: MGL2+ cells exhibit DC phenotype and their depletion activates multiple lymphocyte populations. Fig. S6: Blocking PD-L2 reduces metastasis without affecting primary tumor burden. Fig. S7: PD-L2+ and MGL+ cells are present in human PDAC tissues. Fig. S8: Development and function of CD11b+ DC in PDAC metastasis.

Supplementary Materials and Methods

Context.— The ongoing COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has elicited a surge in demand for serologic testing to identify previously infected individuals. In particular, antibody testing is crucial in identifying COVID-19 convalescent plasma, which has been approved by the Food and Drug Administration under the Emergency Use Authorization for use as passive immunotherapy for hospitalized patients infected with COVID-19. Currently, high-titer COVID-19 convalescent plasma can be qualified by Ortho's Vitros COVID-19 IgG antibody test. Objective.— To explore the use of an efficient testing method to identify high-titer COVID-19 convalescent plasma for use in treating COVID-19–infected patients and track COVID-19 positivity over time. Design.— We evaluated an enzyme-linked immunosorbent assay (ELISA)–based method that detects antibodies specific to the SARS-CoV-2 receptor binding domain (RBD) with individual and pooled plasma samples and compared its performance against the Vitros COVID-19 IgG antibody test. Using the pooled RBD-ELISA (P-RE) method, we also screened more than 10 000 longitudinal healthy blood donor samples to assess seroprevalence. Results.— P-RE demonstrates 100% sensitivity in detecting Food and Drug Administration–defined high-titer samples when compared with the Vitros COVID-19 IgG antibody test. Overall sensitivity of P-RE when compared with the Vitros COVID-19 IgG antibody test and our individual sample RBD-ELISA (I-RE) were 83% and 56%, respectively. When screening 10 218 healthy blood donor samples by P-RE, we found the seroprevalence correlated with the local infection rates with a correlation coefficient of 0.21 (P < .001). Conclusions.— Pooling plasma samples can be used to efficiently screen large populations for individuals with high-titer anti–RBD antibodies, important for COVID-19 convalescent plasma identification.

For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and is the most important prognostic factor. Yet, LN metastases can harbor distinct tumor subclones from those within distant metastases, calling into question their role in distant metastasis. Here, we develop a syngeneic mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that a tumor-intrinsic interferon response program confers enhanced LN metastatic potential by upregulating MHC-I and PD-L1, enabling evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce regulatory T cells (Tregs), and generate tumor-specific immunosuppression that subsequently facilitates colonization of distant tissues. These effects extend to human cancers as well as additional cancers in mice, implicating a conserved mechanism by which malignancies spread to distant sites through systemic suppression of anti-tumor immunity.

Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low dendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg) in vitro and in vivo. Targeted depletion of this DC population in Mgl2DTR hosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell–mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection. Cancer Res; 77(15); 4158–70. ©2017 AACR.

Background/Methods: Visceral adipose tissue (VAT) is associated with increased risk for T2D and CVD. This depot, which accounts for only 5-10% of total fat mass, may play a relatively important role in determining insulin resistance (IR) due to its proximity to gut lymphatics and liver. Immune cells in fat respond to adipocyte stress and bacterial antigens from the gut with cytokine release, adversely affecting hepatic and systemic glucose metabolism. Few human studies have examined inflammation in VAT with regard to systemic IR. We hypothesized that inflammation in VAT vs. SAT would be associated with IR. To test this hypothesis we recruited 25 nondiabetic bariatric surgery patients who underwent whole-body insulin sensitivity testing via steady-state plasma glucose (SSPG) test prior to surgery. Samples of SAT and VAT were harvested for collagenase digestion and flash freezing for flow cytometry and gene expression. Results/Conclusion: The cohort was 80% female, 20% male, age 48±10 years, BMI 43.7±6.1 kg/m2, fasting glucose 103±15 mg/dL. IR was defined as SSPG > 200, and IS as SSPG < 160 mg/dL. Comparison of immune cell profiles revealed greater frequency of macrophages in both SAT and VAT in IR vs. IS subjects, and higher expression of proinflammatory marker CD44 in IR vs. IS subjects. Macrophage number and phenotype (M1, M2, CD44+) did not differ between depots, suggesting systemic rather than localized inflammation contributes to IR. Disclosure T. McLaughlin: Advisory Panel; Self; January. Stock/Shareholder; Self; Eiger BioPharmaceuticals. E.J. Zanley: None. C.I. Petlura: None. L.L. Tolentino: None. O. Choi: None. J. Chuang: None. J. Morton: None. Funding American Heart Association

Chronic intestinal inflammation accompanies familial adenomatous polyposis (FAP) and is a major risk factor for colorectal cancer in patients with this disease, but the cause of such inflammation is unknown. Because retinoic acid (RA) plays a critical role in maintaining immune homeostasis in the intestine, we hypothesized that altered RA metabolism contributes to inflammation and tumorigenesis in FAP. To assess this hypothesis, we analyzed RA metabolism in the intestines of patients with FAP as well as APCMin/+ mice, a model that recapitulates FAP in most respects. We also investigated the impact of intestinal RA repletion and depletion on tumorigenesis and inflammation in APCMin/+ mice. Tumors from both FAP patients and APCMin/+ mice displayed striking alterations in RA metabolism that resulted in reduced intestinal RA. APCMin/+ mice placed on a vitamin A–deficient diet exhibited further reductions in intestinal RA with concomitant increases in inflammation and tumor burden. Conversely, restoration of RA by pharmacologic blockade of the RA-catabolizing enzyme CYP26A1 attenuated inflammation and diminished tumor burden. To investigate the effect of RA deficiency on the gut immune system, we studied lamina propria dendritic cells (LPDC) because these cells play a central role in promoting tolerance. APCMin/+ LPDCs preferentially induced Th17 cells, but reverted to inducing Tregs following restoration of intestinal RA in vivo or direct treatment of LPDCs with RA in vitro. These findings demonstrate the importance of intestinal RA deficiency in tumorigenesis and suggest that pharmacologic repletion of RA could reduce tumorigenesis in FAP patients. Cancer Immunol Res; 4(11); 917–26. ©2016 AACR.

Although all-trans-retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis, whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8(+) Tcells, which were activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8(+) Tcells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.

Background Biallelic variants in PIGW have been suggested to cause infantile spasms and hyperphosphatasia. PIGW encodes for a protein involved in the third step of glycosylphosphatidylinositol (GPI) synthesis. GPI anchored proteins are increasingly recognized as important structures for cellular interactions and neuronal development. Methods Molecular testing of PIGW was performed followed by fluorescence activating cell sorting analysis of granulocytes, lymphocytes, and monocytes, and compared to controls. Findings An infant was homozygous for variants in PIGW (c.199C>G; p.Pro67Ala) with an associated phenotype of infantile spasms, myoclonic seizures, cortical visual impairment, developmental delay, and minor dysmorphic features. Alkaline phosphatase levels ranged from normal to mildly elevated. Flow cytometric studies showed significantly decreased expression of important GPIs, providing functional evidence of pathogenicity. Conclusion Our data provide further evidence of a novel autosomal recessive PIGW-related epilepsy disorder. Flow cytometry provided functional evidence of the pathogenicity of homozygous variants of uncertain significance in PIGW, and supports the use of flow cytometry as a functional tool to demonstrate decreased surface expression of GPI anchored proteins in individuals with variants of unknown significance.

Plasmacytoid dendritic cells (pDCs) are known mainly for their secretion of type I IFN upon viral encounter. We describe a CD2hiCD5+CD81+ pDC subset, distinguished by prominent dendrites and a mature phenotype, in human blood, bone marrow, and tonsil, which can be generated from CD34+ progenitors. These CD2hiCD5+CD81+ cells express classical pDC markers, as well as the toll-like receptors that enable conventional pDCs to respond to viral infection. However, their gene expression profile is distinct, and they produce little or no type I IFN upon stimulation with CpG oligonucleotides, likely due to their diminished expression of IFN regulatory factor 7. A similar population of CD5+CD81+ pDCs is present in mice and also does not produce type I IFN after CpG stimulation. In contrast to conventional CD5-CD81- pDCs, human CD5+CD81+ pDCs are potent stimulators of B-cell activation and antibody production and strong inducers of T-cell proliferation and Treg formation. These findings reveal the presence of a discrete pDC population that does not produce type I IFN and yet mediates important immune functions previously attributed to all pDCs.

Aim The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. Methods Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. Results Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p

BACKGROUND: The Trima Accel displays a “verify WBCs” message if the plateletpheresis product (PLT) may not be leukoreduced (LR). Most blood banks require sensitive white blood cell (WBC) testing of these PLTs by flow or Nageotte. We evaluated how often these PLTs were non-LR by European or US Food and Drug Administration (FDA) criteria and whether sensitive WBC testing is necessary. STUDY DESIGN AND METHODS: Phase 1 reviewed the frequency of this message with various procedure types and the flow WBC results for PLTs with or without the message. Phase 2 assessed how many FDA LR failures were detectable by a hematology analyzer. In Phase 3, PLTs were managed by hematology analyzer results. RESULTS: In Phase 1, 3.8% of PLT-only and 11.1% of PLT-plasma collections had the “verify WBCs” message. Only 1% of “verify” PLTs contained more than 1 × 106 WBCs and only 0.5% were FDA LR failures. In Phase 2, 10 of 670 “verify” PLTs and one nonflagged PLT were FDA LR failures. Six of 11 LR failures had hematology analyzer WBC concentrations of 0.4 × 109/L or higher. In Phase 3, “verify” PLTs were allowed in inventory if hematology analyzer WBC concentration was below 0.4 × 109/L; inventory quality control showed no FDA LR failures by flow. Trima Version 6.0 software lowered the “verify” message frequency in PLT-plasma procedures but not in PLT-only procedures. CONCLUSION: Four percent of Trima PLT collections have the “verify WBCs” message but almost all of these are LR by European and FDA criteria. Fifty percent of FDA LR failures were detectable by a hematology analyzer. Sensitive WBC testing of all “verify WBCs” PLTs may not be necessary to satisfy LR quality assurance requirements.

Objective— The biological mechanisms linking obesity to insulin resistance have not been fully elucidated. We have shown that insulin resistance or glucose intolerance in diet-induced obese mice is related to a shift in the ratio of pro- and anti-inflammatory T cells in adipose tissue. We sought to test the hypothesis that the balance of T-cell phenotypes would be similarly related to insulin resistance in human obesity. Approach and Results— Healthy overweight or obese human subjects underwent adipose-tissue biopsies and quantification of insulin-mediated glucose disposal by the modified insulin suppression test. T-cell subsets were quantified by flow cytometry in visceral (VAT) and subcutaneous adipose tissue (SAT). Results showed that CD4 and CD8 T cells infiltrate both depots, with proinflammatory T-helper (Th)-1, Th17, and CD8 T cells, significantly more frequent in VAT as compared with SAT. T-cell profiles in SAT and VAT correlated significantly with one another and with peripheral blood. Th1 frequency in SAT and VAT correlated directly, whereas Th2 frequency in VAT correlated inversely, with plasma high-sensitivity C-reactive protein concentrations. Th2 in both depots and peripheral blood was inversely associated with systemic insulin resistance. Furthermore, Th1 in SAT correlated with plasma interleukin-6. Relative expression of associated cytokines, measured by real-time polymerase chain reaction, reflected flow cytometry results. Most notably, adipose tissue expression of anti-inflammatory interleukin-10 was inversely associated with insulin resistance. Conclusions— CD4 and CD8 T cells populate human adipose tissue and the relative frequency of Th1 and Th2 are highly associated with systemic inflammation and insulin resistance. These findings point to the adaptive immune system as a potential mediator between obesity and insulin resistance or inflammation. Identification of antigenic stimuli in adipose tissue may yield novel targets for treatment of obesity-associated metabolic disease.

Aims/hypothesis Proinflammatory immune cell infiltration in human adipose tissue is associated with the development of insulin resistance. We previously identified, via a gene expression-based genome-wide association study, the cellsurface immune cell receptor CD44 as a functionally important gene associated with type 2 diabetes. We then showed that, compared with controls, Cd44 knockout mice were protected from insulin resistance and adipose tissue inflammation during diet-induced obesity. We thus sought to test whether CD44 is associated with adipose tissue inflammation and insulin resistance in humans. Methods Participants included 58 healthy, overweight/ moderatelyobese white adultswho met predetermined criteria for insulin resistance or insulin sensitivity based on the modified insulin-suppression test. Serum was collected from 43 participants to measure circulating concentrations of CD44. Subcutaneous adipose tissue was obtained from 17 participants to compare CD44, its ligand osteopontin (OPN, also known as SPP1) and pro-inflammatory gene expression. CD44 expression on adipose tissue macrophage (ATM) surfaces was determined by flow cytometry. Results Serum CD44 concentrations were significantly increased in insulin-resistant (IR) participants. CD44 gene expression in subcutaneous adipose tissue was threefold higher in the IR subgroup. The expression of OPN, CD68 and IL6 was also significantly elevated in IR individuals. CD44 gene expressioncorrelatedsignificantly withCD68andIL6expression. CD44 density on ATMs was associated with proinflammatory M1 polarisation. Conclusions/interpretation CD44andOPNinhumanadipose tissueareassociatedwithlocalisedinflammationandsystemic insulin resistance. This receptor–ligand pair is worthy of further research as a potentially modifiable contributor to human insulin resistance and type 2 diabetes.

Monocytes and T helper (TH) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that TH cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, TH1 and TH17 cells interact with monocytes and instruct these cells to differentiate into TH1- and TH17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, TH2 cells interact with monocytes and elicit the formation of TH2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas TH subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell–induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that TH-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that TH cells govern the formation and function of specialized DC subsets.