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Immunotherapy has increased overall survival of metastatic cancer patients,and cancer antigens are promising vaccine targets. To fulfill the promise,appropriate tailoring of the vaccine formulations to mount in vivo cytotoxic T cell (CTL) responses toward co-delivered cancer antigens is essential. Previous development of therapeutic cancer vaccines has largely been based on studies in mice,and the majority of these candidate vaccines failed to induce therapeutic responses in the subsequent human clinical trials. Given that antigen dose and vaccine volume in pigs are translatable to humans and the porcine immunome is closer related to the human counterpart,we here introduce pigs as a supplementary large animal model for human cancer vaccine development. IDO and RhoC,both important in human cancer development and progression,were used as vaccine targets and 12 pigs were immunized with overlapping 20mer peptides spanning the entire porcine IDO and RhoC sequences formulated in CTL-inducing adjuvants: CAF09,CASAC,Montanide ISA 51 VG,or PBS. Taking advantage of recombinant swine MHC class I molecules (SLAs),the peptide-SLA complex stability was measured for 198 IDO- or RhoC-derived 9-11mer peptides predicted to bind to SLA-1(*)04:01,-1(*)07:02,-2(*)04:01,-2(*)05:02,and/or -3(*)04:01. This identified 89 stable (t½ ≥ 0.5 h) peptide-SLA complexes. By IFN-$\gamma$ release in PBMC cultures we monitored the vaccine-induced peptide-specific CTL responses,and found responses to both IDO- and RhoC-derived peptides across all groups with no adjuvant being superior. These findings support the further use of pigs as a large animal model for vaccine development against human cancer. View Publication

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system,we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes,including TMPRSS2-ERG fusion,SPOP mutation,SPINK1 overexpression,and CHD1 loss. Whole-exome sequencing shows a low mutational burden,consistent with genomics studies,but with mutations in FOXA1 and PIK3R1,as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies. View Publication

Small-cell lung cancer (SCLC),an aggressive neuroendocrine tumor with early dissemination and dismal prognosis,accounts for 15-20{\%} of lung cancer cases and ∼200,000 deaths each year. Most cases are inoperable,and biopsies to investigate SCLC biology are rarely obtainable. Circulating tumor cells (CTCs),which are prevalent in SCLC,present a readily accessible 'liquid biopsy'. Here we show that CTCs from patients with either chemosensitive or chemorefractory SCLC are tumorigenic in immune-compromised mice,and the resultant CTC-derived explants (CDXs) mirror the donor patient's response to platinum and etoposide chemotherapy. Genomic analysis of isolated CTCs revealed considerable similarity to the corresponding CDX. Most marked differences were observed between CDXs from patients with different clinical outcomes. These data demonstrate that CTC molecular analysis via serial blood sampling could facilitate delivery of personalized medicine for SCLC. CDXs are readily passaged,and these unique mouse models provide tractable systems for therapy testing and understanding drug resistance mechanisms. View Publication

Maintenance of blood DC homeostasis is essential to preventing autoimmunity while controlling chronic infection. However,the ability of bacteremic pathogens to directly regulate blood DC homeostasis has not been defined. One such bacteremic pathogen,Porphyromonas gingivalis,is shown by our group to survive within mDCs under aerobic conditions and therein,metastasize from its oral mucosal niche. This is accompanied by expansion of the blood mDC pool in vivo,independently of canonical DC poietins. We presently know little of how this bacteremic pathogen causes blood DC expansion and the pathophysiological significance. This work shows that optimum differentiation of MoDCs from primary human monocytes,with or without GM-CSF/IL-4,is dependent on infection with P. gingivalis strains expressing the DC-SIGN ligand mfa-1. DC differentiation is lost when DC-SIGN is blocked with its ligand HIV gp120 or knocked out by siRNA gene silencing. Thus,we have identified a novel,noncanonical pathway of DC differentiation. We term these PDDCs and show that PDDCs are bona fide DCs,based on phenotype and phagocytic activity when immature and the ability to up-regulate accessory molecules and stimulate allo-CD4(+) T cell proliferation when matured. The latter is dependent on the P. gingivalis strain used to initially educate" PDDCs. Moreover we show that P. gingivalis-infected conventional MoDCs become resistant to apoptosis and inflammatory pyroptosis as determined by levels of Annexin V and caspase-8 -3/7 and -1. Taken together we provide new insights into how a relatively asymptomatic bacteremia may influence immune homeostasis and promote chronic inflammation." View Publication

Diverse hematopoietic progenitors,including myeloid populations arising in inflammatory and tumoral conditions and multipotent cells,mobilized by hematopoietic growth factors or emerging during parasitic infections,display tolerogenic properties. Innate immune stimuli confer regulatory functions to various mature B-cell subsets but immature B-cell progenitors endowed with suppressive properties per se or after differentiating into more mature regulatory B cells remain to be characterized. Herein we provide evidence for innate pro-B cells (CpG-proBs) that emerged within the bone marrow both in vitro and in vivo upon Toll-like receptor-9 activation and whose adoptive transfer protected nonobese diabetic mice against type 1 diabetes (T1D). These cells responded to IFN-$\gamma$ released by activated effector T cells (Teffs),by up-regulating their Fas ligand (FasL) expression,which enabled them to kill Teffs through apoptosis. In turn,IFN-$\gamma$ derived from CpG-proBs enhanced IFN-$\gamma$ while dramatically reducing IL-21 production by Teffs. In keeping with the crucial pathogenic role played by IL-21 in T1D,adoptively transferred IFN-$\gamma$-deficient CpG-proBs did not prevent T1D development. Additionally,CpG-proBs matured in vivo into diverse pancreatic and splenic suppressive FasL(high) B-cell subsets. CpG-proBs may become instrumental in cell therapy of autoimmune diseases either on their own or as graft complement in autologous stem cell transplantation. View Publication

IMPORTANCE Although leukocyte telomere length is associated with mortality and many chronic diseases thought to be manifestations of age-related functional decline,it is not known whether it relates to acute disease in younger healthy populations. OBJECTIVE To determine whether shorter telomeres in leukocytes,especially CD8CD28- T cells,are associated with decreased resistance to upper respiratory infection and clinical illness in young to midlife adults. DESIGN,SETTING,AND PARTICIPANTS Between 2008 and 2011,telomere length was assessed in peripheral blood mononuclear cells (PBMCs) and T-cell subsets (CD4,CD8CD28+,CD8CD28-) from 152 healthy 18- to 55-year-old residents of Pittsburgh,Pennsylvania. Participants were subsequently quarantined (single rooms),administered nasal drops containing a common cold virus (rhinovirus 39),and monitored for 5 days for development of infection and clinical illness. MAIN OUTCOME MEASURES Infection (virus shedding or 4-fold increase in virus-specific antibody titer) and clinical illness (verified infection plus objective signs of illness). RESULTS Rates of infections and clinical illness were 69{\%} (n = 105) and 22{\%} (n = 33),respectively. Shorter telomeres were associated with greater odds of infection,independent of prechallenge virus-specific antibody,demographics,contraceptive use,season,and body mass index (PBMC: odds ratio [OR] per 1-SD decrease in telomere length,1.71 [95{\%} CI,1.08-2.72]; n = 128 [shortest tertile 77{\%} infected; middle,66{\%}; longest,57{\%}]; CD4: OR,1.76 [95{\%} CI,1.15-2.70]; n = 146 [shortest tertile 80{\%} infected; middle,71{\%}; longest,54{\%}]; CD8CD28+: OR,1.93 [95{\%} CI,1.21-3.09],n = 132 [shortest tertile 84{\%} infected; middle,64{\%}; longest,58{\%}]; CD8CD28-: OR,2.02 [95{\%} CI,1.29-3.16]; n = 144 [shortest tertile 77{\%} infected; middle,75{\%}; longest,50{\%}]). CD8CD28- was the only cell population in which shorter telomeres were associated with greater risk of clinical illness (OR,1.69 [95{\%} CI,1.01-2.84]; n = 144 [shortest tertile,26{\%}; middle,22{\%}; longest,13{\%}]). The association between CD8CD28- telomere length and infection increased with age (CD8CD28- telomere length × age interaction,b = 0.09 [95{\%} CI,0.02-0.16],P = .01,n = 144). CONCLUSION AND RELEVANCE In this preliminary study among a cohort of healthy 18- to 55-year-olds,shorter CD8CD28- T-cell telomere length was associated with increased risk for experimentally induced acute upper respiratory infection and clinical illness. View Publication

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances.

During the progression of autoimmune (type 1) diabetes,T cells and macrophages infiltrate the pancreas,disrupt islet function,and destroy insulin-producing beta cells. B-lymphocytes,particularly innate like B-cell populations such as marginal zone B cells and B-1 cells,have been implicated in many autoimmune diseases,and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence,inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110$\delta$ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110$\delta$ is important for B cell migration,activation,proliferation,and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110$\delta$ activity,and p110$\delta$ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity,we tested whether a p110$\delta$ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114,a highly selective small molecule inhibitor of p110$\delta$,reduced the infiltration of inflammatory cells into the pancreatic islets and,accordingly,delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode,IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3K$\delta$ inhibitors could be useful for managing type 1 diabetes. View Publication

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K,which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 $\mu$mol/L (MCF7 cells) and 0.17 $\mu$mol/L (SW620 cells),and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 $\mu$mol/L (MCF7 cells) and more than 10 $\mu$mol/L (SW620 cells). Five-day exposure to 1 $\mu$mol/L KU-0060648 inhibited cell proliferation by more than 95{\%} in MCF7 cells but only by 55{\%} in SW620 cells. In clonogenic survival assays,KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells,but not in DNA-PKcs-deficient cells,thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts,concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold,without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. View Publication

Pulmonary metastasis of breast cancer requires recruitment and expansion of T-regulatory cells (Treg) that promote escape from host protective immune cells. However,it remains unclear precisely how tumors recruit Tregs to support metastatic growth. Here we report the mechanistic involvement of a unique and previously undescribed subset of regulatory B cells. These cells,designated tumor-evoked Bregs (tBreg),phenotypically resemble activated but poorly proliferative mature B2 cells (CD19(+) CD25(High) CD69(High)) that express constitutively active Stat3 and B7-H1(High) CD81(High) CD86(High) CD62L(Low) IgM(Int). Our studies with the mouse 4T1 model of breast cancer indicate that the primary role of tBregs in lung metastases is to induce TGF-$\beta$-dependent conversion of FoxP3(+) Tregs from resting CD4(+) T cells. In the absence of tBregs,4T1 tumors cannot metastasize into the lungs efficiently due to poor Treg conversion. Our findings have important clinical implications,as they suggest that tBregs must be controlled to interrupt the initiation of a key cancer-induced immunosuppressive event that is critical to support cancer metastasis. View Publication