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Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless,many details of the immunopathogenesis of TRALI,particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast,34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia,severe pulmonary edema,and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8(+) T lymphocytes or CD4(+) T cells but not CD19(+) B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia,lung damage,and mortality,suggesting that T lymphocytes were responsible for the protective effect. Taken together,these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions. View Publication

RATIONALE: Chitin is a ubiquitous polysaccharide in fungi,insects,allergens,and parasites that is released at sites of infection. Its role in the generation of tissue inflammation,however,is not fully understood. OBJECTIVES: We hypothesized that chitin is an important adjuvant for adaptive immunity. METHODS: Mice were injected with a solution of ovalbumin and chitin. MEASUREMENTS AND MAIN RESULTS: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo,ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response,Th2 cytokine elaboration,IgE induction,and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2,MyD88,and IL-17A played critical roles in the chitin-induced responses,and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro,CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments,CD4(+) T-cell proliferation,IL-5,IL-13,IFN-γ,and IL-17A production were appreciated. Toll-like receptor-2,MyD88,and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation,whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. CONCLUSIONS: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2,Th1,and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2,MyD88,and IL-17A. View Publication

Gammaherpesviruses,including Kaposi sarcoma-associated herpesvirus (KSHV),establish latency in B cells. We hypothesized that the KSHV latency-associated nuclear antigen (LANA/orf73) provides a selective advantage to infected B cells by driving proliferation in response to antigen. To test this,we used LANA B-cell transgenic mice. Eight days after immunization with antigen without adjuvant,LANA mice had significantly more activated germinal center (GC) B cells (CD19(+) PNA(+) CD71(+)) than controls. This was dependent upon B-cell receptor since LANA did not restore the GC defect of CD19 knockout mice. However,LANA was able to restore the marginal zone defect in CD19 knockout mice. View Publication

NK cells are the dominant population of immune cells in the endometrium in the secretory phase of the menstrual cycle and in the decidua in early pregnancy. The possibility that this is a site of NK cell development is of particular interest because of the cyclical death and regeneration of the NK population during the menstrual cycle. To investigate this,we searched for NK developmental stages 1-4,based on expression of CD34,CD117,and CD94. In this study,we report that a heterogeneous population of stage 3 NK precursor (CD34(-)CD117(+)CD94(-)) and mature stage 4 NK (CD34(-)CD117(-/+)CD94(+)) cells,but not multipotent stages 1 and 2 (CD34(+)),are present in the uterine mucosa. Cells within the uterine stage 3 population are able to give rise to mature stage 4-like cells in vitro but also produce IL-22 and express RORC and LTA. We also found stage 3 cells with NK progenitor potential in peripheral blood. We propose that stage 3 cells are recruited from the blood to the uterus and mature in the uterine microenvironment to become distinctive uterine NK cells. IL-22 producers in this population might have a physiological role in this specialist mucosa dedicated to reproduction. View Publication

OBJECTIVES: Sustained suppression of plasma viremia in HIV-infected individuals is attainable with antiretroviral therapy (ART); however,eradication of virus that would allow discontinuation of ART has been hampered by the persistence of HIV reservoirs. It is of great interest to identify individuals who had received ART for prolonged periods of time with extremely low or undetectable HIV reservoirs and monitor plasma viremia following discontinuation of therapy. METHODS: We measured the size of HIV reservoirs in CD4(+) T cells of individuals on long-term ART and monitored plasma viremia following cessation of ART in one individual with an exceptionally low viral burden after a decade of therapy. RESULTS: We demonstrated undetectable levels of HIV DNA in the blood of eight of 45 infected individuals on long-term ART. Among those eight individuals,the frequency of cells carrying infectious virus was significantly lower in those who initiated ART during the early versus the chronic phase of infection. One individual with undetectable HIV DNA in both blood and tissue and a profoundly low level of infectious virus experienced plasma viral rebound 50 days following discontinuation of ART. CONCLUSIONS: Our data suggest that a significant reduction in the size of viral reservoirs may be achievable in selected individuals who initiate standard ART early in infection. However,given re-emergence of plasma viremia in an individual with an extraordinarily low viral burden,therapeutic strategies aimed at specifically targeting these extremely rare HIV-infected cells with novel interventions may be necessary in order to achieve eradication of virus. View Publication

A small subset of human cord blood CD34+ cells express endothelial protein C receptor (EPCR/CD201/PROCR) when exposed to the hematopoietic stem cell (HSC) self-renewal agonist UM171. In this article,we show that EPCR-positive UM171-treated cells,as opposed to EPCR-negative cells,exhibit robust multilineage repopulation and serial reconstitution ability in immunocompromised mice. In contrast to other stem cell markers,such as CD38,EPCR expression is maintained when cells are introduced in culture,irrespective of UM171 treatment. Although engineered overexpression of EPCR fails to reproduce the effects of UM171 on HSC activity,its expression is required for the repopulating activity of human HSCs. Altogether,our results indicate that EPCR is a reliable and cell culture-compatible marker of UM171-expanded human cord blood HSCs. View Publication

The T cell antigen receptor (TCR) recognizes peptides from pathogenic proteins bound in the major histocompatibility complex (MHC). To convert this binding event into downstream signaling,the TCR complex contains immunoreceptor tyrosine-based activation motifs (ITAMs) that act as docking sites for the cytoplasmic tyrosine kinase ZAP-70. Unique among antigen receptors,the TCR complex uses 10 ITAMs to transduce peptide-MHC binding to the cell interior. Using synthetic,drug-inducible receptor-ligand pairs,it was found that greater ITAM multiplicity primarily enhanced the efficiency with which ligand binding was converted into an intracellular signal. This manifested as an increase in the fraction of cells that became activated in response to antigen,and a more synchronous initiation of TCR-proximal signaling,rather than direct amplification of the intracellular signals. Exploiting these findings,the potency and selectivity of chimeric antigen receptors targeted against cancer were substantially enhanced by modulating the number of encoded ITAMs. View Publication

Proteasome inhibitors bortezomib,carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM),but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites,$\beta$5i,$\beta$1i,and $\beta$2i,which are different from their constitutive $\beta$5c,$\beta$1c,and $\beta$2c counterparts. Bortezomib and carfilzomib block $\beta$5c and $\beta$5i sites. We report here that pharmacologically relevant concentrations of $\beta$5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition,increasing immunoproteasome expression by interferon-$\gamma$ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102,an inhibitor of $\beta$2 sites,dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus,immunoproteasome inhibitors,currently in clinical trials for the treatment of autoimmune diseases,should also be considered for the treatment of MM. View Publication

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. View Publication

Comprehensive identification of factors that can specify neuronal fate could provide valuable insights into lineage specification and reprogramming,but systematic interrogation of transcription factors,and their interactions with each other,has proven technically challenging. We developed a CRISPR activation (CRISPRa) approach to systematically identify regulators of neuronal-fate specification. We activated expression of all endogenous transcription factors and other regulators via a pooled CRISPRa screen in embryonic stem cells,revealing genes including epigenetic regulators such as Ezh2 that can induce neuronal fate. Systematic CRISPR-based activation of factor pairs allowed us to generate a genetic interaction map for neuronal differentiation,with confirmation of top individual and combinatorial hits as bona fide inducers of neuronal fate. Several factor pairs could directly reprogram fibroblasts into neurons,which shared similar transcriptional programs with endogenous neurons. This study provides an unbiased discovery approach for systematic identification of genes that drive cell-fate acquisition. View Publication

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells,resulting in reversal of Treg suppression. Importantly,TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression,which may be used as a strategy to advance tumor immunotherapy. View Publication

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication