Citations

Abstract Regulation of complement activation is essential in the prevention of damage to autologous tissue. This activity is mediated by the presence of specific complement regulatory proteins on the surface of host cells. In humans, one molecule involved in this regulation is a 70-kDa glycoprotein that has been designated decay-accelerating factor (DAF). We present the full-length cDNA sequence and chromosomal localization of the mouse genetic homologue of the human DAF gene. Interestingly, two classes of cDNA clones were obtained that, rather than representing alternately spliced mRNAs, were derived from two separate but closely related linked genes. Both genes encoded proteins with an amino-terminal signal sequence, followed by four short consensus repeats and a domain rich in serine and threonine. Hydrophilicity plots and alignment with human DAF predicted that one gene encoded a glycosylphosphatidylinositol-anchored form of mouse Daf with 64% nucleotide and 47% amino acid identity to human DAF. The product encoded by the second gene was predicted to have an alternate amino-terminal signal sequence and carboxyl-terminal membrane-spanning and cytoplasmic domains. The two mouse Daf genes share 85% nucleotide and 78% amino acid identities, and have been designated Daf-glycosylphosphatidylinositol and Daf-transmembrane to reflect the two alternate mechanisms of membrane attachment. mRNA expression analysis indicated that the two mouse Daf genes were differentially expressed in the adult mouse. Chromosome localization studies mapped the mouse Daf genes to chromosome 1, where they segregated with the C4-binding protein (C4bp) gene.

AbstractIntroductionEmpathy relies on fronto‐cingular and temporal networks that are selectively vulnerable in behavioral variant frontotemporal dementia (bvFTD). This study modeled when in the disease process empathy changes begin, and how they progress.MethodsFour hundred thirty‐one individuals with asymptomatic genetic FTD (n = 114), genetic and sporadic bvFTD (n = 317), and 163 asymptomatic non‐carrier controls were enrolled. In sub‐samples, we investigated empathy measured by the informant‐based Interpersonal Reactivity Index (IRI) at each disease stage and over time (n = 91), and its correspondence to underlying atrophy (n = 51).ResultsEmpathic concern (estimate = 4.38, 95% confidence interval [CI] = 2.79, 5.97; p < 0.001) and perspective taking (estimate = 5.64, 95% CI = 3.81, 7.48; p < 0.001) scores declined between the asymptomatic and very mild symptomatic stages regardless of pathogenic variant status. More rapid loss of empathy corresponded with subcortical atrophy.DiscussionLoss of empathy is an early and progressive symptom of bvFTD that is measurable by IRI informant ratings and can be used to monitor behavior in neuropsychiatry practice and treatment trials.

Abstract Chronic inflammation in the aged closely resembles tumor-induced immune suppression, manifested as a progressive T1→T2 shift and an increased presence of MDSCs. We hypothesize that reversing these age-related phenomena should reduce the elderly’s heightened susceptibility to malignancy. We have now optimized real-time analyses of phosphorylated STATs in murine T cells and MDSCs in order to correlate STAT activation with immune suppression. Both young and old T cells responded to IL-4 stimulation with STAT6 activation and to IFN-γ stimulation with STAT1 activation. Uniquely, however, young T cells also phosphorylated STAT1, STAT3 and STAT5 in response to IL-4, and responded to IL-6 with STAT3 activation, all consistent with a greater susceptibility to T1/T17 differentiation. Regarding MDSCs, both old and young displayed constitutive STAT3 activation which could be enhanced by G-CSF or IL-6 treatment. Both young and old MDSCs activated STAT1 briskly in response to IFN-γ, STAT5 in response to GM-CSF and STAT6 in response to IL-4. However, younger MDSCs, like younger T cells, displayed more diverse responsiveness to IL-4, activating not only STAT6 but also STAT1 and STAT3. While chronic in vivo exposure to T2 cytokines may explain the decreased responsiveness of older MDSCs and T cells to IL-4, paradoxically IL-4 still robustly activated STAT6 in these cells. Such selective responsiveness may be required to preserve the immune suppressive potential of older MDSCs.

Abstract Recent guidelines by the American Society of Clinical Oncologists recommend that obese individuals receive chemotherapy based on their actual body weight (ABW) without dose reduction. Contrary to prior reports, our own data demonstrate that ABW-dosed chemotherapy or immunotherapy is as effective and well tolerated by obese mice as by lean mice. Nonetheless, obese mice develop significantly greater weight loss in a dose-dependent manner during the entire period that they receive therapy, with corresponding quantifiable reductions in visceral fat depots. This disparity is not explained by altered food intake, as both lean and obese mice experience less than a week of anorexia which does not recur after Cycle 1 of treatment. Furthermore, malabsorption is absent. These observations are relevant to multiple classes of chemo agents. Altered pharmacokinetics of chemotherapy agents in adipose tissue have been reported, yet the pharmacodynamics of obesity remain speculative. Fasting blood glucose monitoring demonstrates glucose level variances which are temporally linked to treatment, rendering it likely that chemotherapy and immunotherapy provoke adipose tissue lipolysis, or alternatively cytoreduction or immunomodulation of adipocyte progenitor cells. Understanding these effects could contribute to improved treatment regimens in the setting of obesity.

Abstract IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

Abstract Although tumors from melanoma patients can often provide a source of therapeutically active T cells for adoptive therapy, it has historically proved challenging to employ peripheral blood as the T cell source for any type of cancer. We have observed nonetheless that HER2-specific T cells can be Ag-driven and enriched in culture when PBMCs from HER2-vaccinated breast cancer patients are treated with exogenous IL12. We hypothesized that stimulating innate immunity could further improve PBMC cultures by activating the DC subpopulation more effectively than adding exogenous rIL12. Bulk PBMC exposure to optimized TLR agonists produced vast quantities of IL12 and IL23, and upregulated HLA-DR, B7.1 and CD40 in the DC subpopulation, effects not produced by exogenous IL12. Subsequent IL7 exposure emulated homeostatic proliferation, selectively causing the Ag-driven T cell subset both to proliferate faster and to strongly resist apoptosis. Ag-specificity within two weeks approached 80-100% of both CD4+ and CD8+ T cells for recall Ags and 1-15% for a wide array of MUC1- and HER2-derived peptides, including the ability to distinguish glycoforms. Culture-expanded T cells retained a young CD28+/CD56- phenotype, uniformly expressed ROR-γ during culture, variably produced IL-2 and/or IL-17, and uniformly expressed T-bet and secreted IFN-γ when reexposed to Ag. Such T1/T17 bipotency may be ideal for anti-tumor adoptive therapy and highly efficient for use in Ag discovery.

Abstract Chronic inflammation associated with aging is termed inflamm-aging. It manifests as a rise in many proinflammatory mediators and contributes to a decline in immune function. Unregulated inflammasome activation initiates an inappropriate inflammatory response. It is particularly imperative to understand inflammasome dysregulation in the elderly (&gt;65 yrs), since it is a likely basis of immune dysfunction. Analyses of freshly isolated memory CD4+ and CD8+ splenic T cells demonstrated that even without further activation, inflammasome genes were already significantly up-regulated in old mice compared to young, including factors critical to myeloid-derived suppressor cell (MDSC) expansion including S100a8, S100a9, IL-1β, IL-13 and IL-6. A corresponding 2-3 fold increase in CD11b+Gr1+cells was observed in the spleens of old naïve mice compared to young. Importantly, cells with the CD11b+Gr1+ phenotype from old mice, unlike young, were functionally MDSCs and suppressed T cell proliferation. Therefore, the immunosuppressive environment of the tumor-bearing host also appears to be operative in aging, even in the absence of tumor. We hypothesize that constituitive activation of inflammasome complex results in a chronic inflammatory state, altering immune responses in the elderly similarly to the tumor-bearing state. Preemptive reversal of inflammasome-induced MDSCs may enhance T cell immunosurveillance in the elderly, a population with a heightened susceptibility to malignancy.

Abstract A C57BL/6 mouse transgenic for human MUC1 (MUC1.Tg) was developed to evaluate MUC1-specific tumor immunity in an animal that expresses MUC1 as a normal self protein. Previous studies showed that MUC1.Tg mice, challenged with syngeneic tumors expressing MUC1 (B16.MUC1), developed progressively growing MUC1-positive tumors, whereas wild-type C57BL/6 (wt) mice developed MUC1-negative tumors at a significantly slower rate. The results of a limiting dilution CTL frequency assay were not informative, in that similar numbers of MUC1-specific CTL precursors (CTL) were detected in MUC1.Tg and wt mice. Tumor immunity in vivo was characterized by an adoptive transfer method to evaluate the degree of MUC1 or non-MUC1 tumor immunity in wt or MUC1.Tg mice. The results revealed that wt mice developed protective tumor immunity mediated by MUC1-specific CD4+ lymphocytes, while MUC1.Tg mice were functionally tolerant to MUC1 in vivo. The potential of adoptive immunotherapy to provide immunity to tumors expressing MUC1 and to produce undesirable autoimmunity in recipient MUC1.Tg mice expressing MUC1 as a self Ag was evaluated. Adoptive transfer of immune cells from wt mice primed in vivo with B16.MUC1 tumor cells into MUC1.Tg recipients resulted in significant increases in the survival of MUC1.Tg recipients compared with unmanipulated control MUC1.Tg mice challenged with B16.MUC1 tumor cells. This response was specific for MUC1 since control tumors developed at equivalent rates in recipient or control MUC1.Tg mice. No gross or histologic evidence of autoimmunity was observed in recipient MUC1.Tg mice, indicating that tumor immune responses mediated by MUC1-specific CD4+ lymphocytes spare nontransformed epithelia-expressing MUC1.

Abstract The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.

Abstract A double antibody radioimmunoassay (RIA) was developed to measure IgD in serum and secretions. One IgD myeloma protein was used as radiolabeled antigen and standard with antiserum to a second IgD myeloma protein. The IgD standard and normal sera yielded parallel inhibition curves in the RIA and inhibition was produced by IgD and not by any of the other immunoglobulins. The assay had a lower limit of sensitivity of 0.01 International Unit (I.U.)/ml and modifications increased the sensitivity to 0.0008 I.U./ml. Measurable IgD levels were found in all 112 normal adult sera assayed (geometric mean 13.0 I.U./ml, arithmetic mean 30.1 I.U./ml, median 14.8 I.U./ml, range 0.10 to 202 I.U./ml, 95% of values between 0.19 and 156 I.U./ml. The distribution of IgD in the 112 normal sera appeared trimodal with modes at approximately 0.25 I.U./ml, 5 I.U./ml, and 35 I.U./ml. IgD was measurable in nasal and bronchial washes and human milk, but could not be detected in parotid fluid.