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Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery.
We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed.
Conclusions and Significance
Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines.
Citation: Eger KA, Sundrud MS, Motsinger AA, Tseng M, Kaer LV, Unutmaz D (2006) Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions. PLoS ONE 1(1): e50. doi:10.1371/journal.pone.0000050
Academic Editor: Douglas Nixon, University of California, San Francisco, United States of America
Received: October 9, 2006; Accepted: October 12, 2006; Published: December 20, 2006
Copyright: © 2006 Eger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants AI50953, NS44044, HL68744 to LVK, GM62758 to AAM, T32 HL069765 to KAE, and AI054206 to DU.
Competing interests: The authors have declared that no competing interests exist.
NKT cells play important and multifaceted roles in immune regulation, tumor rejection and resistance to a variety of viral, bacterial, and parasitic pathogens through their rapid secretion of immunoregulatory cytokines and potent cytotoxicity [1]–[4]. NKT cells are characterized by a highly conserved T cell receptor (TCR) in both mice and humans [5]–[9], with human NKT cells most reliably identified by the expression of their invariant TCR, consisting of a Vα24-Jα18 chain preferentially paired with a Vβ11 chain [10]–[12]. NKT cells are present at low and highly variable frequencies in human blood (0.01%–1%) but are present at higher numbers in the liver [13]–[15].
In contrast to conventional T cells, NKT cells recognize glycolipid antigens presented by the non-polymorphic MHC class-I like molecule CD1d [5], [16]–[18]. The marine sponge derived glycosphingolipid α-galactosylceramide (α-GalCer) was the first synthetic NKT ligand identified that specifically stimulated NKT cells in the context of CD1d [19]. More recently, NKT cells have been shown to recognize and respond to an endogenous lysosomal glycosphingolipid, isoglobotrihexosylceramide [20]. Several bacterial derived glycolipids have also been recently shown to activate NKT cells [9], [21]–[25].
NKT cells can be phenotypically subdivided into CD4+ and CD4− subsets. A portion of both human and monkey NKT cells also express CD8 [26]–[31]. While human adult NKT cells are comprised of both CD4+ and CD4− subsets, neonatal NKT cells are almost exclusively CD4+ [32]–[34]. Furthermore, CD4+ NKT cells but not CD4− NKT cells have been shown to develop in the human fetal thymus [32], [34]. It remains unclear whether CD4− NKT cells develop from precursor CD4+ NKT cells or as a separate lineage.
Upon activation, NKT cells rapidly secrete large amounts of cytokines, including interferon gamma (IFN-γ) and IL-4. While some functional differences between human CD4+ and CD4− NKT cells have been described [27], [28], [35], it is presently unclear whether NKT cells are solely comprised of subsets that are hardwired to secrete specific cytokine patterns or if some of these subsets can modify their effector functions during peripheral immune activation, analogous to conventional T cells. In contrast to murine NKT cells, which were reported to be resistant to cytokine polarization in vivo [36], upon activation with either environmentally instructed dendritic cells (DCs) or DC1 and DC2 antigen presenting cells (APCs), human NKT cells have been shown to undergo polarization into cells with Th1- or Th2-like cytokine production profiles, termed NKT1 and NKT2 cells [37], [38]. A strong correlation between CD4 expression and the capacity to produce Th2 cytokines has also been reported [27], [28], [35]. Examination of the expression patterns of chemokine receptors and adhesion molecules, which play critical roles in leukocyte trafficking, have revealed further differences between CD4+ and CD4− NKT cells [35].
In the current study, we demonstrate that neonatal CD4+ (nCD4+), adult CD4+ (aCD4+) and adult CD4− (aCD4−) NKT cells are phenotypically distinct in their expression of effector/memory T cell markers and NK cell markers. Consistent with their more naïve phenotype, we also demonstrate that some, but not all, of the effector functions of human nCD4+ NKT cells can be easily modified through either polarizing cytokines or ectopic expression of the master Th1 and Th2 transcription factors (TFs) T-bet and GATA-3, respectively. By comparison, aCD4+ NKT cells were less permissive, whereas aCD4− NKT cells were largely refractory to these polarizing signals. These findings have implications for harnessing and reprogramming the effector functions of human NKT cells in vivo for novel therapeutic and vaccine approaches.
Phenotypic characterization of human NKT cell subsets
Human NKT cells can be subdivided into CD4+ and CD4− subsets, which display functionally distinct cytokine secretion profiles [27], [28], [35]. We further characterized the expression profiles of a panel of cell surface markers on resting nCD4+, aCD4+ and aCD4− NKT cells. Neonatal NKT cells were predominantly (>90%) CD4+, whereas adult blood NKT cells were either CD4+ or CD4−, with variable levels of CD8 expression (Figure 1A), which is consistent with previous reports [32], [33]. The chemokine receptor CCR7 and the lymph node homing receptor CD62L, which are specifically expressed on naïve and central memory T cells [39], were expressed highly on nCD4+, moderately on aCD4+, and relatively lower on aCD4− NKT cells (Figures 1B and 1C). In contrast, expression of the NK lineage receptors CD94 and CD161 were mostly absent on nCD4+ T cells but detected at moderate and high levels on aCD4+ and aCD4− NKT cells, respectively (Figures 1B and 1C). Expression of the co-stimulatory molecule CD28 was high on all subsets, but statistically higher on nCD4+ NKT cells compared to either adult NKT cell subsets (Figures 1B and 1C). Because we observed heterogeneous expression of the above markers on aCD4+ NKT cells from different donors, we next determined whether these markers were expressed coordinately on cells from the same donor, such that donors with high levels of CCR7 also expressed high levels of CD62L and low levels of CD161, and vice-versa. Because CD161 and CD62L are inversely expressed (Figure 1C), we calculated the ratio of these values. We found a striking negative correlation (−0.7765) between CCR7 expression and the ratio of CD161 and CD62L. The slope of the associated regression line was statistically significant with a p value = 0.008 (Figure 1D). These results suggest that human nCD4+, aCD4+ and aCD4− NKT cells may represent different stages of maturation and that there is wide disparity in the maturation state of the aCD4+ NKT cell pool, which can be phenotypically subdivided into CCR7highCD62LhighCD161low and CCR7lowCD62LlowCD161high populations.
(A) NKT cells in either neonatal or adult PBMC were identified by staining for the invariant TCR using antibodies against Vα24 (FITC) and Vβ11 (unlabelled followed by goat anti-mouse APC).
Double positive cells were identified as NKT cells and gated on for further analysis to determine CD4 (PE) and CD8 (biotin followed by strepavidin-PerCP-Cy5.5) expression.
(B) NKT cell subsets were identified by staining for Vα24, Vβ11, and CD4 as in 1A and then gated on for further analysis. Expression of the indicated cell surface marker on these subsets was then determined.
Combinations of the following antibodies were used, depending on the particular stain: Vα24 (FITC or unlabelled), Vβ11 (unlabelled or PE), CD4 (PE, biotin, or unlabelled), CCR7 (unlabelled), CD62L (PE), CD94 (unlabelled), CD161 (FITC), CD28 (biotin), goat anti-mouse (APC), and strepavidin (PerCP-Cy5.5). One representative donor is shown.
(C) Summary of NKT cell phenotype data for all neonatal and adult donors. P values are indicated.
For comparison of nCD4+ to either aCD4+ or aCD4− subsets, P values were generated using the Wilcoxon rank sum test.
For comparison of aCD4+ to aCD4− subsets, P values were generated using the Wilcoxon signed rank test.
(D) For CD4+ NKT cells of adult donors represented in 1C, the ratio of CD161 (% expression) to CD62L (% expression) was generated and compared to CCR7 expression using linear regression with a resulting equation of y = −19.277X+86.676 and p = 0.008.
Ex vivo cytokine polarization of nCD4+, aCD4+ and aCD4− NKT cell subsets
Conventional CD4+ T helper (Th) cells regulate adaptive immune responses by differentiating into Th1 or Th2 effector subsets, which produce either IFN-γ or IL-4, respectively [40]–[42]. Although many factors contribute to the polarization of naïve Th cell effector functions, cytokine signals during activation are critical in this differentiation process. Specifically, IFN-γ and IL-12 are instrumental in directing Th1 differentiation, whereas IL-4 promotes Th2 differentiation [40]–[42]. As naive Th cells differentiate from naïve to effector/memory subsets, heritable chromatin modifications at cytokine gene loci imprint an effector program which results in Th cells that are less capable of reprogramming their effector functions [43], [44]. Therefore, we investigated the extent to which NKT cell subsets that exhibit distinct memory phenotypes could acquire Th1 or Th2-like effector functions when activated in the presence of polarizing cytokine signals.
Purified human neonatal or adult NKT cells were activated by DCs pulsed with α-GalCer in the presence or absence of conventional Th1- (IL-12, neutralizing IL-4 antibody) or Th2- (IL-4, neutralizing IL-12 and IFN-γ antibodies) polarizing conditions, here termed NKT1 and NKT2 respectively. Following activation, NKT cells were expanded for approximately 2.5 weeks and their effector functions were determined. All NKT cell subsets activated with DCs alone (NKT0) expressed high levels of IFN-γ; compared to adult subsets, NKT0 nCD4+ NKT cells also produced relatively higher levels of IL-4 (Figure 2). When neonatal CD4+ NKT cells were activated under NKT1 conditions these cells displayed increased levels of IFN-γ, however expression of IL-4 was not greatly downregulated as compared to NKT0 cells (Figure 2). Under Th2-like polarization signals, nCD4+ NKT cells were easily programmed towards a NKT2 phenotype, as defined by secretion of high levels of IL-4 and low levels of IFN-γ (Figure 2), similar to conventional naïve CD4+ T cells polarized to Th2 cells under the same conditions (Figure S1). In contrast, aCD4− NKT cells, and to a lesser extent aCD4+ NKT cells, were more resistant to NKT2 polarization (Figure 2).
Neonatal or adult NKT cells were purified and activated under conventional Th0 (medium alone; NKT0), Th1 (NKT1), or Th2 (NKT2) polarizing conditions as described in the methods and expanded in IL-2-containing media.
These cells were re-stimulated for 6–8 hours using DCs pulsed with α-GalCer (100 ng/ml), or alternatively, anti-CD3 and anti-CD28, in the presence of GolgiStop (BD) and were then intracellularly stained with antibodies against IL-4 (PE) and IFN-γ (APC).
NKT cells were also co-stained with anti-Vβ11 (FITC) to exclude contaminating non-NKT cells.
For adult NKT cells, CD4 expression was also assessed by cell surface staining (CD4-biotin followed by strepavidin-PerCP-Cy5.5) to differentiate CD4+ from CD4− NKT cells.
One representative donor out of 5 is shown.
Certain chemokine receptors such as CXCR3 and CCR4 are preferentially expressed on Th1 or Th2 cells, respectively [44], [45]. To further examine the extent of NKT cell polarization, we determined the expression levels of these chemokine receptors on cytokine-polarized NKT cell lines. NKT0 nCD4+ NKT cells expressed increased levels of the Th2-associated receptor CCR4 compared to either adult subset (Figure 3A), which is consistent with their higher levels of IL-4 expression (Figure 2). CCR4 expression was strongly induced on nCD4+ NKT cells under NKT2 polarizing conditions, but only moderately so on aCD4+ and even less so on aCD4− NKT cells (Figure 3A). Expression of Th1-specific CXCR3 was highly expressed on all NKT cell subsets and, in stark contrast to conventional Th cells [44], was not substantially modulated by Th2-like cytokine polarization (Figure 3B). Taken together, these results suggest that nCD4+, and to a lesser extent aCD4+ NKT cells, have an enhanced capacity to modify their effector functions as compared with their CD4− counterparts. However, in contrast to conventional T cells, NKT cells also display some pre-programmed characteristics, which could not be modulated by cytokine polarizing signals.
One representative neonatal or adult sample of 5 is shown for each.
(A) CCR4 expression on polarized NKT cell lines was assayed by staining with anti-CCR4 (unlabelled followed by goat anti-mouse APC) and anti-CD4 (PE) to differentiate between adult CD4+ and CD4− NKT cells and anti-Vβ11 (FITC) to exclude contaminating non-NKT cells.
Isotype control is depicted in light gray.
(B) CXCR3 (unlabelled followed by goat anti-mouse APC) expression on polarized NKT cell subsets were assayed as in 3A.
Reprogramming NKT cell subset effector functions by ectopic expression of the transcription factors GATA-3 and T-bet
In conventional Th cells, Th1 and Th2 differentiation are directed by the master transcription factors (TFs) T-bet and GATA-3, respectively [46]–[48]. Whereas T-bet or GATA-3 are sufficient to program Th1 or Th2 effector function when ectopically expressed in conventional naïve Th cells, these same TFs are insufficient to reprogram the established effector functions of memory Th cell subsets [44]. Therefore, we asked whether bypassing cytokine polarization signals by ectopically expressing T-bet or GATA-3 could overcome the resistance of adult NKT cells to effector function reprogramming. Accordingly, NKT cells were purified as before, activated using DCs pulsed with α−GalCer and simultaneously transduced with a lentiviral vector expressing either T-bet or GATA-3, in conjunction with a reporter cell surface marker, murine CD24 (mCD24). An empty vector expressing mCD24 alone was used as a negative control. Transduced NKT cells were then expanded and assayed for cytokine production and chemokine receptor expression as before, using mCD24 as a marker for transduction. Similar to cytokine polarization, T-bet or GATA-3 expression in nCD4+ NKT cells was sufficient to drive NKT1 or NKT2 polarization, respectively (Figures 4A and 4C). As with the cytokine polarization experiments, ectopic expression of either of these TFs did not modulate CXCR3 expression on nCD4+ cells (Figure 4C). These results suggests that T-bet or GATA-3 can partially function to program the effector function of nCD4+ NKT cells. Ectopic expression of these TFs in either aCD4+ or aCD4− NKT cell subsets was unable to overcome the partial resistance of these subsets to effector function reprogramming by cytokine signals (Figures 4B and 4D). Taken together, these results suggest that, similar to conventional effector/memory CD4+ Th subsets [44], bypassing cytokine receptor signaling does not notably overcome the resistance of aCD4− NKT cells to effector function reprogramming.
NKT cells were activated under non-polarizing conditions as in Figure 2, transduced at the time of activation with either the control lentiviral vector or vectors expressing T-bet or GATA3, and expanded in IL-2-containing media.
One representative donor out of 3 is shown for each.
(A) Intracellular stain of nCD4+ NKT cells was performed as in Figure 2 and cells were co-stained with anti-mCD24 (FITC) to identify transduced cells.
(B) Intracellular cytokine stains of aCD4+ or aCD4− NKT cells were performed as in 4A.
CD4 expression was determined by co-staining with anti-CD4 (biotin followed with strepavidin PerCP-Cy 5.5).
(C) CCR4 or CXCR3 expression on T-bet- or GATA-3-transduced nNKT cells was assayed as in Figures 3A and 3B, and cells were co-stained with anti-mCD24 (FITC) as a marker for transduction.
(D) CCR4 expression of T-bet or GATA-3-transduced aCD4+ or aCD4− NKT cell was examined as in 4C, and cells were stained with anti-CD4 (PE) to differentiate between CD4+ and CD4− NKT cells.
Differential cytotoxic function of human NKT cell subsets
One of the characteristics of NKT cells is their cytotoxic function [49]. We therefore asked whether NKT cell subsets differed in their cytotoxic potential and whether this activity could be regulated by cytokine polarization. For these experiments, we developed a cytotoxicity assay using a murine fibroblast line (NIH 3T3 cells) which endogenously expresses CD1d and can present α-GalCer to activate human NKT cells in a dose-dependent fashion, as measured by up-regulation of the IL-2 receptor α chain, CD25 (Figure 5A). Further, upon TCR stimulation, NKT cells can kill target 3T3 cells, also in an α-GalCer dose-dependent fashion (Figure 5B). Using this assay, we tested the cytotoxic potential of in vitro generated nCD4+, aCD4+ and aCD4− NKT cell lines. We found that aCD4− NKT cells had higher cytotoxic activity against α-GalCer presenting target 3T3 cells compared to either nCD4+ or aCD4+ NKT cells at a variety of effector to target ratios (Figure 5C).
Experiments were performed in triplicate per donor; standard deviation is depicted.
For A, B, C, and D, one representative donor out of three independent experiments is shown. (A) NKT cells were added at an Effector:Target (NKT cell: 3T3 cell) ratio of 1∶1 to adherent 3T3 cells in the presence of different α-GalCer concentrations.
After 18–20 hours, NKT cells were collected and stained with anti-CD25 (unlabelled followed by goat anti-mouse APC) to determine activation.
(B) As in A, NKT cells at an NKT:3T3 of 1∶1 were activated in the presence of varying concentrations of α-GalCer.
After 18–20 hours, cells were collected by trypsinization to remove any 3T3 cells that were still adherent and target 3T3 cells were stained with propidium iodide to determine their viability.
(C) α-GalCer was added at a concentration of 100 ng/ml to adherent 3T3 cells.
NKT cell subsets were then added at different NKT:3T3 ratios. Viability of 3T3 cells after an 18–20 hour incubation period was determined as in 5B.
(D) Polarized nCD4+ NKT cells were generated as in Figure 2.
The cytotoxic capacity of these NKT0, NKT1, and NKT2 lines was assayed as in Figure 5C.
One NKT:3T3 ratio for each donor is shown.
Because conventional Th1 CD4+ Th cell lines can exhibit higher cytotoxicity relative to Th2 cells upon activation (Figure S2) [50], we next asked whether NKT1 and NKT2 cells displayed differences in their cytotoxic effector functions. For this experiment we used nCD4+ NKT cells, since these could be much more readily differentiated into NKT1 or NKT2 type cells (Figures 2 and 3A). In contrast to conventional CD4+ Th1 and Th2 subsets activated with SEB and tested under these conditions (Figure S2), nCD4+ NKT1 and NKT2 cells were similar in their cytotoxic capacity (Figure 5D). These results suggest that, unlike cytokine production and similar to CXCR3 expression, the cytotoxic function of NKT cells appears to be developmentally imprinted.
Here, we have demonstrated that NKT cells from neonates are phenotypically and functionally distinct from adult NKT cell subsets. In comparison to aCD4+ NKT cells, nCD4+ NKT cells exhibit: 1) higher expression of the differentiation markers CCR7 and CD62L, 2) lower expression of the NK cell markers CD161 and CD94, and 3) are more amenable to effector function reprogramming. We also found wide heterogeneity within the aCD4+ NKT cell subset for expression of memory markers. These findings suggest that the pool of CD4+ NKT cells in adult peripheral blood represents a more heterogeneous mixture of cells with respect to their phenotype and stage of maturation. This premise would predict that adult NKT cells are not recent thymic emigrants, but have undergone some form of peripheral maturation. Several lines of evidence are consistent with our hypothesis. In one study, Vα24+CD4+ NKT cell progenitors were observed to be present in the early fetal thymus but rare in the postnatal thymus, again suggesting the possibility that adult CD4+ NKT cells are likely to be peripherally expanded cells rather than recent thymic emigrants [34]; other studies also reported low levels of NKT cells in the postnatal thymus [32], [33]. Analysis of T cell receptor excision circles in NKT cells suggested that aCD4+ NKT cells found in the periphery were a result of both thymic output and peripheral expansion [32]. Despite expression of the memory marker CD45RO, resting neonatal NKT cells were also reported to be functionally immature as defined by their inability to produce IL-4 or IFN-γ upon primary activation and only acquired their effector functions after cell division, implying that in vivo activation and peripheral expansion are necessary to attain functional maturation [32], [51].
Several lines of evidence presented here also clearly suggest that aCD4− NKT cells are more differentiated compared to CD4+ subsets: 1) aCD4− NKT cells displayed lower expression levels of CD62L and CCR7 and increased expression levels of CD94 and CD161, 2) aCD4− NKT cells were predominantly resistant to reprogramming either via polarizing cytokine signals or ectopic expression of T-bet or GATA-3, and 3) aCD4− NKT cells also displayed greater cytotoxic activity compared to either nCD4+ or aCD4+ NKT cells. Based on these findings, we hypothesize that CD4 is a differentiation marker for human NKT cells, such that CD4− NKT cells may possibly represent a product of precursor CD4+ NKT cells which have undergone peripheral maturation. This hypothesis is also supported by the findings that aCD4+ NKT cells contain a higher level of T cell receptor excision circles than aCD4− NKT cells [32] and that CD4− NKT cells are absent in neonates and only begin to accumulate after about 6 months of age [32]–[34].
Conventional Th cells progressively lose the capacity to modify their effector functions as they differentiate from naïve cells to central and effector memory cells [43], [44]. Therefore the flexibility of nCD4+, aCD4+ and aCD4− NKT cell subsets to reprogram effector functions could be a reflection of their differentiation state, as defined by the expression of memory T cell markers and NK cell lineage receptors, as well as their cytotoxic capacity. We speculate that the phenotypic and functional profiles of neonatal CD4+ and adult CD4− NKT cells are analogous to conventional naïve and memory Th cell profiles [39], [43], [44]. However, in contrast to conventional Th cells, several NKT cell effector functions (such as cytotoxity and CXCR3 expression), which can be modulated in conventional T cells, were not responsive to polarizing cytokine signals or forced expression of TFs. We also observed that nCD4+ cells had higher expression levels of IL-4 under NKT1 polarizing conditions compared to conventional Th1 cells, which rapidly silence the IL-4 locus. However, because the aCD4− subset has reduced expression of IL-4 and CCR4 in non-polarizing conditions [27], [28], [35] (Figures 2 and 3A), as well as increased cytotoxicity (Figure 5C), it is conceivable that these functions are modified in vivo as NKT cells are activated by antigens and/or cytokines such as IL-12, which has been speculated to contribute to the peripheral maturation of NKT cells [52]. However, the importance of factors such as co-stimulation, cytokine signals present at the time of activation, strength and length of signal and the nature of the antigen, whether endogenous or foreign, in the differentiation and maturation process of NKT cells remains to be elucidated.
The functional differences between conventional Th and NKT cells described in these studies could potentially result from distinct, lineage-specific transcriptional regulatory mechanisms, or alternatively, may reflect unique developmentally imprinted genetic programs. As such, because the positive selection of NKT cells is mediated by double-positive thymocytes rather than cortical epithelial cells [53], it is possible that the unique developmental program of NKT cells may result in qualitatively different signals that impact subsequent functional programs. Indeed, NKT cell development differs from conventional T cells in the required expression of several transcription and signaling molecules, including T-bet, NF-κB and PKC-θ[54]–[58], thus suggesting unique developmental signals.
While we have shown here that certain functions of NKT cells are likely to be developmentally imprinted, others can be readily modulated, at least in the CD4+ NKT cell subset. It is not clear how NKT cells would acquire different functional profiles in vivo. Although murine NKT cells were reported to be resistant to cytokine polarization in vivo [36], direct ex-vivo cytokine mediated polarization was not assessed and in various other experimental systems evidence suggests that the nature of the antigen [17], [59]–[62], the type of antigen presenting cell [37], [38], [63], and the presence or absence of co-stimulatory signals [64], [65] could all play a role in modulating the functional and phenotypic profiles of NKT cells. For example, distinct structural analogs of α−GalCer can elicit different cytokine secretion profiles by NKT cells [17], [59]–[62]. While both human NKT cell subsets produce IFN-γ, CD4+ NKT cells produce higher levels of Th2 cytokines than CD4− NKT cells, suggesting that NKT cell effector function may also be regulated through the selective recruitment and activation of these subsets [27], [28], [35]. Furthermore, it was recently demonstrated that murine NKT cells from different organs may mediate distinct functions [66], raising the possibility that naïve-like NKT cell subsets may be recruited to different organs where they can undergo divergent effector function programming into distinct effector subsets. Indeed, similar mechanisms appear to be in place for conventional Th cells, whereby specialized, tissue-specific APCs can fine-tune T cell effector function [67].
Dysregulation of NKT cell function has been linked to a variety of immune deficiencies and pathologies. In comparison to normal healthy donors, which possess very low levels of NKT cells in the lung, on average a striking 60% of CD3+ cells in the lungs of bronchial asthma patients were NKT cells, with greater than 90% representing CD4+ NKT cells that expressed IL-4 but little IFN-γ [68]. Other examples of the role of NKT cells in autoimmunity, tumor biology and infection have also been reviewed [4], [9]. Our findings highlight the possibility that therapeutic manipulation of NKT cells to improve immune responses against autoimmune or infectious diseases may be most optimally achieved by targeting specific subsets rather than the entire NKT cell pool. While CD4+ NKT cells are amenable to reprogramming, CD4− NKT cells are functionally static. Conceivably, some autoimmune conditions may be circumvented by activation conditions that preferentially target CD4+ NKT cells and favor the development of strong Th2 responses. Also, the potent anti-tumor or anti-viral activity of NKT cells may be most beneficial under activation conditions that preferentially favor the development of either NKT1 or CD4− NKT cells, which can potently mediate the downstream activation of NK cells and CD8+ cytotoxic T lymphocytes, further amplifying anti-tumor or viral activity. Of note and in contrast to conventional T cells, we were unable to modulate the direct cytotoxic activity of NKT cells. As a better understanding of the mechanisms that regulate NKT cell function is achieved and as novel ligands that can specifically modulate these functions are generated, it may be possible to specifically boost these types of responses in vivo.
In conclusion, our results reveal both imprinted and highly flexible differentiation and effector programs of neonatal NKT cells, whose flexibility progressively diminish in adult NKT cells as they acquire a more differentiated phenotype. These findings suggest that CD4+ and CD4− NKT cells may represent unique differentiation states. While we cannot exclude the possibility that CD4− NKT cells may develop as a separate lineage, our studies in conjunction with previous reports [32], [34], support a linear model of human NKT cell differentiation. Because NKT cells serve as an important link between the innate and adaptive immune system, our results have important implications for the design of novel therapeutic interventions aimed at harnessing the immunoregulatory potential of NKT cells.
Materials and Methods
Purification and preparation of primary human T cell subsets and DCs
PBMC were isolated from either neonatal cord blood or peripheral adult blood from healthy individuals by Ficoll (Pharmacia) density centrifugation. NKT cells were enriched from adult PBMC or neonatal CD4+ T cells by staining with Vα24 antibody, followed by goat anti-mouse magnetic beads (Miltenyi Biotec), and positively sorted by magnetic cell sorting using AutoMACS (Miltenyi Biotec) as described [30]. CD4+ conventional T cells (Th cells) were purified from PBMC using Dynabead anti-CD4 antibodies and magnetic separation, as previously described [44]. Monocyte-derived DCs were prepared as previously described [69], [70]. DCs were matured by stimulation with 10 ng/ml LPS (Sigma) one day prior to T cell activation. All subjects provided written informed consent that was approved by the Vanderbilt Institutional Review Board.
T cell activation and differentiation
Purified Vα24+ NKT cells were stimulated with α-GalCer (100 ng/mL, Kirin Brewery Co., Gunma, Japan) using mature DCs. CD4+ Th cells were activated by DCs using Staphylococcus enterotoxin B (SEB, 20 ng/mL, Sigma). For cytokine polarization, cells were activated in the presence or absence of polarizing cytokine conditions as previously described for conventional CD4+ Th cells [44] under the following conditions: human rhIL-12 (30 ng/ml) and neutralizing anti-IL-4 antibody (0.5 µg/mL) for Th1 conditions and human rhIL-4 (50 ng/mL) and neutralizing anti-IFN-γ (2.5 µg/ml) and anti-IL-12 (2.5 µg/ml) antibodies for Th2 conditions (all from R&D Systems) [44]. Cells were expanded in rhIL-2 containing media. The culture media used in all experiments was as described [70]. For purification of aCD4+ or aCD4− NKT cells, from expanded adult NKT cell lines, cells were stained with Vβ11 (Immunotech) to exclude any contaminating non-NKT cells and CD4 (BD Pharmingen); aCD4+ and aCD4− NKT cells were sorted using flow cytometry (FACS Aria, BD Pharmingen).
Virus production and infections
Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviruses were generated as previously described [70]. Briefly, HEK-293T cells were transfected with lentiviral vectors and VSV-G envelope plasmids. Supernatants were collected 48 h post-transfection and filtered through 0.45 µM filters. Construction and use of lentiviral vectors expressing a marker gene (murine CD24, mCD24) and either GATA-3 or T-bet have also been described [44], [70]. For transduction experiments, NKT and conventional CD4+ Th cells were infected at the time of activation at a multiplicity of infection of 3–8.
Staining and FACS analysis
Cells were stained with the relevant antibody and analyzed using a flow cytometer (FACSCalibur) as described [44]. Analysis was performed using FlowJo software (Tree Star). The following anti-human antibodies were used for staining: Vα24, Vβ11 (Immunotech); CD4, CD8, CD28, CD94, CD161, CD25, CCR4, CXCR3, CD62L (all from BD Pharmingen) and CCR7 (R&D Systems). In some experiments mCD24 antibody (BD Pharmingen) was used as marker for lentivirus-transduced T cells. Secondary goat anti-mouse antibody and streptavidin conjugates were from BD Pharmingen and pure mouse IgG1 was purchased from Caltag.
Cytokine expression assays
For intracellular cytokine analysis, NKT and CD4+ Th cells were stimulated with α−GalCer (100 ng/ml) or SEB (20 ng/ml) pulsed DCs, respectively, or alternatively by cross-linking the TCR with antibodies against CD3 (OKT3, American Type Culture Collection (ATCC)) and CD28 (BD Biosciences), in the presence of 0.67 µl/ml GolgiStop (BD Pharmingen) and incubated for 6–8 hours at 37°C. This was followed by cell surface staining for CD4 to identify cells from either the CD4+ or CD4− lineage and anti-Vβ11 to exclude any contaminating non-NKT cells. Lentiviral transduction was monitored using anti-mCD24. Subsequently, cells were fixed and permeabilized using a commercial kit (BD Pharmingen) according to the manufacturer's instructions. Cells were then stained with the following anti-human cytokine antibodies: IFN-γ and IL-4 (both from BD Pharmingen).
Cytotoxicity Assay
Murine fibroblast cells (NIH-3T3) expressing endogenous murine CD1d were seeded in a 96 well plate at 8,000 cells per well and incubated for 1–3 h to adhere. To activate NKT cells, α-GalCer (between 0.01–100 ng/ml) was added to these cultures. For CD4+ Th cell activations, 3T3 cells ectopically expressing the human class II molecule HLA-DR were pulsed with SEB (20 ng/mL). Effector cells (NKT or Th) were then added at various effector to target ratios (2∶1 to 1∶32). After 18–20 hours, cells were trypsinized, collected and stained with propidium iodide (PI, 10 µg/ml, Sigma) or anti-CD25 and analyzed by flow cytometry. For experiments comparing different NKT or Th lines, a negative control was used for normalization between samples. 3T3 cells were distinguished from NKT or CD4+ Th cells by their distinctive larger forward and side scatter profiles.
Statistical Analysis
Statistical analyses were performed using STATA version 9.0. In all cases, a p-value<0.05 was considered statistically significant. Due to the non-normal nature of the distributions, non-parametric tests were used for all comparisons in order to avoid spurious results. Linear regression was used to characterize the relationship between CCR7 expression and the ratio of CD161∶CD62L expression in aCD4+ NKT cells. A Kruskal-Wallis test was used for the initial comparisons of the distribution of cell surface marker expression profiles of nCD4+, aCD4+, and aCD4− NKT cell subsets, followed by pair-wise comparison tests. For non-matched comparisons, the Wilcoxon rank-sum test was used, and for matched comparisons, the Wilcoxon's signed rank test was performed. It is important to note that the p-values presented are uncorrected for the multiple tests performed in the pair-wise comparisons, yet even with a highly conservative Bonferroni correction, all results except one are still statistically significant.
Supporting Information
Figure S1.
Cytokine polarization of CB CD4+ Th cells. CB CD4+ T helper cells were activated under Th0- Th1- or Th2-polarizing conditions as described in the methods and expanded in IL-2-containing media. These cells were re-stimulated using DCs pulsed with SEB (20 ng/ml) or anti-CD3 and anti-CD28 in the presence of GolgiStop (6-8 hrs) and subsequently stained intracellularly with anti-IL-4 (PE) and anti-IFN-γ (APC).
Figure S2.
Cytotoxic capacity of polarized neonatal Th cells. Polarized Th1 or Th2 neonatal lines were generated as in Figure S1. The cytotoxic capacity upon activation at different Th:3T3 ratios of these lines was assayed as in Figure 5B using 20ng/ml SEB for each. Experiments were performed in triplicate per donor; standard deviation is depicted. One representative donor out of three is shown.
We thank Amanda Johnson and Drs. Geraldine Miller, Jelena Bezbradica, Sebastian Joyce, and Kyra Richter for critical reading of this manuscript. We thank the CFAR Immunology core for FACS sorting and analysis and the Vanderbilt clinical research core and nurses for collection of blood samples. We acknowledge support from the Vanderbilt Flow Cytometry and Cell Sorting Core and from the Human Immunology Core of the Vanderbilt GCRC. We would also like to thank Kirin Brewery Co. (Gunma, Japan) for providing synthetic α-GalCer.
Author Contributions
Conceived and designed the experiments: DU KE. Performed the experiments: KE MT. Analyzed the data: DU KE AM. Contributed reagents/materials/analysis tools: LV MS AM. Wrote the paper: DU KE.
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subset: commoncrawl


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control of a pledged thing if the one giving the security interest fails to perform as agreed. The one receiving a security interest becomes a secured Party, especially if the instrument establishing the security interest is registered. He has rights, which are remedies and defenses that he can use to enforce an agreement if the other party fails to perform as agreed. y Rights includes remedies A remedy is a commercial right for those who acquire that right through an instrument. y Contract Basic contract law => a plaintiff suing on a written contract must produce a signed contract proving he is entitled to relief. y MERS => a. Does not hold financial interest b. Not prepared of equipped for consumer discovery requests c. Does not deliver anyone with any standing to foreclose d. Kansas Supreme Court => is more akin to that of a Straw man than to party possessing all the rights given a buyer e. By statute, assignment of the mortgage carries with it the assignment of the debt . Indeed, in the event that a mortgage loan somehow separates interests of the note and the deed of trust, with the deed of trust lying with some independent entity, the mortgage may become unenforceable. The practical effect of splitting the Deed of Trust from the promissory note is to make it impossible for the holder of the note to foreclose, unless the holder of the deed of trust is the agent of the holder of the note. Without the agency relationship, the [person holding only the note lacks the power to foreclose in the event of default. The person holding the deed of trust will never experiencing default because only the holder of the note is entitled to payment of the underlying obligation. The mortgage loan becomes ineffectual when the mote holder did not also old the deed of trust: f. MERS as straw man lacks standing to foreclose, but SO DOES THE ORIGINAL LENDER, although it was signatory to the deal. The lender lacks standing because title had to pass to the secured parties for the arrangement to legally qualify as a security. The lender has been paid in full and has no further legal interest in the claim. Only the securities holders have skin in the game. But they have no standing to foreclose, because they were not signatories to the original agreement. They cannot satisfy the basic requirement of contract law that a plaintiff suing on a written contract must produce a signed contract proving he is entitled to relief.
unequivocally holds that a notice that fails to identify the holder of the mortgage is defective. See .L. a person specifically authorized by the power of sale or an attorney. 327 Mass. Eno & W.. that it will have someone with authority to execute the relevant paperwork. there is nothing to suggest that the one who expects to receive the mortgage by assignment may undertake any foreclosure activity. (not always easy when all previous assignments have not been recorded at the Registry). c. The plaintiffs were not the present holders of the mortgage at the time of the mortgage and sale. that you will be able to determine the entity that validly holds the mortgage you need assigned. Practice: Real Estate Law. no such documents were included in the record. Memorandum of Decision on Motion for Relief at 7 (Apr. 28 Mass. 244. As the statute recognizes. Bankr Ct. the plaintiffs contention at oral argument that G. that the entity will still be operational. 225 Mass.U. if challenged. §4.259. 217. Bottomly v Kabachnick. g. Moreover. Hovey. D. legal guardian or conservator of those persons acting in the name of those persons. did the party conducting the foreclosure have authority to do so and . Neither bank had been assigned the mortgages at the time notice was published and sale took place. h.L. actually holding something and having the right to be its holder are two very different things. there is nothing in the record to mortgag indicate when the promissory notes were assigned and the record is unambiguously clear that e the mortgage were assigned on the dates referenced herein. that it will be able to find the relevant paperwork. Bank National Association v Ibanez & Wells Fargo v Larace Massachusetts Land Court Department. e. One can become the holder of the mortgage ( an interest in land) only by a writing satisfying the statute of frauds. Thus. (apparently done by the contract documents referencing them generally. 341. Long a. the notice and conduct of the foreclosure sale in the plaintiffs names under the incorrect representation that the plaintiffs were the mortgage holders makes the sale invalid. Blank mortgage assignments failed to name an assignee. Judge J. 2007) y y A mortgage is a contract.S. can it prove that is had such authority? d. that you have a demonstrable right to get it. § 14 s requirement of holder status was satisfied by the assignment of the promissory note secured by the mortgage to the securitized pools. And retroactive assignments do not cure statutory defects. in other words the current mortgagee. Morrissey. A. 7 Case No. Flavin v. Macurda v. these are the mortgagee or his valid assignee. and that it will be able to do so in a timely fashion. 19. the power of sale) has the contractual right to do so at the time of its exercise. While mortgagee has been defined to include assignees of a mortgage.S. To obtain a mortgage assignment you do not actually possess presumes. b. Fuller. It is a fundamental and basic that a party seeking to exercise a contractual right (here. G. Eno & Hovey ) and cases cited therein. the holder of the mortgage needs to be identified in the notice c. 344-345 (1916). In any event. f. along with hundreds or thousands of other Holder such notes) fails. thereby rendering the foreclosure sale void as a matter of law. § 1.50 at 109 (4th ed 2004)(hereafter. Chap. Mass. They were not properly authorized by the mortgage holder at those times. In re Sima Schwartz. so ant argument of based upon them are unsupported and waived. they were ineffective to transfer any interest in the mortgage. U. c. 219 (1951).. in recordable form. 06-42476-JBR. at the least.
with the intention of passing legal title to such property to the trustee (Brown v Spohr 180 NY 201 209). 2010 Judge Schack Foreclosure Judgement and Sale Vacated with Prejudice. y . Valid assignment of mortgage and note b. U. 181 (2d Dept 2006). v Pataki 100 NY2d. the plaintiff must establish the existence of the mortgage and the mortgage note. The plaintiff who has standing. First Union Mortgage Corp. ownership of the mortgage. It is a threshold issue. (2) a designated trustee. 36 AD3d 176. If he fails to do so there is no valid execution of the power and the sale is wholly void ).A. (Stark v Goldberg. KINGS COUNTY Judge Arthur M. Lack of 2. Cert denied 540 US 1017 [2003] held that standing to sue is critical to the proper functioning of the judicial system. The plaintiff who has standing.A. Plaintiff s lack of standing and to vacate the instant judgment of foreclosure and sale for lack of Standing jurisdiction to render judgement because plaintiff Chase lacks Standing. the Court held that standing to sue requires an interest in the claim at issue in the lawsuit that the law will recognize as a sufficient predicate for determining the issue at the litigant s request. the pathway to the courthouse is blocked. Failure to demonstrate ownership of mortgage loan c. 297 AD203 [1d Dept 2002]). A claim for lack of jurisdiction is a condition precedent to the maintenance of the claim. Inc. Court of Appeals ( Saratoga County Chamber of Commerce.S.L. 18 AD3d 527 [ 2d Dept 2005]. foreclosue 3. It is axiomatic that there are four essential elements of a trust: (1) a designated beneficiary. may cross the threshold and seek judicial redress. 183. the plaintiff may not proceed in the action. It is a threshold issue. N. 901. however. 196 AD2d 812 [2d Dept 1993]. recognizing that person seeking to exercise the power must first comply with the terms of the mortgage and with the statutes relating to the foreclosure of mortgages by the exercise of a power of sale ) y WELLS FARGO. If standing is denied. 294 Mass. Standing to sue is critical to the proper functioning of the judicial system.McGreevey. c. Ocwen Federal bank FSB v Miller. v Fern. Inc. Schack a. 19 AD3d 402 [2d Dept 2005]. 298 AD2d 490 [2d Dept 2002]. If standing is denied. If the plaintiff lacks standing to sue. Bank Trust Nat. In Carper v Nussbuam. Failure to name a beneficiary for the Trustee renders the assignment without merit. v George 2010 NY Slip Op 50786(U) May 04. At 484 ( it is familiar law that one who sells under a power must follow strictly its terms. and the defendant s default in the payment. VALID ASSIGN MENT JP Morgan Chase Bank. the pathway to the courthouse is blocked. 812 [2003]. is a motion that can be Vacate entered at any time. (See Household Finance Realty Corp v New York v Wynn. and (4) actual delivery or legal assignment of the property to the trustee. The Court in Campaign v Barba . v FARMER 2008 NY Slip Op 51133(U) [19 Misc 3d 1141 (A)] SUPREME COURT. 19 AD3d 545 [2d Dept 2005. Village Bank v Wild Oaks Holding. v Yahhobi. see also G. N. said mortgage to be duly assigned by an Assigment to be recorded is not an assignment that s valid at the time of notice. Sears Mortgage Corp. (3) a fund or other property sufficiently designated or identified to enable title thereto to pass to the trustee. e. Ass n v Butti.. 23 AD3d 327 [2d Dept 2005]. & sale 4. d. 1. held that to establish a prima facia case in an action to foreclose a mortgage. § 21 ( statutory power of sale in mortgage. 16 AD 3d 408 [2d Dept 2005].
Wells Fargo bank. 15 USC § 1692e(2). or control of any real property. v Marchione.S. Inc. may cross the threshold and seek judicial redress. Federal law prohibits the use of any false. deceptive or misleading representation or Fair means in connection with the collection of any debt including the false Debt representation of the character. misrepresentation.(5) & (10) A. modification or vacatur of a prior judgment or order upon which it is based Whittiker & Stepanek v Deutsch Bank National Trust Company Judge David D. Standing to sue requires an interest in the claim at issue in the lawsuit that the law will recognize as a sufficient predicate for determining the issue at the litigant s request. Violation of FDCPA. or other misconduct of an adverse party (4) lack of jurisdiction to render the judgment or order (5) reversal.32 e Act a. (Caprer v Nussbaum.A. U. no authority to act as an agent Interest d. 100 NY2d 801 812 [2003].. denied 540 US 1017 [2003]) 5.District Court for the Northern District of Ohio Eastern Division Case No. 5015 Relief from judgment or order (a) On motion (1) excusable default. Appointment of Receiver under RC § 2735. amount.however. Thus. Dowd.32 (A)(2) b. a retroactive assignment cannot be used to confer standing upon the assignee in a foreclosure action commenced prior to the execution of an assignment.. RC § 2923.01 y y Supreme Court of Arkansas chief justice Jm Hannah. 69 AD3d 204. 210 {2d Dept 2009]) 7. 3. 1:08CV300 First Amended Class Action Complaint Action for 1. shall attempt to influence a public servant in the discharge of the person s duty. 5015(a)(4) Motion was brought based on this rule by defendant CPLR R. N. (Ohio RICO) § 2923. cert. through a pattern of corrupt activity shall acquire or maintain. (2) newly-discovered evidence. CPLR R. y Kansas Supreme Court Landmark VS Kessler . it hold no property interest MERS b. recording. or otherwise using a materially false or fraudulent writing in a wonton or reckless manner. Jr. 36 AD3d 176. directly or indirectly. (3) fraud.2009 .181 [2d Dept 2006]) 6.03 of revised code stated that no person. (Saratoga County Chamber Of Commerce. Cannot act on its own independent of the direction of the specific lender who holds the repayment interest in the security instrument at the time MERS purports to act. or legal status of any debt and the threat Collect to take any action that cannot legally be taken 15 USC 1692e Practic 2.Aug 28.. knowingly and by filing. Section 2921.. MERS is at most a mere agent of the lender a. any interest in. Ohio RICO states that no person. v Pataki. it s not a necessary party (to an action / proceding) No c.
App. rights. without ownership of the mortgage instrument. there is no evidence of record that establishes that MERS either held the promissory note on was given the authority to assign the note. 170 (1931) e. 3d619 (mo. 275. it must be raised. Hillery. The person holding only the Deed Of Trust will never experience default because only the holder of the note is entitled to payment of the underlying obligation. the mortgage loan became ineffectual when the not holder did not also hold the deed of trust. v. usually a mortgage or deed of trust. as a practical matter becomes unsecured. LLC. Inc. 2009) Issue of standing a. it vests in the transferee all the interest. v. whenever it becomes apparent ). Typically. the note and deed of trust are inseparable. the trial court does not have jurisdiction to grant the requested relief c. Restatement (third) of property (mortgages) 5. either by the parties or by the Court.S. which secures payment on the note by giving the lender the ability to foreclose on the property.. 329 Mo. zd 166. 2008 WL 5170180 (MD. 21 L. There is no evidence in the record or the pleading that MERS held the promissory note or that BNC gave MERS the authority to transfer the promissory note y AURORA LOAN SERVICES. Services.LLC V JUDITH MENDES DA COSTA Case no. In the event that the note and Deed of Trust are split. Ed 313 (1872)]) y Bellistri v Ocwen Loans Serving. 271. splitting if the note and mortgage creates an immediate and fatal flow in the title b.: 09-142-CA Circuit Court Collier County. Louise MUT Life Eng Co. Effectively. 715. 284 S.W. The practical effect of spitting the deed of trust front the promissory note is to make it impossible for the holder of the note too foreclose. 83 U. Lack of Standing i.W. St. 2010 a. If MERS is only the mortgagee. 46 S. unless the holder of the Deed of Trust is the agent of the note without the agency relationship. Standing is a threshold issue a) Hence.3d 619 (Missouri Court of Appeals. LLC 284 S. powers. it does not have enforceable right. CA / 2008) (unpublished opinion) ( For there to be a valid assignment. Florida Judge Rob Crown April 28. 16 wall. the note. 271. Saxon Mort. 2009) . d. An assignment of Deed of Trust separate from the note has no Force. a defect in standing cannot be waived. A mortgage loan consist of (i) a promissory note and (2) security instrument. ( while the note is essential the mortgage is only an accident to the note [quoting Carpenter v Logan.W. the same person holds both the note and the deed of trust. the person holding only the note lacks the powers to foreclose in the event of default. and security conferred by the DOT upon the beneficiary there in and the payee in the notes. Bellistri v Ocwen Loan Servicing.MERS holds no interest a. there must be more than just assignment of the deed alone. If party seeking relief lacks standing. party seeker relief have a legally cognizable interest in the subject matter and that he has a threatened or actual injury b. and when the promissory note is transferred. Walter. ) c.4 comment. the note must also be assigned MERS purportedly assigned both the deed of trust and the promising note However.
704 N. ) b. collect mortgage payments. b) MERS has no substantive rights itself and. Assignment is from MERS to Plaintiff and is completely ineffective. make decisions on whether to extend credit. Lamy. a) As nominee for lender.2006) d) MERS own definition of its very limited role as a nominee Subsequently. does not vest plaintiff with standing. It follows that the Provider would gave lacked standing under these circumstances. If on the date the Provider filed the original statement of claim Mr. When exhibits are attached to a complaint. Assignment i. 913 So. MERS serves in a very limited capacity.2d 769. Joseph had not assigned benefits to the provider. Possession of the Original Note i. plaintiff must necessarily rely upon a valid assignment. Mortgage Electronic Registration Systems. counsel for MERS explained that MERS does not take applications. Id. 2006 WL 2251721 (Sip.b) ( Lack of standing cannot be waived and may be considered by the court sua sponte. [T]he plaintiff s lack of standing at the inception of the case is not a defect that may be cured by the acquisition of standing after the case is filed. or provide any loan servicing functions whatsoever. Inc.W. e) not only are there substantive deficiencies with an assignment from MERS. Ins. hold escrows for taxes and insurance. underwrite loans. c) a nominee of the owner of the note and mortgage may not effectively assign the note and mortgage to another for want of ownership interest in said note and mortgage by the nominee. only Mr. v McGrath Community Chiropractic.S. cannot assign what is does not have. Rather . v Nebraska Department of Banking and Finance. 824 N. the contents of the exhibit control over the allegations of the complaint ii. and the case should have been dismissed. . Moreover language controls over contrary allegations contained in the complaint.Y. 2nd DCA 2005). in and of itself. f) there is no indication on the assignment that the note and mortgage were physically transferred prior to that date. None of the documents identify Plaintiff as holder iii.2d 1281 (Fla. Progessive Exp. Ass n v. Joseph had standing to bring the action.2d 784 (Neb. therefore. possession of the original note. MERS merely tracks the ownership of the lien and is paid for its services through membership fees charged to its members. La Salle Bank Nat.2005). iv. which does not exist. Co. c.
The employer must still honor the check despite the fact that the check was a fraudulent negotiable instrument.. filed May 10. the False Claims Act because the Defendants made false representation in order to avoid payment in full of all recording fees reflecting the establishment and/or transfer of secured interests in real property in the State. punitive damages. J. In discovery either in or out of litigation. the pretender lender. appraisal fraud. thereby. that the real parties in interest are not only not present in court but don t even have actual notice of the proceedings. fraudulent. Bufford in Los Angeles issued a notice last month warning plaintiffs in foreclosure cases to bring the mortgage notes to court and not submit copies. securities fraud. y y y y y y Fraud in the Factum is a type of fraud where misrepresentation causes one to enter a transaction without accurately realizing the risks.y U. After having recorded false. decreased and/or diminished their obligation to pay fees or monies to the counties. such as a promissory note or check. treble damages. BoA. is induced to sign the instrument without a reasonable opportunity to learn of its fraudulent character or essential terms. duties. If the pretenders have a case. etc. they will need to prove it through evidence rather than assumptions and reputations. Predatory lending. If they finally give real documentation. Determination of whether an act constitutes fraud in the factum depends upon consideration of ³all relevant factors. much less service of process.S. this requirement will apply because developments in the secondary market for mortgages and other security interests cause the court to lack confidence that presenting a copy of a promissory note is sufficient to show that movant has a right to enforce the note or that it qualifies as a real party in interest. the proceeds and achieve a windfall. misleading and untruthful documents and. Defendants failed to cure/correct said false. take the house. . These referral documents contain information that will probably show that the would-be forecloser.Morgan. since the investors don t seem to be getting any of the benefit. Bankruptcy Judge Samuel L. you want to see the transmittal documents by which the foreclosure judicial or non-judicial were initiated. you will see for yourself and so will the court. rescission under all available theories of law. [1] This can be when the maker or drawer of a negotiable instrument. Here. Request the original note A. 2010 in Lassen County Courts Statement of the Case: Plaintiffs seek recovery pursuant to California Government Code § 12650 et seq. misleading and untruthful documents with the land records of the counties of this State. the above-named real parties in interest. Do not accept a lost-note affidavit. the notice said. is attempting to sue in its own name. or obligations incurred.´ Fraud in the factum usually voids the instrument under state law and is a real defense against even an holder in due course. exemplary damages. and consequential economic damages. Contrast this with the situation where a trusted employee signs a check without permission. damages. Coutrywide. MERS rules don t allow members to submit lost-note affidavits in place of mortgage notes State of California v MERS. the employer had a reasonable opportunity to avoid the obligation by restricting access to the checks. avoided.P.
and John tries to enforce the deed. If Mom signs the deed believing what he told her." y y .Fraud in the factum is often contrasted with fraud in the inducement. but then John falsely tells her that the bank will foreclose on the property unless she signs it over to him. and John tries to enforce the deed. suppose John tells his mother that he is taking a college course on handwriting analysis. and occurs when A enters into an agreement. knowing that it is supposed to be a contract and (at least having a rough idea) what the agreement is about. or does not understand the nature/content of the agreement." Fraud in the inducement is an equitable defense. but either does not realize that it is supposed to be a contract. Mom can plead "fraud in the factum. Mom can plead "fraud in the inducement. and occurs where A makes/signs an agreement. If Mom signs the deed because of this statement from John. For example. but the reason A signed/made the agreement was because of some false information that B gave to A. suppose John tells his mother to sign a deed giving him her property. because of some false information that B gave to A. y Fraud in the factum is a legal defense. For example. Mom refuses at first. and for his homework he needs her to read and sign a pretend deed.
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Press freedom dives in Hong Kong: survey
Tighter controls on information and self-censorship 'among key reasons'
ucanews.com reporter, Hong Kong
Hong Kong
June 25, 2012
Nearly 87 percent of media workers feel press freedom has worsened, according to a poll released by the Hong Kong Journalists Association yesterday. The survey, conducted between April 18 and May 4, polled 663 reporters, photographers, editors and media management. The results show a marked increase over a previous poll from 2007 in which 58.4 percent of journalists thought press freedom had deteriorated over the last decade. According to the survey, 57.2 percent of those polled said there was "obvious deterioration" in press freedom since Donald Tsang became chief executive in 2005. A clear majority attributed the change to the government's tightened grip on the flow of information (92.7 percent), self-censorship in the industry (71 percent) and political interference by Beijing and the central government's liaison office (67.5 percent). Among the "darkest moments" noted by respondents were police interference in reporting on the Chinese vice-premier Li Keqiang's Hong Kong visit last year, pressure from the liaison office on local media and the distortion of an opinion piece by a pro-Beijing paper during the chief executive election this year. Dominic Lau Chung-yeung, a Catholic journalist in Hong Kong, said the "trend of deterioration" poses a significant threat and has "increased after Hong Kong's handover in 1997." Lau added that the government has reduced the number of press conferences and offers information largely on condition of off-record or anonymous sources. The survey comes amid public outcry over Wang Xiangwei, editor in chief of the South China Morning Post, who has been criticized for downplaying in print the suspicious death of activist Li Wangyang, who was found hanged in his hospital room earlier this month. Public protest and heavy media coverage in other papers ultimately led to a call by the Hong Kong government to investigate Li's death. The latest Press Freedom Index from Reporters Without Borders also showed a sharp drop in press freedom, with Hong Kong dropping in rank from 34 in 2010 to 54 in 2011-12. Related reports Discontent undermines handover anniversary Police and protesters clash over election
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Is methylprednisolone given for laryngitis?
- Posted:
- 25 May 2013 by clopez1105
- Topics:
- methylprednisolone
Answers (2)
25 May 2013
Most laryngitis is treated with rest and perhaps antibiotics if a throat culture comes back positive. Avoid talking to rest the throat but be sure you see a doctor in case it is strep throat.
Methylprednisolone is an anti inflammatory and I do not know if it would work for this. We are not doctors on this site, so you will receive information from various people.
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Small Town's UFO Scare Revealed To Be Alien Hoax (Season 1: Ep 8 on IFC)
In local news, repeated "UFO" sightings in Pennington, IL have turned out to be nothing more than a silly prank perpetrated by visitors from another world.
"Everybody knows we wouldn't have these issues of teams losing if college football just instituted a playoff."
"You'd think they would have changed their ranking after the first loss."
"That just has to happen 17 or 18 more times this season and they'll let Boise State be No. 1."