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The Journal of Immunology, 2007, 179, 819 -829
Copyright © 2007 by The American Association of Immunologists, Inc.
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Antiparallel Segregation of Notch Components in the Immunological Synapse Directs Reciprocal Signaling in Allogeneic Th:DC Conjugates1

Winifred H. Luty*, David Rodeberg{dagger}, Jerome Parness{ddagger} and Yatin M. Vyas2,*

* Division of Pediatric Hematology-Oncology and Bone Marrow Transplantation, {dagger} Division of Pediatric Surgery, {ddagger} Department of Anesthesiology, Children’s Hospital of Pittsburgh, University of Pittsburgh Medical Center, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Direct T cell allorecognition underlies the development of a vigorous immune response in the clinical setting of acute graft rejection. The Notch pathway is an important regulator of Th immune responses, yet the molecular underpinnings of directional Notch signaling, otherwise critical for binary cell fate decisions, are unknown during autologous or allogeneic Th:DC interactions. Using the development of immune synapses (IS) in the allogeneic, human physiological Th:DC interaction, we demonstrate that Th-Notch1 receptor and DC-Notch ligands (Delta-like1, Jagged1) cluster in their apposed central-supramolecular-activation-clusters (cSMAC), whereas DC-Notch1 receptor and Th-Notch ligands cluster in their apposed peripheral-SMAC (pSMAC). Numb, a negative regulator of Notch, is excluded from the IS-microdomains where Notch1 receptor accumulates. This antiparallel arrangement across the partnering halves of the IS supports reciprocal Notch signal propagation in the DC-to-Th direction via the cSMAC and Th-to-DC direction via the pSMAC. As a result, processed Notch1 receptor (Notch-intracellular-domain, NICD1) and its ligands, as well as their downstream targets, HES-1 and phosphorylated-STAT3, accumulate in the nuclei of both cell-types. There is also enhancement of GLUT1 expression in both cell-types, as well as increased production of Th-IFN-{gamma}. Significantly, neutralizing Notch1R Ab inhibits NICD1 and HES-1 nuclear translocation, and production of IFN-{gamma}. In contrast, the IS formed during Ag-nonspecific, autologous Th:DC interaction is immature, resulting in failure of Notch1 receptor segregation and subsequent nuclear translocation of NICD1. Our results provide the first evidence for the asymmetric recruitment of Notch components in the Th:DC immunological synapse, which regulates the bidirectional Notch signal propagation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Notch signaling pathway, which involves local cell-to-cell communication mediated through membrane-bound Notch receptors and their appositionally located ligands, affects pleiotropic, organotypic cellular functions across the range of organisms from helminthes to humans (1, 2). This pathway, traditionally understood to regulate developmental cell fate and pattern formation, is now gaining increasing prominence as a regulator of various functions in the vertebrate hematolymphoid system. The expression of Notch receptor isoforms 1–4 and its ligands, Delta-like (Dll)3 1, 3, 4, or Jagged1–3, both in immature and mature lymphocytes, suggests a role for Notch signaling in lymphocyte development, and its peripheral maturation and differentiation (3, 4, 5). Indeed, individual Notch ligands on the APC can have distinct effects on peripheral Th cell differentiation (6). Specifically, Delta ligands regulate Th1 differentiation, and Jagged ligands, Th2 differentiation. More recent evidence points to a role of Notch in the early induction of IL-4 gene transcription and Th2 phenotype acquisition (7, 8).

Because Notch signaling occurs in the context of ligand-dependent, intramembrane Notch-receptor proteolysis, it requires tight spatio-temporal regulation. Proteins modulating Notch signaling, such as Numb, Deltex1, and several E3 ubiquitin ligases, impact on the functional outcome of Notch activation during binary cell fate decisions (9, 10, 11). During development, for example, differential segregation of Numb, a negative regulator of Notch, into only one of the two daughters of a dividing cell is part of a program to regulate Notch-dependent, binary cell fates (12). This option is unavailable to fully differentiated circulating immune cells, since division of these cells results in distribution of Numb and other regulators to all daughter cells. Indeed, peripheral Th and DC both express identical Notch receptors (NotchR), Notch ligands (NotchL) and Numb (3, 13). The physical proximity of NotchR and NotchL in the plasma membrane of an individual cell can result in the phenomenon of cis-inhibition resulting from the ubiquitination and degradation of the NotchR-L complex (14, 15). It is especially intriguing how Th and DC, which express both NotchR and NotchL on its cell surface, avoid NotchL-dependent cis-inhibition of NotchR signaling upon their conjugation. This inhibition will need to be lifted for productive Notch activation to occur during Th:DC conjugation. During Th:DC interaction, the immunological synapse (IS) formed at the intercellular contact site comprises the structural framework mediating receptor-dependent signals that determines the functional fate of Th (16, 17, 18). Therefore, we posited that Notch proteins are present in the IS, and that NotchL and Numb must be segregated into IS-microdomains that exclude NotchR.

Another important tenet of Notch-dependent outcomes is the "directionality" of the Notch signal propagation. Unidirectional signaling from Notch ligand-expressing cells (signal sending cell, SSC) to Notch receptor-expressing cells (signal receiving cell, SRC) is a key feature of many developmental processes. We, therefore, hypothesized that during the Th:DC interaction there develops a cell type-specific NotchR-NotchL polarity within the partnering halves of the IS, i.e., ThIS and DCIS, that reflects the differential functionality of a SRC (for Th) and a SSC (for DC). Alternatively, we reasoned that both cell types concurrently assume the dual roles of SSC and SRC, yet the directionality of Notch signals would be maintained by the differential segregation of individual Notch components within microdomains of the IS. The present studies were designed to test these hypotheses.

In this study, we investigated the cell-specific, spatio-temporal segregation of Notch signaling components within the two apposed halves of the IS of allogeneic Th:DC conjugates. Strikingly, we find differential segregation of Notch signaling pathway components within the pSMAC and cSMAC of apposed ThIS and DCIS. Our studies support the hypothesis that there is establishment of antiparallel, reciprocal Notch signaling between Th and DC that is guided by Notch component segregation within microdomains of the IS. Our studies also suggest that one of the possible roles of Notch activation in the interacting Th and DC is to prevent activation-induced cell death (AICD) in both cell types while an appropriate immune response is being generated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells

Human CD4+ Th and monocyte-derived CD86+ CD83+ DC were generated from freshly isolated PBMCs obtained either from the Central Blood Bank (Pittsburgh, PA) or from peripheral blood of normal donors (n = 20) per Institutional Review Board regulations. Standard isolation methods were used to generate Th and DC. In brief, for DCs, 5 x 105 CD14+ cells isolated by CD14-positive selection using magnetic cell separation (Miltenyi Biotec) were plated into a 6-well plate in the culture medium containing 500 IU/ml rhIL-4 (Sigma-Aldrich), and 1000 IU/ml rhGM-CSF (Sigma-Aldrich). Between days 5–7 of culture, the DCs were matured by adding 100 µg/ml poly(I:C), 1 µg/ml PGE2 (both from Sigma-Aldrich), 10 ng/ml TNF-{alpha}, 10 ng/ml IL-1beta, and 100 U/ml IL-6 (all obtained from R&D Systems). Using FACS, mature DCs (CD14–, CD83+, CD86+) were phenotypically characterized before conjugation. Similarly, Th cells isolated from PBMC by CD4-positive selection (Miltenyi Biotec) were propagated in the culture medium enriched with 300 IU/ml IL-2. Both IL-2-stimulated and unstimulated Th were included in the described experiments. Using FACS, Th (CD56–, CD3+, CD4+) were also phenotypically characterized before conjugation.

Experimental cellular system

In this study, we used human peripheral monocyte-derived, DC-mediated physiological activation in primary human peripheral Th cells as an in vitro cellular model of acute allograft rejection in human transplants. We have chosen this model of direct MHC allorecognition over the Ag-specific, autologous T cell response to study Notch signaling, since the direct alloresponse is generalized, vigorous, dependent only on MHC disparity, and results from a relatively high frequency of T cells able to respond to a foreign MHC molecule on a DC. In contradistinction to Ag-specific TCR-transgenic mouse models, the frequency of Ag-specific, circulating human Th cells represent an extremely small proportion of the total Th population. This minimizes the likelihood of identifying an Ag-specific autologous Th:DC conjugates in our human cellular system. To show that our allogeneic Th:DC conjugation results in the formation of a productive and mature IS, we have included Ag-nonspecific, autologous Th:DC conjugates as controls. In addition, we generated allogeneic Th cell line, by incubating purified Th cells derived from donor-A with DCs from donor-B for 10–14 days. The donor-A Th cells were then re-challenged with the freshly matured DC from the same donor-B for additional 4–6 days. The Th:DC conjugation experiments were then performed using donor-A Th already in culture with freshly matured DC from donor-B.

Reagents

Primary Abs. Anti-CD86 (clone 2331), anti-CD83 (clone HB15e), anti-CD14 (clone M5E2), anti-pAKT S472/473/474 (clone 104A282, recognizes phosphorylated AKT1, 2, 3), and -CD3 were purchased from BD Pharmingen. Anti-Numb (N-20), anti-Jagged1 (C-20) recognizing a peptide mapped at the carboxy terminus (described in text as JICD1), anti-Delta (C-20) recognizing a peptide mapped at the carboxy terminus (described in text as DICD1), anti-pSTAT3, Y705 (B-7), -Rab11 (H-87), -Talin (C-20), and -{alpha}-tubulin (E-19) (identifies MTOC and tubules) were purchased from Santa Cruz Biotechnology. Anti-GLUT1 (Clone 202915), anti-Jagged1 (clone 188331) recognizing the extracellular domain (described in text as JECD1), and anti-Dll1 (clone 251127), recognizing the Dll protein 1 extracellular domain (described in text as DECD1), were purchased from R&D systems. Anti-Notch1 (clone A6), recognizing the ligand-binding region of Notch1 (described in text as NECD1) was purchased from Lab Vision/Neomarkers. For Notch1R neutralizing experiments, either 20 µg ml–1 of mouse IgG2b (BD Pharmingen) or anti-Notch1R Ab (clone A6), was preincubated with 1 x 106 Th and DC in serum-free medium for 1 h before and throughout the duration of the conjugation experiment, as previously described (19, 20).

Anti-cleaved Notch1, detecting Notch1 only when cleaved between Gly1743 and Val1744, which identifies Notch1 extracellular truncation (NEXT1) or Notch1 intracellular domain (described in text as NICD1) was purchased from Cell Signaling Technology. Anti-HES-1 (AB5702) was purchased from Chemicon International. BODIPY FL phallicidin, a probe for F-actin, was purchased from Invitrogen Life Technologies. Anti-LFA-1 and mouse anti-human LAMP-1 (H4A3, identifies lysosomes) obtained from the Developmental Studies Hybridoma Bank, Department of Biological Sciences (University of Iowa, Ames, IA). DAPI, used to identify the nucleus, was purchased from Sigma-Aldrich. Isotype controls IgG1 and IgG2 were purchased from Sigma-Aldrich or BD Pharmingen. To selectively identify the outer leaflet of DC plasma membrane, DCs fixed on poly-L-lysine slides were stained for 10–15 min with the styryl fluorescent membrane dye, SynaptoGreen (similar to FM1–43), purchased from Biotium. In addition, this dye also marks membranes of recycled endosomes (21), making it an ideal reagent to study Dll1 and Jagged1 membrane localization and recycling.

Because there are no specific reagents that can individually identify extracellular domain (ECD) or intracellular domain (ICD) fragments of the processed Notch ligands, we have relied on the amino-terminal fragment (NTF) and carboxy-terminal fragment (CTF) dual-labeling strategy to distinguish a full-length (when NTF and CTF signals overlap) from a processed ligand fragment (when NTF and CTF signals do not overlap). Therefore, Th:DC conjugates were multiply labeled with the Notch ligand Abs specific either for extracellular NTF (DECD1 or JECD1) or intracellular CTF (DICD1 or JICD1) of Dll1 and Jagged1.

Secondary Abs. Affinity-purified second Abs and species-adsorbed conjugates (FITC, Cy3, Cy5, and AMCA) for multilabeling were purchased from Chemicon International and Invitrogen Corporation (Carlsbad, CA).

The specificity of Ab staining of cellular targets was controlled for in the following manner: first, 2–3 different Abs against the same Ag, and obtained from different vendors, were used for both immunofluorescence and Western blotting. Western blot analysis and uniformity of staining pattern between Abs to the same Ag in our immunofluorescence studies were taken as evidence of specific reactivity. Furthermore, Ag-specific Ab staining patterns were compared with secondary Ab alone as well as with the corresponding isotype-specific Ab. Only those primary Abs whose isotype control Ab either completely failed to stain the cell or exhibited a distinctly different staining pattern from the primary Ab, were included in the present study. We have, therefore, given the specific details of each primary Ab used in our studies.

Th:DC conjugation assay and immunofluorescent cell imaging

Fixed Th:DC conjugates were immunofluorescently labeled as described previously (22) and analyzed at 5, 15, 30, 60 min, and 28 h. Based on the Nomarsky images, only those Th cells that were clearly conjugated with the DCs were selected for further analysis. In all experiments, a digital imaging system with a Zeiss Axioplan 2 (Intelligent Imaging Innovations (3-I)) was used. Images were obtained both in two-dimension (2D) (x-y-axis) and three-dimension (3D) (x-z-axis). A 63x objective with 1.0 magnification was used to acquire all images. The 2D images were used to analyze polarization events in the conjugates, as previously described (22). An event was considered polarized if the majority of the fluorescence of the molecule or organelle of interest was located in the proximal one-third of the cell nearest to the cell:cell contact site. For 3D images, 70–80 z-stack images were taken using a 0.2 µm step size between each plane. After deconvolving the acquired image using a nearest neighbor algorithm, the 3-view tool was implemented, displaying the image simultaneously in xz, xy, and yz axes. The images in yz (side view) and xy (front view) optical planes were used to identify Th:DC conjugates whose contact zones were horizontally aligned with each other and in whom the fluorescent signals of identical proteins in the contact zones could be clearly separated (see Fig. 3a for the example of conjugates included in the study). Such Th:DC conjugates, carefully selected by two independent observers, were included in further analysis of IS by examining the xz optical axis, using the segmentation and statistics capabilities of SlideBook 4.1.0.2 software (3-I). To properly identify the IS for 3D analysis, we delineated the contact area by specifically drawing the mask border along the edges of F-actin fluorescence using the manual masking function of the SlideBook software. By using this manual outlining of the contacting Th and DC halves of the IS, as opposed to the more inclusive fixed shaped mask options, we minimize the extent of cytoplasmic staining that might otherwise be erroneously considered part of the IS. To improve image quality, out-of-focus light was removed from the fluorescent image stacks using the nearest neighbor deconvolution algorithm. The acquired images were then processed using Photoshop 7.0 software (Adobe Systems).


Figure 3
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  		FIGURE 3. Temporal patterning of Notch signaling components in the microdomains of the apposed ThIS and DCIS. Representative fluorescent images of Th:DC conjugates obtained in the optical x-y, y-z and x-z-axis (x-z images described as ThIS or DCIS) are shown in a. The Th side of the IS (i.e., ThIS) and the DC side of the IS (i.e., DCIS) formed in the same Th:DC conjugate is shown for the indicated reagents. These images are representative of the dominant event for each molecule of interest. For clarity, the images shown are representative of the indicated event, irrespective of the time point at which they were acquired. In b, temporal quantitation of each indicated Notch event in the pSMAC and cSMAC of partnered ThIS (in red) and DCIS (in blue) are illustrated in independent histograms. In this figure, x-axis denotes conjugation time and y-axis denotes percentage of conjugates. Data in the figure is representative of between 30 and 55 ThIS and DCIS analyzed for each experimental condition. SEM for each event was calculated from at least three independent experiments. These values for ThIS ranged from ±0.01–0.1 (median: 0.04) for pSMAC events; ±0.01–0.08 (median: 0.06) for cSMAC events. The values for DCIS ranged from ±0.01–0.1 (median: 0.04) for both pSMAC and cSMAC events (error bars not shown). In c, the cartoon depicts the anti-parallel segregation of the key components of Notch signaling pathway (illustrated in distinct color-coded shapes) within the partnering ThIS (top) and DCIS (bottom) during Th:DC interaction. The block arrows traversing through the pSMAC or cSMAC denote the directionality of Notch signaling via the respective IS substructure. SSD, Signal-sending microdomain; SRD, signal-receiving microdomain.

 
ELISA

To quantitate IFN-{gamma}, the culture supernatant was harvested from 1 x 105 Th: 1 x 105 DC coculture at 24 h and measured by the Human Cytokine Premixed LINCOplex Kit (LINCO Research) with the Luminex (100) (Luminex Corporation). In each experiment, supernatant derived from equal number of Th alone and DC alone cell cultures was also included as controls. For Notch1R neutralizing experiments, unconjugated Th and DC as well as the Th:DC mixture were incubated with either the isotype Ab or Notch1R neutralizing Ab for 24 h. The supernatant harvested from the above cell conditions was used for ELISA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Transcellular Notch receptor-ligand interactions result in bidirectional Notch signaling in Th:DC conjugates

To test the hypothesis that conjugated allogeneic Th and DC assume a SSC or SRC phenotype, we first examined the temporal development of transcellular Notch receptor-ligand polarity in the interacting cells. Human allogeneic Th:DC conjugates were analyzed by deconvolution immunofluorescence microscopy for the presence and subcellular localization of Notch1R, Dll1, Jagged1, and Numb from 5 min to 28 h postconjugation, and the results compared with steady state events in unconjugated Th and DC.

To validate our experimental cellular system, we first demonstrated that the allogeneic, but not Ag-nonspecific autologous, Th:DC conjugation results in the formation of a productive, mature monocentric IS. Here, the Th:DC conjugates labeled with reagents for CD3, talin and {alpha}-tubulin (for MTOC) were analyzed at 30 min postconjugation. Fig. 1 demonstrates that in the allogeneic IS, talin segregates in the pSMAC while CD3 and MTOC in the cSMAC. This SMAC configuration is consistent with the formation of mature, productive IS in bonafide conjugates, as previously described for Ag-specific IS (16, 17, 18). In contrast, these proteins fail to form ordered SMAC structures in the Ag-nonspecific autologous Th:DC conjugates, in which the MTOC remains non-polarized (Fig. 1a). In addition, the ordered segregation of Notch1R and the generation of NICD1 are observed only in the allogeneic Th:DC conjugates, but not in the Ag-nonspecific Th:DC conjugates (Fig. 1, b and c). Furthermore, allogeneic Th:DC conjugates demonstrate LFA-1 polarization to the cell:cell contact and segregation to the pSMAC of ThIS, enclosing the centrally located NICD1 (Fig. 1d). Collectively, our results indicate that our in vitro allogeneic Th:DC conjugation results in the formation of a productive IS, similar to the one described during Ag-specific autologous Th:DC interactions (16, 17, 18).


Figure 1
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  		FIGURE 1. Composition of IS in allogeneic and Ag-nonspecific autologous Th:DC conjugates. a, DIC and fluorescent (2D and 3D) images of allogeneic and autologous Th:DC conjugates labeled with the indicated reagents and analyzed at 30 min. In the 3D (ThIS) image panel, one color is removed in each picture for better visualization of the individual events. Arrow indicates the location of MTOC in the conjugated Th (2D image) and cSMAC (3D image). b, x-y (front view), y-z (side view), and x-z (en face view) (ThIS and DCIS) images of autologous Th:DC conjugates labeled with the indicated reagents and analyzed at 30 min. The image on the right side of the panel has the green color removed for better visualization of NECD1 distribution. In c, DIC overlay and 2D fluorescent images of a representative Ag-nonspecific autologous Th:DC conjugate dual-labeled with the indicated reagents and analyzed at 30 min. d, 2D and 3D (ThIS) images of allogeneic Th:DC conjugate dual-labeled with the indicated reagents and analyzed at 30 min. Arrow indicates the location of NICD1 at the contact site. The data in the figure is representative of at least 20 conjugates analyzed for each condition.

 
Th events

In non-permeabilized Th, NECD1 signals are detected using both FACS and imaging studies. In the permeabilized, unconjugated Th, both NECD1 and NEXT/NICD1 assume a circumferential, perimembranous distribution, and their presence is not detected in the nucleus (Fig. 2a). Upon conjugation, however, NECD1 is polarized to the ThIS in ~40% of Th within 5 min of contact with DC, and remains so for up to 30 min (Fig. 2, c and d). The number of NECD1-polarized Th conjugates then drops to 7% at 1 h, and 1% at 28 h. Significantly, NICD1 is detected in the nucleus in 25–33% of Th conjugates during the first hour of conjugation, and remains sustained in ~40% of Th conjugates between 1 and 28 h (Fig. 2, c and d). Given the reported transience of nuclear NICD1 in cells (2), we interpret its sustained presence as resulting from ongoing, conjugation-dependent processing of Notch1R.


Figure 2
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  		FIGURE 2. Quantitation of temporal Notch events in single Th:DC conjugates by immunofluorescence microscopy. Representative DIC overlay and 2D fluorescent images of unconjugated Th (a), DC (b), and Th:DC conjugates (c) multiply labeled with the indicated reagents are shown. The images in c are representative of the dominant event for each molecule of interest. In c, location of polarized NECD1 is indicated by a white arrowhead; in the NICD1 image, the location of the Th-nucleus is identified by a white arrowhead, and that of the DC, by a black arrowhead. Here, the pink pseudocolor within the nucleus of each conjugated cell results from an overlap of red and blue colors. Proteins of interest are shown in either red or green, and the nucleus is blue (DAPI). Temporal (from 5 min to 28 h) quantitation of these Notch events is illustrated in the accompanying graphs (d), where "nd" denotes not done, "uc" (on x-axis) denotes data for the unconjugated Th (in red) or DC (in blue). SEM (SE mean) for each event was calculated from at least three independent experiments and ranged from ±0.01–0.12 (median: 0.03) for all Th events and ±0.01–0.09 (median: 0.02) for all DC events at all time points (error bars not shown). Data in this figure is representative of ≥100 unconjugated Th or DC and between 60 and 320 Th:DC conjugates analyzed for each condition. For clarity, the images shown are representative of the indicated event, irrespective of the time point at which they were acquired. ECD, extracellular domain; ICD, intracellular domain; J, Jagged1; D, Dll1; N, Notch1 receptor.

 
Like NECD1, Numb also assumes a predominantly perimembranous distribution in unconjugated Th (Fig. 2a). Upon conjugation, rapid polarization of Numb (within 5 min) to the ThIS occurs in 32% of the Th conjugates (Fig. 2, c and d), consistent with previous observations made in murine Th:APC conjugates (23). Soon thereafter, <10% of Th conjugates demonstrate Numb polarization, suggesting that Numb is only transiently part of a polarized Notch1R-signaling complex. Unlike NECD1 and Numb, the Notch ligands DECD1 and JECD1 in the conjugated Th remain non-polarized at all time points postconjugation (Fig. 2c). However, processed fragments of these ligands are still detected in the nuclei of conjugated Th, with the most significant rise in nuclear Notch ligand seen with JICD1 between 15 and 60 min (Fig. 2, c and d).

DC events

In permeabilized, unconjugated DCs, NECD1, DECD1 and JECD1 signals are detected in the submembranous region, as well as throughout the cytoplasm, (Fig. 2b). NICD1 signals are detected in all unconjugated DCs, and in ~50% of these cells, NICD1 is constitutively nuclear (Fig. 2, c and d). This indicates that full activation of Notch1R via S2 and S3 cleavages (2) occurs in DC before its conjugation with Th.

In conjugated DC, the classical polarization of NECD1, DECD1 or JECD1 is not observed in ~90% of cells. The presence of nuclear NICD1, seen in ~50% of unconjugated DC, is sustained in the same range of conjugated DCs during the first hour of Th:DC interaction (Fig. 2, c and d). By 28 h, however, ~90% of conjugated DCs demonstrate nuclear NICD1 (Fig. 2d). Finally, we observe a temporal increase in the number of conjugated DCs demonstrating localization of Notch ligands, either full-length and/or their processed fragments, within the nucleus (Fig. 2, c and d). When compared with its partnered Th, the nuclear translocation of JICD1 and DICD1 was both rapid and more pronounced in the conjugated DC (Fig. 2d). In the vast majority of the conjugated DC, Abs that specifically identify CTF of Dll1 or Jagged 1 (i.e., DICD1 or JICD1) were observed in the nucleus, whereas those detected by the NTF (i.e., DECD1 or JECD1) were only infrequently observed. Collectively, this result demonstrates active ligand engagement and processing following DC interaction with Th.

In summary, 2D temporal analysis of Th:DC conjugates demonstrates activation of Notch1R, Dll1, and Jagged1 in both Th and DC, suggesting that Notch signaling during Th:DC interaction is bidirectional.

Anti-parallel segregation of Notch pathway components within IS microdomains

To gain further insight into the development of directionality in Notch signal propagation during the Th:DC interaction, we temporally analyzed the segregation pattern of the components of Notch signaling within the apposed halves of the same IS, i.e., ThIS and DCIS. These results were contrasted with those acquired from autologous, Ag-nonspecific Th:DC conjugates.

In the majority of allogeneic conjugates (55–95%), NECD1 and NICD1 cluster in the cSMAC of the ThIS at 5, 15, and 30 min, whereas DECD1 and JECD1 either cluster in the pSMAC or are absent altogether from this microdomain (Fig. 3, a and b). In contrast, in the majority of the apposed DCIS, NECD1 and NICD1 cluster in the pSMAC, whereas DECD1 and JECD1 cluster in the cSMAC. The two Notch ligands sustain their cSMAC (in DCIS) or pSMAC (in ThIS) locations even at 1 h and 28 h. Therefore, Th-Notch1R and DC-Notch ligands are appositionally clustered in the cSMACs of their respective halves of the IS, whereas DC-Notch1R and Th-Notch ligands are appositionally clustered in the pSMACs (Fig. 3c).

Because Numb is a negative regulator of Notch, we next inquired whether the localization of Numb within the two apposed halves of the IS could further clarify the directionality of Notch signaling within the spatially segregated pathways. After an initial (5 min) clustering of Numb in the cSMAC of ThIS, Numb clusters predominantly in the pSMAC. In contrast, in virtually all the DCIS examined, Numb is located predominantly in the cSMAC (Fig. 3, a and b). Therefore, Numb and Notch1R fluorescent signals are identified in mutually exclusive microdomains of both ThIS and DCIS. This arrangement of Notch1R and Numb in the IS is consistent with a model in which Notch and Numb negatively regulate each other in a reciprocal manner. Taken together, the anti-parallel arrangement of Numb within the two IS is consistent with Numb selectively preventing DC from receiving Notch signals through the center of the IS, and Th through the periphery of the IS.

In a separate experiment, the above Notch1R events were examined in the autologous Ag-nonspecific Th:DC conjugates at 30 min. First, the anti-parallel SMAC segregation of NECD1 observed in the allogeneic IS is not seen in the autologous IS (compare Fig. 1b and Fig. 3a). In the autologous IS, NECD1 and F-actin are evenly distributed in the contacting membranes of both the conjugated Th and DC, indicating lack of segregation. Second, NICD1 is not detected in the nucleus of >90% (n = 20) of the conjugated autologous Th (Fig. 1c), suggesting a lack of full Notch1R activation (i.e., including the S3 cleavage). Therefore, lack of ordered SMAC structures in the ephemeral IS of autologous, Ag-nonspecific Th:DC conjugates results in impaired Notch1R activation. These results suggest that segregation of Notch components into distinct microdomains of the IS is required for full Notch1R activation.

In summary, 3D temporal analysis of Th:DC conjugates demonstrates that Notch1R activation events dominate in the cSMAC of ThIS and pSMAC of DCIS, whereas Notch ligand and Numb events dominate in the cSMAC of DCIS and pSMAC of ThIS. This molecular arrangement allows for the establishment of a structural platform suitable for propagating reciprocal, spatially restricted, Notch signaling across partnering DC and Th.

MTOC polarization in the conjugated Th is associated with translocation of Notch-ligand containing, Rab11-endosomes to the IS

Previous studies in Drosophila have linked trafficking of the internalized Notch ligands in the Rab11+ recycling endosomes with enhanced, membrane-localized, ligand signaling activity (24, 25). In lymphocytes, many intracytoplasmic proteins accumulate around the microtubule-organizing center (MTOC), and the reorientation of MTOC toward the interacting target cell physically transports these proteins to the cell:cell contacting membrane (26). We, therefore, hypothesized that during the Th:DC interaction, intracellular Notch ligands will be recruited to the IS by Rab11+ endosomes in an MTOC-dependent manner.

Our data show that Notch ligands are colocalized with Rab11+ endosomes in both Th and DC, whether conjugated or unconjugated (Fig. 4, a–c). As in the Drosophila system, these results are consistent with active recycling of Notch ligands from internal stores to the cell surface membrane, a process that would maintain Notch ligand signaling "readiness".


Figure 4
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  		FIGURE 4. Notch ligand, Rab11, and MTOC events in Th:DC conjugates. Representative immunofluorescent images of Th:DC conjugates analyzed at 1 h after labeling with the indicated combination of reagents are shown. a, One DC conjugated with multiple Th analyzed at 1 h after triple-labeling with JECD1 (in green), Rab11 (in red), and DAPI (in blue). A magnified view of the DC plasma membrane in contact with one of the many Th is shown (within the rectangular box on the left) to identify the pattern of colocalization of JECD1 with Rab11. b, 2D and 3D (i.e., ThIS or DCIS) images of a Th:DC conjugate analyzed at 1 h after labeling with the indicated reagents. The relationship of Rab11 with JECD1 within the microdomains of the apposed ThIS and DCIS, in the same conjugate, is illustrated in these images. The Th and DC contacting areas are derived from projections of the x-z-axis of the conjugate, and are labeled ThIS or DCIS, respectively. c, Top images are of unconjugated Th or DC triple-labeled with the indicated reagents to identify the location and relationship of DECD1 with MTOC and recycling endosomes. The bottom images shows one DC conjugated with two Th analyzed at 30 min after triple-labeling with the indicated reagents. The arrows indicate the location of MTOC in the conjugated Th and DC. Data in each panel is representative of 30–50 Th:DC conjugates analyzed for each experimental condition. d, A representative unconjugated DC dual-labeled with the indicated reagents to identify the location and relationship of Notch ligands with the outer leaflet of DC plasma membrane and recycling vesicles (see text).

 
Th:DC conjugates were then evaluated at 1 h for the presence of unique patterns of Notch ligands and Rab11 segregation in the two apposed halves of the IS (Fig. 4b). Our 3D analysis reveals that 75% of DCIS demonstrated a predominantly cSMAC coaccumulation of Rab11 with JECD1 (Fig. 4b) or DECD1 (not shown) (n = 32). In contrast, 55% of ThIS demonstrated a predominantly pSMAC coaccumulation of Rab11 and Notch ligands. Furthermore, Notch ligand signals are predominantly below the actin-delimited, perimembranous space (Fig. 3a), indicating that they are restricted components of the plasma membrane. Indeed, FACS and immunofluorescence microscopy of non-permeabilized Th and DC detects Notch ligand signals in only 10–30% of the cell population (n = 10 donors), while FACS and imaging of the same cells upon permeabilization demonstrate the presence of these ligands in nearly 100% of cells (Fig. 2, a and b; FACS data not shown).

To further substantiate that some fraction of Notch ligands is present in the plasma membrane of unconjugated DC, these cells were labeled with SynaptoGreen, a marker of the outer layer of the plasma membrane, along with Abs against Jagged1 (JECD1) or Dll1 (DECD1). We observed partial colocalization of the ligand signals with SynaptoGreen, suggesting the presence of Notch ligand in the outermost layer of the DC plasma membrane (Fig. 4d). Furthermore, since this dye rapidly internalizes and identifies recycling cytoplasmic vesicles, our observations of colocalized distribution of Notch ligands and SynaptoGreen in DC cytoplasm suggests active internalization of surface expressed Notch ligands. Collectively, these results are consistent with the hypothesis that internalized Notch ligands are focally presented to the SMAC structures of IS by Rab11+ recycling endosomes in both Th and DC. Definitive evidence of such a role for Rab11 awaits experiments with Th and DC expressing dominant-negative Rab11.

Others have demonstrated that during development, Rab11 endosomes accumulate around reorienting MTOC in Drosophila sensory organ precursor cells, suggesting that these endosomes are reoriented in conjunction with that of MTOC (25). Furthermore, the MTOC has been shown to guide delivery of lysosomes to the cSMAC of CTL (27). We therefore inquired whether the translocation of Rab11+ endosomes to the IS is accompanied by reorientation of the MTOC-cytoskeleton during the Th:DC interaction. Therefore, Th:DC conjugates triple-labeled with Abs to {alpha}-tubulin, Rab11, and DECD1 or JECD1, were analyzed at 5, 15, 30, and 60 min following conjugation (Fig. 4c). In conjugated Th, polarization of the MTOC toward the ThIS was observed in 35% of conjugates at 5 min, increasing to 50% by 1 h (Fig. 4c, n = 75 for each time point). In conjugated Th, Rab11 and the Notch ligands were colocalized with MTOC, a phenomenon observed at all stages of positional translocation of MTOC from posterior Th uropod to the ThIS. In unconjugated Th, Notch ligands are also colocalized with Rab11 and MTOC (Fig. 4c, top panel), indicating a preformed ligand delivery mechanism.

These results stand in sharp contrast to those obtained in conjugated DC, where Notch ligands do not colocalize with MTOC. Furthermore, the MTOC is infrequently polarized (~15–20%, n = 60) in conjugated DC at all time points. Despite the lack of association with MTOC in DC, Notch ligands are colocalized with Rab11 at the plasma membrane and other subcellular locations along microtubules (Fig. 4c, bottom panel). Therefore, within the time frame of these experiments, our data are consistent with MTOC- and/or microtubule-based delivery of Notch ligands to the IS.

IS microdomains support bidirectional propagation of functionally relevant Notch signals in allogeneic Th:DC conjugates

We next evaluated whether the above-described physical evidence of bidirectional Notch signaling translates into functionally relevant activation signals in both Th and DC. We reasoned that any change in subcellular localization or in the temporal activity of canonical Notch targets in both conjugated Th and DC would suggest vectorial, SMAC-directed, Notch activation.

Notch target activation events in Th

We initially evaluated the temporal relation of the expression and subcellular location of HES-1 in Th:DC conjugates, as compared with unconjugated controls. Nearly all unconjugated primary human Th, either fresh or IL-2 activated, already express HES-1, and in ~50%, HES-1 is constitutively nuclear (n = 75). This result suggests that there exists a mechanism(s) to up-regulate HES-1 in unconjugated Th, which may include CD3/CD28- or serum-dependent activation signals (28, 29). Following Th:DC conjugation, however, the number of Th demonstrating nuclear HES-1 increases to 70–80% at 30–60 min postconjugation (Fig. 5, a and b). Because the nuclear transcription factor STAT3 has been described as a direct target of Notch-mediated HES-1 activation (30, 31), we evaluated the kinetics of nuclear translocation of phosphorylated STAT3 (pSTAT3) in the context of allogeneic Th:DC conjugation. Despite the relatively high background of HES-1 activation in Th, the number of unconjugated Th demonstrating nuclear pSTAT3 (Y705) is only 4% (Fig. 5). Upon conjugation with DC, however, Th-specific nuclear translocation of pSTAT3 rapidly increases and is observed in 90–100% of Th conjugates at 1 h (Fig. 5).


Figure 5
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  		FIGURE 5. Temporal activation of Notch-related targets in the conjugated Th and DC. a, Representative DIC overlay and 2D fluorescent images of Th:DC conjugates labeled with the indicated reagents is shown. In each image the Th and DC are indicated. "pS-AKT" indicates AKT phosphorylated at Serine473 and "pY-STAT3" indicates phosphorylated STAT3 at tyrosine 705. Nucleus is identified by the blue color of DAPI. For clarity, the images shown are representative of the indicated event, irrespective of the time point at which they were acquired. b, Temporal quantitation of each indicated event in the conjugated Th (in red) and DC (in blue) is illustrated in independent histograms. The designation "uc" on the abscissa indicates steady-state events in the unconjugated Th or DC. The data in this panel is representative of 50–75 conjugates analyzed from 2 to 3 independent experiments. SEM for each event ranged from 0.01–0.1 (median: 0.03) for Th events and 0.01–0.08 (median: 0.01) for DC events (error bars not shown).

 
Because Notch responsiveness is also a PI3K-AKT pathway-dependent phenomenon that depends on activated STAT3 (28, 31), and since Notch signaling promotes glucose metabolism through the regulation of AKT (32), we evaluated the temporal changes in phosphorylated-AKT (pAKT, Ser473) and the expression of GLUT1 in Th:DC conjugates. As shown in Fig. 5, the expression of GLUT1 is rapidly up-regulated selectively in the conjugated Th. At 15 min, 90% of the Th conjugates show strong plasma membrane staining for GLUT1. In comparison, unconjugated Th demonstrate weak or absent GLUT1 signals. Similarly, at 5 min, pAKT is polarized to the ThIS in ~40% of Th conjugates, but the number of Th conjugates with polarized pAKT drops rapidly to 7% at 1 h (n = 60, data not shown). Importantly, the number of Th conjugates demonstrating nuclear pAKT increases in parallel with decreasing AKT-polarized conjugates, suggesting the translocation of membrane-bound pAKT to the nucleus (Fig. 5, a and b). By 28 h, Th conjugates regain the steady-state level of AKT activation. This dynamic subcellular pattern of pAKT trafficking is consistent with previous observations (33).

In summary, the temporal progression of cytoplasmic and transcriptional responses to physiological Notch activation reported above are in remarkable agreement with those found for these indicators of Notch signaling in neuronal stem cells (31).

Notch target activation events in DC

Similar events were temporally evaluated in conjugated DC (Fig. 5). Our results demonstrate basal nuclear HES-1 in nearly 100% and nuclear pSTAT3 in ~50% of unconjugated DC, indicating preconjugation activation of these signaling proteins. HES-1 remains nuclear in ~100% of conjugated DCs up to 30 min postconjugation, whereupon it declines somewhat. Significantly, there is a 30–50% increase in the number of DCs demonstrating nuclear pSTAT3 upon conjugation with Th. Similarly, there is a rapid but transient increase in nuclear pAKT and strong GLUT1 expression in the conjugated DC (Fig. 5). Therefore, despite a relatively stable nuclear presence of NICD1 and HES-1 in both unconjugated and conjugated DC, we observe a rapid and significant postconjugation rise in nuclear pSTAT3, pAKT and GLUT1 with similar kinetics to that observed in the partnering Th.

Collectively, the above results indicate that Notch1R activation does not prevent the occurrence of other parallel activation events that are initiated during a productive Th:DC interaction.

Finally, we inquired whether blocking Notch1R will perturb the ability of Th:DC conjugates to a) generate NICD1, b) activate HES-1, and c) produce IFN-{gamma}, a marker for T cell activation in cytokine "neutral" conditions. Th:DC conjugates were incubated in serum-free medium with either anti-Notch1R neutralizing Ab, or IgG2b isotype-specific Ab. NICD1 and HES1 activity were analyzed after 1 h, and IFN-{gamma} after 24 h of incubation with neutralizing Ab (n = 20, 2 independent experiments). Cocultures of Th and DC incubated with both blocking and control Abs continued to form stable conjugates (Fig. 6a). These Th:DC conjugates demonstrated polarization of F-actin (Fig. 6a, arrow) and its appropriate segregation in the pSMAC of ThIS (Fig. 6b), events that are associated with the formation of a productive IS. This result suggests that the remodeling of F-actin cortical cytoskeleton, as a component of SMAC formation, is independent of Notch1R signaling. Importantly, these conjugates revealed a significant diminution of both nuclear NICD1 and nuclear HES-1 only in Th and DC treated with neutralizing anti-Notch1R Ab (Fig. 6a), but not with isotype control Ab (data not shown) (compare Fig. 6 with Figs. 2, 3, and 5). Additionally, treatment with neutralizing Notch1R Ab resulted in ~40% decrease in the amount of IFN-{gamma} in the cell supernatant when compared with the IgG2b control (Fig. 6c), which is consistent with a known role of Notch1R signaling in Th1 differentiation and function (6, 34). Collectively, these results provide direct evidence for the involvement of Notch1R signaling in DC-driven, T cell activation under physiologic conditions.


Figure 6
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  		FIGURE 6. Neutralization of Notch1R in Th:DC conjugates. a, DIC and fluorescent 2D images of Th:DC conjugates incubated with Notch1R neutralizing Ab for 1 h. Arrow indicates the polarized location of F-actin in the conjugated Th. b, The 2D and 3D (ThIS, DCIS) images of Th:DC conjugates incubated with Notch1R neutralizing Ab for 1 h. Arrow in the 2D image indicates the location of infrequently present weak NICD1 signals (compare images in a and b with the NICD1 image in Fig. 2c). The data in the two panels is representative of 20 conjugates analyzed for each condition. c, The bar diagram of the quantitative ELISA for IFN-{gamma} performed on the culture medium harvested from Th:DC conjugates incubated with either the control IgG2b Ab or Notch1R Ab for 24 h. Numbers atop each bar is the actual value in picograms per milliliter.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results offer the first evidence for the development of reciprocal Notch signaling regulated by the IS during a physiological, human allogeneic Th:DC interaction. The temporal changes in the molecular topology of Notch signaling indicates establishment of a platform that directs vectorial DC-to-Th signaling via the cSMAC, and Th-to-DC signaling via the pSMAC, across a partnered IS. Therefore, intramicrodomain IS polarity allows the process of intercellular Notch signaling to remain unidirectional, yet reciprocal. This arrangement, seen only in the allogeneic but not the autologous Ag-nonspecific Th:DC interaction, permits bidirectional intercellular communication, and suggests that Notch patterning in the IS is dependent on productive TCR signaling.

The unidirectionality of Notch activation, so characteristic of Notch signaling in the developmental system, is maintained in the peripheral immune system. However, unlike developmental systems, neither Th nor DC assumes an exclusive SSC or SRC phenotype. Rather, they each assume aspects of both phenotypes. To parallel the SSC and SRC phenotypes seen in developmental systems, we here define the IS microdomain that contains Notch ligands and Numb as the signal-sending domain (SSD) and the microdomain that contains Notch receptor as the signal-receiving domain (SRD) (Fig. 3c). The lateral segregation of Notch1R from NotchL in Th-side or DC-side of the IS may be functionally relevant. Indeed, very recent data indicate that cis-inhibitory Notch receptor-ligand interactions, previously described for intracytoplasmic NotchR-L complexes (14), also occur at the cell surface (15). Therefore, microdomain-imposed physical separation of Notch receptor from Notch ligands during the Th:DC interaction may represent a unique mechanism for preventing spontaneous, local, ligand-induced, cis-inhibition of Notch receptor in the membranes of the interacting immune cells.

In Th:DC conjugates, the molecular anatomy of the two apposing halves of IS favors a model in which functionally relevant Notch signals propagate bidirectionally using both c- and p-SMAC microdomains for signal propagation. In addition to our evidence for cSMAC-based Notch signaling, which is consistent with the cSMAC-driven model for TCR signaling (18, 35), the following key observations point toward a signaling role for the pSMAC. First, the post-30-min incremental increase of NICD1 in the nuclei of conjugated DC and the reciprocal rise in nuclear Jagged1 (JICD1) in the partnered Th cell indicate ongoing Th-to-DC Notch signaling via the pSMAC. Second, the steep temporal rise in the number of conjugated DC demonstrating activation and nuclear translocation of STAT3, an event previously shown to be Notch1-regulated (30), is evidence for pSMAC-driven signaling. Third, the persistent presence of otherwise short-lived HES-1 (29) in the nuclei of conjugated DC strongly supports ongoing, contact-dependent, HES-1 activation. These findings are supported by our ability to inhibit NICD1 formation and its nuclear translocation, as well as HES-1 activation after treatment of both Th and DC with neutralizing anti-Notch1R Ab. Additionally, the rapid, postconjugation rise in nuclear pAKT and GLUT1 surface expression in the conjugated DC, the latter an indicator of full metabolic activation (36, 37), indicates productive DC activation, although the microdomain(s) responsible for this aspect of Th-to-DC signaling has yet to be determined.

Collectively, however, the pSMAC-driven DC events are consistent with productive Th-to-DC Notch signaling. Our observations in the physiological allogeneic Th:DC conjugation, therefore, extend those derived from the hybrid live cell-supported lipid bilayer model in which TCR-pMHC interactions resulted in signaling via the periphery of the ThIS (38, 39).

Although the mechanisms responsible for the ordered segregation of intramembranous and cytoplasmic Notch proteins in the IS are not known, previous work in lymphocytes has demonstrated colocalization of signaling molecules with MTOC (26). MTOCs have also been shown to polarize and associate with both pSMAC and cSMAC microdomains of the IS (27, 40). Because Th and DC Notch ligands are colocalized predominantly with the Rab11 endosomes and the microtubular cytoskeleton, we hypothesize that Notch ligands in Rab11 endosomes are actively transported to the cell surface in an MTOC or microtubule-dependent manner during conjugation. Collectively, therefore, our results suggest a functional role for tubulin- and/or the MTOC-cytoskeleton in Notch signaling by mediating the translocation of ligand-containing endosomes to and/or from the IS, and warrants a detailed evaluation of the mechanistic role of the MTOC-cytoskeleton and the Rab11 recycling subcellular compartment in this process.

A prolonged exchange of activation signals in the Th:DC interaction is often required to elicit an appropriate T cell response (17), which may also stimulate pathways that induce activation-induced cell death (AICD). Recent evidence demonstrates that STAT3 activation is part of an IL-6 receptor-mediated, anti-apoptotic response of T cells in Crohn’s Disease (41). Furthermore, STAT-3-deficient mice demonstrate IL-6-mediated T cell apoptosis (42). Others have shown Notch signaling to be intimately involved in the activation and nuclear translocation of STAT3 in COS-1 cells (30). These studies allow us to suggest a similar "survival" role for the Notch-dependent, rapid activation of STAT3 in the DC-activated Th. Assuming a unity of mechanism across cell types, the rapid nuclear accumulation of pSTAT3 in the conjugated DC presents us with the intriguing possibility that at least one of the functions of the novel Th-to-DC Notch signaling is the inhibition of apoptosis in the conjugated DC for the duration of the individual Th:DC interaction. Indeed, the presence of nuclear NICD1, an inhibitor of proapoptotic Nur77 (43) in the conjugated Th and DC, would collectively suggest the initiation of NICD1-, STAT3-, and AKT-mediated anti-apoptotic mechanisms in both the conjugated Th and DC. Our studies suggest, therefore, that one of the possible roles of Notch activation in both Th and DC is to prevent AICD in interacting immune cells, thereby insuring their viability while an appropriate immune response is generated.

In summary, our data indicate that lineage-specific immune cells communicate bidirectionally using unidirectional Notch signaling platforms of spatially segregated membrane-microdomains of the IS. During Notch-mediated binary cell fate decisions of organogenesis, cells in a homogeneous population acquire a SSC or SRC phenotype in the process of terminal differentiation. In contrast, our observations of bidirectional Notch signaling suggest that the heterologous Th:DC interaction is cooperative, requiring reciprocal information transfer across both cell types to mount an appropriate immune response. Despite activation of the same Notch signaling cascade in these two interacting cell types, these signals must be inducing cell-type specific functional outcomes that result from context-dependent activation of unique, Notch-sensitive targets. This concept of context-dependent Notch targets and readouts may explain the apparent disparity in reported functional Th outcomes (i.e., activation vs inhibition) following activation of Notch signaling (3, 44). Additionally, our data raises the possibility that antiparallel, bidirectional Notch signaling may occur at specific time points even during cell fate decisions of organogenesis. We suggest that this anti-parallel arrangement of Notch receptors and ligands may apply to the mounting of a vigorous adaptive immune response during the autologous, Ag-specific Th:DC interaction, as well.


   Acknowledgments
 
We thank Drs. Janis K. Burkhardt (University of Pennsylvannia, Philadelphia, PA), Mark K. Jenkins (University of Minnesota, Minneapolis, MN), Christian Munz (Rockefellar University, New York, NY), Anuradha Ray (University of Pittsburgh, Pittsburgh, PA), and Arthur Weiss (University of California, San Fancisco, San Francisco, CA) for insightful discussions and/or review of the manuscript. We also thank Dr. Jay Kolls for the use of Luminex ELISA system and deconvolution microscope facility, and Sheryl D’Silva for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant K08AI51402 (to Y.M.V.), R01AR045593 (to J.P.), and from U.S. Immune Deficiency Network (to Y.M.V.). Back

2 Address correspondence and reprint requests to Dr. Yatin M. Vyas, Division of Pediatric Hematology-Oncology and Bone Marrow Transplantation, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213. E-mail address: yatin.vyas{at}chp.edu Back

3 Abbreviations used in this paper: Dll, Delta-like ligand; NECD1, Notch extracellular domain 1; NICD1, Notch intracellular domain 1; NEXT, Notch extracellular truncation; DECD1, Dll1 extracellular domain; JECD1, Jagged1 extracellular domain; HES 1, hairy and enhancer of split 1; GLUT1, glucose transporter 1; IS, immunological synapse; SMAC, supramolecular activation clusters; SSC, signal sending cell; SRC, signal receiving cell; MTOC, microtubule organizing center; CTF, C-terminal fragment; NTF, N-terminal fragment. Back

Received for publication December 21, 2006. Accepted for publication May 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Artavanis-Tsakonas, S., M. D. Rand, R. J. Lake. 1999. Notch signaling: cell fate control and signal integration in development. Science 284: 770-776. [Abstract/Free Full Text]
  2. Schweisguth, F.. 2004. Notch signaling activity. Curr. Biol. 14: R129-R138. [Medline]
  3. Maillard, I., T. Fang, W. S. Pear. 2005. Regulation of lymphoid development, differentiation, and function by the Notch pathway. Annu. Rev. Immunol. 23: 945-974. [Medline]
  4. Radtke, F., A. Wilson, S. J. Mancini, H. R. MacDonald. 2004. Notch regulation of lymphocyte development and function. Nat. Immunol. 5: 247-253. [Medline]
  5. Dallman, M. J., E. Smith, R. A. Benson, J. R. Lamb. 2005. Notch: control of lymphocyte differentiation in the periphery. Curr. Opin. Immunol. 17: 259-266. [Medline]
  6. Amsen, D., J. M. Blander, G. R. Lee, K. Tanigaki, T. Honjo, R. A. Flavell. 2004. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117: 515-526. [Medline]
  7. Tanaka, S., J. Tsukada, W. Suzuki, K. Hayashi, K. Tanigaki, M. Tsuji, H. Inoue, T. Honjo, M. Kubo. 2006. The interleukin-4 enhancer CNS-2 is regulated by Notch signals and controls initial expression in NKT cells and memory-type CD4 T cells. Immunity 24: 689-701. [Medline]
  8. Tu, L., T. C. Fang, D. Artis, O. Shestova, S. E. Pross, I. Maillard, W. S. Pear. 2005. Notch signaling is an important regulator of type 2 immunity. J. Exp. Med. 202: 1037-1042. [Abstract/Free Full Text]
  9. McGill, M. A., C. J. McGlade. 2003. Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. J. Biol. Chem. 278: 23196-23203. [Abstract/Free Full Text]
  10. Lai, E. C.. 2002. Protein degradation: four E3s for the notch pathway. Curr. Biol. 12: R74-R78. [Medline]
  11. Matsuno, K., R. J. Diederich, M. J. Go, C. M. Blaumueller, S. Artavanis-Tsakonas. 1995. Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121: 2633-2644. [Abstract]
  12. Spana, E. P., C. Kopczynski, C. S. Goodman, C. Q. Doe. 1995. Asymmetric localization of numb autonomously determines sibling neuron identity in the Drosophila CNS. Development 121: 3489-3494. [Abstract]
  13. Yamaguchi, E., S. Chiba, K. Kumano, A. Kunisato, T. Takahashi, H. Hirai. 2002. Expression of Notch ligands, Jagged1, 2 and Delta1 in antigen presenting cells in mice. Immunol. Lett. 81: 59-64. [Medline]
  14. Sakamoto, K., O. Ohara, M. Takagi, S. Takeda, K. Katsube. 2002. Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev. Biol. 241: 313-326. [Medline]
  15. Glittenberg, M., C. Pitsouli, C. Garvey, C. Delidakis, S. Bray. 2006. Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis. EMBO J. 25: 4697-4706. [Medline]
  16. Bromley, S. K., W. R. Burack, K. G. Johnson, K. Somersalo, T. N. Sims, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 2001. The immunological synapse. Annu. Rev. Immunol. 19: 375-396. [Medline]
  17. Huppa, J. B., M. M. Davis. 2003. T-cell-antigen recognition and the immunological synapse. Nat. Rev. Immunol. 3: 973-983. [Medline]
  18. Lee, K. H., A. R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma, T. N. Sims, W. R. Burack, H. Wu, J. Wang, et al 2003. The immunological synapse balances T cell receptor signaling and degradation. Science 302: 1218-1222. [Abstract/Free Full Text]
  19. Ostroukhova, M., Z. Qi, T. B. Oriss, B. Dixon-McCarthy, P. Ray, A. Ray. 2006. Treg-mediated immunosuppression involves activation of the Notch-HES1 axis by membrane-bound TGF-beta. J. Clin. Invest. 116: 996-1004. [Medline]
  20. Yasutomo, K., C. Doyle, L. Miele, C. Fuchs, R. N. Germain. 2000. The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404: 506-510. [Medline]
  21. Betz, W. J., G. S. Bewick. 1992. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255: 200-203. [Abstract/Free Full Text]
  22. Huang, W., H. D. Ochs, B. Dupont, Y. M. Vyas. 2005. The Wiskott-Aldrich syndrome protein regulates nuclear translocation of NFAT2 and NF-kappa B (RelA) independently of its role in filamentous actin polymerization and actin cytoskeletal rearrangement. J. Immunol. 174: 2602-2611. [Abstract/Free Full Text]
  23. Anderson, A. C., E. A. Kitchens, S. W. Chan, C. St Hill, Y. N. Jan, W. Zhong, E. A. Robey. 2005. The Notch regulator Numb links the Notch and TCR signaling pathways. J. Immunol. 174: 890-897. [Abstract/Free Full Text]
  24. Wang, W., G. Struhl. 2004. Drosophila Epsin mediates a select endocytic pathway that DSL ligands must enter to activate Notch. Development 131: 5367-5380. [Abstract/Free Full Text]
  25. Emery, G., A. Hutterer, D. Berdnik, B. Mayer, F. Wirtz-Peitz, M. G. Gaitan, J. A. Knoblich. 2005. Asymmetric Rab 11 endosomes regulate {delta} recycling and specify cell fate in the Drosophila nervous system. Cell 122: 763-773. [Medline]
  26. Sancho, D., M. Vicente-Manzanares, M. Mittelbrunn, M. C. Montoya, M. Gordon-Alonso, J. M. Serrador, F. Sanchez-Madrid. 2002. Regulation of microtubule-organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions. Immunol. Rev. 189: 84-97. [Medline]
  27. Stinchcombe, J. C., E. Majorovits, G. Bossi, S. Fuller, G. M. Griffiths. 2006. Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443: 462-465. [Medline]
  28. McKenzie, G., G. Ward, Y. Stallwood, E. Briend, S. Papadia, A. Lennard, M. Turner, B. Champion, G. E. Hardingham. 2006. Cellular Notch responsiveness is defined by phosphoinositide 3-kinase-dependent signals. BMC Cell Biol. 7: 10[Medline]
  29. Hirata, H., S. Yoshiura, T. Ohtsuka, Y. Bessho, T. Harada, K. Yoshikawa, R. Kageyama. 2002. Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298: 840-843. [Abstract/Free Full Text]
  30. Kamakura, S., K. Oishi, T. Yoshimatsu, M. Nakafuku, N. Masuyama, Y. Gotoh. 2004. Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling. Nat. Cell Biol. 6: 547-554. [Medline]
  31. Androutsellis-Theotokis, A., R. R. Leker, F. Soldner, D. J. Hoeppner, R. Ravin, S. W. Poser, M. A. Rueger, S. K. Bae, R. Kittappa, R. D. McKay. 2006. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 442: 823-826. [Medline]
  32. Ciofani, M., J. C. Zuniga-Pflucker. 2005. Notch promotes survival of pre-T cells at the beta-selection checkpoint by regulating cellular metabolism. Nat. Immunol. 6: 881-888. [Medline]
  33. Andjelkovic, M., D. R. Alessi, R. Meier, A. Fernandez, N. J. Lamb, M. Frech, P. Cron, P. Cohen, J. M. Lucocq, B. A. Hemmings. 1997. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272: 31515-31524. [Abstract/Free Full Text]
  34. Minter, L. M., D. M. Turley, P. Das, H. M. Shin, I. Joshi, R. G. Lawlor, O. H. Cho, T. Palaga, S. Gottipati, J. C. Telfer, et al 2005. Inhibitors of {gamma}-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat. Immunol. 6: 680-688. [Medline]
  35. Lin, J., M. J. Miller, A. S. Shaw. 2005. The c-SMAC: sorting it all out (or in). J. Cell Biol. 170: 177-182. [Abstract/Free Full Text]
  36. Chakrabarti, R., C. Y. Jung, T. P. Lee, H. Liu, B. K. Mookerjee. 1994. Changes in glucose transport and transporter isoforms during the activation of human peripheral blood lymphocytes by phytohemagglutinin. J. Immunol. 152: 2660-2668. [Abstract]
  37. Swainson, L., S. Kinet, N. Manel, J. L. Battini, M. Sitbon, N. Taylor. 2005. Glucose transporter 1 expression identifies a population of cycling CD4+ CD8+ human thymocytes with high CXCR4-induced chemotaxis. Proc. Natl. Acad. Sci. USA 102: 12867-12872. [Abstract/Free Full Text]
  38. Varma, R., G. Campi, T. Yokosuka, T. Saito, M. L. Dustin. 2006. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25: 117-127. [Medline]
  39. Mossman, K. D., G. Campi, J. T. Groves, M. L. Dustin. 2005. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310: 1191-1193. [Abstract/Free Full Text]
  40. Kuhn, J. R., M. Poenie. 2002. Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing. Immunity 16: 111-121. [Medline]
  41. Atreya, R., J. Mudter, S. Finotto, J. Mullberg, T. Jostock, S. Wirtz, M. Schutz, B. Bartsch, M. Holtmann, C. Becker, et al 2000. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat. Med. 6: 583-588. [Medline]
  42. Takeda, K., T. Kaisho, N. Yoshida, J. Takeda, T. Kishimoto, S. Akira. 1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell-specific Stat3-deficient mice. J. Immunol. 161: 4652-4660. [Abstract/Free Full Text]
  43. Jehn, B. M., W. Bielke, W. S. Pear, B. A. Osborne. 1999. Cutting edge: protective effects of notch-1 on TCR-induced apoptosis. J. Immunol. 162: 635-638. [Abstract/Free Full Text]
  44. Eagar, T. N., Q. Tang, M. Wolfe, Y. He, W. S. Pear, J. A. Bluestone. 2004. Notch 1 signaling regulates peripheral T cell activation. Immunity 20: 407-415. [Medline]



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