text:
Article| February 04 2002
Differential T Cell Function and Fate in Lymph Node and Nonlymphoid Tissues
Nicola L. Harris,
Nicola L. Harris
Malaghan Institute of Medical Research, Wellington School of Medicine, 6002 Wellington, New Zealand
Victoria Watt,
Victoria Watt
Franca Ronchese,
Franca Ronchese
Graham Le Gros
Address correspondence to Nicola L. Harris, Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand. Phone: 64 4 389 5096; Fax: 64 4 389 5095; E-mail: nharris@malaghan.org.nz
Abbreviations used in this paper: BAL, broncho-alveolar lavage; BMDC, bone marrow dendritic cells; CFSE, carboxyl fluorescein succinimidyl ester; DC, dentritic cells; MCC88–103, moth cytochrome c fragment 88–103; med LN, mediastinal lymph node.
Received: September 07 2001
Revision Received: November 16 2001
Accepted: December 10 2001
https://doi.org/10.1084/jem.20011558
Nicola L. Harris, Victoria Watt, Franca Ronchese, Graham Le Gros; Differential T Cell Function and Fate in Lymph Node and Nonlymphoid Tissues . J Exp Med 4 February 2002; 195 (3): 317–326. doi: https://doi.org/10.1084/jem.20011558
The functions and fate of antigen-experienced T cells isolated from lymph node or nonlymphoid tissues were analyzed in a system involving adoptive transfer of in vitro–activated T cells into mice. Activated T cells present in the lymph nodes could be stimulated by antigen to divide, produce effector cytokines, and migrate to peripheral tissues. By contrast, activated T cells that had migrated into nonlymphoid tissues (lung and airway) produced substantial effector cytokines upon antigen challenge, but were completely unable to divide or migrate back to the lymph nodes. Therefore, activated T cells can undergo clonal expansion in the lymph node, but are recruited and retained as nondividing cells in nonlymphoid tissues. These distinct regulatory events in lymph node and nonlymphoid tissues reveal simple key mechanisms for both inducing and limiting T cell immunity.
The development of an effector T cell immune response involves a defined sequence of events. The first event involves the delivery of Ag from an exposed tissue site to the draining lymph node. This initiates the activation of naive T cells that constantly recirculate from blood to lymph node via specialized high-walled endothelium (1–3), and then back to the blood via lymphatic drainage. The final event that occurs is the differentiation and expansion of effector and memory T cells. These effector T cells express new cell surface molecules that allow them to recirculate back to nonlymphoid tissue sites (3). Here effector T cells influence with other leukocytes such as eosinophils, mast cells, neutrophils, and macrophages, to cause inflammation and to clear Ag.
It is clear that some effector T cells must be retained in the lymphoid organs to contact B cells and orchestrate humoral responses. However, what has not been delineated is the response of effector T cells at nonlymphoid tissue sites when they reencounter Ag. Do they proliferate again, return to the draining lymph node to proliferate, or simply carry out effector functions such as cytokine production? In this study we attempt to determine whether effector T cells present in nonlymphoid organs display similar functions to those effector T cells resident in lymph node, after contact with Ag. We also follow the fate of those cells that enter nonlymphoid tissues.
We generated populations of in vitro–activated T cells that expressed CD62Lmed-lo and CD62Lhi phenotypes, which, after adoptive transfer, localize to nonlymphoid tissue and lymph nodes, respectively. When adoptively transferred mice were challenged with Ag, the activated T cells in both the nonlymphoid tissues and lymph node produced effector cytokines within 2 h. Activated T cells in the lymph node divided extensively after Ag challenge. By contrast, activated T cells in nonlymphoid tissues, such as the lung and airway, could not be induced to divide in vivo under any circumstance, despite being able to divide to the same antigenic stimulus in vitro. Those activated T cells resident in the airway remained fully competent with respect to effector cytokine production, but were apparently unable to return to the draining lymph nodes nor to the circulating pool of lymphocytes.
All mice were bred and maintained at the Animal Facility of the Wellington School of Medicine (Wellington, New Zealand). B10.A mice were originally obtained from The Jackson Laboratory and maintained by brother x sister mating. The −I line 5C.C7 transgenic mice (4, 5) were backcrossed to B10.A mice and maintained by breeding transgenic males to B10.A females. The TCR used to generate the 5C.C7 transgenic strain was derived from the cytochrome c–specific T cell clone 5C.C7. All animal experimental procedures used in this study were approved by the Wellington School of Medicine Animal Ethics Committee, and performed in accordance with the guidelines set by the University of Otago (Dunedin, New Zealand).
T Cell Culture, Carboxyl Fluorescein Succinimidyl Ester Labeling, and Adoptive Transfer.
Naive T cells from 5C.C7 mice were purified from the lymph nodes by gradient centrifugation over 60 and 70% layered Percoll® (Amersham Pharmacia Biotech), followed by depletion of CD8+ T cells and B cells using Dynabeads® (Dynal). CD4+ T cells were incubated on anti-CD3 mAb (2C11, 10 μg/ml) coated six-well plates (Falcon; Becton Dickinson) in complete IMDM plus 20 U/ml recombinant human IL-2 (IL2L6 supernatant), 1,000 U/ml recombinant mouse IL-4 supernatant, 10 ng/ml recombinant human IL-6 and anti-CD28 mAb (1:50 dilution of 37.51 supernatant). Complete IMDM consisted of IMDM (Sigma-Aldrich) with 5% FCS (GIBCO BRL), 2 mM glutamine (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich) and 5 × 10−5 M 2-ME (Sigma-Aldrich). Cells were cultured for 5–6 d with media. Floating cells were removed every 2–3 d and replaced with fresh cIMDM + cytokines. At the end of the anti-CD3 culture, plate-bound cells were removed, washed extensively, and then cultured for another 3–4 d in cIMDM + 20 U/ml IL-2. After this rest period, the percentage of Vα11+Vβ3+ T cells was determined by FACS® analysis. Cells were labeled with carboxyl fluorescein succinimidyl ester (CFSE)* by incubation with 5 μM CFSE (Molecular Probes) for 8 min at room temperature in PBS, followed by quenching of the unlabeled CFSE with excess FCS and extensive washing. Between 5 × 106 and 2 × 107 CFSE+Vα11+Vβ3+ T cells were injected into the tail vein of sex-matched B10.A mice in a total volume of 500 μl IMDM, as indicated for each experiment. Alternatively 106 CFSE+Vα11+Vβ3+ T cells were administered intranasally to naive B10.A mice in a total volume of 30 μl IMDM.
Peptide and Cytokine Challenge.
At the stated times after adoptive transfer, recipient mice were anesthetized by injection of a mixture of Ketamine and Xylazine (Phoenix), and then challenged intranasally with 100 μg moth cytochrome c (MCC)88–103 in 50 μl PBS, or with PBS alone. Alternatively, 100 μg MCC88–103 in 200 μl alum adjuvant (SERVA) was injected into the peritoneum (i.p. challenge). For intranasal administration of IL-2, mice were given an inoculation of 1,000 U recombinant human IL-2 in 50 μl cIMDM, or 50 μl cIMDM alone.
Bone Marrow Dendritic Cells (BMDC) Culture and Administration.
Bone marrow cells from B10.A mice were cultured in six-well plates (Falcon) at 4 × 105 cells/ml in cIMDM containing 20 ng/ml IL-4 and 20 ng/ml GM-CSF, as described previously (6). Cultures were provided with fresh cIMDM + cytokines every 2 d and incubated at 37°C for a total of 7 d. BMDC (106 cells/ml) were loaded with MCC88–103 by incubation for 2 h at 37°C in cIMDM +16 μg/ml MCC88–103. Cells were washed three times with IMDM to remove excess Ag, and then 106 BMDC ± MCC88–103 administered intranasally to anesthetized mice in a total volume of 30 μl IMDM.
Lymphocyte Isolation.
Cell suspensions were made from lymph node or spleen, and RBC lysed by incubation for 5 min at 37°C in 3 ml ammonium-chloride-Tris buffer. Mice used for the preparation of lung lymphocytes were killed and the lungs perfused via the right ventricle of the heart with ∼5 ml PBS to remove circulating blood. Minced lung tissue was incubated for 1 h in cIMDM containing 2.4 μg/ml collagenase type II (GIBCO BRL) and 0.1% DNase 1 (Sigma-Aldrich), and mononuclear cells purified by gradient centrifugation over 30% Percoll®. When lung and airway cells were used for in vitro culture, macrophages were depleted by culturing cells on plastic dishes in cIMDM for 1–2 h at 37°C. Alternatively, lung or liver tissue was minced for 3 min in a Medimachine (Becton Dickinson) in IMDM containing 0.1% DNase 1, and mononuclear cells purified by gradient centrifugation over 30% Percoll®. Lymphocytes were isolated from peripheral blood by collecting 100 μl of tail blood into 1 ml Alsever's solution, followed by a 5–10-min incubation in 3 ml ammonium-chloride-Tris buffer to lyse RBC. Mononuclear cells were purified from airway by broncho-alveolar lavage (BAL). BAL was performed by flushing 3 × 1 ml PBS a total of five times each into the airway. Analysis of cellular infiltration into the airway was performed by BAL and differential cell counting as described previously (7). Intraperitoneal wash mononuclear cells were isolated by flushing 8 ml PBS three times into the peritoneal cavity.
Flow Cytometric Analysis.
FACS® analysis of lymphocytes was performed by staining as described (7). Anti-CD4 (GK1.5) was purified from hybridoma culture supernatant, and conjugated to biotin. Anti–Vα11-PE or Biotin, anti–Vβ3-Biotin, anti–CD44-PE, anti–3G-11-Biotin, anti–CD62L-PE, and Streptavidin-PerCP or -APC were all obtained from PharMingen.
Intracellular Cytokine Detection.
Lymphocytes were cultured for 2 h on uncoated, or anti-CD3 mAb (2C11, 10 μg/ml) coated, 24-well plates (Falcon) in cIMDM and anti-CD28 mAb (1/50 37.51 supernatant) at a concentration of 106 cells/well. Monensin (Calbiochem-Novabiochem) was added to the cultures at a final concentration of 2 μM and the incubation continued for 4 h. Alternatively, lymphocytes were collected directly into cIMDM containing 2 μM monensin, and then incubated at 37°C for 20 min without stimulation. At the end of the culture, cells were harvested, washed once in PBS buffer, and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. Fixed cells were washed and resuspended in FACS® buffer containing anti-Fc receptor mAb (24.G2). Indicated cells were then surface stained with Vα11-Biotin mAb followed by SA-PerCP (PharMingen). After washing to remove unbound antibody, cells were subjected to intracellular cytokine staining in saponin buffer (PBS, 10 mM Hepes, 0.1% saponin, 0.1% BSA, 0.02% Na azide) for 30 min at room temperature. Anti–IL-4–APC, –IL-5–PE, –IFN-γ–APC, and –IL-2–PE were all obtained from PharMingen and used at a 1:100 dilution.
Lymph Node and Nonlymphoid Tissues Select Distinct T Cell Populations after Adoptive Transfer of In Vitro Activated Vα11+Vβ3+ T Cells.
Activated T cells were prepared for adoptive transfer to B10.A recipients according to the following protocol. Naive CD4+ T cells from 5C.C7 mice were purified by gradient centrifugation over Percoll® and negative depletion using Dynabeads®. Between 50 to 70% of CD4+ T cells from 5C.C7 mice expressed both transgenic Vα11 and Vβ3 TCR chains specific for either pigeon cytochrome c fragment 81–104 and I-Ek, or MCC (8, 9). The remaining CD4+ T cells expressed the transgenic Vβ3 TCR chain along with an endogenously rearranged TCRα chain and did not respond to the MCC peptide (unpublished data).
The naive 5C.C7 CD4+ T cells were activated with plate-bound anti-CD3 and anti-CD28 mAb in the presence of IL-2, IL-6, and IL-4. Cells were cultured for 5–6 d, with floating cells removed every 2 d and replaced with fresh media plus additives. Cells were cultured for an additional 2–4 d in IL-2 alone. At the end of this period the large majority of harvested T cells were viable and expressed elevated levels of CD44 (Pgp-1), and medium to low levels of CD62L (L-selectin, Mel-14) (Fig. 1 A). These cells completely lacked expression of 3G-11 (Fig. 1 A), a marker that is lost after the activation of naive cells (10). If restimulated with anti-CD3, these T cells were found to be predominantly IL-4 producers (68%), whereas a smaller number produced IL-2 (33%), IL-5 (18%), and IFN-γ (19%) (unpublished data).
Before adoptive transfer, the activated T cells were labeled with CFSE (11) and injected intravenously into naive syngenic B10.A hosts. 5 d later, cell suspensions were prepared from lymph nodes, lung, and airway tissue of adoptive hosts. Airway cells were recovered by performing a broncho-alveolar lavage. Lung cells included all cells remaining in the airways after lavage, as well as cells resident in the lung tissue itself. Reisolated CFSE+Vα11+Vβ3+ T cells from each tissue were identified by fluorescent staining and examined for the expression of CD44, CD62L, and 3G-11 (Fig. 1 B). CFSE+Vα11+Vβ3+ T cells from the lymph nodes, lung, and airway of adoptive hosts all displayed an activated phenotype (CD44hi, 3G-11lo). Cells recovered from the lung contained two populations that were either CD62Lmed or CD62Llo, whereas the majority of cells recovered from the airway were CD62Llo. In contrast, CFSE+Vα11+Vβ3+ T cells recovered from the lymph nodes were almost all CD62Lhi.
Thus, adoptively transferred activated T cells appear to separate into distinct subpopulations that migrate to either lymph nodes or nonlymphoid tissues according to their expression of high or low levels of CD62L, respectively.
In Vivo Ag Challenge Induces Clonal Expansion of Adoptively Transferred Vα11+Vβ3+ T Cells Migrating to the Lymph Node, but Not Those Migrating to Nonlymphoid Tissue.
We compared the ability of previously activated Vα11+Vβ3+ T cells to divide after contact with Ag in a lymph node, versus contact in a nonlymphoid tissue environment. Mice were injected intravenously with in vitro–activated CFSE+ Vα11+Vβ3+ T cells and then challenged with 100 μg MCC88–103 via the intranasal route. Intranasal challenge is a reliable means of delivering either soluble Ag, infectious agents, or cells to the lower airways, and results in a specific T cell response in the airways, lung, and draining mediastinal lymph node (med LN)(12, 13). Cell division of the adoptively transferred CFSE+Vα11+Vβ3+ T cells at various host tissue sites was followed by FACS® analysis. Although in vitro–activated T cells do not label with CFSE in a sufficiently narrow peak to enable accurate quantitation of cell division, we were able to demonstrate that Vα11+Vβ3+ T cells in the draining med LN divided vigorously in response to MCC88–103 intranasal challenge (Fig. 2 A). Strikingly, Vα11+Vβ3+ T cells isolated from the airway had not divided, even by 90 h after intranasal Ag challenge (Fig. 2 A). Similarly, no division of Vα11+ Vβ3+ T cells from lung tissue was detected (unpublished data). It is important to note that a small proportion (∼0.8%) of T cells in naive B10.A hosts express the Vα11 and Vβ3 TCR chains. These cells are not specific for MCC and can be distinguished from the adoptively transferred cells because they are CFSE negative in the FACS® plots shown (Fig. 2 A).
The Ag-induced accumulation of Vα11+Vβ3+ T cells in the draining med LN and airway was also determined at various time points after intranasal challenge. A marked accumulation of Vα11+Vβ3+ T cells was observed in both the med LN and airway of MCC88–103 intranasally challenged, but not PBS challenged, mice (Fig. 2 B). These data indicate that the MCC88–103-induced Vα11+Vβ3+ T cell accumulation in the airway occurs solely as a result of cellular recruitment from the blood and lymph nodes, since T cells resident in the airway did not divide (Fig. 2 A). Analysis of the inflammatory exudate induced by intranasal MCC88–103 challenge also revealed that eosinophils had been recruited to the airway (Fig. 2 C), an event dependent on local IL-5 production by CD4+ T cells (7, 14, 15). This result indicated that airway Vα11+Vβ3+ T cells had recognized Ag and were producing IL-5. No airway eosinophilia or Vα11+Vβ3+ T cell accumulation was observed in B10.A mice that had been intranasally challenged with MCC88–103, and had not received in vitro–activated Vα11+Vβ3+ T cells (unpublished data).
To further compare the function of T cells encountering Ag in either the lymph nodes or lung and airway tissue, we analyzed in vivo cytokine production by adoptively transferred Vα11+Vβ3+ T cells after intranasal MCC88–103 challenge. Mice were given an intranasal challenge with 100 μg MCC88–103 2 d after receiving an adoptive transfer of in vitro–activated CFSE+Vα11+Vβ3+ T cells. 2 h after the intranasal challenge, lymphocytes were isolated from med LN, lung, and airway tissue. Once isolated, these cells were incubated for 20 min in monensin-containing medium (without stimulation) to inhibit the loss of any cytokines that were being synthesized in vivo at the time of tissue removal. At the end of the incubation period, cells were fixed and cytokine production analyzed by intracellular cytokine staining.
The data in Fig. 3 shows the proportion of CFSE+ T cells that could produce IL-2, IFN-γ, or IL-4 after the intranasal Ag challenge. CFSE+ T cells from either the med LN, or the lung and airway of MCC88–103 challenged mice, were able to produce IL-2, IFN-γ, and IL-4. Slightly more CFSE+ T cells from the lung and airways produced IL-2 as compared to CFSE+ T cells from the med LN after intranasal challenge with Ag (Fig. 3). In the experiment shown (Fig. 3), a greater proportion of CFSE+ T cells in the med LN appeared to be producing IL-4, as compared with lung and airway T cells. However, in other experiments this situation was reversed (unpublished data), indicating there is some background variation.
The percentage of CFSE+ T cells found to produce effector cytokines in these experiments was very low. We believe a much greater number of CFSE+ T cells are capable of producing effector cytokines in the lung, airway, and med LN, but that these are not detected by the intracellular cytokine staining protocol for ex vivo T cells. Indeed we found no IL-5 production in any of the experiments performed. This likely reflects a poor sensitivity of the intracellular cytokine staining protocol used, as the Ag-dependent eosinophilia indicated that significant IL-5 production was occurring in the airways. Our results contrast with those of Lee Reinhardt et al. (16) who reported that production of the effector cytokine IFN-γ is largely confined to the nonlymphoid tissues. However, these authors were investigating memory T cells, and the experimental protocol used involved the coadministration of LPS, which could have significant effects on T cell activation and recirculation patterns. Furthermore, our analysis of IL-4 expression supports our finding that effector cytokines can be produced in lymph node.
Taken together, our data demonstrates that previously activated T cell populations resident in nonlymphoid tissues, such as the lung and airway, do not divide and do not clonally expand when activated by Ag (Fig. 2 A). However, these T cells are not anergic as they are able to produce cytokines (Fig. 3) and initiate profound inflammatory cell exudates in the airway (Fig. 2 C). By contrast, previously activated T cells resident in the draining lymph node undergo rapid cell division after Ag challenge (Fig. 2 A). These lymph node–homing T cells also produce effector cytokines (Fig. 3).
Reisolated Lung and Airway Vα11+Vβ3+ T Cells Can Divide In Vitro.
We questioned whether the lack of detectable division by Ag-challenged T cells in the airway was an intrinsic property of those T cells. In vitro–activated CFSE+Vα11+Vβ3+ T cells were adoptively transferred into naive hosts, and then recovered from the peripheral lymph nodes, lung, and airway, without additional in vivo Ag challenge. These cells were then cultured with T cell–depleted splenocytes from naive B10.A mice ± 10 μg/ml MCC88–103. Cell division was detected by recording the changes in CFSE intensity. Reisolated CFSE+Vα11+Vβ3+ T cells from all tissues could divide in response to MCC88–103 stimulation in vitro (Fig. 4 A). In each of the experiments, previously activated T cells reisolated from the lung and airway were found to divide at a slightly faster rate than those reisolated from peripheral lymph nodes (Fig. 4 A and unpublished data). All the T cells from the lung and airways appeared to divide in vitro.
The ability of reisolated CFSE+Vα11+Vβ3+T cells to produce cytokines after reactivation in vitro was also examined. Lymphocytes were isolated from lymph nodes, lung, and airway, and stimulated for 6 h on plate-bound anti-CD3 plus anti-CD28 mAb. Monensin was added for the last 4 h of culture, after which cells were fixed in 4% paraformaldehyde. The production of IL-4, IL-5, IL-2, or IFN-γ by the reisolated CFSE+Vα11+Vβ3+ T cells was examined by intracellular cytokine staining. The data presented in Fig. 4 B demonstrate that CFSE+Vα11+Vβ3+ T cells from either the lymph nodes, or lung and airway, can produce IL-4, IFN-γ, and IL-2. For each of these cytokines a slightly larger proportion of CFSE+Vα11+Vβ3+ T cells from the lung and airway were cytokine positive, as compared with those from the lymph nodes (Fig. 4 B). Using this technique, IL-5 producing CFSE+Vα11+Vβ3+ T cells could only be detected in small numbers, and only in the lung (Fig. 4 B).
In summary, our data indicate that activated T cells present in the lung and airway are more sensitive to Ag stimulation in vitro (as measured by division and cytokine production) than those from the lymph nodes. Our findings also indicate that it is the microenvironment of the lung and airway that prevents T cell division in vivo (Fig. 2 A), since the inability to divide is not an intrinsic property of the T cells present at these sites.
Immunization With Peptide-loaded BMDC Cannot Induce Division of Airway Resident Vα11+Vβ3+ T Cells.
We considered it likely that the environmental factor limiting T cell division in the lung and airway was APC function. Although dendritic cells (DC) are considered to be the most potent lung APC (17, 18), numerous studies (19, 20) have shown that lung DC exhibit an immature phenotype with a low level of expression of MHC class II and costimulatory molecules. In our laboratory we routinely generate in vitro–cultured bone marrow DC (BMDC) that exhibit a mature phenotype, and express high levels of MHC class II and B7–1 (unpublished data). Lambrecht et al. and Havenith et al. have shown that DC delivered via the intratracheal route can activate naive T cells present in the med LN (13, 21). In addition, we found that CFSE-labeled BMDC delivered intranasally could later be isolated from the lung and med LN tissues, indicating their ability to establish in the airway and to further migrate to local lymph nodes (unpublished data).
To test whether the lack of T cell division in the lung and airway was due to poor APC function by airway resident DC, we challenged mice with in vitro–generated BMDC presenting MCC88–103. As shown in Fig. 5 A, Ag-loaded BMDC induced vigorous division of CFSE+ Vα11+Vβ3+ T cells in the med LN. However, no division of airway CFSE+Vα11+Vβ3+ T cells was observed at 50 or 66 h after intranasal challenge. It is important to note that at the 90-h time point we observed the appearance of CSFEdull Vα11+Vβ3+ T cells in the airway (Fig. 5 A). The level of CSFE staining in the 90-h airway population was comparable to that seen in the med LN at 66 h (Fig. 5 A). This suggested that the CSFEdull T cells present in the airway at 90 h had been recently recruited from the med LN, and were not resident airway T cells that had divided. Interestingly, the adoptively transferred CFSE+Vα11+Vβ3+ T cells had undergone several rounds of division in the med LN of the PBS-challenged mice at 66 h. Although this phenomenon did not occur consistently throughout the experiments, it suggests that in vitro–activated T cells may be capable of dividing in vivo in the absence of Ag. This may reflect the activity of homeostatic mechanisms operating to maintain memory T cells. Taken together, these data indicate that the lack of division observed for airway Vα11+Vβ3+ T cells in Fig. 2 A, cannot be overcome by Ag presentation on BMDC. This suggests that lack of cell division in the airway is not simply a result of reduced airway APC function.
To evaluate whether donor DC transferred Ag to host DC, a separate group of mice were challenged with ethanol-killed BMDC + MCC88–103. Ethanol-fixed BMDC expressed levels of MHC class II that were equivalent to that seen for live BMDC. The conditions used to fix the BMDC were adjusted to avoid the loss of peptide from MHC class II molecules. Intranasal delivery of live BMDC + MCC88–103 induced the accumulation of Vα11+Vβ3+ T cells in both the med LN and the airway (Fig. 5 B). It also led to the development of a profound airway eosinophilia (Fig. 5 C). These responses were not observed after intranasal administration of ethanol-killed BMDC + MCC88–103 (Fig. 5, B and C), indicating that MCC88–103 presentation occurs exclusively by the donor BMDCs.
Exogenous IL-2 Cannot Restore Division of Airway Resident Vα11+Vβ3+ T Cells.
The lack of division of T cells in the airway did not appear to be due to poor airway APC function. We speculated that it could instead be caused by limited local IL-2 production. We have shown in Fig. 3 that CFSE+Vα11+Vβ3+ T cells in the lymph node, lung, and airway, all produced similar levels of IL-2 after intranasal Ag challenge. Nevertheless, it was possible that bystander IL-2 production was confined to the lymph node environment. Therefore, to address the possibility of IL-2 deficiency in the lung environment, we delivered large doses of exogenous IL-2 to the airway of mice. The same mice were also given CFSE+Vα11+Vβ3+ T cells ± MCC88–103 via the intranasal route.
Intratracheal adoptive transfer of lung memory CD4+ T cells has already been shown by Hogan et al. (22) to result in a functional population of cells that remain in the airway for several weeks after transfer. In our experiments, intranasal adoptive transfer of in vitro–activated CFSE+ Vα11+Vβ3+ T cells resulted in a population of airway-resident T cells that were viable and could respond to intranasal-delivered Ag by producing IL-5 and generating an airway eosinophilia (Fig. 6 B). At day 4 after intranasal adoptive transfer, ∼20% of input CFSE+ T cells could be recovered from the airway by broncho-alveolar lavage (BAL). In addition, a significant population of viable CFSE+ T cells that were not isolated by BAL, could be identified in lung and airway tissue that had been subjected to collagenase digestion (Fig. 7 A).
Despite being able to generate an airway eosinophilia after MCC88–103 stimulation, CFSE+Vα11+Vβ3+ T cells administered to the airways remained undivided even 4 d after Ag challenge (Fig. 6 A). The addition of exogenous IL-2 to the airway was not found sufficient to restore cell division (Fig. 6 A). However, IL-2 resulted in a large increase in the number of airway eosinophils seen after MCC88–103 stimulation (Fig. 6 B). This indicates that the adoptively transferred T cells were capable of responding to IL-2 by producing increased amounts of IL-5. These data verify our earlier observations that previously activated T cells present in the airway do not divide, and indicate that this is not due to a lack of IL-2 production.
Airway Resident Vα11+Vβ3+ T Cells Do Not Return To the Circulation.
One of the surprising findings of this study was that while undivided, CFSEbright Vα11+Vβ3+ T cells were abundant in the airway at all time points, undivided cells were lost from the med LN by 50 h and did not return (Fig. 5 A). We hypothesized that T cells in the airway were unable to recirculate back to the draining lymph node, even when it was inflamed.
To test this hypothesis, mice were intranasally administered with in vitro–activated CFSE+Vα11+Vβ3+ T cells. This population contains T cells that are capable of migrating to the lymph node and nonlymphoid tissues after intravenous injection (Fig. 1). 4 d after intranasal transfer of CFSE+Vα11+Vβ3+ T cells, relevant tissues were examined for the presence of Vα11+Vβ3+ T cells, and the number of these cells that were CFSE+ determined. As expected from the previous data, all the Vα11+Vβ3+ T cells recovered by lavage from the airway of nonimmunized mice were CFSE+ (Fig. 7 A). The majority of Vα11+Vβ3+ T cells found in the lung were also CFSE+. In contrast, no Vα11+Vβ3+ T cells recovered from the draining med LN, mesenteric LN, or spleen, were CFSE+ (Fig. 7 A). Nor were any CFSE+Vα11+Vβ3+ T cells found in the liver, an organ that has been suggested to act as a "graveyard" for activated T cells (23).
We were concerned that CFSE+Vα11+Vβ3+ T cells that left the airway might have been distributed to systemic tissues in numbers below our threshold of detection. To address this possibility we challenged mice with MCC88–103 in alum adjuvant via the intraperitoneal route. Intraperitoneal immunization of naive or primed mice results in Ag drainage to the spleen (24), and an accumulation of Ag-specific T cells in both the peritoneum and spleen (unpublished data). Therefore, any CFSE+Vα11+Vβ3+ T cells present in the circulation after intranasal inoculation should be recruited to the peritoneum and spleen after intraperitoneal MCC88–103–alum challenge. At days 2 and 4 after intraperitoneal Ag challenge, we examined the spleen and peritoneal cavity of mice that had received an intranasal transfer of CFSE+Vα11+Vβ3+ T cells. No CFSE+ Vα11+Vβ3+ T cells were detected in either the spleen or the peritoneum at day 2, before any cell division would be expected to occur, or day 4, after intraperitoneal challenge with MCC88–103 in alum (Fig. 7 B).
In summary, these data indicate that adoptively transferred CFSE+Vα11+Vβ3+ T cells immigrating to the lung and airway are unable to recirculate to other tissue sites, even after specific Ag challenge. Together these data support our hypothesis that previously activated T cells resident in (or recruited to) the lung and airway are unable to return to the lymph node or recirculating pool.
Adoptively transferred CFSE-labeled in vitro–activated T cells were found to separate into distinct subpopulations based on their migration to lymph node, lung, or airway tissue. Although we have no understanding of what in vitro factors determine the commitment of naive T cells into each of the activated T cell subpopulations, their migration into lymph nodes correlated with higher levels of expression of CD62L. CD62L is known to be important for lymphocyte entry into lymph nodes (25), and acts as a ligand for MAdCAM-1 that is expressed on high-walled endothelium of the lymph node (26). Our subpopulations of activated T cells closely resemble the proposed TEM and TCM subpopulations of memory T cells proposed by Sallusto et al. (27), with respect to their expression of CD62L. This resemblance in terms of CD62L expression may be explained by the finding that TEM and TCM subpopulations of memory T cells also reside in nonlymphoid or lymphoid tissues, respectively (16, 28). CCR7, a chemokine receptor expressed by naive and central memory (TCM) T cells (27), also controls homing to secondary lymphoid tissues and may be expressed by T cells found to enter lymph node in our experiments.
We compared the functions of the adoptively transferred CFSE+ T cells that migrate to the lymph node, with CSFE+ T cells that migrate to nonlymphoid tissues such as the lung and airway. CFSE+ T cells present in either the lymph nodes, or the lung and airway, could all produce similar levels of cytokines after encountering MCC88–103 in vivo (Fig. 3). CFSE+ T cells in the med LN also divided vigorously in response to MCC88–103 (Fig. 2 A). In striking contrast, CFSE+ T cells in the lung and airway could not be induced to divide upon exposure to MCC88–103 (Fig. 2 A and unpublished data). However, CFSE+ T cells reisolated from either the lymph nodes, or the lung and airway, could divide extensively when restimulated with Ag during in vitro culture (Fig. 4 A). Therefore, the inability to divide is not an intrinsic property of T cells that go to the lung, but is likely to be due to the microenvironment of the lung and airway not being supportive for T cell division.
We endeavored to determine the reason for the lack of in vivo division observed for T cells in the lung and airway. To our surprise, MCC88–103 presentation by donor-mature BMDC could not induce CFSE+ T cells in the airway to divide (Fig. 5 A), although an IL-5–dependent airway eosinophilia was observed (Fig. 5 C). These data strongly indicate that it is not the poor T cell costimulatory function, nor inadequate Ag processing and presentation, of lung and airway APC that is responsible for the absence of T cell division and clonal expansion. Nor was this lack of division found to be the result of limited IL-2 production in the lung environment, since the addition of large amounts of IL-2 could not induce intranasally delivered CFSE+ T cells to divide (Fig. 6 A).
There are several reports that indicate that alveolar macrophages are able to suppress T cell division in vitro (for review see 29). These findings were also extended to lung immune responses (30). In two separate studies, it was demonstrated that alveolar macrophages could mediate this suppressive effect on proliferation through modification of the signaling properties of the IL-2 receptor (31, 32). This effect was found to be mediated by nitric oxide (33, 34). Our results extend these in vitro findings by reporting the key observation that the airway environment does not affect the production of effector cytokines. Although we have not resolved whether the absence of T cell division in the airway is mediated through nitric oxide, there are several other candidate mediators and cytokines, such as IL-10 and TGF-β, which could exert this effect. Alternatively, agents that induce production of pro-inflammatory cytokines, or that have mitogenic properties, may be required for the proliferation of lung and airway T cells. Lastly, factors (other than IL-2) that normally support T cell division in the lymph nodes may be absent from the lung and airway. We are currently investigating all these possibilities in an attempt to further define the mechanism by which the suppression of T cell proliferation in the lung and airway occurs. The question of the physiological significance of the inability of T cells to divide in the lung and airway raises some intriguing possibilities. An important consideration is that suppression of T cell division in peripheral tissues may serve to prevent potentially pathological T cell activation in response to harmless environmental Ag. Additionally, the development of cross-reactive autoimmunity by pathogen-activated T cells in response to sequestered tissue Ag would be avoided in tissue environments where division is prevented.
Our data indicates that activated T cells cannot divide in the lung or airway, although cells that have divided can enter these tissues after exiting the lymph node. We posed the question as to whether these airway-homing T cells can migrate from the airway back to the lymph node or general circulation. The data presented in Fig. 7 indicates that CFSE+ T cells delivered to the airway do not return to the recirculating pool. This failure to migrate out of the airway and lung tissue is likely to be due to an inability of these cells to move across epithelial barriers or into draining-afferent lymphatics. It is not due to the inability of the intranasally transferred cells to survive in the airway microenvironment, since these cells were shown to respond to Ag and induce an airway eosinophilia (Fig. 6 B). It is interesting to note that although intranasally administered, activated T cells cannot leave the airway, BMDC administered intranasally are capable of migrating to the draining med LN and inducing a response here (unpublished data and 13). Our data appears to contrast that of Mackay et al. who reported the presence of CD4+ T cells (35) exhibiting a memory phenotype (3) in popliteal-afferent lymph. We do not rule out the possibility that activated T cells leave other nonlymphoid tissue sites to enter afferent lymphatics and return to the lymph node. However, it would appear that this is not the fate of the large majority of activated T cells resident in the lung and airway.
In the context of devising vaccine strategies or immunotherapeutic interventions, one key implication of our work is that the extent and degree of T cell accumulation at an inflammatory site is tightly regulated by two distinct events. The first is the amplification of T cell number by expansion in the lymph node. The second involves recruitment of T cells to the site of Ag exposure. There is no apparent on-site amplification of the T cell response. In addition, we found that the cells that migrate to the airway are lost to this site, and fail to return to the recirculating pool. Thus, previously activated T cells that have entered the airway appear to be trapped, and must either remain in the airway as long-lived cells or be lost to the mucosal surface. This finding raises the possibility that resting memory T cells may suffer the same fate. The question as to whether memory T cells can recirculate, or whether they form two populations destined solely for lymphoid or nonlymphoid tissues, has important implications for our understanding of secondary immune responses and requires further investigation.
Our studies have focused on the lung, airway and draining mediastinal lymph node, and on an acute immune response. It remains to be determined whether the observations we have made are relevant to other nonlymphoid tissues. It would also be of considerable interest to investigate whether chronic infections and/or autoimmune diseases are subject to the same control mechanisms. Experimental evidence suggests that chronic inflammation involves ectopic formation of de novo lymphoid structures (for reviews see 36, 37) that may allow T cell division to occur. Formation of de novo lymphoid structures occurs in infectious diseases such as Helicobacter pylori–induced gastritis (38) and in autoimmune disorders including Hashimoto's thyroiditis (39), rheumatoid arthritis (40, 41), Sjogren's syndrome (42), and experimental murine diabetes (43). It is also not clear what physiological controls exist that prevent activated T cells from dividing in the lung and airway tissues. Elucidation of such mechanisms would have clear potential for the development of new therapeutics.
The authors thank the personnel of the animal facility of the Wellington School of Medicine for animal husbandry.
This work was supported by grants from the Marsden Fund, Royal Society of New Zealand, Wellington Medical Research Foundation, Health Research Council of New Zealand, and the Wellcome Trust.
Gowans, J.L., and E.J. Knight.
. The route of recirculation of lymphocytes in the rat.
Proc. R. Soc. Lond. B.
Picker, L.J., and E.C. Butcher.
. Physiological and molecular mechanisms of lymphocyte homing.
Annu. Rev. Immunol.
Mackay, C.R., W.L. Marston, and L. Dudler.
. Naive and memory T cells show distinct pathways of lymphocyte recirculation.
J. Exp. Med.
Koh, W.P., E. Chan, K. Scott, G. McCaughan, M. France, and B. Fazekas de St Groth.
. TCR-mediated involvement of CD4+ transgenic T cells in spontaneous inflammatory bowel disease in lymphopenic mice.
J. Immunol.
Seder, R.A., W.E. Paul, M.M. Davis, and B. Fazekas de St Groth.
. The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice.
Garrigan, K., P. Moroni-Rawson, C. McMurray, I. Hermans, N. Abernethy, J. Watson, and F. Ronchese.
. Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors.
Blood.
Harris, N., C. Campbell, G. Le Gros, and F. Ronchese.
. Blockade of CD28/B7 co-stimulation by mCTLA4-Hgamma1 inhibits antigen-induced lung eosinophilia but not Th2 cell development or recruitment in the lung.
Eur. J. Immunol.
Hansburg, D., T. Fairwell, R.H. Schwartz, and E. Appella.
. The T lymphocyte response to cytochrome c.
Fink, P.J., L.A. Matis, D.L. McElligott, M. Bookman, and S.M. Hedrick.
. Correlations between T-cell specificity and the structure of the antigen receptor.
Nature.
Ben-Sasson, S.Z., K. Makedonski, J. Hu-Li, and W.E. Paul.
. Survival and cytokine polarization of naive CD4(+) T cells in vitro is largely dependent on exogenous cytokines.
Lyons, A.B., and C.R. Parish.
. Determination of lymphocyte division by flow cytometry.
J. Immunol. Methods.
Harris, N.L., M. Prout, R.J. Peach, B. Fazekas de St. Groth, and F. Ronchese.
. CD80 costimulation is required for Th2 cell cytokine production but not for antigen-specific accumulation and migration into the lung.
Lambrecht, B.N., R.A. Pauwels, and B. Fazekas de St. Groth.
. Induction of rapid T cell acitvation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model.
Coffman, R.L., B.W. Seymour, S. Hudak, J. Jackson, and D. Rennick.
. Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice.
Science.
Foster, P.S., S.P. Hogan, A.J. Ramsey, K.I. Matthaei, and I.G. Young.
. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model.
Lee Reinhardt, R., A. Khoruts, R. Merica, T. Zell, and M.K. Jenkins.
. Visualizing the generation of memory CD4 T cells in the whole body.
Masten, B.J., J.L. Yates, A.M. Pollard Koga, and M.F. Lipscomb.
. Characterization of accessory molecules in murine lung dendritic cell function: roles for CD80, CD86, CD54, and CD40L.
Am. J. Respir. Cell. Mol. Biol.
Holt, P.G.
. Regulation of antigen-presenting cell function(s) in lung and airway tissues.
Eur. Respir. J.
Cochand, L., P. Isler, F. Songeon, and L.P. Nicod.
. Human lung dendritic cells have an immature phenotype with efficient mannose receptors.
Gonzalez-Juarrero, M., and I.M. Orme.
. Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis.
Infect. Immun.
Havenith, C.E., A.J. Breedijk, M.G. Betjes, W. Calame, R.H. Beelen, and E.C. Hoefsmit.
. T cell priming in situ by intratracheally instilled antigen-pulsed dendritic cells.
Hogan, R.J., W. Zhong, E.J. Usherwood, T. Cookenham, A.D. Roberts, and D.L. Woodland.
. Protection from respiratory virus infections can be mediated by antigen-specific CD4+ T cells that persist in the lungs.
Huang, L., G. Soldevila, M. Leeker, R. Flavell, and N. Crispe.
. The liver eliminates T cells undergoing antigen-triggered apoptosis in vivo.
Immunity.
Olin, T., and T. Saldeen.
. The lymphatic pathways from the peritoneal cavity: a lymphangiographic study in the rat.
Cancer Res.
Gallatin, W.M., I.R. Weissman, and E.C. Butcher.
. A cell-surface molecule involved in organ-specific homing of lymphocytes.
Streeter, P.R., B.T.N. Rouse, and E.C. Butcher.
. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.
Sallusto, F., D. Leing, R. Forster, M. Lipp, and A. Lanzavecchia.
. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.
Masopust, D., V. Vezys, A.L. Marzo, and L. Lefrancois.
. Preferential localization of effector memory cells in nonlymphoid tissue.
. Downregulation of immune responses in the lower respiratory tract: the role of alveolar macrophages.
Clin. Exp. Immunol.
Thepen, T., C. McMenamin, B. Girn, G. Kraal, and P. Holt.
. Regulation of IgE production in presensitised animals: in vivo elimination of alveolar macrophages preferentially increases IgE responses to inhaled allergen.
Clin. Exp. Allergy.
Strickland, D., U.R. Kees, and P.G. Holt.
. Regulation of T-cell activation in the lung - alveolar macrophages induce reversible T-cell anergy in vitro associated with inhibition of interleukin-2 receptor signal transduction.
Immunology.
Upham, J.W., D.H. Strickland, B.W.S. Robinson, and P.G. Holt.
. Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages.
Thorax.
Bingisser, R.M., P.A. Tilbrook, P.G. Holt, and U.R. Kees.
. Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/Stat5 signalling pathway.
Tulic, M.K., J.L. Wale, P.G. Holt, and P.D. Sly.
. Differential effects of nitric oxide synthase inhibitors in an in vivo allergic rat model.
Mackay, C.R., W.G. Kimpton, M.R. Brandon, and N.P. Cahill.
. Lymphocyte subsets show marked differences in their distribution between blood, and afferent and efferent lymph draining peripheral lymph nodes.
Hjelmstrom, P.
. Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines.
J. Leukoc. Biol.
Bachmann, M.F., and M. Kopf.
. On the role of the innate immunity in autoimmune disease.
–F50.
Mazzucchelli, L., A. Blaser, A. Kappeler, P. Scharli, J.A. Laissue, M. Baggiolini, and M. Uguccioni.
. BCA-1 is highly expressed in Helicobacter pylori-induced mucosa-associated lymphoid tissue and gastric lymphoma.
J. Clin. Invest.
–R54.
Soderstrom, N., and A. Biorklund.
. Organization of the invading lymphoid tissue in human lymphoid thyroiditis.
Scand. J. Immunol.
Iguchi, T., and M. Ziff.
. Electron microscopic study of rheumatoid synovial vasculature: Intimate relationship between tall endothelium and lymphoid aggregation.
Wagner, U.G., P.J. Kurtin, A. Wahner, M. Bracketz, D.J. Berry, J.J. Goronzy, and C.M. Weyand.
. The role of CD8+CD40L+ T cells in the formation of germinal centre's in rheumatoid synovitis.
Aziz, K.E., P.J. McCluskey, A. Montanaro, and D. Wakefield.
. Vascular endothelium and lymphocyte adhesion molecules in minor salivary glands of patients with Sjogren's syndrome.
J. Clin. Lab. Immunol.
Ludewig, B., B. Odermatt, S. Landmann, H. Hengartner, and R. Zinkernagel.
. Dendritic cells induce autoimmune diabetes and maintain disease via de novo formation of local lymphoid tissue.
related&metrics
Related & Metrics
View large Download slide
Activation marker expression by pre- and postadoptively transferred Vα11+Vβ3+ T cells. (A) Activated T cells were generated in vitro, and then examined for expression of CD44, 3G-11 and CD62L. Profiles shown are for live Vα11+Vβ3+ T cells. Activation marker expression on naive peripheral blood Vα11+Vβ3+ T cells from 5C.C7 mice is shown as a control. (B) In vitro–activated T cells were CFSE labeled, and 2 × 107 CFSE+Vα11+Vβ3+ T cells injected into the tail vein of naive syngenic B10.A hosts. 2–5 d later, lymphocytes were purified from the peripheral lymph nodes, lung, and airway, and their expression of CD44, 3G-11, and CD62L examined. Profiles shown are for live CFSE+ Vα11+Vβ3+ T cells. Numbers reflect the percentage of cells that fall within the shown boundaries. The data shown are from one representative experiment using six to eight mice per group. Similar results were obtained from three separate experiments.
In vivo accumulation and division of activated Vα11+Vβ3+ T cells in the med LN and airway of mice challenged intranasally with MCC88–103. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 5 × 106 CFSE+Vα11+Vβ3+ T cells injected into the tail vein of naive syngenic B10.A hosts. On that day mice were challenged intranasally with PBS or MCC88–103. Responses were measured at indicated time points after intranasal challenge. (A) Cell division was examined by FACS® analysis of CFSE intensity. Profiles shown are for live Vα11+Vβ3+ T cells. (B) Accumulation of live Vα11+Vβ3+ T cells in the med LN and airway of intranasally challenged mice was examined by FACS®. (C) Eosinophil infiltration into the airway of intranasally challenged mice was examined by BAL and differential cell counting of Giemsa-stained cytospins. The data shown represents mean ± SE of two to three mice per group from one representative experiment. Similar results were obtained from three separate experiments.
In vivo cytokine production by activated Vα11+Vβ3+ T cells in the med LN or lung and airway, after intranasal challenge with MCC88–103. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 7.5 × 106 CFSE+Vα11+Vβ3+ T cells injected into the tail vein of naive syngenic B10.A hosts. The next day mice were challenged intranasally with MCC88–103 or PBS. 2 h later mice were killed and lymphocytes from the med LN, or lung and airway, were incubated for 20 min in cIMDM containing 2 μM monensin. T cells were then fixed and examined for expression of IL-4, IL-2, or IFN-γ using intracellular cytokine staining. Profiles shown are for CFSE+ T cells. The data shown represents mean ± SE of three mice per group from one representative experiment. Similar results were obtained from three separate experiments.
In vitro division and cytokine production by activated Vα11+Vβ3+ T cells reisolated from the lymph nodes, or lung and airway. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 2 × 107 CFSE+Vα11+Vβ3+ T cells injected into the tail vein of naive syngenic B10.A hosts. 2–5 d later lymphocytes were purified from the peripheral lymph nodes, and lung and airway. (A) 5 × 105 lymph node, or lung and airway, cells were cultured with 1.5 × 106 T cell–depleted splenocytes from naive B10.A mice ± 10 μg/ml MCC88–103. After 48 h of culture, T cell division was examined by FACS® analysis of CFSE intensity. Profiles shown are for live Vα11+Vβ3+ T cells. (B) Peripheral lymph node, or lung and airway, T cells were restimulated with anti-CD3 and anti-CD28 in the presence of 2 μM monensin for 6 h. T cells were then examined for expression of IL-4, IL-5, IL-2, or IFN-γ using intracellular cytokine staining. Profiles shown are for CFSE+Vα11+Vβ3+ T cells. In both A and B, lung represents lung and airway cells. The data shown are from representative experiments, using six to eight mice per group. Each experiment was repeated two to three times with similar results.
Effect of intranasal challenge with BMDC + MCC88–103 on division of adoptively transferred Vα11+Vβ3+ T cells. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 5 × 106 CFSE+Vα11+Vβ3+ T cells injected into the tail vein of naive syngenic B10.A hosts. On that day mice were challenged intranasally with 106 ethanol-killed or live BMDC ± MCC88–103. Responses were measured at indicated time points after intranasal challenge. (A) The extent of cell division was examined by FACS® analysis of CFSE intensity. Profiles shown are for live Vα11+Vβ3+ T cells (B) Accumulation of live Vα11+Vβ3+ T cells in the med LN or airway of intranasally challenged mice was examined by FACS®. (C) Eosinophil infiltration into the airway of intranasally challenged mice was enumerated by BAL and differential cell counting of Giemsa-stained cytospins. The data shown represent mean ± SE of three to four mice per group from one representative experiment. Similar results were obtained in two to four separate experiments.
In vivo division of intranasally administered, activated Vα11+Vβ31 T cells, after intranasal challenge with MCC88–103 ± IL-2. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 106 CFSE+Vα11+Vβ3+ T cells administered intranasally to naive syngenic B10.A hosts. On that day mice were challenged intranasally with PBS or MCC88–103. At days 0 and 2 after challenge, mice were additionally treated with an intranasal inoculation of 500 U rhIL-2 in cIMDM, or cIMDM alone. (A) On day 4 after MCC88–103 intranasal challenge, cell division was examined by FACS® analysis of CFSE intensity. Profiles shown are for live Vα11+Vβ3+ T cells. (B) Accumulation of eosinophils in the airway 4 d after MCC88–103 intranasal challenge was determined by BAL and differential cell counting of Giemsa-stained cytospins. The data shown represents the mean ± SE of three to four mice from one representative experiment. Similar results were obtained from two to four separate experiments.
Migration of intranasally administered, activated Vα11+ Vβ3+ T cells. Activated Vα11+Vβ3+ T cells were generated in vitro, CFSE labeled, and 106 CFSE+Vα11+Vβ3+ T cells administered intranasally to naive syngenic B10.A hosts. (A) 4 d later lymphocytes were isolated from the airway, lung, med LN, mes LN, liver, and spleen. (B) Alternatively, recipient mice were immunized intraperitoneally with 100 μg MCC88–103 in 200 μl alum adjuvant, and lymphocytes isolated from the airway, spleen, and peritoneal cavity at days two and four after immunization. Profiles shown are for live Vα11+Vβ3+ T cells. The data shown are from representative experiments, using three mice per group. Each experiment was repeated two to four times with similar results.
Lipopolysaccharide-enhanced, Toll-like Receptor 4–dependent T Helper Cell Type 2 Responses to Inhaled Antigen
Asymmetric Division and Lineage Commitment at the Level of Hematopoietic Stem Cells : Inference from Differentiation in Daughter Cell and Granddaughter Cell Pairs
Blockade of Inflammation and Airway Hyperresponsiveness in Immune-sensitized Mice by Dominant-Negative Phosphoinositide 3-Kinase–TAT


text:
background/reports the/gp/chronicles top/stories
background/report: T.W.O Automatic/Date watches from 1957
In the 1950s, if you wanted to feel the pulse of the time and the market, as a watch manufacturer, you had to have an automatic watch with date window in your collection. It was really in the 50s, when this took off. Nowadays, we admire simple dials without date and even manual wound calibers, but in those days, automatic movements and a date function were the latest fashion.
So it is no surprise that houses like Girard-Perregaux and Jaeger-LeCoultre jumped on that train. By no means were those the first, we have known date complications for much longer and also automatically winding wristwatches but as said, in the mid 50s, they really became the sought after complication everyone wanted to have. So we can look at T.W.O early examples from Girard-Perregaux and Jaeger-LeCoultre.
The Girard-Perregaux Ref 7317 houses the Gyromatic caliber 22 with 39 Jewels, marketed at that time as a breakthrough technology as the Gyrotron design allowed for bi-directional winding. The Jaeger-LeCoultre E 329 on the other hand housed a bumper automatic movement, Cal. 813. There are a lot of similarities but also differences.
Both watches have put some emphasis on the date, GP using a blue print for the date rather than the usual black and JLC decided on a Roulette date with alternating black and white dates. Hop over to the Blomman Watch Report for more details on the JLC E 329. It is probably debatable if the full-rotor winding or the bumper winding is better or more modern. Obviously, the full rotor design has ultimately won the market share, but the bumper winding is also very efficient and provides for that very special feel on the wrist, certainly the romantic choice.
Both cases are similar in size, 34.5mm for the JLC and around 33.5-34 for the GP. Lugs are integrated into the case and while the JLC has a more pronounced bezel, the GP uses some case design tricks to let more light into the dial. Lug width is 17mm. On the GP the lug size should be 18mm, but I fitted a 17mm strap which just seems to fit better due to the tapering lugs.
Tagsbackground/reports • gyromatic • top/stories • watches/not/gp

redpajama_set_name: RedPajamaCommonCrawl


text:
By Mary Ellen Hayward. Baltimore: The Johns Hopkins University Press, 2008; 307 pp., illustrations; cloth, $45.00.
New York built tenements, walk-up apartments of several stories, with no water and few windows in the inner walls. The conditions in these tenements became scandalous via writers like Jacob Riis in the late 19th century.
Baltimore builders learned to build two- or three-story row houses on the main streets, and tiny row houses on the back streets or alleys. In early Philadelphia and Baltimore, builders erected diminutive two-story versions of the grand Federal houses built for wealthier clients. Often a block was subdivided with the larger houses facing outward and an alley running through the middle with more modest dwellings facing it. By laying out blocks with an alley street down the middle, developers could produce more house lots. Some of the earliest alley dwellings had no backyards but abutted the houses built behind them.
In communities where all but the very wealthy had to walk to work, housing was required for every income level within the same geographic area. Substantial merchants and businessmen could afford large three-story houses on major streets. Shopkeepers, craftsmen, and artisans lived in two-story houses nearby. The laboring classes who did the physical work of the community also had to live nearby, occupying more modest houses, which they rented or sometimes were able to buy. This arrangement continued until the advent of the streetcars in the latter 19th century, when workers were able to live further away from their jobs.
Later alley dwellings had small backyards to accommodate a privy. These small working-class row houses accommodated every wave of emigrant that landed in Baltimore, a large native African American population, and African American emigrants coming up from the south. Large numbers of Irish, German, Bohemian, Polish, Jewish, Italian, and Greek immigrants came through Baltimore. Many of them stayed on to found vibrant ethnic neighborhoods, some of which remain to this day. Poor immigrants occupying back alley houses, through thrift saving clubs, were able to make small down payments on purchasing a row house. As people from these groups moved on to more ample housing, newly arrived immigrants took up their places in old Baltimore neighborhoods. Many neighborhoods revolved around ethnic churches, which had a priest, rabbi, or minister who spoke the native language. Extolling the Baltimore row house, George Washington Howard wrote in 1873, in The Monumental City: Its Past History and Present Resources about Baltimore's "utter absence of tenement-houses, with their squalid wretchedness," where "the humblest mechanic or laborer can ensconce his family in a modest dwelling and surround them with the pleasures and comforts of home."(1)
The Irish arrived in large number to Baltimore during the Potato Famine in 1846-50. Ships would arrive in the Baltimore Harbor with many passengers who had died in the Atlantic Passage. Local Irish helped the famine victims find housing near Union Square in west Baltimore. Luckily, the Baltimore and Ohio Railroad was developing great guns at that time, and many of the Irish found work digging the roadbed and laying track for the railroad to Cumberland, Maryland and westward. Many of these Irish laborers followed the railroad westward with their families. Many others stayed in Baltimore.
Germans came after the failure of the 1848 German Democratic movement. They established singing societies, bakeries, breweries, and piano factories, adding to the commercial and cultural life of the city. Many Bohemians who came had a high school education and were skilled woodworkers, carvers, cabinetmakers, or tailors. A Bohemian builder, Frank Novak developed the classic Baltimore row house in the early 20th century, with marble basement and marble steps. German Jewish businessmen built grand row houses along Eutaw Place with magnificent new synagogues. The Italians settled in an area near Fells Point still known as Little Italy.
Well-to-do African Americans, often descended from Maryland freedmen, occupied a fashionable neighborhood in the Druid Hill Park area, moving in after German Jewish settlers, who were denied residence in other white neighborhoods. Baltimore neighborhoods became "red lined" restricting Jewish and African Americans from buying property. Blacks had always lived in the Fells Point and Federal Hill neighborhoods, sharing both principle street and alley dwellings with white residents. The influx of southern blacks after the Civil War swelled these neighborhoods. A "Great Migration" of blacks from the South continued in the years between the World Wars, filling up old neighborhoods and alley dwellings in central Baltimore. A residential housing city ordinance enacted after the Civil War, disallowed blacks from moving into blocks that were 50 percent white. The U.S. Supreme Court overturned this ordinance in 1917, but de facto segregation continued.
Early 20th-century reformers addressed the alley dwellings lacking sanitation facilities, as the city gradually moved to provide sewers and indoor toilet and bathing facilities.
Eastern Europeans, Poles, Czechs, Italians, and Russian Jews arrived to swell the ethic mix and rich diversity of Baltimore neighborhoods. The newcomers often occupied the cheapest alley dwellings vacated by the upwardly mobile previous occupants. The canneries east of Fells Point provided a source of employment for unskilled newly arrived workers. Slum clearance became a preoccupation, and Poe Homes, Baltimore's first public housing project with garden style apartments was built 1940-1941.
In 1967, fighting a new interstate highway that would be built across historic waterfront neighborhood Fells Point and Federal Hill, preservationists rallied to nominate both neighborhoods to a new National Register of Historic Places. The famous dollar house program that followed allowed homesteaders and gentrifiers to renovate waterfront neighborhoods with alley dwellings as well. Very soon alley dwellings and larger homes on surrounding streets, vacated by the city's poor, became chic new homes for young white urban pioneers who desired to live downtown close to their workplaces. While some inner-city neighborhoods have been renovated, several outlying neighborhoods remain unrestored and derelict, creating a challenge to preserve a huge housing stock of Baltimore row houses for all classes. Substantially built, they have provided housing for generations of Baltimoreans.
With meticulous research, Mary Ellen Hayward has re-created the history of working-class Baltimore and the waves of its ethnic emigrants and citizens by studying row houses and their neighborhoods. The book is an extraordinarily focused lens into 19th—and early 20th—century immigration patterns into America, with a rich social history included of the lives and fortunes of all the groups who arrived and struggled to establish themselves, with many ultimately providing themselves and their families a good living and solid housing, if constricted by street entrances, party walls, and limited light and ventilation, yet with quick and easy access to work, markets, church, and temple.
Gary Scott
1. George W. Howard, The Monumental City: Its Past History and Present Resources (Baltimore: J.D. Ehlers & Co., 1873), 35,44, cited on p. 144.


The aim of our scheme is to provide both, Spanish and English children, with a memorable experience. The Spanish children have the opportunity to be immersed in the daily life of a British family in order to practice and improve the language, the knowledge of the culture and a love for the country, whilst the English benefit from so many values learnt through such experience like sharing, caring, empathy, tolerance, patience, generosity, cooperation, communication skills, being a role model, teaching, and so on.
We match the Spanish children and the British families very carefully to make sure they have features in common, such as personalities, style, hobbies,... in order to maximize the possibilities of bonding.
We therefore look for "caring" families with children anywhere in the south of England, who would like to host a Spanish child from June 23rd to July 19th, 2018. A very generous allowance is offered to cover all expenses, and more.
Since 1986 we have been organizing these trips, and we are very proud to say that in general terms they have been extremely successful. Past host families agree with us that it is a fabulous experience for all those involved. We often receive the most gratifying feedback from the families and the participating schools. The children have fun together and all the family benefits from the experience. It allows them to make a Spanish friend, many times turning into a long-life friendship.
Any caring family with children between the ages of 12 and 16 can potentially be a host family for us. Having a spare room for the student is not an essential request, but if sharing, it would have to be with the child of the family, who would be taking the role of the "host brother or sister".
The Spanish child should be made welcome to the same school the host child attends. Most schools in the area are happy to support our programme, but unfortunately a few of them do not. They either cannot see the advantages this experience can add to their pupils' education or they have particular circumstances that do not help. If that was the case with your son or daughters school you could still host if we found a local school the Spanish child could attend with a friend, neighbour or relative of the family.
Click here to see a list of participating schools. If you cannot find your school on the list, please get in touch as it well may be that we have never had dealings with them.
All members of the family over 18 will be requested to be DBS checked.


Age Appropriate Viewing Alternatives for Your Tweens.
As a Netflix Streamteam member I received special perks for my participation. Opinions are all mine.
It's hard to say no to your kids when they ask to watch the "popular shows", especially when "all their friends get to watch them" (which may very well be true). But the truth is many of these shows are made for an adult audience, and although our kids think they are adults, they really aren't. Navigating the rating systems can be hard, especially when some episodes might be okay, while others contain content that's definitely not age appropriate for kids.
This becomes especially problematic for that challenging pre-high school tween age group. I'm not suggesting kids should live in a bubble or not watch shows depicting some of the very challenging issues tweens and teens face today. I'm suggesting that how those issues are presented may not be appropriate for viewing on their own without accompanying conversations about issues that help to put things into context and perspective. Since we can't be there with our kids all the time, what can they watch that doesn't require supervision or conversations?
In other words ... if not that, then what? I have watched, streamed, and hunted high and low for suggestions about popular themes that are age appropriate to help bridge the gap for you. Having said that I always recommend watching the shows yourself so you can decide if the content is appropriate for you and your child.
so not all may be appropriate for you and your child.
Just a reminder to parents: Kids are naturally drawn to animated shows and cartoons, but many are not age appropriate and contain mature content, for example Family Guy (TV-MA). It is important that you look at the content and the ratings prior to letting your kids watch them. What is acceptable for one parent may not be for another. Keep an eye out for a followup post with suggestions for animated shows and cartoon alternatives appropriate for tweens in the near future.
You can get social with Netflix on twitter and catch all the Streamteam adventures by following the #Streamteam hashtag.
Yep, just me Cathy thinking out loud about streaming alternative for tweens.


Adrift in a sea of digital apps for every imaginable function, we often feel our needs are met better today than in any previous era.
Previous Post Epic and private.
Next Post #16: Red Hat Summit, Day 3: Major Hayden lives!


I've seen how rigid compute platforms don't work, and I've also seen how they do. One of our partners, for example, provides software libraries for our IoT edge Gateways. The software speaks many industrial protocols and interfaces to dozens of PLCs and automation devices. It makes it possible for switching hardware to be used as data protocol translators, helping liberate data from those silos.
With all these new devices and sensors coming online and getting put on a common network, we need a way to keep them all monitored and updated. Most current management structures aren't going to be able to handle this.
We're going to need new products to manage this network infrastructure. Our Industrial Network Director and our Fog Director are two unique solutions. They will make it possible to manage the devices and to perform statistical analysis at the edge of the network to slim down the data sets sent on for further business analysis and archiving.
There is a massive risk to putting industrial equipment on a network shared with IT and connected to the global Internet: IT will also be free to share all its malware and hacker attacks. Unfortunately, the practice of keeping the industrial network safe by simply keeping it isolated is coming to an end.
IT personnel are accustomed to large attack surfaces. It's a constant game of cat and mouse in which, for the most part, the good guys manage to keep slightly ahead. But the tools that IT uses to protect data networks don't always apply to industrial systems. You can't just take one machine offline at a random time to patch its operating system, for example – no matter how critical the security issue.
I listened to several concerned industrial experts and customers at Hannover who wanted to learn from Cisco's IT leaders about factory floor security and how to better protect connected operational environments. We've been learning from industrial leaders for years and our current products bring together the best security practices from both sides.
Industrial technology projects are driven by business, not back-office needs. Everything we do with data on the shop floor is aimed at directly improving product quality, boosting manufacturing speed, lowering direct cost, or some other process that directly goes to the bottom line. Operational necessity is the driver of our digital transformation.
While industry has been hardening its machine-to-machine networking to exist in physically punishing environments, IT has built systems that can process massive amounts of information and (for the most part) stay secure and globally connected at the same time. Merging the disciplines is difficult, but both groups have a lot to learn from each other and the new combined industrial/IT networks will be much stronger for it, and more powerful for businesses, too.


Getting a timeshare means you get just that a share in time. When you get the timeshare then this is something that will aid you to get the frequency inside a particular resort. However, after someone time you might think that this is not a reasonable business and if this is the case, then you should get sell the shares.
In the case you are going to enjoy the gains then the selling of these shares have to be done right. The best thing is that you should get all the help you need. Start by understanding the shares and how to make the sales.
Some of the pointers to put in mind is that you should get an agent who will aid you. When you are dealing with such matters then you need to warrant you have chosen the right professional. the another pointer is that you ought to figure out if they can deal with this venture. Find them and discuss the products you are selling is that you have been able to hear their opinion. In case they say they can be able to deal with the sale, some of the pointers to put in mind is that they will be in safe hands.
the agent will take care of the investment needs you have as well as guarantee you do not end p getting work up. They understand that the reason you are making the sale is that you need to make money and this being the case, they strive to warrant you have gotten it within no time.
The another pointer is that when you are doing this, then the agent will aid with the documentation. The thing is that when you are dealing with such matters, then you have to note on your own you might have the challenge to get out of the timeshare. Thus rather than deal with the frustration on your own, then you should note that the agent will be able to get rid of the timeshare.
Since you want to get the best outcome you should take your time in choosing the right agent. When doing this, some of the pointers is they should have done the job for a long time. The thing is that it is paramount for them to be well connected in their line of job. The ting is that if they are connected, then you can be certain you will end up selling the share within no time.
The much they need for this job is another pointer to put in mind. This might be one of the difficult things to do and this is the reason you should get an agent who will aid you. This is why you should not get into this on your own.


text:
Home All Destinations Turkey Itinerary
Turkey Yacht Charter Itinerary
Explore the emerald bays and charming coastal towns on Turkey
Turkey is a beautiful and dynamic country with lively coastal towns, world-famous bazaars, verdant pine forests, and a wealth of archeological sites. Its coast is lined with historic ruins, sandy beaches, quaint fishing villages, hidden coves, and sea caves.
Here is a sample itinerary for an alluring Turkey yacht charter:
Day 1: Bodrum
Bodrum is an enchanting town with a Bohemian vibe owed in part to the dissident artists and writers who were exiled to the area. The castle of St. Peter, built by the Crusaders, is a rich archaeological site and houses the Hall of Underwater Archeology that displays finds from shipwrecks along the coast. Bodrum is also home to one of the Seven Wonders of the Ancient World, the Mausoleum at Halicarnassus, with its pyramid and marble columns. Other impressive sites include the Bodrum Amphitheater and the white windmills. On the east side of the city is the marina and yacht club surrounded by boutiques, clubs, and upscale restaurants.
Day 2: Datca
The seaside village of Datca is home to the ancient port of Knidos, a clear water cove located at the tip of the Doric Peninsula. Known as a center of art and culture in the 4th Century BC, you can wander its terraced streets while exploring the city walls, theaters, and the remains of several temples including the Temple of Aphrodite. There are two harbours where you can drop anchor before spending some time ashore.
Day 3: Symi
Symi is a Greek island known for its harbor town surrounded by beautiful mountains, the beaches, and its historical significance. Once a hub for the shipbuilding and sponge industries, tourism is now its main focus. Some of the sites you'll want to see include The Monastery of the Archangel Michael Panormitis, which is still in use today, the municipal clock tower, and the Kastro, built by the Knights of St. John as part of a Byzantine castle. Symi hosts a festival each year between July and September, where you can experience open air concerts by Greek musicians, as well as dance and theater performances.
Day 4: Marmaris
Marmaris is known as one of Turkey's most beautiful towns and is considered the yachting capital of the country. Located where the Aegean and Mediterranean Seas meet, it has secluded coves, sandy beaches, and lush evergreen forests. It's a popular spot for snorkeling and swimming the azure waters. Visitors can explore ancient ruins, venture inland to discover tiny villages and impressive waterfalls, or wander the many weaving alleyways of the colourful bazaar. Marmaris offers dynamic nightlife, complete with restaurants and bars. In fact, Bar Street is where you have to stop if you want to dance until sunrise.
Day 5: Ekincik
Ekincik is a charming village in itself, but it's also the best base for exploring the Dalyan River, the ancient ruins of Kaunos, and the Lycian tombs. Not far from the city center is the Sultaniye Thermal Baths where you can luxuriate in the natural hot springs. The Beach of Iztuzu is nearby, famous for Caretta Caretta sea turtles, also known as loggerhead turtles.
Day 6: Oludeniz and Gemiler Island
Oludeniz, also known as the Blue Lagoon, is a small village and beach resort located on the southwestern Turquoise coast. One of the most photographed beaches on the Mediterranean and ranked one of the top beaches in the world, Oludeniz is known for its pebbled shores and glistening aquamarine waters. The water is crystal clear, making it perfect for scuba diving or snorkeling. The area is also known as one of the best places in the world for paragliding from Mount Babadag which towers above the beach.
Nearby is Gemiler Island, located just off the coast of Turkey near the city of Fethiye. Anchor at its crescent-shaped bay and explore the ruins of several churches built between the 4th and 6th Centuries AD. Archeologists believe this is where the original tomb of St. Nicholas was located.
Day 7: Gulf of Fethiye
The Gulf of Fethiye holds many places to explore. The port town of Fethiye has a lagoon, marina, Roman amphitheater, and the Lycian tombs in the surrounding hills. Manastir Bay, also known as Cleopatra's Baths or Sunken Bath Bay, holds the legendary baths once used by Cleopatra. The ruins are semi-submerged, and you can explore them either by tender or by snorkeling.
The island of Tersane, once known as Telandria, was the dockyard of the Ottoman fleet during World War I. Once inhabited, you can see the remains of houses and churches. The people who once lived on the island dealt in livestock and fruit; in fact, fruit is still grown here to this day, and you may come across herds of goat.
Day 8: Gocek
Gocek and its lively port is considered by many yachters as one of the best sailing destinations in the Mediterranean. Located at the foot of pine-fringed mountains and overlooking Gocek Bay, this spot is famous for lavish resorts, exquisite restaurants, unique shops, and vivacious bars. The weather is beautiful, and the natural surroundings are breathtaking. Stop in one of the secluded anchorages and spend the afternoon enjoying watersports in the clear, warm water, and then sunbathe on a sandy beach. At night, enjoy a delicious meal followed by some music or dancing.
Featured Turkey Yacht Charters
€115,000 /week
Barracuda Red Sea
121.5ft | Sunseeker
Geosand
138ft | Baglietto
My Toy
140ft | AB Yachts
Rebecca V
84.9ft | Benetti
View All Turkey Yacht Charters


Abstract- This paper examines the average length of a spell of unemployment in the Irish Republic. The analysis is conducted using two measures, one emphasising the flow of persons into unemployment and the other concentrating on the stock of currently unemployed individuals. These measures imply long average durations for the unemployed in Ireland both in absolute terms and relative to their British counterparts. Some of the traditional structural aspects of Irish unemployment, i.e., the high proportion of unskilled, elderly or regionally disadvantaged persons among the unemployed, provide a partial explanation of these long spell lengths.


Why Do We Eat King Cakes During Mardi Gras?
We are in the thick of Mardi Gras season here in south Louisiana. There's already been a ton of Mardi Gras balls and the chicken runs and parades will commence in a matter of days.
For a lot of us, the happiest part of the season is scarfing down on those delicious and decedant treats known as King Cakes.
Sometimes when I have friends visiting from out-of-town I have to stop and think when they ask the question, "Why do y'all make these?"
Our friends from Rickey Meche's Donut King dropped over a king cake yesterday and in it there was a flyer explaining the tradition of king cakes. It does a great job of summarizing the origin of these tasty delights.
The Mardi Gras season officially begins on the Twelfth Night of Christmas, or January 6th known as the Epiphany - or Little Christmas. Since the third century the Epiphany has been observed as the day the Three Wise Men finally found the infant Jesus. King Cakes, therefore were initiated to celebrate this holy day and a small baby doll is placed in each cake as symbol of "Finding the Baby Jesus".
King Cakes are oval shaped and decorated in carnival colors of green, yellow and purple. As the cake is cut, each person looks to see if his piece contains the small baby. The person who finds the baby brings the next King Cake to the next celebration.
So there you go, a brief explanation as to why we eat King Cakes during the Mardi Gras season.


text:
From the Head of School
What We Stand Forarrow
Educating in the 21st Century
Diversity at LMS
Equity and Anti-Bias
Engaging in the World
LMS at a Glance
Montessori at LMSarrow
More on Montessori
Applyingarrow
Why LMS?
Inquire or Apply Online
Curriculum and Programsarrow
Growing Up LMS
Children's House
Lower Elementary
Physical Education and Athletics
Summer Recessarrow
Summer Recess Informationarrow
Summer Recess Downloads
Communityarrow
Alums Return to LMS
Alum Class Notes
Horizons at LMSarrow
Give to HORIZONS at LMS
2019 LMS 5K and Kids' Fun Run
myLMS
Montessori at LMS
Summer Recess
Summer Recess Information
Horizons at LMS
By Biff Maier, LMS Director of Faculty and Curriculum Development
The child will develop a kind of philosophy, which teaches him the unity of the universe. This is the very thing to organize his intelligence and to give him a better insight into his own place and task in the world.
- Maria Montessori, International Montessori Conference. Amsterdam, 1950
Dr. Montessori's view of the world and Earthlings was characterized by a grand cosmic vision. She looked at Earthly existence in the context of the universe and all of its interrelationships. She celebrated order in nature, especially at the level of creation. She described "cosmic agents" that enable cosmic order and maintain ongoing creation. She noted that while these agents generally perform their work unconsciously, human agents have the unique potential to act consciously and purposefully. Montessori talked about a "cosmic plan," which can be understood more clearly if we think of the world as a great, cosmic household, where all the work has been distributed among the family members. In this way we can understand that everything has tasks to fulfill, cosmic work to accomplish.
Dr. Montessori's view of the world and Earthlings was characterized by a grand cosmic vision. She looked at Earthly existence in the context of the universe and all of its interrelationships. She celebrated order in nature, especially at the level of creation. She described "cosmic agents" that enable cosmic order and maintain ongoing creation. She noted that while these agents generally perform their work unconsciously, human agents have the unique potential to act consciously and purposefully.
Montessori talked about a "cosmic plan," which can be understood more clearly if we think of the world as a great, cosmic household, where all the work has been distributed among the family members. In this way we can understand that everything has tasks to fulfill, cosmic work to accomplish.
Inorganic agents "work" according to their inherent nature. The Sun is an inorganic agent providing light and heat energy. The lithosphere provides the ground we stand on and the soil we plant in, as well as the container for the seas and oceans. The water in the hydrosphere and the air in the atmosphere provide the environment for a sphere of life, the biosphere, where plants and animals, the organic cosmic agents, "work" according to their inherent nature. It is a vision of indivisible unity made up of energy, sky, rocks, water, and life. Near the end of her life, Dr. Montessori wrote, "Man's arrival has created a 'psychosphere' on Earth. We must understand that mankind, too, has a task with regard to the Earth on which we live. …His scientific work gradually discloses the secrets of Nature and, moreover, makes use of them, thus creating new possibilities." This reminds me of Teilhard de Chardin's idea of a Noosphere, a sphere of shared intelligence, superimposed upon the biosphere.
Montessori argued that the human experience can only be understood in the context of the evolution of our species. She believed that Homo sapiens has been guided by "finality," the drive toward ever-greater complexity, from the time of our appearance. Further, she argued that to know an individual, we must consider the whole of his or her development. We don't teach a toddler or a middle-schooler, but rather a human being at a particular stage of development. Each individual must be seen in his/her oneness as well as in the developmental differences of diverse stages or "seasons of life."
"Cosmic education" is a term used by Dr. Montessori and her son, Mario, to describe the Montessori elementary curriculum. It responds to the specific developmental characteristics and needs of the human being during this stage of life. It employs the "age of the imagination" to understand reality and to explore the vastness of culture.
Montessori said, "Cosmic Education gives children orientation and guidance in life. It wants to prepare the growing child for the task awaiting him in adult life, so that he will feel at ease in his own environment, in which he will later have to live as an independent being." As children learn to understand the world's evolutionary development, ecological functioning, and the interdependency of various forces, they gain a sense of meaning and purpose. They come to understand and appreciate the importance of collaboration at a cosmic level.
They become aware of the importance of work, but especially of the importance of work that contributes to the well being of others. They come to appreciate how they benefit from the efforts of others, in the past and in the present. Consequently, they begin to seek their own personal ways to contribute.
Lexington Montessori School
130 Pleasant Street, Lexington, MA 02421 | Phone: 781-862-8571


text:
User-accessible Content
From 2021 2020 2019 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 1996 1995 1994 1993 1992 1991 1990 — To 2021 2020 2019 2018 2017 2016 2015 2014 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 1996 1995 1994 1993 1992 1991 1990
You are looking at 1 - 3 of 3 items for
Author or Editor: Michel Ravelonandro x
Sort by RelevanceArticle A - ZArticle Z - AAuthor A - ZAuthor Z - ADate - Old to RecentDate - Recent to Old
Short interfering RNAs are Associated with Virus Resistance in a Woody Perennial Fruit Tree (Prunus domestica)
Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro
We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.
In HortScience Volume 39 (2004): Issue 4 (Jul 2004)
Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica
Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza
Constructs with self-complementary sequences separated by an intron produce "hairpin" RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.
In Journal of the American Society for Horticultural Science Volume 132 (2007): Issue 6 (Nov 2007)
'HoneySweet' (C5), the First Genetically Engineered Plum pox virus–resistant Plum (Prunus domestica L.) Cultivar
Ralph Scorza, Michel Ravelonandro, Ann Callahan, Ioan Zagrai, Jaroslav Polak, Tadeuz Malinowski, Mariano Cambra, Laurene Levy, Vern Damsteegt, Boris Krška, John Cordts, Dennis Gonsalves, and Chris Dardick
In HortScience Volume 51 (2016): Issue 5 (May 2016)


Alexandre Lacazette's late equaliser earned Arsenal an entertaining 1-1 draw against Liverpool on Saturday to underline the Gunners' top-four credentials.
Lacazette ran on to a pass from Alex Iwobi in the 82nd minute and held off goalkeeper Alisson, creating enough space to fire a difficult shot into the far corner.
In a crowd-pleasing heavyweight battle filled with chances at both ends, Liverpool had taken the lead through James Milner in the 61st minute after goalkeeper Bernd Leno failed to control a cross from Sadio Mane, diverting it straight into the path of the Liverpool midfielder.
But the fightback extended Arsenal's unbeaten streak to 14 games and showed they are ready to compete with England's top sides again.
This was arguably Arsenal's most impressive -- and encouraging -- performance under Unai Emery. Unlike most games this season, the Gunners came out full of energy and intent in the first half and maintained a high level throughout the 90 minutes.
A point certainly felt well deserved and Arsenal easily could have scored more if they had been sharper in front of goal. Following the losses to Manchester City and Chelsea to start the season, Emery's team badly needed a result to prove that they are making strides towards challenging their main rivals and they delivered the right kind of statement at the Emirates.
No team will be able to completely contain Liverpool's high-powered attack for 90 minutes but Milner's opener still felt a bit unnecessary. Leno looked unusually shaky in the air throughout the game and can be singled out as the main culprit for the goal, but the defending from Hector Bellerin and Shkodran Mustafi was suspect as well. Arsenal still need to eradicate those defensive weaknesses completely before they can start winning games like these.
8 -- Emery went with his most attack-minded lineup by starting his four biggest stars together for the first time, with Mesut Ozil, Henrikh Mkhitaryan, Pierre-Emerick Aubameyang and Lacazette all getting the nod. It looked like an attempt to fight fire with fire and the quartet managed to peg Liverpool back for large parts of the game and limit the opportunities for the visitors' forwards to shine. His substitutions also worked well, with Iwobi creating the equaliser after replacing Aubameyang.
GK Bernd Leno, 5 -- A mixed bag from the goalkeeper. Good saves to deny deny Virgil Van Dijk in either half but also misread a cross which allowed the defender to head on to the post, and should have done better in controlling the cross that led to the goal.
DF Hector Bellerin, 6 -- His pace down the right was one of Arsenal's main attacking outlets, but he lost track of Mane in the lead-up to Liverpool's opener.
DF Rob Holding, 7 -- Very good in aerial duels and coped well when dealing with Roberto Firmino in close quarters. A composed performance, with the only negative his poor passing at times.
DF Shkodran Mustafi, 7 -- Was near flawless for the opening hour but questions can be raised about his role in Liverpool's goal as well.
DF Sead Kolasinac, 7 -- Couldn't have had a tougher assignment in his first league start of the season in going up against Mohamed Salah, but he managed to use his physicality to great effect against the Egyptian without stepping over the line.
MF Lucas Torreira, 8 -- A one-man wrecking crew in midfield at times, throwing himself about to win the ball back and constantly looking to instigate attacks. Keeps going from strength to strength.
MF Granit Xhaka, 8 -- A perfectly timed sliding tackle on Salah in the box set the tone for the entire team. Helped control the game and finally looked like the midfield general Arsenal need him to be.
MF Mesut Ozil, 7 -- Didn't boss the game but showed a few magical touches to create chances for his teammates. Can't be accused of going missing in this one.
MF Henrikh Mkhitaryan, 6 -- Showed his connection with Aubameyang on a couple of occasions and nearly scored with a header in the first half. Still can't get much reward for his work, though.
FW Pierre-Emerick Aubameyang, 6 -- Arsenal's main danger man in the first half and used his pace to great effect to torment the Liverpool defenders. But faded after the break and was taken off with Arsenal chasing an equaliser.
FW Alexandre Lacazette, 7 -- Came to the rescue with a great strike after a fairly quiet performance up to that point. Should have done better when Ozil set him up with a sublime backheel pass in the first half.
MF Alex Iwobi, 7 -- Gave Arsenal a spark when he came on and did well to set up Lacazette for the equaliser.
MF Aaron Ramsey N/R -- Came on shortly before the goal but didn't have much of an impact off the bench this time.
MF Danny Welbeck N/R -- Injected pace and energy as a late sub and helped create some late chances for the winner.


We would soon be launching our freshly designed website. Its been great to have all the suggestions and feedback from people. We look forward to seeing more positive and fruitful achievements with out new web presence.


Customers and customer expectations have changed in recent years. Browsers and buyers have become more fickle and less loyal. They are more impatient. And they have come to expect more from business in terms of innovation, personalization, and product offerings.
So businesses need to change. They need to rethink how they do business and how they interact with customers. And they need to alter their billing and other processes so they can more effectively compete in the digital economy.
A key part of that change should involve billing, said Mitch Ivey, PwC's Revenue Innovation & Billing Transformation practice leader. In the past, he added, billing and pricing functions were utilities a company would apply to a product just before it was ready to be introduced. Now, however, billing has become core to the value of products, he said. It can serve as a competitive differentiator, a way to grow new revenues, and a tool through which to strengthen customer relationships.
Jean Lawrence, director of product marketing for Oracle Monetization Cloud, added that in the new digital world, subscription-based commerce is a quickly growing model. For example, she noted, Intuit (News - Alert) used to sell software as a one-time transaction. Now its software is available via subscription and through cloud.
So is media like movies and music, she noted. Just take a look at Apple (News - Alert), which started out with a model based on one-time downloads. Now, she said, the company is encouraging customers to buy monthly Apple Music subscriptions, and it had activated 20 million subscribers as of last December.
Businesses are introducing subscription-based solutions to address customer demand and build loyalty, enable recurring revenue streams, and provide them the flexibility to pivot quickly when customer behavior signals a new trend, she said.
The value of subscriptions is not just conjecture. Just look at the Amazon Prime effort. Amazon Prime customers spend twice as much as non Prime customers, she said. A recent Oracle (News - Alert) survey of 293 U.S. executives points to a strong link between companies with subscription-based services and those exceeding their plans for revenue growth.
And an Economist Intelligence Unit study that Oracle commissioned notes the belief by business leaders that customer retention is key to company success. Yet, only 42 percent of those surveyed rank themselves as strong on customer retention.
Why is retention so challenging? Because of competition. Lawrence noted that 44 percent of the executives surveyed say competitors are undercutting prices and 34 percent say new entrants are stealing customers' attention. That means businesses need to work harder at building meaningful relationships with customers, who have increasingly short attention spans, she said.
They can do that by bundling offers, doing more cross selling, offering discounts and reward points, and providing subscription-based services that keep them in constant contact with customers and enable them to see firsthand how those customers are using their services.