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Foxp3+ CD25– CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion
Santiago Zelenay, Thiago Lopes-Carvalho, Iris Caramalho, Maria Francisca Moraes-Fontes, Manuel Rebelo, and Jocelyne Demengeot
PNAS March 15, 2005 102 (11) 4091-4096; https://doi.org/10.1073/pnas.0408679102
Santiago Zelenay
Thiago Lopes-Carvalho
Iris Caramalho
Maria Francisca Moraes-Fontes
Manuel Rebelo
Jocelyne Demengeot
Communicated by N. M. Le Douarin, Académie des Sciences de l'Institut de France, Paris, France, February 3, 2005 (received for review November 17, 2004)
Figures & SI
Expression of the IL-2 receptor α chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (TR), although cells with regulatory properties are also found in the CD4+CD25– subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of TR differentiation, we have evaluated the requirements for CD25 expression by peripheral TR. We first show that in vivo depletion of CD25+ cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25+ TR. Consistently, Foxp3-expressing TR encompassed in the CD25– cell population convert to CD25+ after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on TR is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25– cells constitute a peripheral reservoir of differentiated TR, recruited to the CD25+ pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of TR takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral TR differentiation from naive CD4 T cells, defined as CD25–.
T lymphocyte
Healthy unmanipulated mice bear a significant number of "naturally" activated B and T lymphocytes, which seem to represent physiological autoreactivity because they are equally represented in "germ-free" and "antigen-free" mice (1). Like other antigen-experienced CD4 cells, naturally activated CD4 cells are encompassed in the CD45RBlow pool (2). As demonstrated in several experimental systems using adoptive transfers, CD4+CD45RBlow cells (from now on denoted CD45RBlow) limit the pathological potential of the complementary CD45RBhigh naive cells (3, 4). A subset of CD45RBlow cells expressing the CD25 marker is highly enriched in regulatory T cells (TR) that limit both protective and pathological immune responses (5). Several reports, however, demonstrate that TR are not exclusively contained within the CD25-expressing subset (6–9). Moreover, surface markers and genes that are highly represented or expressed in the CD25+ cells are also found in the CD45RBlowCD25– subpopulation although at a lower frequency or level, while being absent in the CD45RBhigh subset. This is the case for the surface molecules CD103 (8), glucocorticoid-induced tumor necrosis factor receptor (GITR) (10), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (11), Toll-like receptor (TLR)-4, -5, -7, and -8 (12), and the transcription factor Foxp3 (13). Expression of the Foxp3 gene is strictly required for TR development and enough to confer conventional CD4 T cells with regulatory function (13–15). It is to date the only known TR commitment/differentiation factor in mice.
For convenience of experimental design, most current studies use surface expression of CD25 to distinguish "conventional" T cells from TR. This approach seems appropriate, because most in vivo studies successfully associated regulatory activity to this cellular subset, and >95% of the CD25+ cells in a normal mouse express Foxp3, as evaluated in GFP-Foxp3 fusion knock-in mice (16). Clearly, however, the reverse does not apply, because lack of CD25 expression in a cell population cannot be taken for absence of regulatory cells. This reservation is critical, in view of previous claims that regulatory cells can differentiate in the periphery from naive CD4 cells, defined as CD25–.
The CD25 molecule is the α chain of the IL-2 receptor, and its expression results in higher affinity to IL-2 (17). Upon activation, conventional CD4 cells express CD25, while lacking many of the other phenotypic and functional characteristics of TR (18). Similar induction of CD25 expression upon activation may well occur on TR, and it has been proposed that IL-2 promotes acquisition of this marker and functional activation (19). Several groups have reported that CD25+ cells lose CD25 expression upon adoptive transfer in lymphopenic mice, a phenomenon that is less marked if conventional CD4 cells, presumably serving as a source of IL-2, are present and undergoing homeostatic expansion (6, 20). Intriguingly, acquisition of CD25 expression by CD25– cells undergoing homeostatic expansion was also reported, although the nature of the cells contributing to this phenomenon was not assessed (6, 20, 21).
In this study, we investigated the relevance of CD25 surface expression for the definition of TR and, thus, the possibility that they may arise from the naive CD4 pool in the periphery. We show that administration of depleting anti-CD25 mAb in vivo targets both newly activated conventional cells and a limited subset of regulatory T cells. Furthermore, the bulk of Foxp3-expressing T cells encompassed in the CD45RBlowCD25– cell pool convert to a CD25+ phenotype in lymphopenic conditions, and these cells display functional characteristics of TR. Finally, surface expression of CD25 on TR is fully labile because it can be lost and regained without affecting the functional potential of the cells. Taken together, these analyses indicate that a reservoir of TR is contained in the CD45RBlowCD25– population and that such cryptic TR can rapidly be recruited to the CD25+ pool.
Mice. BALB/c, C57BL/6, C57BL/6-Thy1.1, Igha, Gpi1a , C57BL/6 RAG2–/–, B10-PL-MBP-TCR-Tg, and B10-PL-RAG1–/–-MBP-TCR-Tg mice were bred and maintained under specific pathogen-free conditions in our animal house. All animals were used between 4 and 10 weeks of age.
Antibodies and Reagents. Allophycocyanin (APC), CyChrome-, and phycoerythrin (PE)-conjugated anti-CD4 mAb (clone RM4-5), CD45RB-PE (clone 16A), and Thy1.2 biotin (CD90.2) were purchased from BD Biosciences. Thy1.1 (CD90.1) biotin and Alexa Fluor 488-CD25 (clone PC61) were home-made. Biotinylated antibodies were revealed with streptavidin-PE or -APC (BD Biosciences). CD25+ cell depletion was performed with 200 μg of anti-CD25 mAb (clone PC61) injected i.p. As a control, mice received the same amount of rat IgG (Sigma–Aldrich). Depletion was evaluated by using the 7D4 anti-CD25 mAb (BD Biosciences). Pertussis toxin from Bordetella pertussis (Sigma–Aldrich) was injected i.v. (200 ng per mouse).
Thymectomy and Disease Evaluation. Four-week-old BALB/c mice were thymectomized (Tx), and absence of noticeable thymic remnants was confirmed at the end of the experiment. Experimental autoimmune encephalomyelitis (EAE) was scored every 3 days as described (22).
Cell Purification and Transfer. Pooled lymph nodes (LNs) stained with a mixture of anti-CD4-PE and CD25-Alexa mAbs, or with anti-CD4-CyChrome, CD25-Alexa, and CD45RB-PE were purified on a MoFlo High Speed Cell Sorter (Cytomation, Fort Collins, CO). Purity was routinely >98% for CD4+CD25+ cells and >99% for the other CD4 subsets. Cells were suspended in PBS and injected in the retroorbital plexus (100 μl per mouse).
Cell Recovery and Flow Cytometric Analysis. Cell suspensions from spleen or mesenteric LNs were prepared, stained, and washed in PBS containing 2% FCS and 0.01% sodium azide. Propidium iodide was added to the final suspension. Analyses were performed inside a live lymphocyte gate on a FACSCalibur (Becton Dickinson) by using cellquest software. Life lymphocyte counts were deduced from the acquisition of a fixed number of 10-μm latex beads (Coulter) mixed with a known volume of unstained cell suspension.
Cell Cultures and Suppression Assays. Cultures were set in RPMI medium 1640 containing 10% FCS, 100 μg/ml penicillin and streptomycin, 50 μM 2-mercaptoethanol (2-ME), 10 mM Hepes, and 1 mM sodium pyruvate (all purchased from Life Technologies, Grand Island, NY). IL-2 production was as follows: 2.5 × 103 CD4+CD25– cells (target) mixed with 5 × 103 irradiated splenocytes and various numbers of the cell populations under test were stimulated with 0.5 μg/ml anti-CD3 mAb (145.2C11; home-made) for 48 h (U-shape 96-well plate, 100 μl final). Fifty microliters of the supernatant was transferred to 103 CTLL-2 cells, and a saturating amount of IL-2 was added to the last 24 h of a 3-day culture (amplification). For pretreatments, the cells (2 × 106 per well, 24-well plate) were stimulated for 6 days with 1 μg/ml soluble anti-CD3 mAb and a saturating amount of IL-2. Alternatively, the cells (106 per well, six-well plate) were sequentially stimulated with 1 μg/ml and 10 μg/ml plate-bound anti-CD3 mAb for 5 and 3 days, respectively. Standard suppression assays are described in ref. 12. All cultures were set in triplicate, and [3H]thymidine [1 μCi per well (1 Ci = 37 GBq); Amersham Pharmacia Biosciences] was added for the last 6 h.
Real-Time PCR. Total RNA was extracted from 104 to 106 cells by using TriPure (Roche Diagnostics), treated with DNaseI and reverse transcribed by using SuperScript II RT and oligo(dT)12–18 primer (all from Life Technologies). PCRs were performed by using the QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) and the Light Cycler system (Roche), and consisted of 15 min at 95°C and 45 cycles of 15 s at 95°C, 20 s at 61°C (Foxp3), or 55°C [hypoxanthine phosphoribosyltransferase (HPRT)], and 10 s at 72°C. Primer pairs (5′–3′) were: Foxp3, TTCATGCATCAGCTCTCCACT and AAGGTGGTGGGAGGCTGA; HPRT, CCAGCAAGCTTGCAACCTTAACCA and GTAATGATCGTCAACGGGGGAC. The standard curve method was applied for quantification of each amplicon. The normalized values for Foxp3 mRNA were calculated as the quantity of Foxp3 mRNA levels divided by the quantity of HPRT mRNA levels and converted in reference to the CD45RBhighCD25– subset (= 1).
Depleting Anti-CD25 mAb Antibody Targets Both Activated Effector and Regulatory T Cells in Vivo. To establish the contribution of CD25 surface expression to conventional, activated/effector cells and to the TR pool in the same model system, we treated anti-myelin basic protein (MBP) T cell antigen receptor (TCR) transgenic (Tg) mice (23) with a depleting anti-CD25 mAb. Recombination-activating gene (RAG)-deficient monoclonal mice (T/R–) spontaneously develop severe and progressing encephalomyelitis by 2 months of age. In contrast, RAG-competent Tg mice (T/R+) remain healthy, although >90% of the cells are self-reactive, "protected" by TR expressing endogenously encoded TCR (22, 24). Nevertheless, in sick T/R– as in healthy T/R+ mice, ≈5% of all CD4 cells are CD25+ (data not shown). Consistently with the respective phenotype often attributed to activated effector cells and TR (25, 26), the CD4+CD25+ subset detected in T/R– mice expressed higher levels of CD4 and lower levels of CD25 than the CD4+CD25+ T cells observed in T/R+ animals (not shown). Depletion of CD25-expressing cells in T/R– animals prevented EAE when initiated before onset of encephalomyelitis, and interruption of the treatment restored the development of progressing EAE (Fig. 1A ). Similar treatment administered to sick animals did not revert the disease process but prevented its progression (Fig. 1B ). These findings indicate that newly activated cells express CD25 and that CD25+ cells in this system contain mostly pathological cells. Whether they also encompass a small number of TR, in a too small proportion to ensure tolerance, remains to be assessed.
Depletion of both activated and regulatory T cells by anti-CD25 antibody in vivo. T/R– and T/R+ mice were injected with 200 μg of anti-CD25 mAb. (A) One-month-old T/R– animals (n = 7) received five weekly injections and were followed for 4.5 months. The percentage of mice that developed EAE (score ≥2) is represented. (B) Sick T/R– mice (EAE score = 2–3) received a continuous weekly treatment (n = 5). As control, a group of T/R– mice was left untreated (n = 20). Plotted is the percentage of mice alive at 5 months of age and the EAE score of the survivors. Group comparison for the mean EAE score was statistically significant using Student's t test (C) Adult T/R+ mice were treated once with the anti-CD25 mAb (day 0) either alone or together with 200 ng of pertussis toxin (day 0 and 2). Control T/R+ mice were left untreated. EAE level was scored weekly, and plotted is the percentage of mice that developed EAE (score ≥2).
A single injection of the CD25-depleting mAb in healthy T/R+ animals did not lead to significant disease; however, when combined with pertussis administration, ≈55% of the mice developed encephalomyelitis, whereas pertussis alone had no significant effect (Fig. 1C ). This result, by revealing that CD25+ cells in the T/R+ animals encompass efficient TR, confirms previous conclusions drawn from adoptive cell transfer experiments (22). More importantly for the present topic, in vivo depletion of all donor T cells by using mAbs to a Thy-1 allotype in T/R– mice, protected by adoptive transfer of normal CD4 cells, was shown to lead to spontaneous severe-progressing EAE (27) whereas we evidence here that depletion of solely CD25+ cells in T/R+ animals does not induce pathology (Fig. 1C ). In addition, all T/R+ animals that developed disease upon combined injection of anti-CD25 antibody and pertussis stabilized at a stage of partial hind limbs paralysis (level 2) without progressing to more severe stages of the disease (not shown). Together, these results reveal that administration of CD25-depleting mAb did not lead to total abrogation of regulation in T/R+ mice, confirming that a subset of TR is encompassed in the CD25– cell subset.
The CD25 + TR Pool Is Rapidly Restored in the Periphery After in Vivo Depletion. The results above prompted us to assess the composition of the peripheral CD4 cell pool after anti-CD25 mAb administration in vivo. Because T/R+ animals display a low number of TR, we used normal BALB/c mice to ensure accurate analysis. In a first step, to exclude the potential contribution of newly generated thymic CD4 cells, 4-week-old BALB/c mice were thymectomized and treated with the depleting anti-CD25 mAb 1 month later. Two days after treatment, a maximum of 0.5% of the total CD4 cells in peripheral blood lymphocytes (PBLs), spleen, and LNs stained positive with the 7D4 anti-CD25 mAb, representing <5% of the normal frequency of CD4+CD25+ cells in PBLs (Fig. 2A ), spleen and LNs (not shown). In less than a month after depletion, the CD4+CD25+ cell pool progressively recovered normal frequency (Fig. 2 A ). Surprisingly, euthymic mice submitted to the same treatment displayed similar kinetics of CD4+CD25+ cell recovery (not shown), indicating that thymic output is not a major factor for peripheral CD25+ T cell homeostasis as shown recently in newborn Tx mice (28). We next assessed whether the repopulating T cells were recently activated or regulatory CD4+ T cells by monitoring their ability to suppress conventional CD4 cell responses to TCR triggering. As shown in Fig. 2B , CD4+CD25+ cells isolated from mice Tx and treated 3 months before with either anti-CD25 mAb or rat IgG were equally efficient in inhibiting the IL-2 production by normal CD25– cells. These results, consistent with a previous report (29), indicate that the peripheral immune system is autonomous to ensure efficient control of TR homeostasis. This control may result from a substantial expansion of the few CD25+ cells that escaped depletion and/or from proficient conversion of other CD4+ T cell subtypes into a CD4+CD25+ TR phenotype.
CD4+CD25+ TR recover normal levels after in vivo depletion of Tx mice. Adult-Tx BALB/c(n = 7) mice were injected i.p. with 200 μg of anti-CD25 mAb or rat IgG (controls) twice at 1-week intervals. (A) Peripheral blood lymphocytes were analyzed for the frequency of CD25+ among CD4+ cells at different days after the injection. Shown is the percentage of CD25+ among CD4+ cells in treated animals relative to control animals (n = 7). (B) CD4+CD25+ cells were sort-purified from LNs of either CD25+-depleted (CD25+ Tx-depleted) or from rat IgG-injected (CD25+ Tx control) mice 3 months after depletion and tested for their capacity to suppress IL-2 production by stimulated CD4+CD25– cells. Primary cultures consisted in 2.5 × 103 CD4+CD25– cells isolated from normal mice, stimulated alone or in the presence of different numbers of CD25+ cells isolated from Tx-depleted or Tx control mice. As a negative control, CD25– cells (CD25–) were also tested. Shown is the percentage of inhibition [(cpm in control) – (cpm in experiment)/cpm in control] is plotted versus the ratio of the population tested/CD4+CD25– cell number at the origin of the primary culture.
CD25 – Foxp3-Expressing Cells Acquire CD25 Expression upon Homeostatic Expansion. To assess the efficiency of CD4+CD25– cells to convert to a CD25+ TR phenotype, we conducted a phenotypic and functional analysis of these cells either Thy1.1+ CD45RBhighCD25– or Thy1.2+ CD45RBlowCD25– 12 days after adoptive transfer into RAG–/– mice. As reported before (6), a significant fraction of the originally CD45RBlowCD25– cells acquired a CD25+ phenotype (≈4.5% in spleen and 15% in LN) whereas the Thy1.1+ cells (originally CD45RBhighCD25–) expressing CD25 were barely detectable (Fig. 3A ). More than 80% of the original CD45RBhigh cells became CD45RBlow upon adoptive transfer (not shown); however, sequential transfer of total CD4+CD25– cells (Thy1.2+) in these mice did not increase the frequency of Thy1.1+ cells that converted to a CD25+ phenotype (Fig. 3A ). Similar results were obtained in recipients of simultaneous cotransfer, confirming that the two cell subsets are controlled independently for this feature. Finally, Thy1.2+CD45RBlowCD25– cells were mixed with 100-fold fewer Thy1.1+CD4+CD25+ cells before adoptive transfer into RAG–/– animals. This deliberate contamination exceeded that routinely obtained after sort-purification and is similar to that detected in the blood and lymphoid tissues 2 days after administration of depleting anti-CD25 mAb in normal mice. At day 12 posttransfer, Thy1.1+ cells were hardly detectable (average 0.2% and 0.1% of CD4 cells in spleen and mesenteric LN, respectively), whereas the percentage of Thy1.2+ cells expressing the CD25 marker was the same as in single transfer (not shown). It is noteworthy that, inside the CD25+ subset, the contribution of the newly converted Thy1.2+ was therefore >95%. These last results indicate that CD45RBlowCD25– cells undergo efficient conversion to a CD25+ phenotype that largely dominates over the expansion of few contaminating CD4+CD25+ cells. Together these findings demonstrate that acquisition of CD25 surface expression by CD45RBlowCD25– cells in nonimmunized mice is the result of a specific phenotypic modification restricted to a subset of the naturally activated T cell pool.
CD45RBlowCD25– cells that acquire in vivo CD25 expression display a regulatory phenotype. RAG2–/– mice received 3 × 105 CD4+CD25– cells either Thy1.2+CD45RBlow or Thy1.1+CD45RBhigh (Single), or an equal number of both cell subsets (Co), and were analyzed 12 days later. For the sequential transfer (Seq), mice that received 3 × 105 Thy1.1+CD45RBhigh cells at day 0 were injected with 3 × 105 Thy1.2+CD4+CD25– cells at day 12 and analyzed at day 24. (A) FACS analysis of splenocytes. Shown is the percentage of CD25+ cells inside a gate either CD4+Thy1.2+ (originally CD25–45RBlo) or CD4+ Thy1.1+ (originally CD25–45RBlo). (B) Foxp3 mRNA levels determined by real-time PCR. (Left) cDNA prepared from freshly isolated (Fresh) CD4+cells, either CD25+ (25+), CD25–CD45RBhigh (25–45hi), or CD25–CD45RBlow (25–45low) served as controls. (Right) cDNA was prepared from CD4+CD25+ or CD4+CD25– cells sort-purified from pooled spleen and LN of either of the single-transfer recipients (Post-Transfer). (C) IL-2 production upon TCR triggering. Thy1.2+CD4+ cells (originally CD25–CD45RBlow) were sort-purified from co-transferred recipients and fractionated as CD25+ or CD25–. Shown is the proliferation of CTLL-2 cells exposed to supernatants of primary cultures that contained 2 × 103 of either cell subset. Control was CD4+CD25+ cells purified from normal C57BL/6 animals. The background (CTLL-2) and the maximum proliferation (CTLL-2 IL-2) are those of CTLL-2 cells maintained in medium either alone or containing saturating amounts of IL-2. (D) Suppression of IL-2 production. As in C except that primary cultures consisted in 2.5 × 103 CD4+CD25– cells isolated from normal mice, stimulated alone (Targets) or in the presence of 1.25 × 103 CD4+ cells (filled bars) either CD25+ or CD25– purified as in C.
We next assessed whether acquisition of CD25 upon homeostatic expansion is a signature of TR by monitoring the level of Foxp3 mRNA in each CD4 subset before and after adoptive transfer (Fig. 3B ). Strikingly, the originally CD45RBlowCD25– cells that converted to a CD25+ phenotype displayed levels of Foxp3 mRNA comparable with that of freshly isolated CD25+ cells. In contrast, those that remained CD25– expressed Foxp3 to a lower level than freshly isolated CD45RBlowCD25– cells and rather similar to fresh CD45RBhighCD25– cells. We interpret this result as evidence that CD25– Foxp3-expressing cells inside the CD45RBlow subpopulation selectively express CD25 upon homeostatic expansion. The amount of Foxp3 transcripts in the few CD45RBhighCD25– cells that converted to a CD25+ phenotype remains low, indicating that this cell pool contains recently activated cells. Nevertheless, it is remarkable that the little increase of Foxp3 signal in this cellular subset correlates with a decreased signal (average value 0.2) in the remaining CD25– cells, indicating again that Foxp3-expressing cells convert to a CD25+ phenotype upon homeostatic expansion.
Finally, we tested whether acquisition of CD25 expression by CD45RBlowCD25– cells reveals functional suppressor cells. As references, we used freshly isolated CD4+CD25+ cells. Clearly, the CD25+ but not the CD25– cell subset was unresponsive to TCR stimulation as measured by IL-2 production (Fig. 3C ) and suppressed IL-2 production by conventional CD4 cells, to a level comparable with freshly isolated CD4+CD25+ cells (Fig. 3D ). We conclude that the CD45RBlowCD25– cells that acquire in vivo CD25 expression display a regulatory phenotype similar to conventional CD4+CD25+ TR.
In Vitro Induction of CD4 + CD25 – Cells to Suppressor Activity Is Restricted to CD45RBlow Cells. The evidence that CD45RBlowCD25– cells contain Foxp3+ T cells that convert to a CD25+ TR phenotype upon homeostatic expansion prompted us to reevaluate their in vitro suppressor activity. As reported previously (12, 30), freshly isolated CD45RBlowCD25– like CD45RBhighCD25– cells do not show suppressive functions when tested in vitro. However, CD45RBlowCD25– but not CD25–CD45RBhigh cells pretreated with IL-2 and soluble anti-CD3 mAb for 6 days display suppressor function, comparable in efficiency with freshly isolated CD25+ cells (Fig. 4A ). Similarly, using a protocol originally described to induce anergic (31) and suppressor (32) cells in vitro, we evidence that CD45RBlowCD25– but not CD25–CD45RBhigh cells pretreated with immobilized anti-CD3 mAb exhibit suppressor activity (Fig. 4B ). We propose that these pretreatments selectively expand committed TR and thus mimic in part the homeostatic expansion/activation of the CD25– TR we evidenced above.
Activation of CD45RBlowCD25– cells in vitro reveals their regulatory properties. (A) Sorted CD45RBhighCD25– and CD45RBlowCD25– cells (25–45hi and 25–45lo, respectively) maintained for 6 days in culture containing anti-CD3 mAb and IL-2 (IL-2) were washed and cultured with the same number of untreated CD4+CD25– cells, and cultured for another 3 days in the presence of anti-CD3 mAb and antigen-presenting cells. Freshly isolated (Fresh) CD4+CD25+ (25+), CD45RBlowCD25– (25–45RBlo), and CD45RBhighCD25– (25–45hi) cells were used as controls. (B) Sorted CD45RBlowCD25– and CD45RBhighCD25– cells (25–45RBlo and 25–45RBhi, respectively) were stimulated with plate-bound anti-CD3 mAb according to Materials and Methods. The evaluation of their suppressor function was performed by adding them at various ratios to untreated CD4+CD25– cells as in A. Freshly isolated CD4+CD25+ cells (25+fresh) were used as control. The percentage of inhibition of naive T cell proliferation is plotted versus the ratio of the population tested/CD4+CD25– cell number at the origin of the culture, as in Fig. 2B .
CD25 + Regulatory T Cell Expansion and CD25 Surface Expression Are Dissociated. The findings that CD25– TR acquire surface expression of CD25 during homeostatic expansion prompted us to reassess the phenotypic stability of the CD25+ TR isolated from normal mice. As reported before (6, 20, 33), upon adoptive transfer into alymphoid recipients, the vast majority of these cells lose CD25 expression (Fig. 5B ). The number of Thy1.2+ cells (originally CD25+) recovered in such transfer experiments is rather low (Fig. 5A ), and any proliferative advantage to rare CD25– contaminants in the original preparation may explain this result. However, when the purified Thy1.2+CD4+CD25+ cells were voluntarily contaminated with 1% Thy1.1+CD4+CD25– cells, <10% of the recovered CD4 cells were composed of Thy1.1+ cells, and the frequency of CD25-expressing cells was indistinguishable from that obtained in the pure Thy1.2+ cell transfer group (Fig. 5B ). We next confirmed that cotransfer of CD4+CD25– cells at a 1:1 ratio stabilizes to some extent the CD25+ phenotype (33), and further assessed whether the loss of CD25 expression in single transfer is reversible (Fig. 5C ). At day 12 posttransfer of Thy1.2+CD25+ cells, a group of mice was analyzed to ensure that ≈80% of the CD4 cells stained negative for CD25 (Fig. 5B ), whereas another group received 3 × 105 Thy1.1+CD25– cells. After an additional 12 days, on average, 77% of the Thy1.2+ cells were CD25+. Because the Thy1.2+ cells did not expand significantly after this secondary transfer (Fig. 5A ), we favor the interpretation that these cells underwent a reconversion to a CD25+ phenotype. It it noteworthy that the Thy1.2+ cells that were overall CD25– at the time of the second transfer, while converting to a CD25+ phenotype, were also able to control the in vivo expansion of the infused Thy1.1+CD4+CD25– population (Fig. 5A ), indicating that loss of CD25 expression does not correlate with loss of function.
Functional CD4+CD25+ cells can lose and regain CD25 expression. RAG2–/– mice received CD4 cells either Thy1.2+CD25+ or Thy1.1+CD25– alone (Single), together in same number (Co), at a 100:1 ratio (1%), or sequentially (Seq) and were analyzed 12 days after the last transfer. (A) Efficient control of Thy1.1+ expansion by Thy1.2+ cells. The number of Thy1.1+ (Left) and Thy1.2+ (Right) cells recovered from the spleen is shown for each animal. (B) Loss of CD25 expression upon adoptive transfer. CD4+ cells from mice recipient of 3 × 105 Thy1.2+CD25+ cells transferred alone (Single) or together with 3 × 103 Thy1.1+CD25– cells are analyzed for CD25 and Thy1.1 expression. (C) In cotransfer experiments, CD4+CD25– cells maintain and restore CD25 expression on Thy1.2+ CD4+CD25+ cells. Shown is the CD25 expression on Thy1.2+ cells recovered from mice that received an equal number of Thy1.2+CD4+CD25+ and Thy1.1+CD4+CD25– cells either at the same time (Co) or 12 days apart (Seq).
The present work establishes that, upon disruption of homeostasis, CD4 cell subsets contribute to the pool of TR phenotypically defined as CD25+ and Foxp3+ by recruitment from a peripheral reservoir of differentiated CD25– TR. Membrane expression of CD25 appears therefore as a signature of activation equally for conventional T cells and TR. This finding bears several consequences for the potential usage of CD25 as a therapeutic target and for our understanding of TR origin and dynamics.
We show that in vivo depletion of CD25-expressing cells can induce both protection and susceptibility to the very same disease. Depleting anti-CD25 mAbs have already been used to target pathological cells in autoimmune mice and humans. For instance, similarly to what we observed in T/R– animals, clinical trials testing the sustained usage of anti-CD25 mAb in combined therapies for multiple sclerosis gave promising results (34). On the other hand, and similarly to what we report here for the T/R+ animals, a single anti-CD25 mAb administration targets TR and sets a time window where immunization protocols gain in efficiency, an approach explored to improve tumor therapies (35). Short-lasting anti-CD25 mAb administration in mice does not induce or accelerate autoimmune diseases per se unless it is administered early in life or together with self-antigen and strong adjuvants (29, 36). The bulk of our analysis strongly suggests that this temporal limitation and the relative safety of these approaches must rely on the replenishment of the CD25+ TR pool by recruitment of peripheral differentiated CD25– TR. Numerous efforts have been developed worldwide to identify molecular targets that would strictly distinguish all TR from activated cells to improve the efficiency of TR therapeutic depletion protocols. Our results, together with the indication that the number of CD25– cells that can convert to a CD25+ TR phenotype is limited (37), seem to predict that the use of these new tools may lead to an irreversible highly challengeable state of tolerance because it may exhaust the pool of TR.
We established that, upon homeostatic expansion, Foxp3-expressing cells encompassed in the CD45RBlowCD25– subset are the cells contributing to the pool of converted CD25+ TR. We favor the idea that, under similar conditions, the few CD25– TR cells encompassed in the CD45high subpopulation are also those converting to a Foxp3+ CD25+ phenotype. In support of this proposition, it has been shown that Foxp3 expression, although rather low, is detectable in CD45high cells but not in differentiated helper cells (13). Whether acquisition of surface CD25 by Foxp3+ TR is necessary for their regulatory function remains to be formally established. However, we confirmed that CD25 is an activation marker and activated TR are more efficient regulatory cells than untreated cells (12, 19). Our results therefore indicate that, at the steady state, a normal immune system maintains a reservoir of "inactive" TR. Both the frequency of cells converting to a CD25+ phenotype upon homeostatic expansion and their level of Foxp3 mRNA expression indicate that TR contribute to ≈10–20% of the CD45RBlowCD25– cell population, which approximately represents a reservoir of an additional 2 million TR in a normal mouse. This reservoir may serve the purpose of keeping available a large number of inactive TR that could rapidly be recruited to the "active" TR pool upon immune activities. In turn, this subdivision may ensure the robustness of TR-mediated immune regulation while avoiding immunological paralysis by an otherwise excess of regulation. Infections not only trigger immune responses but also often associate with a state of thymic involution and transient lymphopenia, conditions that would favor the recruitment of these cryptic TR.
The original demonstrations that TR are generated intrathymically and selected by recognition of antigens expressed on thymic epithelial cells (TEC) (reviewed in refs. 38 and 39) gained further support from the recent revelation that TR represent a unique differentiative pathway (13–15) that is adopted by CD4 cells with "high avidity" for TEC-antigens (40, 41). The present finding that CD25 expression by TR is fully labile prompted us to propose that, in physiological conditions, peripheral TR and conventional CD4 T cells are strictly compartmentalized according to their previous thymic experience (Fig. 6). Acquisition, maintenance, or loss of CD25 expression by both conventional CD4+ Foxp3– and Foxp3+ TR rely solely on their encounter with activation signals. In uninfected individuals, activation of conventional CD4 cells is limited because their repertoire is biased for low affinity against self-antigens, at least for those encountered in the thymus (41), and does not require exogenous IL-2 (42). The nature of the activating signals for TR remains to be fully established; however, there is little doubt that IL-2 (19), produced mostly by conventional CD4 T cells, and TCR engagement by self-antigen/MHC complexes (16, 40, 41) are crucial. Additional signals are provided by inflammatory cytokines and endogenous ligands binding to the Toll-like receptors expressed by TR (12). Whether the CD25– and CD25+ TR subsets cover different TCR reactivities in normal individuals or whether these cells are highly labile and therefore rapidly switch from an activating to a nonactivating environment remains to be clarified. In addition, activated TR may well limit CD25– TR conversion to a CD25+ phenotype as they do for naive cells, a feature that would explain the remarkable stability of TR CD25+ versus CD25– distribution at the steady state.
CD25 is an activation marker of both TR and conventional CD4 cell subsets. In the thymus, conventional CD4 cells undergo positive selection, and these CD4+Foxp3–cells exit the thymus as naive CD45RBhighCD25–. Once in the periphery, if they receive activating signals, they acquire an activated phenotype characterized by a low level of CD45RB and high level of CD25 expression. Exit from the environment where the activation signals are provided associates with the loss of CD25 expression but maintenance of an antigen experienced CD45RBlow phenotype. Thymic TR commitment and differentiation require interaction with activating ligands (gray area), and TR exit the thymus with a CD25+CD45RBlow phenotype. Once in the periphery, Foxp3 expression is maintained. If specific activating signals are absent or below a certain threshold, CD25 expression is lost but CD45RB expression remains low. Reexposure to activation signals reverts this phenotype, and TR may reacquire a CD25 surface expression.
Finally, our findings that surface expression of CD25 is labile in differentiated Foxp3-expressing TR may serve as a word of caution for the interpretation of experiments aiming at the induction of "naive" peripheral CD4+CD25– T cell differentiation to Foxp3+CD25+ TR.
We thank members of the Instituto Gulbenkian de Ciência Cell Imaging Unit for cell sorting assistance and antibodies production, and we thank the mouse facility team. We thank Dinis Calado and Shohei Hori for their help in real-time PCR optimization. We thank Jorge Carneiro, Werner Haas, and Antonio Coutinho for valuable suggestions. This work was supported by the Fundação para a Ciência e a Tecnologia (FCT), Portugal, with the coparticipation of the Fundo Europeu de Desenvolvimento Regional (FEDER) through Grants POCTI/MGI/43063/2001 and POCTI/MGI/46477/2002 and fellowships to S.Z., T.L.-C., I.C., and M.R.
↵ † To whom correspondence should be addressed. E-mail: jocelyne{at}igc.gulbenkian.pt.
↵ * Present address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294.
Author contributions: S.Z., T.L.-C., I.C., and J.D. designed research; S.Z., T.L.-C., I.C., M.F.M.-F., M.R., and J.D. performed research; S.Z., T.L.-C., I.C., M.F.M.-F., and J.D. analyzed data; and S.Z., T.L.-C., and J.D. wrote the paper.
Abbreviations: TR, regulatory T cells; RAG, recombination-activating gene; TCR, T cell antigen receptor; T/R– and T/R+, anti-myelin basic protein TCR transgenic mice homo- and heterozygous for a null mutation of the RAG-1 gene; Tx, thymectomized; EAE, experimental autoimmune encephalomyelitis; LN, lymph node; PE, phycoerythrin.
Copyright © 2005, The National Academy of Sciences
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Iggy Azalea's Softcore "Mo Bounce" Video: 10 Sexy Screengrabs
Mike Wass @mikewassmusic | March 24, 2017 7:29 pm
Iggy Azalea's Eye-Popping "Mo Bounce" Video
Iggy Azalea's "Mo Bounce" video is every bit as raunchy as the gifs suggested.
Iggy Azalea's "Mo Bounce" video arrived this morning (March 24) and it's every bit as explicit as the teaser gifs suggested. Directed by Lil Internet, who previously helmed Beyonce's "No Angel," the clip doesn't have a narrative as such. It's basically just the Aussie rapper hanging out in Hong Kong, rocking a series of sexy outfits. There's cool streetwear, a pink, Angelyne-inspired ensemble and a sheer, white leotard. That latter allows maximum jiggle when the hitmaker shows off her twerking skills. (That practice really paid off!)
To celebrate the return of Iggy, I've taken screengrabs of the video's 10 sexiest moments. You can peruse the NSFW pics above. It will be interested to see how "Mo Bounce" is received. The song has earned surprisingly good reviews and even won praise from The New York Post. The consensus seems to be that the 26-year-old's return to her edgier, mixtape sound was a wise decision. Time will tell how the furious banger performs on the charts, but Iggy must be glowing after reading all that good press.
Do you love the video? Let us know below, or by hitting us up on Facebook and Twitter!
Iggy Azalea's Sexiest Pics
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"Here it is, I'm gonna give it to you, let's eat some babies for breakfast right now." I'm kinda thinking that's what they're saying in the lyrics. I love the song to pieces apart from the fact that I couldn't understand any of the words.
What a big target he's taking on, conscious thoughts. What is consciousness? We don't know. He tries to envelop where he comes from and where he is, and guage how much of it is conscious and unconscious? Man, Mr. Promise is hitting one of the big issues of our time.
In their picture they've got a bit of sticky tape over their nip nips. They're hard, driving, catchy punk and you wanna dance. One of the good things about dancing is you're doing some exercise and you're burning cholesterol.


Best Vacuum for Pet Hair - Looking for a Good Dog Vacuum Cleaner?"
Between my German Shepherd and Belgian Malinois Shepherd mix, my house was inundated with pet hair. Factor in 2 indoor cats and my hardwood floors resembled a desert with bundles of pet hair 'tumbleweeds' blowing around.
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Keep the Dog, Ditch the Hair!
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Is the Miele vacuum best for pets? The Miele vacuum motor packs 1200 watts of power. This German engineered canister vacuum is an excellent vacuum for pet hair. Electronic suction control with six settings and maneuverability removes hair from even the tightest of spots.
For discount Miele vacuums, reviews and more, get info on the Miele Cat and Dog Vacuum.
A lower priced vacuum designed for pet owners, the Bissell Pet Hair Eraser is excellent at picking up dog hair. A powerful suction and cyclonic technology allows this unit to clean your home effectively. Be aware that some have stated it can clog sometimes and may not be as easy to maneuver as other vacuums.
But, the decent price may make up for some of the inconvenience. Check out the Bissell Pet Hair Eraser Vacuum.


Going on a coastal vacation? Carry sunglasses along with you. Weekend getaway or travelling, keep your sunglasses safe on the go with this beautiful beach print soft case. It will easily slip into your tote bag and occupy no space. This pouch will help to protect your sunnies from minor scratches, dust and sand. Get a matching cleaning cloth along with the soft case.


It's hard to resist the exotic. Especially when it comes to food.
We all flirt with the idea that by eating something from far away a part of us will, momentarily at least, be transported too. So we slave away over unnecessary midweek tagines, buying cookbooks promising food so exotic you'll barely recognise it.
It doesn't get much more exotic than Hawaiian food. Immediately you imagine grassy peaks, the sweet sound of the ukulele, deserted beaches. How could its exported cuisine possibly live up to the dream? Yet Hawaiian food – and in particular, poke, a raw fish dish – has been earmarked as the next big culinary craze.
Black Roe Mayfair, occupying the site of the short-lived but superb Bouillabaisse, wants to capitalise on London's new hunger for poke. Originating in Hawaii, poke is a raw fish salad, a cousin to sushi and sashimi, often featuring tuna.
If Hawaiian food is cool this year, it's going to need some stylish settings. Ironically lit by neon lighting, bedecked with crate-style tabletops and sleek leather, Black Roe is edgy.
And yet as I sit down in my dimly lit booth, I can't help but notice similarities to Bouillabaisse, a more traditional seafood restaurant serving impeccably fresh fish which was quickly closed to make room for Black Roe.
Thankfully, the attention to detail and ethos of Bouillabaisse has been bequeathed to its successor. Cool or not, Black Roe Mayfair is first and foremost a seriously good restaurant. Bringing the colours and aromas of the pacific to central London is a big ask – and they've pulled it off with swagger.
The menu is divided into poke, appetisers and food from the grill. Keen to get to the bottom of the poke craze, we order two different bowls. Both of them things of beauty, adorned with hibiscus flowers, sporting banana leaf fins and showered with sesame seeds.
Classic Ahi Poke (£7.95), a colourful riot of succulent yellowfin tuna chunks in a sesame soy marinade, is a wholehearted success. Chunky and robust, the delicate fish is more than a match for the nutty marinade.
Though more complex, the 'Black Roe' Yellowtail & Ahi Poke (£8.95) is based on the same principle of simple, fresh flavours. A heap of sweet yellowtail mixed together with more rose-coloured chunks of ahi tuna, topped with a signature mound of glittering black roe.
Flecks of onion and parsley bring a welcome tartness to the treacly flavour of the fish. A spicy yuzu sauce adds fire, but bullies the delicate flavour of the fish: the sesame marinade of the Classic Ahi dish is a better fit.
It's not hard to see why poke is such a hit. Scoop a heaping spoonful of fish chunks, sesame seeds and roe, and savour the tender symphony of tropical flavour. Bringing the sour citrus uppercut of ceviche together with the delicate combinations of sushi, it's familiar but while being beguilingly distinct.
Having scraped every last sesame seed from the ornate ceramic bowls, we're well pleased with our introduction to Hawaiian cuisine. But there's more. Ahi tataki toasted brioche (£13.95) is a surreal splash of tropical tones, a miniature Hawaiian island on an oval plate.
Seared tataki tuna slices, earth-brown on the rim and jam-coloured in the middle, are loaded with vines of watercress, bursts of hibiscus, lashings of chilli oil, foothills of crumbly brioche and smoked aubergine, surrounded by an archipelago of chive. It's stunning: for a few moments we don't dare to pick up our cutlery, unwilling to disturb the beauty.
Of course after a few awed murmurs, we dive in. If this dish sounds complicated, the flavour is anything but: zingy, crisp and bold, the fleshiness of the tuna is elaborated by spice and oil and buttressed by the brioche. We scoop, wipe and lick until only a few smears of the masterpiece remain.
Scallops (£14.50) arrive in sea-shell chariots. Sitting in the centre of the shell like a pearl, a mound of scallop wearing a quiff of quinoa crowned with a golden hibiscus is shaded by a bright green stem of cress.
Again, we're delighted: the effort to bring the vibrancy of the Pacific to the tabletop adds so much to the meal. It helps that these scallops are a fine example of the dish: squidgy, tender and redolent with smoke, with a honey-like mango salsa for sweetness.
Blowing everything else out of the water, the octopus is our favourite dish of a very strong bunch. They say that if you don't like an ingredient, it's because you've yet to try it in the right dish. I've never been a big fan of octopus, but at Black Roe every mouthful of it is absolutely superlative (£10.50).
Marinated in soy sauce and cooked in soda water, this is no ordinary octopus. Two cylindrical copper-coloured coils, scorched by that smoke grill, lie beside a bay of lemon celeriac mousse and a ludicrous truffle aioli.
Take a nugget of the octopus, bathe it in the aioli and the mousse, and close your eyes. Each bite unleashes a burst of juiciness, a flurry of sweet, smoky flavour. With the zesty citrus cutting through the charcoal, with the truffle aioli adding subtle earthy tones, every mouthful is both instantly delicious and lingeringly rich. Miraculously free of chewiness, the octopus is beyond tender. By the end we're devastated to see the last chunk disappear from the plate. A total triumph.
The rest of our dishes display the same admirable experimentation, but without quite hitting the same high notes as the octopus or the tataki. Cajun-style blackened sea bass (£14.95) with pineapple salsa is expertly cooked, but the peppery crumb is an uneasy partner to the fish.
By the time the smoky lamb rack (£15.95) arrives, we're overpowered by the smoky flavours. An undeniably effective Kiawe Wood Grill culminates in a nervous reliance on a single dominant flavour. Served with a spicy red coconut salsa and twigs of rosemary, the lamb is as tender as you'd hope, with a punchy paprika seasoning. It's a robust and compelling take on a traditional dish; had we not had so many smoky dishes beforehand, we would've devoured the lamb in half the time.
Dessert is simple and refreshing. We're tempted by a chocolate 'shake 'n' bake' for two (£12.50), but we opt for a simple sorbet. Black Roe have intelligently composed a rejuvenating medley of sorbet flavours, from lemony sorrel to sour yuzu. It's two scoops for £5.95, and a fitting conclusion to the meal.
For all its tropical trimmings, Black Roe Mayfair succeeds because it gets all the basics right: high-quality, fresh food, prepared with imagination and flair, and served by excellent waiting staff. They've managed to bring Hawaii to the table. It's enough to make you fall in love with the exotic all over again.


Unlike other fitness and beauty bloggers, Natasha's Instagram is one that makes you love all of yourself – imperfections and all. Her Instafeed is peppered with fun Yoga and dance routines for health enthusiasts. But it's her 'Beautiful/Still Beautiful' posts that really hit home – it's how she makes body positivity and acceptance the primary message to her hundreds and thousands of followers. Instant love.


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Access to Health Data
Data use registers are a key component of improving transparency and trust in the use of health data for research - they allow patients and members of the public to see and feel confident in how patient data is being requested. They are also very useful for researchers and funders to see what datasets have been made available for research.
The data use register is publicly available to view on the HDR Innovation Gateway
view the data use register
Key Papers and Publications
Until now there have not been any standards for data use registers, making them difficult to access, navigate and review. To address this issue and in our role as the national institute for health data science, the UK Health Data Research Alliance (The Alliance) has created the first set of recommended standards for data use registers.
Read the story behind the latest output from the Alliance
At our first Data Access and Discovery webinar we heard from from Nada Karrar, our Data Access Registers Project Manager, and guest speakers, Angela Coulter, Chair of HDR UK Public Advisory Board, and Victoria Yorke-Edwards, Research Fellow at MRC Clinical Trials Unit UCL.
To watch the event recording, please click here. To view the Q&A from the event, click here. And to continue the...
View the event slides
How can we support adoption of the data use register standard?
In January of 2022 the UK Health Data Research Alliance (the 'Alliance') published recommendations for a data use register standard. The standard outlines five recommendations to support data custodians and controllers responsible for the safe storage, processing and sharing of data, to become more transparent about whom, how and why health data is being used for...
Setting the standards for transparency in the use of health data
UK Health Data Research Alliance publishes first ever recommended standards for data use registers
Why health data access matters to me: My Dad's battle with COVID
"Whilst it saddens me that my dad didn't get the opportunity to benefit from the amazing scientific endeavours of the last two years, it's reassuring that many others have had the chance to...
Improving transparency in the use of health data for research
The use of health data in the research and development of new medicines, therapies and services is essential to the health and wellbeing of society. Controllers and custodians of data play a...
Communication and newsletters
If you would like to sign up to receive more communications about data use registers and the Gateway, please sign up to the Gateway newsletter below.


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A conservative newspaper helped to spearhead an effort to unseal Ellison's divorce papers.
Political enemies of Minnesota's Democratic Rep. Keith Ellison are using every trick to turn what should have been a relatively easy campaign for state attorney general into a nightmare.
A court unsealed the congressman's divorce records on Wednesday under a petition to make them public, MPR News reported.
The petitioners wanted to see if there was documentation that Ellison physically abused his ex-wife, in the hope of substantiating the recent domestic abuse allegations by his ex-girlfriend.
Karen Monahan, the congressman's ex-girlfriend, made the claims in August — just days before Minnesota's primary election. Ellison immediately denied the charge. Monahan claimed to have video evidence that shows Ellison attacking her, but she has declined to release any footage, raising serious doubts about its existence.
Conservative news site Alpha News was one of the petitioners that spearheaded the effort to unseal the divorce records.
However, there was no evidence in the documents that Ellison was abusive toward his ex-wife, Kim Ellison. Surprisingly, the congressman described instances of Kim Ellison physically abusing him.
Meanwhile, social media posts, one of them still active with more than 100,000 shares, shows a picture of Ellison with a fake image that purports to be his ex-girlfriend. However, the woman, whose face was battered and bruised, is not Monahan, Politico reported.
Ellison came into his campaign with significant advantages over his Republican rival, including name recognition, the state's Democratic leanings (Minnesota has not elected a Republican attorney general since 1966) and more than a decade of serving in Congress. But the congressman still finds himself in a tight race, with one poll putting him dead even at 41 percent with his opponent.
Today Botham Shem Jean was supposed to celebrate his 27th story. He was supposed to be a hashtag or a viral story. He wasn't supposed to be gunned down in his own apartment by a police officer on September 6 in Dallas Texas. The St. Lucia native was supposed to be celebrating his life. SEE ALSO: Texas Cop Shoots Unarmed Black Man For Entering His Own Truck The person who shot and killed was Amber Guyger. She was arrested and charged with manslaughter on September 9, more than two days after she killed Jean. After only a few hours in custody, she was released on a bond of $300,000. After five search warrants, her apartment was never searched and she reportedly moved out of the complex the weekend she killed Jean. The 30-year-old was finally fired from the Dallas Police Department on September 24. In honor of Botham Jean, see his life in photos and video. Rest in power.


Really think this would make a better Amtrak scheme than what the Amtrak units actually got.
And heck no, never want design students to come up with a scheme, it would make end up being something with a design just to be a design element and would look forced.
But when it shows up in Philadelphia, you'll be able to cut the ensuing "foam" with a knife!! Guaranteed!
My apologies to anyone who took it that way. Just stating the obvious.
Me personally, I don't think the Septa Sprinter is all that bad looking. I'm actually looking forward to seeing them in action. I will however, miss the AEM-7s when they are gone for good.
liftedjeep wrote: Me personally, I don't think the Septa Sprinter is all that bad looking. I'm actually looking forward to seeing them in action. I will however, miss the AEM-7s when they are gone for good.
You're not going to miss the lone ALP-44?
I should have mentioned the ALP-44 as well. Speaking of which, I do need some better pictures of #2308 before it's retired.
It looks better than that red, white, and blue circus color scheme of the early 198o's.
That will match nicely with the Chinese multi-level cars.
Next to arrive on track one, the Fortune Cookie Flyer to Fox Chase.
Now boarding on track two, the Lo Mein Limited to Lansdale.
NorthPennLimited wrote: It looks better than that red, white, and blue circus color scheme of the early 198o's.
Don't forget the Warminster Wonton Express.
LOVE IT! Much better than in the original rendering. Looks better than even the Amtrak units IMO.
Red? it is Orange and blue.
DutchRailnut wrote: Red? it is Orange and blue.
I think it appears orange because the photographer is somewhat shooting into the sun, and whatever is casting a reflection on the side facing us gives it that illusion. Septa's colors are Red, White & Blue if I'm not mistaken.
Any word on when they are to be shipped east,by Amtrak or the freight RR's?
You know what it looks like? A low budget 1980's toy train version of the real thing.
"from another forum : Siemens will ship from their Sacramento facility, the first 3 MARC Charger locomotives as well as the first SEPTA ACS64.
Union Pacific will pick up the locomotives sometime on Friday."


It started as a rough sketch in my sketchbook then mostly photoshop work... also it's another attempt at painting hair. Hopefully I'll get better at it... but the combination paint smudge tool I used takes forever.
comments and crits always welcome !


Fantastic scenery on your way to Devils Bridge and quite the experience once you do. Go in off season or week day as Devils Bridge gets very crowded. We picked the Long Canyon trailhead to break Mescal into two hikes. We call this one Mescal-Devils Bridge and the northern hike Mescal-Deadman. Download them both for a full day adventure. We measured an elevation change of 443 feet.


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Home News Briefs Benson Tam Returns to iSoftstone Board
Benson Tam Returns to iSoftstone Board
Angela Sormani
Ms. Ling He has resigned as director at IT services and solutions provider iSoftstone and the company has subsequently announced that Benson Bing Chung Tam has rejoined as an independent director. Mr. Tam served as a pre-IPO iSoftStone director from 2007 until December 2010. From 2002 until February 29, 2012, Tam was a partner and head of technology investments at Fidelity Growth Partners Asia (formerly named Fidelity Asia Ventures). Prior to that, he was a partner of Electra Partners Asia from 1998 to 2002 and was the founding director of Hellman & Friedman Asia from 1992 to 1998. Tam worked in M&A corporate finance at S.G. Warburg from 1989 to 1992.
iSoftStone Holdings Limited ("iSoftStone" or the "Company," NYSE: ISS), a leading China-based IT services and solutions provider, today announced that Ms. Ling He resigned as an iSoftstone director effective February 28, 2012. Ms. He joined the iSoftstone Board in 2009 in connection with pre-IPO investments in the Company by SeaBright China Special Opportunities Fund II, L.P., and CSOF Technology Investments Limited. Ms Ling intends to pursue other interests and there are no outstanding disputes between the Company and Ms. Ling relating to iSoftstone's operations, policies or practices.
Mr. Benson Bing Chung Tam has rejoined as an independent Director on the iSoftStone Board of Directors with effect on March 1, 2012. Mr. Tam served as a pre-IPO iSoftStone director from 2007 until December 2010. Mr. Tam will also serve on the Company's Audit Committee.
Mr. T.W. Liu, iSoftStone's Chairman of the Board and Chief Executive Officer, said, "I thank Ms. Ling He for her substantial Board contributions during her two-plus year tenure. Her judgment and guidance were helpful in successfully transitioning iSoftstone to a NYSE listed company."
"It is somewhat rare in any company to be able to welcome back a board member, so I am particularly pleased that Mr. Benson Tam has agreed to rejoin us as an independent Director. Benson's previous contributions to the Board were considerable, so we are very thankful to again have his wise counsel and expertise on the Board."
Mr. Tam is a chartered accountant and prior to the Company's December 2010 IPO served on the Company's audit committee. From 2002 until February 29, 2012, Mr. Tam was a partner and head of technology investments at Fidelity Growth Partners Asia (formerly named Fidelity Asia Ventures). Prior to joining Fidelity Growth Partners Asia, Mr. Tam was a partner of Electra Partners Asia from 1998 to 2002, and was the founding director of Hellman & Friedman Asia from 1992 to 1998. Mr. Tam worked in M&A Corporate Finance at S.G. Warburg from 1989 to 1992.
About iSoftStone Holdings Limited
Founded in 2001, iSoftStone is a leading China-based IT services provider serving both greater China and global clients. iSoftStone provides an integrated suite of IT services and solutions including consulting & solutions, IT services, and business process outsourcing services. The company focuses on industry verticals that include technology, communications, banking, financial services, insurance, energy, transportation, and public sectors. iSoftStone's American depositary shares began trading on the New York Stock Exchange on December 14, 2010.
iSoftStone Holdings Limited
Ms. Sophie Yang
Email: jhyang@isoftstone.com
Mr. Jonathan Zhang
Email: ir@isoftstone.com
Mr. Tom Myers
Email: tmyers@christensenir.com
SOURCE iSoftStone Information Technology (Group) Co., Ltd.


The Care Call Carbon Monoxide Alarm System with Pager quickly alerts carers to the presence of smoke and/or fire, and the Vibrating Pillow Pad vibrates carers awake for night-time monitoring.
The Care Call Smoke Alarm System with Pager and Vibrating Pillow Pad is a complete kit that enables you to be quickly and efficiently alerted to the presence of smoke in your home. The kit comes with three parts – the pager, the charger and the smoke alarm.
The pager has been designed to easily alert you. Simply wear it on your belt, in your pocket or around your neck and the pager will beep if the smoke alarm detects smoke in your home, so there's no risk of missing the alarm.
The included charger will charge the pager at night, ensuring that you can have constant monitoring for the most peace of mind. What's more, the Vibrating Pillow Pad vibrates carers awake if the smoke alarm detects smoke, enabling you to easily monitor your home day and night without losing sleep.
The accompanying Smoke Alarm wirelessly connects to the pager and alerts you to the presence of smoke in your home.


C Minor Chords Musical Soundscape. 128 BPM. Modify/use however you want.
Made with Harmor, FL Studio 11, & Edison.
If you use this in anything, please put it in the comments, me and other people may want to check it out!
I have also credited you. Once again thank you for the sound!
This sample is perfect, thank you very much!


Ibrox Noise: Rangers January report card - how well did Steven Gerrard do?
Rangers January report card - how well did Steven Gerrard do?
So the transfer window has been and gone, and Ibrox Noise decided to give an appraisal and report card of Rangers' business last month. How many of our ratings do you agree with?
It all started with huge fervour as Jermain Defoe and Steven Davis arrived in quickfire succession early on in this window. One was an ex-England international, the other a current Norn Iron international. Theoretically two very fine players, but we were very puzzled to see how Defoe would fit in. Later on, Jordan Jones was added along with Glen Kamara, both on pre-contracts, before Barrow Town's Andy Firth rather suddenly showed up, soon flanked by USA international Matt Polster. Last but not least, at the eleventh hour, Dundee's Glen Kamara was confirmed an immediate signing.
The name was huge, and set Britain's tongue's wagging. It was one of the biggest transfers of the window, period, and almost matched the fever over Steven Gerrard becoming manager. As he's played though, it's been a little less impressive. He's managed a goal, but little else. Hopefully he'll get up to speed but we're struggling to see where he'll fit in in terms of being a starter. Odd one.
Another big name, not as big as Defoe but a fine player and well respected by fans of British football. It was a big coup to get him back from Southampton and he seemed eager to return. His performances have been good – not set the world alight but a smooth and professional level of quality about him and we'll see how he fits in over time.
No one knew who he was. No one still knows who he is. He's been signed for reasons unknown, and he comes from the National League, having been coached in the Liverpool youths by his new manager. It's a curious signing, obviously for goalie depth, but then we've spoken about that already.
Signed for quality depth in defence and midfield, Polster, a one-time US international, does seem to have a bit of something about him – well-rated in the MLS, he's a player who many feel should have achieved more in his career given his talent. Well, he has the chance now.
It's not like Rangers have enough midfielders, right? While some question the signing of yet another man for the middle, this is a top-class Finnish international with a tonne of experience already at only 23. He cost a mere £100,000 and it really could be money well spent. He was very highly rated by Dens' fans till his performances dropped off recently.
Rangers failed to sell anyone this window. No contractual freeings either. On one hand that's good because we didn't lose Alfredo Morelos, James Tavernier or Ross McCrorie (remember Swansea's reported interest) but we also didn't shed any dead wood either. So, that is very disappointing.
Rating: We'd score the retention of our best players 10/10, but the sales of the dross 0. Which one matters more to you? Benefit and a hazard all at the same time.
This was the only way Rangers could get deadwood off the wage bill, even if only temporarily. Some of the loans out we'd actually have preferred to keep – Rossiter, Alnwick, Hardie and suchlike, but others, such as Barjonas, McCrorie and others would not have played enough or got any worthwhile experience staying. It's poor though that Rangers have now got 16 players out on loan – in our lifetimes as Rangers fans we don't recall a loan output that ridiculously high.
Rating: 8 for the fact we got some more deadwood off the payroll for 6 months, 3 for the fact we couldn't sell a single one of them.
Not one mention of Docherty?
over the next 4 months SG and the management team will asses every single player, including the ones on load. Come the next window everyone who doesn't make the grade will go (inc Defoe if he doesn't cut it), we will be left with a more well balanced squad. Watch this space..
It not that simple hence the reason we still have Pena and Herrera stinking the place out( in their absence).
Also think the Manager/Board should have done more to shift some of the Players who aren't gonna play.


We make this at Tru, slice it paper thin and then dry it in the oven to make sweet saffron crostini. We serve it with cinnamon creme brulee and almond risotto or passion fruit mousse.
Make the Pound Cake: Preheat the oven to 350 degrees F. Butter a 6-cup loaf pan and line it with parchment or waxed paper.
In a small saucepan, bring the orange juice and saffron to a boil then turn the heat off immediately and let steep 5 minutes.
In a medium bowl, combine the flour, baking powder, and salt. In a mixer fitted with a whisk attachment (or using a hand mixer), cream the butter. Add 1 cup of the sugar and mix. With the mixer running at low speed, add the eggs one at a time. Working in alternating batches, and mixing after each addition, add the dry ingredients and 1/4 cup of the saffron spiked orange juice, trying to use all the threads in the orange juice. Mix just until smooth.
Pour into the prepared pan and bake until raised in the center and a tester inserted into the center comes out dry and almost clean (a few crumbs are OK), 65 to 75 minutes.
Meanwhile, make the glaze: In a small bowl, stir together the remaining 1/3 cup sugar and the remaining saffron spiked orange juice (about 1/3 cup), until the sugar is dissolved.
When the cake is done, let cool in the pan 15 minutes (it will still be warm). Run a knife around the sides of the pan. Set a wire rack on a sheet pan with sides (to catch the glaze) and turn the cake out onto the rack. Peel off the waxed paper. Using a turkey baster or pastry brush, spread glaze all over the top and sides of the cake and let soak in.
Repeat until all the glaze is used up, including any glaze that has dripped through onto the sheet pan. Let cool at room temperature or, wrapped in plastic wrap, in the refrigerator.
The cake will last up to a week, well-wrapped with plastic wrap.
Make the Lemon Caramel Sauce: Pour the sugar into the center of a deep saucepan. Carefully pour the water around the walls of the pan, trying not to splash any sugar onto the walls. Do not stir; gently draw your finger twice through the center of the sugar, making a cross, to moisten it. Over high heat, bring to a full boil and cook without stirring until amber-colored caramel, about 10 to 15 minutes, swirling the mixture occasionally to even out the color.
Add the lemon juice and vanilla and wait for the caramel to bubble and subside. Let the caramel cool.
To serve, cut the pound cake in thin slices and serve with scoops of cinnamon ice cream and a drizzling of lemon caramel sauce.


It's no secret that as a business owner you have to make sacrifices sometimes, but sleep shouldn't be one of them. A continuous lack of sleep can be harmful to you, your relationships and your business.
The average person needs to have around eight hours of sleep a day, give or take, depending on the individual. You should be aiming for seven to nine hours to be productive and healthy at work.
feel physically and mentally more alert and less likely to make mistakes.
When we sleep, we do more than just physically rest. It's also the time when our brain repairs our bodies, stores memories, and flushes out toxins. Good sleep can give us an overall better sense of health and wellbeing. It makes us less likely to get sick and improves mood, metabolism and our ability to cope with pain.
Keep it consistent: Try to go to bed and get up at roughly the same time every day, even on the days you're not working. According to sleep experts, you should avoid sleeping in, even if you have had a poor night's sleep. Consistency in your sleeping pattern will keep your internal clock regulated. Your internal clock tells your body when to sleep, rise and eat, and also controls body temperature, blood pressure and the daily release of hormones.
Set the right bedroom conditions and turn off your screens: A good sleep environment is dark, comfortable, quiet and slightly cool. Watching television or using your computer or mobile phone in bed should be avoided. Research has shown that screen time before bed suppresses melatonin, the hormone that helps control your sleep and wake cycles. It can also prolong the time it takes to fall asleep and reduce how alert you are the following morning.
Diet and exercise: Try not to eat too close to your bed time. A full stomach can make it difficult to fall asleep. If you are feeling hungry, eat something small and light. Caffeine (tea, coffee and caffeinated soft drinks) and nicotine (cigarettes) and other such stimulants can keep you awake at night. Also try to avoid alcohol as although it may make you drowsy, it can disrupt your sleep. Regular exercise can help you improve your sleep. However, try not to do strenuous exercise too close to bedtime, instead consider lower-intensity workouts, such as yoga or stretches.
Relaxation is key: If you're having trouble winding down, try some relaxing activities such as reading a book, taking a bath, or perhaps try some breathing exercises or meditation. If you find yourself worrying or having stressful thoughts when it's time to sleep, try to set aside some time before bedtime where you can reflect on and manage your concerns. You might like to make a to-do list or just jot down your ideas and thoughts so that they can be put aside until the next day.
Take sleep more seriously: You may feel like sleeping is less important than other activities in your life, but did you know, staying awake for up to 16 hours can have the same impact on your performance as a blood alcohol level of 0.05? After 24 hours that will increase to 0.1 and could increase your risk of an accident or injury.
A-Z fact sheets on sleep-related topics, including healthy sleep as well as common sleep problems.
A personalised six-week program that helps improve your general health and wellbeing by focusing on mood, energy, exercise and sleep.
Article: Is your business robbing you of sleep?
Simple tips for a quality sleep.


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Guide/How To
Smartphone Camera
Android Phone Apps
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Honor Play Review: a reliable gaming companion
By PhoneYear
"Crazy fast, Crazy smart" is the tagline for Honor's first phone with the revolutionary GPU Turbo Technology, Honor Play. It is a respectable mid-range phone with a surprising number of features borrowed from flagships like the Mate 10 and P20 Pro.
With its big screen, a powerful processor, and the Turbo tech, Honor Play is the best choice for your intense PUBG sessions.
But is it worth all the hype? Does the Turbo tech perform as advertised? Is the device worth every penny of your money? Read on to find the answers in our in-depth review of the phone.
Design and Display
We got our hands on the black colored Honor Play, which is a metal unibody, a slim and sleek gadget. We have seen some very cool designs earlier from Honor at the time of Honor 10 and Honor 9 Lite. Still, aesthetically, this phone doesn't stand on par with few of the fantastic designs of the family but instead gives a feel of an Honor 7C successor in terms of build. However, it sits in hand pretty well due to its appropriate heft.
The placement of keys is a usual trait that we usually see on the Honor and Huawei phones. On the right-hand side, you'll find a power key along with a volume rocker, both of which are well within the hand's reach and can be operated single-handedly with ease.
On the left side rests a dual nano-SIM card slot, which can also be used to expand the memory with a microSD card if you wish to use a single SIM. It can be used one-handed, but reaching the top part of the screen is a bit difficult and requires two-hand use.
The device packs a generous 6.3-inch LCD with a 1080 x 2340 resolution, and the iPhone X inspired notch. Well, because it's 2018 and iPhone is probably the culmination of industrial design, so we see a herd of phones copying the same design in one way or the other.
With a 19.5:9 aspect ratio, Honor Play offers a vivid display that furnishes accurate colors and sharpness as much as expected from an LCD Panel.
The viewing angles are pretty good to a certain degree, but as the angle gets smaller, the screen loses its viewability to some extent. The display gets specifically dull when looking from the top. This may not be a serious issue as we have not encountered any problems in normal usage.
Other than that, the screen really struggles in direct sunlight. Even with the brightness cranked to the full, it is pretty hard to browse through the phone outdoors, let alone watch movies or play games in sunlight.
Honor Play features the EMUI 8.2 skin on top of Android 8.1 Oreo. If you have already handled Honor 10, the experience is very similar here. It uses multiple home screens to house all of your icons.
However, if you are addicted to the app drawer, EMUI lets you change the layout and enable the app drawer. There are many other options in the latest EMUI, which include lowering down the display resolution to conserve battery, changing the warmth of display, and the list goes on.
Honor Play Game Mode
The game mode is another useful feature that you can avail so that during those intense and immersive gaming sessions when you are enjoying a PUBG game with your buddies, no one interrupts.
There is also a party mode that lets you connect with 7 other Honor Play devices to stream the same music through all of them, thus creating a surround sound effect.
What we didn't like is the number of preinstalled apps. Honor really needs to address this and rid its phones of the bloatware that comes preinstalled. Consistently using phone's resources, these apps tend to drag the performance of the device down.
Performance and Battery
The highlighting feature of the Honor Play is its Kirin 970 chipset, which is the same that powers Huawei's top-notch phones like the Mate 10 series and the P20 Pro. It's awe-inspiring to see this chip in a device with such a low price tag.
Other specs of the phone include Mali-G72 MP12 GPU, 4GB RAM, and a huge 64GB of internal storage, which should be enough for heavy users, including mobile gamers.
The device also performed well in benchmarking apps and scored decently on AnTuTu and Geekbench.
These amazing specs make the phone sail through the most rigorous and heavy usage easily. In addition to the hardware, as we have earlier mentioned, Honor Play comes with software-based GPU Turbo technology, which is not just gimmickry. It enhances the phone's performance by 60% and decreases battery usage by 30%.
With so much hype, we really wanted to check whether the phone is really "crazy fast" as the company claims.
So we went to play intensive games, and heavy multitasking, and the device turned out to be what the Honor promised it though the company has compromised on design and many other things but has indeed come out with a fantastic gaming phone and that too within a controlled price.
Playing Asphalt 9 on Honor Play
The new Asphalt 9: Legends ran on the device like a charm. We didn't notice any lag, pauses, glitches, or any frame drops during the game.
Honor Play comes with a massive 3750mAh battery that also supports 18W fast charging which can charge up the phone from 0% to 50% in 30 minutes
In our real-life 1 hour test, the phone consumed 13% battery, which is far better than Oppo F7 (which lost 19% in moderate usage). In another heavy usage test, the phone consumed 19% battery with intense gaming and heavy multitasking, which indicates that it packs an outstanding battery with good optimization to fulfill the needs of even the most avid gamers.
The phone packs a dual-camera setup on its back with a 16-megapixel, f/2.0 aperture lens, and a secondary 2-megapixel sensor with f/2.4 that is used to collect the depth in the picture. Upfront, you can find a 16-megapixel camera sitting in the notch.
In auto-mode, the Honor Play turns out to produce true and natural colors and shows decent enough dynamic range in good lighting conditions. The HDR mode does an excellent job of brightening the darker corners of the picture while maintaining the exposure.
Artificial intelligence in the camera enhances the picture and is capable of recognizing 22 objects in 500 different scenarios.
Though this might be true, the AI robs the picture of its natural colors and aggressively treats the image to a point where it starts looking artificial, which is a huge turn-off.
AI Mode
Wide Aperture mode in the Honor Play is not the best we've seen, but it works just fine. You can clearly see some patches where it failed to blur the back and somewhere it excessively did.
Wide Aperture Mode
Portrait Mode
The portrait mode identifies the subject promptly and does an excellent job of distinguishing between the background and the foreground.
Here are some of the more samples of aggressive AI treatment of the phone and also how it performs at night along with HDR Mode.
As expected, Honor Play isn't capable of great low light images, but that's just fine considering the price tag. The AI, however, helps and brightens up the dark corners and gives a better low light experience. With a well-lit subject, the pictures can come out very convincing.
Honor Play can shoot 4K videos at 30fps and 1080p videos at 60fps. Moreover, if you are a photography enthusiast, we really think you should take pictures without the AI on, to avoid the over-saturation and extra brightening of the colors.
As the name suggests, Honor Play is basically a gaming-centric phone which truly justifies its name with ultra-fast and super-smooth gaming. But you don't play games all day, do you?
The phone is a sturdy yet slim and sleek device with a long-lasting metal build, and while this may be a personal preference, with such a build, the Honor Play looks cheaper than it actually is. The camera on the phone failed to impress us, and the AI looked nothing but gimmickry having the photos treated harshly and enhancing the colors unwantedly.
Should you buy it? Well, that depends on your needs and usage pattern. If it's about gaming and performance along with a superb battery, you can go for it with your eyes closed, and you'll not repent.
But if its camera and premium design you are after and willing to compromise on the performance part, Honor Play might not be the phone for you.
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I am trying to find a good ambisonic library for Linux pd. Does anyone have a recommendation? The Hoalib looks perfect for ambisonic decoding and encoding, but I can't find a Linux build for pd anywhere and building from source is not working out as the configure for it seems to have a typo and fails for me every time on Ubuntu Studio 16.04. Does anyone have the externals already built that they could send me or have any tips for building from source?
Thanks for any time and help!