T cells bearing the CD44hi "memory" phenotype display characteristics of activated cells in G1 stage of cell cycle.

T cells capable of anamnestic proliferative responses to antigen in vitro (i.e., "memory" cells) have been shown to display the CD44hi CD45RBlo surface phenotype. To assess the state of activation of these T cells, CD4+ T cells expressing the CD44hi or CD45RBlo phenotype were compared to CD4+ T cells expressing the CD44lo or CD45RBhi phenotype in the context of expression of the "activated" (asialo-GM1hi) vs "resting" (asialo-GM1lo) phenotype and in the context of cell size, total protein content, and total RNA content. Dual fluorescence analysis demonstrated that all CD4+ T cells expressing the CD44hi phenotype also expressed the asialo-GM1hi phenotype associated with cell activation. In vitro proliferative assays confirmed that the CD4+ asialo-GM1hi, the CD4+ CD45RBlo, and the CD4+ CD44hi FACS-sorted populations displayed stronger in vitro responsiveness to stimulation with immobilized anti-CD3 mAB than the CD4+ asialo-GM1lo, CD45RBhi, or CD44lo populations. Acridine orange analysis of sorted CD44hi/lo fractions revealed that the diploid (G1) population of the CD44hi T cells displayed a higher mean RNA content than the CD44lo T cells. Similarly, the CD44hi T cells displayed a higher mean cell size and a higher mean total protein content than the CD44lo CD4+ T cells. Similar results were obtained with asialo-GM1 and CD45RB subsets of CD4+ T cells. The basal rate of protein synthesis, as determined by [3H]leucine incorporation, was approximately 50% higher in the CD44hi small CD4+ T cells than in the CD44lo CD4+ T cells. Based on the knowledge that cell size, total protein and RNA content, and responsiveness to signals inducing proliferation are lowest in G0 stage of cycle and increase through G1 stage of cycle, it appears that the CD44hi CD45RBlo T cells exist in a higher activation state than CD44lo CD45RBhi T cells. The previously demonstrated association of CD44hi CD45RBlo phenotype with memory T cells suggests that the CD44hi memory T cells are maintained in G1 (not necessarily cycling) rather than resting "out of cycle" in G0.     

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.     

A unique hybrid renal mononuclear phagocyte activation phenotype in murine systemic lupus erythematosus nephritis

Renal infiltration with mononuclear cells is associated with poor prognosis in systemic lupus erythematosus. A renal macrophage/dendritic cell signature is associated with the onset of nephritis in NZB/W mice, and immune-modulating therapies can reverse this signature and the associated renal damage despite ongoing immune complex deposition. In nephritic NZB/W mice, renal F4/80(hi)/CD11c(int) macrophages are located throughout the interstitium, whereas F4/80(lo)/CD11c(hi) dendritic cells accumulate in perivascular lymphoid aggregates. We show here that F4/80(hi)/CD11c(int) renal macrophages have a Gr1(lo)/Ly6C(lo)/VLA4(lo)/MHCII(hi)/CD43(lo)/CD62L(lo) phenotype different from that described for inflammatory macrophages. At nephritis onset, F4/80(hi)/CD11c(int) cells upregulate cell surface CD11b, acquire cathepsin and matrix metalloproteinase activity, and accumulate large numbers of autophagocytic vacuoles; these changes reverse after the induction of remission. Latex bead labeling of peripheral blood Gr1(lo) monocytes indicates that these are the source of F4/80(hi)/CD11c(int) macrophages. CD11c(hi)/MHCII(lo) dendritic cells are found in the kidneys only after proteinuria onset, turnover rapidly, and disappear rapidly after remission induction. Gene expression profiling of the F4/80(hi)/CD11c(int) population displays increased expression of proinflammatory, regulatory, and tissue repair/degradation-associated genes at nephritis onset that reverses with remission induction. Our findings suggest that mononuclear phagocytes with an aberrant activation profile contribute to tissue damage in lupus nephritis by mediating both local inflammation and excessive tissue remodeling.     

Breast cancer cells expressing stem cell markers CD44<Superscript>+</Superscript> CD24<Superscript>lo</Superscript> are eliminated by Numb-1 peptide-activated T cells

Cancer stem cells (CSC) are resistant to chemo- and radiotherapy. To eliminate cells with phenotypic markers of CSC-like we characterized: (1) expression of CD44, CD24, CD133 and MIC-A/B (NKG2 receptors) in breast (MCF7) and ovarian (SK-OV-3) cells resistant to gemcitabine (GEM), paclitaxel (PTX) and 5-fluorouracil (5-FU) and (2) their elimination by Numb- and Notch-peptide activated CTL. The number of cells in all populations with the luminal CSC phenotype [epithelial specific antigen⁺ (ESA) CD44^hi CD24^lo, CD44^hi CD133⁺, and CD133⁺ CD24^lo] increased in drug-resistant MCF7 and SK-OV-3 cells. Similarly, the number of cells with expressed MIC-A/B increased 4 times in drug-resistant tumor cells compared with drug-sensitive cells. GEM^Res MCF7 cells had lower levels of the Notch-1-extracellular domain (NECD) and Notch trans-membrane intracellular domain (TMIC) than GEM^Sens MCF7. The levels of Numb, and Numb-L-[P]-Ser²⁶⁵ were similar in GEM^Res and GEM^Sens MCF7 cells. Only the levels of Numb-L (long)-Ser²⁹⁵ decreased slightly. This finding suggests that Notch-1 cleavage to TMIC is inhibited in GEM^Res MCF7 cells. PBMC activated by natural immunogenic peptides Notch-1 (2112–2120) and Numb-1 (87–95) eliminated NICD^positive, CD24^hi CD24^lo MCF7 cells. It is likely that the immunogenic Numb-1 peptide in MCF7 cells originated from Numb, [P]-lated by an unknown kinase, because staurosporine but not wortmannin and MAPK-inhibitors decreased peptide presentation. Numb and Notch are antagonistic proteins which degrade each other to stop and activate cell proliferation, respectively. Their peptides are presented alternatively. Targeting both antagonistic proteins should be useful to prevent metastases in patients whose tumors are resistant to conventional treatments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Notch pathway plays a novel and critical role in regulating responses of T and antigen-presenting cells in aGVHD

Graft-versus-host disease (GVHD) induced by host antigen-presenting cells (APCs) and donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). Notch signaling pathway is a highly conserved cell-cell communication that is important in T cell development. Recently, Notch signaling pathway is reported to be involved in regulating GVHD. To investigate the role of Notch inhibition in modulating GVHD, we established MHC-mismatched murine allo-BMT model. We found that inhibition of Notch signaling pathway by γ-secretase inhibitor in vivo could reduce aGVHD, which was shown by the onset time of aGVHD, body weight, clinical aGVHD scores, pathology aGVHD scores, and survival. Inhibition of Notch signaling pathway by DAPT ex vivo only reduced pathology aGVHD scores in the liver and intestine and had no impact on the onset time and clinical aGVHD scores. We investigated the possible mechanism by analyzing the phenotype of host APCs and donor-derived T cells. Notch signaling pathway had a broad effect on both host APCs and donor-derived T cells. The expressions of CD11c, CD40, and CD86 as the markers of activated dendritic cells (DCs) were decreased. The proliferative response of CD8+ T cell decreased, while CD4⁺ Notch-deprived T cells had preserved expansion with increased expressions of CD25 and Foxp3 as markers of regulatory T cells (Tregs). In conclusion, Notch inhibition may minimize aGVHD by decreasing proliferation and activation of DCs and CD8⁺ T cells while preserving Tregs expansion.     

Beta 1 integrin (CD29) expression on human postnatal T cell subsets defined by selective CD45 isoform expression

The integrin beta 1 (CD29) is a marker for total very late activation Ag integrins on cells, and exhibits considerable fluctuation in cell surface density at various stages of T cell development. We have analyzed beta 1 integrin expression on subsets of human thymus, and on T cells from healthy babies and children, in comparison to healthy adults aged 26 to 75. T cells from adult peripheral blood include a CD29-, a CD29lo, and a CD29hi set. Compared with adults, PBMC T cells from children have reduced numbers of both CD29lo and CD29hi subsets but equivalent numbers of CD29- T cells. The number of CD29hi T cells increases gradually with age, achieving adult levels only at about 26 yr of age; in aged adults (69 to 75 yr), nearly all T cells have a CD29hi phenotype. Most thymocytes and cord blood T cells, in contrast, have a single peak of CD29 staining that is intermediate to the two peaks seen in adults. Multi-negative progenitor and CD45RO- thymocytes (presumptive thymic generative line-age) are 98% CD29hi. Progenitor thymocytes and adult PBMC T cells express equivalent amounts of beta 1 and alpha 4, but progenitors are alpha 5hi, whereas PBMC T cells are alpha 5lo. T cells from children have reduced beta 1hi and alpha 5lo, but nearly comparable numbers of alpha 4hi. This suggests that the major very late activation Ag integrins during childhood may be alpha 5 beta 1 and alpha 4 complexed with an alternate beta chain. In children, the majority of CD29hi cells are also CD45RAhi, in contrast to the pattern in adults, in whom the majority of CD29hi T cells are CD45RA-. This suggests that in children, the main defense against infection may reside in the CD29hi45RAhi T cells, which have not yet made the transition to CD45RO and to bona fide memory status. The proliferative response to tetanus toxoid of 4- to 6-mo-old babies correlates with the number of CD29hi45RAhi T cells, suggesting that it derives at least in part from cells that do not express a "memory" phenotype. These observations show a pattern of alternating high and low density CD29 during T cell development, which is consistent with the idea that CD29 is a marker for functionally defined T cell sets. Analysis of the CD29 expression of CD29hi thymocytes developing in vitro supports this view. We suggest that the intensity of CD29 expression on a T cell varies, dependent upon the microenvironmental interactions required by a differentiating T cell.     

The "early' CD4+CD8+ stage of thymocte differentiation hallmarks the end of a strong positive correlation between extracellular matrix receptor expression and protein tyrosine phosphorylation.

We used irradiation-induced thymic regression/reconstitution to study phosphotyrosine (PTyr) levels and expression of extracellular matrix receptors in thymocyte subsets by flow cytometry. High PTyr levels (PTyr(hi)) characterized cells from the CD4-CD8-(DN)CD25in/hi to the "early" CD4-CD8+(DP)CD25- stage. Correlation indexes (R) between the percentages of these PTyrhi cells and cells with up-regulated expression of alpha4 integrin (alpha4hi) were strongly positive (R= 0.91, P= 0.002, for DN; R= 0.98, P= 0.0001 for DP). At the "early" DP stage, R between PTyrhi cells and cells with up-regulated expression of alpha5 integrin and L-selectin (alpha5hi and L-sel(hi)) also rendered strongly positive (R>0.95, p<0.0003). "Late" expanding DP cells exhibited intermediate PTyr levels (PTyr(in)), associated with a down-regulation of the adhesion receptors assessed. Triple-labeling suggested that in most early CD3-/lo cells, alpha4hi and alpha5hi, but not L-sel(hi) expression preceded a PTyr(hi) content. CD3in/hi-enriched CD8+ cells were also PTyr(hi), but conversely to the immature ones exhibited a tendency for a negative R between PTyr(hi) and alpha4hi (R = -0.93, P = 0.067, n= 4) or alpha5hi cells (R = -0.77, P = 0.23, n = 4). CD4+ cells were either PTyr(hi) or PTyr(in), exhibiting a tendency for a positive R (R = 0.59, P = 0.124, n= 8) between PTyr(hi) and L-sel(hi) cells only. In conclusion, our results associate an up-regulation of alpha4 and alpha5 chains expression with PTyr(hi) levels and, as elsewhere published, with increased adhesion to fibronectin up to the "early" DP stage, but not afterwards.     

Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G₂/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.     

CD14, CD16 and HLA-DR reliably identifies human monocytes and their subsets in the context of pathologically reduced HLA-DR expression by CD14(hi) /CD16(neg) monocytes: Expansion of CD14(hi) /CD16(pos) and contraction of CD14(lo) /CD16(pos) monocytes in acute liver failure

Changes in monocytes and their subsets (CD14(hi) /CD16(neg) , CD14(hi) /CD16(pos) and CD14(lo) /CD16(pos) ) have been described in several diseases. The combination of CD14, CD16 and HLA-DR has been suggested to discriminate monocytes from the CD16(pos) /HLA-DR(neg) NK-cells and neutrophils but no data exist whether this strategy can be used in situations when monocyte HLA-DR expression is pathologically reduced. Monocytes and their subsets were concurrently identified through negative (exclusion of CD66b(pos) neutrophils, CD56(pos) NKcells, CD19(pos) B-cells, and CD3(pos) T-cells) and positive gating (inclusion of monocytes by expression of CD14, CD16, and HLA-DR) strategies on 30 occasions [9 healthy controls (HC) and 21 patients with conditions associated with low monocyte HLA-DR expression]. Bland-Altman and Passing and Bablok regression statistics did not demonstrate any significant measurement bias between the two strategies of monocyte identification. Monocyte subset phenotype was then compared in 18 HC and 41 patients with acute liver failure (ALF). Compared with HC, in ALF, the percentage of CD14(hi) /CD16(pos) monocytes was higher (7% vs 4%) whilst the percentage of CD14(lo) /CD16(pos) was lower (1.9% vs. 7%) (P ≤ 0.001); HLA-DR and CD86 MFIs on all monocyte subsets were lower, whilst CCR5, CD64, and CD11b MFIs were higher (P < 0.05). The relative expression by monocyte subsets of HLA-DR, CCR2, CCR5, CX3CR1, and CD11a was similar in ALF patients and HCs. Repeat analysis of an identical antibody-fluorochrome "backbone" targeting HLA-DR, CD14, and CD16 was assessed in 189 samples across 5 different experiments. There was excellent agreement in the results obtained using the positive gating strategy (interclass correlation coefficients > 0.8). Monocytes and their subsets can be reliably identified using an antibody-fluorochrome "backbone" of HLA-DR, CD14, and CD16. CD16(pos) monocytes continue to constitutively express HLA-DR even in conditions where HLA-DR is pathologically reduced on CD14(hi) /CD16(neg) monocytes. Understanding the changes in monocyte pheontype in ALF and similar clinico-pathological diseases may allow the development of novel biomarkers or therapeutic strategies. ? 2012 International Society for Advancement of Cytometry.     

Down-regulation of Notch1 by gamma-secretase inhibition contributes to cell growth inhibition and apoptosis in ovarian cancer cells A2780

The release of Notch intracellular domain (NICD) is mediated by γ-secretase. γ-Secretase inhibitors have been shown to be potent inhibitors of NICD. We hypothesized that Notch1 is acting as an oncogene in ovarian cancer and that inhibition of Notch1 would lead to inhibition of cell growth and apoptotic cell death in ovarian cancer cells. In this study, expressions of Notch1 and hes1 in four human ovarian cancer (A2780, SKOV3, HO-8910, and HO-8910PM), and one ovarian surface (IOSE 144) cell lines were detected by Western blot and quantitative real-time RT-PCR. The effects of γ-secretase inhibition (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT) were measured by MTT assay, flow cytometry, ELISA and colony-forming assay. Our results showed that Notch1 and hes1 were found in all the four human ovarian cancer and IOSE 144 cell lines, and they were significantly higher in ovarian cancer cells A2780 compared to another four ovarian cells. Down-regulation of Notch1 expression by DAPT was able to substantially inhibit cell growth, induce G1 cell cycle arrest and induce cell apoptosis in A2780 in dose- and time-dependent manner. In addition, hes1 was found to be down-regulated in dose- and time-dependent manner by DAPT in A2780. These results demonstrate that treatment with DAPT leads to growth inhibition and apoptosis of A2780 cells in dose- and time-dependent manner. These findings also support the conclusion that blocking of the Notch1 activity by γ-secretase inhibitors represents a potentially attractive strategy of targeted therapy for ovarian cancer.     

Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Protocadherin-18 is a novel differentiation marker and an inhibitory signaling receptor for CD8+ effector memory T cells

CD8(+) tumor infiltrating T cells (TIL) lack effector-phase functions due to defective proximal TCR-mediated signaling previously shown to result from inactivation of p56(lck) kinase. We identify a novel interacting partner for p56(lck) in nonlytic TIL, Protocadherin-18 ('pcdh18'), and show that pcdh18 is transcribed upon in vitro or in vivo activation of all CD8(+) central memory T cells (CD44(+)CD62L(hi)CD127(+)) coincident with conversion into effector memory cells (CD44(+)CD62L(lo)CD127(+)). Expression of pcdh18 in primary CD8(+) effector cells induces the phenotype of nonlytic TIL: defective proximal TCR signaling, cytokine secretion, and cytolysis, and enhanced AICD. pcdh18 contains a motif (centered at Y842) shared with src kinases (QGQYQP) that is required for the inhibitory phenotype. Thus, pcdh18 is a novel activation marker of CD8(+) memory T cells that can function as an inhibitory signaling receptor and restrict the effector phase.     

Induction of a germinal center phenotype in B cells in vitro by a Th2 cell line

We have investigated the contribution of various stimuli for generating in vitro the changes in surface phenotype characteristic of B cells responding to a T-dependent antigen in a germinal center (GC). We show that, unlike many other stimuli such as B cell mitogens, cytokines, and surrogate antigen, alone or in combination, an alloreactive Th2 clonal line induces splenic B cells to become cell surface peanut agglutinin (PNA)(hi), Ig(lo), CD62L(lo), and CD44(hi) to produce mRNA for M17 and to express a GC-specific transgene even without B cell receptor ligation. Neither proliferation nor prior activation of responding B cells is needed, but B cells from CD45-null mice show reduced efficiency of this induction. These findings open up possibilities for separation and dissection of the various components of the GC response.     

Notch-1 Signaling Regulates Microglia Activation via NF-κB Pathway after Hypoxic Exposure In Vivo and In Vitro

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.     

Host-reactive CD8+ memory stem cells in graft-versus-host disease

Graft-versus-host disease (GVHD) is caused by alloreactive donor T cells that trigger host tissue injury. GVHD develops over weeks or months, but how this immune response is maintained over time is unknown. In mouse models of human GVHD, we identify a new subset of postmitotic CD44(lo)CD62L(hi)CD8(+) T cells that generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8(+) T cells, while self-renewing. These cells express Sca-1, CD122 and Bcl-2, and induce GVHD upon transfer into secondary recipients. The postmitotic CD44(lo)CD62L(hi)CD8(+) T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigens and dendritic cells and require interleukin-15. Thus, their long life, ability to self-renew and multipotentiality define these cells as candidate memory stem cells. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including GVHD.     

NK1.1+ thymocytes. Adult murine CD4-, CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-V beta 8+ subset.

CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.     

CD2 costimulation reveals defective activity by human CD4+CD25(hi) regulatory cells in patients with multiple sclerosis

Studying the activity of homogeneous regulatory T cell (Treg) populations will advance our understanding of their mechanisms of action and their role in human disease. Although isolating human Tregs exhibiting low expression of CD127 markedly increases purity, the resulting Treg populations are still heterogeneous. To examine the complexity of the Tregs defined by the CD127 phenotype in comparison with the previously described CD4(+)CD25(hi) subpopulations, we subdivided the CD25(hi) population of memory Tregs into subsets based on expression of CD127 and HLA-DR. These subsets exhibited differences in suppressive capacity, ability to secrete IL-10 and IL-17, Foxp3 gene methylation, cellular senescence, and frequency in neonatal and adult blood. The mature, short telomere, effector CD127(lo)HLA-DR(+) cells most strongly suppressed effector T cells within 48 h, whereas the less mature CD127(lo)HLA-DR(-) cells required 96 h to reach full suppressive capacity. In contrast, whereas the CD127(+)HLA-DR(-) cells also suppressed proliferation of effector cells, they could alternate between suppression or secretion of IL-17 depending upon the stimulation signals. When isolated from patients with multiple sclerosis, both the nonmature and the effector subsets of memory CD127(lo) Tregs exhibited kinetically distinct defects in suppression that were evident with CD2 costimulation. These data demonstrate that natural and not induced Tregs are less suppressive in patients with multiple sclerosis.