�����ƽ��

B1 cells differ in many ways from conventional B cells, most prominently in the production of natural immunoglobulin, which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here, we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion, efficient T cell stimulation, and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70, both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age, which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease.

In elderly subjects and in patients with chronic inflammatory diseases, there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype, whose origin and functional relevance has not been well characterized. In this study, we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells, and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence, intracellular cytokine expression, ability to interact with endothelial cells, and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p textless 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%, respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209, release of cytokines in response to low-intensity stimulus, and increased capacity to sustain lymphocyte proliferation. Finally, compared with CD14(++)CD16(-) cells, CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary, our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells, with increased inflammatory activity and ability to interact with endothelial cells. Therefore, accumulation of senescent monocytes may explain, in part, the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular bulk degradation pathway that plays critical roles in eliminating intracellular pathogens, presenting endogenous Ags, and regulating T lymphocyte survival and proliferation. In this study, we have investigated the role of autophagy in regulating the endoplasmic reticulum (ER) compartment in T lymphocytes. We found that ER content is expanded in mature autophagy-related protein (Atg) 7-deficient T lymphocytes. Atg7-deficient T cells stimulated through the TCR display impaired influx, but not efflux, of calcium, and ER calcium stores are increased in Atg7-deficient T cells. Treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor thapsigargin rescues the calcium influx defect in Atg7-deficient T lymphocytes, suggesting that this impairment is caused by an intrinsic defect in ER. Furthermore, we found that the stimulation-induced redistribution of stromal interaction molecule-1, a critical event for the store-operated Ca(2+) release-activated Ca(2+) channel opening, is impaired in Atg7-deficient T cells. Together, these findings indicate that the expanded ER compartment in Atg7-deficient T cells contains increased calcium stores, and the inability of these stores to be depleted causes defective calcium influx in these cells. Our results demonstrate that autophagy plays an important role in maintaining ER and calcium homeostasis in T lymphocytes.

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue, activate complement, and induce intestinal damage. Because C3 cleavage products act as ligands for CR2, we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire, we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage, C3 deposition, and inflammation in response to IR. In contrast, similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production, we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore, Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally, adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs.

The T-cell functions of a proliferation-inducing ligand (APRIL, also known as TNFSF13) remain largely undefined. We previously showed that APRIL suppressed Th2 cytokine production in cultured CD4(+) T cells and Th2 antibody responses. Here we show that APRIL suppresses allergic lung inflammation, which is associated with diminished expression of the transcription factor c-maf. Mice deficient in the April gene (April(-/-) mice) had significantly aggravated lung inflammation compared with WT mice in the ovalbumin-induced allergic lung inflammation model. Likewise, blockade of APRIL in WT mice by the APRIL-receptor fusion protein, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI)-Ig, enhanced lung inflammation. Transfer of APRIL-sufficient, ovalbumin-specific, TCR-transgenic CD4(+) T (OT-II) cells to April(-/-) mice restored the suppressive effect of APRIL on lung inflammation. Mechanistically, the expression of the Th2 cytokine transcription factor c-maf, but not GATA-3, was markedly enhanced in April(-/-) CD4(+) T cells at the RNA and protein level and under non-polarizing (Th neutral, ThN) and Th2-polarizing conditions. Since c-maf transactivates the IL-4 gene, the increased c-maf expression in April(-/-) mice readily explains increased Th2 cytokine production. Independent of its effect on IL-4, APRIL suppressed IL-13 expression. APRIL thus may regulate lung inflammation in a dual way, by acting on c-maf expression and by directly controlling IL-13 production.

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID), GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

Although oral dendritic cells (DCs) were shown to induce cell-mediated immunity, the identity and function of the various oral DC subsets involved in this process is unclear. In this study, we examined the mechanisms used by DCs of the buccal mucosa and of the lining mucosa to elicit immunity. After plasmid DNA immunization, buccally immunized mice generated robust local and systemic CD8(+) T cell responses, whereas lower responses were seen by lining immunization. A delayed Ag presentation was monitored in vivo in both groups; yet, a more efficient presentation was mediated by buccal-derived DCs. Restricting transgene expression to CD11c(+) cells resulted in diminished CD8(+) T cell responses in both oral tissues, suggesting that immune induction is mediated mainly by cross-presentation. We then identified, in addition to the previously characterized Langerhans cells (LCs) and interstitial dendritic cells (iDCs), a third DC subset expressing the CD103(+) molecule, which represents an uncharacterized subset of oral iDCs expressing the langerin receptor (Ln(+)iDCs). Using Langerin-DTR mice, we demonstrated that whereas LCs and Ln(+)iDCs were dispensable for T cell induction in lining-immunized mice, LCs were essential for optimal CD8(+) T cell priming in the buccal mucosa. Buccal LCs, however, failed to directly present Ag to CD8(+) T cells, an activity that was mediated by buccal iDCs and Ln(+)iDCs. Taken together, our findings suggest that the mechanisms engaged by oral DCs to prime T cells vary between oral mucosal tissues, thus emphasizing the complexity of the oral immune network. Furthermore, we found a novel regulatory role for buccal LCs in potentiating CD8(+) T cell responses.

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

Adoptive immunotherapy is evolving to assume an increasing role in treating cancer. Most imaging studies in adoptive immunotherapy to date have focused primarily on locating tumor-specific T cells rather than understanding their effector functions. In this study, we report the development of a noninvasive imaging strategy to monitor T-cell activation in living subjects by linking a reporter gene to the Granzyme B promoter (pGB), whose transcriptional activity is known to increase during T-cell activation. Because pGB is relatively weak and does not lead to sufficient reporter gene expression for noninvasive imaging, we specifically employed 2 signal amplification strategies, namely the Two Step Transcription Amplification (TSTA) strategy and the cytomegalovirus enhancer (CMVe) strategy, to maximize firefly luciferase reporter gene expression. Although both amplification strategies were capable of increasing pGB activity in activated primary murine splenocytes, only the level of bioluminescence activity achieved with the CMVe strategy was adequate for noninvasive imaging in mice. Using T cells transduced with a reporter vector containing the hybrid pGB-CMVe promoter, we were able to optically image T-cell effector function longitudinally in response to tumor antigens in living mice. This methodology has the potential to accelerate the study of adoptive immunotherapy in preclinical cancer models.

To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM), we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group, a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells, FISH succeeded in 272 of 301 (90.4%) PCM, but in only 302 of 381 (79.3%) MGUS cases (P textless 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range, 0-9) and MGUS one (range, 0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes, del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001), del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P textless 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients.

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

Retinoic acid (RA) imprints gut-homing specificity on T cells upon activation by inducing the expression of chemokine receptor CCR9 and integrin α4β7. CCR9 expression seemed to be more highly dependent on RA than was the α4β7 expression, but its molecular mechanism remained unclear. In this article, we show that NFAT isoforms NFATc1 and NFATc2 directly interact with RA receptor (RAR) and retinoid X receptor (RXR) but play differential roles in RA-induced CCR9 expression on murine naive CD4(+) T cells. TCR stimulation for 6-24 h was required for the acquisition of responsiveness to RA and induced activation of NFATc1 and NFATc2. However, RA failed to induce CCR9 expression as long as TCR stimulation continued. After terminating TCR stimulation or adding cyclosporin A to the culture, Ccr9 gene transcription was induced, accompanied by inactivation of NFATc1 and sustained activation of NFATc2. Reporter and DNA-affinity precipitation assays demonstrated that the binding of NFATc2 to two NFAT-binding sites and that of the RAR/RXR complex to an RA response element half-site in the 5'-flanking region of the mouse Ccr9 gene were critical for RA-induced promoter activity. NFATc2 directly bound to RARα and RXRα, and it enhanced the binding of RARα to the RA response element half-site. NFATc1 also bound to the NFAT-binding sites and directly to RARα and RXRα, but it inhibited the NFATc2-dependent promoter activity. These results suggest that the cooperativity between NFATc2 and the RAR/RXR complex is essential for CCR9 expression on T cells and that NFATc1 interferes with the action of NFATc2.