Cross-dressed CD8α+/CD103+ dendritic cells prime CD8+ T cells following vaccination

Activation of naïve cluster of differentiation (CD)8⁺ cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8⁺ T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide–MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8⁺ T cells and requires CD8α⁺/CD103⁺ DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8⁺ T-cell responses to viral infection, tumors, and vaccines.Professional antigen-presenting cells (APCs) are typically required to activate naïve cluster of differentiation (CD)8⁺ T cells, either by direct priming or cross-priming. In direct priming, infected (viral infection) or directly transfected (DNA vaccination) APCs synthesize the foreign antigen and use endogenous MHC class I pathways of antigen presentation to present antigen and prime CD8⁺ T cells. In cross-priming, APCs are able to capture, process, and present exogenous antigen onto MHC class I molecules through a process known as cross-presentation (1). Cross-priming has been shown to be an essential pathway for immunity to many viral infections and tumors. Although the pathways that lead to cross-presentation remain incompletely understood, increasing evidence suggests that only certain dendritic cell (DC) subsets are efficient in this process.Cross-dressing involves the transfer of intact MHC class I/peptide complexes between cells without the requirement for further processing, representing an alternative pathway of indirect antigen presentation (2, 3). Although cross-dressed DCs can activate memory CD8⁺ T cells following viral infection in vivo (4), it remains unclear whether cross-dressing can prime naïve CD8⁺ T-cell responses, what DC subtypes are required to prime CD8⁺ T cells by cross-dressing, and how robust this pathway is compared with traditional pathways of indirect antigen presentation. These questions must be addressed before the physiologic relevance of cross-dressing can be evaluated in context.To address these questions, we have taken advantage of Batf3-deficient mice and engineered MHC class I single chain trimer (SCT) constructs. Batf3^−/− mice have a selective loss of CD8α⁺ and CD103⁺ DCs, without abnormalities in other hematopoietic cell types or architecture (5). DCs from Batf3^−/− mice are deficient in cross-presentation, and cytotoxic T lymphocyte (CTL) responses to viral infection and syngeneic tumors are impaired in Batf3^−/− mice. Thus, Batf3^−/− mice represent a valuable model system to study cross-presentation, cross-dressing, and the role of CD8α⁺/CD103⁺ DCs following DNA or cellular vaccination. We have previously engineered completely assembled MHC class I SCT whereby all three components of the complex (heavy chain, β₂m, and peptide) are attached by flexible linkers (6). Through progressive molecular engineering, even peptides with low binding affinities can be successfully anchored in the peptide binding groove by a disulfide trap between the first linker and the heavy chain (7–9). Using these experimental tools, we demonstrate that cross-dressing is a robust pathway of antigen presentation following DNA and cellular vaccination, capable of priming naïve and memory CD8⁺ T cells. In addition, we demonstrate that CD8α⁺/CD103⁺ DCs are required to prime CTLs by cross-dressing.     

Adult human circulating CD34⁻Lin⁻CD45⁻CD133⁻ cells can differentiate into hematopoietic and endothelial cells

A precise identification of adult human hemangioblast is still lacking. To identify circulating precursors having the developmental potential of the hemangioblast, we established a new ex vivo long-term culture model supporting the differentiation of both hematopoietic and endothelial cell lineages. We identified from peripheral blood a population lacking the expression of CD34, lineage markers, CD45 and CD133 (CD34?Lin?CD45?CD133? cells), endowed with the ability to differentiate after a 6-week culture into both hematopoietic and endothelial lineages. The bilineage potential of CD34?Lin?CD45?CD133? cells was determined at the single-cell level in vitro and was confirmed by transplantation into NOD/SCID mice. In vivo, CD34?Lin?CD45?CD133? cells showed the ability to reconstitute hematopoietic tissue and to generate functional endothelial cells that contribute to new vessel formation during tumor angiogenesis. Molecular characterization of CD34?Lin?D45?CD133? cells unveiled a stem cell profile compatible with both hematopoietic and endothelial potentials, characterized by the expression of c-Kit and CXCR4 as well as EphB4, EphB2, and ephrinB2. Further molecular and functional characterization of CD34?Lin?CD45?CD133? cells will help dissect their physiologic role in blood and blood vessel maintenance and repair in adult life.     

Regulatory dendritic cells program B cells to differentiate into CD19hiFcγIIbhi regulatory B cells through IFN-β and CD40L

Regulatory dendritic cells (DCs) play important roles in the induction of peripheral tolerance and control of adaptive immune response. Our previous studies demonstrate that splenic stroma can drive mature DCs to proliferate and further differentiate into a unique subset of CD11b(hi)Ia(low) regulatory DCs, which could inhibit T-cell response, program generation of immunosuppressive memory CD4 T cells. However, the effect of regulatory DCs on B-cell function remains unclear. Here, we report that regulatory DCs can induce splenic B cells to differentiate into a distinct subtype of IL-10-producing regulatory B cells with unique phenotype CD19(hi)FcγIIb(hi). CD19(hi)FcγIIb(hi) B cells inhibit CD4 T-cell response via IL-10. CD19(hi)FcγIIb(hi) B cells have enhanced phagocytic capacity compared with conventional CD19(+) B cells, and FcγRIIb mediates the uptake of immune complex by CD19(hi)FcγIIb(hi) B cells. We found that regulatory DC-derived IFN-β and CD40 ligand are responsible for the differentiation of CD19(hi)FcγIIb(hi) B cells. Furthermore, an in vivo counterpart of CD19(hi)FcγIIb(hi) B cells in the spleen and lymph nodes with similar phenotype and regulatory function has been identified. Our results demonstrate a new manner for regulatory DCs to down-regulate immune response by, at least partially, programming B cells into regulatory B cells.     

ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3− NK cells

Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient’s body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 ± 0.5 CD3⁺ cells/μl with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3⁺ T cell number of 221 × 10³ (0.09%, CD34 selected grafts, n = 187), 900 × 10³ (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 × 10³ (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations. U. Koehl and K. Bochennek contributed equally to the work.     

Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease

Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4⁺ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4⁺ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8⁺ αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8⁺ αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases.     

Differentiation of endothelial cells from human umbilical cord blood AC133−CD14+ cells

Endothelial progenitor cells (EPCs) participate in neovascularization and are consistent with postnatal vasculogenesis. In vitro, they differentiate into endothelial cells (ECs). Prior reports have suggested that circulating human AC133(+) cells have the capacity to differentiate into ECs as progenitor cells. However, recent studies have demonstrated that circulating CD34(-)CD14(+) cells also have EPC-like properties in vitro and in vivo. We tested whether AC133(-)CD14(+) cells from human umbilical cord blood (HUCB) have the potential to differentiate into ECs. The AC133(-)CD14(+) cells were isolated from HUCB by magnetic bead selection and cultured on fibronectin-coated six-well trays in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin growth factor (IGF-1). The AC133(-)CD14(+) cells adhered slightly within 1 day of culture and subsequently underwent a distinct process of morphological transformation to spindle-shaped cells that sprouted from the edge of the cell clusters. After 14 days, the cells formed cord- and tubular-like structures. The AC133(-)CD14(+) cells showed a strong increase in the endothelial marker P1H12 over time, whereas CD14 decreased, and CD45 did not change, respectively. In addition, the cells expressed endothelial markers von Willebrand's factor (vWF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1, VEGFR-2/Flk-1, eNOS, and VE-cadherin, but did not express Tie-2 after 7 days of culture. The present data indicate that AC133(-)CD14(+) cells from HUCB are able to develop endothelial phenotype with expression of endothelial-specific surface markers and even form cord- and tubular-like structures in vitro as progenitor cells.     

Do CD8 effector cells need IL-7R expression to become resting memory cells?

The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into "full" hosts underwent a protracted and unremitting contraction compared with IL-7R-expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell-deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R-deficient effector cells were rapidly lost from IL-15-deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process.     

Human acute myeloid leukemia CD34+CD38? stem cells are susceptible to allorecognition and lysis by single KIR-expressing natural killer cells

The concept of tumor immunosurveillance has raised prospects for natural killer cell-based immunotherapy of human cancer. The cure of acute myeloid leukemia may depend on eradication of leukemic stem cells, the self-renewing component of leukemia. Whether natural killer cells can recognize and lyse leukemic stem cells is not known. To develop strategies that effectively target acute myeloid leukemia-leukemic stem cells, we investigated anti-leukemic effects of human alloreactive single KIR⁺ natural killer cells. Natural killer effectors with KIR specificity mismatched with respect to HLA class I allotype of target cells effectively recognized acute myeloid leukemia-leukemic stem cells defined phenotypically as CD34⁺CD38⁻, while healthy bone marrow-derived CD34⁺CD38⁻ hematopoietic stem cells were spared, as demonstrated by cytotoxicity and hematopoietic colony-forming assays. The HDAC inhibitor valproic acid increased the activating NKG2D ligand-dependent lysis of acute myeloid leukemia-CD34⁺CD38⁻ leukemic stem cells. These results show that alloreactive natural killer cells have the potential to detect and target leukemic stem cells, and thus to improve the treatment outcome in acute myeloid leukemia.     

CD7 expression by CD34+ cells in CML patients,of prognostic significance?

The purpose of the study was to identify a unique immunophenotype of normal or Philadelphia chromosome positive (Ph+) CD34+ cells that might be used to purify normal CD34+ cells from chronic myelogenous leukemia (CML) patients. An immunophenotypical study of CD34+ bone marrow cells of 20 patients with CML at diagnosis and during hydroxyurea treatment, and 39 controls were performed. All patients were Ph+, two patients had variant translocations and three patients displayed cytogenetic signs of clonal evolution. The immature progenitor cell compartment (CD34+ HLA-DR- and CD34+ CD38- cells) was comparable. The CD34+ AC133+ progenitor cell compartment was decreased in CML patients. We found no difference for any of the adhesion molecules examined except for CD62L, where the percentage of CD34+ CD62L+ cells was decreased in CML patients. The number of myeloid progenitors (CD34+ CD33+) was increased at the expense of B-lymphoid progenitors (CD34+ CD10+ and CD34+ CD19+) in CML patients indicating that B-lymphopoiesis is inhibited in CML. The megakaryocytic (CD34+ CD61+) and erythroid (CD34+ CD71+) progenitors were increased in CML patients. The number of CD34+ CD7+ cells was also significantly increased (mean 25.3% vs. 4.9%). However, the level of CD7 expression was quite heterogeneous, and the patients could be separated into two populations according to CD7 expression (more or less than 20% CD7+ CD34+ cells). The Sokal and Hasford risk scores did not differ between CD34+ CD7- CML and CD34+ CD7+ CML, but all patients with signs of disease progression clustered in the CD34+ CD7+ population indicating that the level of CD7 expression on CD34+ cells may be of prognostic importance in CML.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Recently primed CD8+ T cells entering the liver induce hepatocytes to interact with naïve CD8+ T cells in the mouse

Large number of T cells traffic through the liver. In order to examine the effects of such traffic on the phenotype of hepatocytes, we vaccinated mice using DNA vaccines encoding antigens with MHC class I-binding epitopes. Small numbers of activated CD8(+) T blasts (10(5)-10(6)/liver) changed the surface phenotype and cytokine expression profile of hepatocytes (HCs). HCs upregulate surface expression of major histocompatibility complex (MHC) class I molecules and CD1d but not MHC class II molecules Qa-1, CD80, CD86, CD54, or CD95; in addition, they expressed/secreted interleukin (IL)-10 and IL-4 but not IL-1, IL-6, IL-13, interferon (IFN)-gamma, tumor necrosis factor (TNF), IL-4, or IL-27 (i.e., they acquire the HC* phenotype). HCs* (but not HCs) induced specific activation, proliferation, and IFN-gamma, TNF, and IL-13 release of cocultured na?ve CD8(+) T cells. In contrast to the specific activation of na?ve CD8(+) T cells by dendritic cells (DCs), specific CD8(+) T cell activation by HC* was not down-modulated by IFN-alphabeta. Only recently activated CD8(+) T blasts (but not recently activated CD4(+) T blasts or activated cells of the innate immune system, including natural killer T [NKT] cells) induced the HC* phenotype that is prominent from day 10 to day 20 postvaccination (i.e., time points at which peak numbers of recently primed CD8(+) T blasts are found in the liver). In conclusion, recently activated CD8(+) T blasts that enter the liver postimmunization in small numbers can transiently modulate the phenotype of HC, allowing them to activate na?ve CD8(+) T cells with unrelated specificities.     

Expansion of CD4+CD25+ regulatory T cells from cord blood CD4+ cells using the common ��-chain cytokines (IL-2 and IL-15) and rapamycin

Rapamycin has important roles in the modulation of regulatory T cells. We tried to expand CD4(+)CD25(+) regulatory T cells (Treg cells) from umbilical cord blood (CB) CD4-positive cells using interleukin (IL)-15 or IL-2 with transforming growth factor (TGF)-β and rapamycin. We were able to obtain more than 500-fold expansion of CD4(+)CD25(+) cells from CB CD4(+) cells using IL-15 and TGF-β with rapamycin. These expanded CD4(+)CD25(+) cells expressed forkhead box P3 (FoxP3) mRNA at a level about 100-fold higher and could suppress allogeneic mixed lymphocyte culture (MLC) by more than 50%. Early after rapamycin stimulation, CB CD4(+) cells showed increased expression of FoxP3 and a serine/threonine kinase Pim2 and sustained expression of negative phosphoinositide 3-kinase regulator phosphatase and tensin homolog deleted on chromosome 10 (PTEN). On the other hand, CD4(+)CD25(+) cells expanded with rapamycin for 8 days showed much higher levels of FoxP3 mRNA expression and decreased expression of PTEN. A comparison of IL-15 stimulation and IL-2 stimulation showed slightly higher efficiency of IL-15 for expansion of CD4(+)CD25(+) cells, and for FoxP3 expression, IL-15 also showed significantly higher efficacy for inhibition of MLC. The combination of the common γ-chain cytokine IL-15, TGF-β, and rapamycin may be a useful means for expanding Treg cells. Pim2 expression early after stimulation with rapamycin may be important for conferring rapamycin resistance for growth of Treg cells. IL-15 is not less useful than IL-2 for expansion of Treg cells.