CD8alpha+ dendritic cells originate from the CD8alpha- dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 up-regulation.

CD8alpha+ and CD8alpha- dendritic cells (DCs) have been considered as independent DC subpopulations both ontogenetically and functionally during recent years. However, it has been demonstrated that both DC subsets can be generated from a single precursor population, supporting the concept that they do not represent separate DC lineages. By using highly purified splenic CD8alpha- DCs, which were injected intravenously and traced by means of an Ly5.1/Ly5.2 transfer system, this study shows that CD8alpha- DCs acquired the phenotypic characteristics of CD8alpha+ DCs, by a differentiation process involving CD8alpha, DEC-205, and CD24 up-regulation, paralleled by the down-regulation of CD11b, F4/80, and CD4. These data demonstrate that CD8alpha+ DCs derive from CD8alpha- DCs, and strongly support that CD8alpha- and CD8alpha+ DCs represent different maturation or differentiation stages of the same DC population. Therefore, CD8alpha+ DCs would represent the last stage of DC differentiation, playing an essential role in the induction of T-cell responses, due to their antigen-presenting potential, cross-priming ability, and capacity to secrete large amounts of key cytokines such as interferon gamma and interleukin-12.     

CD8alpha-11b+ dendritic cells but not CD8alpha+ dendritic cells mediate cross-tolerance toward intestinal antigens

Cross-presentation is a critical process by which antigen is displayed to CD8 T cells to induce tolerance. It is believed that CD8alpha+ dendritic cells (DCs) are responsible for cross-presentation, suggesting that the CD8alpha+ DC population is capable of inducing both cross-priming and cross-tolerance to antigen. We found that cross-tolerance against intestinal soluble antigen was abrogated in C57BL/6 mice lacking mesenteric lymph nodes (MLNs) and Peyer patches (PPs), whereas mice lacking PPs alone were capable of developing CD8 T-cell tolerance. CD8alpha-CD11b+ DCs but not CD8alpha+ DCs in the MLNs present intestinal antigens to relevant CD8 T cells, while CD8alpha+ DCs but not CD8alpha-CD11b+ DCs in the spleen exclusively cross-present intravenous soluble antigen. Thus, CD8alpha-CD11b+ DCs in the MLNs play a critical role for induction of cross-tolerance to dietary proteins.     

Identification of CD8alpha+CD11c- lineage phenotype-negative cells in the spleen as committed precursor of CD8alpha+ dendritic cells

CD8alpha+ dendritic cells (DCs) represent a functionally distinct DC subset in vivo, which plays a critical role in initiating various cellular immune responses. However, the committed precursor of CD8alpha+ DCs remains to be identified. We reported here that murine splenic CD8alpha+CD11c- lineage phenotype (Lin)- cells could differentiate into CD8alpha+ DCs in vivo after intravenous transplantation. Immunohistochemistry staining showed that donor-derived DCs mainly located in T-cell areas of the spleen. Functionally, these CD8alpha+CD11c-Lin- cell-derived DCs were capable of stimulating allogenic T-cell response, as well as secreting bioactive interleukin 12 p70 and interferon gamma. Freshly isolated CD8alpha+CD11c-Lin- cells expressed CC chemokine receptor (CCR)2, CCR5, and CCR7 messenger RNA, whereas CD8alpha+ DCs derived from CD8alpha+CD11c-Lin- cells further obtained the expression of CCR6 and macrophage-derived chemokine. Flow cytometry analysis showed that CD8alpha+CD11c-Lin- cells were identified in bone marrow and lymph nodes. Moreover, transplanted splenic CD8alpha+CD11c-Lin- cells could also home to thymus and lymph nodes and were capable of developing into CD8alpha+ DCs in these locations. However, CD8alpha+CD11c-Li- cells failed to differentiate into CD8alpha- DCs, T cells, natural killer cells, or other myeloid lineage cells in irradiated chimeras. Taken together, all these findings suggest that CD8alpha+CD11c-Lin- cells are a committed precursor of CD8alpha+ DCs.     

CD8alpha+ mouse spleen dendritic cells do not originate from the CD8alpha- dendritic cell subset

Although previous studies had indicated that the CD8alpha- and CD8alpha+ subtypes of murine dendritic cells (DCs) differ in immediate origin, a recent study found that intravenous transfer of CD8alpha- DCs led to CD8alpha+ DCs in the spleen several days later, suggesting a direct precursor-product relationship. We have repeated these experiments with a balance sheet approach. We find that though a few CD8alpha+ DCs can be generated in such experiments, this is a rare event and could be the result of a contaminant precursor. Most of the immediate precursors of CD8alpha+ DCs are cells that lack the phenotype of a recognizable DC. CD8alpha- DCs and CD8alpha+ DCs are not precursor-product related, though these sublineages may be connected further upstream.     

Tumor rejection by the poliovirus receptor family ligands of the DNAM-1 (CD226) receptor

The poliovirus receptor CD155 and its family member CD112 (nectin-2) are the ligands for the activating cell-surface receptor DNAM-1 on CD8+ T cells and natural killer (NK) cells. Here, we demonstrate that, whereas the RMA tumor grew in syngeneic mice, DNAM-1 ligand-transduced RMA was rejected, in which CD8+ T cells and NK cells played an essential role. Importantly, CD8+ memory cytotoxic T cells to parental RMA were generated in these mice. We found that DNAM-1 was also expressed on CD8alpha+, rather than CD8alpha-, dendritic cells (DCs). Cross-linking DNAM-1 induced maturation of CD8alpha+ DCs. Antigen presentation by these stimulated DCs drove Th1 cells. Moreover, the rejection of DNAM-1 ligand-transduced RMA was canceled in CD4+ T-cell-depleted and major histocompatibility complex class II-deficient mice. Taken together, these results suggest that DNAM-1 ligands stimulate innate immunity by CD8alpha+ DCs as well as NK cells, which efficiently prime cell-mediated tumor-specific immunity.     

Generation of autologous cytotoxic and helper T-cell responses against the B-cell leukemia-associated antigen HB-1: relevance for precursor B-ALL-specific immunotherapy

Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (BALL) occur frequently after primary treatment. Therefore, development of additional treatment modalities to eliminate residual tumor cells is needed. Active immunotherapy using dendritic cells (DCs) loaded with tumor-associated antigens is a promising approach to induce specific T-cell immunity in patients with cancer. In previous studies, we described HB-1 as a B-cell lineage-specific antigen that is recognized by donor-derived cytotoxic T lymphocytes (CTLs) on allogeneic B-ALL tumor cells. Here, we investigated the potential use of the HB-1 antigen as an autologous T-cell vaccine target. To determine whether HB-1-specific CTL precursors are present within the T-cell repertoire, we induced expansion of CD8+ T cells using mature monocyte-derived DCs pulsed with the previously identified HB-1.B44 antigenic peptide. In 6 of 8 donors, CD8+ CTL lines have been generated that exert cytotoxicity against target cells exogenously pulsed with peptide or endogenously expressing the HB-1 antigen. From one of these HB-1-specific T-cell lines, we isolated a CD8+ CTL that produces interferon-gamma on stimulation with B-ALL tumor cells. Interestingly, the HB-1 antigen also induced CD4+ T-helper responses on activation with protein-loaded mature monocyte-derived DCs. We identified 2 novel epitopes recognized in the context of HLA-DR4 and HLA-DR11 with the use of HB-1-specific CD4+ T-cell clones generated from different donors. These present data, that HB-1 induces both helper and cytotoxic T-cell responses, indicate that the HB-1 antigen is a candidate target to induce T-cell-mediated antitumor immunity in patients.     

CD3+, 4+ and/or 8+ T cells and CD3+, 4-, 8- T cells repopulate at different rates after allogeneic bone marrow transplantation

The antigen receptors of the majority of peripheral blood T lymphocytes are constituted of alpha- and beta-chains in association with CD3. The phenotype of those T cell receptor-alpha, beta cells is CD3+, 4+ and/or 8+. The small subset of CD3+, 4-, 8- T cells includes TCR-gamma, delta cells. These two T cell subsets have different TCR gene rearrangement patterns, tissue distributions and mechanisms of antigen recognition. We studied the repopulation of both T cell subsets in 20 allogeneic marrow graft recipients in relation to the type of graft (T cell-depleted versus non-depleted) and the occurrence of active cytomegalovirus (CMV) infection, using three-color immunofluorescence and flow cytometry. The CD3+, 4+ and/or 8+ and CD3+, 4-, 8- T cells had clearly different repopulation patterns. At 1 month post-BMT, they had repopulated the blood to similar levels. Thereafter, the CD3+, 4+ and/or 8+ T cells increased further in number, whereas the CD3+, 4-, 8- T cells stabilized on average between 100 and 200 x 10(6)/l. The nine recipients of T cell-depleted marrow grafts showed a relatively delayed repopulation of their CD3+, 4+ and/or 8+ T cells compared with the 11 recipients of non-depleted marrow. In contrast, the repopulation rate of the CD3+, 4-, 8- T cells was similar in both groups. The occurrence of active CMV infection post-BMT was associated with an increased rate of repopulation of the CD3+, 4+ and/or 8+ T cells, particularly those expressing HNK1, but did not affect the repopulation of the CD3+, 4-, 8- T cells.     

Priming CD8+ T cells with dendritic cells matured using TLR4 and TLR7/8 ligands together enhances generation of CD8+ T cells retaining CD28

TLRs expressed on dendritic cells (DCs) differentially activate DCs when activated alone or in combination, inducing distinct cytokines and costimulatory molecules that influence T-cell responses. Defining the requirements of DCs to program T cells during priming to become memory rather than effector cells could enhance vaccine development. We used an in vitro system to assess the influence of DC maturation signals on priming naive human CD8+ T cells. Maturation of DCs with lipopolysaccharide (LPS; TLR4) concurrently with R848 (TLR7/8) induced a heterogeneous population of DCs that produced high levels of IL12 p70. Compared with DCs matured with LPS or R848 alone, the DC population matured with both adjuvants primed CD8+ T-cell responses containing an increased proportion of antigen-specific T cells retaining CD28 expression. Priming with a homogenous subpopulation of LPS/R848-matured DCs that were CD83(Hi)/CD80+/CD86+ reduced this CD28+ subpopulation and induced T cells with an effector cytokine signature, whereas priming with the less mature subpopulations of DCs resulted in minimal T-cell expansion. These results suggest that TLR4 and TLR7/8 signals together induce DCs with fully mature and less mature phenotypes that are both required to more efficiently prime CD8+ T cells with qualities associated with memory T cells.     

Ontogeny and innate properties of neonatal dendritic cells

We investigated whether a developmental immaturity of the dendritic cells (DCs) compartment could contribute to the high susceptibility to infections observed in newborns. DCs are among the first cells to colonize the spleen, but the ontogeny of DC subsets follows distinct steps. At birth, plasmacytoid DCs and CD4-CD8alpha- DCs are found in the spleen, whereas CD8alpha+ and CD4+ DCs are not present. Then, the CD8alpha+ DC compartment quickly develops and reaches an adult size by day 7, whereas the CD4+ DC compartment slowly increases to become predominant by the age of 3 weeks. The production of interleukin (IL)-12p70 by DCs is particularly efficient after birth, reflecting the stronger capacity of the neonatal CD8alpha- DCs to secrete IL-12 compared with its adult counterpart. Like-wise, neonatal DCs produced type I and II interferons. In vivo, following microbial stimulation, up-regulation of major histocompatibility complexes (MHCs) and of costimulatory molecules on DCs was induced clearly showing the activation of neonatal DCs in the neonatal environment. Therefore, despite a markedly different DC subset composition in early life compared with the adult DC compartment, neonatal DCs are fully competent in their innate immune functions.     

CD8+ T-cell-mediated killing of donor dendritic cells prevents alloreactive T helper type-2 responses in vivo

Accumulating evidence indicates that, in absence of CD8+ T-cell activation, CD4+ T-cell-mediated allograft rejection is associated with a dominant Th2-cell response and eosinophil infiltrates. In this study, we analyzed the mechanisms by which CD8+ T cells regulate alloreactive CD4+ T-cell priming and differentiation into interleukin 4 (IL-4)-producing cells. We showed that interferon gamma (IFN-gamma) production by CD8+ T cells was dispensable for the inhibition of Th2-cell development, as well as tissue eosinophilia and type 2 cytokine production in the rejected grafts. Since we noticed that CD8+ T cells not only suppressed Th2 differentiation, but also down-modulated the overall priming of alloreactive CD4+ T cells, we evaluated whether CD8+ T cells act by limiting the accumulation of donor-derived dendritic cells (DCs) in lymph nodes. We found that indeed, alloreactive CD8+ T cells rapidly eliminated allogeneic DCs from T-cell areas of draining lymph nodes, through a perforin-dependent mechanism. Thus, our data demonstrate that cytotoxic T lymphocyte (CTL)-mediated clearance of allogeneic DCs is a negative feedback mechanism that limits the duration of alloantigen presentation in draining lymph nodes, thereby modulating the amplitude and polarization of the primary alloreactive CD4+ T-cell responses.     

Tumor antigen processing and presentation depend critically on dendritic cell type and the mode of antigen delivery

Dendritic cells (DCs) are being evaluated for cancer immunotherapy due to their unique ability to induce tumor-directed T-cell responses. Here we report that the type of human DC, the mode of activation, and the strategy for delivery of antigen are 3 critical factors for efficient stimulation of tumor-specific CD8+ and CD4+ T cells. Only CD1c+ blood DCs and monocyte-derived DCs (MoDCs) were capable of presenting epitopes of the full-length tumor antigen NY-ESO-1 on both major histocompatibility complex (MHC) class I (cross-presentation) and MHC II, whereas plasmacytoid DCs were limited to MHC II presentation. Cross-presentation was inefficient for soluble protein, but highly efficient for antigen-antibody immune complexes (NY-ESO-1/IC) and for protein formulated with ISCOMATRIX adjuvant (NY-ESO-1/IMX). DC activation with CD40L further enhanced cross-presentation efficiency. The mode of antigen delivery was found to be a determining factor for cytosolic proteolysis by DCs. Immune complexes (ICs) targeted a slow, proteasome-dependent cross-presentation pathway, whereas ISCOMATRIX (IMX) targeted a fast, proteasome-independent pathway. Both cross-presentation pathways resulted in a long-lived, T-cell stimulatory capacity, which was maintained for several days longer than for DCs pulsed with peptide. This may provide DCs with ample opportunities for sensitizing tumor-specific T cells against a broad array of tumor antigen epitopes in lymph nodes.     

HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes

Aim:  Dendritic cells (DCs) pulsed with HBsAg efficiently reverse the immune tolerance to hepatitis B virus (HBV) and induce HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice and healthy volunteers. However, it is not clear whether HBV core antigen (HBcAg)-pulsed DCs can effectively induce CD4⁺ helper T cells polarization into Th1, which contribute to the induction and maintenance of HBV-specific CD8⁺ T cells in chronic hepatitis B (CHB) patients. To address this issue, we conducted this study and investigated whether HBcAg-pulsed DCs could polarize Th1 cells and induce an HBcAg-specific CTL response.
Methods:  HBcAg-pulsed DCs were generated from 21 CHB patients. The capacity of the HBcAg-pulsed DC vaccine to stimulate CD4⁺ and CD8⁺ T cells to produce IFN-γ and IL-4 was estimated by intercellular cytokine staining, and the HBcAg-pulsed DCs derived from 10 humam leucocyte antigen (HLA)-A2⁺ CHB patients were tested for the induction of HBV-specific CTLs from autologous T cells by pentamer staining. The cytotoxicity of these CTLs was evaluated in vitro by flow cytometry.
Results:  The HBcAg-pulsed DCs derived from CHB patients exhibited a stronger capacity to stimulate autologous CD4⁺ and CD8⁺ T cells to release IFN-γ rather than IL-4, which could induce HBV core 18-27 specific CTLs, suggesting a specific cytotoxicity against T2 cells that had been loaded with the HBV core 18-27 peptide in vitro .
Conclusion:  HBcAg-pulsed DC vaccine derived from CHB patients efficiently induced autologous T cell polarization to Th1 and generation of HBV core 18-27 specific CTLs.     

Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8 alpha(+) dendritic cells and reduces experimental acute graft-versus-host disease

Recent evidence suggests that dendritic cells (DCs) can regulate and amplify immune responses. Flt3 ligand (FL)-derived DC function was tested as a stimulator of allogeneic lymphocytes in vitro and in vivo. Treatment of mice with FL dramatically expanded DC number, but DCs isolated from FL-treated mice (FL DCs) were poor stimulators of allogeneic T-cell responses in vitro. Further activation of FL DCs did not restore their stimulatory ability, and FL DCs did not suppress the stimulation of the allogeneic T cells by normal DCs. FL treatment significantly increased the CD8 alpha(+) DC subset, which appeared to be the reason for their poor stimulatory capacity. These observations were confirmed in vivo using a mouse model of acute graft-versus-host disease (GVHD) wherein host DCs play a critical role. FL treatment of recipients before allogeneic bone marrow transplantation dramatically suppressed donor T-cell responses to host antigens, thereby reducing GVHD mortality (P <.01). These data represent a novel strategy that alters host DCs and reduces acute GVHD.     

The differential production of cytokines by human Langerhans cells and dermal CD14(+) DCs controls CTL priming

We recently reported that human epidermal Langerhans cells (LCs) are more efficient than dermal CD14(+) DCs at priming naive CD8(+) T cells into potent CTLs. We hypothesized that distinctive dendritic cell (DC) cytokine expression profiles (ie, IL-15 produced by LCs and IL-10 expressed by dermal CD14(+) DCs) might explain the observed functional difference. Blocking IL-15 during CD8(+) T-cell priming reduced T-cell proliferation by ～ 50%. These IL-15-deprived CD8(+) T cells did not acquire the phenotype of effector memory cells. They secreted less IL-2 and IFN-γ and expressed only low amounts of CD107a, granzymes and perforin, and reduced levels of the antiapoptotic protein Bcl-2. Confocal microscopy analysis showed that IL-15 is localized at the immunologic synapse of LCs and naive CD8(+) T cells. Conversely, blocking IL-10 during cocultures of dermal CD14(+) DCs and naive CD8(+) T cells enhanced the generation of effector CTLs, whereas addition of IL-10 to cultures of LCs and naive CD8(+) T cells inhibited their induction. TGF-β1 that is transcribed by dermal CD14(+) DCs further enhanced the inhibitory effect of IL-10. Thus, the respective production of IL-15 and IL-10 explains the contrasting effects of LCs and dermal CD14(+) DCs on CD8(+) T-cell priming.     

Antigen-induced, tolerogenic CD11c+,CD11b+ dendritic cells are abundant in Peyer's patches during the induction of oral tolerance to type II collagen and suppress experimental collagen-induced arthritis

OBJECTIVE: Although oral tolerance is a well-known phenomenon, the role of dendritic cells (DCs) is not well characterized. This study was conducted to better understand the differential role played by each Peyer's patch DC subset in the induction of oral tolerance to type II collagen (CII) in murine collagen-induced arthritis (CIA). METHODS: CII was fed 6 times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. We compared the proportion of CD11c+,CD11b+ DCs and CD11c+,CD8alpha+ DCs in the Peyer's patches of CII-fed tolerized and phosphate buffered saline-fed nontolerized mice after the induction of CIA. The immunosuppressive properties of each DC subset were determined using fluorescence-activated cell sorter analysis for intracellular interleukin-10 (IL-10) and IL-12 and mixed lymphocyte culture. The ability of each DC subset to induce CD4+,CD25+ T regulatory cells was also examined. Mice were injected with CII-pulsed CD11c+,CD11b+ DCs isolated from Peyer's patches of tolerized mice, and the effect on CIA was examined. RESULTS: The severity of arthritis was significantly lower in tolerized mice. The proportion of CD11c+,CD11b+ DCs was increased in the Peyer's patches of tolerized mice and those DCs exhibited immunosuppressive characteristics, such as increased IL-10 production, inhibition of T cell proliferative responses to CII, and CD4+,CD25+ regulatory T cell induction. Furthermore, the CD11c+,CD11b+ DCs suppressed the severity of arthritis upon adoptive transfer. CONCLUSION: Our observations demonstrate that CD11c+,CD11b+ DCs, which are abundant in Peyer's patches during the induction of oral tolerance to CII, are crucial for the suppression of CIA and could be exploited for immunotherapy of autoimmune diseases.     

Autoimmune-mediated intestinal inflammation-impact and regulation of antigen-specific CD8+ T cells

BACKGROUND & AIMS: Few data exist regarding mechanisms of mucosal CD8+ T-cell reactivity to epithelial-specific antigen. To dissect the immunologic mechanisms underlying CD8+ T-cell dysregulation, reactivity to a self-antigen expressed in intestinal epithelium of mice bearing a major histocompatibility complex class I-restricted T-cell receptor specific for this antigen was studied. In addition, antigen-specific regulatory CD4+ T cells induced in vivo were tested to control these autoreactive CD8+ T cells. METHODS: Transgenic VILLIN-HA mice were mated with CL4-TCR transgenic mice. Alternatively, adoptive transfer of CL4-TCR transgenic CD8+ T cells into VILLIN-HA transgenic mice was performed to mimic spontaneous encounter of neoantigen. Mucosal CD8+ T cells were characterized under different conditions of tolerance, immunopathology, and active immunosuppression. RESULTS: Transgenic CD8+ T cells from VILLIN-HA x CL4-TCR transgenic mice preferentially migrated and expanded in mucosal lymphoid tissues. Although transgenic CD8+ T cells showed signs of T-cell activation, they failed to cause tissue damage. This was accompanied by the induction/expansion of CD4+ and CD8+, Foxp3-expressing T cells. In contrast, adoptive transfer of naive transgenic CD8+ T cells from CL4-TCR transgenic mice into VILLIN-HA transgenic mice induced severe intestinal inflammation with poor clinical course of disease. Transgenic CD8+ T cells secreted vigorous amounts of proinflammatory cytokines like interferon gamma/tumor necrosis factor alpha. Strikingly, this acute wasting disease was significantly ameliorated by cotransfer of antigen-specific regulatory CD4+ T cells. CONCLUSIONS: Epithelial-specific antigen expression is sufficient to trigger severe antigen-specific CD8+ T-cell-mediated intestinal inflammation; this might be controlled by antigen-specific regulatory T cells under physiological conditions.     

Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells

Exosomes are nanovesicles released by leukocytes and epithelial cells. Although their function remains enigmatic, exosomes are a source of antigen and transfer functional major histocompatibility complex (MHC)-I/peptide complexes to dendritic cells (DCs) for CD8(+) T-cell activation. Here we demonstrate that exosomes also are internalized and processed by immature DCs for presentation to CD4(+) T cells. Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing, followed by loading of exosome-derived peptides in MHC-II molecules for presentation to CD4(+) T cells. Targeting of exosomes to DCs is mediated via milk fat globule (MFG)-E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the exosome and alpha(v)/beta(3) integrin, and CD11a and CD54 on the DCs. Circulating exosomes are internalized by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. Internalization of blood-borne allogeneic exosomes by splenic DCs does not affect DC maturation and is followed by loading of the exosome-derived allopeptide IEalpha(52-68) in IA(b) by host CD8alpha(+) DCs for presentation to CD4(+) T cells. These data imply that exosomes present in circulation or extracellular fluids constitute an alternative source of self- or allopeptides for DCs during maintenance of peripheral tolerance or initiation of the indirect pathway of allorecognition in transplantation.     

Surface expression of HLA-DM on dendritic cells derived from CD34-positive bone marrow haematopoietic stem cells

HLA-DM has been known to be largely absent from the cell surface of antigen-presenting cells, accumulating instead in the intracellular compartment. In this study, we demonstrated that a population of HLA-DM-positive (HLA-DM+) dendritic cells (DCs) can be identified in an in vitro culture of CD34+ bone marrow haematopoietic stem cells. CD34+ bone marrow cells of healthy donors were used to generate DCs with the recombinant human cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor alpha (TNF-alpha) and stem cell factor (SCF), both with and without interleukin 4 (IL-4). Flow cytometric analysis demonstrated that HLA-DM+ cells comprised 2.5 +/- 0.9% and 1.8 +/- 0.4% of the CD34+ cell-derived progeny in the presence of GM-CSF, TNF-alpha and SCF after 7 d and 14 d of culture respectively. The number of HLA-DM molecules expressed per HLA-DM+ cell on d 7 was significantly higher than that on d 14 (1410 +/- 47 versus 370 +/- 25, P < 0.05). The addition of IL-4 to the cytokines from the commencement of culture increased the proportion of HLA-DM+ cells and increased the number of HLA-DM molecules per HLA-DM+ cell significantly (P < 0.05). Although most of the HLA-DM+ cells expressed CD1a, CD80 or CD86 antigen, only a small proportion of CD1a+, CD80+ or CD86+ cells expressed HLA-DM. About half the HLA-DM+ cells expressed CD83. The addition of IL-4 resulted in a decrease in the expression of CD83 on the HLA-DM+ cells on d 7. Microscopic evaluations of sorted HLA-DM+ cells revealed the characteristic morphological features of DCs. Primary mixed lymphocyte cultures demonstrated that the HLA-DM+ cells elicited a vigorous proliferation of allogeneic T cells. The level of antigen-specific T-cell activation induced by antigen-pulsed, chloroquine-treated HLA-DM+ cells was substantially higher than that induced by HLA-DM- cells (P < 0.05). These results show that HLA-DM can be used as a useful DC lineage-specific marker, as well as a tool for the characterization of DCs and human immunotherapy.     

Failure of HIV-exposed CD4+ T cells to activate dendritic cells is reversed by restoration of CD40/CD154 interactions

Because interactions between activated CD4+ T cells and antigen-presenting cells (APCs) are crucial for optimal APC function, defective CD4+ T-cell activation may contribute to APC dysregulation in HIV infection. Here, we show that CD4+ T cells exposed during stimulation to noninfectious HIV having functional envelope glycoproteins failed to provide activation signals to autologous dendritic cells (DCs). Consequently, important DC functions, including production of immunoregulatory cytokines (interleukin-12 p40 and interleukin-10) and up-regulation of costimulatory molecules (CD86, CD40, CD83), as well as the capacity to stimulate naive allogeneic T cells, were all adversely affected. The blunted up-regulation of CD154 in CD4+ T cells that were activated in the presence of noninfectious viruses is likely to be the major underlying mechanism for these defects. Addition of recombinant trimeric CD154 could restore production of cytokines by DCs cocultured with HIV-exposed T cells. Moreover, the functional defects mediated by coculture with HIV-exposed T cells were similar to those following antibody blockade of CD40-CD154 interactions. HIV-mediated blunted CD154 expression may thus play an important role in the suppression of cell-mediated immunity seen in HIV infection.     

Expansion of a lymphocyte population co-expressing T4 (CD4) and T8 (CD8) antigens in the peripheral blood of a normal adult male

Approximately 2% to 3% of circulating human T cells co-express CD4 and CD8 (CD4+, CD8+) T-cell antigens. These CD4+, CD8+ cells may be immature precursors that function to replenish functional T-cell subsets. We detected a very high level of CD4+, CD8+ cells in the peripheral blood lymphocytes of a healthy white male, ranging from 21% to 36%. The morphology of his peripheral blood lymphocytes was normal, and he has maintained an elevated level of CD4+, CD8+ cells without clinical disease over a 19-month observation period. The CD4+, CD8+ cells did not possess thymocyte membrane antigen (CD6), nor did they have increased Tac (CD25) antigen. His intact, purified blood T cells had a normal proliferative response to phytohemagglutinin and interleukin-2 (IL-2), provided help for B-cell proliferation at control levels and exposure to IL-2 resulted in generation of cytotoxic cells. However, the purified blood CD4+, CD8+ cells were deficient in these latter functions except for help in B-cell function. Despite defective function, the isolated CD4+, CD8+ cells co-expressed CD2 and CD3. Prolonged in vitro culture of CD4+, CD8+ cells was possible in the presence of recombinant IL-2. The cultured CD4+, CD8+ cells retained the double antigens (CD4 and CD8) throughout a 4-week period. It is likely these cells are less mature than CD4+, CD8- or CD4-, CD8+ T cells as the CD4+, CD8+ have less in vitro function than the former cells, but it is not yet clear if they mature into either CD4+, CD8-, or CD4-, CD8+ cells. Finally, the presence of an expanded, hypofunctioning CD4+, CD8+ cell population in a normal adult male is apparently compatible with excellent clinical health.