CD34+-derived CD11c+ + + BDCA-1+ + CD123+ + DC: expansion of a phenotypically undescribed myeloid DC1 population for use in adoptive immunotherapy

BACKGROUND: DC are commonly defined as HLA-DR+/Lin- cells that can be CD11c+ + + CD123+/ -, termed DC1/myeloid DC that induce a Th1 response, or CD11c- CD123+ + +, termed DC2/lymphoid DC that induce a Th2 response. However, significant heterogeneity within DC preparations is apparent and supports the existence of several distinct DC subpopulations. This study aimed to expand and characterize CD34+ DC for use in immunotherapy. METHODS: CD34+ cells were seeded at 1 x 10(5)/mL and expanded for 14 days in RPMI + 10% autologous plasma supplemented with GM-CSF, IL-4, Flt-3L and SCF. Maturation was induced with TNF-alpha and PGE2 for 2 days. DC were analyzed morphologically, phenotypically with a panel of MAb to lineage and DC markers, and functionally in MLR, T-cell assays and T-cell cytokine secretion by ELISA. RESULTS: Significant cellular expansion was observed: 60+/-5 x 10(6) DC from 1 x 10(6) CD34+ cells (n=28). Phenotypically DC were characterized as HLA-DR+ +, CD11c+ + +, CD80+ +, CD83+, CD86+ +, CD123+ +, CD15+ +, CD33+ +, BDCA-1+ +, CD4+ and Lin-. DC displayed potent allostimulatory capacity and efficient presentation of KLH and tetanus toxin. DC-primed T cells secreted IFN-gamma (Th1); however, no detectable IL-4 (Th2) was noted. DISCUSSION: We present features of CD34+ DC that have not been previously described. The CD34+ DC generated represent a population of myeloid DC functioning as DC1 but phenotypically expressing markers characteristic of both DC1 and DC2. This novel DC population is capable of inducing naive T-cell responses and can be expanded to clinically useful numbers. CD34+-derived DC represent attractive candidates for use in adoptive T-cell immunotherapy.     

Ex vivo enhancement of antigen-presenting firnction of dendritic cells and its application for DC-based immunotherapy

Dendritic cells (DCs) are potent antigen presenting cells that are able to initiate and modulate immune responses and are hence exploited as cellular vaccines for immunotherapy. In particular DCs generated from peripheral blood monocytes (Mo-DCs) have been used with promising results as a new approach for the immunotherapy of cancer. In this study, we have analyzed the changes in the pattern of expression molecules on Mo-DCs during DC maturation using different maturation cytokine combinations and the expansion capacity of an antigen specific CD8+T cells monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. These analyses revealed that the expansion of antigen specific CD8+T cells is the most effective when T cells were activated by fully maturated DCs by culturing monocytes for 5 days in the presence of GM-CSF and IL-4, followed by 2-3 days of maturation with pro-inflammatory mediators including TNFalpha, IL-6, IL-1beta and PGE2. These results pave the way to a more effective immunotherapy using DCs for patients with malignancy, as well as infectious diseases.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.     

Dendritic cells: Potential role in cancer therapy

Dendritic cells (DC) are extremely potent antigen presenting cells, uniquely capable of sensitizing naive T cells to protein antigens and eliciting antigen specific immune responses. Studies of human DC isolated from peripheral blood indicate that these cells can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro. On the basis of these findings we have initiated pilot clinical studies to investigate the ability of DC pulsed ex vivo with tumor associated proteins to stimulate host anti-tumor immunity when re-infused as a vaccine. In the first such study DC pulsed with tumor derived idiotype protein were infused into patients with low grade malignant B cell lymphoma who had failed conventional chemotherapy. The majority of treated patients developed T cell mediated anti-idiotype immune responses and some of the patients experienced tumor regression. These results suggest that DC based immunotherapy is a potentially useful approach to B cell lymphoma and raises the possibility that the approach may prove useful in the treatment of other tumors as well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulatory and conventional CD4+ T cells show differential effects correlating with PD-1 and B7-H1 expression after immunotherapy

Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3- Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-gamma knockout (IFN-gamma-/-) and IFN-gamma receptor knockout (IFN-gammaR-/-) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN-gamma.     

B7 costimulates proliferation of CD4-8+ T lymphocytes but is not required for the deletion of immature CD4+8+ thymocytes.

In addition to TCR-derived signals, costimulatory signals derived from stimulation of the CD28 molecule by its natural ligand, B7, have been shown to be required for CD4+8- T cell activation. We investigate the ability of B7 to provide costimulatory signals necessary to drive proliferation and differentiation of virgin CD4-8+ T-cells that express a transgenic TCR specific for the male (H-Y) Ag presented by H-2Db class I MHC molecules. Virgin male-specific CD4-8+ T cells can be activated either with B7 transfected chinese hamster ovary (CHO) cells and T3.70, a mAb specific for the transgenic TCR-alpha chain that is associated with male-reactivity, or by male dendritic cells (DC). Activated CD4-8+ T cells proliferated in the absence of exogenously added IL-2. IL-2 activity was detected in supernatants of CD4-8+T3.70+ cells that were stimulated with T3.70 and B7+CHO cells. The response of CD4-8+T3.70+ cells to T3.70/B7+CHO or to male DC stimulation were inhibited by CTLA4Ig, a fusion protein comprising the extracellular portion of CTLA4 and human IgG C gamma 1. It has been previously shown that CTLA4Ig binds B7 with high affinity. Staining with CTLA4Ig revealed that DC express about 50 times more B7 than CD4-8+ T cells. CTLA4Ig also specifically blocked the proliferation of male-reactive cells in vivo. We have also used an in vitro deletion assay whereby immature CD4+8+ thymocytes expressing the transgenic male-specific TCR are deleted by overnight incubation with either immobilized T3.70 or male DC to investigate the participation of the CD28/B7 pathway in the negative selection of immature thymocytes. Staining with B7Ig established that both immature murine CD4+8+ and mature CD4-8+ thymocytes express a high level of CD28. However, despite the high expression of CD28 on CD4+8+ thymocytes, it was found that deletion of CD4+8+ thymocytes expressing the male-specific TCR by the T3.70 mAb was not inhibited by B7+CHO cells. Furthermore, the deletion of these thymocytes by DC also was not inhibited by CTLA4Ig. These findings provide evidence that although signaling through CD28 can costimulate a primary anti-male response in mature CD4-8+ T cells, the CD28/B7 pathway does not appear to participate in the negative selection of immature CD4+8+ thymocytes.     

A Potential Protein Adjuvant Derived from Mycobacterium tuberculosis Rv0652 Enhances Dendritic Cells-Based Tumor Immunotherapy

A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from Mycobacterium tuberculosis Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4⁺ and CD8⁺ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8⁺ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.     

Skin TLR7 Triggering Promotes Accumulation of Respiratory Dendritic Cells and Natural Killer Cells

The TLR7 agonist imiquimod has been used successfully as adjuvant for skin treatment of virus-associated warts and basal cell carcinoma. The effects of skin TLR7 triggering on respiratory leukocyte populations are unknown. In a placebo-controlled experimental animal study we have used multicolour flow cytometry to systematically analyze the modulation of respiratory leukocyte subsets after skin administration of imiquimod. Compared to placebo, skin administration of imiquimod significantly increased respiratory dendritic cells (DC) and natural killer cells, whereas total respiratory leukocyte, alveolar macrophages, classical CD4+ T helper and CD8+ T killer cell numbers were not or only moderately affected. DC subpopulation analyses revealed that elevation of respiratory DC was caused by an increase of respiratory monocytic DC and CD11b(hi) DC subsets. Lymphocyte subpopulation analyses indicated a marked elevation of respiratory natural killer cells and a significant reduction of B lymphocytes. Analysis of cytokine responses of respiratory leukocytes after stimulation with Klebsiella pneumonia indicated reduced IFN-γ and TNF-α expression and increased IL-10 and IL-12p70 production after 7 day low dose skin TLR7 triggering. Additionally, respiratory NK cytotoxic activity was increased after 7d skin TLR7 triggering. In contrast, lung histology and bronchoalveolar cell counts were not affected suggesting that skin TLR7 stimulation modulated respiratory leukocyte composition without inducing overt pulmonary inflammation. These data suggest the possibility to modulate respiratory leukocyte composition and respiratory cytokine responses against pathogens like Klebsiella pneumonia through skin administration of a clinically approved TLR7 ligand. Skin administration of synthetic TLR7 ligands may represent a novel, noninvasive means to modulate respiratory immunity.     

Evaluation of monocyte-derived dendritic cells, T regulatory and Th17 cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors

Immunotherapy with dendritic cells (DC) may constitute a new and advantageous option for patients with chronic myeloid leukemia (CML) who respond to therapy with tyrosine kinase inhibitors (TKI), but do not reach complete cytogenetic or molecular remission. In this study, we evaluated the immunophenotype of DC generated from monocytes (Mo-DC) of patients with CML and the influence of TKI therapy on the results of CML-DC generation. We also measured the percentages of T regulatory cells (Tregs) as well as Th17 cells in 19 untreated patients suffering from CML, and in 28 CML patients treated with TKI. We found that DC can be reliably generated from the peripheral blood CD14+ cells of untreated CML patients. But we observed a persistent expression of CD14 monocyte marker on DC from CML patients, together with lower percentages of Mo-DC with expression of CD1a (p = 0.002), CD80 (p = 0.0005), CD83 (p = 0.0004), and CD209 (p = 0.02) compared to healthy donors. There was an adverse correlation between WBC count and the percentage of Mo-DC with co-expression of CD80 and CD86 (R = -0.63; p = 0.03). In patients treated with TKI, we observed higher efficacy of DC generation in seven-day cultures, compared to untreated patients. Expression of CD209 on DC was higher in patients treated with TKI (0.02). The duration of TKI therapy correlated adversely with MFI for CD1a (R = -0.49; p = 0.006) and positively with MFI for CD83 (R = 0.63; p = 0.01). Percentages of CD4+CD25highFoxP3+ cells (p = 0.0002) and Th17 cells (p = 0.02) were significantly higher in untreated CML patients compared to healthy controls. There was a significant correlation between the percentage of Treg cells and the percentage of peripheral blood basophiles (R = 0.821; p = 0.02). There were no changes in Tregs or Th17 cell percentages in CML patients after six months of TKI therapy. However, the expression of intracellular IL-17 in Th17 cells correlated negatively with the time of TKI therapy in the whole group of treated patients (R = -0.516; p = 0.04). We noted a correlation between IL-6 serum level and peripheral blood WBC count (R = 0.492; p = 0.04). There was also an inverse correlation between the serum level of IL-6 and the duration of TKI therapy (R = -0.66; p = 0.03). Taken together, our data shows that mature DC can be generated from CML patients treated with TKI, and that the yield of Mo-DC is higher in patients treated with TKI than in patients with active disease. This should encourage further trials with DC immunotherapy in patients with cytogenetic response after TKI therapy. We also found increased frequencies of T regulatory and Th17 cells in CML patients, which might suggest their potential role in immunity against this disease. Further studies are needed to determine if manipulation of these cell populations might improve the results of DC immunotherapy.     

Impaired circulating myeloid DCs from myeloma patients

BACKGROUND: In clinical trials, cancer patients have received immunotherapy based on DCs generated from leukapheresed blood. It would therefore be an advantage to be able to measure blood levels and estimate the phenotype of DC before leukapheresis, to estimate the yield required for preparation of vaccines, or ex vivo stimulation of T cells for adoptive immunotherapy. METHODS: Recently, circulating lineage negative (Lin-) myeloid DC cells and their precursors have been identified by flow cytometry. We apply this strategy to the screening of blood samples from patients with multiple myeloma, in an attempt to characterize and quantitate the subset. By a direct flow cytometry approach, the blood levels of circulating lineage (CD3, CD19, CD14) negative, CD33++, HLA-DR+ cells were estimated before and following ex vivo cell differentiation, and phenotyped by MAbs with specificity against HLA-DR, HLA-ABC, CD1a, CD11c, CD33, CD40, CD49d, CD49e, CD54, CD80, CD83, and CD86. RESULTS: This study demonstrated that multiple myeloma patients have a 50% reduced blood level of Lin-, CD33++, HLA-DR+ myeloid DC, but a DC-precursor level within normal range. Furthermore, GM-CSF and IL-4 ex vivo stimulated DCs demonstrated an impaired up-regulation of the co-stimulatory molecule CD80 and the adhesion molecule CD54. DISCUSSION: These results may have clinical implications as a predictor for yield and functionality of the harvested DCs to be used in vaccination of myeloma patients.     

Flt3-ligand and granulocyte colony-stimulating factor mobilize distinct human dendritic cell subsets in vivo

Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11c+ DCs induce T cells to produce Th1 cytokines in vitro, whereas the CD11c- plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC subsets, or DC precursors, in the blood. FL increases both the CD11c+ DC subset (48-fold) and the CD11c- IL-3R+ DC precursors (13-fold). In contrast, G-CSF only increases the CD11c- precursors (>7-fold). Freshly sorted CD11c+ but not CD11c- cells stimulate CD4+ T cells in an allogeneic MLR, whereas only the CD11c- cells can be induced to secrete high levels of IFN-alpha, in response to influenza virus. CD11c+ and CD11c- cells can mature in vitro with GM-CSF + TNF-alpha or with IL-3 + CD40 ligand, respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DC-lysosome-associated membrane protein, and they stimulate naive, allogeneic CD4+ T cells efficiently. These two DC subsets elicit distinct cytokine profiles in CD4+ T cells, with the CD11c- subset inducing higher levels of the Th2 cytokine IL-10. The differential mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to manipulate immune responses in humans.     

Immunization with heat shock protein 105-pulsed dendritic cells leads to tumor rejection in mice

Recently, we reported that heat shock protein 105 (HSP105) DNA vaccination induced anti-tumor immunity. In this study, we set up a preclinical study to investigate the usefulness of dendritic cells (DCs) pulsed with mouse HSP105 as a whole protein for cancer immunotherapy in vivo. The recombinant HSP105 did not induce DC maturation, and the mice vaccinated with HSP105-pulsed BM-DCs were markedly prevented from the growth of subcutaneous tumors, accompanied with a massive infiltration of both CD4+ T cells and CD8+ T cells into the tumors. In depletion experiments, we proved that both CD4+ T cells and CD8+ T cells play a crucial role in anti-tumor immunity. Both CD4+ T cells and CD8+ T cells specific to HSP105 were induced by stimulation with HSP105-pulsed DCs. As a result, vaccination of mice with BM-DCs pulsed with HSP105 itself could elicit a stronger tumor rejection in comparison to DNA vaccination.     

Significant virus replication in Langerhans cells following application of HIV to abraded skin: relevance to occupational transmission of HIV

The cellular events that occur following occupational percutaneous exposure to HIV have not been defined. In this study, we studied relevant host cellular and molecular targets used for acquisition of HIV infection using split-thickness human skin explants. Blockade of CD4 or CCR5 before R5 HIV application to the epithelial surface of skin explants completely blocked subsequent HIV transmission from skin emigrants to allogeneic T cells, whereas preincubation with C-type lectin receptor inhibitors did not. Immunomagnetic bead depletion studies demonstrated that epithelial Langerhans cells (LC) accounted for >95% of HIV dissemination. When skin explants were exposed to HIV variants engineered to express GFP during productive infection, GFP+ T cells were found adjacent to GFP+ LC. In three distinct dendritic cell (DC) subsets identified among skin emigrants (CD1a+langerin+DC-specific intercellular adhesion molecule grabbing non-integrin (SIGN)- LC, CD1a+langerin-DC-SIGN- dermal DC, and CD1a-langerin-DC-SIGN+ dermal macrophages), HIV infection was detected only in LC. These results suggest that productive HIV infection of LC plays a critical role in virus dissemination from epithelium to cells located within subepithelial tissue. Thus, initiation of antiretroviral drugs soon after percutaneous HIV exposure may not prevent infection of LC, which is likely to occur rapidly, but may prevent or limit subsequent LC-mediated infection of T cells.     

Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells.     

Dendritic cells induce MUC1 expression and polarization on human T cells by an IL-7-dependent mechanism

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however, the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7, but not IL-2 or IL-4, markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15, but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+, CD8+, CD25+, CD69+, naive CD45RA+, and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7, activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover, DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism.