Autologous Monocytes Research Articles

In vivo tracking of dendritic cell therapy in patients with multiple myeloma

2568 Background: Dendritic cell (DC) immunotherapy is being actively studied in patients (pts) with multiple myeloma (MM). However, the best route of injection of these cells is unknown. The aim of this study was to determine the in vivo distribution of DC when given to pts with MM via varying routes of injection by using nuclear medicine imaging techniques. Methods: Eligible pts had stable or slowly progressive MM not requiring systemic treatment, with elevated serum MUC-1 or positive marrow plasma cell MUC-1 staining. DC were produced by in vitro culture of autologous monocytes collected from pts via apheresis. Cells were stimulated in culture by GM-CSF plus IL-13 and then pulsed with Mannan-MUC1 fusion protein, using the VacCell processor. Prior to injection, DC were labelled with either F-18 FDG or Indium-111 (In). Pts underwent serial PET and SPECT scans, respectively, to track the destination of injected cells which were given by subcutaneous (s.c.), intradermal (i.d.), intranodal (i.n.) or intraven...

F-18 Fluorodeoxyglucose (FDG) PET and Indium-111 SPECT scans to track the biodistribution of macrophage-activated killer (MAK) cells linked with MDX-H210 in patients with epithelial ovarian cancer

2595 Background: MAK cell immunotherapy has demonstrated activity in Phase I/II trials for patients (pts) with relapsed ovarian cancer. However, little is known about the in vivo distribution of these cells following injection. The aim of this study was to track the destination of MAK cells using PET or SPECT imaging after delivery via intravenous (i.v.) or intraperitoneal (i.p.) injection to pts with recurrent epithelial ovarian cancer. Methods: MAK cells were produced by in vitro culture of autologous monocytes, collected from pts via apheresis. The cells were stimulated by γ interferon plus GM-CSF, and then bound to the anti-HER2/neu antibody (MDX-H210). Prior to injection, the MAK cells were labelled with either F-18 FDG or Indium-111. Pts then underwent serial PET or SPECT scans, respectively to track the destination of injected cells. Results: 13 infusions have been performed in 7 pts, 5 of whom had both i.v. and i.p. (Table 1). In all pts after i.v injection, cells passaged firstly through the lungs and then to liver and spleen. Tracking of cells to sites of peritoneal tumor have been demonstrated after 5 of 7 i.v. injections (in 4 pts), using both PET and SPECT. Following i.p administration, cells distributed throughout the peritoneal cavity in 4 of 5 pts (limited due to adhesions in 1 pt), and tracking to tumor sites was also demonstrated in all 4 pts. Cells were still visible up to 96 hours on serial SPECT. No tracking to tumor was observed in 2 pts, most likely due to poor sensitivity of the method for low-volume disease. Conclusions: We have demonstrated that it is possible to label MAK cells being used for immunotherapy with radiotracers and track their progress in vivo following injection using both PET and SPECT scans. Tracking of cells to the tumour target has been observed after both i.p and i.v injection. No significant financial relationships to disclose.

In vivo tracking of dendritic cell therapy in patients with multiple myeloma

2568 Background: Dendritic cell (DC) immunotherapy is being actively studied in patients (pts) with multiple myeloma (MM). However, the best route of injection of these cells is unknown. The aim of this study was to determine the in vivo distribution of DC when given to pts with MM via varying routes of injection by using nuclear medicine imaging techniques. Methods: Eligible pts had stable or slowly progressive MM not requiring systemic treatment, with elevated serum MUC-1 or positive marrow plasma cell MUC-1 staining. DC were produced by in vitro culture of autologous monocytes collected from pts via apheresis. Cells were stimulated in culture by GM-CSF plus IL-13 and then pulsed with Mannan-MUC1 fusion protein, using the VacCell processor. Prior to injection, DC were labelled with either F-18 FDG or Indium-111 (In). Pts underwent serial PET and SPECT scans, respectively, to track the destination of injected cells which were given by subcutaneous (s.c.), intradermal (i.d.), intranodal (i.n.) or intravenous (i.v.) route. Results: 10 infusions of non-matured DC have been performed in 3 pts (Table 1). After i.v injection of In-labeled DC, cells were initially imaged within the lungs on SPECT. 24 hours post injection, most DC had cleared the lungs and were seen within the liver, spleen and axial and proximal appendicular skeleton. DC were still visible until 72 hours. Following s.c., i.n. and i.d. injection, tracking of a fraction of the DC to the draining lymph node was imaged (after s.c = 1 pt out of 3, after i.n = 1 pt, after i.d = 1pt). PET was not useful for tracking, as the FDG-labeling efficiency of DC was low with little retention of tracer in vivo. Future cohorts of pts will compare results for DC after maturation in vitro prior to injection using In labeling only. Conclusion: We have demonstrated that although FDG-labeling of non-matured DC was not feasible, it is possible to label DC with In and track their distribution in vivo. No significant financial relationships to disclose.

Lactobacilli Modulate Proliferation and Cytokine Production of Human Peripheral Blood Natural Killer Cells In Vitro

Natural killer (NK) cells are cells of the nonspecific immune system lysing altered self‐cells. A noncytolytic subset of NK cells may serve a regulatory role by secreting cytokines. Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. Here, we investigated how human gut flora‐derived lactobacilli affect NK cells in vitro, by measuring proliferation and IFN‐γ production of human NK cells upon bacterial stimulation. CD3–CD56+NK cells were isolated from buffy coats by negative isolation using non‐NK lineage‐specific antibodies and magnetic beads. NK cells were incubated with 10μg/ml UV‐inactivated bacteria or 10μg/ml phytohemagglutinin (PHA) for 4 days. Proliferation was assessed by incorporation of radioactive thymidine into NK‐cell DNA. The IFN‐γ concentration was measured by ELISA. Incubation of NK cells with a Lactobacillus acidophilus strain increased the proliferation of the NK cells and induced IFN‐γ production, both to levels comparable to PHA stimulation. The proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the NK cells. In contrast, a Lactobacillus paracasei strain caused the NK cells to proliferate only in the presence of monocytes. These results demonstrate that various strains of lactobacilli have the capacity to activate NK cells in vitro, in a monocyte‐dependent or ‐independent way. Hence, the encounter of NK cells with lactic acid bacteria will affect NK‐cell activation. Such activation of NK cells may potentially skew an on‐going or subsequent immune response towards a Th1 response.

The differential interaction of p38 MAP kinase and tumor necrosis factor-α in human alveolar macrophages and monocytes induced by Mycobacterium tuberculois

Cellular signaling by TNF-α is mediated through activation of mitogen activated protein (MAP) kinases. In particular, p38 MAP kinase is activated in mononuclear phagocytes and may be important in sustaining TNF-α activity. Here, we compared the activation and mutual regulation of p38 MAP kinase and TNF-α by MTB in human alveolar macrophages (AM) and blood monocytes (MN). AM and autologous MN were prepared, and stimulated by MTB at 1:1 (bacteria/cell). MAP kinase activation was assessed by immunoprecipitation and kinase activity. TNF-α mRNA was assessed by real-time RT-PCR, and TNF-α immunoreactivity was assessed by ELISA. MTB-induced p38MAP kinase rapidly in AM as compared to MN, and inhibition of p38 MAP kinase by SB203580 reduced both TNF-α mRNA and protein. Activation of ERK (1/2) by MTB followed similar kinetics in both AM and MN. TNF-α produced by MTB sustained p38 MAP kinase activation in MN only. These data suggest that interaction of resident pulmonary macrophages and the more immature MN with MTB differ with regard to both p38 MAP kinase activation and TNF-α expression.

Costimulatory ligand 4-1BBL (CD137L) as an efficient adjuvant for human antiviral cytotoxic T cell responses.

Effective adjuvants capable of inducing strong cytotoxic T cell responses in humans are lacking. In this study, we tested 4-1BBL as an adjuvant for activation of human memory antiviral CD8 T cell responses ex vivo. A recombinant replication-defective 4-1BBL adenovirus was used to convert autologous monocytes into efficient antigen-presenting cells after overnight incubation, bypassing the need to generate dendritic cells. Together with viral peptides, 4-1BBL led to robust memory responses of human Epstein-Barr virus- and influenza virus-specific cytotoxic T cells, with expansion of peptide-specific CD8 effector cells; up-regulation of Bcl-x(L), granzyme A, and perforin; enhanced cytotoxic activity; and increased cytokine production. The response was significant even at a 100-fold lower peptide dose, compared with responses obtained with control adenovirus. Adenovirus-delivered B7.1 also expanded and activated virus-specific CD8 T cells, but 4-1BBL was more effective in driving the T cells toward a more fully differentiated CD27(-) effector state. Thus, 4-1BBL is a promising adjuvant for human memory CD8 T cells and will likely be most effective in the boost phase of a prime-boost strategy.

Transplantation of monocytes: a novel strategy for in vivo augmentation of collateral vessel growth.

Therapeutic augmentation of collateral vessel growth (arteriogenesis) is of particular clinical interest. Because monocytes localize to areas of collateral growth and create a highly arteriogenic environment through secretion of multiple growth factors, we tested the hypothesis that monocyte "homing" can therapeutically be exploited. We have used a rabbit model of arteriogenesis to investigate the therapeutic potential of transplanted rabbit monocytes that were either ex vivo stimulated or adenovirally transduced to express a transgene encoding an arteriogenic growth factor. The monocytes were intravenously injected 24 hr or 7 days after ligation of the animal's right femoral artery. Seven days after transplantation collateral flow was determined with a doppler flow probe and collateral vessels were quantified angiographically. Whereas transplantation of allogeneic cells (same species) resulted in a strong promotion of arteriogenesis, most likely through induction of local inflammation and recruitment of recipient monocytes, transplantation of autologous cells (same animal) was not able to significantly augment collateralization. However, when autologous monocytes were used as vehicles to deliver granulocyte macrophage-colony stimulating factor as therapeutic transgene, collateralization was strongly augmented. Their localization to the site of collateral development posttransplantation was demonstrated by ex vivo transduction with beta-galactosidase. Because isolation of monocytes is clinically widely available their ex vivo engineering and transplantation represents an intriguing new strategy for therapeutic arteriogenesis.

Regulation of Interleukin 12 p40 and p70 Production by Blood and Alveolar Phagocytes During Severe Sepsis

Paradoxically, the host response to severe sepsis may lead to immunosuppression, thereby favoring nosocomial infections. We examined the role of the two IL-12 isoforms, bioactive IL-12p70 and regulatory IL-12p40, in 16 patients with severe sepsis. We compared the capacity of purified blood and alveolar phagocytes [polymorphonuclear neutrophils (PMN) and monocytes/macrophages] to secrete each isoform. Blood monocytes had normal basal secretions. In contrast, a marked imbalance was observed after ex vivo stimulation by lipopolysaccharide plus IFN-γ, with significantly lower IL-12p70 production and higher IL-12p40 production. Conversely, stimulated IL-12p40 production by the patients' blood PMN tended to be impaired, as was their cell-surface β2 integrin and L-selectin expression, known as markers of cell activation. In the patient's bronchoalveolar lavage fluid, the production of both IL-12 isoforms after ex vivo stimulation was significantly lower with alveolar macrophages than with autologous blood monocytes and significantly higher with alveolar PMN than with autologous blood PMN. This sheds new light on the potential role of PMN in local modulation of inflammation, via secretion of the anti-inflammatory IL-12 p40 subunit. The imbalance between the bioactive and regulatory IL-12 isoforms, which is probably designed to control excessive inflammation, may also make septic patients more susceptible to nosocomial infection.

Brief hypoxia conditions monocytes to protect reperfused cardiocytes against cell death via the CD11b receptor

Background: Traditionally leukocytes have been viewed as the mediators and effectors of cell injury after tissue ischemia and reperfusion through the indiscriminate release of toxic cytokines and oxygen free radicals. This can be detrimental to functioning of the transplanted heart when reperfused after implantation. Paradoxically, evidence now suggests that certain cytotoxic cytokines and even oxygen free radicals can be cytoprotective in smaller concentrations. This study sought to determine whether cultured human monocytes can be pre-conditioned by brief hypoxia to protect cardiomyocytes from cell death after hypoxia and re-oxygenation. Methods: Cultured human monocytes were exposed to transient hypoxia (10 minutes), after which we determined CD11b expression using flow cytometry. The 3 control groups comprised immunoglobulin G–negative controls, fMLP-positive controls, and virgin monocyte (VM) controls. We studied chick embryonic ventricular myocytes in a flow-through chamber while controlling flow rate, pH, O 2, and CO2 tension. We quantified cardiomyocyte viability using propidium iodide (5 μmol/liter). Cell systems of cardiomyocytes alone, cardiomyocytes and VM, human monocytes exposed to transient hypoxia before coculture with cardiomyocytes (PCHM-cardiomyocyte), and PCHM cocultured with anti-CD11b antibodies for 30 minutes before coculture with cardiomyocytes (CDHM-cardiomyocyte) were subjected to 1 hour of hypoxia and 3 hours of re-oxygenation, and cell death was determined. The experiment was repeated and the cell systems fixed, stained, and examined for monocytes adhering to cardiomyocytes. Results: CD11b expression increased significantly with both transient hypoxia and fMLP (18.39% ± 4.116%, n = 5, and 37.04% ± 7.783%, n = 5, respectively, p < 0.05 vs VM 100%, n = 5). Coculture of cardiomyocytes with VM had no effects on cardiocyte death (40.6% ± 6.1%, n = 6) compared with controls (46.5% ± 4.0%, n = 10). The PCHM cocultured with cardiomyocytes significantly decreased cardiomyocyte death (25.2% ± 4.7%, n = 6, p < 0.05). This protection was abrogated by the addition of CD11b-blocking antibodies to PCHM before coculture with cardiomyocytes (51.0% ± 6.1%, n = 6, p < 0.05). The PCHM showed increased adhesion to cardiomyocytes (5.4 ± 0.38/high-power [HP] field vs 0.67 ± 0.24/HP field in VM, p < 0.05). The increased adhesion was abolished by CD11b-blocking antibody (0.78 ± 0.28 vs 0.67 ± 0.24 cells/HP field, p < 0.05). Conclusion: These results suggest that monocytes activated by transient hypoxia protect cardiomyocytes during hypoxia and re-oxygenation through expression of CD11b receptors. These cells seem to adhere to myocytes through this receptor to achieve this effect. The exact mechanism is unclear and requires further study. Autologous recipient monocytes may be pre-conditioned to protect the donor heart during reperfusion.

CD40 ligand trimer enhances the response of CD8+ T cells to Mycobacterium tuberculosis.

We investigated the effect of recombinant CD40 ligand trimer (CD40LT) on the functional capacity of peripheral blood CD8(+) T cells from healthy tuberculin reactors that were cultured with Mycobacterium tuberculosis-infected autologous monocytes. CD40LT enhanced the capacity of M. tuberculosis-responsive CD8(+) T cells to produce IFN-gamma by increasing the number of IFN-gamma-producing CD8(+) T cells and the amount of IFN-gamma produced per cell. CD40LT-induced IFN-gamma production was dependent on production of IL-12 and IL-18, but did not require IL-15. CD40LT up-regulated expression of the transcription factors phosphorylated CREB and c-Jun, both of which have been previously shown to stimulate IFN-gamma mRNA transcription by binding to the IFN-gamma promoter. CD40LT also enhanced the capacity of CD8(+) T cells to lyse M. tuberculosis-infected monocytes, and increased CTL activity was associated with higher expression of perforin and granulysin, but not of Fas ligand. We conclude that CD40LT can enhance CD8(+) T cell effector function in response to M. tuberculosis.

The apoptotic ligands TRAIL, TWEAK, and Fas ligand mediate monocyte death induced by autologous lupus T cells.

Individuals with systemic lupus erythematosus show evidence of a significant increase in monocyte apoptosis. This process is mediated, at least in part, by an autoreactive T cell subset that kills autologous monocytes in the absence of nominal Ag. We have investigated the apoptotic pathways involved in this T cell-mediated process. Expression of the apoptotic ligands TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and Fas ligand on lupus T cells was determined, and the role of these molecules in the monocyte apoptotic response was examined. We report that these apoptotic ligands mediate the autologous monocyte death induced by lupus T cells and that this cytotoxicity is associated with increased expression of these molecules on activated T cells, rather than with an increased susceptibility of lupus monocytes to apoptosis induced by these ligands. These results define novel mechanisms that contribute to increased monocyte apoptosis characterizing patients with lupus. We propose that this mechanism could provide a source of potentially antigenic material for the autoimmune response and interfere with normal clearing mechanisms.

Single-cell analysis: a novel approach to tumour necrosis factor-alpha synthesis and secretion in sarcoidosis.

Tumour necrosis factor (TNF)-alpha is thought to be a key early cytokine in the pathogenesis of sarcoidosis, despite conflicting data. Largely the product of mononuclear phagocyte activation, it is unclear whether TNF-alpha production at disease sites is a feature of all mononuclear phagocytes that accumulate there or whether it is secreted by a subset of these cells. Using the reverse haemolytic plaque assay, the aims of this study were to determine if the upregulation of TNF-alpha could be confirmed and to investigate whether this was monocyte or macrophage specific. The reverse haemolytic plaque assay allows the measurement of cytokine production at a single cell level. A greater number of alveolar macrophages produced TNF-alpha compared to autologous monocytes in sarcoidosis but not in controls and, based on cell size, it was confirmed that this was the product of more mature macrophages and that the secretion of TNF-alpha by monocytes and macrophages was heterogeneous: not all monocytes and macrophages secrete TNF-alpha. No differences in the average levels of TNF-alpha secretion by peripheral blood monocytes or alveolar macrophages were observed. This study has demonstrated that a subset of mononuclear phagocytes, mature macrophages, are responsible for tumour necrosis factor secretion and this could have implications for targeted management in sarcoidosis in the future.

Mechanisms for monocyte activation in co-culture with autologous tumor spheroids

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24 h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2 min) or actinomycin-D ( 1 μ g/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100 mM) or mannose (100 mM), and to some extent galactose (100 mM), but not fructose (100 mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human β 1-integrin (anti-CD29) antibody (2 μ g/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and β 1-integrin.

Mechanisms for monocyte activation in co-culture with autologous tumor spheroids

Biopsies from carcinoma tissue and benign control mucosa from head and neck squamous cell carcinoma patients were used to establish fragment (F)-spheroids in vitro. We have previously shown that autologous monocytes co-cultured with F-spheroids in vitro secrete interleukin (IL)-6 upon 24 h in co-culture. Presently, the aim was to study the mechanisms of this monocyte secretion. Paraformaldehyde (0.1% for 2 min) or actinomycin-D ( 1 μ g/ml for 24 h) pre-treatment of the F-spheroids abolished the monocyte IL-6 co-culture response. Addition of glucose (100 mM) or mannose (100 mM), and to some extent galactose (100 mM), but not fructose (100 mM) to the co-cultures, partly inhibited the monocyte IL-6 co-culture response, but such addition did not inhibit the in vitro monocyte lipopolysaccharide (LPS)-generated IL-6 secretion. When mannose was added to the co-cultures, monocyte IL-6 mRNA expression was eradicated in malignant co-cultures and reduced to a low level in benign co-cultures. Addition of mouse anti-human β 1 -integrin (anti-CD29) antibody (2 μ g/ml) diminished the IL-6 co-culture response but not the monocyte LPS-generated IL-6 response. In conclusion, the monocyte IL-6 co-culture response is dependent on live spheroids and to some extent on direct contact with the F-spheroids, possibly via lectin-like receptor(s), the mannose receptor and β 1 -integrin.

Activation of human NK cells by staphylococci and lactobacilli requires cell contact-dependent costimulation by autologous monocytes.

NK cells are instrumental in innate immune responses, in particular for the early production of gamma interferon (IFN-gamma) and other cytokines necessary to control certain bacterial, parasitic, and viral infections. NK cell-mediated effector functions are controlled by a fine balance between distinct receptors mediating activating and inhibitory signals; however, little is known about activating receptors on NK cells and their corresponding ligands. Several studies have shown that commensal lactobacilli isolated from the human gastrointestinal tract activate human mononuclear cells and are potent inducers of IFN-gamma and monocyte-derived interleukin 12 (IL-12). NK cell activation was shown for Lactobacillus johnsonii La1. In this study the cellular mechanisms of in vitro NK cell activation by gram-positive bacteria were analyzed. Staphylococcus aureus- and L. johnsonii La1-mediated activation of CD3(-) CD16(+) CD56(+) human peripheral blood NK cells, including expression of the activation antigen CD69 and secretion of IFN-gamma, required cell contact-dependent costimulation by autologous monocytes. S. aureus- and L. johnsonii-preactivated monocytes retained their capacity to induce NK cell activation. In contrast, cytokine-primed monocytes completely failed to induce NK cell activation unless bacteria were present. This suggests that phagocytosis of bacteria provided additional coactivation signals on accessory cells that may differ from those induced by tumor necrosis factor and IFN-gamma. Blocking of costimulatory molecules by B7.1, B7.2, and IL-12 but not CD14 monoclonal antibodies inhibited S. aureus- and L. johnsonii-induced effector function of NK cells. Our data suggest an important role for accessory cell-derived signals in the process of NK cell activation by gram-positive bacteria.

Role of N-glycans and SDF-1α on the coassociation of CD4 with CXCR4 at the plasma membrane of monocytic cells and blood lymphocytes

CXCR4 is a coreceptor, along with CD4, for human immunodeficiency virus type 1 (HIV-1). Trimolecular complexes between HIV-1 glycoprotein (gp)120, CD4 and CXCR4 constitute a prerequisite for HIV entry. We studied whether CD4 is associated with CXCR4 on CD4+ CXCR4+ cells. Using the conformation-dependent anti-CXCR4 mAb 12G5, CD4 was coimmunoprecipitated with CXCR4 from the membrane of U937 cells which support HIV-1 LAI efficient infection, and from that of peripheral blood lymphocytes (PBL). CD4 association with CXCR4 increased upon PBL coculture for 5 days with autologous monocytes, decreased upon treatment of the cells or the CD4-CXCR4 complex with either N-glycanase or stromal cell derived factor-1α (SDF-1α) and was abolished by incubation of the cells with both, N-glycanase and SDF-1α. This indicates that glycans are partly involved in CD4 association with CXCR4 and may partly explain the inhibitory effect of SDF-1α on HIV infection.

Immunological characterization of a gammadelta T-cell stimulatory ligand on autologous monocytes.

Bovine gammadelta T cells are stimulated to proliferate by autologous monocytes. This is referred to as the autologous mixed leucocyte reaction (AMLR). It has been shown previously that the stimulatory component is constitutively expressed on the monocyte plasma membrane and is a protein or has a protein moiety. Here we showed that gammadelta T-cell responses to the monocytes requires interaction with the T-cell receptor because Fab1 fragments of a monoclonal antibody (mAb) that reacts with the delta chain of the T-cell receptor blocked proliferation in the AMLR. Monocyte molecules involved in stimulation were also characterized further by biochemical and immunological methods. A mAb, named M5, was generated by immunizing mice with bovine monocytes and shown to block the ability of monocytes to stimulate in the AMLR. Treatment of monocytes or monocyte membranes with high salt, chelating agents or phospholipase C did not affect their ability to stimulate gammadelta T-cell proliferation or reactivity with mAb M5 indicating the ability of monocytes to stimulate does not involve peripheral membrane components or a glycosyl-phosphatidylinsositol (GPI)-anchored components. Hence it was concluded that the stimulation occurred as a result of intergral membrane proteins including that recognized by mAb M5. The ligand for mAb M5 was on all bovine monocytes and to a lower level on granulocytes but not on lymphocytes. MAb M5 also reacted with sheep monocytes but not with human monocytes or murine macrophages, in agreement with a previous reports that sheep monocytes but not human or mouse mononuclear phagocytes have the capacity to stimulate bovine gammadelta T cells in in vitro cultures. The level of expression of the M5 ligand was not altered by gamma-irradiation or culture of monocytes with lipopolysaccharide but it was decreased following culture with interferon-gamma-containing cell culture supernatants.

Amplification of low-frequency antiviral CD8 T cell responses using autologous dendritic cells.

To utilize the potent antigen-presenting capacity of mature dendritic cells (MDC) in order to develop a rapid, sensitive method for quantifying antigen-specific CD8 T cells present at low frequency in peripheral blood. Peripheral blood mononuclear cells (PBMC) were obtained from seven HIV-1-positive individuals with low to moderate CD8 T cell responses, including five on highly active antiretroviral therapy (HAART). IFN-gamma ELISPOT assays were performed using either monocytes or MDC to present antigens expressed by recombinant vaccinia viruses (r-VV). Peripheral blood-derived monocytes were cultured for 5-6 days in the presence of IL-4 and granulocyte macrophage colony-stimulating factor, then matured in monocyte-conditioned medium. MDC were infected with r-VV and co-cultured in an ELISPOT assay with autologous monocyte-depleted PBMC. Relative to autologous monocytes, MDC amplified detection of antigen-specific CD8 T cells by 2-30-fold in response to antigens from HIV-1, Epstein-Barr virus and cytomegalovirus. Furthermore, antigenic specificities were revealed that had not been detected using standard ELISPOT of PBMC. This assay will prove useful for the detection of memory T cells present at low frequency, and may be of interest for identifying subdominant cytotoxic T lymphocyte epitopes. This method may have broad applications for the detection of antiviral CD8 T cell responses in patient populations in whom such responses have been difficult to detect, including HIV-1-seropositive individuals with advanced disease or undergoing HAART.

Monocyte-fibronectin interactions, via alpha(5)beta(1) integrin, induce expression of CXC chemokine-dependent angiogenic activity.

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene alpha, and growth-related oncogene gamma) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MG(FN-)) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MG(FN-) revealed a significant decrease in GRO-alpha and GRO-gamma compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via alpha(5)beta(1) integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via alpha(5)beta(1) integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.

CD85/LIR-1/ILT2 and CD152 (Cytotoxic T Lymphocyte Antigen 4) Inhibitory Molecules Down-Regulate the Cytolytic Activity of Human CD4+T-Cell Clones Specific forMycobacterium tuberculosis

Antigen-specific cytolytic CD4+ T lymphocytes control Mycobacterium tuberculosis infection by secreting cytokines and by killing macrophages that have phagocytosed the pathogen. However, lysis of the latter cells promotes microbial dissemination, and other macrophages engulf the released bacteria. Subsequently, CD4+ T-cell-mediated killing of macrophages goes on, and this persistent process may hamper control of infection, unless regulatory mechanisms maintain a subtle balance between lysis of macrophages by cytolytic CD4+ cells and activation of cytolytic CD4+ cells by infected macrophages. We asked whether inhibitory molecules expressed by CD4+ cytolytic T lymphocytes could play a role in such a balance. To this end, human CD4+ T-cell clones specific for M. tuberculosis were produced that displayed an autologous major histocompatibility complex class II-restricted lytic ability against purified protein derivative (PPD)-pulsed antigen-presenting cells. All T-cell clones expressed CD152 (cytotoxic T-lymphocyte antigen 4 [CTLA-4]) and CD85/leukocyte immunoglobulin-like receptor 1 (LIR-1)/immunoglobulin-like transcript 2 (ILT2) inhibitory receptors, but not CD94 and the killer inhibitory receptor (or killer immunoglobulin-like receptor [KIR]) p58.2. CD3-mediated activation of the clones was inhibited in a redirected killing assay in which CD152 and CD85/LIR-1/ILT2 were cross-linked. Specific antigen-mediated proliferation of the clones was also sharply reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked by specific monoclonal antibody (MAb) followed by goat anti-mouse antiserum. In contrast, blockade of the receptors by specific MAb only increased their proliferation. Production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by the T-cell clones was also strongly reduced when CD152 and CD85/LIR-1/ILT2 were cross-linked. The lytic activity of the T-cell clones against PPD-pulsed autologous monocytes or Epstein-Barr virus-activated B cells was increased by blockade and decreased by cross-linking of the receptors. These results indicate that CD152 and CD85/LIR-1/ILT2 play a role in the regulation of the antigen-specific activity of CD4+ cytolytic T lymphocytes against PPD-presenting cells.