Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.

Checkpoint inhibitors have revolutionized cancer treatment. However, only a minority of patients respond to these immunotherapies. Here, we report that blocking the inhibitory NKG2A receptor enhances tumor immunity by promoting both natural killer (NK) and CD8+ T cell effector functions in mice and humans. Monalizumab, a humanized anti-NKG2A antibody, enhanced NK cell activity against various tumor cells and rescued CD8+ T cell function in combination with PD-x axis blockade. Monalizumab also stimulated NK cell activity against antibody-coated target cells. Interim results of a phase II trial of monalizumab plus cetuximab in previously treated squamous cell carcinoma of the head and neck showed a 31% objective response rate. Most common adverse events were fatigue (17%), pyrexia (13%), and headache (10%). NKG2A targeting with monalizumab is thus a novel checkpoint inhibitory mechanism promoting anti-tumor immunity by enhancing the activity of both T and NK cells, which may complement first-generation immunotherapies against cancer.

Increased monocyte turnover from bone marrow correlates with severity of SIV encephalitis and CD163 levels in plasma.

Cells of the myeloid lineage are significant targets for human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in monkeys. Monocytes play critical roles in innate and adaptive immunity during inflammation. We hypothesize that specific subsets of monocytes expand with AIDS and drive central nervous system (CNS) disease. Additionally, there may be expansion of cells from the bone marrow through blood with subsequent macrophage accumulation in tissues driving pathogenesis. To identify monocytes that recently emigrated from bone marrow, we used 5-bromo-2'-deoxyuridine (BrdU) labeling in a longitudinal study of SIV-infected CD8+ T lymphocyte depleted macaques. Monocyte expansion and kinetics in blood was assessed and newly migrated monocyte/macrophages were identified within the CNS. Five animals developed rapid AIDS with differing severity of SIVE. The percentages of BrdU+ monocytes in these animals increased dramatically, early after infection, peaking at necropsy where the percentage of BrdU+ monocytes correlated with the severity of SIVE. Early analysis revealed changes in the percentages of BrdU+ monocytes between slow and rapid progressors as early as 8 days and consistently by 27 days post infection. Soluble CD163 (sCD163) in plasma correlated with the percentage of BrdU+ monocytes in blood, demonstrating a relationship between monocyte activation and expansion with disease. BrdU+ monocytes/macrophages were found within perivascular spaces and SIVE lesions. The majority (80-90%) of the BrdU+ cells were Mac387+ that were not productively infected. There was a minor population of CD68+BrdU+ cells (<10%), very few of which were infected (<1% of total BrdU+ cells). Our results suggest that an increased rate of monocyte recruitment from bone marrow into the blood correlates with rapid progression to AIDS, and the magnitude of BrdU+ monocytes correlates with the severity of SIVE.

Recently infiltrating MAC387(+) monocytes/macrophages a third macrophage population involved in SIV and HIV encephalitic lesion formation.

Monocytes/macrophages are critical components of HIV and SIV encephalitic lesions. We used in vivo BrdU labeling and markers specific to stages of macrophage differentiation or inflammation to define macrophage heterogeneity and to better define the role of macrophage populations in lesion formation and productive infection. Lesions were heterogeneously composed of resident macrophages (CD68(+)HAM56(+)), perivascular macrophages (CD163(+) CD68(+)MAC387(-)), and recently infiltrated MAC387(+) CD68(-)CD163(-) monocytes/macrophages. At 24 and 48 hours after BrdU inoculation, 30% of MAC387(+) monocytes/macrophages were BrdU(+), consistent with their being recently infiltrated. In perivascular cuffs with low-level SIV replication, MAC387(+) monocytes/macrophages outnumbered CD68(+) macrophages. Conversely, lesions with numerous SIV-p28(+) macrophages and multinucleated giant cells had fewer MAC387(+) monocytes/macrophages. The MAC387(+) cells were not productively infected nor did they express detectable CCR2, unlike perivascular macrophages. Overall, we found that the proportion of MAC387(+) cells tends to be higher than the proportion of CD68(+) macrophages in the brain of animals with mild encephalitis; the ratio was reversed with more severe encephalitis. These results suggest that development of SIV and HIV encephalitis is an active and ongoing process that involves the recruitment and accumulation of: i) nonproductively infected MAC387(+) monocytes/macrophages that are present with inflammation (potentially M1-like macrophages), ii) CD163(+) perivascular macrophages (consistent with M2-like macrophages), and iii) CD68(+) or HAM56(+) resident macrophages. The latter two populations are cellular reservoirs for productive infection.

Alterations in brain metabolism during the first year of HIV infection.

Migration of both uninfected and infected monocytes into the brain during acute HIV infection likely initiates metabolic changes that can be observed with magnetic resonance spectroscopy (MRS). Herein, we measured changes in brain metabolism during the first year of HIV infection and examined the relationship of these metabolite levels to CD16+ monocyte populations measured in the blood. MRS was performed on nine HIV+ subjects identified during acute HIV infection and nine seronegative control subjects. HIV+ subjects were examined within 90 days of an indeterminate Western blot, then again 2 and 6 months later, during early infection. Blood samples were collected for plasma viral RNA and monocyte subset quantification. HIV+ subjects were identified with acute viral ailment and did not display severe cognitive deficits such as dementia or minor cognitive motor disorder. Changes in lipid membrane metabolism (choline levels) in the frontal cortex and white matter were observed during the initial year of HIV infection. Greater numbers of CD16+ monocytes were associated with lower N-acetylaspartate levels and higher choline levels in the brain. These results suggest that HIV infection induces metabolic changes in the brain early during infection and that these changes may be related to monocyte dynamics in the periphery.

Genetically modified CD34+ hematopoietic stem cells contribute to turnover of brain perivascular macrophages in long-term repopulated primates.

Studies in rodents have shown that brain perivascular macrophages are derived from bone marrow precursors. Less is known about the origin and turnover of perivascular cells in the human central nervous system. We took advantage of non-human primates reconstituted with autologous CD34+ hematopoietic stem cells that had been transduced with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) to study the ontogeny of brain macrophages of rhesus macaques. Flow cytometry and immunohistochemistry/fluorescence microscopy showed long-term reconstitution of monocytes/macrophages in the blood, lymphoid, and brain tissues 4 years post-transplant. In the brain, EGFP+ cells were detected in the choroid plexus, cerebellum, and cerebrum, where the percent engraftment between animals reflected the percentage of EGFP+ monocytes in the blood. Morphology and location of brain EGFP+ cells exclusively in the vicinity of blood vessels were consistent with perivascular macrophages. Up to 85% of brain EGFP+ cells expressed CD163, a marker of perivascular macrophages, and greater than 70% were CD68+ macrophages. These findings clearly demonstrate that a subpopulation of CD163+/CD68+ brain perivascular macrophages in rhesus macaques are renewed by CD34+ hematopoietic stem cell-derived precursors and exhibit a continuous long-lasting turnover. Because perivascular macrophages are significant targets of productive HIV/simian immunodeficiency virus infection in the brain, these observations point to hematopoietic stem cells as targets of both HIV/simian immunodeficiency virus infection and potential gene therapy.

Evaluation of a 12-color flow cytometry panel to study lymphocyte, monocyte, and dendritic cell subsets in humans.

Monitoring changes in human immune cell populations such as lymphocytes, monocytes, and dendritic cells (DCs) during infectious diseases like human immunodeficiency virus (HIV) is crucial. However, difficulties to identify rare or heterogeneous cell populations can be limiting. For example, to accurately measure DC subsets, eight flow cytometry parameters are ideal. The aim of this work was to analyze the phenotype of human lymphocyte, monocyte, and DC subsets using a single 12-color flow cytometry panel. After erythrocyte lysis, blood from healthy human volunteers was washed and labeled with a cocktail of 12 antibodies. Samples were analyzed on a Becton-Dickinson FACSAria equipped with three lasers. Data were compared with lineage-specific panels using 5-8 Ab combinations per lineage. Acquired data were analyzed using FlowJo software. Our 12-color panel allows for the identification of the following major subsets of circulating cells in a single tube: CD4+ and CD8+ T lymphocytes, B lymphocytes, NK cells, NKT cells, monocyte subsets (CD14 and/or CD16), and five nonoverlapping HLA-DR+Lin- subsets: CD34+ hematopoietic stem cells, CD123+ plasmacytoid DC, and three subsets of CD11c+ myeloid DC expressing either CD16, CD1c (BDCA-1), or CD141 (BDCA-3). We have developed a single flow cytometry panel that allows for simultaneous detection of the lymphocyte and monocyte cell populations and all known DC subsets. Studying these major players of the immune system in one single panel may give us a broader view of the immune response during HIV infection and the ability to better define the role of individual cell types in Acquired Immune Deficiency Syndrome (AIDS) pathogenesis. (c) 2010 International Society for Advancement of Cytometry.

Human CD34+ CD11b- cord blood stem cells generate in vitro a CD34- CD11b+ subset that is enriched in langerin+ Langerhans dendritic cell precursors.

OBJECTIVE: We investigated whether the expression of CD11b on precursors derived in vitro from CD34+ hematopoietic stem cells was related to their ability to generate CD11b- and CD11b+ Langerhans dendritic cells (LC). METHODS: Human CD34+ cells purified from cord blood were cultured with FLT3 ligand, thrombopoietin, and stem cell factor (FTS) for 2 weeks, analyzed, and sorted by FACS. Sorted fractions were cultured as above, or differentiated into LC with GM-CSF, IL-4, and TGF-beta1 (G4-TGF) for 6 days. The capacity of LC to internalize langerin and dextran was assessed. RESULTS: Ex vivo, human CD34+ cells were CD11b- and mostly CLA+. After 2 weeks of culture with FTS, CD34- CLA- CD11b- and CD34- CLA- CD11b+ cells emerged. CD11b- cells were the most ancestral because they were the only ones to proliferate with FTS, and constantly generated CD11b+ cells. Both CD11b- and CD11b+ sorted cells generated E-cadherin+ langerin+ LC after incubation with G4-TGF. The former fraction contained 46% +/- 15% of E-cadherin+ and 10% +/- 5% of langerin+ cells, whereas in the latter fraction these values reached respectively 66% +/- 23% and 30% +/- 16% (mean +/- SD, n = 7, p < 0.056). Looking at functional properties, CD11b- and CD11b+ LC were similar in terms of langerin and dextran endocytosis. By contrast, only CD11b+ LC internalized fluorescent LPS. CONCLUSION: Human CD34+ CD11b- cells differentiate in FTS culture into a CD34- CD11b- precursor that in turn generates CD34- CD11b+ cells. These cells are enriched in LC precursors compared to CD34- CD11b- cells. Both CD11b- and CD11b+ LC are generated in vitro, and each fraction may assume different functions in inflammatory situations.

A method for obtaining simian immunodeficiency virus RNA sequences from laser capture microdissected and immune captured CD68+ and CD163+ macrophages from frozen tissue sections of bone marrow and brain.

Laser capture microdissection (LCM) is used to extract cells or tissue regions for analysis of RNA, DNA or protein. Several methods of LCM are established for different applications, but a protocol for consistently obtaining lentiviral RNA from LCM captured immune cell populations is not described. Obtaining optimal viral RNA for analysis of viral genes from immune-captured cells using immunohistochemistry (IHC) and LCM is challenging. IHC protocols have long antibody incubation times that increase risk of RNA degradation. But, immune capture of specific cell populations like macrophages without staining for virus cannot result in obtaining only a fraction of cells which are productively lentivirally infected. In this study we sought to obtain simian immunodeficiency virus (SIV) RNA from SIV gp120+ and CD68+ monocyte/macrophages in bone marrow (BM) and CD163+ perivascular macrophages in brain of SIV-infected rhesus macaques. Here, we report an IHC protocol with RNase inhibitors that consistently results in optimal quantity and yield of lentiviral RNA from LCM-captured immune cells.

Haematopoietic stem cells and mesenchymal stem cells as tools for present and future cellular therapies.

Postnatal stem cells are present in many adult tissues, and are thought to ensure homoeostasis by replacing functionally declining cells by newly differentiated ones. Postnatal stem cells used as such or after in vitro manipulation hold out strong hopes for reconstructive therapies. For instance, the grafting of native haematopoietic stem cells (HSC) restores haematopoiesis in genetically deficient individuals or in lethally conditioned leukaemic patients, and systemic injection of in vitro amplified mesenchymal stem cells (MSC) induces recovery of bone growth in patients with osteogenesis imperfecta. Moreover, cells differentiated in vitro from postnatal stem cells exhibiting a specific function can also be used for cell therapy. Myeloid dendritic cells (DC) derived from cultures of HSC may induce tumour-specific cytotoxic T lymphocytes to eradicate the tumour via antigen recognition. In addition, long-lived MSC has been engineered to secrete specific proteins coded by a transgene and used as a source of therapeutic molecules in vivo. All these approaches require large quantities of cells that cannot be obtained (with the exception of HSC) directly from the donor. In vitro procedures allowing the production of therapeutic cells from postnatal stem cells are needed and are at present under development. Below we discuss the rationale and methods currently available for generation of therapeutic cells derived from haematopoietic and mesenchymal stem cells.

Distinct phenotype, longitudinal changes of numbers and cell-associated virus in blood dendritic cells in SIV-infected CD8-lymphocyte depleted macaques.

Loss of circulating CD123+ plasmacytoid dendritic cells (pDCs) during HIV infection is well established. However, changes of myeloid DCs (mDCs) are ambiguous since they are studied as a homogeneous CD11c+ population despite phenotypic and functional heterogeneity. Heterogeneity of CD11c+ mDCs in primates is poorly described in HIV and SIV infection. Using multiparametric flow cytometry, we monitored longitudinally cell number and cell-associated virus of CD123+ pDCs and non-overlapping subsets of CD1c+ and CD16+ mDCs in SIV-infected CD8-depleted rhesus macaques. The numbers of all three DC subsets were significantly decreased by 8 days post-infection. Whereas CD123+ pDCs were persistently depleted, numbers of CD1c+ and CD16+ mDCs rebounded. Numbers of CD1c+ mDCs significantly increased by 3 weeks post-infection while numbers of CD16+ mDCs remained closer to pre-infection levels. We found similar changes in the numbers of all three DC subsets in CD8 depleted animals as we found in animals that were SIV infected animals that were not CD8 lymphocyte depleted. CD16+ mDCs and CD123+ pDCs but not CD1c+ mDCs were significantly decreased terminally with AIDS. All DC subsets harbored SIV RNA as early as 8 days and then throughout infection. However, SIV DNA was only detected in CD123+ pDCs and only at 40 days post-infection consistent with SIV RNA, at least in mDCs, being surface-bound. Altogether our data demonstrate that SIV infection differently affects CD1c+ and CD16+ mDCs where CD16+ but not CD1c+ mDCs are depleted and might be differentially regulated in terminal AIDS. Finally, our data underline the importance of studying CD1c+ and CD16+ mDCs as discrete populations, and not as total CD11c+ mDCs.

High expression levels of BLyS/BAFF by blood dendritic cells and granulocytes are associated with B-cell dysregulation in SIV-infected rhesus macaques.

Dendritic cells (DCs) modulate B-cell survival and differentiation, mainly through production of growth factors such as B lymphocyte stimulator (BLyS/BAFF). In recent longitudinal studies involving HIV-1-infected individuals with different rates of disease progression, we have shown that DCs were altered in number and phenotype in the context of HIV-1 disease progression and B-cell dysregulations were associated with increased BLyS/BAFF expression in plasma and by blood myeloid DCs (mDCs) in rapid and classic progressors but not in HIV-1-elite controllers (EC). Suggesting that the extent to which HIV-1 disease progression is controlled may be linked to BLyS/BAFF expression status and the capacity to orchestrate B-cell responses. Herein, longitudinal analyses of simian immunodeficiency virus (SIV)-infected rhesus macaques also revealed increased expression of BLyS/BAFF by blood mDCs as soon as day 8 and throughout infection. Strikingly, granulocytes presented the highest BLyS/BAFF expression profile in the blood of SIV-infected macaques. BLyS/BAFF levels were also increased in plasma and correlated with viral loads. Consequently, these SIV-infected animals had plasma hyperglobulinemia and reduced blood B-cell numbers with altered population frequencies. These data underscore that BLyS/BAFF is associated with immune dysregulation in SIV-infected rhesus macaques and suggest that BLyS/BAFF is a key regulator of immune activation that is highly conserved among primates. These findings emphasize the potential importance of this SIV-infected primate model to test whether blocking excess BLyS/BAFF has an effect on the overall inflammatory burden and immune restoration.

Soluble CD163 and monocyte populations in response to antiretroviral therapy and in relationship with neuropsychological testing among HIV-infected children.

BACKGROUND: Monocytes play a central role in HIV neuropathogenesis, but there are limited data on monocyte subsets and markers of monocyte activation in perinatally HIV-infected children. OBJECTIVE: To determine the relationship between monocyte subsets, the sCD163 monocyte activation marker, and neuropsychological performance among perinatally HIV-infected children initiating antiretroviral therapy (ART). METHODS: ART-naïve children from the PREDICT study were categorised into two groups: those on ART for ≥24 weeks (ART group, n =201) and those untreated (no ART group, n =79). This analysis used data from the baseline and week 144 including sCD163 and frequencies of activated monocytes (CD14+/CD16+/HLA-DR+), perivascular monocytes (CD14+/CD16+/CD163+ and CD14low/CD16+/CD163+), and neuropsychological testing scores: Verbal and Performance Intelligence Quotient (VIQ and PIQ), Beery Visuomotor Integration (VMI) and Children's Color Trails 2 (CT2). RESULTS: Baseline demographic and HIV disease parameters were similar between groups. The median age was 6 years, CD4 was 20% (620 cells/mm3), and HIV RNA was 4.8 log10. By week 144, the ART vs the no ART group had significantly higher CD4 (938 vs 552 cells/mm3) and lower HIV RNA (1.6 vs 4.38 log10 copies/mL, P <0.05). sCD163 declined in the ART vs no ART group (median changes -2533 vs -159 ng/mL, P <0.0001). Frequencies of all monocyte subsets declined in the treated but not the untreated group (P <0.05). Higher CD14+/CD16+/HLA-DR+ percentage was associated with higher VIQ, Beery VMI and CT2 scores. Higher percentages of CD14+/CD16+/CD163+ and CD14low/CD16+/CD163+ were associated with higher CT2 and VIQ, respectively. CONCLUSION: ART significantly reduced sCD163 levels and frequencies of activated and perivascular monocytes. Higher frequencies of these cells correlated with better neuropsychological performance suggesting a protective role of monocyte-macrophage immune activation in perinatal HIV infection in terms of neuropsychological function.

Flt3+ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia.

BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.

Minocycline inhibition of monocyte activation correlates with neuronal protection in SIV neuroAIDS.

BACKGROUND: Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline's functions are not well defined. METHODS: Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy ((1)H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied. RESULTS: Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14(lo)CD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/Cr (neuronal injury) and circulating CD14+CD16+ and CD14(lo)CD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation. CONCLUSION: Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation.

Immunophenotyping of lymphocyte, monocyte and dendritic cell subsets in normal rhesus macaques by 12-color flow cytometry: clarification on DC heterogeneity.

Monitoring changes in rhesus macaque immune cell populations during infectious disease is crucial. The aim of this work was to simultaneously analyze the phenotype of rhesus macaque lymphocyte, monocyte and dendritic cell (DC) subsets using a single 12-color flow cytometry panel. Blood from healthy non-infected rhesus macaques was labeled with a cocktail of 12 antibodies. Data were compared to three smaller lineage specific panels and absolute and relative percentages of cells were compared. Our 12-color panel allows for the identification of the following major subsets: CD4+ and CD8+ T lymphocytes, B lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, monocyte subsets and four non-overlapping Lin-HLA-DR+ cell subsets: CD34+ hematopoietic stem cells, CD11c- CD123+ plasmacytoid DC, CD11c+ CD16+ and CD11c(-)(/dim) CD1c+ myeloid DC. The development of a multiparameter flow cytometry panel will allow for simultaneous enumeration of mature lymphocyte, NK cells, monocyte and DC subsets. Studying these major players of the immune system in one panel may give us a broader view of the immune response during SIV infection and the ability to better define the role of each of these individual cell types in the pathogenesis of AIDS.

TNF-alpha induces the generation of Langerin/(CD207)+ immature Langerhans-type dendritic cells from both CD14-CD1a and CD14+CD1a- precursors derived from CD34+ cord blood cells.

CD34+ cell-derived hematopoietic precursors amplified with FLT3-ligand, thrombopoietin and stem cell factor became, after a 6-day induction with GM-CSF, IL-4 and TGF-beta1, HLA-DR+, CD1a+, CD83-, CD86-, CD80- cells. A fraction of them expressed Langerin, Lag, and E-cadherin, resembling epidermal Langerhans cells (LC). TNF-alpha added for the last 3 days only marginally induced CD83 expression, but strikingly increased the proportion of immature Langerin+CD83- LC. Langerin+CD83+ and Langerin+CD83- cells were functionally distinct, the former internalizing less efficiently Langerin than the latter. Both CD1a-CD14- and CD1a-CD14+ cells sorted from FLT3-ligand, thrombopoietin and stem cell factor cultures responded to TNF-alpha by an increase of Langerin+ cells. Thus, TNF-alpha rescued LC precursors irrespective of their commitment to the monocytic lineage. When added to GM-CSF, IL-4 and TGF-beta1 containing-cultures, LPS or IL-1beta also induced significant numbers of Langerin+CD83- immature cells displaying a low allostimulatory activity, while CD40-ligand largely promoted highly allostimulatory Langerin-CD83+ cells. Altogether, these data show that in contrast to CD40-ligand, which induced LC maturation even in presence of TGF-beta1, nonspecific proinflammatory factors such as TNF-alpha, IL-1 or LPS, essentially induced immature LC generation, and little cell activation in the presence of TGF-beta1.