Penelope A. Morel

Dendritic Cells Mediate NK Cell Help for Th1 and CTL Responses: Two-Signal Requirement for the Induction of NK Cell Helper Function

Early stages of viral infections are associated with local recruitment and activation of dendritic cells (DC) and NK cells. Although activated DC and NK cells are known to support each other’s functions, it is less clear whether their local interaction in infected tissues can modulate the subsequent ability of migrating DC to induce T cell responses in draining lymph nodes. In this study, we report that NK cells are capable of inducing stable type 1-polarized “effector/memory” DC (DC1) that act as carriers of NK cell-derived helper signals for the development of type 1 immune responses. NK cell-induced DC1 show a strongly elevated ability to produce IL-12p70 after subsequent CD40 ligand stimulation. NK-induced DC1 prime naive CD4+ Th cells for high levels of IFN-γ, but low IL-4 production, and demonstrate a strongly enhanced ability to induce Ag-specific CD8+ T cell responses. Resting NK cells display stringent activation requirements to perform this novel, DC-mediated, “helper” function. Although their interaction with K562 cells results in effective target cell killing, the induction of DC1 requires a second NK cell-activating signal. Such costimulatory signal can be provided by type I IFNs, common mediators of antiviral responses. Therefore, in addition to their cytolytic function, NK cells also have immunoregulatory activity, induced under more stringent conditions. The currently demonstrated helper activity of NK cells may support the development of Th1- and CTL-dominated type 1 immunity against intracellular pathogens and may have implications for cancer immunotherapy.

Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model.

This article describes an in vivo imaging method for visualizing and quantifying a specific cell population. Cells are labeled ex vivo with a perfluoropolyether nanoparticle tracer agent and then detected in vivo using 19F MRI following cell transfer. 19F MRI selectively visualizes only the labeled cells with no background, and a conventional 1H image taken in the same imaging session provides anatomical context. Using the nonobese diabetic mouse, an established model of type 1 diabetes, 19F MRI data were acquired showing the early homing behavior of diabetogenic T cells to the pancreas. A computational algorithm provided T cell counts in the pancreas. Approximately 2% of the transferred cells homed to the pancreas after 48 hr. The technique allows for both unambiguous detection of labeled cells and quantification directly from the in vivo images. The in vivo quantification and cell trafficking patterns were verified using 19F spectroscopy and fluorescence microscopy in excised pancreata. The labeling procedure did not affect T‐cell migration in vivo. This imaging platform is applicable to many cell types and disease models and can potentially be used for monitoring the trafficking of cellular therapeutics. Magn Reson Med 58:725–734, 2007.

Receptor-mediated endocytosis of iron-oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging

Dendritic cells (DCs) function as antigen presenting cells in vivo and play a fundamental role in numerous diseases. New methods are described for high‐efficiency intracellular labeling of DCs with superparamagnetic iron‐oxide (SPIO) utilizing a receptor‐mediated endocytosis (RME) mechanism. Bone marrow‐derived DCs or a fetal skin‐derived DC line were incubated with SPIO conjugated to anti‐CD11c monoclonal antibody (mAb) under conditions favoring RME. These cells exhibited approximately a 50‐fold increase in uptake relative to DCs incubated with SPIO without the mAb. Flow cytometry studies assaying cell surface markers showed a down‐modulation of CD11c, but no other changes in phenotype. Immunological function of the DCs was unmodified by the labeling, as determined by cytokine secretion assays. The RME mechanism was confirmed using electron microscopy, endocytosis inhibition assays, and incubation experiments with SPIO conjugated to mAbs against accessory molecules that are not expressed on DCs. Labeled DCs were injected into murine quadriceps and monitored in vivo for several days using MR microimaging at 11.7 T. DCs were observed to remain within the muscle for >24 hr. The use of RME is an efficient way to label immune cells for in vivo MRI and can be applied to a wide variety of cell types. Magn Reson Med 49:1006–1013, 2003.

Dominant role of antigen dose in CD4+Foxp3+ regulatory T cell induction and expansion

The definitions of tolerogenic vs immunogenic dendritic cells (DC) remain controversial. Immature DC have been shown to induce T regulatory cells (Treg) specific for foreign and allogeneic Ags. However, we have previously reported that mature DC (mDC) prevented the onset of autoimmune diabetes, whereas immature DC (iDC) were therapeutically ineffective. In this study, islet-specific CD4+ T cells from BDC2.5 TCR-transgenic mice were stimulated in the absence of exogenous cytokine with iDC or mDC pulsed with high- or low-affinity antigenic peptides and examined for Treg induction. Both iDC and mDC presenting low peptide doses induced weak TCR signaling via the Akt/mammalian target of rapamycin (mTOR) pathway, resulting in significant expansion of Foxp3+ Treg. Furthermore, unpulsed mDC, but not iDC, also induced Treg. High peptide doses induced strong Akt/mTOR signaling and favored the expansion of Foxp3neg Th cells. The inverse correlation of Foxp3 and Akt/mTOR signaling was also observed in DO11.10 and OT-II TCR-transgenic T cells and was recapitulated with anti-CD3/CD28 stimulation in the absence of DC. IL-6 production in these cultures correlated positively with Ag dose and inversely with Treg expansion. Studies with T cells or DC from IL-6−/− mice revealed that IL-6 production by T cells was more important in the inhibition of Treg induction at low Ag doses. These studies indicate that the strength of Akt/mTOR signaling, a critical T cell-intrinsic determinant for Treg vs Th induction, can be controlled by adjusting the dose of antigenic peptide. Furthermore, this operates in a dominant fashion over DC phenotype and cytokine production.

In vivo cytometry of antigen-specific t cells using 19F MRI.

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A 19F MRI‐based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen‐specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal 19F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen. The apparent T‐cell numbers were calculated in the LN from the time‐lapse 19F MRI data. The effect of in vivo T‐cell division on the 19F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and 19F MRI is broadly applicable to studies of whole‐body cell biodistribution. Magn Reson Med, 2009.

Immunogenetic risk and protective factors for the idiopathic inflammatory myopathies: distinct HLA-A, -B, -Cw, -DRB1, and -DQA1 allelic profiles distinguish European American patients with different myositis autoantibodies.

Abstract: The idiopathic inflammatory myopathies (IIM) are systemic connective tissue diseases defined by chronic muscle inflammation and weakness associated with autoimmunity. We have performed low to high resolution molecular typing to assess the genetic variability of major histocompatibility complex loci (HLA-A, -B, -Cw, -DRB1, and -DQA1) in a large population of European American patients with IIM (n = 571) representing the major myositis autoantibody groups. We established that alleles of the 8.1 ancestral haplotype (8.1 AH) are important risk factors for the development of IIM in patients producing anti-synthetase/anti-Jo-1, -La, -PM/Scl, and -Ro autoantibodies. Moreover, a random forests classification analysis suggested that 8.1 AH-associated alleles B*0801 and DRB1*0301 are the principal HLA risk markers. In addition, we have identified several novel HLA susceptibility factors associated distinctively with particular myositis-specific (MSA) and myositis-associated autoantibody (MAA) groups of the IIM. IIM patients with anti-PL-7 (anti-threonyl-tRNA synthetase) autoantibodies have a unique HLA Class I risk allele, Cw*0304 (pcorr = 0.046), and lack the 8.1 AH markers associated with other anti-synthetase autoantibodies (for example, anti-Jo-1 and anti-PL-12). In addition, HLA-B*5001 and DQA1*0104 are novel potential risk factors among anti-signal recognition particle autoantibody-positive IIM patients (pcorr = 0.024 and p = 0.010, respectively). Among those patients with MAA, HLA DRB1*11 and DQA1*06 alleles were identified as risk factors for myositis patients with anti-Ku (pcorr = 0.041) and anti-La (pcorr = 0.023) autoantibodies, respectively. Amino acid sequence analysis of the HLA DRB1 third hypervariable region identified a consensus motif, 70D (hydrophilic)/71R (basic)/74A (hydrophobic), conferring protection among patients producing anti-synthetase/anti-Jo-1 and -PM/Scl autoantibodies. Together, these data demonstrate that HLA signatures, comprising both risk and protective alleles or motifs, distinguish IIM patients with different myositis autoantibodies and may have diagnostic and pathogenic implications. Variations in associated polymorphisms for these immune response genes may reflect divergent pathogenic mechanisms and/or responses to unique environmental triggers in different groups of subjects resulting in the heterogeneous syndromes of the IIM. Abbreviations: AH = ancestral haplotype, DM = dermatomyositis, EA = European Americans, HVR3 = third hypervariable region, IBM = inclusion body myositis, IIM = idiopathic inflammatory myopathies, MAA = myositis-associated autoantibodies, MHC = major histocompatibility complex, MSA = myositis-specific autoantibodies, PM = polymyositis, RF = random forests, RSP = restrictive supertype patterns, SRP = signal recognition particle.

Regulatory Th2 response induced following adoptive transfer of dendritic cells in prediabetic NOD mice

We previously demonstrated that immunotherapy with dendritic cells (DC) prevented diabetes development in prediabetic NOD mice and that this effect was optimal when using a stimulatory DC population generated from bone marrow cells cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IL‐4. In this study, we have investigated the mechanism by which GM‐CSF‐ and IL‐4‐cultured DC prevent diabetes in prediabetic NOD mice. Histological analysis of pancreatic tissue from DC‐treated mice revealed a reduction in the severity of insulitis compared to controls. Analysisof the T cell response in DC‐treated mice suggested a general shift towards a Th2‐dominated response, as determined by cytokine production following either concanavalin A or anti‐TCR stimulation. Furthermore, sorted CD45RBlo CD25+ CD4+ T cells from the spleen of DC‐treated mice produced high amounts of Th2 cytokines following anti‐TCR stimulation, suggesting that these cells are responsible for the apparent Th2 shift. We conclude that DC therapy may have corrected the immunoregulatory defect in the NOD mouse, thus restoring a balance between pathogenic Th1 cells and protective Th2 cells.

FcγRIIa, Not FcγRIIb, Is Constitutively and Functionally Expressed on Skin-Derived Human Mast Cells

The expression of FcγR by human skin-derived mast cells of the MCTC type was determined in the current study. Expression of mRNA was analyzed with microarray gene chips and RT-PCR; protein by Western blotting and flow cytometry; function by release of β-hexosaminidase, PGD2, leukotriene C4 (LTC4), IL-5, IL-6, IL-13, GM-CSF, and TNF-α. FcγRIIa was consistently detected along with FcεRI at the mRNA and protein levels; FcγRIIc was sometimes detected only by RT-PCR; but FcγRIIb, FcγRI, and FcγRIII mRNA and protein were not detected. FcγRIIa-specific mAb caused skin MCTC cells to degranulate and secrete PGD2, LTC4, GM-CSF, IL-5, IL-6, IL-13, and TNF-α in a dose-dependent fashion. FcεRI-specific mAb caused similar amounts of each mediator to be released with the exception of LTC4, which was not released by this agonist. Simultaneous but independent cross-linking of FcεRI and FcγRIIa did not substantially alter mediator release above or below levels observed with each agent alone. Skin MCTC cells sensitized with dust-mite-specific IgE and IgG, when coaggregated by Der p2, exhibited enhanced degranulation compared with sensitization with either IgE or IgG alone. These results extend the known capabilities of human skin mast cells to respond to IgG as well as IgE-mediated signals.

Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage‐colony stimulating factor (GM), GM + interleukin (IL)‐4, or GM + IL‐15 and generated three distinct DC populations. The GM + IL‐4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced ow levels of IL‐12p70. GM and GM + IL‐15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL‐15 DCs produced high levels of IL‐12p70 and interferon (IFN)‐γ, whereas GM DCs produced only high levels of IL‐12p70. Naive T cells stimulated with GM + IL‐4 DCs secreted high levels of IL‐4 and IL‐5 in addition to IFN‐γ. In contrast, the GM + IL‐15 DCs induced higher IFN‐γ production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL‐12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL‐12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC‐based vaccines in immunotherapy of cancer and other clinical conditions.

Dendritic Cells Promote Macrophage Infiltration and Comprise a Substantial Proportion of Obesity-Associated Increases in CD11c+ Cells in Adipose Tissue and Liver

Obesity-associated increases in adipose tissue (AT) CD11c+ cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)–induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow–derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD.