Localization of Distinct Peyer's Patch Dendritic Cell Subsets and Their Recruitment by Chemokines Macrophage Inflammatory Protein (Mip)-3α, Mip-3β, and Secondary Lymphoid Organ Chemokine

We describe the anatomical localization of three distinct dendritic cell (DC) subsets in the murine Peyer's patch (PP) and explore the role of chemokines in their recruitment. By two-color in situ immunofluorescence, CD11b+ myeloid DCs were determined to be present in the subepithelial dome (SED) region, whereas CD8α+ lymphoid DCs are present in the T cell–rich interfollicular region (IFR). DCs that lack expression of CD8α or CD11b (double negative) are present in both the SED and IFR. By in situ hybridization, macrophage inflammatory protein (MIP)-3α mRNA was dramatically expressed only by the follicle-associated epithelium overlying the SED, while its receptor, CCR6, was concentrated in the SED. In contrast, CCR7 was expressed predominantly in the IFR. Consistent with these findings, reverse transcriptase polymerase chain reaction analysis and in vitro chemotaxis assays using freshly isolated DCs revealed that CCR6 was functionally expressed only by DC subsets present in the SED, while all subsets expressed functional CCR7. Moreover, none of the splenic DC subsets migrated toward MIP-3α. These data support a distinct role for MIP-3α/CCR6 in recruitment of CD11b+ DCs toward the mucosal surfaces and for MIP-3β/CCR7 in attraction of CD8α+ DCs to the T cell regions. Finally, we demonstrated that all DC subsets expressed an immature phenotype when freshly isolated and maintained expression of subset markers upon maturation in vitro. In contrast, CCR7 expression by myeloid PP DCs was enhanced with maturation in vitro. In addition, this subset disappeared from the SED and appeared in the IFR after microbial stimulation in vivo, suggesting that immature myeloid SED DCs capture antigens and migrate to IFR to initiate T cell responses after mucosal microbial infections.

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Peyer's Patch Dendritic Cells Process Viral Antigen from Apoptotic Epithelial Cells in the Intestine of Reovirus-infected Mice

Journal of Experimental Medicine ◽

10.1084/jem.20041132 ◽

2004 ◽

Vol 200 (2) ◽

pp. 235-245 ◽

Cited By ~ 106

Author(s):

Marina N. Fleeton ◽

Nikhat Contractor ◽

Francisco Leon ◽

J. Denise Wetzel ◽

Terence S. Dermody ◽

...

Keyword(s):

T Cells ◽

Epithelial Cells ◽

Cd4 T Cells ◽

Cell Protein ◽

Peyer’S Patch ◽

Peyer's Patch ◽

Antigen Uptake ◽

Cross Presentation

We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (σ1) and nonstructural (σNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both σ1 and σNS, indicating productive viral replication. In contrast, σ1, but not σNS, was detected in the subepithelial dome (SED) in association with CD11c+/CD8α−/CD11blo DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained σ1, but not σNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, σ1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4+ T cells in vitro. These studies show that CD8α−/CD11blo DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4+ T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

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Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽

10.1182/blood.v81.6.1497.1497 ◽

1993 ◽

Vol 81 (6) ◽

pp. 1497-1504 ◽

Cited By ~ 33

Author(s):

VF Quesniaux ◽

GJ Graham ◽

I Pragnell ◽

D Donaldson ◽

SD Wolpe ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Inhibitory Effect ◽

In Vivo Model ◽

Hematopoietic Progenitors ◽

Inflammatory Protein ◽

Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

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Antigen‐Specific Production of RANTES, Macrophage Inflammatory Protein (MIP)–1α, and MIP‐1β In Vitro Is a Correlate of Reduced Human Immunodeficiency Virus Burden In Vivo

The Journal of Infectious Diseases ◽

10.1086/315849 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1247-1250 ◽

Cited By ~ 24

Author(s):

John Ferbas ◽

Janis V. Giorgi ◽

Shamim Amini ◽

Kathie Grovit‐Ferbas ◽

Dorothy J. Wiley ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Specific Production ◽

Inflammatory Protein ◽

Immunodeficiency Virus ◽

Macrophage Inflammatory Protein

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Reinforcement of Cancer Immunotherapy by Adoptive Transfer of Cblb-Deficient Cytotoxic T Lymphocytes Combined with a Dendritic Cell Vaccine

Blood ◽

10.1182/blood.v116.21.957.957 ◽

2010 ◽

Vol 116 (21) ◽

pp. 957-957

Author(s):

Christina Lutz-Nicoladoni ◽

Patrizia Stoizner ◽

Magdalena Pircher ◽

Stephanie Wallner ◽

Anna Maria Wolf ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dendritic Cell ◽

Cancer Immunotherapy ◽

Cytotoxic T Lymphocytes ◽

Adoptive Transfer ◽

Ex Vivo ◽

Dc Vaccine

Abstract Abstract 957 Introduction: Various approaches to induce immunological rejection of tumors including transfer of autologous tumor infiltrating lymhocytes (TIL) after ex vivo clonal expansion or application of ex vivo transduced antigen specific T cell (TCR) transgenic T cells have been elaborated. In general, adoptive T cell transfer (ATC) has been combined with lympho-depleting agents (e.g. cyclophosphamide). However, the therapeutic efficacy of these cancer immunotherapy approaches is limited due to insufficient in vivo activation, expansion and survival of transferred effector immune cells, which is mainly due to suppressive mileu signals and immune evasion mechanisms induced by TGF-β. The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is assumed to confer TGF-β resistance. Thus we performed a proof-of-concept study evaluating Cbl-b targeting as “intracellular adjuvant” strategy to improve ATC for cancer immunotherapy. Material and Methods: We first tested the in vitro sensitivity of CTL towards TGF-β mediated immuno-suppressive cues and then in vivo evaluated the anti-tumor reactivity of cblb-deficient cytotoxic T lymphocytes (CTL) in murine tumor models alone or in combination with a dendritic cell (DC) vaccine. Results: Cblb-deficient CTL are hyper-responsive to TCR/CD28-stimulation in vitro and protected from the negative cues induced by TGF-β as determined by quantification fo IFN-g secretion and quantification of their proliferative capacity. Unexpectedly, adoptive transfer of polyclonal, non TCR-transgenic cblb-deficient CD8+ CTL, however, is not sufficient to reject B16ova or EG7 tumors in vivo, which is in clear contrast to previous reports using lymphopenic animals receiving adoptively transferred TCR-transgenic T cells. Thus, we next evaluated in vivo re-activation of adoptively transferred cblb-deficient T cells by a DC vaccine (i.e. SIINFEKL-pulsed DC). In strict contrast to ATC monotherapy, this approach now markedly delays tumor outgrowth and significantly increase survival rates, which is paralleled by an increased CTL infiltration rate to the tumor site and an enrichment of ova-specific and IFN-g-secreting CTL in the draining lymph nodes. Moreover, compared to wild-type CTL, cblb-deficient mice vaccinated with the DC vaccine show an increased cytolytic activity in vivo. Conclusions: In summary, we provide experimental evidence that genetic inactivation of cblb in polyclonal, non-TCR transgenic adoptively transferred CTL might serve as a novel “adjuvant approach”, suitable to augment the effectiveness of anti-cancer immunotherapies using ATC in immune-competent recipients. Disclosures: No relevant conflicts of interest to declare.

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TACI-BLyS signaling via B-cell–dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo

Blood ◽

10.1182/blood-2004-12-4708 ◽

2006 ◽

Vol 107 (2) ◽

pp. 594-601 ◽

Cited By ~ 31

Author(s):

Yaiza Diaz-de-Durana ◽

George T. Mantchev ◽

Richard J. Bram ◽

Alessandra Franco

Keyword(s):

B Cells ◽

Dendritic Cell ◽

B Cell ◽

B Lymphocyte ◽

Costimulatory Molecule ◽

Early Signal ◽

Maturation In Vitro ◽

Deficient Mice

AbstractWe demonstrated that B-cell–dendritic cell (DC) interactions via transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) and B-lymphocyte stimulator (BLyS) provide an early signal critical to generate adequate numbers of mature antigen presenting cells (APCs) to prime naive CD8+ T cells (CTLs) in vivo. Evidence that B cells are required for efficient CTL generation in mice and that reconstitution with wild-type but not TACI-knockout B cells restored normal CTL responses support our conclusion. Moreover, low doses of a TACI fusion protein (TACI-Fc) that express the extracellular domain of TACI (amino acid [aa] 1-126) restored CTL priming in B-cell–deficient mice in vivo and induced DC maturation in vitro. In fact, following interactions with B cells, splenic DCs rapidly express the CD86 costimulatory molecule, to an extent comparable to the exposure to antigenic stimuli. BLyShigh peptide-pulsed bone marrow–derived DCs, used as vaccines in vivo, cannot generate CTLs in B-cell–deficient and TACI-deficient mice, strongly supporting a need for B-cell–DC cooperation through TACI-BLyS during CTL first encounter with antigens in vivo.

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Mature Dendritic Cells Infiltrate the T Cell-Rich Region of Oral Mucosa in Chronic Periodontitis: In Situ, In Vivo, and In Vitro Studies

The Journal of Immunology ◽

10.4049/jimmunol.167.8.4693 ◽

2001 ◽

Vol 167 (8) ◽

pp. 4693-4700 ◽

Cited By ~ 134

Author(s):

Ravi Jotwani ◽

Anna Karolina Palucka ◽

Montasr Al-Quotub ◽

Mahyar Nouri-Shirazi ◽

Jay Kim ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Oral Mucosa ◽

Chronic Periodontitis ◽

In Vitro Studies ◽

Rich Region

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Deciphering the Mechanisms of Improved Immunogenicity of Hypochlorous Acid-Treated Antigens in Anti-Cancer Dendritic Cell-Based Vaccines

Vaccines ◽

10.3390/vaccines8020271 ◽

2020 ◽

Vol 8 (2) ◽

pp. 271

Author(s):

Michele Graciotti ◽

Fabio Marino ◽

HuiSong Pak ◽

Petra Baumgaertner ◽

Anne-Christine Thierry ◽

...

Keyword(s):

T Cell ◽

Dendritic Cell ◽

Hypochlorous Acid ◽

T Cell Responses ◽

Cd4 T Cell ◽

Tumor Lysate ◽

Cell Responses ◽

Significant Difference

Hypochlorous acid (HOCl)-treated whole tumor cell lysates (Ox-L) have been shown to be more immunogenic when used as an antigen source for therapeutic dendritic cell (DC)-based vaccines, improving downstream immune responses both in vitro and in vivo. However, the mechanisms behind the improved immunogenicity are still elusive. To address this question, we conducted a proteomic and immunopeptidomics analyses to map modifications and alterations introduced by HOCl treatment using a human melanoma cell line as a model system. First, we show that one-hour HOCl incubation readily induces extensive protein oxidation, mitochondrial biogenesis, and increased expression of chaperones and antioxidant proteins, all features indicative of an activation of oxidative stress-response pathways. Characterization of the DC proteome after loading with HOCl treated tumor lysate (Ox-L) showed no significant difference compared to loading with untreated whole tumor lysate (FT-L). On the other hand, detailed immunopeptidomic analyses on monocyte-derived DCs (mo-DCs) revealed a great increase in human leukocyte antigen class II (HLA-II) presentation in mo-DCs loaded with Ox-L compared to the FT-L control. Further, 2026 HLA-II ligands uniquely presented on Ox-L-loaded mo-DCs were identified. In comparison, identities and intensities of HLA class I (HLA-I) ligands were overall comparable. We found that HLA-II ligands uniquely presented by DCs loaded with Ox-L were more solvent exposed in the structures of their source proteins, contrary to what has been hypothesized so far. Analyses from a phase I clinical trial showed that vaccinating patients using autologous Ox-L as an antigen source efficiently induces polyfunctional vaccine-specific CD4+ T cell responses. Hence, these results suggest that the increased immunogenicity of Ox-L is, at least in part, due to qualitative and quantitative changes in the HLA-II ligandome, potentially leading to an increased HLA-II dependent stimulation of the T cell compartment (i.e., CD4+ T cell responses). These results further contribute to the development of more effective and immunogenic DC-based vaccines and to the molecular understanding of the mechanism behind HOCl adjuvant properties.

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Natural pattern-recognition-receptor agonists as adjuvants for in situ vaccination lymphoma immunotherapy.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.123 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 123-123 ◽

Cited By ~ 1

Author(s):

Mark Aleynick ◽

Paul Peng ◽

Linda Hammerich ◽

Ranjan Upadhyay ◽

Netonia Marshall ◽

...

Keyword(s):

Pattern Recognition ◽

T Cell ◽

Immune Activation ◽

Pattern Recognition Receptor ◽

Receptor Agonists ◽

Prophylactic Vaccines ◽

Recognition Receptor

123 Background: In patients with low-grade lymphoma, in situ vaccination has yielded both partial and complete remissions in clinical trials. Though clinical responses have been observed with multiple pattern recognition receptor agonists (PRRa), the optimal immune stimulant is unknown. We hypothesize that natural PRRa, such as the attenuated pathogens or subunits found in common prophylactic vaccines, could target multiple PRR in a physiologically relevant context and lead to a more robust activation of dendritic cells (DCs) versus synthetic PRRa. Methods: 20 vaccines, including BCG, Typhim Vi, MMR-II, etc. were screened in vitro, where DC phenotype and function were evaluated by flow cytometry. Flt3L-mobilized DC ability to phagocytose, process, present, and cross-present soluble protein or tumor derived antigen, were assessed using CRISPR gene-edited, β2m(-/-) GFP-lymphoma cells and a novel GFP-specific (‘JEDI’) CD8 T cell system. Vaccine mechanism of immune activation was elucidated using a library of PRR-null macrophage cell lines. Potent vaccines were also evaluated in vivo in a Flt3L-primed in situ vaccination using the A20 murine lymphoma model. Results: Several vaccines induced robust DC activation and several showed significant increases in subsequent T cell activation, proliferation, and tumor killing, suggesting increased antigen processing and cross-presentation by DCs. Some vaccines, either as single agents or in combination, were significantly more effective than synthetic PRRa in activating DCs and inducing a T cell response. In vivo, vaccine combination therapies induced tumor regression in a majority of animals, suggesting synergistic immune activation. Conclusions: This data suggests prophylactic vaccines are effective clinical-grade DC activators and can be repurposed for use in the in situ vaccination maneuver, with immediate translation into the clinic. Additionally, by extensive in vitro evaluation in parallel with in vivo studies, this work aims to identify a predictive in vitro molecular immune signature that correlates closely with adjuvant efficacy in vivo.

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Acid-induced Lung Injury

Anesthesiology ◽

10.1097/00000542-200312000-00013 ◽

2003 ◽

Vol 99 (6) ◽

pp. 1323-1332 ◽

Cited By ~ 19

Author(s):

Lilly Madjdpour ◽

Sita Kneller ◽

Christa Booy ◽

Thomas Pasch ◽

Ralph C. Schimmer ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Expression Pattern ◽

Binding Activity ◽

Pyrrolidine Dithiocarbamate ◽

Inflammatory Protein ◽

Nf Kappab ◽

Macrophage Inflammatory Protein

Background Aspiration of acidic gastric contents leads to acute lung injury and is still one of the most common clinical events associated with acute lung injury. This study was performed to assess acid-induced lung inflammation in vitro and in vivo with respect to the time pattern of activated transcription factor nuclear factor-kappaB (NF-kappaB) and proinflammatory molecules. Methods L2 cells (alveolar epithelial cells) were exposed for various periods to a medium with a pH of 6. In the in vivo model, 1 ml/kg of 0.1 n acidic solution was instilled into the lungs of rats. NF-kappaB binding activity and expression pattern of inflammatory mediators were determined. Blocking studies were performed with the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Results In vitro NF-kappaB binding activity showed a biphasic expression pattern with a first peak at 1 h and a second one at 6-8 h. In acid-injured rat lungs, NF-kappaB binding activity was confirmed in a biphasic manner with a first increase at 0.5-2 h (608 +/- 93% and 500 +/- 15%, respectively, P < 0.05) and a second peak at 8 h (697 +/- 35% increase, P < 0.005). Whole lung mRNA for macrophage inflammatory protein-1beta and macrophage inflammatory protein-2 showed a similar expression pattern, which could explain the biphasic neutrophil recruitment. Intratracheal pyrrolidine dithiocarbamate attenuated lung injury as evidenced by a reduction of neutrophil accumulation and expression of inflammatory mediators. Conclusions These data suggest that NF-kappaB binding activity plays a key role in molecular and cellular events in acid-induced lung injury.

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