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1.
J Leukoc Biol ; 107(2): 323-339, 2020 02.
Article in English | MEDLINE | ID: mdl-31749181

ABSTRACT

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c- , CD304+ , CD85g+ , and myeloid DC that are CD11c+ . The CD11c+ DC are readily classified as CD1c+ DC and CD141+ DC. Monocytes are broadly divided into the CD14+ CD16- (classical) and CD14dim CD16+ subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dim CD16+ monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dim CD16+ monocytes (CD16+ Mo), and CD14- CD16+ DC (CD16+ DC). We sought to identify the functional and kinetic relationship of CD16+ DC to CD16+ Mo. We demonstrate that differentiation of CD16+ DC and CD16+ Mo during activation with IFNγ in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+ DC in both auto- and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+ Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.


Subject(s)
Antigen-Presenting Cells/immunology , Biomarkers/analysis , Dendritic Cells/immunology , Graft vs Host Disease/immunology , Monocytes/immunology , Myeloid Cells/immunology , Receptors, IgG/metabolism , Antigen-Presenting Cells/metabolism , Antigens, Surface/metabolism , Cell Differentiation , Cell Lineage , Dendritic Cells/metabolism , GPI-Linked Proteins/metabolism , Graft vs Host Disease/diagnosis , Graft vs Host Disease/metabolism , HLA-DR Antigens/metabolism , Hematopoietic Stem Cell Transplantation , Humans , Lectins, C-Type/metabolism , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/therapy , Membrane Glycoproteins/metabolism , Monocytes/metabolism , Multiple Myeloma/immunology , Multiple Myeloma/therapy , Myeloid Cells/metabolism , Receptors, CCR5/metabolism , Receptors, Cell Surface/metabolism , Transplantation, Homologous
2.
Mol Oncol ; 13(10): 2107-2120, 2019 10.
Article in English | MEDLINE | ID: mdl-31338922

ABSTRACT

Antibody-based therapy in acute myeloid leukemia (AML) has been marred by significant hematologic toxicity due to targeting of both hematopoietic stem and progenitor cells (HSPCs). Achieving greater success with therapeutic antibodies requires careful characterization of the potential target molecules on AML. One potential target is CD300f, which is an immunoregulatory molecule expressed predominantly on myeloid lineage cells. To confirm the value of CD300f as a leukemic target, we showed that CD300f antibodies bind to AML from 85% of patient samples. While one CD300f monoclonal antibody (mAb) reportedly did not bind healthy hematopoietic stem cells, transcriptomic analysis found that CD300f transcripts are expressed by healthy HSPC. Several CD300f protein isoforms exist as a result of alternative splicing. Importantly for antibody targeting, the extracellular region of CD300f can be present with or without the exon 4-encoded sequence. This results in CD300f isoforms that are differentially bound by CD300f-specific antibodies. Furthermore, binding of one mAb, DCR-2, to CD300f exposes a structural epitope recognized by a second CD300f mAb, UP-D2. Detailed analysis of publicly available transcriptomic data indicated that CD34+ HSPC expressed fewer CD300f transcripts that lacked exon 4 compared to AML with monocytic differentiation. Analysis of a small cohort of AML cells revealed that the UP-D2 conformational binding site could be induced in cells from AML patients with monocytic differentiation but not those from other AML or HSPC. This provides the opportunity to develop an antibody-based strategy to target AMLs with monocytic differentiation but not healthy CD34+ HSPCs. This would be a major step forward in developing effective anti-AML therapeutic antibodies with reduced hematologic toxicity.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Epitopes/immunology , Leukemia, Myeloid, Acute/drug therapy , Receptors, Immunologic/immunology , Cell Line, Tumor , Humans , Leukemia, Myeloid, Acute/immunology , Molecular Targeted Therapy , Monocytes/drug effects , Monocytes/immunology , Receptors, Immunologic/antagonists & inhibitors
3.
PLoS One ; 14(5): e0216368, 2019.
Article in English | MEDLINE | ID: mdl-31075107

ABSTRACT

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with ~20,000 new cases yearly. The disease develops in people of all ages, but is more prominent in the elderly, who due to limited treatment options, have poor overall survival rates. Monoclonal antibodies (mAb) targeting specific cell surface molecules have proven to be safe and effective in different haematological malignancies. However, AML target molecules are currently limited so discovery of new targets would be highly beneficial to patients. We examined the C-type lectin receptor CD302 as a potential therapeutic target for AML due to its selective expression in myeloid immune populations. In a cohort of 33 AML patients with varied morphological and karyotypic classifications, 88% were found to express CD302 on the surface of blasts and 80% on the surface of CD34+ CD38- population enriched with leukemic stem cells. A mAb targeting human CD302 was effective in mediating antibody dependent cell cytotoxicity and was internalised, making it amenable to toxin conjugation. Targeting CD302 with antibody limited in vivo engraftment of the leukemic cell line HL-60 in NOD/SCID mice. While CD302 was expressed in a hepatic cell line, HepG2, this molecule was not detected on the surface of HepG2, nor could HepG2 be killed using a CD302 antibody-drug conjugate. Expression was however found on the surface of haematopoietic stem cells suggesting that targeting CD302 would be most effective prior to haematopoietic transplantation. These studies provide the foundation for examining CD302 as a potential therapeutic target for AML.


Subject(s)
Antigens, Neoplasm/metabolism , Antineoplastic Agents, Immunological/pharmacology , Blast Crisis , Drug Delivery Systems , Lectins, C-Type/metabolism , Leukemia, Myeloid, Acute , Neoplastic Stem Cells , Receptors, Cell Surface/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Blast Crisis/drug therapy , Blast Crisis/metabolism , Blast Crisis/pathology , Female , HL-60 Cells , Hematopoietic Stem Cell Transplantation , Hep G2 Cells , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/therapy , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Xenograft Model Antitumor Assays
4.
Front Oncol ; 9: 50, 2019.
Article in English | MEDLINE | ID: mdl-30788290

ABSTRACT

Monoclonal antibodies targeting co-inhibitory immune checkpoint molecules have been successful in clinical trials of both solid and hematological malignancies as acknowledged by the 2018 Nobel Prize in Medicine, however improving clinical response rates is now key to expanding their efficacy in areas of unmet medical need. Antibodies to checkpoint inhibitors target molecules on either T cells or tumor cells to stimulate T cells or remove tumor mediated immunosuppression, respectively. However, many of the well-characterized T cell immune checkpoint receptors have their ligands on antigen presenting cells or exert direct effects on those cells. Dendritic cells are the most powerful antigen presenting cells; they possess the ability to elicit antigen-specific responses and have important roles in regulation of immune tolerance. Despite their theoretical benefits in cancer immunotherapy, the translation of DC therapies into the clinic is yet to be fully realized and combining DC-based immunotherapy with immune checkpoint inhibitors is an attractive strategy. This combination takes advantage of the antigen presenting capability of DC to maximize specific immune responses to tumor antigens whilst removing tumor-associated immune inhibitory mechanisms with immune checkpoint inhibition. Here we review the expression and functional effects of immune checkpoint molecules on DC and identify rational combinations for DC vaccination to enhance antigen-specific T cell responses, cytokine production, and promotion of long-lasting immunological memory.

5.
Semin Cell Dev Biol ; 86: 77-88, 2019 02.
Article in English | MEDLINE | ID: mdl-29454038

ABSTRACT

The ability of immune therapies to control cancer has recently generated intense interest. This therapeutic outcome is reliant on T cell recognition of tumour cells. The natural function of dendritic cells (DC) is to generate adaptive responses, by presenting antigen to T cells, hence they are a logical target to generate specific anti-tumour immunity. Our understanding of the biology of DC is expanding, and they are now known to be a family of related subsets with variable features and function. Most clinical experience to date with DC vaccination has been using monocyte-derived DC vaccines. There is now growing experience with alternative blood-derived DC derived vaccines, as well as with multiple forms of tumour antigen and its loading, a wide range of adjuvants and different modes of vaccine delivery. Key insights from pre-clinical studies, and lessons learned from early clinical testing drive progress towards improved vaccines. The potential to fortify responses with other modalities of immunotherapy makes clinically effective "second generation" DC vaccination strategies a priority for cancer immune therapists.


Subject(s)
Dendritic Cells/immunology , Dendritic Cells/transplantation , Immunotherapy , Neoplasms/immunology , Neoplasms/therapy , Humans , T-Lymphocytes/immunology
6.
Oncoimmunology ; 7(4): e1419114, 2018.
Article in English | MEDLINE | ID: mdl-29632738

ABSTRACT

Only modest advances in AML therapy have occurred in the past decade and relapse due to residual disease remains the major challenge. The potential of the immune system to address this is evident in the success of allogeneic transplantation, however this leads to considerable morbidity. Dendritic cell (DC) vaccination can generate leukemia-specific autologous immunity with little toxicity. Promising results have been achieved with vaccines developed in vitro from purified monocytes (Mo-DC). We now demonstrate that blood DC (BDC) have superior function to Mo-DC. Whilst BDC are reduced at diagnosis in AML, they recover following chemotherapy and allogeneic transplantation, can be purified using CMRF-56 antibody technology, and can stimulate functional T cell responses. While most AML patients in remission had a relatively normal T cell landscape, those who had received fludarabine as salvage therapy have persistent T cell abnormalities including reduced number, altered subset distribution, failure to expand, and increased activation-induced cell death. Furthermore, PD-1 and TIM-3 are increased on CD4T cells in AML patients in remission and their blockade enhances the expansion of leukemia-specific T cells. This confirms the feasibility of a BDC vaccine to consolidate remission in AML and suggests it should be tested in conjunction with checkpoint blockade.

7.
Haematologica ; 103(4): 655-665, 2018 04.
Article in English | MEDLINE | ID: mdl-29351987

ABSTRACT

Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83-T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma.


Subject(s)
Antigens, CD/blood , Biomarkers, Tumor/blood , Hodgkin Disease/drug therapy , Immunoglobulins/blood , Membrane Glycoproteins/blood , Molecular Targeted Therapy/methods , Adolescent , Adult , Aged , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antigens, CD/immunology , Female , Hodgkin Disease/diagnosis , Humans , Immunoglobulins/immunology , Male , Membrane Glycoproteins/immunology , Middle Aged , Salvage Therapy/methods , T-Lymphocytes/cytology , Young Adult , CD83 Antigen
8.
FASEB J ; 32(1): 478-487, 2018 01.
Article in English | MEDLINE | ID: mdl-28928247

ABSTRACT

Previous studies demonstrated that endogenous glucocorticoid signaling in osteoblasts promotes inflammation in murine immune arthritis. The current study determined whether disruption of endogenous glucocorticoid signaling in chondrocytes also modulates the course and severity of arthritis. Tamoxifen-inducible chondrocyte-targeted glucocorticoid receptor-knockout (chGRKO) mice were generated by breeding GRflox/flox mice with tamoxifen-inducible collagen 2a1 Cre (Col2a1-CreERT2) mice. Antigen-induced arthritis (AIA) and K/BxN serum transfer-induced arthritis (STIA) were induced in both chGRKO mice and their Cre-negative GRflox/flox littermates [wild type (WT)]. Arthritis was assessed by measurement of joint swelling and histology of joints collected at d 14. Neutrophil activity and gene expression patterns associated with cartilage damage were also evaluated. In both arthritis models clinical (joint swelling) and histologic indices of inflammatory activity were significantly greater in chGRKO than in WT mice. The STIA model was characterized by early up-regulation of CXCR2/CXCR2 ligand gene expression in ankle tissues, and significant and selective expansion of splenic CXCR2+ neutrophils in chGRKO arthritic compared to WT arthritic mice. At later stages, gene expression of enzymes involved in cartilage degradation was up-regulated in chGRKO but not WT arthritic mice. Therefore, we summarize that chondrocytes actively mitigate local joint inflammation, cartilage degradation and systemic neutrophil activity via a glucocorticoid-dependent pathway.-Tu, J., Stoner, S., Fromm, P. D., Wang, T., Chen, D., Tuckermann, J., Cooper, M. S., Seibel, M. J., Zhou, H. Endogenous glucocorticoid signaling in chondrocytes attenuates joint inflammation and damage.


Subject(s)
Arthritis, Experimental/metabolism , Chondrocytes/metabolism , Glucocorticoids/metabolism , Animals , Arthritis, Experimental/etiology , Arthritis, Experimental/pathology , Bone and Bones/metabolism , Bone and Bones/pathology , Chemokines/genetics , Chemokines/metabolism , Inflammation Mediators/metabolism , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, Transgenic , Receptors, Glucocorticoid/deficiency , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Signal Transduction , Up-Regulation
9.
J Immunol ; 197(12): 4613-4625, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27837105

ABSTRACT

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.


Subject(s)
Antigens, CD/metabolism , Dendritic Cells/immunology , Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation , Immunoglobulins/metabolism , Membrane Glycoproteins/metabolism , Monocytes/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Acute Disease , Animals , Antigens, CD/genetics , Antigens, Viral/immunology , Cells, Cultured , Glycosylation , Humans , Immunoglobulins/genetics , Lymphocyte Activation , Membrane Glycoproteins/genetics , Mice , RNA Isoforms/genetics , RNA, Messenger/genetics , Transplantation, Homologous , CD83 Antigen
10.
Oncoimmunology ; 5(6): e1168555, 2016 Jun.
Article in English | MEDLINE | ID: mdl-27471645

ABSTRACT

There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

11.
J Immunol ; 197(3): 885-98, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27316686

ABSTRACT

C-type lectin receptors play important roles in immune cell interactions with the environment. We described CD302 as the simplest, single domain, type I C-type lectin receptor and showed it was expressed mainly on the myeloid phagocytes in human blood. CD302 colocalized with podosomes and lamellopodia structures, so we hypothesized that it played a role in cell adhesion or migration. In this study, we used mouse models to obtain further insights into CD302 expression and its potential immunological function. Mouse CD302 transcripts were, as in humans, highest in the liver, followed by lungs, lymph nodes (LN), spleen, and bone marrow. In liver, CD302 was expressed by hepatocytes, liver sinusoidal endothelial cells, and Kupffer cells. A detailed analysis of CD302 transcription in mouse immune cells revealed highest expression by myeloid cells, particularly macrophages, granulocytes, and myeloid dendritic cells (mDC). Interestingly, 2.5-fold more CD302 was found in migratory compared with resident mDC populations and higher CD302 expression in mouse M1 versus M2 macrophages was also noteworthy. CD302 knockout (CD302KO) mice were generated. Studies on the relevant immune cell populations revealed a decrease in the frequency and numbers of migratory mDC within CD302KO LN compared with wild-type LN. In vitro studies showed CD302KO and wild-type DC had an equivalent capacity to undergo maturation, prime T cells, uptake Ags, and migrate toward the CCL19/CCL21 chemokines. Nevertheless, CD302KO migratory DC exhibited reduced in vivo migration into LN, confirming a functional role for CD302 in mDC migration.


Subject(s)
Chemotaxis, Leukocyte/physiology , Dendritic Cells/metabolism , Lectins, C-Type/metabolism , Receptors, Cell Surface/metabolism , Animals , Cell Line , Cell Separation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunohistochemistry , Mice , Mice, Knockout , Polymerase Chain Reaction
12.
Immunol Cell Biol ; 94(5): 447-57, 2016 05.
Article in English | MEDLINE | ID: mdl-26791160

ABSTRACT

Human plasmacytoid dendritic cells (pDCs) were considered to be a phenotypically and functionally homogeneous cell population; however, recent analyses indicate potential heterogeneity. This is of major interest, given their importance in the induction of anti-viral responses and their role in creating immunologically permissive environments for human malignancies. For this reason, we investigated the possible presence of human pDC subsets in blood and bone marrow, using unbiased cell phenotype clustering and functional studies. This defined two major functionally distinct human pDC subsets, distinguished by differential expression of CD2. The CD2(hi) and CD2(lo) pDCs represent discontinuous subsets, each with hallmark pDC functionality, including interferon-alpha production. The rarer CD2(hi) pDC subset demonstrated a significant survival advantage over CD2(lo) pDC during stress and upon exposure to glucocorticoids (GCs), which was associated with higher expression of the anti-apoptotic molecule BCL2. The differential sensitivity of these two human pDC subsets to GCs is demonstrated in vivo by a relative increase in CD2(hi) pDC in multiple myeloma patients treated with GCs. Hence, the selective apoptosis of CD2(lo) pDC during stress represents a novel mechanism for the control of innate responses.


Subject(s)
CD2 Antigens/metabolism , Dendritic Cells/metabolism , Stress, Physiological , Apoptosis/drug effects , Bone Marrow/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Survival/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Glucocorticoids/pharmacology , Humans , Ligands , Lymph Nodes/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Immunologic/metabolism , Stress, Physiological/drug effects , Toll-Like Receptors/metabolism
13.
Pharmacol Rev ; 67(4): 731-53, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26240218

ABSTRACT

Although the earliest­rudimentary­attempts at exploiting the immune system for cancer therapy can be traced back to the late 18th Century, it was not until the past decade that cancer immunotherapeutics have truly entered mainstream clinical practice. Given their potential to stimulate both adaptive and innate antitumor immune responses, dendritic cells (DCs) have come under intense scrutiny in recent years as pharmacological tools for cancer immunotherapy. Conceptually, the clinical effectiveness of this form of active immunotherapy relies on the completion of three critical steps: 1) the DCs used as immunotherapeutic vehicles must properly activate the antitumor immune effector cells of the host, 2) these immune effector cells must be receptive to stimulation by the DCs and be competent to mediate their antitumor effects, which 3) requires overcoming the various immune-inhibitory mechanisms used by the tumor cells. In this review, following a brief overview of the pivotal milestones in the history of cancer immunotherapy, we will introduce the reader to the basic immunobiological and pharmacological principles of active cancer immunotherapy using DCs. We will then discuss how current research is trying to define the optimal parameters for each of the above steps to realize the full clinical potential of DC therapeutics. Given its high suitability for immune interventions, acute myeloid leukemia was chosen here to showcase the latest research trends driving the field of DC-based cancer immunotherapy.


Subject(s)
Dendritic Cells/metabolism , Immunotherapy, Active/methods , Neoplasms/immunology , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer/methods , Antibodies, Monoclonal , Antigens, Neoplasm/immunology , Apoptosis , Cancer Vaccines/immunology , Cell Culture Techniques , Cytokines/biosynthesis , Dendritic Cells/immunology , Dose-Response Relationship, Drug , Drug Administration Routes , Humans , Killer Cells, Natural/immunology , Major Histocompatibility Complex/immunology , Signal Transduction
14.
Front Microbiol ; 6: 1520, 2015.
Article in English | MEDLINE | ID: mdl-26834705

ABSTRACT

Induction of inducible nitric oxide synthase in mononuclear phagocytes by IFN-γ and innate tumor necrosis factor (TNF) provide the basis for an effective immune response to the intracellular parasite Leishmania (L.) major. In previous experiments, we observed a fatal visceral form of leishmaniasis in L. major-infected C57BL/6 TNF(-/-) mice. To further delineate the protective function of TNF and its receptor requirements, we comparatively assessed L. major-infected C57BL/6 mice that were either deficient for membrane and soluble TNF (Tnf (-) (/) (-)), for soluble TNF alone (memTnf(Δ/Δ) ), or the TNF receptors type 1 (Tnfr1 (-) (/) (-)) or type 2 (Tnfr2 (-) (/) (-)). We detected locally and systemically increased levels of the cytokine IFN-γ in the absence of the TNF-TNFR1-signaling pathway. An analysis of transcription factors and cytokines revealed that activated Tnf (-) (/) (-) CD4(+) T cells displayed a highly active Th1 phenotype with a strong usage of the T cell receptor Vß5.1/2. From these data we conclude that the fatal outcome of L. major infection in Tnf (-) (/) (-) mice does not result from a skewed or deficient Th1 differentiation.

15.
Immunol Cell Biol ; 91(5): 335-9, 2013 May.
Article in English | MEDLINE | ID: mdl-23588497

ABSTRACT

The CC-chemokine receptor 6 (CCR6) is expressed constitutively at an intermediate level on naïve B cells and is upregulated after activation on pregerminal center (GC) B cells. We hypothesized that it could be involved in the events leading to GC reaction and high-affinity antibody production, and therefore investigated the potential role of CCR6 in B-cell differentiation in vivo. After antigenic challenge of CCR6-/- mice with the T-cell-dependent antigen nitrophenyl-keyhole limpet hemocyanin (NP-KLH), GC B-cell development was found to be accelerated and the number of GC had increased significantly compared with control mice, but the antibodies produced by CCR6-/- B cells were on average of lower affinity. We conclude from these data that the CCR6/CCL20 axis has an important role in regulating the kinetics and efficiency of the GC reaction.


Subject(s)
B-Lymphocytes/immunology , Germinal Center/immunology , Receptors, CCR6/metabolism , Animals , Antibody Affinity/genetics , Antibody Formation/genetics , Chemokine CCL20/metabolism , Gene Expression Regulation , Germinal Center/cytology , Haptens , Hemocyanins/immunology , Immunomodulation , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, CCR6/genetics , Receptors, CCR6/immunology , Up-Regulation
16.
J Immunol ; 188(12): 6258-66, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22615203

ABSTRACT

In the absence of TNF, the normally resistant C57BL/6 (B6.WT) strain develops a fatal, progressive form of leishmaniasis after infection with Leishmania major. It is not yet understood which TNF activity or the lack thereof is responsible for the dramatic progression of leishmaniasis in TNF-negative (B6.TNF(-/-)) mice. To elucidate the underlying mechanisms resulting in the fatal outcome of L. major infection in this gene-deficient mouse strain, we analyzed the monocytic component of the inflammatory infiltrate in the draining popliteal lymph node and the site of the infection using multicolor flow cytometry. The leukocytic infiltrate within the draining lymph node and footpad of B6.TNF(-/-) mice resembled that of B6.WT mice over the first 2 wk of cutaneous L. major infection. Thereafter, the B6.TNF(-/-) mice showed an increase of CD11c(+)Ly-6C(+)CCR2(+) monocytic dendritic cells within the popliteal lymph node in comparison with B6.WT mice. This increase of inflammatory dendritic cells was paired with the accumulation of a novel CD11b(+)Ly-6C(low)CCR2(low) population that was not present in B6.WT mice. This B6.TNF(-/-)- and B6.TNFR1(-/-)-specific cell population was CD115(+)Ly-6G(-)iNOS(-), not apoptotic, and harbored large numbers of parasites.


Subject(s)
Leishmaniasis, Cutaneous/immunology , Macrophages/immunology , Macrophages/parasitology , Tumor Necrosis Factor-alpha/immunology , Animals , Antigens, Ly/immunology , Disease Models, Animal , Flow Cytometry , Immunomagnetic Separation , Leishmania major/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/parasitology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Phenotype
17.
PLoS One ; 7(12): e51851, 2012.
Article in English | MEDLINE | ID: mdl-23284789

ABSTRACT

Dendritic cells (DCs) are the quintessential antigen-presenting cells of the human immune system and play a prime role in coordinating innate and adaptive immune responses, explaining the strong and still growing interest in their application for cancer immunotherapy. Much current research in the field of DC-based immunotherapy focuses on optimizing the culture conditions for in vitro DC generation in order to assure that DCs with the best possible immunogenic qualities are being used for immunotherapy. In this context, monocyte-derived DCs that are alternatively induced by interleukin-15 (IL-15 DCs) have attracted recent attention due to their superior immunostimulatory characteristics. In this study, we show that IL-15 DCs, in addition to potent tumor antigen-presenting function, possess tumoricidal potential and thus qualify for the designation of killer DCs. Notwithstanding marked expression of the natural killer (NK) cell marker CD56 on a subset of IL-15 DCs, we found no evidence of a further phenotypic overlap between IL-15 DCs and NK cells. Allostimulation and antigen presentation assays confirmed that IL-15 DCs should be regarded as bona fide myeloid DCs not only from the phenotypic but also from the functional point of view. Concerning their cytotoxic activity, we demonstrate that IL-15 DCs are able to induce apoptotic cell death of the human K562 tumor cell line, while sparing tumor antigen-specific T cells. The cytotoxicity of IL-15 DCs is predominantly mediated by granzyme B and, to a small extent, by tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) but is independent of perforin, Fas ligand and TNF-α. In conclusion, our data provide evidence of a previously unappreciated role for IL-15 in the differentiation of human monocytes towards killer DCs. The observation that IL-15 DCs have killer DC capacity lends further support to their implementation in DC-based immunotherapy protocols.


Subject(s)
Antigen-Presenting Cells/immunology , CD56 Antigen/immunology , Cytotoxicity, Immunologic/immunology , Dendritic Cells/immunology , Interleukin-15/immunology , Myeloid Cells/immunology , Neoplasms/immunology , Cell Differentiation , Dendritic Cells/cytology , Flow Cytometry , Granzymes/immunology , Granzymes/metabolism , Humans , Killer Cells, Natural/immunology , Monocytes/cytology , Monocytes/immunology , Neoplasms/metabolism , Neoplasms/pathology
18.
Pathology ; 43(6): 605-15, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21897329

ABSTRACT

The recent Food and Drug Administration (FDA) approval of a cellular therapy to treat castration resistant prostate cancer has reinforced the potential of cellular therapy to consolidate current pharmacological approaches to treating cancer. The emergence of the cell manufacturing facility to facilitate clinical translation of these new methodologies allows greater access to these novel therapies. Here we review different strategies currently being explored to treat haematological malignancies with a focus on adoptive allogeneic or autologous transfer of antigen specific T cells, NK cells or dendritic cells. These approaches all aim to generate immunological responses against overexpressed tissue antigens, mismatched minor histocompatability antigens or tumour associated antigens. Current successes and limitations of these different approaches will be discussed with an emphasis on challenges encountered in generating long term engraftment, antigen selection and implementation as well as therapeutic immune monitoring of clinical responses, with examples from recent clinical trials.


Subject(s)
Cell Transplantation/methods , Hematologic Neoplasms/therapy , Cell Transplantation/trends , Clinical Trials as Topic , Dendritic Cells/immunology , Dendritic Cells/transplantation , Hematologic Neoplasms/immunology , Humans , Immunotherapy , Natural Killer T-Cells/immunology , Natural Killer T-Cells/transplantation , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/transplantation
19.
ChemMedChem ; 6(5): 848-58, 2011 May 02.
Article in English | MEDLINE | ID: mdl-21472992

ABSTRACT

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of-and mode of cell death induced by-dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)(2) }(2) {µ-bb(n) }](4+) (Rubb(n)), where phen is 1,10-phenanthroline, bb(n) is bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n=2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bb(n) ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ-Rubb(16) complex displayed the highest cytotoxicity of the series, with an IC(50) value of 5 µM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bb(n) bridge of the metal complex. ΔΔ-Rubb(16) entered the L1210 cells by passive diffusion (with a minor contribution from protein-mediated active transport), inducing cell death via apoptosis. Additionally, metal-complex uptake in leukaemia cells was approximately 16-times that observed in healthy B cells highlighting that the bb(n) series of complexes may have potential as selective anticancer drugs.


Subject(s)
Coordination Complexes/chemistry , Ruthenium/chemistry , Adenosine Triphosphate , Animals , Apoptosis , Calorimetry , Cell Line, Tumor , Coordination Complexes/toxicity , Flow Cytometry , Mice , Phenanthrolines/chemistry , Thermodynamics
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