TIRC7 and HLA-DR axis contributes to inflammation in multiple sclerosis

2014 ◽  
Vol 20 (9) ◽  
pp. 1171-1181 ◽  
Author(s):  
JM Frischer ◽  
M Reindl ◽  
B Künz ◽  
T Berger ◽  
S Schmidt ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
Ex Vivo ◽  
Peripheral Blood Lymphocytes ◽  
Cytokine Expression ◽  
Hla Dr ◽  
T Cell Clone

Background and objective: Interactions between TIRC7 (a novel seven-transmembrane receptor on activated lymphocytes) and its ligand HLA-DR might be involved in the inflammatory process in multiple sclerosis (MS). Methods: Methods comprised immunohistochemistry and microscopy on archival MS autopsies, proliferation-, cytokine-, and surface-staining assays using peripheral blood lymphocytes (PBLs) from MS patients and an in vitro model. Results: TIRC7 was expressed in brain-infiltrating lymphocytes and strongly correlated with disease activity in MS. TIRC7 expression was reduced in T cells and induced in B cells in PBLs obtained from MS patients. After ex vivo activation, T cell expression of TIRC7 was restored in patients with active MS disease. The interaction of TIRC7+ T lymphocytes with cells expressing HLA-DR on their surface led to T cell proliferation and activation whereas an anti-TIRC7 mAb preventing interactions with its ligand inhibited proliferation and Th1 and Th17 cytokine expression in T cells obtained from MS patients and in myelin basic protein-specific T cell clone. Conclusion: Our findings suggest that TIRC7 is involved in inflammation in MS and anti-TIRC7 mAb can prevent immune activation via selective inhibition of Th1- and Th17-associated cytokine expression. This targeting approach may become a novel treatment option for MS.

scholarly journals Mildly oxidized low-density lipoproteins suppress the proliferation of activated CD4+ T-lymphocytes and their interleukin 2 receptor expression in vitro

1998 ◽  
Vol 330 (2) ◽  
pp. 659-666 ◽  
Author(s):  
Sylvie CASPAR-BAUGUIL ◽  
Majed SAADAWI ◽  
Anne NEGRE-SALVAYRE ◽  
Mogens THOMSEN ◽  
Robert SALVAYRE ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Clone ◽  
Peripheral Blood Lymphocytes ◽  
Cd4 T Cell ◽  
Activated T Cells ◽  
T Cell Clone ◽  
Activated T Lymphocytes

Activated T-lymphocytes are present in early atherosclerotic lesions where they may interact with oxidized low-density lipoproteins (oxLDLs). In this study the non-specific effect of oxLDLs on the activation of T-cells in vitro was investigated. LDLs were oxidized by UV irradiation and characterized by a low level of lipid peroxidation and only slight apolipoprotein B modification. Peripheral blood lymphocytes from normal individuals were stimulated in vitro with the polyclonal activator phytohaemagglutinin in the presence of various doses of LDLs and oxLDLs. LDLs enhanced the proliferation of peripheral blood lymphocytes at doses up to 100 μg/ml but were inhibitory at 200 μg/ml, whereas low doses of oxLDLs (over 10 μg/ml) inhibited the proliferation. OxLDLs also inhibited the proliferative responses of an alloreactive CD4+ T-cell line immortalized by Herpes virus saimiri and an influenza haemagglutinin-specific CD4+ T-cell clone. Viability tests using Trypan Blue exclusion or expression of Apo2.7, an apoptosis marker, did not indicate any significant cell death at doses up to 100 μg/ml oxLDL. At this concentration, cell-cycle analysis showed an accumulation of cells at the G1/S interface in the CD4+ cell clone, without significant DNA fragmentation. The expression of the activation antigen CD25 on T-lymphocytes (on phytohaemagglutinin-activated T-cells and on CD4+ T-cell clone), requisite to the commitment of activated T-cells from G1 phase to S phase, was also inhibited by oxLDLs whereas expression of other activation antigens such as CD69 and HLA-DR was unchanged. In conclusion, these data show that mildly oxidized LDLs inhibit the proliferation and CD25 expression of activated T-lymphocytes and suggest that oxLDLs may slow down the T-cell response in atherosclerotic lesions.

scholarly journals Anti-CD20 therapy depletes activated myelin-specific CD8+T cells in multiple sclerosis

2019 ◽  
Vol 116 (51) ◽  
pp. 25800-25807 ◽  
Author(s):  
Joseph J. Sabatino ◽  
Michael R. Wilson ◽  
Peter A. Calabresi ◽  
Stephen L. Hauser ◽  
Jonathan P. Schneck ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antibody Therapy ◽  
Myelin Proteins ◽  
T Cell Epitopes ◽  
Control Subjects ◽  
Anti Cd20

CD8+T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+CD8+T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+T cells in MS, indicates these cells may be attractive targets in MS therapy.

scholarly journals IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Marie-Line Puiffe ◽  
Aurélie Dupont ◽  
Nouhoum Sako ◽  
Jérôme Gatineau ◽  
José L. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Lymphocytic Choriomeningitis ◽  
T Cell Clone

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

Induction of ‘Tolerance’ in a T-Cell Clone from a Mild Hemophilia Patient

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3526-3526
Author(s):  
David W. Scott ◽  
Elizabeth Kadavil ◽  
Ai-Hong Zhang ◽  
Ruth A. Ettinger ◽  
Kathleen Pratt
Keyword(s):  
T Cell ◽  
B Cells ◽  
Hemophilia A ◽  
Cell Clone ◽  
Tolerance Induction ◽  
Hla Dr ◽  
Cell Clones ◽  
T Cell Clone ◽  
Pre Treatment

Abstract A major obstacle in the treatment of Hemophilia A is that patients can develop an inhibitory immune response to therapeutic doses of coagulation factor VIII (fVIII). Over the last decade, we have developed a B-cell delivered gene therapy approach to prevent the development of inhibitory antibodies (“inhibitors”) in fVIII knockout mice (see Lei and Scott, Blood105: 4865, 2005). In our murine platform, activated primary spleen B cells or bone marrow cells are transduced with a retroviral vector encoding the fVIII A2 and/or C2 domain fused to an IgG heavy chain, and these cells are injected systemically into immunocompetent fVIII knockout animals. The recipients are rendered specifically tolerant to the encoded C2 and A2 domains, as evidenced by a >90% reduction of inhibitor titers, even in primed animals. To help evaluate the potential of this approach for translation, we are developing in vitro models for tolerance induction using human T-cell clones isolated from subjects with mild hemophilia A. The clones are isolated by single-cell sorting of CD4+ cells that are labeled by fluorescent HLA-DR tetramers complexed with peptides containing fVIII epitopes, followed by expansion with HLA-DR mismatched peripheral blood mononuclear cells (PBMC), phytohaemagglutinin, and interleukin-2. Our initial model utilizes a T-cell clone from an individual with mild hemophilia A due to fVIII missense genotype A2201P, which recognizes an HLA-DRA-DRB1*0101-restricted epitope within a synthetic peptide corresponding to fVIII residues 2194–2213. All of the antigen-specific T-cell clones isolated from this subject secreted interferon-gamma (IFN-γ) when stimulated by fVIII2194–2213 presented by irradiated HLA-DR-matched PBMCs or with plate-bound anti-CD3. Because of their robust response to a clinically relevant epitope in fVIII, one of these clones that expanded well in culture was chosen for initial testing of a modified gene therapy platform similar to that developed using the murine hemophilia A model. HLA-matched peripheral blood B cells were activated with antibodies to IgM or with CD40L-expressing fibroblasts and then transduced with a modified retroviral vector containing the human C2 domain sequence in-frame with the IgG sequence. These B cells were cultured with the hemophilic T-cell clone. After pre-treatment (“tolerance-induction step”), the cells were washed and then stimulated by plate-bound anti-CD3. The subsequent IFN-γ response (measured by ELIspots and ELISA) was dramatically reduced compared to the response of same T-cell clone cultured with mock-transduced B cells. The post-treatment reduction in IFN-γ secretion was equivalent to that induced after soluble anti-CD3 pre-treatment, a known method to induce T-cell anergy in vitro. Interestingly, IL-10 was produced during the tolerance induction (pre-treatment) phase, most likely from the activated B cells. Preliminary, parallel experiments with B cells transduced with a “gutless” adenovirus vector expressing C2-Ig did not result in a similar down-regulation of the T-cell response, suggesting that this non-integrating method of expressing antigens for tolerance is not effective, at least in this system. These results are the first to demonstrate in vitro modulation of cytokine responses using DR-restricted, fVIII-specific T cells from a hemophilia A subject. Further investigations using T-cell clones from hemophilic subjects with and without anti-fVIII antibodies will allow us to explore mechanisms of tolerance and may also suggest novel approaches to reduce inhibitor titers.

Cytotoxicity Is Not Required for the Anti-Leukemia Efficacy of Human H-Y-Specific CD4+ T Lymphocytes In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3553-3553
Author(s):  
Attilio Bondanza ◽  
Lothar Hambach ◽  
Zohara Aghai ◽  
Monica Casucci ◽  
Bart Nijmeijer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Clone ◽  
Scid Mice ◽  
Helper T Cell ◽  
T Cell Clone ◽  
Control Clone

Abstract Abstract 3553 Poster Board III-490 Introduction Minor histocompatibility antigens (mHag) play a major role in the graft-versus-leukemia (GvL) effect following HLA-matched allogeneic hemopoietic cell transplantation (allo-HCT). Clinically, the GvL effect coincides with the emergence of mHag-specific CD8+ cytotoxic T lymphocytes (CTL). Experimentally, targeting a single mHag with human CD8+ CTL has a major anti-leukemia effect in NOD/scid mice. Altogether, these observations suggest that mHag-specific cytotoxicity by CD8+ T cells is an important component of the GvL effect. In contrast, little is known on the contribution of mHag-specific CD4+ T cells. Female-to-male allo-HCT is characterized by a low rate of leukemia relapse, indicating that H-Y-encoded mHag are potent leukemia-regression antigens. Earlier, we described a DRB3*0301-restricted H-Y mHag epitope inducing CD4+ helper T-cell responses in H-Y-mismatched HLA-matched allo-HCT. Aim: The aim of this study is to elucidate the role of mHag-specific human CD4+ T lymphocytes on the GvL effect. Methods The ALL-CM leukemia cell line, derived from a male (i.e. H-Y+) HLA-A0201+, DRB30301+ patient, reproducibly engrafts in NOD/scid mice after administration of 10×106 cells. Both an HLA-A0201-restricted H-Y-specific CD8+ CTL clone and the DRB30301-restricted H-Y-specific CD4+ helper T-cell clone that we earlier described were used to investigate the anti-leukemia efficacy of CD8+ and CD4+ T cells in NOD/scid mice. Results In vitro, the CD8+ H-Y specific CTL clone was highly cytotoxic against the ALL-CM leukemia. The H-Y specific CD4+ helper T-cell clone did not lyse the leukemia, but produced IFN-γ upon recognition. Infusion of the H-Y-specific CD8+ CTL clone (25×106 cells/mouse) 3 days after ALL-CM leukemia challenge significantly delayed leukemia progression by 3 weeks compared to a CMV-specific CD8+ CTL control clone (p<0,001). Despite no measurable in vitro cytotoxicity, the H-Y-specific CD4+ helper T-cell clone (25×106 cells/mouse) delayed leukemia progression by 2 weeks compared to a leukemia non-reactive HLA-DR1-specific CD4+ helper T-cell control clone (p<0,001). In vitro co-incubation of the H-Y-specific CD4+ helper T-cell clone did not influence leukemia proliferation but induced up-regulation of MHC-class I and II, CD80, CD86 and CD40. In vitro, pre-incubation of leukemia cells with the H-Y-specific CD4+ helper T-cell clone irradiated did not improve the in vivo anti-leukemia efficacy of the H-Y-specific CD8+ CTL clone. Co-infusion of the H-Y specific CD4+ helper T-cell clone did not augment the in vivo persistence of the H-Y-specific CD8+ CTL T-cell clone. Nevertheless, the co-infusion resulted in a delay in leukemia progression of approximately 5 weeks, suggesting an additive, non overlapping anti-leukemia mechanism. Conclusions Minor Hag-specific human CD4+ T lymphocytes may contribute to the GvL effect through a direct, non cytotoxic mechanism, which could be additive to that of CD8+ CTL. The nature of this non cytotoxic GvL effect is currently under investigation. A.B. and L.H. equally contributed to this study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vivo Survival of  Viral Antigen–specific T Cells that Induce Experimental Autoimmune Encephalomyelitis

1998 ◽  
Vol 188 (9) ◽  
pp. 1725-1738 ◽  
Author(s):  
Rafael L. Ufret-Vincenty ◽  
Laura Quigley ◽  
Nancy Tresser ◽  
Seong Hee Pak ◽  
Ameer Gado ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
T Cell ◽  
Viral Antigen ◽  
Low Dose ◽  
Cell Clone ◽  
Viral Peptide ◽  
Autoreactive T Cell ◽  
T Cell Clone

A peptide derived from the human papillomavirus L2 protein is recognized by a myelin basic protein (MBP)-specific T cell clone from a multiple sclerosis patient and by MBP-specific autoantibodies purified from multiple sclerosis brain tissue. We now show in mice that low doses of this papillomavirus peptide were optimal in selecting a subpopulation of papillomavirus peptide–specific T cells that cross-reacted with MBP(87–99) and with an unrelated viral peptide derived from the BSLF1 protein of Epstein-Barr virus (EBV). These low dose viral peptide– specific T cell lines were highly encephalitogenic. Splenocytes from mice transferred with viral peptide–specific T cells showed a vigorous response to both the papillomavirus and MBP peptides, indicating that viral antigen–specific T cells survived for a prolonged time in vivo. The EBV peptide, unable to prime and select an autoreactive T cell population, could still activate the low dose papillomavirus peptide–specific cells and induce central nervous system (CNS) autoimmunity. Cytokine profiles of papillomavirus peptide–specific encephalitogenic T cells and histopathology of CNS lesions resembled those induced by MBP. These results demonstrate conserved aspects in the recognition of the self-antigen and a cross-reactive viral peptide by human and murine MBP-specific T cell receptors. We demonstrate that a viral antigen, depending on its nature, dose, and number of exposures, may select autoantigen-specific T cells that survive in vivo and can trigger autoimmune disease after adoptive transfer.

scholarly journals The interaction between CD8+ cytotoxic T cells and Leishmania-infected macrophages.

1991 ◽  
Vol 174 (3) ◽  
pp. 499-505 ◽  
Author(s):  
L E Smith ◽  
M Rodrigues ◽  
D G Russell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Clone ◽  
Cytotoxic T Cells ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
T Cell Clone ◽  
Vitro Model ◽  
Leishmania Parasites

Leishmania is resident within the macrophages of its vertebrate host. In any intramacrophage infection, where the pathogen is present in a form capable of mediating cell to cell transmission, the contribution of a cytotoxic T cell response to protective immunity is questionable. This study presents data from an in vitro model designed to elucidate the outcome of an interaction between CD8+, cytotoxic T cells and infected macrophages. Experiments were conducted with an H-2d-restricted, cytotoxic CD8+ T cell clone and Leishmania parasites present in mixed macrophage cultures, with the parasites confined to either histocompatible BALB/c macrophages, or incompatible CBA macrophages. Initial experiments indicated that the viability of Leishmania was unaffected by the lysis of its host macrophage by cytotoxic T cells. However, extended experiments showed that the parasites were killed between 24 and 72 h. The same results were obtained regardless of whether the parasites were resident in the target, BALB/c, macrophages or the bystander, CBA, macrophages. Addition of neutralizing, anti-IFN-g antibody to the cultures ablated most of the leishmanicidal behavior, indicating that parasite death was attributable to macrophage activation, resulting from cytokine secretion from the T cells following the initial recognition event.

scholarly journals Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia

Blood ◽  
1997 ◽  
Vol 89 (10) ◽  
pp. 3691-3699 ◽  
Author(s):  
Shinji Nakao ◽  
Akiyoshi Takami ◽  
Hideyuki Takamatsu ◽  
Weihua Zeng ◽  
Naomi Sugimori ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Mononuclear Cells ◽  
Cell Clone ◽  
Cytotoxic T Cells ◽  
Hematopoietic Cells ◽  
T Cell Clone ◽  
Cd34 Cells

Abstract The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Immunotargetting of the Wilms’ Tumor WT1 Antigen for Dendritic Cell and B-Cell-Based Vaccination of Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2541-2541
Author(s):  
Zwi N. Berneman ◽  
Ann Van Driessche ◽  
Peter Ponsaerts ◽  
Liquan Gao ◽  
Hans J. Stauss ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Dendritic Cell ◽  
Mhc Class I ◽  
Wilms Tumor ◽  
Cell Clone ◽  
Class I ◽  
T Cell Clone

Abstract The Wilms’ tumor antigen (WT1) is overexpressed in almost all leukemias and in several solid tumors. Overexpression of WT1 blocks the normal differentiation and enhances proliferation of hematopoietic progenitor cells. WT1 is also used in the detection of minimal residual disease. Using WT1-specific MHC class I tetramers, we were able to detect ex vivo low numbers of WT1-specific CD8+ T cells in the peripheral blood or bone marrow of leukemia patients, but not of healthy donors. In one particular donor we could detect up to 24% WT1 tetramer positive cells at the time of diagnosis. WT1 tetramer positive cells were present in all types of leukemia, except for CLL, and also in patients with MDS. Because WT1 plays an important role in leukemogenesis, it could serve as an antigenic target for dendritic cell-based immunotherapy. We used the mRNA electroporation strategy that allows presentation of multiple WT1 epitopes by MHC class I molecules, irrespective of the HLA haplotype. Monocyte-derived DC (Mo-DC) were electroporated with in vitro transcribed WT1 mRNA. RT-PCR and Western blot analysis showed that WT1 RNA and protein, respectively, was present for up to 5 days in WT1-electroporated DC, but not in mock- or EGFP mRNA-electroporated Mo-DC. Importantly, using a CD8+ T cell clone that secretes IFN-gamma upon recognizing the HLA-A2 immunodominant WT1126–134 epitope, we showed that WT1 mRNA-electroporated Mo-DC processed the WT1 protein via the MHC class I pathway and presented the WT1 epitope to the T cells in an HLA- and antigen-specific manner. Since Mo-DCs are a non-expandable source of antigen-presenting cells, we also used proliferating CD40-activated B (CD40-B) cells as inducers for WT1-specific T cell immunity. CD40-B cells were expanded to high numbers from a limited amount of peripheral blood and subsequently electroporated with WT1 mRNA. In T cell clone activation experiments, WT1 mRNA-electroporated CD40-B cells were as efficient as Mo-DC in presenting the WT1 epitope in a MHC class I-restricted manner. Based on these results, we are currently focusing on the in vitro (re)activation of autologous WT1-specific cytotoxic T cells of leukemia patients using WT1-loaded autologous Mo-DC or CD40-B cells and on the immunological parameters to break immune tolerance against the WT1 tumor self antigen.

Ex Vivo Generation Of Functional Plasmacytoid and Myeloid Dendritic Cells Is Strongly Promoted By The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2025-2025
Author(s):  
Soley Thordardottir ◽  
Hangalapura Basav N. ◽  
Tim Hutten ◽  
Marta Cossu ◽  
Jan Spanholtz ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cell Responses ◽  
Aryl Hydrocarbon ◽  
Hla Dr ◽  
Cd34 Cells

Abstract The prominent role of dendritic cells (DCs) in T cell activation is the rational for DC-based immunotherapy of cancer and infectious diseases. In cancer, DC therapy aims to induce tumor-specific effector T cell responses that can reduce or eliminate the tumor, and to develop immunological memory to control tumor relapse. So far, the vast majority of DC vaccination studies have been performed with DCs differentiated from monocytes (Mo-DCs) that are loaded with tumor-associated antigens (TAAs) or minor histocompatibility antigens (MiHA). This strategy has been reported to induce the expansion of antigen-specific CD4+ and/or CD8+ T cells in the majority of patients, however only a fraction of the patients develop clinical responses. Strategies to improve the potency of DC-based vaccines are to increase the stimulatory and migratory capacity of Mo-DCs, or to use alternative DC subtypes, such as naturally circulating plasmacytoid DCs (pDCs), BDCA1+ myeloid DCs (mDCs) or BDCA3+ mDCs. These DC subsets are potent inducers of antigen-specific T cell responses, and are therefore attractive cells to exploit for DC-based therapy. However, since their frequency in blood is very low, it is a challenge to obtain high enough numbers for immunotherapy. It would be advantageous if DCs, which are phenotypically and functionally similar to blood pDCs and mDCs, could be generated from CD34+ hematopoietic progenitor cells (HPCs). Interestingly, recent findings have indicated that the aryl hydrocarbon receptor (AhR) not only regulates toxic effects of environmental contaminants, but also plays a role in modulating hematopoiesis and the immune system. For instance, it has been reported that StemRegenin 1 (SR1), a small molecule inhibitor of AhR, promotes the ex vivo expansion of human CD34+ HPCs that are able to effectively engraft immunodeficient mice. Furthermore, differentiation of Langerhans cells and monocytes in vitro from HPCs can be inhibited by the addition of the AhR agonist VAF347. In light of these data, we investigated if we could generate DC subsets from CD34+ HPCs by supplementing SR1. Therefore, we cultured CD34+ HPCs in medium containing SCF, Flt3L, IL-6, TPO supplemented with 1 μM SR1 or DMSO as control. Interestingly, addition of SR1 explicitly promoted the emergence of pDCs (CD11c-HLA-DR+CD123hiBDCA2+BDCA4+ cells), BDCA1+ mDCs (Lin1-HLA-DR+BDCA1+BDCA3- cells) and BDCA3+ mDCs (Lin1-HLA-DR+BDCA1-BDCA3+ cells). After three weeks of culture, the frequency of these DC subsets was significantly higher in cultures with SR1 compared to control conditions; 2.9% vs. 0.04% for pDCs, 4.6% vs. 0.5% for BDCA1+ mDCs and 1.1% vs. 0.1% for BDCA3+ mDCs (n=3-5 donors). The average yield after three weeks of culture with SR1 starting from 105 CD34+ UCB cells was 3.8x106 pDCs, 5.3x106 BDCA1+ mDCs and 1.2x106 BDCA3+ mDCs (n=3-5 donors). Furthermore, SR1 also promoted the differentiation of DC subsets from CD34+ cells obtained from peripheral blood of G-CSF-mobilized donors. The average frequency of DCs in these SR1-cultures was 4.7%, 3.8% and 0.9% for pDCs, BDCA1+ and BDCA3+ mDCs, respectively (n=3 donors), which is comparable to the frequency obtained from UCB CD34+ cells. But the expansion potential of G-CSF-mobilized blood CD34+ HPCs was lower than that of UCB CD34+ cells, resulting in average DC yields of 0.6x106, 0.5x106 and 0.1x106 from 105 CD34+ cells (n=3). Flow cytometry analysis demonstrated that the SR1-induced pDCs and mDCs are phenotypically comparable to their naturally occurring counterpart in blood. Furthermore, the ex vivo-generated pDCs potently responded to stimulation with TLR7 and TLR9 ligands by secreting high amounts of IFN-α and upregulating CD83, CD80, CD86 and CCR7. The HPC-mDC subsets also upregulate CD80 and CD83 upon TLR3, TLR4 or TLR7/8 ligation. Finally, both the ex vivo-generated pDCs and mDCs induced potent allogeneic T cell responses and activated CD8+ effector T cells against hematopoietic-restricted MiHA. These findings demonstrate that our SR1 culture system not only allows detailed study of DC differentiation and molecular regulations in vitro, but it also offers the opportunity to evaluate the in vivo efficacy of cultured DC subsets upon vaccination into patients with cancer and viral infections. Disclosures: Spanholtz: Glycostem Therapeutics: Employment.

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