Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II-restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T-cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM-derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.

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ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming

Blood ◽

10.1182/blood-2005-01-0220 ◽

2005 ◽

Vol 106 (1) ◽

pp. 216-223 ◽

Cited By ~ 300

Author(s):

Elodie Segura ◽

Carole Nicco ◽

Bérangère Lombard ◽

Philippe Véron ◽

Graça Raposo ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Mhc Class Ii ◽

T Cell Responses ◽

Class Ii ◽

Naive T Cells ◽

Naïve T Cells ◽

Cell Responses

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes, which transfer functional major histocompatibility complex (MHC)–peptide complexes to other DCs. Since immature and mature DCs induce different functional T-cell responses (ie, tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that exosomes secreted by lipopolysaccharide (LPS)–treated mature DCs are 50- to 100-fold more potent to induce antigen-specific T-cell activation in vitro than exosomes from immature DCs. In vitro, exosomes from mature DCs transfer to B lymphocytes the ability to prime naive T cells. In vivo, only mature exosomes trigger effector T-cell responses, leading to fast skin graft rejection. Proteomic and biochemical analyses revealed that mature exosomes are enriched in MHC class II, B7.2, intercellular adhesion molecule 1 (ICAM-1), and bear little milk-fat globule–epidermal growth factor–factor VIII (MFG-E8) as compared with immature exosomes. Functional analysis using DC-derived exosomes from knock-out mice showed that MHC class II and ICAM-1 are required for mature exosomes to prime naive T cells, whereas B7.2 and MFG-E8 are dispensable. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.

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Generation of murine dendritic cells from flt3-ligand–supplemented bone marrow cultures

Blood ◽

10.1182/blood.v96.9.3029 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3029-3039 ◽

Cited By ~ 293

Author(s):

Kenneth Brasel ◽

Thibaut De Smedt ◽

Jeffery L. Smith ◽

Charles R. Maliszewski

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Culture System ◽

Antigen Processing ◽

Class Ii ◽

Interferon Α ◽

Flt3 Ligand ◽

Gm Csf

Abstract Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

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Generation of murine dendritic cells from flt3-ligand–supplemented bone marrow cultures

Blood ◽

10.1182/blood.v96.9.3029.h8003029_3029_3039 ◽

2000 ◽

Vol 96 (9) ◽

pp. 3029-3039 ◽

Cited By ~ 2

Author(s):

Kenneth Brasel ◽

Thibaut De Smedt ◽

Jeffery L. Smith ◽

Charles R. Maliszewski

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Culture System ◽

Antigen Processing ◽

Class Ii ◽

Interferon Α ◽

Flt3 Ligand ◽

Gm Csf

Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

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Small amounts of superantigen, when presented on dendritic cells, are sufficient to initiate T cell responses.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.633 ◽

1993 ◽

Vol 178 (2) ◽

pp. 633-642 ◽

Cited By ~ 91

Author(s):

N Bhardwaj ◽

J W Young ◽

A J Nisanian ◽

J Baggers ◽

R M Steinman

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Dendritic Cell ◽

Mhc Class Ii ◽

Cell Receptor ◽

Class Ii ◽

Cell Mediated Immunity ◽

Quiescent T Cells ◽

Antigen Presenting

Dendritic cells are potent antigen-presenting cells for several primary immune responses and therefore provide an opportunity for evaluating the amounts of cell-associated antigens that are required for inducing T cell-mediated immunity. Because dendritic cells express very high levels of major histocompatibility complex (MHC) class II products, it has been assumed that high levels of ligands bound to MHC products ("signal one") are needed to stimulate quiescent T cells. Here we describe quantitative aspects underlying the stimulation of human blood T cells by a bacterial superantigen, staphylococcal enterotoxin A (SEA). The advantages of superantigens for quantitative studies of signal one are that these ligands: (a) engage MHC class II and the T cell receptor but do not require processing; (b) are efficiently presented to large numbers of quiescent T cells; and (c) can be pulsed onto dendritic cells before their application to T cells. Thus one can relate amounts of dendritic cell-associated SEA to subsequent lymphocyte stimulation. Using radioiodinated SEA, we noted that dendritic cells can bind 30-200 times more superantigen than B cells and monocytes. Nevertheless, this high SEA binding does not underlie the strong potency of dendritic cells to present antigen to T cells. Dendritic cells can sensitize quiescent T cells, isolated using monoclonals to appropriate CD45R epitopes, after a pulse of SEA that occupies a maximum of 0.1% of surface MHC class II molecules. This corresponds to an average of 2,000 molecules per dendritic cell. At these low doses of bound SEA, monoclonal antibodies to CD3, CD4, and CD28 almost completely block T cell proliferation. In addition to suggesting new roles for MHC class II on dendritic cells, especially the capture and retention of ligands at low external concentrations, the data reveal that primary T cells can generate a response to exceptionally low levels of signal one as long as these are delivered on dendritic cells.

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Donor CD4+CD25+ T Cells Require Syngeneic MHC Class II for Initial Support of MHC-Mismatched Hematopoietic Engraftment.

Blood ◽

10.1182/blood.v104.11.251.251 ◽

2004 ◽

Vol 104 (11) ◽

pp. 251-251 ◽

Cited By ~ 1

Author(s):

Alan Hanash ◽

Robert B. Levy

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Allogeneic Bone Marrow Transplantation ◽

Class Ii ◽

Allogeneic Bone ◽

Inherited Disorders ◽

Donor Chimerism

Abstract Despite the potential to cure both acquired and inherited disorders involving the hematopoietic compartment, application of allogeneic bone marrow transplantation (BMT) is limited by the frequent and severe outcome of Graft vs. Host Disease (GVHD). Unfortunately, efforts to reduce GVHD by purging the donor graft of T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic engraftment without inducing GVHD could increase the potential for donor-recipient matching and decrease treatment associated risks. We have observed that GVHD-suppressive donor CD4+CD25+ T cells are capable of supporting allogeneic hematopoietic engraftment, as demonstrated by initial donor progenitor activity and long-term chimerism and tolerance. Using a murine MHC mismatched model transplanting 0.5–2x106 GFP+ C57BL/6 (B6) T cell-depleted bone marrow cells into 7.0 Gy sublethally irradiated BALB/c recipients, splenic CFU assessment demonstrated that co-transplantation of 1x106 B6 CD4+CD25+ T cells lead to increased donor lineage-committed GM (p<.01) and multi-potential HPP (p<.05) progenitors seven days post-BMT compared to transplantation of BM alone. Furthermore, co-transplantation of CD4+CD25+ T cells lead to lymphoid and myeloid chimerism in peripheral blood (lineage specific mean donor chimerism ± SE: B220, 67.7±15.2 vs. 0.3±0.3; CD4, 38.3±10.5 vs.0.9±0.9; CD8, 48.3±11.0 vs. 1.0±1.0; Mac-1, 58.8±16.5 vs. 0.3±0.3) and the presence of donor GM and HPP progenitors in recipient marrow two months post-BMT (mean CFU chimerism ± SE: CFU-GM, 54.5±12.8 vs. 0.0; CFU-HPP, 63.0±17.8 vs.0.0). Donor chimerism persisted six months post-BMT and was associated with tolerance to donor and host antigens by acceptance of donor and host skin grafts >50 days post-homotopic grafting. Characterization of the initial invents of engraftment support demonstrated that augmentation of donor progenitors did not require CD4+CD25+ T cell IL-10, as co-transplantation of B6-wt and B6-IL-10−/− CD4+CD25+ T cells both significantly increased total CFU-GM (mean CFU±SE: BM alone, 657.5±248.2; BM + wt, 1972±331.5; BM + IL-10−/−, 1965±401.7; both p<.05 vs. BM alone). Assessment of the antigenic requirements for activation of progenitor support demonstrated that donor CD4+CD25+ T cells did not require alloreactivity to support progenitors, as BALB/c x B6 F1 CD4+CD25+ T cells significantly increased B6 CFU-GM in BALB/c recipients (p<.001 vs. BM alone). However, B6 CD4+CD25+ T cells failed to augment C3H/HeJ CFU-GM in BALB/c recipients (p>.05 vs. BM alone), suggesting that donor CD4+CD25+ T cells might require recognition of syngeneic MHC for progenitor support. Indeed, augmentation of donor CFU-GM was abrogated when B6 CD4+CD25+ T cells were co-transplanted with B6-MHC class II−/− marrow into BALB/c recipients (p>.05 vs. BM alone). In conclusion, donor CD4+CD25+ T cells capable of promoting long-term engraftment and tolerance do not require IL-10 for support of initial donor progenitor activity, however progenitor support does require co-transplantation with syngeneic MHC class II expressing marrow. Donor CD4+CD25+ T cells may thus represent a useful alternative to unfractionated T cells for promotion of engraftment following allogeneic hematopoietic transplantation.

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Severe and multifaceted systemic immunosuppression caused by experimental cancers of the central nervous system requires release of non-steroid soluble mediators

10.1101/2020.03.24.006825 ◽

2020 ◽

Author(s):

K Ayasoufi ◽

CK Pfaller ◽

L Evgin ◽

RH Khadka ◽

ZP Tritz ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Class Ii ◽

Cd4 T Cell ◽

Unknown Etiology ◽

Thymic Involution ◽

Cell Counts ◽

Hallmark Feature

AbstractImmunosuppression of unknown etiology is a hallmark feature of glioblastoma (GBM) and is characterized by decreased CD4 T cell counts and down regulation of MHC class II expression on peripheral blood monocytes in patients. This immunosuppression is a critical barrier to the successful development of immunotherapies for GBM. We recapitulated the immunosuppression observed in GBM patients in the C57BL/6 mouse and investigated the etiology of low CD4 T cell counts. We determined that thymic involution was a hallmark feature of immunosuppression in three distinct models of CNS cancer, including mice harboring GL261 glioma, B16 melanoma, and in a spontaneous model of Diffuse Intrinsic Pontine Glioma (DIPG). In addition to thymic involution, we determined that tumor growth in the brain induced significant splenic involution, reductions in peripheral T cells, reduced MHC class II expression on hematopoietic cells, and a modest increase in bone marrow resident CD4 T cells with a naïve phenotype. Using parabiosis we report that thymic involution, declines in peripheral T cell counts, and reduced MHC class II expression levels were mediated through circulating blood-derived factors. Conversely, T cell sequestration in the bone marrow was not governed through circulating factors. Serum isolated from glioma-bearing mice potently inhibited proliferation and functions of T cells both in vitro and in vivo. Interestingly, the factor responsible for immunosuppression in serum is nonsteroidal and of high molecular weight. Through further analysis of neurological disease models, we determined that the aforementioned immunosuppression was not unique to cancer itself, but rather occurs in response to CNS injury. Noncancerous acute neurological insults also induced significant thymic involution and rendered serum immunosuppressive. Both thymic involution and serum-derived immunosuppression were reversible upon clearance of brain insults. These findings demonstrate that CNS cancers cause multifaceted immunosuppression and pinpoint circulating factors as a target of intervention to restore immunity.Short SummaryCNS cancers and other brain-injuries suppress immunity through release of non-steroid soluble factors that disrupt immune homeostasis and dampen responses of the peripheral immune system.Graphical Abstract

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Dendritic Cell Cross Presentation of RBC Antigens in-Vivo Is Not Affected By RBC Storage Duration and Requires Red Pulp Macrophage "Help" in-Vitro

Blood ◽

10.1182/blood.v128.22.3845.3845 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3845-3845

Author(s):

Amy Tang ◽

Abdelhadi Rebbaa ◽

Steven L Spitalnik ◽

Eldad A. Hod ◽

Stuart Phillip Weisberg

Keyword(s):

Bone Marrow ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Refrigerated Storage ◽

Marrow Graft ◽

Cross Presentation ◽

Fresh Rbcs ◽

Red Pulp

Abstract BACKGROUND: Chronic transfusion is associated with an increased risk of bone marrow graft rejection. In prior studies with mouse models, minor histocompatibility antigens in red blood cell (RBC) and platelet products were presented in the context of recipient MHC class I to prime recipient cytotoxic T cells; these represent cross presentation and cross priming, respectively. Once primed in the recipient, these T cells may attack allogeneic bone marrow grafts. Thus, understanding the mechanism of transfusion-induced T cell cross priming may lead to new methods to reduce bone marrow graft rejection in chronically transfused patients. Cross presentation is performed by a specialized subset of antigen presenting cells (APCs) - CD11b lo, XCR1+ dendritic cells (XCR DCs). Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Fluorescent tracking of transfused RBCs showed that splenic red pulp macrophages (RPMs) ingest the majority of damaged RBCs, but RPMs are weak APCs. Splenic dendritic cells, including XCR DCs, also display increased uptake of stored vs. fresh RBCs. These data suggested that refrigerated storage may increase cross presentation of RBC antigens, thereby enhancing T cell cross priming. AIMS: To compare T cell cross priming after transfusion of fresh and refrigerator-stored RBCs, a mouse model was used with transgenic OVA-carrying RBCs as the antigen source and transgenic naive MHC class I-restricted (H-2Kb) OVA-specific T cells (OT-1) as responders. In parallel, an in-vitro system was established to determine the cellular elements required for cross presentation of RBC antigens. METHODS: Purified CD8 T cells from OT-1 mice were loaded with the cell proliferation tracking dye Cell Trace Far Red (CTFR) and adoptively transferred (4 x 10e6 per mouse) into cohorts of 8-12 week old C57BL/6 GFP+ mice (n=3 per group). The next day mice were transfused with 400 uL of fresh (<24 hours old), or stored RBCs (14 days old) from transgenic HOD mice (RBCs express surface HEL, OVA, and Duffy antigens). After 96 hours, flow cytometry was used to assess OT-1 cell proliferation by dye dilution and the expression of activation markers CD44, CD122 and CD62L. Control transfusions with non-HOD GFP+ RBCs confirmed the antigen specificity of the response, and transfusions of HOD blood that had undergone RBC lysis ruled out direct antigen presentation by MHC identical white cells in the transfusate. For the in-vitro cross presentation assay, 1x10e5 CTFR-loaded OT-1 cells were plated in U-bottom 96 well plates with all possible combinations of fresh or stored HOD RBCs (50 x 10e6 per well), bone marrow Flt3L-derived C57BL/6 murine dendritic cells (Flt3L-DCs, 5 X 10e4 per well), and magnetically selected BALB/c (H-2Kd) RPMs (5 x 10e4 per well). MHC mismatch rules out antigen presentation by BALB/c RPMs (H-2Kd) to OT-I T cells (H-2Kb). RESULTS: Transfusion of both fresh and stored HOD RBCs induced vigorous proliferation and activation of OT-1 cells. After 4 days, no differences were seen in the proliferation and activation profiles of OT-1 cells in mice receiving fresh (78±4% CD44hi, CTFR diluted) vs. stored (79±5%) HOD RBCs. The in-vitro cross presentation assay showed weak to absent OVA-specific OT-1 proliferation with co-cultures including fresh or stored HOD RBCs alone, and fresh or stored HOD RBCs plus Flt3L-DCs. In contrast, cultures including RPMs and Flt3L-DCs showed enhanced OVA-specific OT-1 proliferation with fresh (29±10% CTFR diluted) and stored (25±2%) HOD RBCs. CONCLUSION: Cross priming by fresh RBCs was stronger than expected based on prior studies showing minimal RBC uptake by XCR DCs after fresh GFP + RBC transfusion. Cross priming in this system may be more sensitive due to the high frequency of antigen specific T cells and, thus, is saturated by the small antigen load delivered by fresh transfusion. It is also possible that direct uptake of RBCs by DCs harms the cross-presentation machinery and offsets the effect of increased RBC antigen delivery. Optimal cross-presentation of RBC antigen may require cooperation with cells functionally specialized for metabolizing RBC by-products. Indeed, the in-vitro study shows RPMs promote DC-mediated cross presentation of antigens on fresh and stored RBCs. Thus, RPMs may share ingested RBC antigen with adjacent DCs and increase DC activation by secreting inflammatory cytokines. . Figure Figure. Disclosures No relevant conflicts of interest to declare.

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Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH

Open Biology ◽

10.1098/rsob.190235 ◽

2020 ◽

Vol 10 (2) ◽

pp. 190235 ◽

Cited By ~ 1

Author(s):

Stephanie J. Crowley ◽

Patrick T. Bruck ◽

Md Aladdin Bhuiyan ◽

Amelia Mitchell-Gears ◽

Michael J. Walsh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Class Ii ◽

Tumour Immunity ◽

Tumour Rejection

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

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IMMU-19. EVALUATING EFFECTS OF REVERSING DISTINCT FACETS OF IMMUNOSUPPRESSION IN EXPERIMENTAL GBM

Neuro-Oncology ◽

10.1093/neuonc/noab196.378 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi95-vi96

Author(s):

Katayoun Ayasoufi ◽

Zachariah Tritz ◽

Cori Fain ◽

Roman Khadka ◽

Fang Jin ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Patient Survival ◽

Combined Therapy ◽

Class Ii ◽

Thymic Involution ◽

Soluble Factors

Abstract Glioblastoma is associated with severe and multifaceted immunosuppression affecting all immune organs. Immunosuppression in GBM is a critical barrier to the success of immunotherapies and patient survival. We demonstrated that immunosuppression in the GL261-model of experimental GBM presents with significant thymic and spleen atrophy, MHCII downregulation, presence of potent immunosuppressive factors in serum, and sequestration of T-cells in the bone marrow. Parabiosis studies determined that soluble factors mediate immunosuppression by inhibiting T-cell proliferation, thymic involution, and loss of peripheral T-cells. In contrast, bone marrow T-cell sequestration was not mediated through soluble factors. While the immunosuppression in GBM is severe, a causative link between each facet of immunosuppression and overall survival is lacking. We used two strategies to block T-cell sequestration into the bone marrow and evaluated the extent survival was impacted in experimental GBM. First, we evaluated the extent a novel and off-the-shelf combination immunotherapy that uses extended 1/2-life IL-2 and anti-PD-1 reverses bone marrow T-cell sequestration. Sham treatment or anti-PD1 monotherapy did not alter T-cell sequestration in the bone marrow and animals had no enhanced survival. Extended 1/2-life IL-2 monotherapy and combination strategy both prevented T-cell sequestration into the bone marrow. However, only combined therapy, which also prevented MHC class II downregulation, improved survival. Second, we determined that glioma-bearing adrenalectomized mice do not present with bone marrow T-cell sequestration. However, sera of glioma-bearing adrenalectomized mice is as immunosuppressive as glioma-bearing controls. Blocking bone marrow T-cell sequestration in the presences of serum immunosuppression led to no survival benefit in glioma-bearing adrenalectomized mice compared to controls. In short, bone marrow T-cell sequestration alone does not correspond with overall survival in experimental glioma. Importantly, a concerted effort to reverse MHC class II downregulation and define inhibitory circulating factors may have the highest impact in immunotherapeutic efficacy and improving patient survival.

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Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽

10.1182/blood.v104.11.2529.2529 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2529-2529

Author(s):

William K. Decker ◽

Dongxia Xing ◽

Sufang Li ◽

Simon N. Robinson ◽

Hong Yang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

Class Ii ◽

T Cell Response ◽

Class I ◽

Cross Presentation ◽

Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p<0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p<0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p<0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p<0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p<0.03) and a 187% increase in total IFN-γ secretion (p<0.03). Unloaded (p<0.01) and mRNA (p<0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

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