Increased generation of dendritic cells from myeloid progenitors in autoimmune-prone nonobese diabetic mice

Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.     

Intrinsic in vitro abnormalities in dendritic cell generation caused by non-MHC non-obese diabetic genes

Genes outside the MHC create a general susceptibility to autoimmunity in non-obese diabetic (NOD) mice. In this study, we describe marked differences in dendritic cell generation, in vitro, caused by non-MHC NOD genes. Bone marrow cells from NOD.H-2k mice cultured in vitro with GM-CSF and IL-4 generated a reduced yield of dendritic cells when compared to bone marrow cells from B10.H-2k mice. This was due to failure to pass through successive rounds of cell division and elevated levels of apoptosis in NOD.H-2k precursor cells. This aberrant response to GM-CSF and IL-4 was unique to the NOD.H-2k background when compared to bone marrow cells from other H-2k congenic strains, and coculture experiments showed that it was cell-autonomous. Overall, the results described in this study demonstrate a striking effect of non-MHC NOD genes on dendritic cell generation from myeloid precursors derived from the NOD.H-2k strain. These results identify a useful genetic model to explore the regulation of dendritic cell formation. Conceivably, the dysregulation of the dendritic cell system described here may contribute to the generalized defects in self-tolerance in the NOD strain.     

Bone marrow precursors of nonobese diabetic mice develop into defective macrophage-like dendritic cells in vitro

The NOD mouse spontaneously develops autoimmune diabetes. Dendritic cells (DC) play a crucial role in the autoimmune response. Previous studies have reported a defective DC generation in vitro from the NOD mouse bone marrow (BM), but a deviated development of myeloid precursors into non-DC in response to GM-CSF was not considered. In this study, we demonstrate several abnormalities during myeloid differentiation of NOD BM precursors using GM-CSF in vitro. 1) We found reduced proliferation and increased cell death in NOD cultures, which explain the previously reported low yield of DC progeny in NOD. Cell yield in NOR cultures was normal. 2) In a detailed analysis GM-CSF-stimulated cultures, we observed in both NOD and NOR mice an increased frequency of macrophages, identified as CD11c(+)/MHCII(-) cells with typical macrophage morphology, phenotype, and acid phosphatase activity. This points to a preferential maturation of BM precursors into macrophages in mice with the NOD background. 3) The few CD11c(+)/MHCII(high) cells that we obtained from NOD and NOR cultures, which resembled prototypic mature DC, appeared to be defective in stimulating allogeneic T cells. These DC had also strong acid phosphatase activity and elevated expression of monocyte/macrophage markers. In conclusion, in this study we describe a deviated development of myeloid BM precursors of NOD and NOR mice into macrophages and macrophage-like DC in vitro. Potentially, these anomalies contribute to the dysfunctional regulation of tolerance in NOD mice yet are insufficient to induce autoimmune diabetes because they occurred partly in NOR mice.     

Altered dendritic cells (DC) might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic (NOD) mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes, an auto-immune disease characterized by the destruction of insulin-secreting beta-cells by autoreactive T cells. Defects in development and/or functions of dendritic cells (DC) might be critical in eliciting the auto-immune reaction to beta cells in this model. In this paper, DC differentiation in NOD mice was investigated in vitro using bone marrow-derived progenitors (BM-DC) in the presence of GM-CSF and IL-4 or spleen-derived progenitors in the presence of GM-CSF and early acting cytokines such as Flt-3L and IL-6 (SPL-DC). In both culture systems, the absolute number of NOD DC generated was strongly reduced as compared to control strains. In addition, both BM-DC and SPL-DC from NOD mice show defective differentiation into mature DC in conventional culture conditions as indicated by low expression of MHC class II and CD80 molecules among CD11c positive cells and low capacity to stimulate allogeneic T cells. However, DC achieved full maturation when exposed to LPS, except for MHC class II expression that remained decreased. Ex vivo analysis confirmed an unusual phenotype of NOD DC. Both sets of results are thus consistent with a specific defect of DC maturation in these mice.     

Lineage divergence at the first TCR-dependent checkpoint: preferential γδ and impaired αβ T cell development in nonobese diabetic mice

The first TCR-dependent checkpoint in the thymus determines αβ versus γδ T lineage fate and sets the stage for later T cell differentiation decisions. We had previously shown that early T cells in NOD mice that are unable to rearrange a TCR exhibit a defect in checkpoint enforcement at this stage. To determine if T cell progenitors from wild-type NOD mice also exhibit cell-autonomous defects in development, we investigated their differentiation in the Notch-ligand-presenting OP9-DL1 coculture system, as well as by analysis of T cell development in vivo. Cultured CD4 and CD8 double-negative cells from NOD mice exhibited major defects in the generation of CD4 and CD8 double-positive αβ T cells, whereas γδ T cell development from bipotent precursors was enhanced. Limiting dilution and single-cell experiments show that the divergent effects on αβ and γδ T cell development did not spring from biased lineage choice but from increased proliferation of γδ T cells and impaired accumulation of αβ T lineage double-positive cells. In vivo, NOD early T cell subsets in the thymus also show characteristics indicative of defective β-selection, and peripheral αβ T cells are poorly established in mixed bone marrow chimeras, contrasting with strong γδ T as well as B cell repopulation. Thus, NOD T cell precursors reveal divergent, lineage-specific differentiation abnormalities in vitro and in vivo from the first TCR-dependent developmental choice point, which may have consequences for subsequent lineage decisions and effector functions.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Immature and Maturation-Resistant Human Dendritic Cells Generated from Bone Marrow Require Two Stimulations to Induce T Cell Anergy In Vitro

Immature dendritic cells (DC) represent potential clinical tools for tolerogenic cellular immunotherapy in both transplantation and autoimmunity. A major drawback in vivo is their potential to mature during infections or inflammation, which would convert their tolerogenicity into immunogenicity. The generation of immature DC from human bone marrow (BM) by low doses of GM-CSF (lowGM) in the absence of IL-4 under GMP conditions create DC resistant to maturation, detected by surface marker expression and primary stimulation by allogeneic T cells. This resistence could not be observed for BM-derived DC generated with high doses of GM-CSF plus IL-4 (highGM/4), although both DC types induced primary allogeneic T cell anergy in vitro. The estabishment of the anergic state requires two subsequent stimulations by immature DC. Anergy induction was more profound with lowGM-DC due to their maturation resistance. Together, we show the generation of immature, maturation-resistant lowGM-DC for potential clinical use in transplant rejection and propose a two-step-model of T cell anergy induction by immature DC.     

Cutting edge: impaired transitional B cell production and selection in the nonobese diabetic mouse

Developing B cells undergo selection at multiple checkpoints to eliminate autoreactive clones. We analyzed B cell kinetics in the NOD mouse to establish whether these checkpoints are intact. Our results show that although bone marrow production is normal in NOD mice, transitional (TR) B cell production collapses at 3 wk of age, reflecting a lack of successful immature B cell migration to the periphery. This yields delayed establishment of the follicular pool and a lack of selection at the TR checkpoint, such that virtually all immature B cells that exit the bone marrow mature without further selection. These findings suggest that compromised TR B cell generation in NOD mice yields relaxed TR selection, affording autoreactive specificities access to mature pools.     

Dendritic Cells Transduced to Express Interleukin 4 Reduce Diabetes Onset in Both Normoglycemic and Prediabetic Nonobese Diabetic Mice

Background

We and others have previously demonstrated that treatment with bone marrow derived DC genetically modified to express IL-4 reduce disease pathology in mouse models of collagen-induced arthritis and delayed-type hypersensitivity. Moreover, treatment of normoglycemic NOD mice with bone marrow derived DC, genetically modified to express interleukin 4 (IL-4), reduces the onset of hyperglycemia in a significant number of animals. However, the mechanism(s) through which DC expressing IL-4 function to prevent autoimmune diabetes and whether this treatment can reverse disease in pre-diabetic NOD mice are unknown.

Methodology/Principal Findings

DC were generated from the bone marrow of NOD mice and transduced with adenoviral vectors encoding soluble murine IL-4 (DC/sIL-4), a membrane-bound IL-4 construct, or empty vector control. Female NOD mice were segregated into normoglycemic (<150mg/dL) and prediabetic groups (between 150 and 250 mg/dL) on the basis of blood glucose measurements, and randomized for adoptive transfer of 10⁶ DC via a single i.v. injection. A single injection of DC/sIL-4, when administered to normoglycemic 12-week old NOD mice, significantly reduced the number of mice that developed diabetes. Furthermore, DC/sIL-4, but not control DC, decreased the number of mice progressing to diabetes when given to prediabetic NOD mice 12–16 weeks of age. DC/sIL-4 treatment also significantly reduced islet mononuclear infiltration and increased the expression of FoxP3 in the pancreatic lymph nodes of a subset of treated animals. Furthermore, DC/sIL-4 treatment altered the antigen-specific Th2:Th1 cytokine profiles as determined by ELISPOT of splenocytes in treated animals.

Conclusions

Adoptive transfer of DC transduced to express IL-4 into both normoglycemic and prediabetic NOD mice is an effective treatment for T1D.     

Effect of human granulocyte-macrophage colony stimulating factor (hGM-CSF) on lymphoid and myeloid differentiation of sorted hematopoietic stem cells from hGM-CSF receptor gene transgenic mice

Bone marrow lineage-negative (Lin(-)) c-Kit(+) Sca-1(+) hematopoietic cells from human GM-CSF receptor gene transgenic mice were cultured on established bone marrow stromal cell (TBR59) layers and on semisolid medium. In the semisolid assay, an increasing number of larger colonies were observed in the presence of hGM-CSF. By coculture with the stromal cells, cobblestones containing myeloid and lymphoid lineages of cells were formed from the stem cell enriched fraction, and addition of hGM-CSF strongly stimulated formation of the cobblestones containing both lineages. Repeating passages of the cobblestones on TBR59 stromal cells in the presence of hGM-CSF gradually decreased cobblestone formation and inversely increased macrophages and granulocytes, while mast cells were generated when the cells derived from the semisolid assay were cultured in a liquid medium containing hGM-CSF. These results consistently suggest that cytokines such as GM-CSF may costimulate the immature hematopoietic cells at their stroma-dependent phase before lineage commitment, and after commitment that occurs by an intrinsic program of the cells, they may stimulate maintenance and maturation of progenitor cells.     

ORGANIZATION OF HAEMOPOIETIC STEM CELLS: THE GENERATION-AGE HYPOTHESIS

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 12 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half, to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of dendritic cell differentiation in the bone marrow mediates sustained immunosuppression after polymicrobial sepsis

Murine polymicrobial sepsis is associated with a sustained reduction of dendritic cell (DC) numbers in lymphoid organs and with a dysfunction of DC that is considered to mediate the chronic susceptibility of post-septic mice to secondary infections. We investigated whether polymicrobial sepsis triggered an altered de novo formation and/or differentiation of DC in the bone marrow. BrdU labeling experiments indicated that polymicrobial sepsis did not affect the formation of splenic DC. DC that differentiated from bone marrow (bone marrow-derived DC [BMDC]) of post-septic mice released enhanced levels of IL-10 but did not show an altered phenotype in comparison with BMDC from sham mice. Adoptive transfer experiments of BMDC into naive mice revealed that BMDC from post-septic mice impaired Th1 priming but not Th cell expansion and suppressed the innate immune defense mechanisms against Pseudomonas bacteria in the lung. Accordingly, BMDC from post-septic mice inhibited the release of IFN-γ from NK cells that are critical for the protection against Pseudomonas. Additionally, sepsis was associated with a loss of resident DC in the bone marrow. Depletion of resident DC from bone marrow of sham mice led to the differentiation of BMDC that were impaired in Th1 priming similar to BMDC from post-septic mice. Thus, in response to polymicrobial sepsis, DC precursor cells in the bone marrow developed into regulatory DC that impaired Th1 priming and NK cell activity and mediated immunosuppression. The absence of resident DC in the bone marrow after sepsis might have contributed to the modulation of DC differentiation.     

A self MHC class II beta-chain peptide prevents diabetes in nonobese diabetic mice

We explored T cell responses to the self class II MHC (I-Ag7) beta-chain-derived peptides in diabetic and prediabetic nonobese diabetic (NOD) mice. We found that one of these immunodominant epitopes of the beta-chain of I-Ag7 molecule, peptide 54-76, could regulate autoimmunity leading to diabetes in NOD mice. T cells from prediabetic young NOD mice do not respond to the peptide 54-76, but T cells from diabetic NOD mice proliferated in response to this peptide. T cells from older nondiabetic mice or mice protected from diabetes do not respond to this peptide, suggesting a role for peptide 54-76-specific T cells in pathogenesis of diabetes. We show that this peptide is naturally processed and presented by the NOD APCs to self T cells. However, the peptide-specific T cells generated after immunization of young mice regulate autoimmunity in NOD mice by blocking the diabetogenic cells in adoptive transfer experiments. The NOD mice immunized with this peptide are protected from both spontaneous and cyclophosphamide-induced insulin-dependent diabetes mellitus. Immunization of young NOD mice with this peptide elicited T cell proliferation and production of Th2-type cytokines. In addition, immunization with this peptide induced peptide-specific Abs of IgG1 isotype that recognized native I-Ag7 molecule on the cell surface and inhibited the T cell proliferative responses. These results suggest that I-Abetag7(54-76) peptide-reactive T cells are involved in the pathogenesis of diabetes. However, immunization with this peptide at young age induces regulatory cells and the peptide-specific Abs that can modulate autoimmunity in NOD mice and prevent spontaneous and induced diabetes.     

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8⁺ T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists.     

Essential role for cyclin D3 in granulocyte colony-stimulating factor-driven expansion of neutrophil granulocytes

The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.     

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.