Lymphoid Dendritic Cells Research Articles

A Case Report of Long-term Remission of Ulcerative Colitis After Lymphocyto-plasmapheresis

Ulcerative colitis (UC) is a chronic inflammatory bowel disease of the colorectum, with mucosal infiltration by activated leukocytes, which are the result of complex interactions between lymphocytes, antigen, and dendritic cells (DCs). We carried out lymphocyto-plasmapheresis (LCPA) in a UC patient with the aim of removing lymphocytes, DCs and inflammatory cytokines (ICKs). A 42-year-old female with UC in moderate activity phase was submitted to 5 weekly LCPA treatments. Before and after LCPA we monitored: (i) the percentage of T, B, NK lymphocytes, monocytes, and peripheral blood lymphoid and myeloid DCs; (ii) the T lymphocyte subpopulations; (iii) the ICKs; and (iv) the immune complexes (IC). We achieved the interruption of all pharmacological therapies, and so far the clinical and histological remission has lasted for 24 months. The flow cytometric assessment of the leukocyte subpopulations did not show any relevant variation of their numbers after LCPA, while TNFalpha, IL-6, IL-12 and serum IgG-C1q ICs decreased. In the present case, the contemporary depletion of plasma, lymphocytes and DCs, allowed LCPA to emerge as an efficient alternative to UC pharmacological therapy without affecting the number of white blood cells, DCs and leukocyte subpopulations that were assessed. Further studies are needed both to address LCPA mechanism of action and optimal apheresis protocol, and to compare this form of therapy to a placebo control group.

Kinetics of murine decidual dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells (APC) capable of induction of primary immune responses as well as immunologic tolerance. Myeloid and lymphoid subsets of murine DCs are able to shift cytokine responses of T cells toward Th2 and Th1 profiles respectively. Thus, DCs would be suitable candidates to mediate the balance of maternal immune responses to conception. We analyzed pregnancy-related variations in uterus and splenic DCs in a murine model. C57BL/6-mated Balb/c female mice with vaginal plugs were scarified at early, middle, and late pregnancy. Frozen sections of uterus and spleen at each stage of pregnancy were immunostained with CD11c- and MHC-II-specific antibodies. Two-color immunohistochemistry was also carried out using anti-CD11c and one of the antibodies against CD11b, CD8alpha, CD86, and DEC-205. Using morphometric analysis, the average density of DCs and relative percentage of myeloid (CD11c+, CD11b+) and lymphoid DCs (CD11c+, CD8a+) were determined at each stage. Our results showed that DCs are present throughout the pregnancy in decidua. The average density of decidual DCs at early pregnancy was significantly higher relative to middle and late gestation or to those of endometrial DCs of non-pregnant mice. Interestingly, the average density of decidual and splenic DCs, followed the same variations at different stages of pregnancy. The relative percentage of decidual lymphoid DCs (LDC) was significantly higher at mid-gestation when compared with other stages of pregnancy or non-pregnant mice. Inversely, the frequency of myeloid DCs (MDC) and the MDC/LDC ratio were statistically lower at the middle stage of pregnancy. A majority of decidual DCs expressed MHC-II and CD86. At early pregnancy, DCs were more concentrated subadjacent to the luminal epithelial layers, whereas at mid-or late gestation, DCs were randomly distributed in the stroma and around the epithelium. Mid-pregnancy period was a critical point with regard to splenic DCs kinetics, as both the average density of DCs and the frequency of MDCs decreased significantly when compared with early or late pregnancy, although the relative percentage of splenic LDCs did not change. Our data suggest that the balance of MDC and LDC is finely tuned throughout pregnancy, pointing an eminent immunoregulatory role of DCs in the maintenance of pregnancy.

In Vivo Permanent Marking of Cells with RAG1 Activation History Demonstrates Only a Minor Contribution of the Lymphoid Pathway to Dendritic Cell Development.

Dendritic cells (DCs) have been shown to arise from both myeloid and lymphoid pathways, by evaluating the in vivo reconstituting potential of myeloid- and lymphoid-committed progenitor populations. It has been suggested that these “myeloid” or “lymphoid” DCs may constitute independent DC compartments with different functions. However, evaluation of DC development after conventional adoptive transfer experiments may not correctly represent normal DC development including lineage contribution toward the formation of the DC pool. In order to accurately evaluate the distribution and the function of DCs originated from each lineage, it is ideal to mark their origin in vivo in steady-state hematopoiesis. By crossing RAG1-Cre knockin with yellow fluorescence protein (YFP)-floxed reporter lines, we developed a mouse line in which cells with a history of RAG activation should be permanently marked with YFP as a result of lymphoid commitment. The YFP expression started at the common lymphoid progenitor (CLP) stage (~35%), and the percentages of YFP+ cells progressively increased as they differentiate into mature lymphocytes. More than 99.5% of mature T and B cells were marked by YFP, while <0.5% of granulocyte/monocyte progenitors (GMPs) or granulocytes were YFP+, indicating that this system efficiently and exclusively marks cells of lymphoid origin. We found that only 4.1, 4.6, and 9.3% of DCs were YFP+ in the spleen, the lymph nodes, and the thymus, respectively. This is the formal evidence that the vast majority of DCs in steady-state hematopoiesis originate from stages that have committed to the myeloid lineage, even in the thymus. We then profiled the gene expression of the YFP+ and YFP- DCs at a genome wide level by DNA microarray analysis. Unexpectedly, the gene expression profiles of myeloid and lymphoid-derived DCs are virtually identical. These results collectively suggest that myeloid and lymphoid DCs might use a common developmental program that can be activated even after myeloid or lymphoid commitment. Thus, the DC developmental program is unique, which should be independent of either myeloid or lymphoid commitment program.

A Randomized Trial To Evaluate the Impact of Cytokines (G-CSF or GM + G-CSF) on Dendritic Cell and T-Cell Content and Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant.

Introduction: Optimal cellular immunity following allogeneic HPC transplant is a balance between effective anti-tumor immunity and the avoidance of life threatening GvHD. Our group previously demonstrated that DC content following allogeneic BMT can impact EFS (Waller et al, Blood 2001), and that mobilization cytokines can impact DC content of the autologous PBSC graft (Lonial et al, BBMT 2004). The current trial was designed with the hypothesis that the use of G + GM-CSF will result in fewer tolerogenic DC2 cells within the graft, and increased Th1 phenotype among the mobilized T-cell when compared with normal donors mobilized with G-CSF alone.Methods: 43 normal donors were randomized to receive G-CSF alone (7.5 mcg/kg BID) or G-CSF (7.5mcg/kg qd) and GM-CSF (7.5 mcg/kg qd) which was administered for 5 days or longer if additional days of apheresis were necessary. Side effects between the 2 cytokine regimens were documented using a questionnaire that was administered within 2 weeks of successful collection. Graft content was evaluated using multi-color flow cytometry, and pre-mobilization blood was collected to test for T-cell and DC content as well as T-cell function. DC1 (myeloid DC) were defined as Lin−/HLA-DR+/CD11C+/CD123−, while DC2 (lymphoid DC) were defined as Lin−/HLA-DR+/CD11C−/CD123+. T-cell proliferation was assessed using thymidine incorporation assays following mitogen exposure, and T-cell activation profile was assessed using ELISA assays of secreted cytokines and intracellular cytokine measurements following mitogen stimulation.Results: 19 patients received G+GM-CSF and 24 patients received G-CSF alone. All 43 donors were successfully mobilized, though more patients in the G-CSF arm required multiple days of collection (mean number of collections 1.4 G-CSF vs 1.1 G+GM-CSF, p=0.06). There was no difference in %CD34+ or CD34+ content of the grafts between groups. There was a marked reduction in DC2 content as measured by percent (0.33% vs 0.45%, p=.03) absolute DC2 content (1.98e6 vs 3.66e6, p=0.0003), and delivered DC2 dose e6/kg (2.8 vs 5.2, p=.0002) for the donors randomized to received the combination of GM/G-CSF vs G-CSF alone. There was no statistical difference in DC1 content measured in an analogous fashion. Additionally, grafts collected with G-CSF alone had significantly greater numbers of total T-cells than compared with G+GM-CSF (379e6/kg vs 230e6/kg, p=0.0007). This percent reduction in total T-cell content was identical to the reduction in the CD4 and CD8 subsets between the 2 randomized groups. Interestingly, cells collected from the recipients of G+GM-CSF on the day of mobilization secreted higher amounts of IL-2 at rest, though when stimulated with PMA, G-CSF mobilized cells secreted significantly higher amounts of IL-2. Other secreted cytokines were measured, and to date do not show a clear trend towards a difference. Side effects between the 2 randomized arms were similar with 1 patient in the G-GM-CSF arm having to stop growth factors secondary to bone pain and fever.Conclusion: The combination of G+GM-CSF mobilizes significantly fewer DC2 cells as well as 40–50% fewer T-cells, and is more likely to result in a successful single PBSC collection than the use of G-CSF alone. Further laboratory evaluation of the graft, survival, and GvHD data following transplant will also be presented.

CCL21-induced immune cell infiltration

Cellular immune responses can be initiated via peptide presentation by specialized antigen presenting cells, dendritic cells (DCs), which stimulate naïve T cells. The trafficking of DCs and T cells is regulated by chemokines such as CCL21. CCL21 is normally expressed in the lymphoid organs and coordinates the interactions between DCs and T cells, thereby contributing to the initiation of T cell responses. In order to comprehend the mechanisms of CCL21 activity and to utilize CCL21 optimally in therapy, understanding the kinetics of the responses of various cell types to CCL21 would be beneficial. Therefore, in this study, we injected mice subcutaneously (s.c.) with CCL21 and examined the DC and T cell infiltration of the local draining lymph node. CCL21 injection resulted in significantly increased numbers of lymphoid and myeloid DCs and effector T lymphocytes in the local node at 4 days. Furthermore, at 4 days small lymphoid-like structures were visible in the injection areas. These results provide guidance for the optimal timing of CCL21 use in combination with vaccines.

Increased antigen presentation and Th1 polarization in genetically histamine-free mice

Histamine is a well-known inflammatory mediator exerting various immunomodulatory effects and affecting the development of antigen-specific immune responses. Dendritic cells (DCs) are the most potent antigen-presenting cells specialized for capture, uptake, transport, processing and presentation of antigens to T cells. Using a genetically histamine-free [histidine decarboxylase knockout (HDC-/-)] mouse model, we examined the effects of histamine on DC-mediated antigen presentation. Applying an in vitro antigen presentation assay, we found that spleen DCs, derived from HDC-/- mice, display a higher efficiency in antigen presentation compared with wild-type cells. Flow cytometric characterization of DCs disclosed that this difference was not due to an altered distribution of DCs between or within the major functional sub-populations (assessed by CD11b and CD4 as myeloid and CD8alpha and DEC205 as lymphoid DC markers) or major changes in the co-stimulatory molecule profile (CD40, CD80, CD86). However, real-time PCR analysis of in vivo CFA-induced IL-12p35, IFNgamma, IL-10 and IL-4 expression showed that DCs matured in a histamine-free environment exhibit significantly elevated levels of IL-12p35 and IFNgamma mRNA. In vitro investigations confirmed that isolated DCs, developed in the absence of histamine, exhibit indeed a predominantly T(h)1-polarized cytokine pattern, as they show elevated levels of IFNgamma mRNA upon LPS stimulation. Similar difference was found at the protein level by ELISA, as well. Our study demonstrates that histamine interferes with antigen presentation and alters the cytokine profile of DCs.

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Recipient Memory-Like Lymphocytes Remain Unresponsive to Graft Antigens After CAMPATH-1H Induction with Reduced Maintenance Immunosuppression

Treatment with CAMPATH-1H at the time of transplantation allows reduced maintenance immunosuppression. We hypothesized that CAMPATH-1H induction would modulate the response of repopulating leukocytes to donor alloantigens. The phenotype, proliferative and stimulatory capacity of peripheral blood leukocytes from 14 renal transplant recipients treated with CAMPATH-1H and reduced immunosuppression with mycophenolate mofetil and tacrolimus were investigated for the first six months after transplantation. The impact of immunosuppressive drugs on leukocytes that escape depletion was also evaluated in vitro. CAMPATH-1H therapy caused a significant decrease in the number of B and T cells, with CD4 T central memory cells being the most resistant to depletion. The recovery of CD8 T cells was faster than that of CD4 T cells. Lymphopenia correlated with a decrease in both proliferative and effector responses, however, the recipient T cells remained responsive to third-party antigens. Dendritic cells (DC) were also depleted but to a lesser extent than lymphocytes; lymphoid DC were more resistant than myeloid DC; these changes correlated with decreased allostimulatory capacity. One of the patients experienced rejection that was treated successfully. The rejection was associated with a high proportion of CD4 T effector memory cells and myeloid DC, increased proliferation and enhanced effector activity to donor antigens. In vitro studies confirmed that the reduced dose of immunosuppressive drugs used could prevent activated T cells from switching to the effector compartment, suppressing both their proliferation and effector activity. CAMPATH-1H induction combined with reduced maintenance immunosuppression is sufficient to control the effector phase of immune response to donor antigens.

Quantitative analysis of the expression of glycosylphosphatidylinositol‐anchored proteins during the maturation of different hematopoietic cell compartments of normal bone marrow

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) are a heterogeneous group of proteins deficiently expressed in patients with paroxysmal nocturnal hemoglobinuria. Up till now, no study has been reported in which the exact patterns of expression of a large number of GPI-AP are quantitatively evaluated in normal bone marrow (BM) cells classified according to their lineage and maturation stage. In the present study, we have quantitatively analyzed the expression of eleven different GPI-AP (CD14, CD16, CD24, CD48, CD52, CD58, CD59, CD66b, CD87, CD109 and CD157) during maturation of the neutrophil, monocytic, erythroid, lymphoid, basophil and plasmacytoid dendritic cells (DC) lineages in normal BM as a frame of reference for the understanding of the abnormal patterns of expression of GPI-AP observed in the BM of PNH patients. Our results show that expression of most GPI-AP varies during normal BM maturation, different profiles being frequently observed depending on the cell lineage or the GPI-AP analyzed. Overall, these results provide a detailed map GPI-AP expression during normal hematopoietic differentiation, which could serve as a basis for the identification and characterization of changes occurring in PNH.

Differences in circulating dendritic cell subtypes in peripheral, placental and cord blood in African pregnant women

Dendritic cells (DC) are important for induction of primary immune responses and immunological tolerance. Changes in the frequency of DC subsets were analyzed in peripheral blood from pregnant women (mPB) and compared to placental blood (PB) and cord blood (CB). DCs were identified by flow cytometry in whole blood as lineage negative and HLA-DR-positive cells. Different DC subtypes were identified with CD123 and CD11c markers. In these data, the percentage of DC was significantly lower in mPB, PB and CB than in control women, but the absolute number of DC was higher in CB, suggesting that numbers of DC in CB do not explain the decrease of the immune response in newborn infants. Myeloid DCs (MDC) decreased in all compartments of pregnant women compared to control women, especially in mPB where MDC became lower than lymphoid DCs. An increase of less differentiated DC was observed in mPB and CB from pregnant women. DCs in pregnant women were mainly immature DC with a proportion of CD83-positive DC, identical as control women. The levels of IFNγ, TNFα, IL-2, IL-4, IL-5 and IL-10 were not different in the three compartments (mPB, PB, CB). In conclusion, the phenotype and subset of DCs were different in pregnant women than in control women, suggesting a role in maintenance of immune tolerance against the fetus. The distribution of DC subsets was different in mPB, PB and CB. Their role in the regulation of immune response remains to be elicited.

Analysis of endometrial myeloid and lymphoid dendritic cells during mouse estrous cycle

This study was performed to evaluate the frequency and localization of endometrial myeloid (CD11c + CD11b +) and lymphoid (CD11c + CD8α +) dendritic cells (DCs) at different stages of murine estrous cycle. To address the systemic effect of ovarian hormones fluctuations during estrous cycle, the same variables were studied in splenic DCs as well. Stages of the estrous cycle of Balb/c mice were determined by examination of vaginal smears. Frozen sections of uterus and spleen at each stage of estrous cycle were stained for CD11c and MHC-II. Two-color immunohistochemistry was also carried out using anti-CD11c with one of the antibodies against CD11b, CD8α, CD86, and DEC-205. The average density of DCs and relative percentage of myeloid and lymphoid DCs (MDCs and LDCs) were determined at each stage of estrous cycle by morphometric analysis. Our results showed that DCs were present throughout the estrous cycle in mice endometrium, but their frequency was highest at estrus and lowest at proestrus ( P < 0.005). The lymphoid subset of DCs was more prominent at estrus relative to those at other stages ( P < 0.005). Conversely, the relative percentage of myeloid DCs at estrus was significantly lower compared to other stages ( P < 0.005). Nearly all endometrial and splenic DCs expressed CD86 and MHC-II. At proestrus, and particularly at estrus, DCs were more concentrated subadjacent to the luminal and glandular epithelial layers with some scattered throughout the stroma whereas, at metestrus and diestrus, DCs were randomly distributed in stroma and around the glandular and luminal epithelial layers. The number and immunophenotype of splenic DCs were not statistically different between stages of estrous cycle. Our results suggest that endometrial but not splenic myeloid and lymphoid DCs are influenced by steroid hormones during estrous cycle.

Efficient T Cell Activation via a Toll-Interleukin 1 Receptor-Independent Pathway

Here, we describe a previously unrecognized pathway for activation of antigen-specific adaptive immune responses that was independent of Toll-Interleukin 1 Receptor signaling and directed toward detection of antigens expressed by apoptotic cells. This pathway is represented within Flt-3 Ligand-derived dendritic cells (DCs) that represent immature lymphoid DCs, but not within GM-CSF-treated bone marrow-derived dendritic cells. Exposure of these DCs to apoptotic cells resulted in production of type I interferon and favored the development of cytotoxic T cell responses. The N-Ethyl-N-Nitrosourea-induced germline mutation 3d (Unc3b1(3d/3d)) abolished both MHC class I and II responses elicited by this pathway, whereas a null allele of Cd36 selectively abolished class II responses. We propose that this mode of adaptive immune activation evolved to permit the sensitive detection of intracellular microbial infections, particularly viral infections, which frequently induce apoptotic cell death, but may also be important in transplantation, autoimmunity, and vaccine development.

Serum pancreatic lipase [EC 3.1.1.3] activity, serum lipid profile and peripheral blood dendritic cell populations in normolipidemic males with psoriasis

The purpose of the study was to explore serum pancreatic lipase activity and the serum lipid profile in relation to peripheral blood dendritic cell subsets and disease severity in males with psoriasis. Material and methods The study population consisted of 22 normolipidemic males with psoriasis and 12 aged-matched and body mass index (BMI)-matched healthy males. The percentages of peripheral blood dendritic cell (DC) subsets were evaluated using appropriate monoclonal antibodies and flow cytometry. The serum pancreatic lipase activity and the lipid profile were determined using standard enzymatic and colorimetric techniques. Results Pancreatic lipase activity was increased ( p = 0.56421), high-density lipoprotein (HDL)-cholesterol concentration ( p = 0.00584) was significantly decreased, triglyceride ( p = 0.00766) and VLDL-cholesterol ( p = 0.00765) levels were significantly increased in serum of psoriatic patients compared to controls. The serum pancreatic lipase activity showed significant correlation with serum triglyceride ( r = 0.42; p = 0.04721) and serum VLDL-cholesterol levels ( r = 0.42; p = 0.04721) in psoriatic individuals. In psoriatic patients the percentage of myeloid DCs was increased ( p = 0.54932), the percentage of lymphoid DCs was decreased ( p = 0.14210) and myeloid DC/lymphoid DC ratio was significantly increased ( p = 0.03569) compared to healthy individuals. Conclusion The direct cause of the abnormal lipid profile in psoriasis and its relationship with the immune system disturbances remains unclear. The reciprocal relationship between serum pancreatic activity and serum triglyceride level appears to confirm the hypothesis about abnormal lipid metabolism in psoriasis.

Immune regulatory activity of liver‐derived dendritic cells generated in vivo

Hepatic tolerance is demonstrated by spontaneous acceptance of liver allografts in mice. Hepatic dendritic cells (DC) play a crucial role in determining immunity or tolerance. In this study, we adopted an approach to transfect gene(s) into the mouse liver by tail-vein injection of plasmid-carrying genes. Transfection with GM-CSF expanded liver CD11c+ myeloid DC (LMDC), while liver B220+CD11c- lymphoid DC (LLDC) were expanded after transfection of IL-3 and CD40L. Flow analysis revealed that these liver DC subsets were phenotypically mature following overnight culture. However, in contrast to LMDC, LLDC induced hyporesponsiveness in allogeneic T-cells, with suppressed secretion of both IL-2 and IFN-gamma, and prolonged cardiac allograft survival. This immune regulatory DC population in the liver may play a role in modulating T-cell immunity in the liver.

Regulatory dendritic cells modulate immune responses via induction of T‐cell apoptotic death

We describe a regulatory lymphoid dendritic cell (LDC) population propagated from mouse liver nonparenchymal cells (NPC) in IL-3 and anti-CD40 monoclonal antibody that are phenotypically mature, and induce T-cell hyporesponsiveness by promoting T-cell apoptotic death, which is partially caspase-dependent, but is unlikely to be mediated by soluble factor(s). In vivo administration of liver LDC significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner. This is associated with enhanced T-cell death in secondary lymphoid organs.

Dendritic cells heterogeneity and its role in cancer immunity

Dendritic cells are the most potent 'professional' antigen-presenting cells, with high ability of primary immune response initiation. Dendritic cells originate from bone marrow progenitors, which circulate in peripheral blood and subsequently give rise to immature dendritic cells, which reside in peripheral tissues. When dendritic cells encounter danger signals, they undergo differentiation and maturation; thereafter they migrate to lymphatic tissues, where they synapse with T-cells and initiate primary immune response. The immune response efficiency is determined by Th1/Th2 balance. Although dendritic cells represent a rare group of leukocytes, their functional and phenotypical heterogeneity confer a great challenge to immunologists. In this review, the myeloid and lymphoid development pathways of dendritic cells, are discussed. The heterogeneity of dendritic cells will be reviewed, based on their anatomical locations, phenotypes and functions. This section focuses on blood and lymphoid tissue dendritic cells. Subsequently, the roles of dendritic cells in the immunity of cancer and how cancer bypasses the dendritic cell-mediated immune responses, are discussed.

A Randomized Trial To Evaluate the Impact of Cytokines (G-CSF or GM + G-CSF) on Dendritic Cell and T-Cell Content and Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant.

Introduction: Optimal cellular immunity following allogeneic HPC transplant is a tenuous balance between effective anti-tumor immunity and the avoidance of life threatening GvHD. The events necessary for potentially curative GvL are related to tumor burden, T-cell content, NK cell content, and dendritic cell content. Our group previously demonstrated that DC content following allogeneic BMT can impact EFS (Waller et al, Blood 2001), and that cytokines used during mobilization impact DC content of the autologous graft (Lonial et al, BBMT 2004). The current trial was designed with the hypothesis that the use of G + GM-CSF will result in fewer tolerogenic DC2 cells within the graft, and increased Th1 phenotype among the mobilized T-cells.Methods: 40 normal donors were randomized to receive G-CSF alone (7.5 mcg/kg BID) or G-CSF (7.5mcg/kg qd) and GM-CSF (7.5 mcg/kg qd) for 5 days or longer if additional apheresis were necessary. Side effects between the 2 cytokine regimens were documented using a questionnaire that was administered within 2 weeks of successful collection. Graft content was evaluated using multi-color flow cytometry, and pre-mobilization blood was collected to test for T-cell and DC content as well as T-cell function. T-cell proliferation was assessed using thymidine incorporation assays following mitogen exposure, and T-cell activation profile was assessed using ELISA assays of secreted cytokines following mitogen stimulation. DC1 (myeloid DC) were defined as Lin-/HLA-DR+/CD11C+/CD123−, while DC2 (lymphoid DC) were defined as Lin-/HLA-DR+/CD11C−/CD123+.Results: 18 patients received G+GM-CSF and 22 patients received G-CSF alone. All 40 donors were successfully mobilized, though more patients in the G-CSF arm required multiple days of collection (mean number of collections 1.5 G-CSF vs 1.0 G+GM-CSF, p=0.01). Donors mobilized with G-CSF alone had a higher overall cell count (765e8/ml compared with 615e8/ml for G+GM-CSF, p=0.04), though the CD34 content of the grafts were comparable. Again, there was a significant reduction in delivered DC2 content of graft mobilized with G+GM-CSF when compared with G-CSF alone (5.1e6/kg compared with 2.85e6/kg, p=0.001), with a trend towards fewer DC1 cells as well (2.6e6/kg vs 1.7e6/kg, p=0.05). Overall, the DC1:DC2 ratio favors DC1 among donors mobilized with G+GM-CSF. Additionally, grafts collected with G-CSF alone had nearly twice the total T-cell content of grafts mobilized with G+GM-CSF (420e6/kg vs 220e6/kg, p=0.0001). The reduction in total T-cells was evenly distributed between CD4 and CD8 cells with similar magnitude of reduction in both subsets amongst those receiving G+GM-CSF. Interestingly, grafts collected from the recipients of G+GM-CSF produced more IL-2 at rest, and secreted significantly more IL-12 in response to mitogen. Toxicity between the 2 arms were similar with 1 patient in the G+GM-CSF arm having to stop growth factors due to bone pain and fever.Conclusion: The combination of G+GM-CSF mobilizes fewer DC2 cells as well as 40–50% fewer T-cells, and is more likely to result in a successful single PBSC collection than the use of G-CSF alone. Preliminary data suggests that the combination of G+GM-CSF shifts T-cells towards a Th1 phenotype, despite fewer overall T-cells. Further laboratory evaluation of the graft, survival, and GvHD data following transplant will also be presented.

Post-Transplant Administration of Flt3 Ligand Enhances Innate Cellular-Mediated Immune Responses to Toll-Like Receptor Agonists in Syngeneic Bone Marrow Transplant Mice.

Innate cellular-mediated immunity (iCMI) responses are initiated through activation of pattern recognition receptors like Toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). FL (Flt3 ligand) is a hematopoietic growth factor that promotes the expansion of dendritic cells (DCs) and natural killer (NK) cells, key immunomodulatory effector cells involved in host defense. We have previously shown that reconstituted splenocyte iCMI following murine syngeneic BMT occurs in distinct phases in which donor-derived, innate effector cell recovery correlates with inducible cytokine production. Therefore, we studied whether post-transplant administration of FL could enhance reconstitution of innate effector cells and their cytokine responses to established TLR agonists. After split-fraction radiation with 12 Gy 137Cs, C57BL/6 recipient mice were tail-vein injected with 5x106 unselected BM cells from congeneic donor mice. Transplant mice were injected intraperitoneally (IP) with either 10μg FL/100ml 0.1%MSA/PBS (FL) or 100ml 0.1%MSA/PBS alone (Sham) for 10 consecutive days starting the first day after transplant (D1). Non-transplant C57BL/6 mice served as controls and were similarly treated with IP injections. At 2 (D12) and 10 days (D22) post-FL and sham therapy, splenocytes were harvested and pooled per group and were characterized phenotypically by flow cytometry and functionally via ex vivo cell-culture stimulation with established PAMP and cell-specific agonists. Unselected splenocytes were cultured in triplicate in the following stimulation conditions: media only; Staphylococcus aureus consorbin (SAC, TLR-2); polyinosine-polycytidylic acid (polyI:C, TLR-3); Salmonella enteriditis lipopolysaccharide (LPS, TLR-4); anti-CD40 alone; anti-CD40 plus recombinant IL-4 (rIL-4); combination anti-CD40, rIL-4, and anti-IL-10; R848 (TLR7/8); unmethylated CpG oligodeoxynucleotide 1826 (CpG, TLR-9); and lastly, combination rIL-12 and rIL-18. 24h cell-culture supernatants were assessed for cytokine levels using standard ELISA. Three main observations were noted. First, FL therapy increased total numbers of splenocyte innate effector cells in both control and D12 transplants by 6.2- and 3.2-fold, respectively. Of effector cell expansion, DC increases were the most dramatic (16.4 and 9.7-fold for control and D12 transplants, respectively). Specifically, numbers of reconstituted lymphoid, myeloid and plasmacytoid DCs were 17-, 7- and 5-fold higher, respectively, in FL than sham D12 transplants. Reconstituted NK cells had only modest increases (2-fold) after FL therapy. Second, levels of TLR-2 and TLR-9 induction of IL-12p70 and IFN-γ were 3 to 5-fold higher and DC-specific p70 induction was 5-fold higher in FL than sham D12 mice. Finally, FL augmentation of cellularity and cytokine responses was not long-lasting, as post-FL and sham D22 transplant mice had similar levels of innate effector cells and inducible cytokines. Together, these results show that post-transplant FL immunotherapy transiently enhances reconstituted iCMI and cytokine responses to PAMP agonists. Therapy targeting iCMI response could potentially be used to improve anti-microbial host defense during absent or incomplete adaptive immune recovery.

Mouse Lymphoid Tissue Contains Distinct Subsets of Langerin/CD207+ Dendritic Cells, Only One of Which Represents Epidermal-Derived Langerhans Cells

Langerin/CD207 is a C-type lectin associated with formation of Birbeck granules (BG) in Langerhans cells (LC). Here, we describe a monoclonal antibody (mAb 205C1) recognizing the extracellular domain of mouse langerin. Cell-surface langerin was detected in all epidermal LC, which presented a uniform phenotype. Two subpopulations of langerin+ cells were identified in peripheral lymph nodes (LN). One population (subset 1) was CD11c(low/+)/CD8alpha(-/low)/CD11b+/CD40+/CD86+. The other population (subset 2) was CD11c(high)/CD8alpha+/CD11b(low), and lacked CD40 and CD86. Only subset 1 was fluorescein 5-isothiocyanate (FITC+) following painting onto epidermis, and the appearance of such FITC+ cells in draining LN was inhibited by pertussis toxin. Mesenteric LN, spleen, and thymus contained only a single population of langerin+ DC, corresponding to peripheral LN subset 2. Unexpectedly, BG were absent from spleen CD8alpha+ DC despite expression of langerin, and these organelles were not induced by mAb 205C1. Collectively, we demonstrate that two langerin+ DC populations (subsets 1 and 2) co-exist in mouse lymphoid tissue. Subset 1 unequivocally identifies epidermal LC-derived DC. The distribution of subset 2 indicates a non-LC origin of these langerin+ cells. These findings should facilitate our understanding of the role played by langerin in lymphoid organ DC subsets.

CD11c+ Dendritic Cells Are Required for Survival in Murine Polymicrobial Sepsis

CD11c+ dendritic cells (DCs) are APCs that link innate and adaptive immunity. Although DCs are lost from spleen and lymph nodes in sepsis, their role in outcome remains unclear. Transgenic mice (B6.FVB-Tg(.Itgax-DTR/EGFP.57)Lan/J) expressing the diphtheria toxin (DT) receptor on the CD11c promoter (DCKO mice) received 4 ng/kg DT, which resulted in depletion of 88-95% of mature myeloid and lymphoid DCs, with less depletion (75%) of plasmacytoid DCs. Pretreatment of DCKO mice with DT resulted in reduced survival in sepsis compared with saline-pretreated DCKO mice (0 vs 54%; p < 0.05) or DT-treated wild-type littermates (0 vs 54%; p < 0.05). This increased mortality was not associated with either increased bacteremia or plasma cytokine concentrations. Intravenous injection of 10(7) wild-type DCs improved survival in DCKO mice (42 vs 0%; p = 0.05). These data confirm that DCs are essential in the septic response and suggest that strategies to maintain DC numbers or function may improve outcome.