Identification and Characterization of Canine Dendritic Cells Generated In Vivo

Article Outline

• Abstract
• Introduction
• Materials and Methods
• Results
  • FL Treatment Increases Peripheral Blood Monocyte Numbers and the Allostimulatory Potential of Unfractionated Peripheral Blood Leukocytes
  • Purification of Putative Dendritic Cells from Whole Blood
  • CD11c+/HLA-DR+/CD14−/DM5− Cells are Potent Stimulators in Allogeneic Mixed Lymphocyte Culture
  • Morphology of CD11c+/HLA-DR+/CD14−/DM5− Cells by Transmission Electron Microscopy
• Discussion
• Acknowledgments
• References
• Copyright

Abstract 

Emerging evidence suggests that host dendritic cells (DC) initiate and regulate graft-versus-host and graft-versus-tumor reactions after allogeneic hematopoietic cell transplantation (HCT). Even though decades of experimentation in the preclinical canine HCT model have substantially improved our understanding of the biology and safety of HCT in human patients, the in vivo phenotype of potent antigen-presenting cells in dogs is poorly defined. Therefore, peripheral blood leukocytes were obtained from dogs treated with recombinant human Flt3-ligand and phenotypically distinct cell populations, including putative DC, were purified by 4-color flow-cytometry and tested for their stimulatory potential in allogeneic mixed lymphocyte cultures (MLC). Cells characterized by surface expression of CD11c and HLA-DR, and absence of expression of CD14 and DM5, a marker of mature granulocytes, were found to be highly potent stimulators in allogeneic MLC. In contrast, all other immunophenotypically different cell populations tested had either weak or absent allostimulatory potential. Transmission electron microscopy of CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells revealed the morphology similar to that described for DC in humans and ex vivo-generated canine DC, including long cytoplasmic extensions, discrete lysosomes, and an abundant Golgi apparatus and endoplasmatic reticulum. In summary, CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells obtained from canine peripheral blood have functional and morphologic characteristics similar to those of human myeloid DC.

Key Words: Canine, Dendritic cells, Transplantation, Allogeneic, Flt3-ligand

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Introduction 

Dendritic cells (DC) comprise a specialized system for presenting antigen to naïve or quiescent T cells, and consequently play a central role in the induction of T cell and B cell immunity in vivo [1]. Immature DC capture microbial or viral antigens in peripheral tissues and migrate to lymphoid organs where, after maturation, they display antigen-derived peptides in the context of MHC molecules. Antigen-specific T cells are activated through recognition of (1) an antigen-specific signal (antigen-major histocompatibility complex complex; “signal 1”) and (2) a “nonspecific” costimulatory signal (“signal 2”) displayed on the DC surface. Given that DC change in phenotype during their lifespan and cell surface markers otherwise characteristic for distinct hematopoietic cell lineages are not specific for identifying DC, it is difficult to use these markers for ontogenic deductions. Thus, the question whether DC originate from a separate lineage or belong to the monocyte/macrophage family is still unresolved.

More recently, host DC have been identified as the key initiators and regulators of graft-versus-host (GVH) and graft-versus-tumor (GVT) effects after allogeneic hematopoietic cell transplantation (HCT) [2, 3, 4]. After allogeneic HCT, host DC may be replaced by donor cells at a rate depending upon the (1) intensity of the preparative regimen, (2) the capacity of self-renewal of host DC, and (3) the recruitment of new donor-derived DC precursors. Hence, the replacement kinetics of host DC by donor DC might influence transplantation outcomes including graft-versus-host disease (GVHD), host-versus-graft reactions that may lead to graft rejection, and GVT effects that maintain remission of the underlying malignancy.

Despite the emerging role of host DC in initiating GVHD and GVT responses after allogeneic HCT, potent antigen-presenting cells analogous to those occurring in humans in vivo have not been defined for the canine model. This is of interest because the random-bred dog model has been extremely useful for improving the safety and our understanding of the biology of human HCT. For example, most of the GVHD prophylactic regimens, such as methotrexate (MTX)/cyclosporine (CSP) [5] and mycophenolate mofetil (MMF)/CSP [6], have been developed in dogs. More recently, the dog model has helped pioneer nonmyeloablative HCT regimens for human transplantation [7].

In the current study we used a panel of canine-specific and crossreacting antihuman monoclonal antibodies (mAbs) to identify DC in peripheral blood of dogs. We identified a CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cell population with functional and morphologic characteristics similar to those described for human myeloid DC.

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Materials and Methods 

The Institutional Animal Care and Use Committee of the Fred Hutchinson Cancer Research Center (FHCRC) approved this study. Standard care was provided as described previously [8, 9]. Beagles or miniature mongrel-beagle crossbreeds (n = 10) were given daily subcutaneous injection of 100 μg/kg recombinant human Flt3-ligand (FL; Amgen, Seattle, WA). Peripheral blood leukocytes (PBL) were obtained from whole blood before and after 10 days of FL treatment, hemolyzed, and cryopreserved for future testing. The following monoclonal antibodies (mAbs) were used for flow cytometric analyses and cell sorting of putative DC: primary murine mAbs used in the study were: antihuman HLA-DR-APC (G46-6), antihuman CD14-PerCP-Cy5.5 (M5E2) (BD Biosciences, San Jose, CA); anticanine DM5-FITC [10], anticanine CD3-FITC (17.6F9) [11], anticanine CD34-FITC (1H6) [12], anticanine CD21-PE (CA2.1D6), anticanine CD4-biotin (1E4) [13], and anticanine CD11c-biotin (CA11.6A1) [14]. Unidirectional mixed lymphocyte cultures (MLC) using peripheral blood mononuclear cells (PBMC) isolated by density-gradient centrifugation were conducted according to established methods [15]. Transmission electron microscopy of sorted CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells was performed using a JEM-1010 electron microscope (JEOL Inc., Peabody, MA).

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Results 

FL Treatment Increases Peripheral Blood Monocyte Numbers and the Allostimulatory Potential of Unfractionated Peripheral Blood Leukocytes 

To determine whether FL treatment changed the composition and thereby increased the allostimulatory potential of PBL, 10 dogs were given daily subcutaneous injections of FL (100 μg/kg/day). After 10 days of FL treatment, we observed a median 1.85-fold increase in PBL (P = .003), which was largely attributable to mobilization of monocytes (median increase, 8.1-fold; P < .001) and granulocytes (median increase, 1.63-fold; P = .03) (Table 1). Additional immunophenotypical analyses of cell populations in peripheral blood of 2 dogs before and after FL treatment showed that the number of CD34⁺ hematopoietic progenitor cells and CD14⁺/DM5⁻ monocytes increased 10.2-fold and 10.6-fold, respectively. There was no significant change in lymphocyte and platelet counts, and hematocrit. Compared to untreated control dogs, FL treatment increased the allostimulatory potential of PBL in MLC by a median of 150% (range: 36%-900%; P = .002; n = 5) (Figure 1).

Table 1. Absolute and Relative Changes in Cell Numbers in the Peripheral Blood of 10 Dogs Treated with Flt3-Ligand

Complete blood counts were obtained prior to (pre-FL) and at the completion of FL treatment (post-FL) (100 μg/kg/day × 10 days). Shown are absolute cell numbers, and relative changes of morphologically characterized cell populations pre- and post-FL treatment.

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• Figure 1. 

  Allostimulatory potential of unsorted peripheral blood leukocytes obtained from dogs before (pre-FL) and after (post-FL) completion of 10 days of Flt3-ligand (FL) treatment. Different numbers of stimulators cells (5-20 × 10⁴) obtained from dogs pre-FL and post-FL (100 μg/kg/day given subcutaneously for 10 days) were incubated with constant numbers (1 × 10⁵) of allogeneic, third-party responder cells as described in Materials and Methods. Shown is one representative experiment of 5 experiments. Compared to untreated control dogs, FL treatment increased the MLC allostimulatory potential of unsorted peripheral blood leukocytes by a median of 150% (range: 36%-900%).

Purification of Putative Dendritic Cells from Whole Blood 

To identify the cell population accounting for the increased allostimulatory potential of unfractionated PBL after FL treatment, phenotypically different cell populations were sorted from post-FL blood and used as stimulator cells in allogeneic MLC. Purity of sorted cell populations was ≥97%. Figure 2A and B shows the change in forward-scatter/side-scatter (FSC/SSC) distribution of PBL after 10 days of FL treatment. The increased white blood cell count after FL treatment was largely attributable to the 10-fold increase in cells with intermediate FSC/SSC characteristics, which were CD14⁺ monocytes. Putative canine DC were sorted from post-FL blood using a 3-step approach. First, cell debris was excluded by establishing a gate according to FSC/SSC characteristics (Gate 1, Figure 3A). Second, a gate was established (Gate 2) to include cells that were CD14⁻ and DM5⁻, thereby excluding monocytes and granulocytes (Figure 3B). Finally, HLA-DR⁺/CD11c⁺ cells were collected (R5, Figure 3C). Although the CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cell population comprised <0.1% of pre-FL PBL, the median percentage of these cells among post-FL PBL was 0.66% (range: 0.16%-1.90%; P = .026; n = 7).

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• Figure 2. 

  Fluorescence activated cell sorting of phenotypically distinct cell populations from peripheral blood of FL-treated dogs. FSC/SSC characteristics of peripheral blood leukocytes before (A) and after (B) 10 days of FL treatment (100 μg/kg/day). Phenotypically distinct cell populations were sorted from post-FL peripheral blood as described in Materials and Methods. The following gates were combined with Gate 1, which was established according to FSC/SSC characteristics: Gate 2 for B lymphocytes (C); Gates 3 and 4 for monocytes and granulocytes, respectively (D); and Gates 5 and 6 for CD4 and CD8 T cells, respectively (E). Purity of sorted cell populations was ≥97%. CD34⁺ progenitor cells were not used as stimulator cells in MLC (F).

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• Figure 3. 

  Phenotypic characterization of putative dendritic cells (DC). Putative canine DC were sorted from post-FL blood using a 3-step approach. First, cell debris was excluded by establishing a gate according to FSC/SSC characteristics (Gate 1) (A). Second, Gate 2 was chosen to exclude all cells that were CD14⁺ (monocytes) and DM5⁺ (granulocytes) (B). Third, HLA-DR⁺/CD11c⁺ cells (R5) were collected (C). Thus, putative DC were defined as live gated cells that were HLA-DR⁺/CD11c⁺/DM5⁻/CD14⁻.

CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ Cells are Potent Stimulators in Allogeneic Mixed Lymphocyte Culture 

Sorted CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ putative DC were subsequently tested for their ability to stimulate third-party responders in allogeneic MLC in comparison to sorted monocytes, granulocytes, and B lymphocytes (Figure 4). All stimulator populations were irradiated (22 Gy) prior to initiation of MLC. Even at low stimulator cell numbers of 5 × 10³/well, putative DC had strong allostimulatory potential, which increased with increasing numbers of stimulator cells (Figure 4). Although sorted B lymphocytes (CD21⁺/HLA-DR⁺) had weak allostimulatory potential, monocytes (CD14⁺/DM5⁻) and granulocytes (DM5⁺/CD14⁻) did not appreciably stimulate responder cell proliferation.

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• Figure 4. 

  Functional characterization of sorted cell populations in mixed lymphocyte cultures. Phenotypically distinct cells populations were sorted from FL-treated dogs as described in Materials and Methods. Varying numbers (5-20 × 10³) of irradiated stimulator cells were placed into mixed lymphocyte cultures using constant numbers (1 × 10⁵) of allogeneic, third-party responder cells. Shown is 1 of 6 representative experiments.

Morphology of CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ Cells by Transmission Electron Microscopy 

Sorted putative DC (CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻) were characterized by long cytoplasmic processes by transmission electron microscopy (Figure 5). They displayed irregularly shaped nuclei and multivesicular bodies. In contrast to macrophages and monocytes, which frequently displayed large and dark phagocytic vacuoles, putative DC had only small, if any, lysosomal organelles. Putative DC were further characterized by their abundant Golgi apparatus and endoplasmic reticulum.

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• Figure 5. 

  Transmission electron microscopy of sorted putative dendritic cells. CD11c⁺/HLA-DR⁺/DM5⁻/CD14⁻ cells were sorted and examined by transmission electron microscopy as described in Materials and Methods. The purity of the sorted population was 97%. In contrast to macrophages, which frequently displayed large and dark phagocytic vacuoles (A; closed arrowhead), putative DC were characterized by long cytoplasmic processes (B,C), only small, if any, lysosomal organelles (B,C), and an abundant Golgi apparatus and endoplasmic reticulum (D). Multivesicular bodies (A and C; open arrowheads).

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Discussion 

Although the mouse has provided important insights into issues of hematopoiesis and HCT, there are serious limitations when extrapolating transplantation studies from mice to humans. In mice, for example, it is relatively easy to cross histocompatibility barriers, even including H2, without consistently encountering donor-graft rejection or fatal GVHD, and murine T cell-depleted (TCD) grafts are much easier to accomplish than those in dogs or humans [16, 17, 18]. In contrast, the random-bred dog model has been extremely useful for improving safety and our understanding of the biology of human HCT, because outcomes in this large animal model closely predict those in human transplant recipients.

More recent studies in mice suggest that DC initiate and regulate GVH and GVT reactions after allogeneic HCT [2, 3, 4]. It has been shown, for example, that (1) host and not donor antigen-presenting cells are required for the induction of GVHD in a model in which CD8 T cells interact with minor MHC molecules presented in the context of class I, and (2) that donor lymphocyte infusions mediate superior GVT effects in mixed compared to full donor chimeras. Both observations support the critical role of host antigen-presenting cells in initiating and regulating GVH responses after allogeneic HCT. Canine DC, however, are poorly characterized, and most of the published literature on canine DC is based on cells generated by ex vivo culture using combinations of hematopoietic growth factors including granulocyte-monocyte colony-stimulating factor (GM-CSF), tumor-necrosis-factor-α (TNF-α), FL, and interleukin (IL)-4 [19, 20, 21]. In this study, we therefore sought to identify and characterize potent antigen-presenting cells that occur in dogs in vivo to apply this knowledge to future studies in the canine HCT model.

In humans, DC comprise <1% of PBMCs, making the study of these cells difficult. Two phenotypically distinct DC subtypes have been described. Myeloid DC (or DC1) express HLA-DR, supposedly originate from myeloid marrow precursors and, therefore, express myeloid antigens including CD11c, and require the presence of GM-CSF for their survival [22, 23, 24]. Myeloid DC produce high levels of IL-12 when stimulated with TNF-α or CD40L, and drive T cell differentiation into Th1 [22]. Lymphoid DC (or DC2) have been described in human peripheral blood and lymphoid tissues as CD11c⁻/HLA-DR⁺/CD4⁺/IL-3Rα⁺ plasmacytoid cells. DC2 cells depend on IL-3 and not on GM-CSF for their survival and differentiation [24, 25]. They have been designated DC2 because, after appropriate activation, they can induce T cell differentiation into Th2 cells [22].

Applying the panel of canine-specific or crossreacting mAbs available to us, we found that cells with an immunophenotype similar to that of myeloid DC in humans (CD11c⁺/HLA-DR⁺/CD14⁻) were, under steady-state conditions, also extremely rare in the peripheral blood of dogs. To facilitate the isolation of these rare cells in numbers sufficient for further functional and morphologic characterization, dogs were treated with recombinant human FL, a hematopoietic growth factor known to crossreact with canine cells [21, 26]. FL has been shown to expand both the number of myeloid and lymphoid DC subsets in mice and humans [27, 28, 29]. Ten days of FL treatment resulted in a doubling of the total white blood cell (WBC) count, which was largely attributable to an approximately 10-fold increase in the number of CD14⁺ monocytes. Moreover, FL treatment led to the emergence of a distinct CD11c⁺/HLA-DR⁺/CD14⁻ cell population, a surface marker profile consistent with that of myeloid DC in humans. These putative DC were further defined by the absence of DM5 expression, a marker highly expressed on mature granulocytes of dogs.

As few as 5 × 10³ irradiated CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells elicited strong proliferative T cell responses in unidirectional, allogeneic MLC. At responder-stimulator ratios of 20:1, CD21⁺/HLA-DR⁺ B cells and CD14⁺/DM5⁻ monocytes were at least 20-fold less effective than putative DC in stimulating allogeneic T cells. CD11c^dim/HLA-DR⁺/CD14⁻/DM5⁻ were also strong stimulators in MLC (not shown), but further characterization of these cells as the possible equivalent of plasmacytoid DC in humans was limited by the lack of canine-specific reagents (ie, anti-IL-3Rα⁺). Transmission electron microscopy of CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells revealed a morphology similar to that described for myeloid DC in humans and for ex vivo-generated canine DC, including long cytoplasmic extensions, discrete lysosomes, and an abundant Golgi apparatus and endoplasmatic reticulum [20, 21, 30, 31].

Whether phenotype and function described for canine DC generated in vivo after FL treatment can be extrapolated to DC present under steady-state conditions remains unknown. The majority of FL-mobilized DC1 and DC2 in healthy human volunteers and cancer patients have been shown to be phenotypically and functionally more immature than their steady-state counterparts, which was evidenced by decreased IL-12 production and allostimulatory potential [32, 33, 34]. Their overall immunophenotype (CD11c⁺/HLA-DR⁺/CD14⁻), however, was not different. If FL treatment also resulted in expansion of relatively immature DC in dogs, the allostimulatory potential of their steady-state counterparts would be expected to be even greater. Thus, the use of FL-generated CD11c⁺/HLA-DR⁺/CD14⁻/DM5⁻ cells in our study might have resulted in an underestimation of the allostimulatory potential of steady-state DC in dogs.

In conclusion, the identification of highly potent antigen-presenting cells in the peripheral blood of dogs, which may represent the equivalent of myeloid DC in humans, will enable us to apply this knowledge to the design and interpretation of future studies in the canine HCT model.

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Acknowledgments 

We thank Beth Bell, Daniel Hirschstein, Jeff Smith, Jane Jin, and Julie Hill at Amgen Inc., Seattle, WA, for flow cytometric data collection and cell sorting support, and Lori Peterson and Norm Boiani for mouse anticanine CD11c antibody purification and biotin conjugation. Peter Moore kindly provided hybridomas for the production of anticanine CD3, CD4, and CD21 mAbs. We thank the staff and all investigators of the canine facility at FHCRC who provided excellent care for the dogs on this study, and Bobbie Schneider for performing the transmission electron microscopy. This work was supported in part by grants DK064715 of the National Institutes of Health, DHHS, Bethesda, MD, and by Amgen, Inc., Seattle, WA.

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References 

1. Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S. Antigen presentation and T cell stimulation by dendritic cells. (Review) Annu Rev Immunol. 2002;20:621–667
   • View In Article
   • MEDLINE
   • CrossRef
2. Shlomchik WD, Couzens MS, Tang CB, et al. Prevention of graft versus host disease by inactivation of host antigen-presenting cells. Science. 1999;285:412–415
   • View In Article
   • MEDLINE
   • CrossRef
3. Mapara MY, Kim YM, Marx J, Sykes M. Donor lymphocyte infusion-mediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graft-versus-leukemia effects after conversion to full donor chimerism. Transplantation. 2003;76:297–305
   • View In Article
   • MEDLINE
   • CrossRef
4. Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood. 2002;100:1903–1909
   • View In Article
   • MEDLINE
   • CrossRef
5. Deeg HJ, Storb R, Weiden PL, et al. Cyclosporin A and methotrexate in canine marrow transplantation: engraftment, graft-versus-host disease, and induction of tolerance. Transplantation. 1982;34:30–35
   • View In Article
   • MEDLINE
   • CrossRef
6. Yu C, Seidel K, Nash RA, et al. Synergism between mycophenolate mofetil and cyclosporine in preventing graft-versus-host disease among lethally irradiated dogs given DLA-nonidentical unrelated marrow grafts. Blood. 1998;91:2581–2587
   • View In Article
   • MEDLINE
7. Storb R, Yu C, Wagner JL, et al. Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation. Blood. 1997;89:3048–3054
   • View In Article
   • MEDLINE
8. Storb R, Kolb HJ, Deeg HJ, et al. Prevention of graft-versus-host disease by immunosuppressive agents after transplantation of DLA-nonidentical canine marrow. Bone Marrow Transplant. 1986;1:167–177
   • View In Article
   • MEDLINE
9. Lee RS, Kuhr CS, Sale GE, et al. FTY720 does not abrogate acute graft-versus-host disease in the DLA-nonidentical unrelated canine model. Transplantation. 2003;76:1155–1158
   • View In Article
   • MEDLINE
   • CrossRef
10. Sandmaier BM, Schuening FG, Bianco JA, et al. Biochemical characterization of a unique canine myeloid antigen. Leukemia. 1991;5:125–130
    • View In Article
    • MEDLINE
11. Georges GE, Storb R, Brunvand MW, et al. Canine T cells transduced with a herpes simplex virus thymidine kinase gene: a model to study effects on engraftment and control of graft-versus-host disease. Transplantation. 1998;66:540–544
    • View In Article
    • MEDLINE
    • CrossRef
12. McSweeney PA, Rouleau KA, Wallace PM, et al. Characterization of monoclonal antibodies that recognize canine CD34. Blood. 1998;91:1977–1986
    • View In Article
    • MEDLINE
13. Cobbold S, Metcalfe S. Monoclonal antibodies that define canine homologues of human CD antigens: summary of the First International Canine Leukocyte Antigen Workshop (CLAW). Tissue Antigens. 1994;43:137–154
    • View In Article
    • MEDLINE
    • CrossRef
14. Danilenko DM, Moore PF, Rossitto PV. Canine leukocyte cell adhesion molecules (LeuCAMs): characterization of the CD11/CD18 family. Tissue Antigens. 1992;40:13–21
    • View In Article
    • MEDLINE
    • CrossRef
15. Raff RF, Storb R, Ladiges WC, Deeg HJ. Recognition of target cell determinants associated with DLA-D-locus-encoded antigens by canine cytotoxic lymphocytes. Transplantation. 1985;40:323–328
    • View In Article
    • MEDLINE
    • CrossRef
16. Korngold R, Sprent J. Lethal graft-versus-host disease after bone marrow transplantation across minor histocompatibility barriers in mice (Prevention by removing mature T cells from marrow). J Exp Med. 1978;148:1687
    • View In Article
    • MEDLINE
    • CrossRef
17. Kolb HJ, Rieder I, Rodt B, et al. Antilymphocytic antibodies and marrow transplantation. Transplantation. 1979;27:242–245
    • View In Article
    • MEDLINE
    • CrossRef
18. Martin PJ, Hansen JA, Buckner CD, et al. Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts. Blood. 1985;66:664–672
    • View In Article
    • MEDLINE
19. Goodell EM, Blumenstock DA, Bowers WE. Canine dendritic cells from peripheral blood and lymph nodes. Vet Immunol Immunopathol. 1985;8:301–310
    • View In Article
    • MEDLINE
    • CrossRef
20. Ibisch C, Pradal G, Bach JM, Lieubeau B. Functional canine dendritic cells can be generated in vitro from peripheral blood mononuclear cells and contain a cytoplasmic ultrastructural marker. J Immunol Methods. 2005;298:175–182
    • View In Article
    • MEDLINE
    • CrossRef
21. Hägglund HG, McSweeney PA, Mathioudakis G, et al. Ex vivo expansion of canine dendritic cells from CD34⁺ bone marrow progenitor cells. Transplantation. 2000;70:1437–1442
    • View In Article
    • MEDLINE
    • CrossRef
22. Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999;283:1183–1186
    • View In Article
    • MEDLINE
    • CrossRef
23. Olweus J, BitMansour A, Warnke R, et al. Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin. Proc Natl Acad Sci USA. 1997;94:12551–12556
    • View In Article
24. Kohrgruber N, Halanek N, Groger M, et al. Survival, maturation, and function of CD11c- and CD11c+ peripheral blood dendritic cells are differentially regulated by cytokines. J Immunol. 1999;163:3250–3259
    • View In Article
    • MEDLINE
25. Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med. 1997;185:1101–1111
    • View In Article
    • MEDLINE
    • CrossRef
26. Yunusov MY, Georges GE, Storb R, et al. FLT3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease. Transplantation. 2003;75:933–940
    • View In Article
    • MEDLINE
27. Maraskovsky E, Pulendran B, Brasel K, et al. Dramatic numerical increase of functionally mature dendritic cells in FLT3 ligand-treated mice. (Review) Adv Exp Med Biol. 1997;417:33–40
    • View In Article
    • MEDLINE
28. Pulendran B, Lingappa J, Kennedy MK, et al. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol. 1997;159:2222–2231
    • View In Article
    • MEDLINE
29. O’Keeffe M, Hochrein H, Vremec D, et al. Effects of administration of progenipoietin 1, Flt-3 ligand, granulocyte colony-stimulating factor, and pegylated granulocyte-macrophage colony-stimulating factor on dendritic cell subsets in mice. Blood. 2002;99:2122–2130
    • View In Article
    • MEDLINE
    • CrossRef
30. Pelletier M, Tautu C, Landry D, Montplaisir S, Chartrand C, Perreault C. Characterization of human thymic dendritic cells in culture. Immunology. 1986;58:263–270
    • View In Article
    • MEDLINE
31. Makala LH, Nagasawa H. Dendritic cells: a specialized complex system of antigen presenting cells. (Review) J Vet Med Sci. 2002;64:181–193
    • View In Article
    • MEDLINE
    • CrossRef
32. Mosca PJ, Hobeika AC, Colling K, et al. Multiple signals are required for maturation of human dendritic cells mobilized in vivo with Flt3 ligand. J Leukoc Biol. 2002;72:546–553
    • View In Article
    • MEDLINE
33. Maraskovsky E, Daro E, Roux E, et al. In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood. 2000;96:878–884
    • View In Article
    • MEDLINE
34. Morse MA, Nair S, Fernandez-Casal M, et al. Preoperative mobilization of circulating dendritic cells by Flt3 ligand administration to patients with metastatic colon cancer. J Clin Oncol. 2000;18:3883–3893
    • View In Article
    • MEDLINE