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1.  Antigen-pulsed CD8α+ Dendritic Cells Generate an Immune Response after Subcutaneous Injection without Homing to the Draining Lymph Node  
Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8α homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8α+) subset induces tolerance, whereas the myeloid-derived (CD8α−) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4+ T cells in vivo, purified CD8α+ or CD8α− dendritic cells were injected subcutaneously into normal mice. In contrast to CD8α− dendritic cells, the CD8α+ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8α+ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide–major histocompatibility complex complexes to migratory host CD8α− dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8α+ and CD8α− dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8α− dendritic cells by CD8α+ dendritic cells.
PMCID: PMC2192915  PMID: 9927521
dendritic cell; T cell; antigen presentation; tolerance
2.  Borrelia burgdorferi-pulsed dendritic cells induce a protective immune response against tick-transmitted spirochetes. 
Infection and Immunity  1997;65(8):3386-3390.
Borrelia burgdorferi-pulsed dendritic cells and epidermal cells were able to initiate the production of anti-outer surface protein A (OspA) antibody in vitro with normal T and B cells from either BALB/c or C3H/HeJ mice. Inhibition of anti-B. burgdorferi antibody production was observed after 3 days, but not after 2 days, of exposure of the antigen-presenting cells to tumor necrosis factor alpha +/- granulocyte-macrophage colony-stimulating factor. Furthermore, splenic dendritic cells pulsed in vitro with live B. burgdorferi spirochetes and then adoptively transferred into naive syngeneic mice mediated a protective immune response against tick-transmitted spirochetes. This protection appeared not to be due to killing of spirochetes in the feeding ticks, since ticks fed to repletion on B. burgdorferi-pulsed dendritic cell-sensitized mice still harbored live spirochetes. Western blot analysis of the sera collected from dendritic cell-sensitized mice demonstrated that the mice responded to a limited set of B. burgdorferi antigens, including OspA, -B, and -C compared to control groups that either had received unpulsed dendritic cells or were not treated. Finally, mice in the early stage of B. burgdorferi infection were able to develop anti-OspA antibody following injection with B. burgdorferi-pulsed dendritic cells. Our results demonstrate for the first time that adoptive transfer of B. burgdorferi-pulsed dendritic cells induces a protective immune response against tick-transmitted B. burgdorferi and stimulates the production of antibodies specific for a limited set of B. burgdorferi antigens in vivo.
PMCID: PMC175479  PMID: 9234802
3.  Resting Respiratory Tract Dendritic Cells Preferentially Stimulate T Helper Cell Type 2 (Th2) Responses and Require Obligatory Cytokine Signals for Induction of  Th1 Immunity  
The Journal of Experimental Medicine  1998;188(11):2019-2031.
Consistent with their role in host defense, mature dendritic cells (DCs) from central lymphoid organs preferentially prime for T helper cell type 1 (Th1)-polarized immunity. However, the “default” T helper response at mucosal surfaces demonstrates Th2 polarity, which is reflected in the cytokine profiles of activated T cells from mucosal lymph nodes. This study on rat respiratory tract DCs (RTDCs) provides an explanation for this paradox. We demonstrate that freshly isolated RTDCs are functionally immature as defined in vitro, being surface major histocompatibility complex (MHC) II lo, endocytosishi, and mixed lymphocyte reactionlo, and these cells produce mRNA encoding interleukin (IL)-10. After ovalbumin (OVA)-pulsing and adoptive transfer, freshly isolated RTDCs preferentially stimulated Th2-dependent OVA-specific immunoglobulin (Ig)G1 responses, and antigen-stimulated splenocytes from recipient animals produced IL-4 in vitro. However, preculture with granulocyte/macrophage colony stimulating factor increased their in vivo IgG priming capacity by 2–3 logs, inducing production of both Th1- and Th2-dependent IgG subclasses and high levels of IFN-γ by antigen-stimulated splenocytes. Associated phenotypic changes included upregulation of surface MHC II and B7 expression and IL-12 p35 mRNA, and downregulation of endocytosis, MHC II processing– associated genes, and IL-10 mRNA expression. Full expression of IL-12 p40 required additional signals, such as tumor necrosis factor α or CD40 ligand. These results suggest that the observed Th2 polarity of the resting mucosal immune system may be an inherent property of the resident DC population, and furthermore that mobilization of Th1 immunity relies absolutely on the provision of appropriate microenvironmental costimuli.
PMCID: PMC2212375  PMID: 9841916
dendritic cell; lung; function; T helper cell type 1; T helper cell type 2
4.  Role of CD80 and CD86 in Host Immune Responses to the Recombinant Hemagglutinin Domain of Porphyromonas gingivalis Gingipain and in the Adjuvanticity of Cholera Toxin B and Monophosphoryl Lipid A 
Vaccine  2007;25(33):6201-6210.
The gingipains of Porphyromonas gingivalis have been implicated in the virulence of this bacterium, and antibodies to the hemagglutinin/adhesin domain (HArep) of the gingipains have been shown to protect against P. gingivalis colonization. However, the cellular mechanisms involved in host responses to HArep have not been elucidated. The purpose of the present study was to determine the functional role of CD80 and CD86 in mediating systemic and mucosal immune responses to the recombinant HArep derived from the gingipain Kgp (Kgp-HArep) after intranasal (i.n.) immunization. We also investigated the effect of the mucosal adjuvants the B subunit of cholera toxin (CTB) and monophosphoryl lipid A (MPL) on the functional role of the costimulatory molecules for the induction of systemic and mucosal responses to Kgp-HArep. The in vivo functional roles of CD80 and CD86 were assessed in C57BL/6 wild-type (wt), CD80-/-, CD86-/- and CD80/CD86-/- mice following intranasal immunization with Kgp-HArep with or without adjuvant. Serum IgG and mucosal IgA antibody responses were induced following i.n. immunization of mice with Kgp-HArep, and were potentiated by CTB or MPL. A differential requirement of CD80 and/or CD86 was observed for systemic IgG anti-Kgp-HArep responses following the primary and secondary immunization with antigen alone or antigen + adjuvant. Compared to wt and CD80-/- mice, CD86-/- mice had reduced serum IgG anti-Kgp-HArep responses following the second immunization with antigen alone or antigen + CTB, whereas similar levels of serum IgG anti-Kgp-HArep antibody activity were observed in wt, CD80-/- and CD86-/- mice immunized with antigen + MPL. Analysis of the serum IgG subclass responses revealed that CD80 influenced both Th1- and Th2-like IgG subclass responses, while CD86 preferentially influenced a Th2-associated IgG subclass response to Kgp-HArep. Mucosal IgA anti-Kgp-HArep responses in saliva and vaginal washes were diminished in CD86-/- mice. In vitro stimulation of murine bone marrow-derived dendritic cells with Kgp-HArep, CTB and MPL resulted in an up-regulation of CD80 and especially CD86 expression. Taken together, our results demonstrate that CD80 and CD86 can play distinct as well as redundant roles in mediating a systemic immune response and that CD86 plays a unique role in mediating a mucosal response to Kgp-HArep following immunization via the i.n. route alone or with adjuvant.
doi:10.1016/j.vaccine.2007.05.066
PMCID: PMC2699271  PMID: 17629367
Costimulatory molecules; Porphyromonas gingivalis gingipain; mucosal immunization; mucosal adjuvants
5.  Dendritic cell progenitors phagocytose particulates, including bacillus Calmette-Guerin organisms, and sensitize mice to mycobacterial antigens in vivo 
Dendritic cells, while effective in sensitizing T cells to several different antigens, show little or no phagocytic activity. To the extent that endocytosis is required for antigen processing and presentation, it is not evident how dendritic cells would present particle-associated peptides. Evidence has now been obtained showing that progenitors to dendritic cells can internalize particles, including Bacillus Calmette-Guerin (BCG) mycobacteria. The particulates are applied for 20 h to bone marrow cultures that have been stimulated with granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce aggregates of growing dendritic cells. Cells within these aggregates are clearly phagocytic. If the developing cultures are exposed to particles, washed, and "chased" for 2 d, the number of major histocompatibility complex class II-rich dendritic cells increases substantially and at least 50% contain internalized mycobacteria or latex particles. The mycobacteria-laden, newly developed dendritic cells are much more potent in presenting antigens to primed T cells than corresponding cultures of mature dendritic cells that are exposed to a pulse of organisms. A similar situation exists when the BCG- charged dendritic cells are injected into the footpad or blood stream of naive mice. Those dendritic cells that have phagocytosed organisms induce the strongest T cell responses to mycobacterial antigens in draining lymph node and spleen. The administration of antigens to GM- CSF-induced, developing dendritic cells (by increasing both antigen uptake and cell numbers) will facilitate the use of these antigen- presenting cells for active immunization in situ.
PMCID: PMC2191128  PMID: 7688024
6.  Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo 
PLoS ONE  2014;9(7):e101644.
Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors.
doi:10.1371/journal.pone.0101644
PMCID: PMC4109917  PMID: 25058148
7.  Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines  
Antigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic murine tumor models that bone marrow (BM)- derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J.I., T. Zorina, W.J. Storkus, C. Celluzzi, L.D. Falo, C.J. Melief, T. Ildstad, W.M. Kast, A.B. DeLeo, and M.T. Lotze. 1995. Nature Med. 1:1297-1302). However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony- stimulating factor-stimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W.J., H.J. Zeh III, R.D. Salter, and M.T. Lotze. 1993. J. Immunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 x 10(5) tumor peptide-pulsed DC dramatically suppressed the growth of weakly immunogenic tumors in day 4 to day 8 established MCA205 (H-2b) and TS/A (H-2d) tumor models, when applied in three biweekly intravenous injections. Using the immunogenic C3 (H-2b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4+ or CD8+ T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor- derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) gamma production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DC- induced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1- associated cytokines (such as IL-12, tumor necrosis factor alpha, IFN- gamma), and eventually, but not initially, blocked by anti-mIL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weakly immunogenic tumors, and serves as a basis for designing clinical trials in cancer patients.
PMCID: PMC2192415  PMID: 8551248
8.  Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions 
The Journal of Experimental Medicine  2009;206(13):3101-3114.
Chemokine receptor CX3CR1+ dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103+ DCs in the mucosa, which, through their ability to synthesize retinoic acid (RA), appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103+ DC, CX3CR1+ cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103+ DCs, but not CX3CR1-expressing cells, migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover, CX3CR1+ cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103+ DC. These findings indicate that selectively CD103+ DCs serve classical DC functions and initiate adaptive immune responses in local LNs, whereas CX3CR1+ populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens.
doi:10.1084/jem.20091925
PMCID: PMC2806467  PMID: 20008524
9.  CD4+ CD25+ Regulatory T Cells Control T Helper Cell Type 1 Responses to Foreign Antigens Induced by Mature Dendritic Cells In Vivo 
Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II–restricted interferon γ–producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.
doi:10.1084/jem.20030654
PMCID: PMC2194073  PMID: 12874259
primary response; T helper cell type 1/type 2 balance; regulation; inflammation; Toll-like receptors
10.  Freshly Isolated Peyer's Patch, but Not Spleen, Dendritic Cells Produce Interleukin 10 and Induce the Differentiation of T Helper Type 2 Cells 
Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c+ DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98–100% pure CD11c+ DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide–specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-γ (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-γ production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.
PMCID: PMC2195574  PMID: 10432286
antigen presentation; cytokine; mucosal immunity
11.  T cells that are naturally tolerant to cartilage-derived type II collagen are involved in the development of collagen-induced arthritis 
Arthritis Research  2000;2(4):315-326.
The immunodominant T-cell epitope that is involved in collagen-induced arthritis (CIA) is the glycosylated type II collagen (CII) peptide 256-270. In CII transgenic mice, which express the immunodominant CII 256-270 epitope in cartilage, the CII-specific T cells are characterized by a partially tolerant state with low proliferative activity in vitro, but with maintained effector functions, such as IFN-γ secretion and ability to provide B cell help. These mice were still susceptible to CIA. The response was mainly directed to the glycosylated form of the CII 256-270 peptide, rather than to the nonglycosylated peptide. Tolerance induction was rapid; transferred T cells encountered CII within a few days. CII immunization several weeks after thymectomy of the mice did not change their susceptibility to arthritis or the induction of partial T-cell tolerance, excluding a role for recent thymic emigrants. Thus, partially tolerant CII autoreactive T cells are maintained and are crucial for the development of CIA.
Introduction:
A discussion is ongoing regarding the possible role of cartilage-directed autoimmunity as a part of the pathogenesis of rheumatoid arthritis (RA). One possibility is that the association of RA with shared epitope-expressing DR molecules reflects a role for major histocompatibility complex (MHC) class II molecules as peptide receptors, and that the predilection of the inflammatory attack for the joint indicates a role for cartilage as a source of the antigenic peptides. A direct role for CII in the development of arthritis is apparent in the CIA model, in which a definite role for MHC class II molecules and a role for CII-derived peptides have been demonstrated [1,2,3]. Remarkably, it was found that the identified MHC class II molecule in the CIA model Aq has a structurally similar peptide binding pocket to that of the shared epitope, expressing DR4 molecules [4]. In fact, DR4 (DRB1*0401) and DR1 (DRB1*0101) transgenic mice are susceptible to CIA because of an immune response to a peptide that is almost identical to that which is involved in Aq-expressing mice [5,6]. They are both derived from position 260-273 of the CII molecule; the peptide binds to the Aqmolecule with isoleucine 260 in the P1 pocket, but with phenylalanine 263 in the P1 pocket of the DR4 and DR1 molecules.
Although these findings do not prove a role for CII in RA, they show that such recognition is possible and that there are structural similarities when comparing mouse with human. However, there are also strong arguments against such a possibility. First, arthritis can evolve without evidence for a cartilage-specific autoimmunity, as seen with various adjuvant-induced arthritis models [7,8] and in several observations using transgenic animals with aberrant immunity to ubiquitously expressed proteins [9,10,11]. Moreover, the MHC association in the adjuvant arthritis models correlates with severity of the disease rather than susceptibility [7,8], as has also been observed in RA [12]. Second, it has not been possible to identify the CII-reactive T cells from RA joints, or to achieve a strong and significant CII proliferative response from T cells derived from RA joints. Most recently these negative observations were corroborated using DR4+CII peptide tetramer reagents [13]. On the other hand, it has also been difficult to isolate autoreactive CII-specific T cells from CIA, and it can be anticipated that, even in the CIA model, T cells that are specific for CII will be hard to find in the joints [4].
We believe that the explanations for these observations in both experimental animals and humans are related to tolerance. The CIA model in the mouse is usually induced with heterologous CII, and is critically dependent on an immune response to the glycosylated CII peptide 256-270, which is bound to the MHC class II Aq molecule. In CII transgenic mice, expressing the heterologous (rat) form of the immunodominant CII 256-270 epitope in cartilage, we observed partial T-cell tolerance. This tolerance is characterized by a low proliferative activity, but with maintained effector functions such as production of IFN-γ and the ability to give help to B cells to produce anti-CII IgG antibodies [14]. Interestingly, these mice were susceptible to arthritis. However, a possibility was that T cells that had newly emerged from the thymus and that were not yet tolerized when the mice were immunized with CII led to the induction of arthritis. We have now addressed this possibility and found that induction of tolerance occurs within a few days, and that mice lacking recent thymic emigrants (ie thymectomized mice) display partially tolerant T cells and susceptibility to arthritis to the same extent as nonthymectomized mice. In addition we found that T cells that are reactive with the nonmodified peptides are relatively more affected by tolerance than T cells that are reactive with the more immunodominant glycosylated variants.
Objectives:
To investigate the possibility that T cells that are naturally tolerant to the cartilage protein CII are involved in the development of arthritis, and to exclude a role for nontolerized recent thymic T-cell emigrants in the development of arthritis.
Materials and methods:
A mutated mouse CII, expressing glutamic acid instead of aspartic acid at position 266, was expressed in a transgenic mouse called MMC (mutated mouse collagen) that has been described earlier [14]. The mice were thymectomized, or sham-operated, at 7 weeks of age and allowed to recover for 4 weeks before being immunized with rat CII in complete Freund's adjuvant. Arthritis development was recorded and sera analyzed for anti-CII IgG, IgG1 and IgG2a levels. To assay T-cell effector functions, other MMC and control mice were immunized in the hind footpads with rat CII in complete Freund's adjuvant, and the draining popliteal lymph nodes were taken 10 days later. The lymph node cells (LNCs) were used for proliferation assay, IFN-γ enzyme-linked immunosorbent assay (ELISA) and B-cell enzyme-linked immunospot (ELISPOT). For the proliferation assay, 106 cells were put in triplicate cultures in microtitre wells together with antigen and incubated for 72h before thymidine-labelling and harvesting 15-18h later. For IFN-γ ELISA analysis, supernatant from the proliferation plates was removed before harvesting and used in an ELISA to quantify the amount of IFN-γ produced [15]. B-cell ELISPOT was performed to enumerate the number of cells producing anti-CII IgG [16].
T-cell lines that were reactive towards rat CII were established by immunization with rat CII. An established T-cell line that was reactive with CII and specific for the CII 256-270 peptide was restimulated with freshly collected, irradiated, syngenic spleen cells and rat CII for 3 days followed by 2 weeks of IL-2 containing medium. Immediately before transfer, the cells were labelled with the cytoplasmic dye 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) [17]. Labelled cells (107) were injected intravenously into transgenic MMC mice and nontransgenic littermates. The mice were killed 4 days after cell transfer, and the concentration of CFSE-labelled cells was determined by flow cytometry.
Results and discussion:
To investigate whether and how quickly CII-reactive T cells will encounter CII in vivo, an established T-cell line that is reactive towards rat CII was labelled with the cytoplasmic dye CFSE and transferred into MMC-QD and control mice. Four days later the mice were killed, and it was found that MMC-transgenic mice had dramatically fewer CFSE-labelled cells in the spleen than did nontransgenic littermates (0.11% compared with 0.57%). Similarly, reduced numbers of CFSE-positive cells were observed in blood. This indicates that the T cells encountered the mutated CII that was present in the cartilage of MMC mice, but not in the nontransgenic littermates. Presumably, CII from cartilage is spread by antigen-presenting cells (APCs) to peripheral lymphoid organs. This observation also suggests that newly exported T cells from the thymus will be tolerized to CII in the periphery within less than 4 days.
To further investigate whether the MMC mice harbours naïve or tolerized T cells, the mice were immunized with CII at different time points after thymectomy that were well in excess of the times required for their encounter with CII. After 10 days, the response was analyzed in vitro towards both the nonglycosylated and the glycosylated CII 256-270 peptides as well as towards purified protein derivative. The galactosylated form of the peptide (Fig. 1) was used because this is the most immunodominant modification [18]. In contrast to control mice, LNCs from transgenic mice did not proliferate significantly towards the nonglycosylated peptide, indicating that these cells have been specifically tolerized, which is in accordance with earlier observations [14]. A reduced, but still significant proliferation was also observed toward the immunodominant glycosylated CII peptide. Most important, however, was that the proliferative response in the MMC mice did not decrease after thymectomy. Similarly, a significant IFN-γ production towards the glycosylated CII peptide was observed in the MMC mice. The response was somewhat reduced compared with that observed in nontransgenic littermates, and this was especially true for the response toward the nonglycosylated peptide. Again, no decrease in the MMC response by thymectomy was observed. Taken together, the T-cell response in transgenic mice was reduced in comparison with that in the nontransgenic littermates. Furthermore, the response in transgenic animals did not decrease by thymectomy (4 or 8 weeks before immunization), showing that autoreactive T cells are still maintained (and partially tolerized) with significant effector functions at least up to 8 weeks after thymectomy, excluding a exclusive role for recent thymic emigrants in the autoimmune response towards CII. To investigate whether thymectomized mice, lacking recent CII-specific thymic emigrants, were susceptible to CIA, mice were immunized with CII 4 weeks after thymectomy and were observed for arthritis development during the following 10 weeks. Clearly, the thymectomized MMC mice were susceptible to arthritis (five out of 18 developed arthritis; Fig. 2), and no significant differences in susceptibility between thymectomized and sham-operated mice, or between males and females, were seen. In accordance with earlier results [14], MMC transgenic mice had a significantly reduced susceptibility to arthritis as compared with the nontransgenic littermates (P < 0.0001 for arthritic scores, disease onset and incidence). All mice were bled at 35 days after immunization, and the total levels of anti-CII IgG were determined. Transgenic mice developed levels of anti-CII IgG significantly above background, but the antibody titres were lower than in nontransgenic littermates (P < 0.0001). No effect on the antibody levels by thymectomy was observed, nor did thethymectomy affect the distribution of IgG1 versus IgG2a titres,indicating that the observed tolerance is not associated with a shift from a T-helper-1- to a T-helper-2-like immune response. These findings show that T cells that are specific for a tissue-specific matrix protein, CII, are partially tolerized within a few days after thymus export and that these tolerized cells are maintained after thymectomy. Most important, mice that lack newly exported CII reactive T cells are still susceptible to CIA, suggesting that the partially tolerant T cells are involved in development of arthritis.
In the light of these data it is possible to explain some of the findings in RA. T-cell reactivity to CII has been shown in RA patients, but with a very weak proliferative activity [19,20]. This is fully compatible with observations in mouse and rat CIA when autologous CII, and not heterologous CII, are used for immunization. This is particularly true if the responses are recorded during the chronic phase of disease, in which the antigen-specific T-cell responses seem to be suppressed in both humans and experimental animals. These observations were confirmed in a recent report [21] in which it was shown that CII-reactive T-cell activity could be detected in RA patients if IFN-γ production but not proliferation was measured. In the present studies in mice the strongest response is seen towards post-translational modifications of the peptide. Because the T-cell contact points are the same whether the peptide is bound to DR4 or to Aq, it is fully possible that post-translational modifications of the peptide also plays a significant role in humans [22]. The fact that IgG antibodies specific for CII are found in many RA patients could be explained by maintained B-cell helper functions of CII-reactive T cells. In fact, it has been reported [23,24] that the occurrence of IgG antibodies to CII is associated with shared epitope DR4 molecules. These observations are thus compatible with a role for CII reactivity in RA. To avoid any confusion, it needs to be stressed that RA is a heterogeneous syndrome in which not only CII, but also other cartilage proteins and other mechanisms are of importance. Such a pathogenic heterogeneity is reflected by the multitude of experimental animal models that have demonstrated how many different pathways may lead to arthritis [25].
PMCID: PMC17814  PMID: 11056672
autoimmunity; rheumatoid arthritis; T lymphocytes; tolerance; transgenic
12.  In Vivo Detection of Dendritic Cell Antigen Presentation to CD4+ T Cells 
The Journal of Experimental Medicine  1997;185(12):2133-2141.
Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4+ T cells specific for an OVA peptide–I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.
PMCID: PMC2196354  PMID: 9182685
13.  Humoral response to Porphyromonas (Bacteroides) gingivalis in rats: time course and T-cell dependence. 
Infection and Immunity  1992;60(9):3579-3585.
In this study, we describe the time course and T-cell dependence of the serum antibody response to the periodontopathogen Porphyromonas (Bacteroides) gingivalis in an experimental rat model. Normal Fischer rats were challenged by a local injection of P. gingivalis (2 x 10(8) bacteria) into gingival tissue or by the administration of a similar number of bacteria by the intravenous (i.v.) route on days 0, 2, and 4. Serum antibody activity was detected within 1 week and peaked at 8 weeks after gingival challenge. A similar but lower response was seen for rats challenged by the i.v. route. The response in both groups of rats was mainly of the immunoglobulin G (IgG) isotype; some IgM but no IgA antibody activity was detected. Analysis of the IgG subclass revealed mainly IgG2c in animals challenged locally in the gingiva with P. gingivalis, whereas IgG2b predominated in rats challenged by the i.v. route. The importance of T cells in the response was established by demonstrating the absence of serum IgG antibodies in nude rats after a local challenge of gingival tissue with P. gingivalis. Nude rats given purified splenic T cells from normal rats immunized systemically with P. gingivalis prior to a local gingival challenge showed a rapid appearance of serum antibody activity that peaked between 4 and 6 weeks. This initial peak occurred 2 to 4 weeks earlier than that seen in normal animals. Fluorescence-activated cell sorter analysis of splenic lymphoid cells from these nude rats revealed a helper T-cell population. The levels of serum IgG antibodies in nude rats given nonimmune T cells rose slowly, and the antibodies were mainly of the IgG2a and IgG2b subclasses. Nude rats given immune T cells showed a rapid increase primarily in IgG2b antibody levels following a local gingival challenge. These findings suggest that the immune helper T-cells contributed to the rapid development of the response to P. gingivalis. Furthermore, it is likely that the IgG subclass response to P. gingivalis in these nude rats was related to the splenic origin of the T cells used for adoptive transfer.
PMCID: PMC257364  PMID: 1323534
14.  Flexibility of Mouse Classical and Plasmacytoid-derived Dendritic Cells in Directing T Helper Type 1 and 2 Cell Development 
Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4+ T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.
doi:10.1084/jem.20021908
PMCID: PMC2193804  PMID: 12515817
dendritic cell; Th1; Th2; TLR; cytokines
15.  H-2 restriction of virus-specific T-cell-mediated effector functions in vivo. II. Adoptive transfer of delayed-type hypersensitivity to murine lymphocytic choriomeningits virus is restriced by the K and D region of H-2 
In mice, primary footpad swelling after local infection with lymphocytic choriomeningitis virus (LCMV) and delayed-type hypersensitivity (DTH) adoptively transferred by LCMV immune lymphocytes are T-cell dependent. Nude mice do not develop primary footpad swelling, and T-cell depletion abrogates the capacity to transfer LCMV-specific DTH. Effector T cells involved in eliciting dose- dependent DTH are virus specific in that vaccinia virus-immune lymphocytes could not elicit DTH in LCMV-infected mice. The adoptive transfer of DTH is restricted to H-2K or H-2D compatible donor- recipient combinations. Distinct from the fowl-gamma-globulin DTH model, I-region compatibility is neither necessary nor alone sufficient. Whatever the mechanisms involved in this K- or D-region associated restriction in vivo, it most likely operates at the level of T-cell recognition of "altered self" coded in K or D. T cells associated with the I region (helper T cells and DTH-T cells to fowl- gamma-globulin) are specific for soluble, defined, and inert antigens. T cells associated with the K and D region (T cells cytotoxic in vitro and in vivo for acute LCMV-infected cells, DTH effector T cells, and anti-viral T cells) are specific for infectious, multiplying virus. The fact that T-cell specificity is differentially linked with the I region or with the K and D regions of H-2 may reflect the fundamental biological differences of these antigens. Although it cannot be excluded that separate functional subclasses of T-effector cells could have self-recognizers for different cell surface structures coded in I or K and D, it is more likely that the antigen parameters determine whether T cells are specific for "altered" I or "altered" K- or D-coded structures.
PMCID: PMC2190399  PMID: 1085340
16.  Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness▿ †  
Infection and Immunity  2009;77(3):1165-1174.
Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.
doi:10.1128/IAI.01129-08
PMCID: PMC2643653  PMID: 19139189
17.  Mechanisms of acquired thymic tolerance in experimental autoimmune encephalomyelitis: thymic dendritic-enriched cells induce specific peripheral T cell unresponsiveness in vivo 
Experimental autoimmune encephalomyelitis (EAE), an experimental model for the study of multiple sclerosis, is an autoimmune disease of the central nervous system that can be induced in a number of species by immunization with myelin basic protein (MBP). MBP-reactive CD4+ T cells, predominantly expressing the V beta 8.2 T cell receptor (TCR), migrate from the peripheral lymphoid organs and initiate the inflammatory response in the brain. We have previously shown that a single intrathymic injection of MBP or its major encephalitogenic peptide (p71-90), but not a nonencephalitogenic peptide (p21-40), induces antigen-specific systemic tolerance and inhibits the induction of EAE in Lewis rats. In this study, we investigated the mechanisms of induction and maintenance of acquired thymic tolerance in this model. First, we investigated which thymic cell is responsible for "induction" of systemic tolerance. Thymic dendritic-enriched cells, isolated by plastic adherence, when incubated in vitro with p71-90 and injected intravenously into Lewis rats, were capable of preventing the development of EAE, but his protection was lost in thymectomized recipients. In addition, intravenous injection of thymic dendritic cells isolated from animals that had been previously injected intrathymically with p71-90 but not p21-40 also prevented the development of EAE. Second, to determine the "effector" mechanisms involved in acquired thymic tolerance, we compared TCR expression in the brains of animals with actively induced EAE with TCR expression in animals that received intrathymic injection of p71-90 or p21-40. Using a semiquantitative reverse transcription-polymerase chain reaction (RT- PCR) technique, we found increased expression of CD4 and V beta 8.2 message in brains of immunized animals compared with those of naive animals. In animals intrathymically injected with p71-90 but not p21- 40, CD4 and V beta 8.2 transcript levels were significantly reduced compared with immunized controls. Immunohistologic studies of brain tissue and spleens with specific V beta 8.2 and control V beta 10 monoclonal antibodies confirmed these observations in vivo. These findings, taken together with recent data demonstrating that activated T cells circulate through the thymus, suggest that interaction of thymic dendritic cells with specific TCR of activated peripheral T cells can lead to inactivation of these antigen-specific cells and confirm the role of V beta 8.2-expressing T cells in EAE.
PMCID: PMC2192120  PMID: 7543136
18.  Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs 
Journal of Clinical Pathology  2000;53(7):518-524.
Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues.
Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared.
Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels.
Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.
Key Words: dendritic cell • Escherichia coli • immunohistochemistry
doi:10.1136/jcp.53.7.518
PMCID: PMC1731227  PMID: 10961175
19.  Dendritic Cells Infiltrating Tumors Cotransduced with Granulocyte/Macrophage Colony-Stimulating Factor (Gm-Csf) and Cd40 Ligand Genes Take up and Present Endogenous Tumor-Associated Antigens, and Prime Naive Mice for a Cytotoxic T Lymphocyte Response 
We transduced BALB/c-derived C-26 colon carcinoma cells with granulocyte/macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (CD40L) genes to favor interaction of these cells with host dendritic cells (DCs) and, therefore, cross-priming. Cotransduced cells showed reduced tumorigenicity, and tumor take was followed by regression in some mice. In vivo tumors were heavily infiltrated with DCs that were isolated, phenotyped, and tested in vitro for stimulation of tumor-specific cytotoxic T lymphocytes (CTLs). BALB/c C-26 carcinoma cells express the endogenous murine leukemia virus (MuLV) env gene as a tumor-associated antigen. This antigen is shared among solid tumors of BALB/c and C57BL/6 mice and contains two epitopes, AH-1 and KSP, recognized in the context of major histocompatibility complex class I molecules H-2Ld and H-2Kb, respectively. DCs isolated from C-26/GM/CD40L tumors grown in (BALB/c × C57BL/6)F1 mice (H-2d×b) stimulated interferon γ production by both anti–AH-1 and KSP CTLs, whereas tumor-infiltrating DCs (TIDCs) of BALB/c mice stimulated only anti–AH-1 CTLs. Furthermore, TIDCs primed naive mice for CTL activity as early as 2 d after injection into the footpad, whereas double-transduced tumor cells required at least 5 d for priming; this difference may reflect direct DC priming versus indirect tumor cell priming. Immunohistochemical staining indicated colocalization of DCs and apoptotic bodies in the tumors. These data indicate that DCs infiltrating tumors that produce GM-CSF and CD40L can capture cellular antigens, likely through uptake of apoptotic bodies, and mature in situ to a stage suitable for antigen presentation. Thus, tumor cell–based vaccines engineered to favor the interaction with host DCs can be considered.
PMCID: PMC2195555  PMID: 10429676
dendritic cells; cross-priming; granulocyte/macrophage colony-stimulating factor; CD40 ligand; tumor antigens
20.  Functional subclasses of T-lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen 
The Journal of Experimental Medicine  1975;141(6):1376-1389.
Ly alloantigens coded by two unlinked genetic loci (Ly-1 and Ly-2/Ly-3) are expressed on lymphoid cells undergoing thymus-dependent differentiation. Peripheral Thy-1+ cells from C57BL/6 mice can be divided into three subclasses on the basis of differential expression of Ly-1, Ly-2, and Ly-3; about 50% express all three Ly antigens (Ly - 123+), about 33% only Ly-1 (Ly-1+), and about 6-8% Ly-2 and Ly-3 (Ly- 23+). Cells of the Ly-123+ subclasses are the first peripheral Thy-1+ cells to appear in ontogeny, and are reduced in the periphery shortly after adult thymectomy. In contrast, Ly-1+ and Ly-23+ subclasses appear later in the peripheral tissues than do Ly-123+ cells, and are resistant to the early effects of adult thymectomymperiheral lymphoid populations depleted of Ly-1+ cells and Ly-123+ cells (and thereby enriched for Ly-23+ cells) were incapable of developing significant helper activity to SRBC but generated substantial levels of cytotoxic activity to allogeneic target cells. The same lymphoid populations, depleted of Ly-23+ cells and Ly-123+ cells (and thereby enriched for Ly- 1+ cells), produced substantial helper responses but were unable to generate appreciable levels of killer activity. These experiments imply that commitment of T cells to participate exclusively in either helper or cytotoxic function is a differentiative process that takes place before they encounter antigen, and is accompanied by exclusion of different Ly groups, Lu-23 or Ly-1 respectively, from TL+Ly-123+ T-cell precursors. It is yet to be decided whether the TL-phase by Ly-123+ subclass is a transitional form or a separately differentiated subclass with a discrete immunologic function.
PMCID: PMC2189856  PMID: 1092798
21.  The effect of antigen dose on CD4+ T helper cell phenotype development in a T cell receptor-alpha beta-transgenic model 
The Journal of Experimental Medicine  1995;182(5):1579-1584.
The dose of foreign antigen can influence whether a cell-mediated or humoral class of immune response is elicited, and this may be largely accounted for by the development of CD4+ T helper cells (Th) producing distinct sets of cytokines. The ability of antigen dose to direct the development of a Th1 or Th2 phenotype from naive CD4+ T cells, however, has not been demonstrated. In this report, we show that the antigen dose used in primary cultures could directly affect Th phenotype development from naive DO11.10 TCR-alpha beta-transgenic CD4+ T cells when dendritic cells or activated B cells were used as the antigen- presenting cells. Consistent with our previous findings, midrange peptide doses (0.3-0.6 microM) directed the development of Th0/Th1-like cells, which produced moderate amounts of interferon gamma (IFN-gamma). As the peptide dose was increased, development of Th1-like cells producing increased amounts of IFN-gamma was initially observed. At very high (> 10 microM) and very low (< 0.05 microM) doses of antigenic peptide, however, a dramatic switch to development of Th2-like cells that produced increasing amounts of interleukin 4 (IL-4) and diminishing levels of IFN-gamma was observed. This was true even when highly purified naive, high buoyant density CD4+ LECAM-1hi T cells were used, ruling out a possible contribution from contaminating "memory" phenotype CD4+ T cells. Neutralizing anti-IL-4 antibodies completely inhibited the development of this Th2-like phenotype at both high and low antigen doses, demonstrating a requirement for endogenous IL-4. Our findings suggest that the antigen dose may affect the levels of endogenous cytokines such as IL-4 in primary cultures, resulting in the development of distinct Th cell phenotypes.
PMCID: PMC2192218  PMID: 7595228
22.  Interleukin-12 Production Is Required for Chlamydial Antigen-Pulsed Dendritic Cells To Induce Protection against Live Chlamydia trachomatis Infection 
Infection and Immunity  1999;67(4):1763-1769.
Immunization with dendritic cells pulsed ex vivo with antigens has been successfully used to elicit primary antigen-specific immune responses. We report that mouse bone marrow-derived dendritic cells pulsed with inactivated chlamydial organisms induced strong protection against live chlamydial infection in a mouse lung infection model. Either the dendritic cells or chlamydial organisms alone or macrophages similarly pulsed with chlamydial organisms failed to induce any significant protection. These observations suggest that dendritic cells can efficiently process and present chlamydial antigens to naive T cells in vivo. Mice immunized with the chlamydia-pulsed dendritic cells preferentially developed a Th1 cell-dominant response while mice immunized with the other immunogens did not, suggesting a correlation between a Th1 cell-dominant response and protection against chlamydial infection. We further found that dendritic cells produced a large amount of interleukin 12 (IL-12) upon ex vivo pulsing with inactivated chlamydial organisms, which may allow the dendritic cells to direct a Th1 cell-dominant response. Dendritic cells from mice deficient in the IL-12 p40 gene failed to produce IL-12 after a similar ex vivo pulse with chlamydial organisms, and more importantly, immunization with these dendritic cells failed to induce a Th1 cell-dominant response and did not induce strong protection against chlamydial infection. Thus, the ability of dendritic cells to efficiently process and present chlamydial antigens and to produce IL-12 upon chlamydial-organism stimulation are both required for the induction of protection against chlamydial infection. This information may be useful for the further design of effective chlamydial vaccines.
PMCID: PMC96526  PMID: 10085016
23.  In vivo modulation of CD1 and MHC class II expression by sheep afferent lymph dendritic cells. Comparison of primary and secondary immune responses 
The Journal of Experimental Medicine  1989;170(4):1303-1318.
The experiments described in this article characterize the phenotypic and functional changes in afferent lymph cell populations that occur as a result of in vivo immune stimulation. During the primary immune response (in antigen-naive sheep) there are very transient increases in level of CD1 expression by subpopulations of dendritic cells (DC) but no alterations in cell kinetics or MHC class II expression. In contrast, secondary antigenic challenge (in primed sheep) into the drainage area of an afferent lymphatic causes profound changes in the cell output, characterized by a greater than threefold drop in total cell output on days 1-3 followed by an approximate fivefold rise on day 5. There is also a substantial increase in both the proportion of MHC class II-positive T lymphocytes (from 28 to 54%) and in the quantitative expression of class II by both DC and lymphocytes. Class II expression by DC increases five- to sixfold by day 5, while the level of expression of class II on lymphocytes approximately doubles. The increase in CD1 expression during the secondary response is more prolonged than during the primary response, being detectable between days 2 and 6 after challenge. The rise in class II affects the whole DC population, in contrast to CD1 where the increase affects only a subpopulation of cells. In terms of functional properties, afferent lymph DC isolated during a primary response show no alteration of their activity, whereas DC taken 4-5 d after secondary challenge are up to fivefold more active in their ability to present soluble antigen to primed autologous T cells and to antigen-specific cell lines as well as to stimulate in the MLR. The relative expression of class II correlates temporally with an increased capacity of DC to present antigen. Monoclonal anti-class II antibodies totally inhibit the in vitro assays but anti-CD1 antibodies have no effect. The previous paper has demonstrated that afferent DC can associate with antigen in vivo and can present that antigen to antigen-specific T cells. This article extends our knowledge of DC biology and demonstrates that DC, activated during secondary in vivo immune responses, have an enhanced ability to present an antigen, unrelated to that used for challenge, to specific T cell lines. This enhancement correlates directly with quantitative variation of expressed class II and not CD1 and suggests that this variation in class II expression plays a physiological role in in vivo immune regulation.
PMCID: PMC2189469  PMID: 2477489
24.  Allosuppressor and allohelper T cells in acute and chronic graft-vs.- host disease. II. F1 recipients carrying mutations at H-2K and/or I-A 
By induction of a graft-vs.-host reaction (GVHR) in nonirradiated H-2- different F1 mice, one can induce stimulatory pathological symptoms, such as lymphadenopathy and hypergammaglobulinemia, combined with the production of autoantibodies characteristic of systemic lupus erythematosus (SLE). Alternatively, the GVHR can lead to the suppressive pathological symptoms, such as pancytopenia and hypogammaglobulinemia, characteristic of acute GVH disease (GVHD). Whether stimulatory or suppressive symptoms are induced by a GVHR depends, in our view (2-4), on the functional subset of donor T cells activated in the F1 host. The purpose of the present study was to investigate whether class I and/or class II H-2 alloantigens can selectively trigger, out of a pool of unselected donor T cells, those subpopulations of T cells responsible for the stimulatory and suppressive GVH symptoms, respectively. For the induction of the GVHR, 10(8) lymphoid cells from C57BL/6 (B6) donors were injected into three kinds of F1 hybrid mice, which had been bred from H-2 mutant strains on a B6 background. Whereas the I-A-disparate (B6 X bm12)F1 recipients exclusively developed stimulatory GVH symptoms, including SLE-like autoantibodies and immune complex glomerulonephritis, the K locus- disparate (B6 X bm1)F1 recipients showed neither clearly stimulatory nor clearly suppressive GVH symptoms. In marked contrast, the (bm1 X bm12)F1 recipients, which differ from the B6 donor strain by mutations at both K and I-A locus, initially developed stimulatory GVH symptoms, but rapidly thereafter showed the suppressive pathological symptoms of acute GVHD and died. Moreover, spleen cells obtained from (B6 X bm12)F1 mice injected with B6 donor cells helped the primary anti-sheep erythrocyte (SRBC) response of normal (B6 X bm12)F1 spleen cells in vitro, whereas spleen cells (bm1 X bm12)F1 mice injected with B6 donor cells strongly suppressed the primary anti-SRBC response of normal (bm1 X bm12)F1 spleen cells. Spleen cells from the K locus-disparate (B6 X bm1)F1 recipients also suppressed the primary anti-SRBC of normal (B6 X bm1)F1 spleen cells; this suppression, however, was weak when compared with the suppression induced by spleen cells from GVH (bm1 X bm12)F1 mice. Taken together, these findings indicate that a small class II (I- A) antigenic difference suffices to trigger the alloreactive donor T helper cells causing SLE-like GVHD. In contrast, both class I (H-2K) and class II (I-A) differences are required to trigger the subsets of donor T cells responsible for acute GVHD. It appears that alloreactive donor T helper cells induce the alloreactive T suppressor cells, which then act as the suppressor effector cells causing the pancytopenia of acute GVHD. These findings may help to understand the variability of GVH-like diseases caused by a given etiologic agent, their cellular pathogenesis, and association with certain HLA loci.
PMCID: PMC2186941  PMID: 6218218
25.  Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha [published erratum appears in J Exp Med 1996 Mar 1;183(3):1283] 
The Journal of Experimental Medicine  1995;182(4):1111-1119.
Several cytokines, especially granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha), have been identified that foster the development of dendritic cells from blood and bone marrow precursors in suspension cultures. These precursors are reported to be infrequent or to yield small numbers of dendritic cells in colony-forming assays. Here we readily identify dendritic cell colony-forming units (CFU-DC) that give rise to pure dendritic cell colonies. Human CD34+ bone marrow progenitors were expanded in semi- solid cultures with serum-replete medium containing c-kit-ligand, GM- CSF, and TNF-alpha. The addition of TNF-alpha to GM-CSF did not alter the number of typical GM colonies but did generate pure dendritic cell colonies that accounted for approximately 40% of the total colony growth. When the two distinct types of colonies were plucked from methylcellulose and tested for T cell-stimulatory activity in the mixed leukocyte reaction, the potency of colony-derived dendritic cells exceeded that of CFU-GM progeny from the same cultures by at least 1.5- 2 logs. Immunophenotyping and cytochemical staining of the CFU-DC- derived progeny was also characteristic of dendritic cells. Other myeloid cells were not identified in these colonies. The addition of c- kit-ligand to GM-CSF- and TNF-alpha-supplemented suspensions of CD34+ bone marrow cells expanded CFU-DCs almost 100-fold by 14 d. We conclude that normal human CD34+ bone marrow cells include substantial numbers of clonogenic progenitors, distinct from CFU-GMs, that can give rise to pure dendritic cell colonies. These CFU-DCs can be expanded for several weeks by in vitro culture with c-kit-ligand, and their differentiation requires exogenous TNF-alpha in addition to GM-CSF. We speculate that this dendritic cell-committed pathway may in the steady state contribute cells to the epidermis and afferent lymph, where dendritic cells are the principal myeloid cell type, and may increase the numbers of these specialized antigen-presenting cells during T cell-mediated immune responses.
PMCID: PMC2192302  PMID: 7561684

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