The role of mast cells in the elicitation of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE), a T cell-mediated autoimmune disease can be transferred with lymphoid cells from actively immunized rats into naive recipients. In the mouse, previous studies have suggested a role for histamine/serotonin in the development of active EAE. We have found that myelin basic protein-reactive cells transfer a biphasic skin test response to naive rats analogous to what has been described in the mouse contact dermatitis system, where mast cell sensitization by Ag-specific T cell factors is required for the induction of skin test responses. Treatment of cell recipients with the serotonin receptor antagonists, cyproheptadine or methysergide, blocked or significantly reduced the development of EAE. Furthermore, it was found that treatment with cyproheptadine was effective in blocking clinical disease when administered day 3 to day 6 after cell transfer. In contrast, cyproheptadine treatments before induction of paralysis day 0 to 3, failed to alter the course of clinical disease. The inhibitor of mast cell degranulation, proxicromil, was also found to effectively block the elicitation of adoptively transferred EAE and was also found to be effective when administered just before the onset of clinical disease. Reserpine, a compound known to deplete mast cells of vasoactive amines by forcing granule contents into the cytoplasm where they are degraded by cell enzymes, was also effective in blocking both active and adoptively transferred EAE. Disease inhibition was found to be partially reversed with pargyline, an inhibitor of monoamine oxidase. In addition lymphocytes from treated animals were capable of transferring disease to naive recipients and appeared to have normal activity as assessed by Ag-or mitogen-driven proliferation in addition to IL-2 production.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

Oral tolerance in experimental autoimmune encephalomyelitis. III. Evidence for clonal anergy.

We have recently reported that experimental autoimmune encephalomyelitis (EAE) can be suppressed by the oral administration of myelin basic protein (MBP). The oral introduction of 20 mg MBP together with a trypsin inhibitor results in inhibition of EAE clinical signs, decreased CNS histopathologic changes and dramatically reduced MBP-specific proliferative responses in fed and challenged Lewis rats. In the present study, we have investigated the mechanism underlying MBP-induced oral tolerance in EAE. Neither lymphoid cells (lymph node cells, spleen cells, Peyer's patch lymphocytes, thymocytes) nor humoral elements derived from tolerant donors were capable of transferring the tolerance to naive recipients. Moreover, lymphoid cells obtained from orally tolerant donors exhibited a marked decrease in their capacity to transfer EAE to naive recipient rats, even after in vitro activation with MBP or Con A. We observed that EAE could be readily transferred into orally tolerant rats using MBP-specific encephalitogenic T cell lines. In vitro cell mixing studies showed that the proliferation of lymphocytes from MBP-sensitized donors was not inhibited by the addition of lymphoid cells from tolerant donors, arguing against the role of a suppressor cell. Investigation of MBP-stimulated lymphokine production showed that both IL-2 and IFN-gamma levels were substantially decreased in spleen and lymph node cell cultures from MBP-fed rats compared to vehicle-fed control animals. Furthermore, limiting dilution analyses revealed that MBP-fed rats exhibited a profound decrease in MBP-reactive, IL-2-secreting lymphocytes relative to control animals. Thus, because lymphocytes from MBP-fed rats neither proliferate nor secrete IL-2 or IFN-gamma in response to MBP and we can find no compelling evidence for the role of suppressor cells, we propose that the oral administration of MBP results in a state of clonal anergy.     

Macrophages and nitric oxide as the possible cellular and molecular basis for strain and gender differences in susceptibility to autoimmune central nervous system inflammation

PVG rats are resistant to actively induced experimental autoimmune encephalomyelitis (EAE) and this appears to be directly related to high and sustained systemic levels of reactive nitrogen intermediates(RNI) following sensitization for EAE when compared to the highly susceptible Lewis rat. An apparent cellular basis for the different EAE susceptibility between the two rat strains is described. Spleens of PVG rats have increased monocyte/macrophage numbers(NO producing cells) and lower erythrocyte (NO scavengers) to nucleated spleen cell ratios compared with Lewis rats. Splenectomy demonstrated the pivotal role of the spleen in resistance to EAE as splenectomized PVG rats were rendered completely susceptible to disease induction.It was further demonstrated that EAE resistance in PVG rats is limited only to females and that only female PVG rats have increased splenic macrophage and an enhanced NO production following immunization. The males are fully susceptible to EAE and their spleen cell populations are similar to those of Lewis rats of either gender. Despite being resistant to active disease induction, immunized female PVG rats can generate EAE effector cells that are capable of passively transferring disease.Furthermore, female PVG rats are fully susceptible to passively transferred EAE. Thus, there appears to be no defect in the female PVG target tissue or in the processing or presentation of antigen,but a block at the level of effector cell expansion and/or recirculation and transmigration into the target tissue in actively induced EAE.     

Correlation of cytotoxicity with experimental allergic encephalomyelitis.

Cytotoxicity of immune lymph node cells in experimental allergic encephalomyelitis (EAE) was maximal 9 days after injection of encephalitogenic emulsion. The ability of these cells to passively transfer EAE was also maximal at this time. Immune spleen cells were more cytotoxic than lymph node cells 9 days after injection; however, these cells did not passively transfer EAE. Twelve days after injection of encephalitogenic emulsion immune spleen cells passively transferred EAE with resulting mild histopathologic lesions. At this time the spleen cells were 50% more cytotoxic than comparable lymph node cells. Cyclophosphamide suppressed the development of clinical EAE and the development of cytotoxic lymphoid cells. It also reduced clinical signs and cytotoxic activity of lymph node cells. Spleen cell cytotoxic activity was enhanced by Cyclophosphamide. It was concluded that cytotoxic activity of lymph node and spleen cells was correlated with the ability of these cells to produce EAE. Lymph node cell populations differed qualitatively and/or quantitatively from immune spleen cell populations in EAE. Capacity to passively transfer EAE coincided with the maximal Cytotoxicity of the lymphoid cells from each tissue.     

Effect of cyclophosphamide on suppressor cell activity in mice unresponsive to EAE.

Protection against experimental allergic encephalomyelitis (EAE) was induced in susceptible mice of (SJL/J X BALB/c)F1 hybrid, by injection of either mouse spinal cord homogenate, the small mouse basic protein, or Cop 1 in incomplete Freund's adjuvant, before EAE induction. It was demonstrated that the unresponsiveness induced by the three antigens is mediated by suppressor T cells residing in the spleen cell population and can be adoptively transferred to normal syngeneic recipients. Low dose of cyclophosphamide (20 mg/kg) administered 2 days before the encephalitogenic challenge abrogated the unresponsiveness to EAE and reverted the protected mice sensitive to disease induction. Cyclophosphamide was also active on adoptively transferred unresponsiveness, thus donors that had been treated with cyclophosphamide were unable to further transfer unresponsiveness to EAE. These results indicate the elimination by cyclophosphamide of suppressor cells that interfere with the effector mechanisms leading to EAE.     

Adoptive transfer of experimental allergic encephalomyelitis with activated spleen cells: Comparison of in vitro activation by concanavalin A and myelin basic protein

Adoptive transfer of experimental allergic encephalomyelitis (EAE) was carried out in Lewis rats using splenic lymphocytes incubated in vitro with either concanavalin A (Con A) or myelin basic protein (MBP). Requirements were established for sensitization of donors, culture conditions, numbers of transferred cells, and incubation period of EAE in recipients. These were strikingly similar whether Con A or MBP was used. In addition, cellular proliferation in vitro was not required in either system, but proliferation after transfer to the recipient was essential for the development of clinical signs and histological lesions. These methods have potential value for analyzing mechanisms of immune induction in this classic model of autoimmune disease.     

Adoptive transfer of experimental allergic encephalomyelitis: conditions influencing memory and effector cell development.

The cellular transfer of clinical experimental allergic encephalomyelitis (EAE) with immune spleen cells is only accomplished following lymphoid cell stimulation during an intervening in vitro culture activation period. Recipients of these cells recover from the ensuing adoptively transferred paralytic episode and subsequently respond to active challenge with myelin basic protein (BP)-CFA in an accelerated time frame consistent with the presence of memory cells in the initial cell transfer inoculum. We have found that the addition of anti-CD4 antibody or dexamethasone during the activation period inhibits the development of the transfer active EAE effector cell subpopulation, but does not alter the in vitro development and subsequent expression of the BP-specific memory cell subpopulation. Additional experiments also suggest the development of memory cells in the absence of effector cell activity. PMA + ionomycin when used as a stimulus during the culture activation period leads to effector and memory cell development. The stimulation response is dose dependent, in that a reduced concentration of PMA + ionomycin does not lead to EAE effector cell development; however, at these reduced levels of PMA + ionomycin, memory cell development still occurred. Additional evidence which supports the concept of independent development of memory cells and effector cells was obtained with a BP-specific cell line. Following recovery from cell line-mediated clinical EAE, as well as following adoptive transfer of the cell lines in the precursor stage, cell recipients did not develop an early onset of active EAE when subsequently immunized with BP-CFA. Thus the BP-specific T-cell line appears to contain the precursors of the effector cell subpopulation but does not appear to contain the BP memory cell subpopulation. Collectively these observations suggest the existence of distinct T-cell subsets or pathways of development that are followed during the response to BP as measured by the development of clinical EAE.     

Correlation between active rosette formation and delayed cutaneous hypersensitivity in experimental allergic encephalomyelitis

The effect of encephalitogenic myelin basic protein, BP, on active rosette-forming T cells (ARFC) was compared to that of nonencephalitogenic peptide S42, a synthetic analogue of the tryptophan region of BP. Depression of ARFC by these antigens was reversible within 24 h after a second dose of the antigen into the skin, or after in vitro incubation of lymphocytes with the sensitizing antigen (Ag-ARFC). The ratio of Ag-ARFC to ARFC rose with time following the sensitization but fell shortly before the clinical onset of experimental allergic encephalomyelitis in animals sensitized with BP. In contrast, the Ag-ARFC/ARFC ratios for animals sensitized with peptide S42 reached plateau levels from which they did not drop. The kinetics of the Ag-ARFC/ARFC responses paralleled those for delayed-type skin hypersensitivity (DTH) in the respective animals. The DTH responses rose following sensitization and fell shortly after the appearance of clinical signs of EAE. The results of this study provide in vitro and in vivo evidence for sensitization to myelin basic protein, and focus attention on the ARFC as a measure for an immunologically active cell population which may be quantitated by antigenic stimulation.Abbreviations used in this report EAE experimental allergic encephalomyelitis - DTH delayed-type skin hypersensitivity - ARFC active rosette-forming T cells - Ag-ARFC antigen-stimulated active rosette-forming T cells - TRFC total rosette-forming T cells     

The nature of the defect in experimental allergic encephalomyelitis (EAE)-resistant Lewis (Le-R) rats

Recently, a colony of Lewis rats has been described which is resistant to experimental allergic encephalomyelitis (EAE). These rats, termed Le-R, are still histocompatible with other Lewis rats. The genetic defect which results in EAE-resistance was shown not to be linked to the RT1.B (Ir) region of the MHC. Myelin basic protein (BP)-sensitization of Le-R rats induces cells capable of mounting a proliferative response to BP in culture but incapable of transferring EAE after culture with BP. The present study demonstrates that the latter deficiency can be overcome either by incorporating lipopolysaccharide (LPS) in the BP-culture medium or by simultaneous transfer of LPS-activated antigen-nonspecific spleen cells with the BP-sensitized cells. The BP-sensitized Le-R cells fail to transfer EAE due to their inability to initiate lesions in the CNS. LPS, working through an antigen-nonspecific cell or cell products, can correct the defect in the Le-R cells such that the antigen-specific cells become capable of initiating CNS lesions which lead to development of clinical EAE.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.

Bone marrow (BM)-derived dendritic cells (DC) are potent stimulators of naive CD4+ T cell activation. Because DC are efficient at Ag processing and could potentially present self Ags, we investigated the role of DC in the presentation of an encephalitogenic peptide from myelin basic protein (Ac1-11) in the induction of experimental autoimmune encephalomyelitis (EAE). To determine if DC could prime for EAE, we transferred DC pulsed with Ac1-11 or with medium alone into irradiated mice in combination with CD4+ T cells isolated from a mouse transgenic for a TCR specific for Ac1-11 + I-Au. Mice transferred with Ac1-11-pulsed DC developed EAE 7-10 days later, whereas mice receiving medium-pulsed DC did not. By day 15, all mice given peptide-loaded DC had signs of tail and hind limb paralysis, and by day 20 infiltration of Ac1-11-specific CD4+ T cells was detected in the brain parenchyma. We also demonstrated interactions between Ac1-11-pulsed DC and Ac1-11-specific T cells in the lymph nodes 24 h following adoptive transfer of both cell populations. These data show that DC can efficiently present the self Ag myelin basic protein Ac1-11 to Ag-specific T cells in the periphery of mice to induce EAE.     

Inhibition of allergic encephalomyelitis in rats by treatment with sulfated polysaccharides

A number of sulfated polysaccharides were tested for their ability to inhibit passively induced experimental allergic encephalomyelitis (EAE) in rats. Heparin and fucoidan both completely inhibited passive EAE even when treatment was begun 3 days after transfer of cells. Pentosan sulfate was partially inhibitory whereas chondroitin-4-sulfate had no effect. Inhibition was not merely due to killing of the cells since active sensitization 14 days after cell transfer resulted in an early onset of disease indicating the persistence of transferred cells as memory cells. Although all the inhibitory polysaccharides are anticoagulants, it would appear that this function alone is not the reason for inhibition since a heparin preparation devoid of anticoagulant activity also partially inhibited EAE. Actively induced EAE was also significantly delayed by treatment with heparin. The results are discussed in terms of the polysaccharides inhibiting the enzymatic dependent movement of lymphocytes across central nervous system vascular endothelium.     

Adoptive transfer of experimental allergic encephalomyelitis: incubation of rat spleen cells with specific antigen.

Spleen cells from myelin basic protein (BP)-sensitized donor rats appear to be incapable of adoptively transferring experimental allergic encephalomyelitis (EAE) directly to normal recipients. It has been reported that in vitro incubation with concanavalin A (Con A) activates rat spleen cells so that they are capable of transferring EAE. We report here that incubation with specific antigen, BP, also permits transfer of disease with spleen cells. Data are presented in which activation of EAE spleen cells by Con A is compared with activation by BP. Cellular proliferation does not appear to be necessary for in vitro activation with specific antigen.     

Suppression of experimental allergic encephalomyelitis by 6-hydroxyphthalaldehydic acid, O-(p-chlorobenzyl)oxime (EN3638)

EN3638 is a new oxime derivative of salicylic acid that has immunosuppressive properties. Oral administration of the compound to rats during the incubation period of experimental allergic encephalomyelitis (EAE) delayed the onset of clinical signs. EN3638 was effective in both ordinary and hyperacute forms of EAE. Doses of 50, 100, or 150 mg/kg daily, 250 mg/kg three times a week, or 400 mg/kg twice a week suppressed both clinical and histologic evidence of EAE during the course of therapy (as long as 4 weeks) and for 8 or more days thereafter. Clinical EAE developed after a full incubation period after discontinuance of EN3638, probably due to the persisting depot of antigen in oil. When EAE was produced without an oily depot, a single dose suppressed the disease for at least 5 weeks in some rats. EN3638 was effective when given only in the second half of the incubation period but not when given at the time that EAE lesions and signs develop. Passive transfer experiments suggested that the drug prevented and even reversed sensitization to neural antigens. It had only slight effect on fully sensitized lymphoid cells or on the recruitment of nonimmune inflammatory cells in the nervous system, and it was not acting as a source of salicylate or as an adrenocortical stimulator.     

The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.     

Prevention of experimental autoimmune encephalomyelitis in Lewis rats by treatment with an anti-rat CD5 antibody (OX19).

Treatment of Lewis rats with a single dose of OX19 antibody, specific for rat CD5, uniformly prevented the development of experimental autoimmune encephalomyelitis (EAE). This protective effect had several notable characteristics: (1) it persisted for at least 10 days; (2) it could be achieved with either high doses of the antibody (> 200 micrograms) or lower doses (100-200 micrograms), which did not deplete T cell populations; and (3) the treated animals were able to mount comparable T cell responses to both myelin basic protein and myelin-unrelated antigens. In addition, antibody treatment consistently prevented the development of adoptively transferred EAE, suggesting that enhanced suppressor cell activity may have contributed to the protection. Antibodies such as OX19 appear capable of blocking the development of EAE, and perhaps other autoimmune diseases as well.     

Recipient contributions to serial passive transfer of experimental allergic encephalomyelitis

EAE can be adoptively transferred into normal recipients by the transfer of BP-specific EAE effector cells. After cell transfer, a series of ill-defined events occur in the recipient that culminate in the development of paralysis associated with neural tissue damage. We investigated the subsequent recipient response to the adoptively transferred disease and examined the role that recipient lymphocytes play in the development of adoptively transferred EAE. Recipient involvement was assessed by the transfer of EAE through a series of normal F1 animals as recipients and at the endpoint of the experiment, determining the MHC restriction pattern of the BP-sensitive cells present. The serial transfer of EAE from BP-CFA-sensitized LEW----(LEW X F-344)F1----(LEW X P2)F1, and from BP-CFA sensitized LEW----(LEW X F344)F1----(LEW X F-344)F1, resulted in the development of BP-sensitive cells in the spleens of the secondary recipients that were able to transfer disease into normal LEW recipients. To test directly for the development of host-derived BP-sensitive cells that might arise in the F1 animal, the serial transfer of EAE from LEW----(LEW X ACI)F1----(LEW X ACI)F1 was performed. When BP-sensitive cells obtained from the secondary (LEW X ACI)F1 recipient animal were transferred into either normal LEW and ACI, or irradiated LEW and ACI animals as final recipients, transfer of disease was successful only into the LEW parental. These results suggest that the development of passive EAE is due solely to the transferred BP-sensitive cells originating from the actively immunized donor, and that no host-derived lymphocytes are recruited into the pool of EAE effector precursor cells found in the spleen of animals after the development of adoptively transferred EAE.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer''s patch (PP). Common dendritic cell progenitors (CDPs, lin^- c-kit^lo CD115⁺ Flt3⁺) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar^-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1⁺ and recipients being CD45.2⁺. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar^-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.