Reprints Available Directly from the Publisher Photocopying Permitted by License Only In-vitro Differentiation of Mature Dendritic Cells from Human Blood Monocytes Hla-dr

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-y to differentiate monocyte-derived DC in vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. HLA-DP/, and HLA-DQ/. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD c/CD45R0 blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-y and IL-4 indicates that these DC may conform to mature mucosal DC.


1987;  Peter
et al., 1987; Kabel et al., 1989; Thomas et al.,  1993; Zhou and Tedder, 1996).

Thus, large numbers of DC can now be generated from myelomonocytic progeny.It should be noted, however, that this does not rule out the possible differentiation of DC from nonmyeloid lineages *Corresponding author.Present address: Robert Gieseler, Division of Clinical Endocrinology, Department of Medicine, University Clinics, Hufelandstrasse 55, D-45122 Essen Germany.RG and DH contributed equally to this work and should be considered first co-authors.(Steinman and Cohn, 1973;Steinman, 1991), perhaps even common with T-cell progenitors (Ardavin et al.,  1993; O'Neill, 1994).Future studies will help to classify such observations, which may, for the time being, be regarded as hin s toward the complex regulation and integration of antigen specificity.

In any case, depending on the nature and composi- tion of environmental stimuli, monocytes may be induced to differentiate into either of the two major classes of professional APC, that is, DC or macro- phages (Mq) (Peters et al., 1991, 1996).Because DC and Mq are crucial for directing the course of immunity, the genetic program underlying their developmental dichotomy is to be regarded as a major cornerstone of immune regulation.

Moreover, as to the diverse conditions in the various anatomic sites, local monocyte differentiation should even result in a broad spectrum of phenotypes, rather than only two idealized cell populations.Indeed, this deduction has long been verified empiri- cally, suggesting that the actual tissue shapes its characteristic APC compartment (e.g., Franklin et al., 1986).This is consistent with the finding that, fo example, DC obtained from spleen and Peyer's patches profoundly differ in their preferential activa- tion of T-helper 1 (Thl) or Th2 cells (Everson et al.,  1996).

Studying the effect of growth factors and cytokines on isolated monocytes thus appears suitable to identify signals controlling the development of dis- crete myeloid APC types.As to the thought pathway sketched before, such molecules would induce basic DC or Mq differentiation as well as fine-tune their tissue-specific maturation.The past years then brought about several culture systems for the genera- tion f monocyte-derived DC (MoDC) in the presence of selected factors (cf.Peters et al., 1996).One of the generally accepted protocols employs a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), leading to CD l a and CD14or CD14 'im DC that are able to induce mycobacterium-and tetanus-toxin-specific T-cell proliferation, as well as mixed leukocyte responses (Porcelli et al., 1992; Ruppert et al., 1993; Romani et   al., 1994; Sallusto and Lanzavecchia, 1994; Steinbach  et al., 1995; Kiertscher and Roth, 1996).

We then proceeded to question whether interferon y (IFN-y), which upregulates the expression of major histocompatibility complex (MHC) class II mole- cules, might support the functionality of MoDC.This working hypothesis could be verified when culturing monocytes with GM-CSF, IL-4, and IFN-T, which gave rise to DC strongly supporting a mixed leuko- cyte reaction (Xu et al., 1995).

This paper now pre ents a phenotypic character- ization of freshly isolated monocytes, and the MoDC (including both DC and veiled cells) generated from them by action of GM-CSF, IL-4, and IFN-y.

Specifically, we focused on the expression of myeloid-type markers, MHC class I and II antigens, selected adhesion molecules, as well as antigens delineating certain subsets and maturity stages of DC in vivo.The resultant phenotypic profile now allows to classify the ontogenetic root of such cells, to pinpoint their stage of development and to discuss their possible counterpart in vivo.Additional morphological and functional data underscore the mature stage of the MoDC obtained with this culture system.


RESULTS


Morphology and Differentiation of MoDC in


Culture

Monocytes cultured for 6 days in the presence of GM- CSF, IL-4, and IFN-y exhibited three principal morphologies.The majority of cells (> 60%) weakly adhered to the gas-permeable Teflon surface of the culture flasks.These cells revealed numerous dendritic projections and occasional veillike cytoplasmic structures.The

erhans c
lls in situ.

Starting with the third day of culture, part of such cells began to detach and gave rise to a second population of suspended cells that, 1 to 2 days later, plateaued at --30% of the total cell number.These nonadherent cells had ovoid, bluntly branched cell bodies exhibiting abundant cytoplasmic veils.Veil structures appeared to mainly bud from such projec- tions (Figure 1), which m y correspond to the dendritiform ramifications during the adherent cell stage.

Third, we observed a small proportion of--5% strongly adherent Mq.The majority of these cells were stretched in shape with a broad footlike projection on one of the cell poles.Mq) were larger than dendritic and veiled cells and, unlike these, exhibited abundant perinuclear lysosomes and occa- sional vacuolization.These Mq revealed some very interesting features deserving special co sideration and are presented in detail elsewhere (Gieseler et al.,  1998).

The Phenotype of MoDC Depending on their mean fluorescence intensities (AMFI; see Materials and Methods), we here defined the expression of markers as dim (AMFI 10-100), + (AMFI > 100-1000), ++ (AMFI > 1000-5000) or +++ (AMFI > 5000).


b FIGURE

Scanning electron micrographs of veiled cells derived from blood monocytes.Veiled cells were collected from supern

ants of cells cultured
for 6 days in the presence of GM-CSF, IL- 4, and IFN-% and processed as indicated. (a) Several cells immobilized on poly-L-lysine exhibit a spectrum of veil structures and cytoplasmic processes.Original magnificat

n 1500.Th
bar represents 10/xm. (b) The typical appearance of a suspended cell in culture conforms to blood dendritic cells (Knight et al., 1986) or veiled cells from afferent lymph (Barfoot et al., 1989) or colonic lamina propria (Pavli et al., 1993).Original magnification 3000.

The bar indicates 5/xm.


Myeloid Antigens

Cultured cells did not proliferate, that is, they did not form clones or incorporate [3H]thymidine (not shown).Hence, DC and veiled cells generated in the presence of cytokines clearly were descendants of monocytes, as shown by their expression of the myeloid lineage marker CD33 (throughout the mye- loid life cycle) as w ll as CD68 (Figure 2).Th

gh often being co
sidered a Mq>specific marker, CD68 may also be expressed by DC (Beelen et al., 1993).However, CD68 expression on MoDC was lower than that observed on serum-cultured Mq (Figures 2B  and 2C).Another marker still regarded as a hall- mark of Mq is CD14, that is, the receptor for complexes of LPS/LPS-binding protein.Yet, in vivo, DC in distinct sites, such as mucosal tissue, may well express CD14, as was shown by Graeme-Cook et al.  (1993).Therefore, supposing a common origin of DC and Mq, CD68 and CD14 may be subject to exogenous environmental regulation in either of these populations and their site-specific subsets.Earlier, Thomas et al. (1993) showed that blood DC are CD 14dimCD33bright.Accordingly, in confirming and extending results from Ruppert et al., (1993), Sallusto  and Lanzavecchia (1994) as well as Kiertscher and CD32 CD33

Fluorescence (Arbitrary Units) FIGURE 2 Expression of myeloid antigens as determined by flow cytometry.The panel compares antigen expression by monocytes (row A), monocytes cultured for 6 days with serum alone (row B), and DC and veiled cells derived from monocytes by action of GM-CSF/ IL-4/IFN-y (row C).The mAb are detailed in Table I.Each graph is representative of four to six experiments carried out.Histograms depict the number of cells (maximal scaling equals to 200 cells) exhibiting various fluorescence intensities (AMFI) with either control mAb (bold lines on the left) or tested antigens (faint lines).Percentages of antigen-positive cells in Fig. 6 were calculated from these data using the C30 program.

Roth (1996) obtained with GM-CSF and IL-4, we here show complete downregulation of CD14 on 28.4% of MoDC as well as a CD14 m phenotype of the remaining cells (Figure 2C).Also, the Fcy receptors (FcR) type I, II, and IIIA (CD64, CD32, CD16a) were found downregulated.Very faint expression of CD 16 and CD64 (Figure 2C) by 33.9% or 24.6% of cells (Figure 6) cultured with GM-CSF/ IL-4/IFN-y was partly due to the small Mq sub- population that coemerged from the starter monocytes (see earlier), and some of the MoDC, but not the veiled cells.An interesting result was the CD32

expression in 78.4% of cell
(Figures 2C and 6), because this conforms to blood DC expressing the maturity marker CD83 (Zhou and Tedder, 1995).The remaining cells were CD32-.Thus, according to the preceding definition, the myeloid antigen pattern detected on MoDC generated i the presence of GM-CSF, IL-4, and IFN-y was CD 14'm/-, CD 16a-mm, CD32m/-, CD33+, CD64-/m, and CD68/.Double statements indicate the expression by different stages of maturity, with the major portion mentioned first and the minor portion in the second position.


DC and Maturation Antigens

Certain populations of DC or subsets thereof can be discerned by several markers.The most prominent may be CDla, which is expressed on immature epidermal Langerhans cells, but becomes abrogated when the cells drain to the lymph nodes and mature.

Here, 9.0-32.6% of MoDC were CDla 'im (Figures 6 and 3C).We did not follow CDla expression during the previous days of culture.However, judging from the maturity markers determined in this study, the majority of MoDC cultured for 6 days appear to be in a mature state that, therefore, does not conflict with the loss of C integrin Cx-chain CD1 l c and CD45R0 a splicing variant of the leukocyte common antigen.CD45R0 is not only expressed by activated or memory T and B cells, but also by mature blood DC (Zhou and Tedder, 1995) as well as DC differentiated from monocytes in the presence of GM-CSF, IL-4, and tumor necrosis factor c (TNF-c0 (Zhou and Tedder, 1996).In 1994, O'Doherty et al. (1993) showed that a subpopulation of blood DC express both, CDllc and CD45R0, which reasonably is taken as circumstantial evidence
C CDla CD1 lc CD40 CD45R0 CD 71
Fluorescence (Arbitrary Units) FIGURE 3 Expression of markers delineating subsets of DC, mature stages of DC differentiation, and cell activation.All specifications are as given in the caption to Figure 2.

for their mature stage--even more so, when bearing in mind that isolated blood DC, when cultured, reveal a CD1 l c+CD45R0 phenotype.Approaching almost 100% of cultured cells, the MoDC generated in this present study were also CDllc+CD45R0 double-positive (Figures 3C and 6).One further antigen clearly expressed by DC, but only weakly by monocytes, is the B-cell mark

CD40 (e.g., Zhou  and Tedder, 1995 and 1996).H
re, almost 100% of MoDC were CD40 (Figures 3C and 6).Further,  Andreesen et al. (1984) showed that CD71, a receptor for complexes of Fe/transferritin, is expressed depending on the degree of myelomonocytic differentiation.We have therefore chosen this marker as additional proof for a mature stage of the MoDC.As opposed to only marginal expression of CD71 by low numbers of fresh monocytes, close to 100% of the MoDC, and veiled cells were strongly positive for this receptor (Figures 3 and 6).The total status of antigens chosen as markers of DC subsets, as well as the degree of maturity and activation of MoDC was CD 1 a-/dim, CD 11 c+, CD40+, CD45R0+, and CD71+.


Antigen-presenting and Costimulatory Molecules

Gene products encoded by the MHC are prerequisite for the effective presentation of processed antigen.Brooks and Moore (1988) showed that HLA-DR, -DP, and-DQ are strongly expressed by blood DC, whereas monocytes are only weakly positive for class II products of the DP and DQ subloci.We here confirmed this finding for freshly isolated monocytes (Figure 4A), and cells cultured with GM-CSF, IL-4, and IFN-y exhibited a class-II phenotype similar to that observed with blood DC (Figure 4C).Also, although culture may lead to the expression of higher levels of HLA-DP and-DQ (Brooks and Moore,  1988, and Figure 4B), the percentages of monocytes positive for these gene products were evidently lower than those detected with the MoDC, of which virtually all cells coexpressed HLA-DR,-DP, and -DQ (Figure 6).Importantly, veiled cells suspended in culture expressed even higher amounts of HLA-DR and -DP, that is, a peak of strong DR expression on the far right of the histogram, and a shoulder of DP expression at AMFI --700 (Figure 4C).Similarly, the expression of MHC class I (HLA-A,-B,-C) strongly HLA-A,-B,-C HLA-DR Fluorescence (Arbitrary Units) FIGURE 4 Flow-cytometric detection of MHC class and class II as well as of costimulatory molecules.In a portion of GM-CSF/ IL-4/IFN-y cells (row C), MHC class and HLA-DR expression exceeded the logarithmic AMFI scale (i.e., >10000).These cells form seperate peaks that accumulate on the far right of the histograms.All specifications are as given for Fig. 2.

increased with the time of culture (Figure 4).Since it is known now that class I molecules not only serve to present endogenous products but also may participate in the presentation of exogenous antigens by Mq and DC populations (Rock et al., 1993), an increase in class I obviously may augment the APC's antigen- presenting capacity.Moreover, paralleling the obser- vations for class II, veiled cells revealed extremely upregulated class I express

n, as reflected on
he far right of the AMFI log scale (Figure 4C).Therefore, in strongly expressing MHC products, such cells qualify as potent APC.This is further underscored by the upregulation of costimulatory molecules B7.1 (CD80) and B7.2 (CD86) on MoDC, as compared to thei starting population, or to cells cultured without cytokines (Figure 4).While being absent from immature DC (McLellan et al., 1995), the maturation of DC implies upregulation of CD86 as primary and CD80 as secondary costimulatory molecules to inter- act with their T-cell ligands CD28 or CTLA-4.

Regular T-cell activation depends on these inter- actions, for their absence leads to anergy or apoptosis of the T cells involved (McLellan et al., 1995;  Thompson, 1995).The breadth of the peak for CD86 indicates that, at this point of time, MoDC at l ast reach the B7.2-dependent stage of maturity (Figure 4C).As will be shown in what follows, such cells are potent stimulators of an llogeneic mixed-leukocyte reaction, whereas it is characteristic for immature DC that they are quiescent.Taken together, the MHC/ costimulator phenotype of MoDC was MHC class i++/+++, HLA_DR++/+++, HLA-DP HLA-DQ CO80 dim (but stronger than on Mqg, and CD86+/++.


Adhesion Molecules

To conclude phenotypic characterization, we selected adhesion molecules, which, similar to B7.1 and B7.2, are essential for the successful stimulation of antigenspecific T cells by APC.Consistently, the Ig super- family adhesion molecules CDlla (cL-chain) and CDllb (cM-chain), the /-chain CD18, as well as intercellular adhesion molecules (ICAM) (CD54) and 3 (CD50) were all upregula

d and expressed by -90-100% of a
l MoDC Figures 5C and 6).As to CD 1 lb, this molecule not only is a receptor for C3bi.For whereas CD1 a and CD18 constitute the leuko- cyte function-associated molecule (LFA-1), CD 11 b and CD18 can similarly associate as Mac-1.Both LFA-1 and Mac-1 may compete for interaction with ICAM-1 (Lub et al., 1996).We therefore will not speculate at the present time which function(s) of C CD18 CD50 CD54

Fluorescence (Arbitrary Units)

FIGURE 5 Cellular expression of selected adhesion molecules.For specifications, see above.

CD b may be utilized by DC.However, it has been shown that cM-chains are expressed by subpopula- tions of DC (Franklin et al., 1986;Robinson et al., 1986), and not exclusively by M q).Interestingly, ICAM-1 appears to be involved in DC migration and

te DC-depen
ent T-cell stimulation (Starling et al.,  1995).Therefore, this molecule may play a special role for mature DC, which would complement the previous picture.Indeed, ICAM-1 was found expressed by MoDC, and its broadened peak indicates different membrane densities of CD54 in the less mature DC and the more mature veiled cells.The pattern of selected adhesion molecules on MoDC was CD 11 a+, CD 11b++, CD 18+, CD50+, and CD54+.


The Functional Capacity of MoDC

To test the APC's potency to induce allospecific stimulation, we ran one-way mixed leukocyte cultures (MLC).Of the MoDC, both their adherent DC and suspended veiled-cell subsets were transferred into microwells and cocultured with pools of T lympho- cytes.We observed the clustering of MoDC and T cells, as is typical of mature lymphoid DC (Austyn, 1987).MoDC induced a very vigorous proliferation of allogeneic T cells, which was not the case with fresh monocytes as stimulator cells (Figure 7).These data complement those presented in our previous study (Xu et al., 1995).Also, we recently were able to show that MoDC generated by action of GM-CSF, IL- 4, and IFN-y are as well able to stimulate autologous antigen-specific T-cell proliferation (Soruri et al.,  1998).


DISCUSSION

The present study shows that DC and veiled cells can be generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-y.Phenotypically, such cells are CDla-mim, CDlla+, CDllb++, CDllc+, CD14dm/-, CD16a-/dim, CD18+, CD32ainu-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71 +, CDS0dim, CD86+/++, MHC class I++/+++, HLA-DR++/+++, HLA-DP+, and HLA-DQ+.Specifically, the nonadher- ent cells exhibited morphologies that strongly resem- bled veiled-cell types that are in transit to, or encoun- tered within, lymphoid tissues (examples are given in Knight et al., 1986; Barfoot et al., 1989; and Pavli et al.,  1993).It is consistent with previous results that these MoDCmand, above all, the veiled cells--morphologically, phenotypically, and functionally represent a mature stage of differentiation.Irrespective of the cell Data of fresh monocytes (empty bars) and cells cultured in the presence of GM- CSF/IL-4/IFN-y (solid bars) are given as means, with error bars indicating maxima and minima among the donors (n 4 to 6).

lineage, CD45R0 is only expressed by activate or memory-type immunocytes.O'Doherty et al. (1993)  were the first to show maturation of isolated blood DC into CD45R0 cells.They concluded that the highly stimulatory CD1 lc+CD45R0 double-positive blood DC subset represents antigen-primed mature DC, which are possibly en route to lymphoid organs, while the less active CD1 l c-CD45R0-subset apparently comprises immature blood DC (O'Doherty et al., 1994).Similarly, in addition to demonstr ting that DC generated by action of GM-CSF, IL-4, and IFN-y are potent stimulators of allogeneic mixed leukocyte 80 '20 '40 :80 '60 :320 Stimulator Responder Ratio FIGURE 7 MoDC are potent stimulators of an allogeneic mixed- leukocyte reaction.Graded numbers (6.25 10 to 104) of fresh monocytes or MoDC generated by action of GM-CSF/IL- 4/IFN-y were coincubated with 2 105 allogeneic T cells.As opposed to monocytes (broken line, means and open symbols), the MoDC (solid line, means and closed symbols) were potent stimulator cells (n 5).T-cell controls revealed baseline incorpora- tion of [3H] thymidine (315 dpm).APC controls did not proliferate in any of the experiments.Consistent to all experiments, Mq only stimulated marginally at a stimulator:responder ratio of 1:160 (529 dpm), and ceased to provoke T-cell proliferation at 1:320 (231 dpm).

reactions (Xu et al., 1995), we now show that such cells coexpress CD45R0 and CD 11 c.Another feature identifying mature DC is their CD14dimCD33brigtt phenotype, as opposed to functionally immature CD14dimCD33oim DC (Thomas et al., 1993; Thomas  and Lipsky, 1994).Likewise, DC derived from monocytes under GM-CSF, IL-4 and IFN-T were CD14im/-CD33+.Zhou and Tedder (1995) showed that CD14 monocytes, when cultured with GM-CSF, IL-4, and TNF-ce, develop into CD83 DC.CD83 belongs to the immunoglobulin superfamily and is distinctive for mature blood DC.Of interest, in the absence of proliferation, the yield of these MoDC was similar to the number of monocytes cultured (Zhou and Tedder,  1996), which corroborates previous results (Peters et  al., 1991), and is in line with the fact that bone- marrow precursors must pass a transient monocyte stage before acquiring DC characteristics (Gieseler et  al., 1991).It was also observed that prolonged culture of MoDC leads to their transition into large, round Mq-like cells (Zhou and Tedder, 1996).This matches the finding that highly stimulatory DC, as derived from human monocytes in the presence of a placental medium supplement, can be triggered to develop into Mq by action of M-CSF, whereby accessory activity is completely abrogated from such cultures (Gieseler, 1987).A serial monocyte DC Mq transition could as well be achieved by the addition of serum to human or rat DC derived from yeloid precursors (Gieseler, 1987; Najar et al., 1990; Gieseler et al.,  1991; Peters et al., 1991).

As to their course of differentiation, DC generated in the presence of GM-CSF, IL-4, and TNF-ce first downregulate CD14, then become CDla+, again downregulate CDla, and eventually express CD83.At this stage, the MoDC additionally acquire CD45R0 (Zhou and Tedder, 1996).Hence, cells differentiated with this cytokine mix reveal a transient CDla Langerhans cell phenotype and further proceed to develop into a mature interdigitating DC type.

Similarly, a mature DC phenotype, which may delineate cells in transit to or present within lymphoid organs, can also be induced with a cytokine cocktail employing GM-CSF, IL-4, and IFN-T.Whether these cells express CD83 is currently investigated (CD83specific mAb was kindly provided by F. Tedder, Duke University Medical Center, Durham, NC).First preliminary results indicate that they start to acquire CD83 on the sixth day of culture.Do these MoDC relate to Langerhans cells?In vivo, skin DC express Mac-1 as well as Fc receptors (Steinman, 1991).Functionally, Mac-1 competes with LFA-1 to interact with ICAM-1 (Lub et al., 1996), hich obviously plays a regulatory role in cell cooperation, and the Fc receptors may actively participate in the phagocytosis of antigens--a func- tion clearly demonstrated for Langerhans cells (Reis e Sousa et al., 1993).These markers were likewise detected on the MoDC.That FcR were found downregulated indicates the MoDC's progression from immature cells that take up antigen to ma ured cells that may present processed antigenic material.This notion is not only supported by the parallel upregulation of MHC and costimulatory molecules, but also by the observation that such cells induce antigen-specific T-cell responses (Soruri et al., 1998).Another indication may be their CDla -/im status.Yet, there is a further interpretation that results from t

ed by Thl and Th2 cytokines IFN-y a
d IL-4.Langerhanslike cells are not only present in skin, but also throughout mucosa- associated lymphoid tissue.Interestingly, enteric immune compartment provides a unique environment where the low-density subsets of both ce//3-TCR and T/6-TCR intraepithelial lymphocytes spontaneously produce the Thl mediator IFN-T (Yamamoto et al.,   1993), whereas DC from Peyer's patches pre- dominantly induce the production of Th2 cytokines such as IL-4 (Everson et al., 1996).We therefore suggest that monocyte-derived cells obtained by action of GM-CSF, IL-4, and IFN-y may resemble mucosal enteric DC.The retention of low amounts of CD32 FcyR II) on these cells, s opposed to human colonic DC in situ (Pavli et al., 1993), may well be due to the low concentration of cytokines employed.We currently address this issue in our experiments.

Most interestingly, and in contrast to epidermal Langerhans cells, DC freshly isolated from the colonic lamina propria are potent MLR stimulators (Pavli et al., 1993).This feature identifies mucosal Langerhanslike cells as mature DC and is in line with their CDla-negative phenotype (Graeme-Cook et al., 1993).It therefore is suggested that the mucosal- based lympho d tissue functions as a genuine lymphoid organ that directly borders to the outer world.

That microenvironmental conditions imprint on the local cells' character is vividly demonstrated by experiments employing GM-CSF.This factor not only supports the differentiation of DC fr m mono- cytic precursors; it also effects the functional matura- tion of immature DC.Accordingly, GM-CSF induces isolated skin Langerhans cells to stimulate an immune response (Witmer-Pack et al., 1987), whereas, in contrast to this, liver-derived immature DC are induced to mature into tolerogenizing DC (Rastell ni  et al., 1995).Therefore, the functional repertory initiated by GM-CSF (and, probably, other factors) on terminal DC maturation obviously depends on the microenvironmental conditions shaping their imma- ture precursors.

Correspondingly, to provide artificial microenvir- onments in vitro may allow to mimick conditions encountered in discrete tissues and has the potential to generate site-specific (and funct onally diverse) DC subsets.Should the MoDC described in this study approximate to mucosal-type DC, they may serve as a starting point from, which to proceed toward an optimized culture protocol.

Future prospects for such cells are evident.Some involve the establishment of in-vitro systems to mimick the tolerogenizing properties of the oral mucosa, which might be employed in the treatment of autoaggression.Others may allow a more specific investigation or treatment of mucosal-based immuno- pathologic disorders, such as the chronic inflamma- tory bowel diseases.


MATERIALS AND METHODS

Isolation and Culture of Monocytes Bu fy coats from healthy donors were kindly provid- ed by the Blood Bank of the University Clinics and suspended 1:1 in phosphate-buffered saline (PBS) without Ca

2+ and Mg 2+ (Flow, Meckenheim, FRG).The suspensions were centrifuged through Lymphoprep (p 1.077 g/ml; Nycomed, Oslo) at 800 g for 20 rain at RT. Interphases containing peripheral blood mononuclear cells (PBMNL) were collected and washed several times with cold PBS to remove platelets.PBMNL were then suspended in RPMI 1640 (Biochrom, Berlin), supplemented with 2 mM L-glutamine (Biochrom), and 100 U/ml penicillin plus 100 U/ml treptomycin (Pen/Strep; Flow) at 5% normal human serum (NHS).

Five-milliliter volumes of 2.5 107 cells were placed in hydrophobic 50-mm Teflon Petriperm dish- es (Bachhofer, Reutlingen, FRG), precoated with human plasma that was obtained from whole blood preparations.The PBMNL were incubated for adher- ence under occasional agitation.After 40-45 rain, nonadherent cells were rinsed off, and successful enrichment of monocytes (> 95%) was verified microscopically.On

verage, 10% of the initial ce
l suspension was adherent under these conditions, that is, --2.5 106 monocytes remained per culture dish.

Monocytes were then added fresh RPMI 1640 with Pen/Strep and 5% NHS, containing GM-CSF (100 IU/ml), IL-4 (100 IU/ml), and IFN-y (50 IU/ml) (Genzyme, Cambridge, UK).The cells were incubat- ed for 6 days to differentiate into MoDC.Monocytes were also cultured without cytokines.

For phenotypical and functional analyses, freshly isolated monocytes or adherent cells in culture were detached by 1-hr incubation with cold PBS/0.01%EDTA.Suspended cells were collected as well.After several washes at 350 g in the cold, cells were adjusted to concentrations detailed in the respective method.

The viability of enriched monocytes or cultured cells was determined with propidium iodide at 5 mg/ ml.The substance intercalates with cellular DNA when infiltrating dead or damaged cells.Incubated cells were immediately analyzed flow-cytometrically for red fluorescence (I 570 nm) of nuclei.General viability was > 95

occasional cul
ures exhibiting lower viability rates were excluded from the experiments.

Isolation, Pooling, and Storage of T Cells Sheep blood was diluted 1:1 with PBS (RT) and washed several times for 12 min at 163

g an
RT.

The erythrocyte pellet was then suspended in RPMI 1640 and stored overnight at 4C. Red cells were used for CD2-dependent rosetting of T cells.To enhance erythrocyte-T-cell interaction, nonadherent PBMNL were washed and suspended at 2 107/ml in 1% (w/v) polyethylene glycol dissolved in RPMI 1640/5% NHS.The suspension was then added 10% (v/v) erythrocyte solution.. Cells were carefully mixed, sedimented for 10 min at 350 g, and T-cell rosetting was carried out for hr in the dark at 4C. Successively, pellets were carefully resuspended with 12 l PBS (RT) to preserve rosettes, layered onto Lymphoprep gradients, and centrifuged for 18 min at 800 g.While discarding the supernatant and nonrosetted interphase cells, pelleted T-cell  erythrocyte rosettes were resuspended in PBS and washed three times.Rosettes were then suspended in 5-10 ml erythrocyte lysis buffer (Gieseler et al., 1991) and incubated for 15-30 min at 37C.Thereafter, T lymphocy es were obtained by several washes to remove red-cell debris.T-cell's viability was > 95% in all of the cases, as determined by trypan blue exclusion.

To employ T cells as allogeneic MLC responder cells, we prepared T-cll pools from four to six donors.Aliquots of 2.5 107 T cells per 450 /xl FCS (PAA Biologics, Marburg, FRG) were then portioned into precooled cryotubes (Nunc, Wiesba- den, FRG).Immediately after adding 50 /xl DMSO each, the tubes were frozen to-70C at 1-2C/min.Pools were used no later than 30 days after torage.Viability of thawed T-cell pools, as determined by trypan blue exclusion, was --90%.


Scanning Electron Microscopy

To preserve general cell morphology and veil expression, we avoided time-consuming preparatory steps and physical alterations.Round 8-mm cover- slips were precoated with 0.01% poly-L-lysine in deionized water and covered with droplets o