discussion

918 28 th F O R U M I N I M M U N O L O G Y

PRICKE'r'r, T.C.R., McKENZIE, J.L. & HART, D.N.J. (1988), Characterization of interstitial dendritic cells in human liver. Transplantation, 46, 754-761.

RAMIREZ, G., BRAATHEN, L.R., KUNZE, R.O.F. & GELDERBLOM, H. (1988), In vitro infection of human epidermal Langerhans cells with HIV. Advanc. exp. h4ed. Biol., 237, 901-905.

ROMAN1, N., LENZ, A., GLASSL, H., STOSSEL, H., STANZL, U., MAJDIC, O. • SCHULER, G. (1989a), Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. J. invest. Dermatol., 93, 600-609.

ROMANI, N., KOIDE, S., CROWLEY, M., WITMER-PACK, M., LIVINGSTONE, A.M., FATHMAN, e.G., INABA, K. & STEINMAN, R.M. (1989b), Presentation of exogenous protein antigens by dendritic ceils to T-cell clones: intact protein is presented best by immature, epidermal Langerhans cells. J. exp. Med., 169, 1169-1178.

ROMANI, N., WITMER-PACK, M., CROWLEY, M., KOIDE, S., SCHULER, G., INABA, K. & STEIN- MAN, R.M. (1988), Langerhans cells as immature dendritic cells. CRC Press, Boca Raton, FL, June 1990.

SCHULER, G. & STEINmAN, R.M. (1985), Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J. exp. Med., 161, 526-546.

SHIMADA, S., CAUGHMAN, S.W., SHARROW, S.O., STEPHANY, D. 8£ KATZ, S.I. (1987), Enhanced antigen-presenting capacity of cultured Langerhans cells is associated with marked- ly increased expression of la antigen. J. lmmunol., 139, 2551-2555.

STEINMAN, R.M. & INABA, K. (1989), Immunogenicity: role of dendritic cells. Bioassays, 10, 145-152.

TSCHACHLER, E., GROH, V., PoPOVlC, M., MANN, D.L., KONRAD, K., SAFAI, B., ERON, L., DIMAR- zo VERONESE, F., WOLFF, K. & STINGL, G. (1987), Epidermal Langerhans cells - - a target for HTLV-III/LAV infection. J. invest. DermatoL, 88, 233-237.

VAN VOORHIS, W.C., HAIR, L.S., STEINMAN, R.M. & KAPLAN, G. (1982), Human dendritic cells: Enrichment and characterization from peripheral blood. J. exp. Med., 155, 1172-1187.

VAN VOORHIS, W.C., VALINZKY, J., HOFFMAN, E., LUBAN, J., HAIR, L.S. & STEINMAr:, R.M. (1983), The relative efficacy of human monocytes and dendritic cells as accessory cells for T-cell replication. J. exp. Med., 158, 174-191.

WILDERS, M.M., SMINIA, T. & JANSE, E.M. (1983), Ontogeny of non-lymphoid and lymphoid cells in the rat gut with special reference to large mononuclear Ia-positive dendritic cells. Immunology, 50, 303-314.

WITMER-PACK, M.D., OLlVIER, W., VALINSKY, J., SCHULER, G. & STEINMAN, R.M. (1987), Granulocyte/macrophage colony-stimulating factor is essential for the viabilit:~ ~rtd function of cultured murine epidermal Langerhans cells. J. exp. Med., 166, 1484-1498.

WITMER-PACK, M.D., VALINSKY, J., OLIVER, W. & STEINMAN, R.M. (1988), Quantitation of SUl- face anti.gens on cultured routine epidermal Langerhans cells: rapid and selective increase m the level of surface MHC products, d. invest. DermatoL, 90, 387-394.

YOUNG, J.W. & STE~NMAN, R.M. (1988), Accessory cell requirements for the mixed leukocyte reaction and polyclonal mitogens, as studied with a new technique for enriching blood dendritic cells. Cell Immunol., 111, 167-182.

DISCUSSION

W.E. Bowers and E.M. Goode|l:

1. m The functional requirements for dendritic cells in different species may differ. For example, the point has been made that, in mouse, the dendritic cell is the unique accessory cell that ac- tivates resting T cells but, once ac- tivated, any Ia+ accessory cell can restimulate the T cell. In rat, the fin-

dings of Green and Jotte (1985) and our laboratory indicate that Ia+ accessory cells other than dendritic cells, particularly macrophages, are not active in restimulating either T-cell blasts or memory cells generated in an ML R or mitogenic responses.

2 . - Not enough information has been obtained to conclude firmly that the Langerhans cell is a highly effective antigen-presenting cell and poor sen- sitizer, in contrast to the dendritic cell

L Y M P H O I D D E N D R I T I C C E L L S 9 i 9

where these functions are reciprocally expressed. Data conflicting with these conclusions (Hauser and Katz, 1988; Shimada et al., 1987) are not adequate- ly presented. If the conclusions about the efficiency of antigen processing by the Langerhans cells and their subse- quent trafficking patterns are true, one would expect that the preferred route of immunization would be the skin.

A considerable l i te ra ture has established the ability of splenic and lymph node dendritic cells to elicit T-cell responses and antibody formation in vitro; the discrepancy between these f'm- dings and the concept that the dendritic cell does not process/present antigen well have not been addressed.

3. - - One of the major difficulties in studying dendritic cells is their low frequency in all tissues. This fact makes it necessary to enrich for dendritic cells through elaborate and often lengthy purification procedures. The function of dendritic cells may be altered through these procedures. If dendritic cells are not purified, the cell populations con- taining dendrit ic cells are often heterogeneous as is the case with the cell populations collected from the thoracic duct after mesenteric lymphadenectomy. Even though the frequency of dendritic cells may be increased, the ex- treme morphological heterogeneity of the cells causes problems in the positive ident i f icat ion of dendri t ic cells. Reagents that can be used to identify and purify dendritic cells from freshly isolated tissues are greatly needed for continued progress in understanding the role of dendritic cells in the immune system.

4. - - The lineage of dendritic cells and its relationship to macrophage/ monocytes still remains to be established. Antigens common to Langerhans cells and macrophages is not conclusive evidence for a shared lineage. For example, the distribution of Thy-I does not mean that brain and T cells are related. If macrophages and dendritic cells do have a common lineage, they have a branch point prior to the cell that responds to M-CSF (Klinkert and Stef- fen, 1988).

G. Kraal:

It is clear that the definition of a dendritic leukocyte is expanding. From the in vitro isolated non-adherent spleen cell, it now encompasses the Langerhans cell, the veiled cell and, according to most authors of this Forum, also the in- terdigitating cell in the T-cell areas of lymphoid organs. Although the migration studies of isolated dendritic cells and the skin painting experiments as reported in the contributions of Sigbjorn Fossum and Stella Knight are quite convincing, both Jonathan Austyn and Ralph Steinman are still uncomfor- table in stating that IDC are the in vivo DC of the lymphoid organs. Part of it may be due to the fact that DC are not a static population and undergo differentiation, so that not all IDC at a given time in a T-cell dependent area of a lymph node or spleen have the same properties on which basis they are isolated in vitro, e.g. the absence of Fc receptors. Therefore, upon isolation, the in vitro dendritic leukocyte population may not comprise all of the inter- digitating cells.

That not all dendritic cells are equal is also clear from the maturation steps which are needed, as outlined by Nikoiaus Romani and Geroid Schuier for the Langerhans cell and by Jonathan Austyn for DC at other non-lymphoid sites. From what Romani and Schuler report, it is not the amount of MHC molecules which determines the unique ability to stimulate a primary T-cell response. Perhaps as part of the different ia t ion, other glycosylation pathways are followed, leading to the distinctive MHC class II molecules on mature DC with fewer sialic acid moieties, as described by Kees Melief. It would be interesting to look at the MHC molecules on Langerhans cells in terms of sialic acids. More knowledge of the steps involved in Langerhans dendritic cell maturation, and also of the earlier precursor differentiation as studied by Bill Bowers and Estelle Goodell, may lead us to the intriguing possibilities, outlined by Ralph Stein- man, of manipulating DC in such a way that they can help in protective immunity and vaccination.


An important point which emerges from these contributions is the mobility of the dendritic leukocyte. Whether it is the migration of Langerhans cells or other immature dendritic cells from non-lymphoid sites to lymph nodes or spleen (what to think of this fascinating concerted movement of LC after skin painting, as reported by Stella Knight ?) or the mobility within a T-cell- dependent area to reach the appropriate T cells, all of it dearly indicates that DC are a population of mobile cells. This emphasizes their importance as initia- tors of immune responses and may strengthen the concept that, as a population, they could very well be the first line of defence in our immuno- logical surveillance.

N. Romani and G. Schuler:

The eight papers accumulated in this Forum present state of the art views of dendritic cells (DC) from different standpoints (in vitro, in vivo). We feel that the following four aspects are worth commenting on.

1. ~ Functional in vitro data (paper by Melief).

DC are extraordinarily potent stimulators of resting/memory T lymphocytes. This fact, originally described by the Steinman group, is convincingly confirmed here. It is true for CD4 + as well as for CD8 + T cells. In particular, DC are outstandingly good stimulators for CD8 + T cells: CD4 + T cells, are no longer needed to provide help in this in ,,itro system. Does this mean that DC are efficient processors of the viral proteins used in Melief's studies as well as in Macatonia's experiments (see paper by Knight) ? Are these data therefore in contrast to our findings that DC as well as cLC are inactive in antigen processing ? We believe not, mainly because we were looking at MHC class-II- restricted proliferative responses, whereas Melief has been studying class- I-restricted ¢ytotoxic T-cell responses. These pathways are different . Moreover, different numbers of con-

taminating processor cells (macrophages, B cells) in t?,e DC and/or responder T-cell populations may ex- plain seemingly conflicting results. The obvious question of how the virus enters the DC arises: is it by phagocytosis of virus particles or is it by fusion of the virus to the plasma membrane? Clearly, this issue still needs much work.

The unique capacity of DC to stimulate resting T cells is thought to de- pend on their capacity to cluster T cells in an antigen-independent manner. As a possible molecular basis, Melief offers the different sialylation patterns of DC as compared to other types of antigen- presenting cells. DC have less sialic acid on their surface and are therefore less negatively charged. As a consequence, they are believed to aggregate more readily with lymphocytes because the electrostatic repulsion forces between the cells are lower. In preliminary experiments, we observed that removal of sialic acid moieties from the surface of freshly prepared LC (i.e. weak stimalators of resting T cells) by neur~minidase treatment does not render them more immunostimulatory. Therefore, we think that there is another (possibly additional) possibility: a distinct but as yet undefined

~ l u O L ~ , J t l l l ~ llllLICl~l~,Lll~ O i l LII~; LJI~., [ d l l U

cLC) surface might be responsible for the efficient antigen-independent clustering of DC with T cells. We and others are currently approaching this problem by trying to raise monoclonal antibodies against this molecule.

2. ~ Phenotypical and ontogenetical aspects (papers by Kraal and Bowers).

Kraal compares the phenotypical profiles of DC and LC. We would like to add that, in man, there are some other markers that are expressed by DC but not by (most) resident LC: CD40 and particular HLA-DQ epitopes (RFD1, 8C4).

By FACS analyses, Crowley et al. (1989) have delineated a minor subset of spleen DC that has a reciprocal phenotype, as compared to the bulk of the spleen DC, with respect to three

L Y M P H O I D D E N D R I T I C C E L L S 92!

markers" 33D1+, J l l D - and NLDC-145- (most DC) vs. 33D1-, J I ID÷ and NLDC-145 ÷ (subset). In this respect, the phenotype of the DC subset is identical to LC (both resident as well as cultured) and thymic DC. These authors speculate that the subset might correspond to the interdigitat- ing cells (IDC) of the periarteriolar sheets, i.e. these DC might represent fixed, longer-lived IDC - - analogous to resident LC. The bulk of DC, however, which is 33D1 +, would con- sist of migratory DC - - those DC that have arrived from the periphery charged with antigenic peptides, have matured and are now ready to sensitize the T cells of the lymph node. It will be interesting to further phenotype this DC subset and also to functionally characterize it.

3. - - Functional in vivo data (papers by Fossum, Austyn and KnighO.

Evidence is accumulating for the scenario where immature DC (e..g. resident LC) pick up antigen m the periphery, process it and sensitize T cells in the respective draining lymph nodes. It should be emphasized, however, that the formal proof is still lacking. In other words, is it really so that the very same cells that sit in the epidermis as LC are the ones that sensitize the T cells in the node? The in

vitro data are certainly compatible with, if not supportive of, this hypothesis. In vivo, it is hard to unequivocally trace a cell on its way through the body. Un- fortunately, LC markers are only LC- specific in the epidermis; outside this tissue, they are shared with other types of haematopoietic cells. We also know that the standard contact sensitizers used in these studies penetrate easily down into the dermis. So it might well be that dermal cells migrate to the lymph nodes, or it might even be that free reagent reaches the lymph node. This is admittedly the reasoning of a devil's advocate but nevertheless, these points have to be considered.

4 . - Clinical aspects (paper by Steinman).

Here we would simply like to em- phasize the fact that most of what we know about DC has been derived from studying experimental animals. This is par t icular ly pronounced for LC research. The fascinating clinical possibilities described by Steinman should make it worthwhile to go through the difficulties of LC/DC research in the human system, such as poor and irregular supplies of tissue, still unsatisfactory enrichment procedures or inconsistencies due to working with individuals rather than with strains. It will be rewarding!

Reference.

CROWLEY, M., INABA, K., WITMER-PACK, M. & STE!NMAN, R.M. (!989), The cell surface of mouse dendritic cells" FACS analyses of dendritic cells from different tissues including thymus. Cell. Immunol., 118, 108-125.


J.M. Austyn:

Quite recently, I heard an eminent molecular biologist refer, with perhaps a hint of skepticism, to the "cottage in- dustry" that has grown up around the dendritic cell. I have also, on more than one occasion, heard an equally/eminent cell biologist wonder out loud if the dendritic cell might in fact be "just a fun- ny sort of mac rophage" . These remarks, presumably made in jest, made me think aJbout one or two more serious points which are quite pertinent to this Forum. Fundamentally, we still have difficulty in defining the various stages of development of the lineage that we like to refer to as dendritic leukocytes (others in this Forum have used the term "dendritic cells" in this general sense). Moreover, we still seem quite a way from explaining precisely how these cells handle antigens and induce immune responses.

The precise point at which dendritic leukocytes diverge from other haematopoietic lineages may become more accessible through studies similar to those described by Bowers. Certainly, it would be most advantageous to be able to grow populations of dendritic cells from bone marrow cultures, but definition of the requisite growth factor(s) is essential. One wonders whether epithelia could also play an important role in the development of dendritic leukocytes. For example, the Langerhans cell in situ shares a number of features in common with monocytes and macrophages, as discussed by Romani and Schuler, but its precursor has not been defined. Might cells resembling Langerhans cells develop if bona f ide monocytes were cultured with epithelial cells, or could this be used as the basis for an assay to define another precursor in blood ? Of all cells of this lineage, it seems that Langerhans cells might be situated close to or at the presumptive branch point with myeloid cells, but the fact that differentiation only occurs in one direction (towards the mature DC) argues at least that this cell is developmentally already committed.

From our own point of view, a particularly important issue is the nature of

the dendritic cell in blood• We feel fairly cenfident that a migration pathway for dendrit ic leukocytes f rom non- lymphoid tissues into the spleen exists. However, Steinman argues that mature dendritic cells cannot be isolated from fresh human blood, while Bowers can obtain these cells from dog but not rat blood. One of the major difficulties with our earlier studies (Kupiec- Weglinski et al., 1958) wa~ that we traced migration of mature cells isolated from spleen, since these were the most accessible to study, although our more recent work with heart allografts (Larsen et al., 1990) implies that the route is physiologically relevant, albeit only demonstrated in a transpW, antation setting. Clearly, it would be of interest to isolate these particular cells and examine their characteristics (e.g. immunostimulatory capacity) but this may prove unfeasible. One might expect these cells to resemble those described by MacPherson and others in afferent lymph or in central lymph after mesenteric lymphadenectomy and ir- radiation. Another area that requires more study is the nature of the cytokines that induce maturation and migration of dendritic leukocytes in vivo; the importance of GM-CSF, IL-I and TNF- alpha have been noted by several in this Forum, but others may also be im- n f l r f o n * ~ v u L L~J~I&L •

Some particularly important questions relating to the life cycle of dendritic cells have been raised by Fossum. As yet, we have no explanation for the discrepancy between his findings and ours relating to the T-dependency of migration from the blood into the spleen. Fossum notes the ability of certain types of dendritic leukocyte (e.g. in- terdigitating cells in situ) to phagocytose particles, and both Steinman and Romani and Schuler comment on the ability of Langerhans cells to handle protein antigens, presumably after end docytosis. It is clearly important to define how these cells endocytose particles and molecules. Perhaps this can be mediated via coraplement receptors and/or Fc receptors, and "natural antibodies" might be involved (Steinman, pers. comm.), since uptake must presumably occur before an immune

L Y M P H O I D D E N D R I T I C C E I , L S 923

response is generated, and therefore before specific antibodie~ have been produced. Knight discusses the cytological changes in Langerhans cells exposed to contact sensitizers and wonders whether antigen itself may in part promote dendritic cell development. This idea should be testable, although it is not clear how a dendritic cell could determine what is and what is not an antigen, and at present we prefer the idea that development is more likely to be triggered by locally produced inflammatory cytokines.

It is unfortunate, but perhaps telling, that there is little in this Forum relating directly to the presumptive dendritic cell-derived activation signals for T cells. So far, as noted by Steinman, it seems unlikely that IL-1 and IL-6 are involved in this process, and while both he and Kraal note cell surface molecules that might be relevant, such as LFA-I, ICAM-1, etc., no real consensus has emerged. One of the more intriguing recent observations, described by Melief, is that the MHC molecules on dendritic cells are relatively desialylated, and that cleavage of sialic acid from the surface of other cell types can increase their potency as accessory cells in certain responses. It will be fascinatip7 to see how this area develops, especla:ly in r ~ l ~ t ; r ~ n t r l r ~ t h ~ r c t i t r l ; ~ c ~ n t h ~ ; n t ~ r ~ _ • ~,,, t , ~ , g 1 , J • • • A t . i J V l , a J t ~ , l . ~ l , t . a ~ . a A ' ~ o ~ . l a A ~ A l ~ . , , A A . I L ~ . ~ , , A L a V ~

tion of antigenic peptides with MHC molecules. Clearly, future attempt'~ to produce monoclonal antibodies to functional determinants of other cell surface molecules may not only give new in- sights into mechanisms of T-cell activation by dendritic cells, but also produce reagents that can be used to define cells of this lineage more precisely.

The great importance of dendritic cells in the induction of immune responses is underlined once again in the contributions of Knight (contact sen- sitivity), Melief (cytotoxic T-cell responses) and Steinman (clinical situa- tions). It will be interesting to see whether the ability to overcome some types of Ir gene defect by immunization with dendritic cells, as detailed by Melief, will find a future clinica~ ap- plication, although it is important to define why this is possible in some

responses but not others (is it simply a case of the actual precursor frequency of specific T cells, or more?). The possibility of using dendritic cells as "adjuvants" for inducing responses to other exogenous antigens is noted by Steinman, who also suggests that den- dritk: cells might express the antigens relevant to autoimmunity. Perhaps one of the most topical issues in a clinical setting is the possibility (or likelihood) that dendritic cells can be infected by HIV-1. As noted by others, it seems quite conceivable that epidermal Langerhans cells or related cells in the genitourinary tract, for example, can be " ~ . . . . if --';";-* ~n~,~,,ted by HIV-I, so, we - , . v . , expect these cells to migrate via lymph or blood and transport the virus centrally (into nodes or spleen). We await with great interest publication of studies on the immunostimulatory function of dendritic cells from patients with AIDS.

There can be no doubt that studies on the role of precisely defined populations of dendritic leukocytes in clinical disease will become one of the Big Businesses of immunology in the future.

C.J.M. Melief:

Several discussants have brought forward the notion of a reciprocal rela- tienship between "immature" dendritic cells (DC), such as Langerhans cells (LC) from skin and mature DC in the lymph nodes, in that LC are much bet- ter than DC at processing a protein antigen such as myoglobin for presentation to T-cell clones. On the other hand, DC and cultured LC were both good at presenting the appropriate specific peptide fragment (Romani et aL, 1989, J. exp. Med. 169, 1169). These findings should be contrasted, however, with the obser,:ation that mature DC become readily infected in vivo and in vitro by a variety of viruses. With the exception of HIV, whirl• may affect DC function, other virus infections of DC result in excellent viral antigen presentation to T cells, at least to CD8 + T cells. Indeed, in the work of Macatonia et al. 19~,9 (J. exp. Med. 169, 1255) intact influenza virus and an im-

924 28 th FOR U M I N I M M U N O L O G Y

munodominant T-cell epitope of influenza virus were both efficiently presented by spleen DC to generate a primary in vitro CTL response against the same epitope.

In our own work, spleen DC were approximately 100 times more efficient than spleen cells in the presentation of Sendai virus to generate a virus-specific CTL response of primed T cells (Kast et al., J. Immunol., 140, 3186, 1988), directed against a single immunodomi- nant peptide encoded by the Sendai virus nucleoprotein gene (Kast and Melief, unpublished observations). Thus, DC are perfectly capable of uptake, replication, processing and presentation of viral antigens. Indeed, one would hope that DC could rapidly induce virus-specific responses in the most expedient way, whether they first en- counter virus in the lymph node, spleen or blood, all locations reached by virulent viruses before efficient immunity has developed. Preferential processing by an immature precursor of DC at these locations would only retard specific induction of immunity, a dangerous situation in the race against time between virus and host.

The proposed cooperation between LC and DC in which the LC process and the DC present is obviously unlikely in those virus infections in which the "porte d'entr6e" is not the skin. For another reason, too much emphasis on the failure of DC to process native antigen seems unwarranted at this time. Everyone agrees that DC are among the most powerful s t imulators of allospecific lymphocyte responses. Evidence is accumulat ing that allospecific reactions are directed against a variety of peptides processed from endogenous proteins, both MHC and non-MHC, including tissue-specific antigens. Whether "empty" MHC molecules can be recognized by allospecific T ceils is at present not known. Also, recent studies with a tumour cell mutant indicate that intact peptide processing is needed for optimal cell surface expression, correct folding and [32m associations of class I MHC molecules (Townsend et al., 1989, Nature, 340, 443). Indeed, peptides are

likely to cause the correct conformation of MHC molecules for stable cell surface expression. It is therefore unlikely that DC would be poor processors of antigens, since their cell surface MHC class I and II expression is higher than that of all other types of APC tested. These abundant MHC molecules have a decay rate. Therefore, during turnover, these MHC molecules have to be constantly charged with intracellularly generated peptides. This turnover also argues against the idea that all MHC molecules of DC are occupied by peptides processed during a previous stage of differentiation.

Thus, while I do not contest the notion that in the skin, LC could have a speciali~ed antigen uptake, carrier and processi~g function, this does not con- tradict a major contribution of DC in the uptake processing and presentation of many antigens on location in the blood or lymphoid organs.

Further work is clearly needed to establish what DC can and cannot do and with which other cells they cooperate in a given foreign antigen in- vasion.

The failure of DC to properly process and present native myoglobin to T cells could be due to failure of ~,~yuglobin to be taken up by DC. Viruses are apparently readily taken up and actively use the biosynthetic pathway of DC to allow optimal viral peptide entry into MHC class I molecules. Several studies have shown that only actively synthesized viral epitopes are efficiently presented to CD8+ cells in the context of class I MHC. Whether viruses use specific receptors on DC or take advantage e.g. of the low cell surface sialic acid oc- cupation of DC remains unknown.

Alternatively, DC may lack a prope~" machinery for processing of myoglobU~ and presentation in the context of class II MHC, even though enough myoglobin is taken up by the cell. I,~t this regard, a recent paper showed that a T-cell epitope recognized by both class-I- and class-II-specific T cells, when introduced in target cells after in- troduction in a synthetic minigene, was

L Y M P H O I D D E N D R I T I C C E L L S 925

only recognized by class I but not class II MHC-res t r i c ted T cells (Sweetsen et al., 1989, Nature, 342, 180). In general, the picture is emerging that the class I and class II MHC peptide processing pathways are different. As stated in my contribution to the Forum, future research should be directed at whether and how various particulate and soluble antigens enter DC and follow specialized processing pathways for class I and class II MHC presentation in DC. The antigen presentation machinery of DC should be compared with that of other types of APC and with precursors of DC. In the next decade, the subcellular and molecular details of antigen processing and presen- tat ion bv DC can hopeful ly be elucidated to a large extent.

S.C. Knight:

These papers not only summarize work in specialist areas but also describe much that is new or work in progress. It is evidence of the embryo nature of this field that the emphasis throughout is on the basic biology and life-history of the dendritic cells (DC) and the identification of many basic questions still to answer. Studies of more functional and clinically important aspects of DC frequently have to be deferred while more fundamenta l questions are answered. Basic questions asked and discussed by Bowers and Goodall relate to the bone-marrow origin of the DC and identify several areas where clarification is needed. They state that "almost nothing is known about the relationships between cells that differen- tiate into DC and those belonging to known haematopoietic lineages" and suggest that if the precursors "belong to the granulocyte/macrophage line then they branch from the maturation pathway prior to the point of action of M-CFS". Information is now available on these points from studies on stem cells in human bone marrow and peripheral blood (Reid, Fryer, Clifford, Kirk and Knight, submitted). Small colonies of dividing DC with their distinctive appearance and phenotype (including expression of CDla) were

obtained in methylcellulose cultures. The numbers were similar to those in rat bone marrow reported by Bowers and Goodall with fewer (1-2 per 105 mononuclear cells) in peripheral blood. Some DC were also found in mixed colonies with macrophages, suggesting an early divergence of these cell lineages at this stem cell stage. Bowers and Goodall also suggest that "antigen presentation would be important to study". The small colonies of DC from the human bone-marrow cultures have been picked out and used as stimulants in mixed leukocyte cultures in hanging drops. Using as few as 40 cells added to 105 allogeneic lymphocytes, potent proliferative responses were obtained. The findings of Bowers and Goodall of DC stem cells in the bone marrow in the rat is thus confirmed from studies of human cells.

Steinman discussed the view that identification and separation of human DC is particularly vital. We agree with this idea very strongly, particularly as our evidence suggests that there may be a higher percentage of DC precursors in human peripheral blood than is obvious from the small numbers of well-purified cells obtained with current techniques. Like Steinman, we have adapted our ~ r i o i n ~ l e ~ n ~ r ~ t i ~ n f ~ p h r l ; ~ l l ~ ue ;na

hypertonic metrizamide. Whole mononuclear cells from peripheral blood are cultured overnight on plastic and the non-adherent cells centrifuged over metrizamide. At this stage we obtain cells with around 25 °70 DC and as many as 2-3 070 of the original mononuclear cells are DC. A further purification by removing more adherent cells takes us to 50-70 070 DC but reduces the yield considerably, giving numbers comparable to those obtained by Steinman's technique. However, there is an obvious loss of DC during the final purification step and this means that there is still considerable room for improvement in separation procedure.

In two clinical areas where DC may have importance, as suggested by Stein- man - - autoimmunity and HIV infection - - there is now evidence to support this view. The first comes from studies

926 28 th FOR U M I N I M M U N O L O G Y

of experimental allergic thyroiditis in- duced in mice by injection of mouse thyroglobulin in complete Freund's ad- juvant. These mice have autoantibodies to thyroglobulin and infiltrating mononuclear cells in their thyroids. Purified DC from the spleens of these animals initiate similar symptoms in naive recipients. Similar findings were obtained using normal DC pulsed in vitro with thyroglobulin (Knight et al., 1988). These results, confirming and expanding similar findings in a rat model of experimental allergic encephalomye- litis (Knight et al., 1983) provide evidence for the view that DC are involved both in ir2tiation and promotion of autoimmunity.

The importance of DC in HIV infection has now been studied in depth

(Macatonia, Patterson and Knight, in preparation). These studies show infection of 3-21% of DC from peripheral blood of HIV-infected individuals using in situ hybridization for viral DNA, although < 0.20 % of labelled lymphocytes were found. The DC were infected, depleted from peripheral blood and failed to 9resent antigens and these defects preceded changes in T-cell number and function. The DC defect may thus underlie the immunosup- pressive effects of the virus.

From the work reported in these papers, it is clear that many basic questions on DC biology remain to be answered and that such studies will open the way to assessing the full potential of these cells in regulating both normal and abnormal immune responses.

References.

KNIGHT, S.C., FARRANT, J., CHAN, J., BRYANT, A., BEDFORF, P.A. & BATEMAN, C. (1988), In- duction of autoimmunity with dendritic cells: studies on thyroiditis in mice. Clin. Immunol. Immunopath., 48, 277-289.

KNIGHT, S.C., MARTIN, J., STACKPOOLE, A. • CLARKE, J. (1983), Induction of immune responses in vivo with small numbers of veiled (dendritic) cells. Proc. nat. Acad. Sci. (Wash.), 80, 6032-6035.

KEY-WORDS: Dendri t ic leukocyte, T lymphocyte , Immunogenes i s ; Phagocytosis, Endocytosis, Contact hypersensitivity, Langerhans cells; Forum.

DEDICATION

I would r, ~ to dedicate this Forum to the m e m o r y o f Professor J .H. Humphre:, whose wit, enthusiasm and insight prov ided inspiration to all who knew him

Gordon MacPherson

discussion

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