INTRODUCTION

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An antigen-presenting cell (APC) can be defined as any cell that expresses MHC or related molecules (e.g., CD1) that bind antigenic components such as peptides, and can be recognized by one class of T cell or another. Thus cytotoxic T cells and helper T cells generally recognize peptide-class I and -class II MHC complexes, respectively, on the surface of the APC. Most if not all mammalian cells can function as APCs, permitting T cells to survey their surfaces and mount an appropriate response if they contain, or have acquired, foreign antigens such as microbes. In general, activated T cells can respond to any type of APC expressing the relevant peptide-MHC complexes for which their T cell receptors are specific.

Dendritic cells (DCs) are a class of specialized APCs that not only can express peptide-MHC complexes for T cell recognition, but can have the additional capacity to deliver so-called costimulatory signals to naive T cells, thereby triggering T cell activation and the initiation of many adaptive (lymphocyte-mediated) immune responses; this may be referred to as the sensitizing function of DCs. Remarkably, evidence is also mounting that DCs may have crucial roles in the converse process, that of induction of T cell tolerance, through deletion or inactivation of antigen-specific T cells; this can be referred to as the tolerizing function of DCs.

Accumulating evidence has demonstrated the existence of distinct subsets or lineages of DCs, each potentially having a different immunological function; different maturation stages of DCs, manifested by phenotypic and functional plasticity; distinct migration pathways of DCs, permitting homing to and localization in distinct anatomical subcompartments, in relation to their various functions; and the effects of microbiological agents on these cells, including the means by which microbes can subvert the immune response by acting at the level of the DC.

The three main sections of this article provide a background and a summary of our data together with speculations on future directions in five different, although related, research areas: first, chemokines, human immunodeficiency virus (HIV), and malaria; second, cancer immunotherapy; and third, transplantation tolerance. For general reviews see References 1-3.

	DENDRITIC CELLS, CHEMOKINES, HIV, AND MALARIA

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DCs can be grown in culture from progenitors contained within bone marrow or blood. DCs generated from human CD34⁺ progenitors differentiate in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus tumor necrosis factor  (TNF-) along two distinct pathways (1,2). In one, a CD1a⁺CD14 intermediate gives rise to CD1a⁺CD14 cells resembling Langerhans cells (LCs), the DCs of skin epidermis. In the other, a CD1aCD14⁺ bipotential intermediate (that can alternatively give rise to macrophages if cultured in macrophage CSF [M-CSF]) generates CD1a⁺CD14 cells that have thus been termed myeloid DCs. The CD34⁺ progenitors for these two pathways can apparently be distinguished depending on whether they do, or do not, respectively express the cutaneous lymphocyte-associated antigen (CLA) antigen, a ligand for E-selectin. The precise physiologic correlates of the two types of DC are not known with certainty, but it seems likely that one (the LC type) populates nonvascularized epithelia such as skin epidermis as LCs, whereas the other (myeloid DCs) may populate vascularized tissues such as heart and kidney as interstitial DCs. While both cell types can stimulate primary T cell responses in vitro, only the myeloid (presumptive interstitial) DCs can induce naive B cells to differentiate into IgM-secreting cells after CD40 ligation and provision of exogenous interleukin 2 (IL-2) in culture (4). It has therefore been speculated that the LC type of DC may be primarily involved in the induction of "cell-mediated immunity" (to use a historical term) whereas myeloid (interstitial) DCs may stimulate T cell help and antibody production by B cells, so-called "humoral immunity," against foreign antigens. On the face of it this would make sense, given the relative inaccessibility of antibodies to pathogens entering the skin epidermis, for example, compared with those in the interstitial spaces of vascularized tissues.

DCs residing within nonlymphoid tissues (e.g., LCs of skin and interstitial DCs of heart and kidney) have specialized capacities for internalization and processing of foreign antigens such as microbes that gain access to these sites, but they have little or no costimulatory activity. Under normal circumstances, naive T cells do not have access to these areas and it thus seems to make sense that LCs and interstitial DCs can migrate to secondary lymphoid tissues, where it is thought they interact with antigen-specific naive T cells and induce primary T cell responses; for example, epidermal LCs can migrate via lymph to regional lymph nodes, while interstitial DCs can migrate via blood to spleen as well (5). A potent stimulus for DC migration from nonlymphoid sites is provided by a variety of microbial products and/or cytokines generated locally in response to them (6). In response to such stimuli, nonlymphoid DCs can load newly synthesized MHC molecules (e.g., class II) with antigenic peptides and display these complexes at the cell surface, and also begin to express the costimulatory molecules required for T cell activation. These cellular responses, which seem to accompany DC migration from the periphery into central lymphoid tissues, and which can also be demonstrated in vitro, have been termed maturation. Arguably, by virtue of their capacity to respond directly to microbial stimuli and signals of innate immunity (e.g., inflammatory cytokines), and thereby to acquire the capacity to interact with and stimulate responses of T and B cells, DCs provide a central link between innate and adaptive immunity.

While it is possible to grow relatively large numbers of DCs from bone marrow or blood DC progenitors (see above), a generally more convenient source of cells for many experimental and certain immunotherapeutic procedures (see below) are those DCs derived from monocytes (1, 2). When cultured in the presence of GM-CSF plus IL-4 (or IL-13), human CD14⁺ monocytes differentiate into cells resembling immature DCs with potent antigen uptake and processing capability; similar cells have been derived in mouse (7). On subsequent exposure to microbial products such as lipopolysaccharide (LPS), cytokines such as TNF-, or signals mimicking contact with T cells such as CD40 ligation, these DCs mature into cells with potent sensitizing functions. Although not proven, other DC progenitors (see above) may be responsible for maintaining the basal levels of DC subsets in normal tissues. In contrast, it seems possible that peripheral blood monocytes can provide a rapidly mobilized source of macrophages or DCs, depending on the local cytokine milieu, after recruitment to sites of infection or inflammation (8). Most of our investigations of human DCs have utilized monocyte-derived DCs (see below).

Chemotaxis and Transendothelial Migration of DCs in Response to Chemokines

Chemokines have chemotactic activity for leukocytes, and play important roles in leukocyte activation and trafficking to sites of inflammation (9). All human chemokines identified to date can be categorized according to their cysteine motifs close to the N terminus. The two major classes are the CC and CXC chemokines, in which the first two cysteines are adjacent or separated by a single residue, respectively, and minor classes of C and CX3C chemokines have also been identified. It is thought that chemokines, such as those produced at inflammatory sites, bind to proteoglycans on cell surfaces and establish concentration gradients in that area. Leukocytes sense such gradients by virtue of chemokine receptors, members of the G protein-coupled receptor superfamily. Nearly all receptors are specific for one class of chemokine only, but a given receptor can bind several chemokines within that class, and multiple chemokines can often bind to more than one receptor; for a summary of more recent nomenclature, see Reference 10.

Chemotaxis and transendothelial migration (TEM) of DCs in response to various chemokines were studied in vitro, using, respectively, bare transwells and transwell inserts on which monolayers of an immortalized human microvascular endothelial cell line (HMEC-1) were established (11). Our studies demonstrated that immature DCs, generated from monocytes in the presence of GM-CSF and IL-4, exhibited potent chemotaxis and TEM in response to the CC chemokines macrophage inflammatory protein 1 (MIP-1), MIP-1, RANTES (regulation on activation, normal T cell expressed and secreted), and monocyte chemotactic protein 3 (MCP-3), but weak responses to MIP-3 and the CXC chemokine stromal cell- derived factor 1 (SDF-1). The potential significance of these findings is that immature DCs seem to respond preferentially to "inducible" chemokines that are produced at inflammatory sites, for example.

In dramatic contrast to immature DCs (see above), after DC maturation was induced by culture in LPS, TNF-, or IL-1, responses to the former CC chemokines were reduced or abolished, whereas responses to MIP-3 and SDF-1 were markedly enhanced. These changes in chemokine responsiveness correlated with changes in chemokine receptor expression by the cells: after maturation expression of CCR5 (binds MIP-1, MIP-1, and RANTES) was reduced, while that of CXCR4 (binds SDF-1) was enhanced. Other investigators (e.g., References 11 and 12) have reported similar findings, for example, that increased responsiveness of mature DCs to MIP-3 (also known as EBI1 ligand chemokine, ELC) and the CC chemokine 6Ckine (also known as secondary lymphoid tissue chemokine, SLC) correlated with upregulation of the appropriate receptor (CCR7). Thus mature DCs seem to respond preferentially to "constitutive" chemokines that are expressed in T cell-rich areas of secondary lymphoid tissues to which these cells home.

Taken together, we have suggested (11) at least two stages for regulation of DC migration in which one set of (inducible) chemokines regulates recruitment into or within peripheral, nonlymphoid tissues, and another set of (constitutive) chemokines controls entry to central lymphoid tissues. It is of note that DCs can themselves secrete most if not all the chemokines mentioned above, particularly after exposure to maturation stimuli. Interestingly, chemokine receptors such as CCR5 are downregulated on exposure to the respective endogenous or exogenous (inducible) chemokines, whereas expression of CCR7 is not thus regulated by its respective (constitutive) chemokines (13). Hence it has been suggested that DCs can self-regulate their own migratory behavior and also recruit other cells (e.g., T cells) necessary for the induction of immune responses.

M-Tropic HIV Can Induce and Modulate DC Chemotaxis

Together with CD4, chemokine receptors can function as coreceptors for HIV-1 entry into target cells such as macrophages, T cells, and DCs (14, 15). Although molecular mechanisms are not completely understood, it is thought that on binding to CD4, gp120 of the viral envelope undergoes a conformational change that promotes coreceptor binding. Residues of the V3 loop of gp120, together with other regions of the molecule, are critical for gp120 binding to the respective coreceptor (see below). The interaction of gp120 with coreceptor may then lead to another conformational change eventually leading to viral fusion (via gp41) with the plasma membrane of the target cell.

Although by no means an absolute rule, the distinct tropisms of different HIV-1 isolates for various types in culture correlate broadly with the particular chemokine receptors expressed by those cells. Thus macrophage-tropic (M-tropic) or non-syncytium-inducing isolates tend to utilize CCR5 as a coreceptor, whereas T cell line-tropic (T-tropic) isolates that are generally syncytium inducing tend to utilize CXCR4. This explains earlier findings in which chemokines such as MIP-1, MIP-1, and RANTES were shown to suppress infection by M-tropic but not T-tropic strains of HIV-1, whereas the converse pattern was exhibited by SDF-1 (14). Given that chemotaxis of immature and mature DCs can be induced by ligation of CCR5 and CXCR4, respectively, we investigated whether binding of HIV-1 isolates could induce or modulate DC chemotaxis (16).

Immature DCs were again generated by culture of monocytes in GM-CSF and IL-4. When tested in bare transwell assays, DCs underwent chemotaxis in response to supernatants of M-tropic BaL HIV derived from PM1 cells, but not to those of T-tropic IIIB HIV derived from H9 cells. To determine whether chemoattractant activity was likely due to virus, supernatants were concentrated across a 100-kD molecular mass cutoff membrane. Strong chemotactic activity was exhibited by the concentrate of BaL, but little by the virus-free concentrate of PM1 supernatant or the filtrate. Chemotaxis did not occur in response to the concentrate of filtrate of IIIB or to virus-free concentrate of H9 cells.

Recombinant BaL gp120 was expressed in a baculovirus system and chemotactic activity for DCs was compared with that of IIIB gp120. Strong chemotactic activity for immature DCs was exhibited by BaL gp120, as well as by recombinant gp160 of the M-tropic JRFL strain of HIV. In contrast, chemotaxis did not occur in response to IIIB gp120, or to recombinant gp120 from the T-tropic MN and SF2 strains of HIV. We therefore concluded that M-tropic, but not T-tropic, strains of HIV can be chemotactic for immature DCs and that this activity resides in the gp120 component of the viral envelope.

Pretreatment of DCs with CC chemokines MIP-1, MIP-1, or RANTES was found to diminish or abolish chemotactic responses to all of these chemokines, but had no effect on responses to SDF-1. Pretreatment of DCs with BaL viral supernatant likewise diminished responses to these CC chemokines but had no effect on responses to SDF-1. In contrast, pretreatment with IIIB viral supernatant had no effect on CC chemokine responses but increased responses to SDF-1. Pretreatment of DCs with recombinant BaL gp120, or coincubation during the chemotaxis assays, abolished responses to the CC chemokine MIP-1. In contrast, pretreatment with IIIB gp120 was without effect on responses to the CC chemokine RANTES, but increased or had no effect on responses to SDF-1; similar findings were made for mature DCs. We concluded that binding of M-tropic HIV to immature DCs results in a dramatically impaired ability to respond to CC chemokines, whereas binding of T-tropic HIV can enhance chemotaxis to SDF-1, and that this is dependent on expression of the respective gp120 molecules (16).

Our findings suggest that chemotaxis of immature DCs toward HIV in peripheral sites (e.g., mucosae) after infection with M-tropic strains may facilitate infection of these cells and dissemination of the virus in the periphery. In addition, the profound inhibition of DC chemotaxis seen in response to CC chemokines, but not to SDF-1, may result in local arrest (and hence greater probability of infection) prior to migration into lymphoid tissues. It has been shown that explosive production of HIV occurs in aggregates or syncytia of DCs with T cells (17). It is intriguing that early HIV-1 infection is associated with M-tropic strains but that progression to acquired immunodeficiency syndrome (AIDS) is often linked to selection for T-tropic strains, and it is tempting to speculate that the relative expression of CCR5 versus CXCR4 on immature versus mature DCs generates selective pressure for this change in HIV-1 tropism.

Malaria-infected Erythrocytes Modulate the Maturation of DCs

Subversion of APC function, and hence host immunity, has been described at various levels for a number of viruses and some bacteria. We have identified a mechanism by which another pathogen, the malaria parasite (Plasmodium falciparum), can modulate DC function (18). It was already known that the ability of malaria-infected erythrocytes to adhere to vascular endothelium is related to disease pathogenesis; binding can be mediated via an interaction between parasite proteins such as P. falciparum erythrocyte membrane protein 1 (PfEMP-1) and thrombospondin, CD36, or CD54, for example. When immature DCs were incubated with erythrocytes infected with parasite lines that can adhere to these molecules, the latter two of which are also expressed by DCs, the cells were found to bind and internalize the erythrocytes and to become refractory to a subsequent potent maturation stimulus (LPS). Consequently, there was a dramatic decrease in the ability of the DCs to stimulate T cell responses to malarial and other antigens. In contrast, coincubation with erythrocytes infected with parasite lines that did not express PfEMP-1 antigens, and that did not adhere to the DCs, was without effect.

It had been assumed that the adhesive properties of malaria parasites have evolved to mediate their sequestration to the endothelium of peripheral tissues, and so to reduce destruction by splenic macrophages. Our findings led us to suggest instead that adhesion of parasite-infected erythrocytes to dendritic cells confers a selective advantage on the parasite in that the acquisition of antimalarial immunity is delayed through modulation of DC function. Clearly, identification of the immunomodulatory component(s) of the malaria parasite may lead to the development of new agents to downregulate immune responses in autoimmunity or allergy to our own benefit.

	DENDRITIC CELL-BASED IMMUNOTHERAPY OF CANCER

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There is considerable international interest in the possibility that the potent sensitizing functions of DCs could be utilized for the induction of antitumor responses in cancer patients, and a number of clinical trials to this end have been or are being performed in various centers worldwide. It has been proposed that the administration of DCs pulsed with tumor antigens may overcome afferent defects at this level and thus permit the generation of effective antitumor immunity in cancer patients. Certainly, a large number of experimental studies in murine systems have suggested that this should be feasible, and some encouraging results have been obtained from the few clinical trials completed to date (19). However, most trials require the identification of specific tumor antigens and the definition of antigenic peptides that can bind to the particular MHC alleles expressed by each patient, in order to use peptide-pulsed DCs as an immunotherapeutic regimen.

To attempt to develop a more "universal" DC-based immunotherapeutic approach, we have performed a phase I trial of autologous dendritic cells injected into skin metastases of patients with stage IV disease of potentially any primary malignancy (Chao, D., A. Harris, and J. M. Austyn, unpublished data). The rationale of this study was that autologous DCs injected into metastatic skin lesions may acquire tumor antigens locally before migrating to regional nodes (for example), where they could potentially trigger responses of tumor antigen-specific T cells. Patients underwent leukapheresis, and autologous DCs were grown from blood monocytes in the presence of GM-CSF and IL-4. After maturation in monocyte-conditioned medium, and pulsing with tetanus toxoid as a recall antigen (in order to assess any possible induction or enhancement of immunity to a "model" antigen), DCs were injected into a single accessible metastatic skin lesion at 2-wk intervals a total of three times.

To date (April 1998 to August 1999) eight patients have been treated, of whom seven received two or more immunizations and were therefore fully assessible. Of the eight patients, there were six cases of metastatic melanoma, one of breast carcinoma, and one of colorectal cancer. There were no significant clinical toxicities associated with any of the immunizations, the worst being Grade II pyrexia and skin inflammation in one patient. Delayed-type hypersensitivity skin tests showed an increased response to tetanus toxoid in only two patients but, importantly, in vitro proliferation assays revealed enhanced responses in five. This suggests that mature DCs can apparently migrate from a skin lesion and induce or enhance T cell immunity to a defined antigen. The challenge now, assuming this approach is developed further, will be to facilitate the acquisition of tumor antigens by the administered DCs, possibly by inducing tumor cell apoptosis in skin lesions because antigens from apoptotic cells may be presented particularly well after uptake by DCs (22).

	DENDRITIC CELLS AND THE INDUCTION OF TRANSPLANTATION TOLERANCE

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In addition to the definition of distinct progenitors that can give rise to LCs and myeloid DCs in culture, and of techniques for generating DCs from peripheral blood monocytes, a distinct DC progenitor of lymphoid origin has been identified in human bone marrow and mouse thymus (1, 2). In the latter case, a CD4^lo progenitor has been isolated that can generate T cells, B cells, natural killer (NK) cells and DCs, but not myeloid cells, in vivo. Such lymphoid DCs express CD8 homodimers, and the DEC-205 molecule. In the human, a related cell type may be the so-called lymphoblastoid T cells of lymphoid tissues, which give rise to DCs after isolation and culture in IL-3 and ligation of CD40. Because these cells can be identified within and close to the high endothelial cells of tonsils, for example, it may be that lymphoid DCs directly populate secondary lymphoid tissues from the blood, without a period of prior residence within peripheral tissues. It has been suggested that such cells may play an important role in the induction of peripheral tolerance to self antigens (23), and in the induction of central tolerance within the thymus. Conceivably, it may become possible to utilize or manipulate these cells for the induction of tolerance in immunotherapy of autoimmune diseases and allergy.

We have defined conditions for growth of stably immature, maturation-resistant DCs from mouse bone marrow (24). An important parameter was the use of low concentrations of GM-CSF and the absence of IL-4. These cells are presumably of myeloid origin, have an immature phenotype, have little or no costimulatory activity in culture assays, and do not mature in response to LPS. Remarkably, intravenous administration of as few as half a million of these cells of donor strain to allogeneic mice 7 d before transplantation resulted in indefinite (> 50 d) survival of fully vascularized, heterotopic cardiac allografts.

To investigate the mechanism of tolerance induction by these cells, tolerizing DCs were compared with "conventional" sensitizing DCs for their capacity to induce T cell responses in a transgenic system (Suri, R., N. Kukutsch, S. Fowler, F. Powrie, and J. M. Austyn, unpublished data). The two types of DC were pulsed with a relevant ovalbumin peptide before separate intravenous administration to recipient mice that had been reconstituted with DO11.10 T cells, expressing a transgenic T cell receptor specific for the ovalbumin peptide-MHC complex. Administration of sensitizing DCs induced marked clonal expansion of the transgenic T cells whereas tolerizing DCs did not. Remarkably, however, both cell types induced broadly similar T cell activation, in terms of blastogenesis, proliferation (measured using a carboxyfluorescein diacetate succinimidyl ester [CFSE] label), and phenotypic profiles, and similar cytokine secretion. We therefore currently assume that T cells activated by the tolerizing DCs undergo rapid apoptosis in vivo.

Migration studies using fluorochrome labels demonstrated that both tolerizing and sensitizing DCs home to T cell-rich areas of spleen after intravenous administration, although the sensitizing cells may persist somewhat longer in these sites. Importantly, high-power microscopy of the spleens of recipients of tolerizing DCs revealed that the fluorochrome appeared to be associated with apoptotic, rather than intact, cells. We are currently investigating whether an important property of the tolerizing population is to undergo apoptosis within lymphoid tissues, after which their contents may be internalized by other DCs (possibly lymphoid DCs?) and presented for the deletion of antigen-specific T cells or the induction of regulatory T cells. Such T cell deletion or regulation may contribute to the state of tolerance seen in our transplantation studies (see above).