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Functions of myeloid and lymphoid dendritic cells
Immunology Letters 72 (2000) 101 – 105
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Functions of myeloid and lymphoid dendritic cells Alexander D. McLellan *, Eckhart Ka¨mpgen Department of Dermatology, Uni6ersity of Wuerzburg, Joseph-Schneider Str.2, Wuerzburg 97080, Germany Received 24 January 2000; accepted 26 January 2000
Abstract The bone marrow derived dendritic cell (DC) is an essential antigen presenting cell (APC) for the initiation of primary, T cell based immune responses. DC are a heterogenous haematopoietic lineage, in that many subsets from different tissues show different surface phenotypes, but the ability to stimulate antigen specific naı¨ve T cell proliferation appears to be shared between these DC subsets. It has been suggested that the so called myeloid and lymphoid-derived subsets of DC perform distinct stimulatory or tolerogenic functions. However, recent data has blurred this apparent distinction of DC subset function and shown that both subsets are at least capable of stimulatory and possibly even tolerogenic functions. Thus, the immunoregulatory potential of DC may depend less on ontology than on recent activatory or downregulatory stimuli. © 2000 Elsevier Science B.V. All rights reserved.
Historically, the concept of the lymphoid-derived DC dates from the identification of a shared precursor for DC and T cells in the murine thymus [1] and CD8 expressing DC in the spleen and thymus of adult mice [2]. These DC expressed CD8a homodimers in high density, but unlike mature CD8 T cells, with absent, or low level, expression of beta chains. Originally suggested to perform tolerogenic functions, even highly purified fractions of CD8a cells were capable of inducing significant T cell proliferation (albeit at lower levels than CD8a- cells). Further studies revealed that CD8a+ were at least equilavent to CD8a− DC in stimulating both CD4 and CD8 responses in vivo and in vitro [3 – 6]. A surprising recent finding is that, CD8a+, but not CD8a−, DC produce significant levels of IL-12 and prime to Th1 T cell responses [3 – 7]. In man, Grouard et al. [8] and Olweus et al. [9] identified CD4+/IL-3R+/CD11c− DC in both cord and peripheral blood, tonsil and bone marrow. Although these authors concluded differently as to whether these Abbre6iations: APC, antigen presenting cell; DC, dendritic cells; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; L, ligand; LC, Langerhans cell; TNF, tumour necrosis factor. * Corresponding author. Tel.: +49-931-2012725; fax: + 49-9312012700. E-mail address: [email protected] (A.D. McLellan)
DC were myeloid or lymphoid in origin, this human DC subset is often referred to as ‘lymphoid’ or plasmacytoid since they show low levels of CD13 and CD33 antigens, display prominent parallel arrays of endoplasmic reticulum and other morphological features resembling plasma cells. Nevertheless, these DC appear to derive from a M-CSFR+ progenitor capable of generating granulocytes as well as IL-3R+ DC [9]. This DC population may be identical to a CD4+ human thymic DC subset described earlier by Shortman’s group [10]. Both murine thymic DC precursors [11] and the human plasmacytotoid [8,9] DC show a dependence on IL-3 rather than GM-CSF for survival and development into mature DC. Interestingly, these CD4+/IL-3R+/ CD11c− DC, in contrast to their lymphoid-derived murine counterparts, were suggested to produce a predominantly Th2 response, while monocyte derived DC stimulated a Th1 phenotype in responding T cells [12]. Thus, if this human DC subset really is lymphoid in origin, then there is not a species consistent definition of the T cell stimulating functions of myeloid and lymphoid-derived DC. In mouse, there are other interesting differences between these two DC subtypes: For example, freshly isolated CD8a+ DC express higher levels of NLDC145 (a 205KD lectin-like molecule, with as yet unknown specificity [13]) than the CD8a− DC. Two groups have suggested that upon isolation and reinjection, the
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lymphoid-derived DC subset has a poor or absent afferent lymph homing ability [5,6]. Others have suggested that there is no inherent difference in migratory capacity between the two subsets [7,14] Also, in RelB knock out mice, CD8a− DC are lacking from spleen, despite normal numbers of CD8a+ DC [15]. Ikaros knock out mice lack B cells and show defects in T cell development, yet make an excess of monocytes and macrophages. Investigation of DC populations showed that the spleen and thymus of B cell deficient Ikaros−/− mice were also deficient in CD8a− DC and had greatly reduced numbers of CD8a+ DC [16]. Furthermore, a dominant negative Ikaros mutant which has defects in all lymphocytes including T cells, has neither CD8a− nor CD8a+ DC. Which leads into the striking conclusion that both CD8a+ and CD8a− may be of lymphiod origin, but that CD8a+ may be closer to T cell lineage than to B cells. Recently, Klatzmann and collegues have described a CD4+ DC population in lymph node that appears to be part of the myeloid subset [17]. Surprisingly, these CD4+/CD8a− DC are also present as a major population in spleen, making up 50% of CD11c+ low density fresh spleen cells (Fig. 1). In contrast to CD8a+ , DC, CD4+ DC express CD11b. Both CD4+ and CD8a+ subsets lack T cell (Thy1.2, CD3), B cell (CD19) markers (Fig. 1) and the F4/80, NK1.1 and Gr1 antigens (not shown). Since this CD4+ DC subset was originally excluded from earlier analyses by use of CD4 mAb to deplete contaminating T cells, the actual ratio of lymphoid to myleoid-derived DC present in spleen may be closer to 1:3, a figure somewhat lower
than earlier reported estimates of 1:1 [12] (see Fig. 1). Interestingly, we have found that a subset of CD4+/ CD8a−/CD11c+ DC clearly stains for NLDC145 (Fig. 1), and that NLDC-145 expression is upregulated on CD4+ by overnight culture, demonstrating that expression of this marker is not exclusive to CD8+ DC. Moreover, although the expression of CD8a is stable upon DC culture, CD4 expression is lost rapidly from DC following overnight culture ([18] and ADM & EK; manuscript in preparation). Thus, our results do not contradict the original description of cultured spleen DC as CD4− [19]. The function of this major CD4+/ CD11c+, DC subset and their anatomical location awaits further analysis. Langerhans cells (LC) provide more puzzlement as to the origins of various DC subsets. LC are extremely potent stimulators of T cells, undoubtedly bone marrow derived [20,21] and are widely thought to represent the ‘gold standard’ of myeloid DC. However, two reports show that both in vivo and in vitro, LC upregulate significant CD8a upon maturation and migration from the skin [22,23]. Therefore, there is a possibility that migrated LC, could form at least a part of the CD8a+ IDC subset in lymph node [23]. Rel B deficient mice, although lacking CD8a− DC, still possess LC, as do Ikaros dominat negative mutant mice. LC development has been shown to possess an absolute requirement for TGFb, though DC in secondary lymphoid tissue do not appear to be affected by TGFb deficiencies [24]. Together, these studies further suggest that LC may be a DC subset distinct from those identified so far in spleen and thymus.
Fig. 1. CD4+ and CD8a+ DC present in fresh spleen suspensions. Low density spleen DC were gradient enriched by 14.1% Nycodenz (Nycomed, Pharma Oslo, Norway) gradient centrifugation from fresh spleen as described by Vremec et al. [2]. At this stage DC (30 – 40% of low density cells) were not further purified by immunodepletion, but immediately labelled in three colours for CD11c (plus FITC-anti-hamster Ig; FL-1), the indicated monoclonal antibodies (mAb; plus PE-anti-rat Ig; FL-2) followed by blocking in 10% rat serum and addition of Cy5-PE conjugated CD4 (FL-3). Dot plots show DC gated for CD11c positivity (as indicated in the histogram; solid line =CD11c, dotted line =isotype control staining) and by size and forward scatter to exclude dead cells and debris (not shown). The percent of events in each quadrant of the dot plots is indicated. The mAb clones and their source were as follows: CD4 (GK1.5; Southern Biotechnology Associates, Eching, Germany), NLDC-145; (G Kraal, Free University, Amsterdam, The Netherlands [13]). Rat IgG (R35-95; IgG2a irrelevant mAb control) and CD3 (17A2) were from Pharmingen, (Hamburg, Germany). CD11c (N418), CD8a (53-6.72), CD11b (M1/70.15), CD19 (1D3) and Thy1.2 (30-H-12) were from The American Type Culture Collection, Manasas, VA.
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Methods to expand DC-like cells from various progenitor or precursors sources in both man and mouse have added to the debate on the origins of DC. The generation in vitro of antigen presenting cells with exceptional T cell stimulatory capacity is possible from pro- and mature B cells [25,26], monocytes [27,28], various population of immature haematapoietic cells [1,29 –31] and even granulocytes [32]. Except in the case of mature B cells, these resulting stimulatory populations were dubbed ‘DC’ or ‘DC-like’. However, these findings at least show us that because DC are a cell type primarily defined by their function that is, the ability to stimulate naı¨ve T cell proliferation, there is considerable latitude in the literature as to which cells fulfill the criteria of ‘DC’. This is an important consideration when extrapolating in vitro data of DC ontology to an in vivo situation. The function of CD95 dependent thymic deletion of T cells has also been ascribed to lymphoid-derivedDC [33]. However, this feature may not be intrinsic to the DC itself, but rather to their placement in the thymic environment. For example, Matzinger and Guerder showed that allogeneic myeloid spleen DC (only myeloid DC appear to be effectively isolated by standard spleen DC isolation procedures [2,34]) are also highly effective veto cells for immature thymocytes in thymic organ culture [35]. This did not seem to be due to indirect transfer of allo-antigen to endogenous lymphoid-derived thymic DC, as suggested [36], since other allogeneic cell types were not as effective as DC in inducing thymocyte deletion and such deletion was allogeneically class restricted [35]. Similarly, the deletion of thymocytes to soluble antigen is maintained equally well by immunostimulatory bone marrow derived DC and thymic DC [37]. Therefore, the ability of DC to delete thymocytes seems to relate more to the intrinsic property of thymocytes to apoptose after binding with high affinity to class I and class II presented antigens, rather than to the type of DC lineage. The possibility that lymphoid-derived DC might delete autoreactive thymocytes via a CD95 dependent pathway also seems unlikely since Fas deficient lpr mice show normal thymic T cell deletion. Therefore, no CD95L+ veto DC or other cell seems to be required for thymic selection [38]. DC have been implicated in peripheral tolerance [39,40], though they may not be unique in this capability [41–43]. For effective intracellular surveillance, class I is expressed by all normal nucleated cells and thus it follows that the tolerance of CD8 cells may also be negatively regulated by many different cell types. The work of Kurts et al. [44] suggests that some of the cells involved in peripheral CD8 tolerance must be capable of soluble antigen capture and class I mediated crosspresentation of these antigens. Since DC are known to be capable of cross-presenting soluble antigens (albeit
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in a stimulatory fashion), this suggests at least a partial role for DC in CD8 tolerance. For induction of CD4+ tolerance the situation is even more suggestive of a role for DC, as in the periphery, class II expression is mostly limited to haematopoietic APC (B cells, macrophages and DC). This expression pattern of class II antigens narrows the identity of a putative CD4+ T cell tolerising cell to an APC that almost certainly is bone barrow derived [45]. Adoptive transfer of immature GM-CSF derived DC, or in vivo targeting of DC, has been shown to mediate antigen specific tolerance [39,40], showing that otherwise immunostimulatory DC also have the potential to induce peripheral tolerance. Lymphoid-derived DC in the T cell areas express high amounts of class II/self-peptide complexes [34]. This has further fueled expectations that lymphoidderived DC might perform a tolerogenic role through the engagement and deletion of self-reactive T cells. High expression of self peptides on MHC molecules would be a useful prerequisite for a tolerising cell that might deliver signal one in absence of signal two to delete or anergise autoreactive T cells. However, the expression of MHC/self-peptides itself is not unique to this cell type, since the stability of both class I and class II molecules depends on MHC complexing with peptides that are almost exclusively self-derived [46–48]. Deletion of mature T cells has been suggested to occur via direct, CD95 dependent, antigen specific contact with lymphoid-derived DC [34,49]. For example, T cell hybridomas underwent marked apoptosis following antigen specific interaction with preparations containing interdigitating lymphoid-derived DC [34]. In the periphery, deletion of autoreactive CD8 cells to soluble antigen occurs through a Fas dependent mechanism of crosspriming dubbed ‘cross-tolerance’ by Kurts et al. [44]. However, a specific DC phenotype, or lineage, involved in this process has yet to be directly identified. In our hands, neither CD8a+ nor CD8a− splenic DC stain for CD95L, as determined by monoclonal antibody staining, although we cannot rule out the possibility that DC activation might induce functional CD95L surface expression. In any case, T cells appear to possess all the essential elements for self-destruction following TCR ligation, without the need for a dedicated tolerising APC. Therefore it is very likely that T cells in the situations described above are dying through activation induced cell death (AICD), via autocrine CD95: CD95L mechanisms, as described for normal T cells and T cell hybridomas [50–52]. In these studies, limiting dilution analysis of TCR-activated T cells, showed that intercellular contact was not required for AICD, but that death was still blocked by soluble CD95 antagonists. Such cell suicide mechanisms may therefore be effected hours following APC: T cell contact would occur independently of CD95L expressed on the APC.
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For CD8 tolerance, the availability of CD4+ antigenspecific help greatly influences the survival of TCR stimulated cytotoxic T cells [53]. Thus, TCR ligation of a CD8+ T cell without CD4 help encourages AICD and may be a more important factor in cytotoxic T cell survival than the lineage of antigen presenting cell involved in the initial stimulation. It is not known how the presense of antigen specific CD4+ T cells might prevent death of expanding CD8+, but it is likely that this process involves a CD4+ T cell-mediated, APC ‘licensing’ step [54]. Therefore, CD4+ T cells may affect the quality and levels of costimulation delivered by the APC for CD8+ expansion. In fact, the induction of both CD4+ and CD8+ T cell tolerance may be regulated solely by the ability of any APC to withold the second (costimulatory) signal during antigen presentation [39,40,55,56]. Some authors have proposed that the placement itself of lymphoid-derived DC in the T cell areas of lymph nodes and spleen is an important mechanism for the control of self-antigen specific T cells [34,36,57]. In this proposition, T cell area DC present antigen to T cells in a tolerogenic fashion, before the immunostimulatory myeloid DC (placed outside of the T cell area at the marginal zone) have had time to migrate into T cell area and present antigen. Adjuvants, such as LPS, stimulate the migration of the myeloid DC into the T cell area where they present their antigens in a stimulatory fashion, perhaps dominating over tolerance induction by the lymphoid-derived DC subset [36]. Although, enough evidence supporting this concept has yet to be furnished, it remains a fascinating proposition that an anatomical ‘handicap race’ between two DC subsets may be the key to anergic or deletional control of anti-self T cells. However, it should be noted that this is contentious, since Sousa et al. [7,58] found that both CD8a+ and CD8a− DC took up foreign antigen and mobilised into the T cell areas following microbial product stimulation. DC differently activated by manipulations of the cytokine mileu, or even by T cell contact, have now been reported that have significant tolerising capacity [59 – 62]. For example, UV irradiation resulting in immunosuppression appears to act via local APC cell function, perhaps stimulating their production of IL-10 [63]. UV or IL-10 modified APC mediate tolerance through anergy induction or suppressor cell skewing in T cells [59,64]. Together, these recent data suggest that recent activation or suppressive events may have the largest impact on whether a DC belongs to a tolerising or activating subset. Other studies have suggested that a non-DC may be responsible for the downregulation of T cell responses. A CD11b+/Gr1+ cell, that deleted expanding CD8+ T cells via a CD95-independent mechanism, was normally present in spleen at very low frequencies, but increased in numbers following immu-
nization [42,43]. This CD11b+/Gr1+ cell influx was GM-CSF dependent, initially paralelled effector cell expansion, but later caused cytotoxic downregulation. Depletion of Gr1+ cells prior to immunisation maintained the CTL frequency and cytotoxic abilities of the responding CD8 T cells [42,43]. Similarly, B cells may play a role in T cell tolerance. Following soluble HEL protein immunisation, B cells display similar ratios of HEL peptides to class II levels as do DC [48,65]. Although B cells display less class II on a cell to cell basis, they may be very effective tolerising cells, since they are present in lymph nodes in much greater numbers compared to DC [48,56]. In conclusion, future studies may indeed reveal different ontological origin for subsets of DC performing different biological functions. In the absence of functional data, it can only be speculated whether the induction of T cell tolerance by a putative DC subset would be through anergic or deletional mechanisms. Alternatively, we may find that these tolerising functions can be performed by the same DC type, but is dependent on recent activation events. Appropriate induction of T cell tolerance or activation would be ensured by allowing DC behaviour to be influenced by environmental signalling at the time of antigen encounter. In contrast, having distinct DC subsets differently perform tolerising or activating functions would require the correct DC being in the right place at the right time and therefore seems a less compelling option for an evolving immune system to chose.
Acknowledgements We gratefully acknowledge Professor AG and PM McLellan and L Fearnley for critical reading of the manuscript, the support of Professor Eva-B Bro¨cker and C Linden for technical assistance. We thank the laboratory of Professor Ken Shortman for their development of techniques allowing insights into this field. ADM is funded by a grant from the Bundesministerium fu¨r Bildung und Forschung (IZKF Wu¨rzburg 01 KS 9603).
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Functions of myeloid and lymphoid dendritic cells Alexander D. McLellan *, Eckhart Ka¨mpgen Department of Dermatology, Uni6ersity of Wuerzburg, Joseph-Schneider Str.2, Wuerzburg 97080, Germany Received 24 January 2000; accepted 26 January 2000
Abstract The bone marrow derived dendritic cell (DC) is an essential antigen presenting cell (APC) for the initiation of primary, T cell based immune responses. DC are a heterogenous haematopoietic lineage, in that many subsets from different tissues show different surface phenotypes, but the ability to stimulate antigen specific naı¨ve T cell proliferation appears to be shared between these DC subsets. It has been suggested that the so called myeloid and lymphoid-derived subsets of DC perform distinct stimulatory or tolerogenic functions. However, recent data has blurred this apparent distinction of DC subset function and shown that both subsets are at least capable of stimulatory and possibly even tolerogenic functions. Thus, the immunoregulatory potential of DC may depend less on ontology than on recent activatory or downregulatory stimuli. © 2000 Elsevier Science B.V. All rights reserved.
Historically, the concept of the lymphoid-derived DC dates from the identification of a shared precursor for DC and T cells in the murine thymus [1] and CD8 expressing DC in the spleen and thymus of adult mice [2]. These DC expressed CD8a homodimers in high density, but unlike mature CD8 T cells, with absent, or low level, expression of beta chains. Originally suggested to perform tolerogenic functions, even highly purified fractions of CD8a cells were capable of inducing significant T cell proliferation (albeit at lower levels than CD8a- cells). Further studies revealed that CD8a+ were at least equilavent to CD8a− DC in stimulating both CD4 and CD8 responses in vivo and in vitro [3 – 6]. A surprising recent finding is that, CD8a+, but not CD8a−, DC produce significant levels of IL-12 and prime to Th1 T cell responses [3 – 7]. In man, Grouard et al. [8] and Olweus et al. [9] identified CD4+/IL-3R+/CD11c− DC in both cord and peripheral blood, tonsil and bone marrow. Although these authors concluded differently as to whether these Abbre6iations: APC, antigen presenting cell; DC, dendritic cells; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; L, ligand; LC, Langerhans cell; TNF, tumour necrosis factor. * Corresponding author. Tel.: +49-931-2012725; fax: + 49-9312012700. E-mail address: [email protected] (A.D. McLellan)
DC were myeloid or lymphoid in origin, this human DC subset is often referred to as ‘lymphoid’ or plasmacytoid since they show low levels of CD13 and CD33 antigens, display prominent parallel arrays of endoplasmic reticulum and other morphological features resembling plasma cells. Nevertheless, these DC appear to derive from a M-CSFR+ progenitor capable of generating granulocytes as well as IL-3R+ DC [9]. This DC population may be identical to a CD4+ human thymic DC subset described earlier by Shortman’s group [10]. Both murine thymic DC precursors [11] and the human plasmacytotoid [8,9] DC show a dependence on IL-3 rather than GM-CSF for survival and development into mature DC. Interestingly, these CD4+/IL-3R+/ CD11c− DC, in contrast to their lymphoid-derived murine counterparts, were suggested to produce a predominantly Th2 response, while monocyte derived DC stimulated a Th1 phenotype in responding T cells [12]. Thus, if this human DC subset really is lymphoid in origin, then there is not a species consistent definition of the T cell stimulating functions of myeloid and lymphoid-derived DC. In mouse, there are other interesting differences between these two DC subtypes: For example, freshly isolated CD8a+ DC express higher levels of NLDC145 (a 205KD lectin-like molecule, with as yet unknown specificity [13]) than the CD8a− DC. Two groups have suggested that upon isolation and reinjection, the
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lymphoid-derived DC subset has a poor or absent afferent lymph homing ability [5,6]. Others have suggested that there is no inherent difference in migratory capacity between the two subsets [7,14] Also, in RelB knock out mice, CD8a− DC are lacking from spleen, despite normal numbers of CD8a+ DC [15]. Ikaros knock out mice lack B cells and show defects in T cell development, yet make an excess of monocytes and macrophages. Investigation of DC populations showed that the spleen and thymus of B cell deficient Ikaros−/− mice were also deficient in CD8a− DC and had greatly reduced numbers of CD8a+ DC [16]. Furthermore, a dominant negative Ikaros mutant which has defects in all lymphocytes including T cells, has neither CD8a− nor CD8a+ DC. Which leads into the striking conclusion that both CD8a+ and CD8a− may be of lymphiod origin, but that CD8a+ may be closer to T cell lineage than to B cells. Recently, Klatzmann and collegues have described a CD4+ DC population in lymph node that appears to be part of the myeloid subset [17]. Surprisingly, these CD4+/CD8a− DC are also present as a major population in spleen, making up 50% of CD11c+ low density fresh spleen cells (Fig. 1). In contrast to CD8a+ , DC, CD4+ DC express CD11b. Both CD4+ and CD8a+ subsets lack T cell (Thy1.2, CD3), B cell (CD19) markers (Fig. 1) and the F4/80, NK1.1 and Gr1 antigens (not shown). Since this CD4+ DC subset was originally excluded from earlier analyses by use of CD4 mAb to deplete contaminating T cells, the actual ratio of lymphoid to myleoid-derived DC present in spleen may be closer to 1:3, a figure somewhat lower
than earlier reported estimates of 1:1 [12] (see Fig. 1). Interestingly, we have found that a subset of CD4+/ CD8a−/CD11c+ DC clearly stains for NLDC145 (Fig. 1), and that NLDC-145 expression is upregulated on CD4+ by overnight culture, demonstrating that expression of this marker is not exclusive to CD8+ DC. Moreover, although the expression of CD8a is stable upon DC culture, CD4 expression is lost rapidly from DC following overnight culture ([18] and ADM & EK; manuscript in preparation). Thus, our results do not contradict the original description of cultured spleen DC as CD4− [19]. The function of this major CD4+/ CD11c+, DC subset and their anatomical location awaits further analysis. Langerhans cells (LC) provide more puzzlement as to the origins of various DC subsets. LC are extremely potent stimulators of T cells, undoubtedly bone marrow derived [20,21] and are widely thought to represent the ‘gold standard’ of myeloid DC. However, two reports show that both in vivo and in vitro, LC upregulate significant CD8a upon maturation and migration from the skin [22,23]. Therefore, there is a possibility that migrated LC, could form at least a part of the CD8a+ IDC subset in lymph node [23]. Rel B deficient mice, although lacking CD8a− DC, still possess LC, as do Ikaros dominat negative mutant mice. LC development has been shown to possess an absolute requirement for TGFb, though DC in secondary lymphoid tissue do not appear to be affected by TGFb deficiencies [24]. Together, these studies further suggest that LC may be a DC subset distinct from those identified so far in spleen and thymus.
Fig. 1. CD4+ and CD8a+ DC present in fresh spleen suspensions. Low density spleen DC were gradient enriched by 14.1% Nycodenz (Nycomed, Pharma Oslo, Norway) gradient centrifugation from fresh spleen as described by Vremec et al. [2]. At this stage DC (30 – 40% of low density cells) were not further purified by immunodepletion, but immediately labelled in three colours for CD11c (plus FITC-anti-hamster Ig; FL-1), the indicated monoclonal antibodies (mAb; plus PE-anti-rat Ig; FL-2) followed by blocking in 10% rat serum and addition of Cy5-PE conjugated CD4 (FL-3). Dot plots show DC gated for CD11c positivity (as indicated in the histogram; solid line =CD11c, dotted line =isotype control staining) and by size and forward scatter to exclude dead cells and debris (not shown). The percent of events in each quadrant of the dot plots is indicated. The mAb clones and their source were as follows: CD4 (GK1.5; Southern Biotechnology Associates, Eching, Germany), NLDC-145; (G Kraal, Free University, Amsterdam, The Netherlands [13]). Rat IgG (R35-95; IgG2a irrelevant mAb control) and CD3 (17A2) were from Pharmingen, (Hamburg, Germany). CD11c (N418), CD8a (53-6.72), CD11b (M1/70.15), CD19 (1D3) and Thy1.2 (30-H-12) were from The American Type Culture Collection, Manasas, VA.
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Methods to expand DC-like cells from various progenitor or precursors sources in both man and mouse have added to the debate on the origins of DC. The generation in vitro of antigen presenting cells with exceptional T cell stimulatory capacity is possible from pro- and mature B cells [25,26], monocytes [27,28], various population of immature haematapoietic cells [1,29 –31] and even granulocytes [32]. Except in the case of mature B cells, these resulting stimulatory populations were dubbed ‘DC’ or ‘DC-like’. However, these findings at least show us that because DC are a cell type primarily defined by their function that is, the ability to stimulate naı¨ve T cell proliferation, there is considerable latitude in the literature as to which cells fulfill the criteria of ‘DC’. This is an important consideration when extrapolating in vitro data of DC ontology to an in vivo situation. The function of CD95 dependent thymic deletion of T cells has also been ascribed to lymphoid-derivedDC [33]. However, this feature may not be intrinsic to the DC itself, but rather to their placement in the thymic environment. For example, Matzinger and Guerder showed that allogeneic myeloid spleen DC (only myeloid DC appear to be effectively isolated by standard spleen DC isolation procedures [2,34]) are also highly effective veto cells for immature thymocytes in thymic organ culture [35]. This did not seem to be due to indirect transfer of allo-antigen to endogenous lymphoid-derived thymic DC, as suggested [36], since other allogeneic cell types were not as effective as DC in inducing thymocyte deletion and such deletion was allogeneically class restricted [35]. Similarly, the deletion of thymocytes to soluble antigen is maintained equally well by immunostimulatory bone marrow derived DC and thymic DC [37]. Therefore, the ability of DC to delete thymocytes seems to relate more to the intrinsic property of thymocytes to apoptose after binding with high affinity to class I and class II presented antigens, rather than to the type of DC lineage. The possibility that lymphoid-derived DC might delete autoreactive thymocytes via a CD95 dependent pathway also seems unlikely since Fas deficient lpr mice show normal thymic T cell deletion. Therefore, no CD95L+ veto DC or other cell seems to be required for thymic selection [38]. DC have been implicated in peripheral tolerance [39,40], though they may not be unique in this capability [41–43]. For effective intracellular surveillance, class I is expressed by all normal nucleated cells and thus it follows that the tolerance of CD8 cells may also be negatively regulated by many different cell types. The work of Kurts et al. [44] suggests that some of the cells involved in peripheral CD8 tolerance must be capable of soluble antigen capture and class I mediated crosspresentation of these antigens. Since DC are known to be capable of cross-presenting soluble antigens (albeit
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in a stimulatory fashion), this suggests at least a partial role for DC in CD8 tolerance. For induction of CD4+ tolerance the situation is even more suggestive of a role for DC, as in the periphery, class II expression is mostly limited to haematopoietic APC (B cells, macrophages and DC). This expression pattern of class II antigens narrows the identity of a putative CD4+ T cell tolerising cell to an APC that almost certainly is bone barrow derived [45]. Adoptive transfer of immature GM-CSF derived DC, or in vivo targeting of DC, has been shown to mediate antigen specific tolerance [39,40], showing that otherwise immunostimulatory DC also have the potential to induce peripheral tolerance. Lymphoid-derived DC in the T cell areas express high amounts of class II/self-peptide complexes [34]. This has further fueled expectations that lymphoidderived DC might perform a tolerogenic role through the engagement and deletion of self-reactive T cells. High expression of self peptides on MHC molecules would be a useful prerequisite for a tolerising cell that might deliver signal one in absence of signal two to delete or anergise autoreactive T cells. However, the expression of MHC/self-peptides itself is not unique to this cell type, since the stability of both class I and class II molecules depends on MHC complexing with peptides that are almost exclusively self-derived [46–48]. Deletion of mature T cells has been suggested to occur via direct, CD95 dependent, antigen specific contact with lymphoid-derived DC [34,49]. For example, T cell hybridomas underwent marked apoptosis following antigen specific interaction with preparations containing interdigitating lymphoid-derived DC [34]. In the periphery, deletion of autoreactive CD8 cells to soluble antigen occurs through a Fas dependent mechanism of crosspriming dubbed ‘cross-tolerance’ by Kurts et al. [44]. However, a specific DC phenotype, or lineage, involved in this process has yet to be directly identified. In our hands, neither CD8a+ nor CD8a− splenic DC stain for CD95L, as determined by monoclonal antibody staining, although we cannot rule out the possibility that DC activation might induce functional CD95L surface expression. In any case, T cells appear to possess all the essential elements for self-destruction following TCR ligation, without the need for a dedicated tolerising APC. Therefore it is very likely that T cells in the situations described above are dying through activation induced cell death (AICD), via autocrine CD95: CD95L mechanisms, as described for normal T cells and T cell hybridomas [50–52]. In these studies, limiting dilution analysis of TCR-activated T cells, showed that intercellular contact was not required for AICD, but that death was still blocked by soluble CD95 antagonists. Such cell suicide mechanisms may therefore be effected hours following APC: T cell contact would occur independently of CD95L expressed on the APC.
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For CD8 tolerance, the availability of CD4+ antigenspecific help greatly influences the survival of TCR stimulated cytotoxic T cells [53]. Thus, TCR ligation of a CD8+ T cell without CD4 help encourages AICD and may be a more important factor in cytotoxic T cell survival than the lineage of antigen presenting cell involved in the initial stimulation. It is not known how the presense of antigen specific CD4+ T cells might prevent death of expanding CD8+, but it is likely that this process involves a CD4+ T cell-mediated, APC ‘licensing’ step [54]. Therefore, CD4+ T cells may affect the quality and levels of costimulation delivered by the APC for CD8+ expansion. In fact, the induction of both CD4+ and CD8+ T cell tolerance may be regulated solely by the ability of any APC to withold the second (costimulatory) signal during antigen presentation [39,40,55,56]. Some authors have proposed that the placement itself of lymphoid-derived DC in the T cell areas of lymph nodes and spleen is an important mechanism for the control of self-antigen specific T cells [34,36,57]. In this proposition, T cell area DC present antigen to T cells in a tolerogenic fashion, before the immunostimulatory myeloid DC (placed outside of the T cell area at the marginal zone) have had time to migrate into T cell area and present antigen. Adjuvants, such as LPS, stimulate the migration of the myeloid DC into the T cell area where they present their antigens in a stimulatory fashion, perhaps dominating over tolerance induction by the lymphoid-derived DC subset [36]. Although, enough evidence supporting this concept has yet to be furnished, it remains a fascinating proposition that an anatomical ‘handicap race’ between two DC subsets may be the key to anergic or deletional control of anti-self T cells. However, it should be noted that this is contentious, since Sousa et al. [7,58] found that both CD8a+ and CD8a− DC took up foreign antigen and mobilised into the T cell areas following microbial product stimulation. DC differently activated by manipulations of the cytokine mileu, or even by T cell contact, have now been reported that have significant tolerising capacity [59 – 62]. For example, UV irradiation resulting in immunosuppression appears to act via local APC cell function, perhaps stimulating their production of IL-10 [63]. UV or IL-10 modified APC mediate tolerance through anergy induction or suppressor cell skewing in T cells [59,64]. Together, these recent data suggest that recent activation or suppressive events may have the largest impact on whether a DC belongs to a tolerising or activating subset. Other studies have suggested that a non-DC may be responsible for the downregulation of T cell responses. A CD11b+/Gr1+ cell, that deleted expanding CD8+ T cells via a CD95-independent mechanism, was normally present in spleen at very low frequencies, but increased in numbers following immu-
nization [42,43]. This CD11b+/Gr1+ cell influx was GM-CSF dependent, initially paralelled effector cell expansion, but later caused cytotoxic downregulation. Depletion of Gr1+ cells prior to immunisation maintained the CTL frequency and cytotoxic abilities of the responding CD8 T cells [42,43]. Similarly, B cells may play a role in T cell tolerance. Following soluble HEL protein immunisation, B cells display similar ratios of HEL peptides to class II levels as do DC [48,65]. Although B cells display less class II on a cell to cell basis, they may be very effective tolerising cells, since they are present in lymph nodes in much greater numbers compared to DC [48,56]. In conclusion, future studies may indeed reveal different ontological origin for subsets of DC performing different biological functions. In the absence of functional data, it can only be speculated whether the induction of T cell tolerance by a putative DC subset would be through anergic or deletional mechanisms. Alternatively, we may find that these tolerising functions can be performed by the same DC type, but is dependent on recent activation events. Appropriate induction of T cell tolerance or activation would be ensured by allowing DC behaviour to be influenced by environmental signalling at the time of antigen encounter. In contrast, having distinct DC subsets differently perform tolerising or activating functions would require the correct DC being in the right place at the right time and therefore seems a less compelling option for an evolving immune system to chose.
Acknowledgements We gratefully acknowledge Professor AG and PM McLellan and L Fearnley for critical reading of the manuscript, the support of Professor Eva-B Bro¨cker and C Linden for technical assistance. We thank the laboratory of Professor Ken Shortman for their development of techniques allowing insights into this field. ADM is funded by a grant from the Bundesministerium fu¨r Bildung und Forschung (IZKF Wu¨rzburg 01 KS 9603).
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