DEC-205 as a marker of dendritic cells with regulatory effects on CD8 T cell responses

Vadim Kronin, Li Wu, Schiaoching Gong1, Michel C Nussenzweig1 and Ken Shortman

The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia
1 The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10021, USA

Correspondence to: K. Shortman


    Abstract
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 Abstract
 Introduction
 References
 
We have previously reported that a population of lymphoid-related CD8{alpha}+ DEC-205+ dendritic cells (DC) from mouse spleen have `regulatory' effects on the T cells they activate. CD8 T cells produce IL-2 and give a sustained proliferative response to allogeneic CD8{alpha} DEC-205 splenic DC, but produce little IL-2 and give a limited response to allogeneic CD8+ DEC-205+ splenic DC. Although CD8{alpha} and DEC-205 correlate closely among splenic DC, lymph nodes (LN) include a large population of CD8{alpha}low DEC-205high DC. By i.v. transfer of purified thymic early lymphoid precursors into irradiated recipient mice we now demonstrate that these CD8{alpha}low but DEC-205high LN DC can be the progeny of a lymphoid precursor population, apparently corresponding to the CD8{alpha}high DEC-205high DC progeny of the same precursors in spleen and thymus. By culture of the separated, purified DC with allogeneic CD8 T cells we demonstrate that the CD8{alpha}low DEC-205high DC of LN are also functionally equivalent to the CD8{alpha}high DEC-205high DC of spleen. Therefore, DEC-205 but not CD8{alpha} serves to segregate functionally distinct DC types in LN. However, DC isolated from the spleens of genetically manipulated DEC-205null mice and separated on the basis of CD8{alpha} expression have a similar capacity to stimulate CD8 T cells as their heterozygous littermate controls, with the CD8{alpha}+ but now DEC-205null DC still giving restricted responses. In conclusion, high expression of DEC-205 appears to be a good marker of the lymphoid-related regulatory type of DC, but DEC-205 itself is not responsible for transmitting negative signals to the T cells.

Keywords: CD8, DEC-205, IL-2 production, regulatory dendritic cells


    Introduction
 Top
 Abstract
 Introduction
 References
 
We have previously reported that certain antigen-presenting dendritic cells (DC) have a capacity to regulate the response of the T cells they activate (13). In mouse spleen two distinct DC populations may be separated on the basis of CD8{alpha} expression, the CD8{alpha}+ DC being apparently of lymphoid origin and the CD8{alpha} DC being apparently of myeloid origin (1,46). Although these two DC types appear equivalent in their ability to present antigen and activate T cells into cell cycle, they differ in the subsequent events which determine the extent and duration of activated T cell proliferation, the CD8{alpha}+ DC having distinct negative effects (2,3). In particular, CD8 T cells when stimulated by CD8{alpha}+ DC produce very little IL-2, which restricts their subsequent expansion, whereas the same CD8 T cells when stimulated by CD8{alpha} DC produce substantial free IL-2 in the culture supernatant, sufficient to support extended cell expansion (3). This difference in induced cytokine output by CD8 T cells is determined within the first day of culture with the appropriate DC (7). This `regulatory' effect could not be attributed to differences between the DC in signals from co-stimulator molecules such as B7-1 or B7-2 which are equivalent on the two populations nor to differences in soluble factors such as IL-12—some new signaling system appears to be involved (7).

The possibility that CD8{alpha} itself on the DC might transmit negative signals to the T cells was considered (8); however, by using CD8 `null' mice and surrogate markers for the separation of two DC populations, it was demonstrated that the regulatory effects were independent of DC CD8 expression (9). This focussed attention on DEC-205 whose surface expression correlates closely with CD8{alpha} on mouse spleen DC, as well as on mouse thymus DC which are predominantly CD8{alpha}+ DEC-205+ (5). DEC-205 served as the surrogate marker for CD8{alpha} in the above experiments. DEC-205, a characteristic marker of interdigitating DC in the T cell areas of lymphoid organs, is recognized by the mAb NLDC-145 (10). Structurally DEC-205 contains 10 distinct lectin-like domains and it may be implicated in the uptake of carbohydrate-containing molecules into the DC antigen-presentation pathway (11). There is nothing so far to suggest DEC-205 is involved in transmitting signals to T cells and the NLDC-145 mAb has no obvious blocking effects (9). However, this possible role of DEC-205 had not been critically examined.

Accordingly, in the present study we first assessed the value of DEC-205 as a marker of those DC which induce only restricted CD8 T cell responses, extending the study from spleen to lymph nodes (LN) where the tight correlation between CD8{alpha} and DEC-205 expression on DC breaks down (5). We then directly tested the role of DEC-205 in transmitting signals regulating CD8 T cell proliferation, by using DC isolated from DEC-205null mice, produced in the Rockefeller Institute laboratory (12).

When DC were isolated from spleen and analyzed by immunofluorescent staining and flow cytometry there was a tight correlation between CD8{alpha} expression and DEC-205 expression, resulting in two predominant populations, CD8{alpha}+ DEC-205+ and CD8 DEC-205 (Fig. 1Go). This confirmed our previous studies (5). As would be expected from this correlation either marker could be used to segregate the two functionally distinct DC populations. Thus, as shown in Fig. 2Go, the DC isolated according to low expression of either CD8{alpha} or DEC-205 induced better proliferation of allogeneic CD8 T cells than did the DC isolated according to high expression of either CD8{alpha} or DEC-205. This applied over a wide DC dose range. These cultures were pulsed at day 3, the time when IL-2 availability begins to limit proliferation in cultures stimulated by CD8{alpha}+ DC; the difference is more pronounced at later time points (3).



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Fig. 1. The relationship between DEC-205 and CD8{alpha} expression in DC isolated from spleen and LN. DC were first extracted and enriched as described elsewhere (3,4). Briefly, tissues were cut into pieces, digested at 22°C for 25 min with collagenase DNase, EDTA treated to break up DC–T cell complexes and then the lightest 3–5% of the cells selected using Nycodenz density centrifugation. DC were further enriched by incubating with a cocktail of mAb against cells other than DC followed by immunomagnetic bead depletion. In the present studies, in order to ensure macrophage depletion this cocktail included low levels of anti-Mac-1 and anti-FcRII to deplete only cells expressing high levels of these surface antigens. We have since found this leads to some DC loss, particularly of CD8{alpha} DC. However, subsequent experiments omitting these depletion reagents and using only F/480 to eliminate macrophages have given similar results to those in this figure, except for an increase in the relative level of DEC-205 CD8{alpha} DC. The final DC preparation, 70–90% pure, was stained in three fluorescent colors with anti-CD11c or anti-class II MHC to distinguish DC, with NLDC-145 to stain DEC-205 (10) and with anti-CD8{alpha}, using directly conjugated or biotinylated mAb as described elsewhere (3,5); propidium iodide was added to the wash solution to stain dead cells. The DC were analyzed on a FACStar Plus instrument (Becton Dickinson, San Jose, CA), gating for CD11cbright or class II MHCbright cells with the high forward and side light scatter characteristics of DC, and gating against propidium iodide-staining dead cells. The resultant fluorescent distribution of DEC-205 versus CD8{alpha} staining is given. For sorting of DC fractions a similar approach was taken, generally with only two-color staining for CD11c versus either CD8{alpha} or DEC-205.

 


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Fig. 2. Cell dose–response of the capacity of different splenic DC fractions to induce sustained proliferation in allogeneic CD8 T cells. Splenic DC were isolated from C57BL/6 mice, and sorted into CD8{alpha}+ and CD8{alpha} populations, or into DEC-205+ and DEC-205 populations, as illustrated in Fig. 1Go. Various numbers of the DC were cultured with 20,000 pure CBA CD8 T cells isolated from LN as described previously (3). Cultures were in 200 µl modified RPMI 1640–10% FCS medium in V-bottom wells of 96-well culture trays, for 3 days at 37°C in a humidified 10% CO2-in-air incubator, as described previously (3). The cultures were then pulsed with 1 µCi of [3H]thymidine for 6 h, the cells harvested on glass fiber filters then the radioactivity incorporated into cellular DNA determined using a gas scintillation ß-counter. Results represent the mean ± SEM of triplicate cultures, from an experiment typical of two (DEC-205) or five (CD8{alpha}) such DC dose–response assays. The differences obtained were more pronounced at later culture times.

 
However, as shown in Fig. 1Go, the correlation between CD8{alpha} and DEC-205 expression is not as close amongst the DC isolated from LN, confirming our previous analyses (5). As well as the CD8{alpha}+ DEC-205+ and CD8{alpha} DEC-205 DC populations an additional group of CD8– to low DC expressing high levels of DEC-205 is apparent. One possibility is that some of these were lymphoid-derived or lymphoid-related DC that had failed to acquire CD8{alpha} expression. We had previously observed that DC which develop in culture from the early thymic lymphoid precursor population lack surface CD8{alpha} expression (13), although the DC progeny of the same precursors found in thymus or spleen following i.v. transfer to irradiated recipient mice all express high levels of CD8{alpha} (6). To test whether the LN DC progeny of a lymphoid precursor population could include DC lacking CD8{alpha} expression, the early thymic lymphoid precursor population was purified from Ly 5.2 mice and transferred i.v. into irradiated Ly 5.1 recipient mice, following our earlier procedures (6) except that the Ly 5.2+ DC progeny in the LN, as well as in the thymus and spleen, were analyzed. As shown in Fig. 3Go, the DC progeny of the thymic lymphoid precursor population in the LN were predominantly CD8– to low, although they were all DEC-205high. In confirmation of our previous studies, the progeny of these precursors in the thymus and spleen of the same mice all expressed high levels of both CD8{alpha} and DEC-205 (data not shown).



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Fig. 3. The expression of CD8{alpha} and DEC-205 on the LN DC progeny of thymic lymphoid precursors. The early thymic precursors (the `low CD4' precursors) were purified from 4- to 6-week-old C57BL/6 (Ly 5.2) mice and 30,000 were transferred i.v. into irradiated 7- to 8-week-old C57BL/6 Ly 5.1-Pep3b (Ly 5.1) mice, as described in detail elsewhere (6). After 4 weeks, the LN (mesentery, aortic, axillary) of recipient mice were pooled, the DC enriched, and then stained in four fluorescent colors and analyzed for expression of class II MHC, of Ly 5.2, and of DEC-205 and CD8{alpha}, as described elsewhere (6). The distribution of DEC-205 and CD8{alpha} is presented for cells gated for high class II MHC and Ly 5.2 expression, with the high forward and side light scatter of DC. The broken line represents the background fluorescence omitting only the relevant mAb. The results are typical of four separate experiments sampling the LN DC progeny from 2 to 6 weeks after precursor cell transfer.

 
To determine if these distinct subgroups of DC in LN showed differential effects on CD8 T cell proliferation, as found for splenic DC, LN DC were isolated and separated on the basis of CD8{alpha} expression, then cultured with allogeneic CD8 T cells. As shown in Fig. 4Go, in contrast to splenic DC, there was little difference in the extent of induced T cell proliferation between the CD8{alpha} and CD8+ LN fractions. Both types of LN DC induced a lower level of proliferation compared to splenic CD8{alpha} DC, especially at the crucial later time points when IL-2 became limiting. However, when the LN DC were separated on the basis of DEC-205 expression, the results were similar to those seen when splenic DC were separated on the basis of DEC-205 expression or CD8{alpha} expression; the responses to both DC fractions were similar up to day 2.5 of culture, but after this time the response of the DEC-205+ DC stimulated cultures fell off rapidly (Fig. 4Go).



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Fig. 4. Kinetics of the response of CD8 T cells to different DC fractions from allogeneic spleen or LN. DC were purified from C57BL/6 mouse spleen or LN and sorted into various fractions as in Fig. 1Go. CD8 T cells were purified from CBA mouse LN as in Fig. 2Go. The DC (500/well) were cultured with CD8 T cells (20,000/well) as in Fig. 2Go. The cultures were pulsed for 6 h with [3H]thymidine at the times indicated and incorporation of radioactivity into cellular DNA measured as in Fig. 2Go. Results are the means ± SEM of pooled data from two to six individual experiments, each experiment involving three cultures per time point. Cultures with T cells alone gave very low counts (see Fig. 2Go), stimulation indices at the peak being always in excess of 100.

 
It seemed that in LN DEC-205, rather than CD8{alpha}, was marking the `regulatory' DC population which stimulated the CD8 T cells into cycle but induced only limited IL-2 production and hence restricted proliferation. Based on the results of Figs 2 and 3GoGo, it could be deduced that the CD8{alpha} fraction from LN would include the more stimulatory CD8{alpha} DEC-205 DC of the type found in spleen, together with some `regulatory', lymphoid-related DC that were DEC-205+ but had failed to express CD8{alpha}, unlike their splenic counterparts. The excess of these CD8{alpha} DEC-205+ DC seemed likely to account for the reduced responses to LN CD8{alpha} DC. To check this, CD8{alpha} DC were isolated from LN, segregated by DEC-205 expression then cultured with allogeneic CD8 T cells. As shown in Fig. 4Go, this view was confirmed, the CD8{alpha} DEC-205+ LN DC giving the curtailed proliferative response resembling that of the splenic CD8{alpha}+ DEC-205+ DC.

The conclusion from these experiments was that DEC-205 served as a better marker of the DC population giving curtailed CD8 T cell responses than did CD8{alpha}. This fitted with our previous findings that CD8{alpha} itself did not give the negative signals restricting CD8 T cell cytokine production, but posed the question of whether DEC-205 was serving to transmit regulatory signals. To test this possibility, we isolated DC from DEC-205null mice and compared their CD8 T cell stimulatory ability with that of DC from littermate DEC-205-expressing heterozygotes. In these experiments only splenic DC were studied, since only in spleen could CD8{alpha} be used as a surrogate marker for DEC-205, to segregate the two functional types of DC. DC were found to be present at near normal total numbers in the spleens of DEC-205null mice, although the numbers of CD8{alpha}+ DC recovered was a little higher (49,000 ± 9000 per spleen compared to 39,000 ± 2000 for the littermates) and the number of CD8 DC recovered was lower (7000 ± 3000 per spleen compared to 12,000 ± 5000 for the littermates). The functional tests on these isolated DC are shown in Fig. 5Go.



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Fig. 5. Kinetics of the response of CD8 T cells to allogeneic splenic DC purified from DEC-205 `null' mice or from heterozygous littermate control mice. Conditions were similar to Fig. 4Go. The results are means ± SEM from a single experiment, representative of three experiments performed.

 
The ability of the two DC fractions from the spleens of DEC-205null mice to stimulate allogeneic CD8 T cells was similar to that of normal mice. In both DEC-205null and normal mice, the CD8{alpha} DC induced a more extended and higher CD8 T cell proliferative response, while the response to the CD8{alpha}+ DC was curtailed. It was concluded that the absence of DEC-205 had little effect on the ability of the DC to interact with allogeneic CD8 T cells in culture, and that DEC-205 itself was not responsible for negative signalling leading to reduced cytokine output and reduced proliferation by CD8 T cells. The one caveat on this conclusion is that the role of DEC-205 could be redundant, other related molecules taking over the function when DEC-205 is absent.

Overall it is clear that DEC-205 can serve as a useful marker of the type of DC inducing in CD8 T cells only restricted cytokine production and a curtailed proliferative response (9); in many circumstances it may be a better marker for this population than CD8{alpha}. This type of `regulatory' DC is likely to be of lymphoid origin, since similar DC can be produced artificially by transfer of the thymic lymphoid precursor population (Fig. 3Go) (6). However, it is clear that DEC-205 itself does not govern the nature of DC interaction with the T cells. Its influence on the earlier steps of antigen uptake and processing remains to be determined, since in this study we have only considered responses to endogenous alloantigens.

Surface DEC-205 expression is clearly not an absolute marker of regulatory DC, since even the population we have designated DEC-205 includes some DC with low surface expression and we have found all DC stain positive for intracellular DEC-205 if permeabilized (5). In addition, the surface expression of DEC-205 increases on all DC if they are cultured for a short period (5). However, even under these circumstances when all DC are clearly surface positive, the level of DEC-205 expression still serves to distinguish the populations (5). The fact that the `regulatory' or less stimulatory type of DC expresses the highest levels of DEC-205 now presents a paradox, since these are the DC believed to be concentrated in the T cell areas of spleen and LN (10), and these might have been expected to be the most stimulatory DC type.


    Acknowledgments
 
This work was supported by the Cooperative Research Centre for Vaccine Technology, Queensland Institute for Medical Research, by a Human Frontier Science Program Grant RG-237-97, and by the National Health and Medical Research Council, Australia. We thank David Vremec for the surface phenotype analysis of normal DC populations, and Dora Kaminaris, Jennie Parker and Frank Battye for assistance with flow cytometry.


    Abbreviations
 
DC dendritic cell
LN lymph node

    Notes
 
Transmitting editor: M. Feldmann

Received 7 June 1999, accepted 3 February 2000.


    References
 Top
 Abstract
 Introduction
 References
 

  1. Shortman, K. and Caux, C. 1997. Dendritic cell development: multiple pathways to nature's adjuvants. Stem Cells. 15:409.[Abstract/Free Full Text]
  2. Süss, G. and Shortman, K. 1996. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J. Exp. Med. 183:1789.[Abstract]
  3. Kronin, V., Winkel, K., Süss, G., Kelso, A., Heath, W., Kirberg, J., von Boehmer, H. and Shortman, K. 1996. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J. Immunol. 157:3819.[Abstract]
  4. Vremec, D., Zorbas, M., Scollay, R., Saunders, D. J., Ardavin, C. F., Wu, L. and Shortman, K. 1992. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J. Exp. Med. 176:47.[Abstract]
  5. Vremec, D. and Shortman, K. 1997. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes on incubation and differences between thymus, spleen and lymph nodes. J. Immunol. 159:565.[Abstract]
  6. Wu, L., Li, C.-L. and Shortman, K. 1996. Thymic dendritic cell precursors: relationship to the T-lymphocyte lineage and phenotype of the dendritic cell progeny. J. Exp. Med. 184:903.[Abstract]
  7. Winkel, K. D., Kronin, V., Krummel, M. F. and Shortman, K. 1997. The nature of the signals regulating CD8 T cell proliferative responses to CD8{alpha}+ or CD8{alpha}- dendritic cells. Eur. J. Immunol. 27:3350.[ISI][Medline]
  8. Sambhara, S. R. and Miller, R. G. 1991. Programmed cell death of T cells signaled by the T cell receptor and the alpha3 domain of class I MHC. Science 252:1424.[ISI][Medline]
  9. Kronin, V., Vremec, D., Winkel, K., Classon, B. J., Miller, R. G., Mak, T. W., Shortman, K. and Süss, G. 1997. Are CD8+ dendritic cells veto cells? The role of CD8 on dendritic cells in the regulation of CD4 and CD8 T cell responses. Int. Immunol. 9:1061.[Abstract]
  10. Kraal, G., Breel, M., Janse, M. and Bruin, G. 1986. Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody. J. Exp. Med. 163:981.[Abstract]
  11. Jiang, W., Swiggard, W. J., Heufler, C., Peng, M., Mirza, A., Steinman, R. M. and Nussenzweig, M. C. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375:151.[ISI][Medline]
  12. Guo, M., Gong, S., Maric, S., Misulovin, Z., Pack, M., Steinman, R. M. and Nussenzweig, M. C. 1999. A monoclonal antibody to human DEC-205. Submitted.
  13. Saunders, D., Lucas, K., Ismaili, J., Wu, L., Maraskovsky, E., Dunn, A., Metcalf, D. and Shortman, K. 1996. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte-macrophage colony-stimulating factor. J. Exp. Med. 184:2185.[Abstract/Free Full Text]