We generated DC populations by culturing bone marrow from RAG−/−
mice in standard cultures containing GM-CSF and IL-4. We chose RAG−/−
mice because memory T cells accumulate in the bone marrow of conventional mice (19
), and these can greatly influence the DCs’ propensity to produce cytokines (9
). After six days of culture, we stimulated the DCs with LPS, waited 24-27 hours (the period in which they have been described to become “exhausted” (1
), washed and then re-stimulated them in various ways. We began with an analysis of IL-12p75 production by the DCs, as IL-12p75 (also known as IL-12p70, which we will hence call “IL-12”) was one of the main cytokines analyzed in the original study defining exhaustion (1
), and it is thought to be the primary cytokine driving TH
shows that mouse bone marrow-derived DCs behave similarly to the human blood monocyte-derived DCs used in the original “exhaustion” study. Resting DCs make large amounts of IL-12 after stimulation with LPS plus IFN-γ. However, when re-stimulated 27 hours after a previous LPS stimulation, the DCs make no IL-12, even in the presence of IFN-γ. Thus, like their human counterparts (1
), LPS stimulated mouse DCs appear to become “exhausted”.
Under normal circumstances, however, a DC would be very unlikely to remain isolated from T cells for very long after stimulation. A resting DC that encountered LPS in the periphery would migrate within a few hours to a draining secondary lymphoid organ, where it would encounter both local and migratory T cells. We therefore asked if signals from T cells might change the responses of “exhausted” DCs, using CD4 T+ cells from RAG−/− 5C.C7 TCR Tg mice, which are specific for pigeon cytochrome c [PCC]. To separately test the effects of naïve versus previously stimulated T cells, we isolated naïve T cells from unprimed mice and used them immediately, or cultured them with PCC and APCs for one to several weeks to generate effector/memory T cells. We found that the presence of T cells did indeed change the behavior of the DCs. , composite of 44 separate experiments, shows that activated T cells (but not naïve T cells) helped both resting and “exhausted” DCs to produce large amounts of IL-12. Thus, the DCs seemed to be only conditionally “exhausted”. They did not respond to LPS plus IFN-γ, or to naïve T cells, but they did respond to IL-12-inducing signals from activated T cells.
Because DCs from RAG−/− mice make very little or no IL-12 when stimulated with LPS alone, we considered the possibility that they might not be sufficiently “exhausted”. It might be necessary, for example, to generate conditions, in which a large amount of IL-12 is made, in order to generate the negative feedback conditions leading to “exhaustion”. To see if a stronger primary IL-12-inducing stimulus might induce a more profoundly “exhausted” state that could not be overcome by the addition of T cells, we stimulated resting DCs with LPS plus IFN-γ, a stimulus that induces large amounts of IL-12 (,). shows that these cells, which we call “hammered” DCs, behaved like “exhausted” DCs. They were unable to produce IL-12 when re-stimulated with LPS plus IFN-γ, but made copious amounts in response to activated T cells.
shows that IL-12 production is not an exception, as pre-stimulated DCs can also produce other pro-inflammatory cytokines when given T cell help. The pattern of TNFα expression, for example, mirrored that of IL-12. “Exhausted” and “hammered” DCs were unable to produce TNFα in response to further stimulation with LPS, or with LPS plus IFN-γ, but responded well to help from T cells. IL-1α behaved somewhat differently, in that “exhausted” and “hammered” DCs actually made more cytokine than resting DCs, and much more in the presence of T cells. Finally, IL-12p40, the subunit that contributes to both IL-12 and IL-23, was made in approximately equivalent amounts by all three sets of DCs.
These data suggest that LPS-activated DCs are not inexorably programmed to follow a particular path to “exhaustion”. Nor, as previously suggested, do they necessarily become skewed towards TH
2 responses, (1
), or anti-microbial responses (2
). Instead, it seems that the DCs lose their responsiveness to LPS, but remain perfectly functional and capable of making pro-inflammatory cytokines if given other appropriate stimuli, such as the signals mediated by activated (but not by naïve) T cells.
Dissecting the T cell signals that overcome “exhaustion”
To begin to dissect the mechanism by which T cells induce “exhausted” DCs to produce cytokines, we added individual soluble and cell-surface T-cell derived molecules, singly and in combination, to DCs stimulated in various ways. We began with the cell surface molecule, CD40L, which is expressed by activated T cells, and rapidly up-regulated by naïve T cells (20
). In the original report on DC “exhaustion”, J558 cells transfected with CD40L were unable to elicit IL-12 from “exhausted” human DCs (1
). We were surprised, therefore, to find that CD40L-transfected NIH-3T3 cells were occasionally able to elicit IL-12 from both resting and “exhausted” mouse DCs. Because this effect was sporadic, we studied it further and found that DCs rigorously washed from their growth/differentiation medium were unable to respond to the CD40L stimulus, and that even small amounts of the medium were sufficient to restore the response, suggesting that the medium contained soluble products able to synergize with CD40L to elicit IL-12. Because both IL-4 and GM-CSF are soluble T-cell-derived constituents of the DC growth/differentiation medium that may remain in varying residual amounts from experiment to experiment, and because the combination of IL-4 plus CD40L has been shown to synergize with LPS to elicit IL-12 from resting DCs (10
), we asked if these cytokines would also synergize with CD40L to stimulate “exhausted” DCs. We cultured resting, “exhausted” or “hammered” DCs alone, or with various cellular stimuli (3T3 cells, transfected or not with CD40L, or effector T cells). For each of these co-culture conditions, we added various combinations of the cytokines, IL-4, IFN-γ and GM-CSF.
, shows that mouse DCs, like their human counterparts, did not produce IL-12 in response to any combination of T-cell-derived cytokines in the absence of CD40L (mock, and also see , where the DCs were grown in the presence of IL-4 and GM-CSF, but have no source of CD40L except when T cells are added), or to CD40L alone (grey bars). Thus, CD40L was essential but not sufficient. In its presence, different DCs responded to different combinations of soluble T cell-derived cytokines. For example, resting DCs made a small amount of IL-12 in response to CD40L plus IL-4 (as previously shown (7
), red bar). “Exhausted” DCs made more, and hammered DCs made even more, suggesting that pre-stimulation with LPS plus IFN-γ strongly sensitizes a DC to further instructions from T cells. The responses of the “exhausted” DCs were further enhanced by the addition of GM-CSF, and even more by the addition of GM-CSF plus IFN-γ. Overall, the data show that none of the DCs were truly “exhausted”. All three populations were capable of making copious amounts of IL-12 in response to intact activated T cells, and different types of activation rendered them differentially sensitive to particular combinations of T cell products. The combination of CD40L plus IL-4 was particularly illuminating, in that “exhausted” DCs were more sensitive to this combination than resting DCs, and “hammered” DCs were even more responsive. These data indicate that an activated DC is prepared to respond to signals that its resting, tissue-resident counterpart cannot.
CD40L and IL-4 play an important role in IL-12p75 production by DCs
TH2 cells produce an inhibitor
The synergy of IL-4 with CD40L in activating IL-12 in “exhausted” DCs suggested that TH
2 cells might be more potent inducers of IL-12 than other classes of T cells. To test this, we used a classic representative of a TH
2 T-cell clone, D10.G4. (21
) (specific for Conalbumin, hereafter called “D10”), comparing it with the TH
1 clone, AE7 (specific for PCC (22
) and to activated TH
0 cells. shows that recently activated TH
0 T cells and the AE7 TH
1 clone and were both very effective at inducing IL-12 from “exhausted” DCs. To our surprise, however, the D10 TH
2 clone was not, even though it expresses CD40L (Fig. S1)
and is known to produce large amounts of IL-4 (Fig. S2)
). Reasoning that long term culture may select for mutant D10 cells that have lost some of their original functions, we generated short term polarized T cells, using standard polarizing culture conditions to differentiate resting T cells into effector TH
2 or TH
17 populations. When added to “exhausted” DCs, the TH
0 and TH
1 cells induced production of IL-12, but the TH
2 and TH
17 cells did not ().
TH0 and TH1 cells induce IL-12p75 production but TH2 cells do not
Given that CD40L plus IL-4 was a sufficient combination to induce IL-12 from “exhausted” and “hammered” DCs, the fact that TH
2 cells were unable to do the same, in spite of their production of IL-4, suggested that they might also be producing an inhibitory factor. We therefore titrated the supernatant from D10 (stimulated with anti-CD3 and anti CD28 so as to preclude anything made by APCs) into a co-culture of “exhausted” DCs with TH
0 effector T cells (which would normally induce good levels of IL-12), and found that the supernatant did indeed produce a dose-dependent inhibition of IL-12 production (). Using Multiplex cytokine arrays, we analyzed the cytokines produced by D10 and found that, in addition to copious amounts of typical TH
2-type cytokines (such as IL-4, 5 and 13) and some pro-inflammatory cytokines (such as IL-6), the supernatant also contained large amounts of IL-10 (Fig. S2
), which has been established to suppress secretion of IL-12 by newly activated DCs (24
). To see if the inhibitory activity in the supernatant was due solely to the IL-10, we passed it over an anti-IL-10 affinity purification column, and tested the depleted supernatant for inhibitory activity on co-cultures of “exhausted” DCs and activated TH
0 cells. shows that the anti-IL-10 column removed the suppressive activity and allowed for production of IL-12, (while an isotype control column did not), and shows that the suppressive activity of the depleted supernatant could be restored by the addition of recombinant IL-10. Thus, IL-10 can suppress the positive stimulation of IL-12 that is normally produced by TH
0 T cells. Altogether, these data suggest that the production of IL-12 is under tight control by T cells, with both positive and negative T-cell-derived regulatory elements (manuscript in preparation).
Other DC-derived cytokines
Thus far, we had focused on the control of IL-12, which is the primary cytokine that led to the idea of “exhaustion”. However, the originators of the “exhaustion” concept did not limit it to IL-12, but also extended it to other cytokines (1
). To test this view, we allowed TH
1 and TH
2 cells to interact with resting, “exhausted” or “hammered” DCs and measured the production of a wide variety of cytokines using multiplex arrays. shows that, even in the absence of T cells (grey bars), not all DC-derived cytokines show the classic “exhaustion” pattern after LPS stimulation. For example, IL-12p40, IL-1α and IL-6 were produced at low levels by resting DCs, and in higher amounts by both “exhausted” and “hammered” DCs (p
values all < 0.03). Thus, inflammatory cytokine production is not extinguished in LPS-stimulated DCs. “Exhaustion” seems to be specific to certain cytokines, such as IL-12 and TNFα.
The presence of T cells made significant changes in these patterns, and the changes varied depending on the effector class of the T cell, and on the activation state of the DC. TH
0 cells, for example, had little effect on the production of IL-12p40, but induced “exhausted” and “hammered” DCs to produce IL-12, and stimulated all three DC populations to increase production of IL-1α, IL-6 and TNFα (p
values for all <0.05 when comparing DCs cultured with TH
0 T cells versus medium controls). TH
1 T cells behaved generally like somewhat more potent versions of TH
0 cells, except that they also triggered an increase in IL-12p40 production,
a strong increase in IL-12 by resting DCs, and a decrease in IL-12 by “hammered” DCs when compared to TH
0 cells. TH
2 cells, while causing a ten-fold increase in the production of IL-6 (compared to medium alone), generated minor or no increases in IL-12p40 or p75, IL-1α, or TNFα. These data suggest that it may be time to change the view that the presence of T cells should merely amplify the pattern of DC cytokine production that has already been programmed by stimulation through TLRs (26
). Instead, it appears that different types of T cells can make significant changes in the pattern of cytokines produced by DCs while showing few differences in their own proliferative responses to these DCs (Fig. S3
To molecularly define the T cell’s role in shaping cytokine production from “exhausted” and “hammered” DCs, we stimulated the three types of DCs with the artificially reconstituted T cell components, CD40L plus/minus IL-4, or with activated T cells. shows that the three types of DCs responded differently. CD40L alone was sufficient to stimulate IL-6 production from all three types of DCs; but IL-12p40, TNFα and IL-23 mainly from “exhausted” and “hammered” DCs, emphasizing the idea that “exhausted” and “hammered” DCs are in fact uniquely primed to receive some T cell signals that do not affect resting DCs, and underscoring the importance of using multiple markers to examine DC activation. The addition of CD40L plus IL-4 increased the production of IL-12. The combination actually decreased the production of IL-12p40 by “exhausted” and “hammered” DCs, when compared to the effect of CD40L alone, as has been previously shown for resting DCs stimulated with LPS (28
). Activated T cells were stronger inducers of IL-12, IL-1α and TNFα than their isolated components of CD40L plus/minus IL-4, but weaker inducers of IL-12p40 and IL-23, suggesting that there are T-cell-derived controlling elements yet to be found. Altogether, these data support the concept that DCs are not pre-programmed indelibly by their initial stimulation to produce a particular set of cytokines, only to be enhanced by T cell signals. They change, depending on what signals they encounter.
We also measured T cell cytokine production (), comparing the relative stimulatory capacities of the resting, “exhausted” and “hammered” DCs. Although it has been suggested that “exhausted” DCs tend to promote TH2, or non-inflammatory responses, we found that the pattern was not as clear as previously thought (). All three DC types stimulated TH0 cells with roughly similar potencies. To see if the cytokine patterns were permanently fixed, we also tested the DCs with known TH1 and TH2 cells, to see if any one of the DC types would pair best with particular T effectors. We found that neither resting, nor “exhausted” nor “hammered” DCs paired best with any particular effector T cell type. When tested with TH1 cells, for example, resting DCs were more efficient at eliciting IL-2 and IL-3 production, but the three types of DCs were equivalent at inducing other cytokines. When tested with TH2 cells, “exhausted” DCs induced twice as much IL-13 as did resting or hammered DCs, while resting DCs induced ten fold more IL-3 than the other DCs, and all three types of DCs induced similar amounts of IL-4, IL-5 and IL-10. Thus, when a large array of cytokines was tested, each DC set promoted a different array of cytokines, and neither “exhausted” nor “hammered” DCs seemed to preferentially promote the production of TH2 cytokines.
Production of various T cell cytokines in the presence of resting, “exhausted” and “hammered” DCs
Collectively, these data confirm that LPS-stimulated DCs are neither “exhausted” (1
), nor paralyzed (29
) nor tolerized (2
) after stimulation with LPS or LPS plus IFN-γ. Nor do they have any one set response pattern. Instead, the DCs seem to move from a state in which they are responsive to some environmental signals (e.g. LPS), to one in which they are highly responsive to a different set of environmental signals (e.g. signals from T cells ). The resulting DC cytokine production depends on the combined effects of the original activating signal(s) and the T-cell derived signal(s) they receive later.
Overview of changes in DC sensitivity