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Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL 32610
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The reconstitution of NZM2410 immunopathology was achieved by the triple congenic combination of Sle1/Sle2/Sle3 in B6.TC mice, which resulted in 100% mortality by 12 mo of age (15). Despite the likely existence of NZM2410 SLE-susceptibility loci other than Sle1, Sle2, or Sle3, the B6.TC strain constitutes a better model to analyze lupus pathogenesis as the fully penetrant autoimmune disease develops on a normal genetic background with only 
6% (i.e., Sle1/Sle2/Sle3) of the parental NZM2410 genome. We are making progress in the identification of the genetic polymorphism corresponding to Sle1 and Sle2 (9, 16, 17, 18). Consequently, the B6.TC model will provide in the near future a link between phenotypic defects on one hand and causative genes and interactions between these genes on the other hand. In addition, B6 and B6.TC share the same H-2b haplotype, which allows functional experiments that have been difficult in the BWF1 or NZM2410 models.
Although most of the attention has been focused on the role of B and T cells in lupus, there is also evidence that dendritic cells (DCs) have abnormal phenotypes in lupus patients and mouse models of SLE. The number of circulating plasmacytoid DCs (pDCs) is reduced in lupus patients (19, 20), and the number of total DCs is increased in the lymphoid organs of lupus-prone mice (21, 22, 23, 24). Abnormal costimulatory profiles have also been reported in lupus patients (25, 26, 27) and in BWF1 or NZM2410 mice (28). DC functions have also been implicated in the pathogenesis of lupus. Injections of BWF1 syngeneic DCs that have been exposed to apoptotic material accelerated disease (29). FAS-mediated DC apoptosis has been recently shown to be critical for the maintenance of self tolerance (30). Furthermore, injections of Sle3-expressing DCs into normal B6 mice were sufficient to induce the production of anti-DNA Abs (13).
The role of DCs in regulating T cell tolerance and T cell activation has been abundantly documented. More recent studies have clearly established that DCs also play an essential role in the dominant suppression of autoimmunity by regulatory T cells (Treg). DCs are necessary for Treg selection in the thymus and expansion in the periphery, as reviewed in Ref. 31 . Imaging studies have also shown that DCs are a necessary intermediate by which Treg exert their regulatory functions (32, 33). As for most autoimmune diseases, Treg deficiencies have been described in lupus patients (34, 35). Treg play a key role in maintaining tolerance to DNA in a transgenic model (36). In spontaneous models of lupus, tolerance induction to nuclear Ag with histone peptides was dependent on the generation of Ag-specific Treg in the (NZB x SWR)F1 mouse (37). Moreover, we have shown that the NZM2410 Sle1 locus was associated with a reduction of CD4+CD25+CD62L+Foxp3+ Treg numbers (8). In the present study, we examined DC phenotypes in B6.TC mice and their effect on Treg functions. We confirmed that DCs accumulate in lymphoid organs, due at least in part to a greater production from the bone marrow (BM) and decreased apoptosis, and that they have a reduced expression of costimulatory molecules. More importantly, B6.TC DCs induce a greater proliferation in CD4+ T cells and inhibit Treg regulatory functions. These phenotypes are at least partly mediated through overproduction of IL-6. A TLR-dependent inhibition of Treg functions has already been shown to be mediated through IL-6 production by DCs (38). Our results show DCs from lupus-prone mice function in a similar proinflammatory mode as normal DCs that have been exposed to microbial products. A greater CD4+ T cell-stimulatory function has been associated with Sle3-expressing DCs (13) and most likely contributes to this phenotype in TC DCs. Here we show, however, that the inhibition of Treg regulatory functions through IL-6 maps to Sle1. Therefore, our results suggest that autoreactive T cells in B6.TC mice result from the interplay of low Sle1-expressing Treg numbers, inhibition of their functions by Sle1-expressing DCs, and hyperstimulation of CD4+ T cells by Sle3-expressing DCs.
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Materials and Methods |
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C57BL/6J (B6), BALB/c, and C57BL/6-Tg (TcraTcrb)425Cbn/J (B6.OT-II) mice were obtained from The Jackson Laboratory and subsequently bred at University of Florida (Gainesville, FA). The production of the triple congenic B6.Sle1.Sle2.Sle3 (B6.TC) (15) and B6.Sle1 (3) mice has been described previously. All experiments were conducted according to protocols approved by the University of Florida Institutional Animal Care and Use Committee. Female mice were used at 6–8 or at 2–3 mo old as indicated, with mice aged matched within a same experiment.
Generation of DCs and DC phenotyping
BM single-cell suspensions were cultured in complete RPMI 1640 containing 10% FCS, 10 ng/ml GM-CSF, and 10 ng/ml IL-4 (R&D Systems). Every 2 days, 50% of the culture medium was replaced with fresh medium. After 6 days, the cultured cells were harvested and ascertained by flow cytometry, then CD11c+ cells were purified using magnetic microbeads according to the manufacturer’s (Miltenyi Biotec) instructions. To assess activation levels, BM-derived DCs were cultured for 24 h with medium alone, LPS (Sigma-Aldrich), CpG oligodeoxynucleotide 2336 (Coley Pharmaceutical), or TRL7 ligand Imiquimod R837 (InvivoGen), all at 1 µg/ml). For cytokine assays, CD11c+ DCs (106 cells/ml) were stimulated with LPS for 13 h (intracellular assay) or with LPS, CpG, or TLR7L (all at 1 µg/ml) for 24 h (supernatant assay). The supernatants were harvested and stored at –80°C until assayed with commercial ELISA kits (BD Systems). To quantify apoptosis, cells were stimulated with LPS (1 µg/ml) for 24 h, stained with annexin V and 7-aminoactinomycin D (BD Biosciences Pharmingen) and examined by flow cytometry.
Flow cytometry
Single-cell suspensions were treated with FcR block (2.4G2) and then directly stained with mAbs to mouse B220 (RA3-6B2), CD3 (145-2C11), CD4 (RM4-5), CD11b (M1/70), CD11c (HL3), CD25 (7D4), CD19 (1D3), CD40 (HM40-3), CD62L (MEL14), CD80 (16-10A1), CD86 (GL1), I-Ab (AF6-120.1), NK1.1 (PK126), TER119, or isotype controls (all from BD Pharmingen). PE-conjugated anti-CD3, CD19, NK1.1, and TER119 Abs were used to gate out CD11clow T cells, B cells, NK cells, and erythroblasts, respectively. Biotinylated Abs were revealed with streptavidin PerCP-Cy5.5. At least 30,000 cells per sample were analyzed on a BD FACSCalibur. To assay for intracellular cytokine production, GolgiPlug reagent (BD Pharmingen) was added in the last 5 h of stimulation; then cells were stained with the appropriate Abs to gate on the specific population of interest, and with anti-IL-6 Ab from an intracellular staining kit (BD Pharmingen).
T cell isolation and proliferation assays
Splenic CD4+ T cells were purified from 2 to 3 mo old B6, B6.TC, BALB/c or B6.OT-II mice by negative selection using the CD4+ T cell isolation kit (MACS; Miltenyi Biotec) and the purity was typically 90–95%. Splenic CD4+ CD25– effector T (Teff) cells and CD4+ CD25+ Treg cells were purified from B6 using the CD4+CD25+ Regularity T cell isolation kit (MACS; Miltenyi Biotec), and the purities were 90–95% and >95%, respectively.
All DCs used in the proliferation and suppression assays were obtained from a 6-day BM culture from 2- to 3-mo-old mice. For allogeneic T cell proliferation assays, BALB/c CD4+ T cells (5 x 105) were labeled with CFSE (Invitrogen Life Technologies) and incubated with B6 or TC BM-derived DCs (5 x 104) in a 37°C incubator with 5% CO2 in 0.5 ml of complete RPMI 1640 (10% FCS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME). For OVA-specific proliferation assays, CFSE-labeled B6.OT-II CD4+ T cells (5 x 105) were incubated with B6 or TC BM-derived DCs (5 x 104) prepulsed with 1 µg/ml OVA323–339 peptide (ISQAVHAAHAEINEAGR, synthesized at the University of Florida Protein Core) for 2 h in complete RPMI 1640. After 72 h of culture, the degree of CD4+ T cell division was gauged from the serial dilution of CFSE.
For syngeneic assays, irradiated (30 Gy) B6 or TC BM DCs (1 x 104 or 1 x 103) were cocultured with CD4+ or CFSE-stained CD4+CD25– Teff cells (1 x 105), with or without CD4+CD25+ Treg cells (5 x 104) at a Treg:Teff ratio ranging from 0:1 to 1:8 in wells precoated with anti-CD3 (1 µg/ml; BD Biosciences) for 72 h in complete RPMI 1640. T cell proliferation was measured by adding 1 µCi of [3H]thymidine to each well for the last 16 h of culture. In some assays, anti-IL-6 (MP5-20F3; BD Pharmingen) was added at 5 µg/ml to neutralize IL-6. An irrelevant matched Ab (murine IgG1) was used at the same concentration as control. In other assays, IL-6 (R&D Systems) was added in graded amounts as indicated in the text. IL-2 and IL-6 levels were measured in supernatants after 48 h (IL-2) or 72 h of culture by sandwich ELISA (BD Biosciences).
Statistics
Statistical significance was always evaluated between the values obtained for the lupus-prone mice and B6 controls. After verification of normal distribution with D’Agostino and Pearson omnibus normality tests, we used either one-tailed t tests when two groups were compared (B6 vs (B6 vs B6.TC and B6.Sle1)). Statistical significance was obtained when p was 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The percentage and absolute number of CD11c+ cells were significantly higher in all the lymphoid organs of B6.TC mice (Fig. 1, A and B, and Table I). On average, the percentage of CD11c+ cells was 2.67 and 3.60 times higher in B6.TC spleen and cervical lymph nodes than in B6, respectively. The difference was even more striking with absolute numbers (3.68- and 4.17-fold higher for the spleen and the lymph node, respectively), due to the lymphoid hyperplasia that is characteristic of lupus-prone mice, including B6.TC (15). Interestingly, the percentage of DCs was significantly decreased in the peritoneal cavity, mostly likely as a consequence of the expansion of B-1a cells in this compartment (18). Both numbers (Fig. 1, C and D, and Table I) and percentages (data not shown) of plasmacytoid (CD11clowB220+; pDCs) and myeloid (CD11chighCD11b+ B220–; MDCs) DCs were 3–4 times higher in the spleen and lymph nodes of B6.TC mice. In the BM, the number of MDCs was also significantly higher in B6.TC, but the number of pDCs was 1.5 times lower than in B6. This latter finding suggests an enhanced recruitment of pDCs to the secondary lymphoid organs, which parallels the relative paucity of circulating pDCs and recruitment to inflammatory sites in SLE patients (39). DC accumulation in lymphoid organs was age-dependent, as the differences were not significant at 2–3 mo of age, although individual B6.TC mice already showed higher values (data not shown). A higher percentage of CD11c+ cells were also generated from B6.TC BM cultures, and this phenotype was observed in both old (Fig. 1E and Table I) and young mice (data not shown). Moreover, these BM-derived DCs showed a significantly decreased apoptotic death in response to LPS stimulation (Fig. 1F).
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TC DCs induce a greater proliferative response in CD4+ T cells
We compared the effect of TC and B6 DCs on CD4+ T cells in three different experimental conditions. In all assays, T cells and DCs were obtained from young 2- to 3-mo-old mice. In an allogeneic mixed lymphocyte reaction, BALB/c CD4+ T cells responded more vigorously to TC than B6 DCs (Fig. 3A). In a syngeneic mixed lymphocyte reaction, B6 CD4+ T cells proliferated significantly more to anti-CD3 stimulation in the presence of TC DCs than B6 DCs (Fig. 3B). The difference was not due to the genetic disparity between the B6 and B6.TC strains that could induce alloreactivity between TC DCs and B6 T cells, because the same results were obtained using either B6 or TC CD4+ T cells (Fig. 3C). Therefore, we used the more abundant B6 mice as the source of T cells for the rest of the experiments. Finally, we tested the stimulatory effects of the TC DCs in an Ag-specific assay. CD4+ T cells purified from B6.OT-II mice, which carry a transgenic TCR specific for the OVA323–339 peptide (40), proliferated significantly more to TC than to B6 DCs pulsed with the OVA peptide (Fig. 3, D and E). These results indicated that TC DCs induce a greater proliferative response in CD4+ T cells regardless of the nature of the stimulation.
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We have previously shown that the Sle1 locus was associated with a reduced number of CD4+ CD25+ CD62L+ Foxp3+ Treg (8). The percentage of CD4+ T cells that expressed both CD25 and CD62L was significantly decreased in B6.TC spleens as compared with B6 (2.06 ± 0.27 vs 5.27 ± 0.31%, respectively; p < 0.0001), and this reduction was comparable with that we have described in B6.Sle1 mice (8), suggesting that Sle2 and Sle3 do not contribute to this phenotype. To assess whether TC DCs affect Treg functions without the potential confounding factor of B6 and TC Treg disparity, we used the same B6-derived CD4+ CD25– (Teff) and Treg in all assays. As expected, B6 BM-derived DCs supported Treg-mediated suppression of Teff proliferation and IL-2 production (Fig. 4). Confirming the results presented earlier with total CD4+ T cells, TC DCs were associated with a greater Teff survival as measured by the percentage of CFSE-positive CD4+ cells, and a greater IL-2 production (Fig. 4, B and C, left bars). Teff proliferation did not differ, however, in the presence of B6 or TC Dcs (data not shown). In the presence of Tregs, TC DCs resulted in a decreased inhibitory capability, especially in the lower Treg:Teff ratios (Fig. 4A) and a still significantly higher percentage of live CD4+ T cells (Fig. 4B). Finally, the production of IL-2 was reduced to 38.28% in the presence of TC DCs as compared with 21.72% in the presence of B6 DCs (p < 0.03) (Fig. 4C). Overall, these results demonstrate that TC DCs impede the regulatory function of Treg.
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It has been shown that DC-produced IL-6 inhibits Treg-mediated suppression in a TLR4-dependent manner (38). We assessed whether IL-6 was also responsible for the TC DC effect on Treg cells. Purified BM-marrow derived CD11c+ DCs produced significantly more IL-6 in B6.TC than in B6 mice (an average of 2-fold more when exposed to LPS (1.75-fold increase), CpG (1.80-fold increase) and TLR7L (1.60-fold increase) (Fig. 5, A and B). When cocultured with Teff cells, TC DCs produced also significantly more IL-6 than B6 DCs (Fig. 5E, left bars). The addition of Treg cells decreased the amount of IL-6 produced by both TC and B6 DCs, which corresponds to the recently described modulation of immature DC function by Treg (41), but IL-6 levels were still significantly higher for TC DCs (Fig. 5E, right bars). The addition of a neutralizing anti-IL-6 Ab to the suppression assays restored Treg inhibitory effect on T cell proliferation in the presence of TC DCs, but it had no effect in the presence of B6 DCs (Fig. 5C). The effect of anti-IL-6 was dose dependent with a maximum effect reached starting at 2.5 µg/ml (Fig. 5F). Interestingly, blocking IL-6 had no effect on the proliferation of Teff cells alone, in the presence of either B6 or TC DCs (data not shown), although large amounts of IL-6 were produced in these cultures (Fig. 5E). We confirmed that the addition of exogenous IL-6 (0.1 µg/ml) abrogated Treg suppression with B6 DCs as previously reported (38), and this effect was similar with TC DCs (Fig. 5D). Interestingly, higher concentrations of IL-6 promoted a significantly higher proliferation in the presence of TC DCs than B6. The spontaneous production of IL-6 by TC DCs in the presence of T cells (Fig. 5E) may be sufficient to not only inhibit Treg suppressive ability but also induce substantial proliferation. Overall, these results suggest that the high level of IL-6 produced by TC DCs exerts a specific function in overcoming Treg inhibitory functions.
Sle1 contributes to TC DC inhibition of Treg-mediated suppression
Sle3-expressing DCs induce hyperactivation and higher proliferation in CD4+ T cells (13). Although Sle3-expressing DCs produce a high level of proinflammatory cytokines, their production of IL-6 is not different from that of B6. Because we have shown that IL-6 is a key factor in TC DC inhibition of Treg-mediated suppression, we deduced that Sle3 expression was not sufficient to mediate this phenotype. IL-6 is produced at high levels by Sle1-expressing B cells and macrophages, and is linked to STAT3 and ERK activation (42). We therefore postulated that Sle1 contributed to the TC DCs phenotype. Similarly to TC DCs, a greater percentage (Table I) and number (data not shown) of DCs were found in the lymphoid organs of B6.Sle1 mice, and Sle1 BM-cultures produced significantly more DCs than B6. All parameters measured were, however, intermediate between the B6 and B6.TC values, suggesting that Sle1 is not the only contributor to the increased TC DC number and percentage in lymphoid organs. Contrary to TC DCs, however, Sle1-BM-derived immature DCs expressed a normal to elevated level of activation markers as shown for CD86 (Fig. 6A). The same results were obtained for CD80 and I-A, with and without LPS stimulation (data not shown). Similarly again to TC DCs, Sle1 DCs showed a significantly decreased apoptotic response (Fig. 6B) and an increased production of IL-6 (Fig. 6C) when exposed to LPS. This increased IL-6 production was associated with an inhibition of Treg functions, because B6 Treg cell ability to suppress B6 Teff cell proliferation were similarly inefficient in the presence of either TC or Sle1 DCs (Fig. 6D). Furthermore, neutralizing of IL-6 significantly increased Treg suppressive effect in the presence of Sle1 DCs (Fig. 6D). Overall, although both Sle3 and Sle1 are expressed in TC DCs, it is Sle1 that plays a major role in TC DC inhibition of Treg-mediated suppression through IL-6 production.
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Discussion |
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(Ref. 46 and L. Morel, unpublished observations). Therefore, the role played by pDCs and type I IFN in B6.TC pathogenesis has yet to be defined. BM cultures also showed that B6.TC mice have a greater propensity to produce MDCs. In addition, TC DCs are relatively resistant to apoptosis. Overall, these multiple factors, altered recruitment, increased BM output, and decreased apoptosis, contribute to DC accumulation in lymphoid organs. Similar phenotypes were found with Sle1-expressing DCs, but at a lower level than for B6.TC, suggesting the involvement of another locus. Sle3 expression resulted only in a small increase in peripheral DC numbers, although a significant increase in macrophage numbers was observed (13). The effect of Sle2 on DCs has not been examined yet. Overall, these results suggest that Sle1 expression, enhanced by interactions with Sle3 and possibly Sle2, plays a major role in increasing the number of peripheral DCs, and that this phenotype is associated with pathogenesis. Apoptosis resistance, which is mediated by both Sle1 (this report) and Sle3 (13) may be a susceptibility factor by itself given that it has been recently demonstrated that FAS-mediated DC apoptosis is essential for the maintenance of systemic self-tolerance (30). The reduction in expression level of activation markers on TC DCs is overall similar to what has been described in lupus patients (25, 26, 27) and more recently in NZM2410, B6.TC parental strain (28). Some differences in relative CD40 and CD86 expression between B6.TC and NZM2410 may be due to the non-Sle loci. In both NZM2410 and B6.TC, however, the activation defect is age dependent, with little difference showed by young mice. It should be noted that the reduction in CD80, CD86, and I-A expression by TC DCs does not correspond to a global defect in costimulatory molecule expression, since TC B cells express a significantly higher levels of these molecules (15). TC DCs showed increased activation levels as they are produced in the BM, then contrary to B6, activation levels remain the same for pDCs or significantly decreases for MDCs, as they move to peripheral lymphoid organs. The reason for this failure to mature is currently unknown. Lower expression of costimulatory markers has also been observed on DCs in the NOD mouse, and it has been suggested that it interferes with the establishment of T cell tolerance by immature DCs (31). Interestingly, a recent report showed that NOD APCs (APCs) were at least partially responsible for suboptimal Treg functions in that strain (47), suggesting that low costimulatory expression on APCs and deficient Treg functions may be linked in more than one model. Less mature DCs are also less able to expand Treg cells in the periphery, due to the greater dependence of Treg proliferation on CD28-CD80-CD86 interactions (48). It is therefore possible that the lower number of Treg cells observed in B6.TC mice is the consequence of the lower expression of CD80 and CD86 by TC DCs. Arguing against this hypothesis, however, is the fact Sle1 DCs express normal to higher levels of CD80/CD86 (Fig. 6A) and B6.Sle1 mice have yet decreased Treg numbers (8). Experiments are ongoing to determine whether the Treg cell deficiency associated with Sle1 is T cell intrinsic or whether DCs are involved. Interestingly, Sle3 expression resulted in increased levels of costimulatory molecules on DCs (13), showing that the overall TC activation status results from synergistic interactions between the two loci and could not be predicted from the phenotypes of the individual loci.
TC DCs induced a greater proliferation in CD4+ T cells either with polyclonal or Ag-specific stimulation. The lower expression of activation markers on TC DCs suggests that other factors are involved. We are currently investigating the role of costimulation in B6.TC. The high production of IL-6, which clearly affects proliferation as shown in Fig. 5 is a good candidate for the effect of TC DCs on CD4+ T cell proliferation. The difference between the effect of TC and B6 DC was greater when purified CD4+ CD25– T cells were cocultured with Treg, cells, which represents an enrichment of the Treg:Teff ratio found in the total CD4+ T cell population. The enhanced proliferation induced by TC DCs in Treg:Teff cocultures most likely affected Teff cells, since Treg proliferation requires high amounts of exogenous IL-2 (48). TC DCs may also induce Treg proliferation, as it has been shown that BM-derived LPS-activated DCs can reverse Treg anergy and induce their proliferation in the absence of exogenous IL-2 (49). TC DCs would therefore produce the same effect, but without the TLR stimulation. Furthermore, TC DCs were able to specifically block Treg down-regulation of Teff IL-2 secretion, which is a phenotype that can be uncoupled from Treg anergy (49, 50). Our results suggest that TC DCs affect both Treg anergy and Treg suppressive function, which will have to be further investigated. The exact mechanism by which TC DCs block Treg suppression is unclear at this point. One of the mechanisms by which Treg suppress Teff function is by preventing their interaction with DCs (33). It is therefore possible that TC DCs engage in interactions with T cells that are more difficult to disrupt than the B6 DC-T cell interactions. This mechanism would not, however, account for the increased proliferation that we observed in the presence of Treg, suggesting that TC DCs affect directly Treg functions. Kubo et al. (50) have shown that exogenous IL-1 and IL-6 can act cooperatively to reverse Treg anergy and induce Treg proliferation. Although the amount of IL-6 used in this study was 10 times higher than the amount of IL-6 produced by TC DCs in our suppression assays, it will be necessary to determinate the effect of TC DCs and their IL-6 production on Treg cells themselves.
Kubo et al. (50) have also described a blockade of Treg regulatory functions with LPS-activated BM-derived DCs from BALB/c mice. In this study, the blockade was not dependent on IL-6 and was associated with high levels of CD80 and CD86 expression. Clearly, the events that are triggered by TC DCs are supported by different mechanisms that require IL-6 and occur despite a low CD80 and CD86 expression. The natural TLR-dependent production of IL-6 which occurs in normal mice in response to pathogens is achieved in the B6.TC and B6.Sle1 mice in the absence of specific infection, or in response to the commensal flora. The mechanism supporting IL-6 overexpression in these mice is unknown. In B6.Sle1 mice, IL6 overexpression is associated with Ras-ERK activation, but it is unclear whether it acts upstream or downstream of IL-6 and whether it corresponds to a primary defect or is secondary to an enhanced BCR signaling (42). Further analyses of signaling pathways in the various cellular compartments involved (B cells, macrophages, and DCs) will be necessary to answer these questions. Regardless of the mechanism for its overproduction, IL-6 or Ras-ERK inhibition was sufficient to block the in vitro production of antinuclear Abs by Sle1 splenocytes (42). It is possible that restoration of Treg functions was involved in these assays, which we are currently testing. It will also be of interest to assess whether the Ras-ERK pathway is involved in the DC/Treg interactions in B6.TC, which would broaden the targets of a Ras-ERK inhibition therapeutic approach.
The evidence for an association between excessive production of IL-6 and lupus is abundant both in patients and in murine models (51). This cytokine has pleiotropic functions including terminal differentiation of B cells into plasma cells, T cell growth, and tissue damage, all of which are highly relevant to lupus pathology. Our study suggests that an additional mechanism by which IL-6 may promote autoimmunity may be an interference with the regulatory functions of Treg cells. Overall, our results suggest that the effects of a reduced number of Treg cells in B6.TC mice are compounded by their DC-induced impaired function, which is predicted to significantly weaken the protective capacity of this regulatory compartment.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported in part by National Institutes of Health Grant RO1-AI045050 (to L.M.). 
2 Address correspondence and reprint requests to Dr. Laurence Morel, Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610-0275. E-mail address: morel{at}ufl.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; DC, dendritic cell; pDC, plasmacytoid DC; MDC, myeloid DC; CD4+CD25+ Treg, regulatory T cell; BM, bone marrow; Teff, effector T cell.
Received for publication June 12, 2006. Accepted for publication October 18, 2006.
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D. Vallois, C. H. Grimm, P. Avner, C. Boitard, and U. C. Rogner The Type 1 Diabetes Locus Idd6 Controls TLR1 Expression J. Immunol., September 15, 2007; 179(6): 3896 - 3903. [Abstract] [Full Text] [PDF]
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P. Deshpande, I. L. King, and B. M. Segal Cutting Edge: CNS CD11c+ Cells from Mice with Encephalomyelitis Polarize Th17 cells and Support CD25+CD4+ T cell-Mediated Immunosuppression, Suggesting Dual Roles in the Disease Process J. Immunol., June 1, 2007; 178(11): 6695 - 6699. [Abstract] [Full Text] [PDF]
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) and B6 (•) cultured for 24 h in medium alone (med), LPS, CpG, or TLR7L of I-Ab. A, CD80; B, CD86; C, CD40; D, I-A. Gates used in A–D are shown on the respective histograms in E. Significance between B6 and TC for each culture condition was evaluated with t tests. *, p < 0.05; **, p < 0.001; ***, p < 0.001. E, Representative FACS histograms of I-Ab, CD80, CD86, and CD40 in immature BM-derived CD11c+ cells. Black and gray lines represent TC and B6 DCs, respectively. Gray-filled histograms show the isotype controls. F, Means and SEs of CD80 mean fluorescence intensity (mfi) on pDCs (rectangles) and MDCs (circles) in the BM, spleen (Spl), and lymph node (LN). Mice 8–10 mo old, filled symbols for B6 and open symbols for B6.TC, n = 8–11. For each tissue and each DC subset, the mean value was significantly different between B6 and B6.TC at least at p < 0.05 as determined with t tests.
, B6; 
, B6.TC DCs. Means and SEs of four mice per group. t tests: *, p < 0.05; **, p < 0.01; ***, p < 0.001). Data representative of three independent experiments performed on 2- to 3-mo-old mice.