T cell hyperactivity in lupus as a consequence
of hyperstimulatory antigen-presenting cells (original) (raw)
Phenotypes of novel congenic/transgenic mice. As summarized in Table 1, B6.Sle3 mice (without the Sle5 locus) exhibited elevated CD4/CD8 ratios, as well as the serological and pathological phenotypes previously described in B6.Sle3/Sle5 congenics (22, 23). To further study the cellular origins of these phenotypes, the OVA-specific TCR Tg, OT-II (29), was successfully bred onto B6.Sle3 mice. As observed previously in B6.Sle3 congenics (22), B6.Sle3.OT-II mice also possessed low but significant levels of ANAs (Figure 1A), but not glomerulonephritis (data not shown). B6.OT-II and B6.Sle3.OT-II Tg mice exhibited similar numbers of T cells and B cells in their spleens and nodes (data not shown). Importantly, the B6.OT-II and B6.Sle3.OT-II Tg mice expressed both the V_β_5 and the V_α_2 TCR transgenes on about 75% of their peripheral CD4+ T cells (Figure 1B). Ex vivo, B6.Sle3.OT-II TCR Tg T cells were not more activated (based on CD69 expression), compared with B6.OT-II TCR Tg T cells (Figure 1C).
Serological and cellular phenotypes in B6.Sle3.OT-II mice. (A) Serum levels of antinuclear autoantibodies were assessed in 9- to 12-month-old B6.OT-II and B6.Sle3.OT-II mice (n = 8 mice per group). The dashed line indicates the mean serum levels of ANAs in B6.Sle1.FASlpr control mice exhibiting full-blown lupus (27). (B and C) The spleens of 3- to 6-month-old B6.OT-II and B6.Sle3.OT-II mice (9–12 mice per group) were also examined for the prevalence of Vβ5+, Vα2+, and CD4+ transgenic T cells (B), and their activation status, based on the surface expression of CD69 (C). Spleens of both strains did not differ in their total numbers of splenocytes, or splenic CD4 T cells. Mean ± SEM is shown in all plots. The depicted P values were derived using the Student’s t test.
Phenotypes of B6.Sle3 and B6.Sle3/Sle5 congenics, and OT-II transgenics
Sle3 impacts T cell function: in vitro and in vivo studies. Although B6.OT-II and B6.Sle3.OT-II mice exhibited similar numbers of Tg T cells, _Sle3_-bearing Tg T cells were hyperproliferative, as displayed in Figure 2A, with an attendant reduction in activation-induced cell death (data not plotted), consistent with previous findings (23). Previous studies had also shown that the impact of Sle3 on T cells was not dependent on T cell–intrinsic Sle3 expression (28). Hence, we designed an in vivo adoptive transfer experiment to confirm this, using the OT-II TCR Tg model. In essence, B6.OT-II Tg T cells were adoptively transferred into B6 or B6.Sle3 hosts, after which the recipient mice were challenged with an immunogenic dose of OVA. In resonance with the earlier report (28), the administration of immunogenic OVA led to a greater degree of T cell expansion in vivo in B6.Sle3 hosts, with an attendant increase in serum IgG anti-OVA Abs (Figure 2B). After challenge with OVA, T cells in both types of hosts were equally activated (>90% of Tg T cells expressed CD69; data not plotted). Finally, _Sle3_-bearing T cells appeared relatively recalcitrant to peripheral tolerance induction, as depicted in Figure 2, C and D. Thus, when B6.OT-II mice were first exposed to their cognate antigen under a tolerizing regime, their T cells were not able to mount a proliferative response when subsequently challenged with an immunogenic form of OVA. In contrast, the presence of Sle3 abrogated effective peripheral tolerance in this experimental model (Figure 2D).
Functional responsiveness of B6.Sle3.OT-II T cells. (A) B6.OT-II and B6.Sle3.OT-II splenocytes were cultured with various stimuli and assessed for proliferation. Stimulation index was cpmexperiment/cpmno-antigen. The cpmno-antigen values ranged from 2,000 to 5,000. Each dot represents an independent spleen sample. Horizontal bars indicate the respective group means. The data shown were reproduced in 2 additional studies (Supplemental Table 1 [supplemental material available online with this article; doi:10.1172/JCI23049DS1]). (B) B6.OT-II splenic T cells were transferred i.v. into 2-month-old B6 or B6.Sle3 mice on D0 and challenged with an immunogenic form of OVA on D1, as described in Methods. Seven days after transfer, the numbers of Tg T cells in the recipient spleens (left) and serum IgG anti-OVA levels (right) were assessed. Horizontal bars indicate group means. The displayed data were pooled from 2 independent studies using 5 mice per strain. (C) B6.OT-II and B6.Sle3.OT-II mice (n = 5 per group) were challenged with OVA323–339 in incomplete Freund’s adjuvant on D0; splenocytes were isolated on D5 and assessed for their proliferative response to OVA323–339. The vertical bars represent the SEM of triplicate cultures. Data shown are representative of 2 independent studies. Observed differences were not statistically significant. (D) B6.OT-II and B6.Sle3.OT-II mice (n = 5 mice per group) were challenged first with tolerogenic OVA on D0 and then with immunogenic OVA on D10 and examined as described in Methods. The vertical bars represent the SEM of triplicate cultures. In a second confirmatory study (data not shown), the fold difference in cpm between the 2 strain groups was 1.9 (P < 0.04, n = 4 each), at an OVA stimulation dose of 1,000 nM.
Sle3 encodes aberrant myeloid-lineage cells. Given that previous allotype-marked BM transfer experiments (28) and the adoptive transfer studies described above (Figure 2B) indicated that the _Sle3_-associated phenotypes may not be T cell intrinsic, we asked whether quantitative or qualitative differences in _Sle3_-bearing cells of myeloid origin, including various APC subsets, may be responsible for the phenotypic differences noted above. Spleens, lymph nodes, and peripheral blood from 9- to 12-month-old B6 and B6.Sle3 mice were examined for the numbers and phenotypes of DCs, macrophages, and neutrophils, by flow cytometry, as illustrated in Figure 3, A and B. As noted in Table 2, myeloid-lineage cells from B6.Sle3 spleens demonstrated several quantitative and qualitative differences. The most consistent difference was the expanded percentages of macrophages in 9- to 12-month-old B6.Sle3 spleens, which was noted in multiple experiments (Table 2). Since B6 and B6.Sle3 mice possessed similar numbers of splenocytes, these differences in percentages also translated to differences in the absolute numbers of these cells. Although splenic neutrophils were also expanded in numbers, these differences fell short of statistical significance. There were no significant differences in the numbers of splenic myeloid DCs, though the numbers of lymphoid DCs and plasmacytoid DCs tended to be relatively higher (Table 2, and data not shown).
Splenic and BM myeloid cell subsets in B6 and B6.Sle3 mice. (A and B) Representative CD11b/CD11c expression profiles observed in 9- to 12-month-old B6 and B6.Sle3 collagenase-treated spleens. The cells in region R2, also expressing CD8, were gated as lymphoid DCs, whereas the cells in region R3 were gated as myeloid DCs. Cells that were in region R4 were gated as macrophages; these cells also exhibited high side scatter and F4/80 expression. Cells in region R5 were gated as neutrophils, and these cells also exhibited high Gr-1 levels. The prevalence of these cells in the spleens of 9- to 12-month-old B6 and B6.Sle3 mice (n = 10–15 each), and the expression levels of CD40, CD80, CD106 (VCAM-1), and FcR on these 4 gated populations, are detailed and statistically compared in Table 2. (C) The respective percentages of the different myeloid cell populations in 2-month-old B6 and B6.Sle3 spleens. (D–F) Expression levels (mean fluorescence intensities, mfi) of various surface markers on 2-month-old splenic CD11b+++, CD11c+++ myeloid DCs (D); on BM M-CSF–cultured macrophages, gated on F4/80+ cells (E); and on BM GM-CSF–cultured DCs, gated on CD11c+ cells (F). For the latter 2 studies, the cells were phenotyped with or without LPS pretreatment (10 ng/ml, 24 hours). For all studies, each dot represents data derived from an individual mouse. Data are representative of 2–3 independent studies each. Depicted P values were computed by comparison of the B6.Sle3 values with the B6 control values, using the Student’s t test. The horizontal bars indicate the respective group means.
Phenotype of DCs, macrophages, and neutrophils in B6 and B6.Sle3 spleens
In addition, DCs, macrophages, and neutrophils isolated from B6.Sle3 spleens were evidently more activated/mature, based on the expression of several surface markers (Table 2). Thus, for example, although the myeloid DCs were not expanded in numbers in B6.Sle3 spleens, they exhibited increased surface levels of CD40, CD80, CD86, CD54, CD106 (VCAM-1), and FcR (CD16/32), with similar differences being noted on B6.Sle3 lymphoid DCs, macrophages, and neutrophils (Table 2). In particular, the surface levels of CD106 were about twice as high on all 4 cell subsets examined, compared with the corresponding levels in B6-derived cells. Similar changes were also noted in the lymph nodes and peripheral blood of B6.Sle3 mice (Table 3, and data not shown). Interestingly, B6.Sle3 lymph node–derived DCs, macrophages, and neutrophils displayed CD40 and I-Ab levels that were severalfold higher than the respective expression levels on the B6 controls.
Phenotype of DCs, macrophages, and neutrophils in B6 and B6.Sle3 lymph nodes
As noted above in the spleens and nodes of older B6.Sle3 mice, one could also discern the heightened activation of different myeloid-lineage cells in young (i.e., 2-month-old) B6.Sle3 mice (Figure 3, C and D). Likewise, macrophages cultured from B6.Sle3 BM (using M-CSF) showed similar phenotypic differences, with these differences becoming amplified following LPS stimulation (Figure 3E). This was particularly pronounced with respect to CD80 expression. B6._Sle3_-derived BM-cultured DCs revealed similar phenotypic differences, when the DCs were elicited using GM-CSF (Figure 3F). On the other hand, DCs that were cultivated using GM-CSF plus IL-4 yielded mixed results: B6-derived DCs appeared to be more activated in 2 experiments, the inverse pattern was noted in a third experiment, and no differences were noted in a fourth experiment (data not shown). The reason for this variability with DCs elicited by GM-CSF plus IL-4 is not presently clear.
Sle3 myeloid cells exhibit altered cytokine profiles and impaired apoptosis. B6._Sle3_-derived macrophages and DCs also exhibited altered cytokine production profiles, compared with the B6 controls, as displayed in Figure 4. Unmanipulated splenic DCs isolated from B6.Sle3 congenics, as well as BM-derived DCs (cultured using GM-CSF), produced more IL-12 but less IL-6 in culture (Figure 4, A and B). In addition, B6.Sle3 DCs also hypersecreted IL-1β and showed a variable difference in TNF-α production (Figure 4B; and see Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI23049DS1). Although IL-12, IL-6, and TNF-α were elevated in B6.Sle3 macrophage supernatant, IL-1β secretion was variably increased (Figure 4, C–E, and Supplemental Figure 1).
Cytokine secretion profiles of B6.Sle3 DCs and macrophages. (A and B) CD11c magnetic bead–purified splenic DCs from 2- to 3-month-old B6 and B6.Sle3 mice (n = 4 per group; A), as well as CD11c magnetic bead–purified DCs cultured from B6 or B6.Sle3 BM (using GM-CSF alone, for 7 days; n = 8–9 mice per group; B), were incubated for 24 hours, with or without 10 ng/ml LPS. Depicted are the IL-12 (p70), IL-6, and IL-1β levels in 24-hour culture supernatant. Each dot represents data derived from splenic or BM DCs of an individual mouse; where the positions of the dots overlap, this has been represented using single dots only, for clarity. Similar findings were noted in a second confirmatory study (Supplemental Figure 1A). (C–E) In addition, 24-hour IL-12, IL-6, and TNF-α secretion by B6- or B6._Sle3_-derived BM-cultured macrophages in response to LPS was assessed by ELISA. Each dot represents data derived from the BM macrophages of an individual mouse. The horizontal bars represent group means. The data shown were reproduced in 2–3 additional experiments (Supplemental Figure 1B). The depicted P values in A–E were computed by comparison of the B6.Sle3 values with the B6 control values, using the Student’s t test.
Besides the elevated production of proinflammatory cytokines, B6._Sle3_-derived myeloid cells also revealed another interesting phenotypic difference — impaired apoptosis (Figure 5, A and B). This difference was demonstrated for both BM-derived DCs and macrophages (Figure 5, A and B). Stimulation with LPS did not alter this phenotypic difference (data not shown). These findings are in line with our recent observation that B6.Sle3 mice also exhibit reduced apoptosis of neutrophils in vitro and in vivo, in an infection challenge model (B. Mehrad and C. Mohan, manuscript submitted for publication). However, Sle3 did not impact the phagocytic potential of macrophages (B. Mehrad and C. Mohan, manuscript submitted for publication). Interestingly, when _Sle3_-bearing DCs were OVA-pulsed and cocultured with OT-II TCR Tg T cells, the T cells also demonstrated reduced apoptosis with a concomitant increase in activation, as gauged by the surface expression of CD69 (Figure 5, C and D), compared with T cells cocultured with B6 DCs.
Impaired apoptosis in B6.Sle3 macrophages and DCs. (A and B) DCs and macrophages were cultured from the BM of B6 and B6.Sle3 mice, and incubated without any stimuli for 24 hours. Following culture, the extent of apoptosis was gauged by flow cytometry using annexin V and 7-amino-actinomycin D (7-AAD), after gating on CD11c-expressing DCs or F4/80+ macrophages. Each group and bar represent data gleaned from 6–7 individual mice from each strain. P values were computed by comparison of the B6.Sle3 values with the B6 control values, using the Student’s t test. The shown horizontal bars indicate the respective group means. (C and D) In addition, DCs cultured from 2-month-old B6 or B6.Sle3 BM were pulsed with 1,000 nM OVA323–339, washed, and cocultured (10,000 per well) with OVA-specific B6.OT-II TCR Tg T cells for varying times, as indicated. After culture, the fraction of T cells that were apoptotic (based on expression of annexin V; C) and the mean fluorescence intensity (mfi) of CD69 on the Tg T cells (D) were assessed by flow cytometry. The respective B6 and B6.Sle3 values were compared using the Student’s t test (*P < 0.05; **P < 0.001).
Sle3 APCs are superior at costimulating T cells. In keeping with the above findings, the more activated/mature phenotype of B6._Sle3_-derived DCs was also accompanied by an enhanced T cell costimulatory capacity. When BM-derived DCs or splenic DCs were cocultured with OVA-reactive B6.OT-II TCR Tg T cells, _Sle3_-bearing DCs induced a greater degree of T cell division and proliferation, in a manner dependent on antigen dose and APC number (Figure 6). Similar results were noted when T cell alloresponsiveness was assayed (data not shown). Although the difference in thymidine incorporation attained statistical significance, the difference in cell division (as assayed by CFSE dilution) failed to attain significance within individual experiments. In both the antigen-specific response experiments and the alloresponse studies, the use of high numbers of B6-derived DCs tended to dampen the proliferative response of the responding T cells (Figure 6, F and G, and data not shown); in contrast, _Sle3_-bearing DCs continued to be hyperstimulatory even when high numbers of DCs were used for costimulation.
B6.Sle3 DCs are superior APCs for T cell stimulation. (A–D) DCs cultured from 2-month-old B6 (A and C) or B6.Sle3 (B and D) BM, using GM-CSF plus IL-4 for 7 days, were pulsed with OVA323–339, and cocultured with CFSE-labeled, OVA-specific B6.OT-II TCR Tg T cells (A and B) or non-Tg T cells (C and D), and the fraction of T cells that had undergone cell division was assessed by flow cytometry. The histograms pertain to coculture studies that were performed with 10,000 OVA-pulsed DCs, and examined by flow cytometry 96 hours after stimulation. (E) Similar results were obtained using OVA-pulsed CD11c bead–purified splenic DCs from B6 and B6.Sle3 mice cocultured with B6.OT-II or B6.Sle3.OT-II T cells for 96 hours; however, any apparent differences noted failed to attain statistical significance. The data depicted in E are representative of 3 independent experiments. In addition, OVA-specific B6.OT-II TCR Tg T cells were cocultured with varying numbers of unpulsed B6 (white dots) or B6.Sle3 (black dots) BM-cultured DCs and 1,000 nM OVA323–339 (F), or with 50,000 unpulsed BM-derived DCs and varying doses of OVA323–339 (G). In both experiments, proliferation was assessed 96 hours after culture, by assaying of 3H-thymidine incorporation. The data portrayed in F and G are representative of 3 independent experiments; an additional experiment is displayed in H, where the proliferation of B6.OT-II and B6.Sle3.OT-II TCR Tg T cells in response to varying numbers of OVA-pulsed B6 or B6.Sle3 splenic CD11c bead–purified DCs was assessed. Shown P values were computed by comparison of the B6.Sle3 values with the B6 control values, using the Student’s t test.
These differences were most apparent when B6.OT-II TCR Tg T cells were used for the cocultures; B6.Sle3.OT-II Tg T cells behaved fairly similarly irrespective of whether B6 or B6.Sle3 DCs were used for stimulation (Figure 6, E and H). In all experiments, the extent of cell division of the Tg T cells failed to approximate 100%, possibly because of the expression of non-Tg TCR since these studies were executed on a _RAG_-sufficient background. Finally, T cells cocultured with B6.Sle3 DCs were demonstrated to produce more IFN-γ, compared with T cells cocultured with B6-derived DCs, in some experiments but not others (data not shown), consistent with the elevated IL-12 production by B6.Sle3 DCs.
Sle3 DCs recapitulate the in vivo phenotypes attributed to Sle3. Given that the Sle3 locus facilitated the generation of APCs that were evidently more mature, proinflammatory, and costimulatory, we next asked whether this cellular phenotype might indeed be responsible for the T cell and serological phenotypes noted in B6.Sle3 mice. Importantly, the adoptive transfer of _Sle3_-bearing DCs (compared with B6-derived DCs) into young B6 hosts, when coupled with LPS coadministration, led to elevated splenic CD4/CD8 ratios (Figure 7A) and elevated serum autoantibody levels (Figure 7B), 2 cardinal _Sle3_-associated phenotypes (22, 23). In these adoptive transfer studies, the maximal autoantibody levels were noted on day 40 (D40), i.e., about 4 weeks after the last administration of DCs (see legend to Figure 7). However, this serological outcome was not sufficient for renal disease to ensue (data not shown). The transfer of DCs alone, without any added LPS, or the administration of LPS alone, was insufficient to elicit the above phenotypes (data not shown). These studies demonstrate that _Sle3_-bearing DCs appear to be sufficient to recreate the _Sle3_-associated immunophenotypes.
Adoptively transferred B6.Sle3 DCs recreate _Sle3_-associated phenotypes. DCs cultured from B6 or B6.Sle3 BM (16–18 mice per strain, pooled from 4 independent experiments) were adoptively transferred into 2-month-old B6 mice on D0, D7, and D14, with coadministration of LPS on D1, D8, and D15. Plotted in A are the terminal splenic CD4/CD8 ratios in the host mice on D20 or D60; since the outcomes were similar at both time points, the results from D20 and D60 experiments have been pooled. Plotted in B are the serum IgG anti–single-stranded DNA Abs (left) and IgG anti–double-stranded DNA Abs (right) at serial time points during the 60-day study period, after the transfer of B6 or B6.Sle3 DCs, with coadministered LPS. All P values shown were computed by comparison of the B6.Sle3 values with the B6 control values, using the Student’s t test. The horizontal bars indicate the respective group means. The data shown were reproduced in 2 additional studies, as portrayed in Supplemental Figure 2.