Endogenous collagen peptide activation of CD1d-restricted NKT cells ameliorates tissue-specific inflammation in mice (original) (raw)
Collagen peptide mCII707–721 activates CD4+TCRαβ+ T cells in an MHC I/II–independent manner. We immunized B10.Q mice with mCII707–721 and observed a strong and specific immune response, as measured by rechallenge of LN cells (LNCs) in vitro with mCII707–721 (Figure 1A). No response was induced in the absence of antigen (media) or by using a nonrelated peptide control, the mouse collagen type I α 1 chain peptide mCI707–721, which shares high homology with mCII707–721. mCI707–721 was chosen as the negative control because this 15-aa peptide is similar to mCII707–721 in size and sequence, differing in only 4 aa positions. It also does not elicit any T cell proliferation. Purified protein derivative (PPD) has been used as a positive control (recall antigen) for accuracy of immunization and proliferation, given that it was used for all the in vivo assays. PPD reactivity showed a proliferation index (mean ± SD) of 12.8 ± 2.1. We also immunized C57BL/6 mice (H2b, 6 mice per group) with mCII707–721 and observed a similar immune response as with B10.Q (proliferation indexes: media = 1; mCI707–721 = 1.25 ± 0.23; mCII707–721 = 3.68 ± 0.32, P ≤ 0.001 versus each negative and positive control; PPD = 12.78 ± 2.09). This indicates that the mCII707–721–reactive response is not strain specific. However, to control for possible strain-specific effects, the remainder of the study utilized only mice of B10.Q background.
Immunodominant mouse collagen type II peptide mCII707–721 is CD1d restricted and activates CD4+TCRαβ+ NKT cells. (A) Significant proliferative response was observed after in vitro peptide stimulation of LNCs from mCII707–721–immunized mice (WT B10.Q). Controls are medium or control peptide mCI707–721. Proliferation index (PI) was calculated by normalizing all cpm values to media control as 1. (B) PI in response to mCII707–721 in KO mice, all B10.Q background. Proliferation was significantly reduced compared with WT littermates in Tcrab–/–, CD4–/–, and CD1d–/– mice, indicating that the mCII707–721 peptide response depends on CD1d-restricted CD4+TCRαβ+ T cells, but not classical MHC I/II presentation. (C) PI in response to mCII707–721 in B10.Q LNCs. Blocking with anti–MHC II and anti–MHC I antibodies produced similar levels of PI compared with mCII707–721 with control antibody. PI was calculated by normalizing all cpm values to media control as 1. (D) In vitro rechallenge response to mCII707–721, abrogated by anti-CD1 (1B1, 10 μg/ml) or anti-NK1.1 (PK136, 10 μg/ml), indicating that CD1d-dependent NKT cells were operative in the response. PI was calculated by normalizing all cpm values to media control as 1. Data are mean ± SD, n = 6. **P ≤ 0.01; ***P ≤ 0.001.
We characterized the T cell response toward the mCII707–721 peptide, using KO mice of genotypes MHC II–/–, TAP1–/–, CD1d–/–, Tcrab–/–, Tcrgd–/–, CD4–/–, and CD8–/–, all backcrossed to the same genetic background, B10.Q. These were immunized and LNCs were rechallenged with or without antigens in vitro. Proliferative response to mCII707–721 required TCRαβ expression, CD4+ T cells, and CD1d, but was not dependent on TCRγδ expression or CD8+ T cells (Figure 1B). No difference in proliferation was observed among the various KO mice and WT B10.Q mice in response to control peptides used in the assay (data not shown).
We investigated potential antigen-presenting molecules for mCII707–721 using MHC II–/– mice and TAP-deficient mice (TAP1–/–), which lack transporter associated with antigen processing, important for presentation of MHC I–restricted peptides. By utilizing these mice (Figure 1B), as well as monoclonal antibodies to block MHC class I and II (Figure 1C), sustained proliferation to mCII707–721 was observed. These data indicate that the mCII707–721 response was neither MHC II– nor MHC I–restricted. An MHC II–binding assay also verified that the mCII707–721 peptide did not bind to H-2q (data not shown). These results demonstrate that the T cell response to mCII707–721 was dependent on CD4 and TCRαβ, but unexpectedly, did not involve MHC I or II presentation.
mCII707–721 binds to CD1d and activates NKT cells. Mice were immunized with mCII707–721, and recall antigen reactivity of LNCs or splenocytes was measured 10–13 days after immunization. As shown in Figure 1B and Figure 1D, the proliferative response to mCII707–721 was essentially completely diminished when mice genetically lacking CD1d-dependent NKT cells (CD1d–/– mice) were investigated or monoclonal antibodies to CD1d or NK1.1 were added to cultures prior to restimulation with mCII707–721. No effect on mCI707–721 or PPD was observed (data not shown). Similarly, depletion of NK1.1+ cells, or antibody blocking of CD1d in vivo prior to subsequent immunization with mCII707–721, resulted in reduced proliferative response to mCII707–721 (data not shown). Thus, the response to mCII707–721 was confirmed to be CD1d restricted.
The binding capacity of CD1d for mCII707–721 was determined using recombinant soluble dimeric mouse CD1d immunoglobulin and biotinylated mCII707–721. Significant binding of biotinylated mCII707–721 to CD1d was observed, while no binding of the negative control, biotinylated mCI707–721, was detected. Excess nonbiotinylated mCII707–721 peptide effectively competed for binding of biotinylated mCII707–721 to CD1d (Figure 2A). Figure 2B shows that the interaction was dose dependent and saturable. Using αGalCer with biotinylated mCII707–721 revealed that at certain concentrations it competes for the binding to CD1d, indicating partial competition for the binding site (Figure 2C). PBS (non-CD1d-Ig dimer) is used for negative control.
mCII707–721 binds CD1d. (A) The binding capacity of mCII707–721 to CD1d was determined using ELISA plates coated with CD1d-Ig dimer, with biotin-labeled anti-CD1d (bio–anti-CD1) (positive control), biotin-labeled mCII707–721 peptide, or biotin-labeled mCI707–721 (negative control). Excess nonlabeled mCII707–721 peptide with biotin-labeled mCII707–721 peptide was used for competitive binding. Significant differences were seen in wells with biotin-labeled mCII707–721 peptide compared with negative control or excess nonlabeled peptide. (B) mCII707–721 binding to CD1d was concentration dependent, with a maximum binding capacity of 0.6 μg/ml. Ratio of binding is the fluorometer OD of the sample divided by the positive control OD. (C) αGalCer and biotinylated-labeled mCII707–721 peptide compete for binding to CD1d. Biotinylated-labeled mCII707–721 peptide binding to CD1d is considered as 100% binding. Data are mean ± SD, n = 3–4. **P ≤ 0.01; ***P ≤ 0.001.
mCII707–721 activates a heterogeneous NKT cell population. To investigate the frequency of NKT cell expansion in response to mCII707–721, WT B10.Q mice were treated in vivo with mCII707–721 and CFA and 13 days later, splenocytes were analyzed. mCII707–721 is capable of significantly inducing NKT cell expansion (gated on CD4+NK1.1+ T cells) compared with all other control groups, namely vehicle-treated (CFA), mCI707–721, and naive mice (Figure 3, A and B).
mCII707–721 induces NKT cell expansion. (A and B) Splenocytes were taken from naive WT (B10.Q) mice and in vivo CFA-, mCI707–721–, and mCII707–721–treated mice. Single-cell suspensions were made and then stimulated with 100 μg/ml mCII707–721 for 48 hours. n = 3 mice per group. (A) A representative FACS staining shows total gated lymphocytes (upper row) and gated CD4+NK1.1+ NKT cells (lower row). (B) Percentage of CD4+NK1.1+ NKT cells from indicated groups. Data are mean ± SD, n = 3. **P ≤ 0.01; ***P ≤ 0.001. (C) Proportion of NK1.1+ T cells in cell line during the first stimulation of LNCs (57%), and after 2 (65%) and 3 (88%) in vitro stimulation cycles with mCII707–721. NK1.1+ T cells enriched after each stimulation, indicating response and proliferation after mCII707–721 exposure. Data are mean ± SD. n = 3. (D) mCII707–721–specific cell line characterized for TCR usage after 3 stimulation cycles with a significantly higher proportion of Vα14-Jα18 by real-time qPCR compared with control BQMOG79–96–specific T cells (top panel). Vα14 PCR product in agarose gel is shown (bottom panel). Data are mean ± SD. n = 3. ***P ≤ 0.001. (E) Splenocytes were taken from mCII707–721–immunized mice treated with 100 μg/ml mCII707–721 for 48 hours. FACS staining shows TCR Vβ usage. Percentages were computed relative to the sum of all of the 15 chains. Data are mean ± SD, n = 3.
To further characterize the mCII707–721–responding T cells, a peptide-specific cell line was established by subjecting LNCs from mice immunized with mCII707–721 to different cycles of restimulation and resting. Expression of the NK1.1 marker was confirmed after each round of antigenic stimulation. Figure 3C shows that 57% of the CD4+ mCII707–721–specific T cells in the primary cell line were NK1.1+ T cells. This percentage increased to 65% after the second stimulation and to 88% after the third, indicating enrichment of NKT cells upon antigenic stimulation.
NKT cells can utilize a diverse combination of Vα and Vβ chains, with evidence suggesting that any of the Vβ chains can pair with the Vα14 invariant chain (14). The TCR Vα usage of the mCII707–721–specific NKT line was characterized by real-time quantitative PCR (qPCR) and FACS. A conventional T cell line specific to the CNS self-antigenic peptide myelin oligodendrocyte glycoprotein 79–96 (MOG79–96) was included for comparison. The mCII707–721–specific NKT cell line showed higher expression of Vα14-Jα18 than the conventional T cell line by real-time quantitative PCR (qPCR) (Figure 3D). Using a panel of Vβ TCR antibodies to stain mCII707–721–specific NKT cells showed that these cells utilize polyclonal and diverse Vβ chains (Figure 3E). Although some skewing toward usage of Vβ8.2 is seen, mCII707–721 reactivity is heterogeneous, not clonal.
Activation of mCII707–721–reactive NKT cells requires TCR and costimulatory signaling. To examine TCR signaling in the mCII707–721–specific response, we investigated ZAP-70 phosphorylation in the mCII707–721–specific NKT cell line. NKT cells stimulated with mCII707–721 expressed phosphorylated ZAP-70 on the cell membrane, indicating TCR engagement and signaling (Figure 4A). Furthermore, blocking CD1d by adding a neutralizing antibody to the culture medium completely inhibited the appearance of phosphorylated ZAP-70 on the cell surface. Thus, interaction among CD1d, mCII707–721, and TCR is operative in the activation of mCII707–721–specific NKT cells.
mCII707–721–specific NKT cell activation engages TCR and requires costimulation. (A) Activation of mCII707–721–specific NKT cells operated through TCR signaling. Percentage of phosphorylated ZAP70-positive cells in NK1.1+ T cells by FACS shows a significant increase over nonstimulated cells upon mCII707–721 stimulation in vitro, with abrogation by blocking anti-CD1 (1B1, 10 μg/ml). (B) Costimulatory signals for proliferation after in vitro stimulation of LNCs from mCII707–721–immunized mice. Blocking with antibodies to costimulatory proteins significantly reduced proliferation. Data are mean ± SD, n = 4. **P ≤ 0.01; ***P ≤ 0.001.
The requirement for costimulatory signals in the activation of mCII707–721–specific NKT cells was studied using antibodies or fusion proteins to block the B7- and CD40-dependent pathways in peptide-stimulated LNC cultures from mCII707–721–immunized mice. Blocking either the CD40/CD40L or the B7/CD28 pathways led to significant inhibition of the proliferative response (Figure 4B) and showed that B7.1, but not B7.2, was the crucial ligand for CD28 on the NKT cells. The data support the interpretation that B7.1-CD28 and CD40-CD40L, in addition to CD1d-TCR ligation, are the necessary signals for mCII707–721–induced NKT cell activation.
mCII707–721–specific NKT cells produce cytokines. Cytokine production by the mCII707–721–specific NKT cells was measured by ELISA for IFN-γ, IL-2, IL-4, IL-10, TGF-β1, and TNF-α in peptide-stimulated LNC cultures from mCII707–721–immunized mice. TGF-β1, IFN-γ, and IL-4 production was significantly increased compared with cells stimulated with the negative control media and mCI707–721 (Figure 5A). IL-2 and IL-10 production was undetectable (data not shown). Interestingly, TNF-α production was significantly reduced upon mCII707–721–specific NKT cell activation (Figure 5B). The kinetics of cytokine production resembled those of conventional T cells, i.e., peaking at 72 hours after restimulation in vitro and showing no early burst of cytokines (data not shown).
mCII707–721–specific NKT cells produce cytokines. (A) mCII707–721–specific cytokine production in primary LNCs from mCII707–721–immunized WT mice measured by ELISA. Significantly elevated levels of TGF-β1, IFN-γ, and IL-4 (n = 3) in response to mCII707–721 are seen, but (B) TNF-α levels were lowered. Data are mean ± SD, n = 6. *P ≤ 0.05; **P ≤ 0.01.
To investigate whether NKT cells are the direct source of cytokine production, we treated WT B10.Q mice with mCII707–721 plus CFA in vivo. As control groups, mice were treated with mCI707–721 plus CFA; an additional group received only vehicle (CFA), and finally a group of mice did not receive any treatment (naive). Splenocytes were dissected and single-cell suspensions treated in vitro with mCII707–721 for 48 hours. Cells were then stained for intracellular cytokines and analyzed using FACS. As shown in Figure 6, mCII707–721–specific NKT cells are capable of intracellular production of TGF-β1, IFN-γ, IL-4, and TNF-α. However, the levels of TNF-α intracellularly (Figure 5B) exhibited a trend similar to the levels seen as assessed by ELISA (Figure 7D), i.e., reduction in the in vivo mCII707–721–treated group compared with controls.
mCII707–721–specific NKT cells produce cytokines intracellularly. Intracellular staining indicates that mCII707–721 induces TGF-β1, IFN-γ, and IL-4 production (A–C), but not increased TNF-α (D). (A–D) Splenocytes were taken from mCI707–721– and mCII707–721–immunized mice, then stimulated with mCII707–721 for 48 hours. Anti-CD3 (10 μg/ml) and anti-CD28 (2 μg/ml) with brefeldin A were added to cell cultures for 5 hours before staining. FACS profile shows intracellular staining of TGF-β1, IFN-γ, IL-4, and TNF-α. Cells were gated on CD4+NK1.1+ cells.
mCII707–721–specific NKT cells produce cytokines. (A–D) Higher TGF-β1, IFN-γ, and IL-4 production and lower TNF-α production were seen from mCII707–721–immunized splenocytes compared with all other control groups in WT mice but not in CD1d–/– mice. Spleens were taken from CFA, mCI707–721–, and mCII707–721–immunized mice, then treated with mCII707–721 for 48 hours. Supernatants were taken for ELISAs of TGF-β1, IFN-γ, IL-4, and TNF-α. Data are mean ± SD, n = 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Cytokine production of mCII707–721–specific NKT cells is CD1d dependent. Splenocytes from similarly treated B10.Q WT and CD1d–/– mice were analyzed for the production of cytokines in response to mCII707–721. As depicted in Figure 7, A–C, only WT mice that were treated in vivo with mCII707–721 showed significant capacity to produce TGF-β1, IFN-γ, and IL-4, as measured by ELISA. Moreover, mCII707–721–specific TGF-β1, IFN-γ, and IL-4 production was significantly reduced in CD1d–/– compared with WT mice, though interestingly, a significantly lower TNF-α production was observed (Figure 7D).
mCII707–721–specific NKT suppression of T cells is Fas-FasL dependent. In vitro suppression of T cells was investigated by activating syngeneic splenocytes with plate-bound anti-CD3 (here referred to as responder cells), which were then cocultured with the mCII707–721–specific NKT cell line. Responder cells proliferated vigorously as indicated by a high proliferation index (Figure 8). However, mCII707–721–specific cells significantly inhibited proliferation of anti-CD3–stimulated responder T cells, compared with the control conventional MOG79–96 T cell line. Since the mCII707–721–specific NKT cells produced TGF-β1, IFN-γ, and IL-4 upon activation, we assessed the role of these cytokines in T cell suppression. To block these cytokines, neutralizing antibodies were added to mCII707–721–specific NKT cell cultures prior to coculture with anti-CD3–activated splenocytes. The neutralizing antibodies did not affect the significant decrease in responder T cell proliferation (Figure 8A), indicating that the suppressive effect of mCII707–721–specific NKT cells was independent of their cytokine production.
mCII707–721–specific NKT cells have a suppressive function. (A and B) Suppressor assay was performed using in vitro–stimulated anti-CD3 splenocytes (responder cells) from B10.Q mice, cocultured for 64 hours with either mCII707–721–specific NKT cell line or B10.Q-restricted MOG79–96–specific T cell line (control) at a 1:1 ratio. Proliferation index was calculated by normalizing all cpm values to media control as 1. (A) Blocking IFN-γ, IL-4, TGF-β1, and TGF-β receptor with antibodies (20 μg/ml) had no effect on NKT cell suppression compared with isotype-matched controls; therefore suppression did not operate through cytokine release from mCII707–721–specific NKT cells. Data are mean ± SD, n = 3. *P ≤ 0.05; ***P ≤ 0.001. (B) Significantly reduced proliferation of anti-CD3–stimulated responder cells was observed when cultured with the mCII707–721–specific NKT cells (with or without Transwell system) compared with control cells. Proliferation of the responder cells was restored when a Transwell system inhibited cell-cell contact. Data are mean ± SD, n = 2–3. **P ≤ 0.01; ***P ≤ 0.001. (C) Cell death by annexin V+ FACS analysis of cocultured splenocytes (CFSE labeled), showing that the mCII707–721–specific NKT cell line induced significantly elevated cell death levels compared with the control cells. Anti-FasL (20 μg/ml) before coculturing revealed that splenocyte killing was mediated by FasL interaction. Data are mean ± SD, n = 3. **P ≤ 0.01. (D) Splenocytes (CFSE labeled) from WT mice stimulated with plate-bound anti-CD3 and cocultured for 48 hours with NKT cells purified from mCII707–721–immunized WT mice or lpr mice. Cell death was determined by 7AAD staining. Data are mean ± SD, n = 10 mice per group. **P ≤ 0.01.
Since these soluble cytokines seem to exert no major effects in the context of mCII707–721–specific NKT cell suppression, we investigated to determine whether cell-cell contact was required. As depicted in Figure 8B, the suppressive function of the mCII707–721–specific NKT cells was dependent on cell-cell contact, since separating direct cell-cell interaction using a Transwell system abrogated their suppressive effect.
We investigated whether mCII707–721–specific NKT cells could induce apoptosis, as measured by annexin V staining of anti-CD3–activated responder cells. Upon coculture, mCII707–721–specific NKT cells significantly induced apoptosis of anti-CD3–activated responder cells (Figure 8C). As Fas-FasL is one of most common mechanisms for induction of apoptosis (15), we used FasL-blocking antibody to determine whether Fas-FasL is involved in apoptosis induction. Cell death induced by mCII707–721–specific NKT cells was Fas-FasL dependent, since adding FasL-blocking antibody to the cultures inhibited apoptosis (Figure 8C). To confirm this result, we purified mCII707–721–specific NKT cells from mCII707–721–immunized Fas receptor–deficient mice (lpr mice). Anti-CD3–activated responder cells were cocultured with mCII707–721–specific NKT cells from lpr or WT mice, and cell death quantified as 7-aminoactinomycin D–positive (7AAD-positive) cells by FACS. Figure 8D shows that the cytotoxic function of mCII707–721–specific NKT cells was Fas-FasL dependent.
IL-17A is not required for mCII707–721–specific NKT function. NKT cells are reported to produce IL-17 (16), so we stained splenocytes from mCII707–721–immunized mice for IL-17A. Upon immunization with mCII707–721, 37.3% of NKT cells produced IL-17A (Figure 9A). However, IL-17A did not appear to play a role in the suppressive function of these cells. CFSE-labeled CD4+ T cells were cocultured with mCII707–721–specific NKT cells in an anti-CD3–coated plate, with or without neutralizing IL-17A antibody. After 48 hours, cell death was analyzed with 7AAD staining on gated CD4+ T cells. The percentage of 7AAD+ T cells was similar with or without IL-17A blocking, suggesting that IL-17A was not involved in mCII707–721–specific NKT cells’ suppressive function (Figure 9B).
IL-17A from mCII707–721–specific NKT cells does not affect suppression. (A) FACS with CD4, NK1.1, IL-17A antibodies and LIVE-DEAD marker from B10.Q WT mouse splenocytes 10 days after immunization with mCII707–721. One representative FACS is shown, with the percentage of IL-17A cells in CFSE-labeled splenocytes, CD4+ T cells, and CD4+ NK1.1+ NKT cells. (B) Single-cell spleen suspensions from B10.Q WT mice immunized with mCII707–721 stained for CD4 and NK1.1, purified by FACS. CD4+ T cells were stimulated with plate-bound anti-CD3 and cocultured for 48 hours with NKT cells with or without anti–IL-17A. Cell death was determined by 7AAD staining.
mCII707–721–specific NKT cells prevent Th1- and Th2-mediated responses. The in vivo suppressive capacity of mCII707–721–specific NKT cells was investigated using B10.Q mice vaccinated with mCII707–721 or the negative control peptide mCI707–721, prior to induction of a delayed-type hypersensitivity (DTH) reaction. Mice vaccinated with mCII707–721 developed significantly less inflammation than the control group (Figure 10, A and B). Moreover, the antiinflammatory effect of mCII707–721 vaccination was dependent upon activation of CD1d-restricted mCII707–721–specific NKT cells, since lack of this population in CD1d–/– mice resulted in significant abrogation of the antiinflammatory effect (Figure 10C). These results support the in vitro findings that CD1d-restricted mCII707–721–specific NKT cells suppress T cell activation and hence inhibit Th1-polarized DTH inflammation.
mCII707–721–specific NKT cells suppress Th1 cell–mediated immune responses. (A and B) In vivo activation of mCII707–721–specific NKT cells significantly reduced Th1-driven inflammation due to DTH induced by rat CII, assessed by histopathological scoring of tissue inflammation. Data are mean ± SD, n = 6 mice per group. *P ≤ 0.05. Original magnification, ×100. (C) DTH response in WT (B10.Q) is significantly lower than in CD1d–/– mice. While mCII707–721 vaccination dampens DTH reaction, the mCI707–721–negative control peptide is inadequate to exert antiinflammatory effects. DTH response (ear swelling) was calculated by subtracting the thickness of the right ear from that of the left ear. Data are mean ± SEM, n = 4–5 mice per group. *P ≤ 0.05.
We next studied the effect of CD1d-restricted mCII707–721 NKT cells on the Th2-mediated immune response. Mice were vaccinated as above and then to provoke a Th2 response were injected with OVA emulsified in alum. Interestingly, a downregulated Th2 response was observed in the mCII707–721–vaccinated group compared with control. A significant decrease in IL-4, IL-5, and IL-13 production was detected in bronchoalveolar lavage fluid (BALF) after OVA rechallenge in WT mice. Moreover, we observed that vaccination with mCII707–721 repressed IgE production in BALF of WT mice. Furthermore, mCII707–721–specific NKT cell activation and the antiinflammatory effects of such activation were determined to be CD1d restricted, as CD1d–/– mice lacked such capacity (Figure 11). Taken together, the observations show that the mCII707–721–specific CD1d-restricted NKT cells are involved in immune regulation of both Th1-mediated cellular responses and Th2-mediated humoral immune responses.
Activation of mCII707–721–specific CD1d-restricted NKT cells in vivo significantly reduces Th2-mediated responses to OVA. (A–D) Expression of IL-4, IL-5, IL-13, and IgE were all significantly reduced in BALF of mCII707–721–vaccinated B10.Q mice compared with all control-vaccinated groups of mice. CD1d–/– mice lack such mCII707–721–mediated antiinflammatory effects. Data are mean ± SD, n = 4–5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Activation of mCII707–721–specific NKT cells ameliorates CIA. The mCII707–721 peptide is a major epitope in mouse collagen type II, so mCII707–721–specific cells might be crucial for regulating CIA. To test our hypothesis, B10.Q mice were vaccinated with either mCII707–721 or control peptide 10 days prior to CIA induction. We found that mCII707–721 vaccination ameliorated the severity of arthritis compared with the control (Figure 12A). The incidence in the mCII707–721–treated group was 74% compared with 93% in the control group. Both clinical signs of disease and histopathological studies revealed that mCII707–721–vaccinated mice had less joint inflammation than control peptide–vaccinated mice (Figure 12B). Prevention of inflammation in these mice was associated with significantly reduced numbers of CD4+ T cells in synovial infiltration (mean ± SD; 12 ± 3 vs. 37 ± 7, n = 10, P ≤ 0.05) and reduced production of IFN-γ, IL-4, and TNF-α (Figure 12, C and D), as determined by joint tissue immunohistochemistry staining. These results support the possibility of controlling arthritis by activation of mCII707–721–specific CD1d-restricted NKT cells.
Activation of mCII707–721–specific NKT cells suppresses arthritis. (A) Prevaccination of B10.Q mice with mCII707–721 significantly reduced severity by CIA clinical scoring compared with control peptide (mCI707–721) vaccination (14 mice per mCI707–721 group, 19 mice per mCII707–721 group; *P ≤ 0.05). (B) Histological arthritic scores by H&E staining of joints. Data are mean ± SD, n = 5. **P ≤ 0.01. (C) Histological scores of percentage of IFN-γ–, IL-4–, and TNF-α–positive cells were significantly reduced in the mCII707–721–vaccinated group. Data are mean ± SD, n = 5. *P ≤ 0.05. (D) Suppression of arthritis was associated with reduced CD4+ T cell–infiltrating cells, IFN-γ–, and IL-4–producing cells by immunohistochemistry staining of joints. Red-brown color shows positively stained cells. Scale bar: 100 μm.
Vaccination with mCII707–721 ameliorates EAE. The DTH and OVA sensitization experiments indicated that the suppressive quality of the mCII707–721–specific NKT cells was not antigen or tissue specific. Therefore, we evaluated the effect of vaccination with mCII707–721 on EAE, a well-described Th1-mediated autoimmune disease affecting the CNS and a widely used experimental model for MS. EAE was induced in B10.Q WT mice vaccinated with mCII707–721 by immunization with MOG79–96. EAE progression was significantly suppressed in mCII707–721–vaccinated mice, resulting in low mean clinical scores compared with the control group vaccinated with mCI707–721 (Figure 13A). Although no differences were seen between the 2 groups in disease incidence (both groups had 89% disease), affected mice in the mCII707–721–vaccinated group recovered entirely, while control group mice exhibited lingering symptoms up to 1 month after disease induction. In support, the CNS of mCII707–721–vaccinated mice showed significantly less demyelination than control mice (Figure 13, B and C), which correlated well with the clinical findings and the general inflammation observed in the CNS (data not shown).
Activation of mCII707–721–specific NKT cells suppresses EAE neurological deficits. (A) Prevaccination of B10.Q mice with mCII707–721 significantly reduced EAE clinical scoring compared with control peptide (mCI707–721) vaccination. n = 9–10 mice per group. *P ≤ 0.05; **P ≤ 0.01. (B and C) Demyelination in CNS was significantly reduced in mCII707–721–vaccinated mice. (B) Representative sections of spinal cord from a mCII707–721–vaccinated mouse (left) and control (right). Arrows show demyelinated areas in white matter. Original magnification, ×25. (C) Demyelination was calculated as mm2 of demyelination per 100 mm2 spinal cord, n = 3 mice per group. Data are median ± SD. **P ≤ 0.01.
These data strongly support the capacity of mCII707–721–specific NKT cells to suppress a variety of inflammatory conditions, not limited to the Th1/Th2 paradigm nor restricted to collagen-tissue specificity.