Direct CD1d-mediated stimulation of APC IL-12 production and protective immune response to virus infection in vivo

Abstract

CD1d-restricted NKT cells rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d(+) APC maturation. However, therapeutic exploitation of NKT cells has been hampered by their paucity and defects in human disease. NKT cell-APC interactions can be modeled by direct stimulation of human APCs through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d(+) but not CD1d knockout murine splenocytes in vitro. Moreover, all of the CD1d mAbs tested also induced IL-12 as well as both IFN-gamma and IFN-alpha in vivo from CD1d(+) but not CD1d-deficient recipients. Unlike IFN-gamma, CD1d-induced IFN-alpha was at least partially dependent on invariant NKT cells. Optimal resistance to infection with picornavirus encephalomyocarditis virus is known to require CD1d-dependent APC IL-12-induced IFN-gamma as well as IFN-alpha. CD1d ligation in vivo enhanced systemic IL-12, IFN-gamma, and IFN-alpha and was protective against infection by encephalomyocarditis virus, suggesting an alternative interpretation for previous results involving CD1d "blocking" in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d F(ab')(2)s in vivo, whereas an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately "downstream," CD1d mAbs were protective later during infection than the invariant NKT cell agonist alpha-galactosylceramide. These data indicate that NKT cells can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as an adjuvant.

Fig. 1. IL-12 production following anti-CD1d treatment of splenocyte cultures in vitro

A - F. C57BL/6J WT or CD1d KO mice splenocyte bioactive p70 IL-12 production from replicate cultures following treatment with 3 different anti-CD1d mAbs (1B1, 19G11, and / or 3C11) or appropriate isotype controls in vitro. Control stimuli: LPS directly stimulates IL-12 production, α-Galcer acts indirectly via iNKT cells leading to monocytic cell activation, but otherwise similar to anti-CD1d directly. C. IFN-γ from expt. 1B. D - F: Independent experiment with 1B1 CD1d mAb, demonstrating specific induction by CD1d mAb of potent cytokine responses, including IFN-α (E).

Fig. 2. Increased systemic cytokine production following anti-CD1d treatment in vivo

Serum cytokines were determined at 24 hr. post-injection of 50μg anti-CD1d mAb 1B1, isotype control mAb, LPS, or α-Galcer. Results shown from individual mice. Note, no significant responses above background were detected with CD1d KO mice. Jα18 KO mice with intact CD1d were able to respond comparably to WT in IL-12 and IFN-γ production. However, IFN-α levels were intermediate, suggesting indirect contribution to such responses by iNKT cells. A - C: anti-CD1d mAb 1B1 or isotype control mAbs with WT C57BL/6J, N12 CD1d KO, or N10 Jα18 KO mice treated as shown. D - F: Further experiment with 1B1 CD1d mAb compared to negative control isotype and other positive control stimuli (lines indicate baseline values).

Fig. 3. Decreased disease following treatment of virus infection with anti-CD1d mAb in vivo

Male C57BL/6J mice were infected with EMCV-D and followed as described previously (30,33). 50μg anti-CD1d mAbs or isotype control mAbs were given once / animal one day prior to infection. Results show individual animal glucose tolerance test and paralysis score data. A.,B. 3C11 anti-CD1d eliminating mild diabetes (A.) and paralysis (B.) of relatively-resistant 7 week old mice to naturally normal levels of uninfected controls. CD1d KO mice included for comparison in the same experiment. C.,D. Anti-CD1d mAbs 3C11, 1B1 and 3C11, or isotype control mAbs reduced diabetes (C.) as well as severe paralysis (D.) of more sensitive 5 week old mice. As previously (30,33), graph lines shown define hyperglycemia as values > 3 times S.D. over mean value of un-infected controls. So for glucose data, lines indicate frank hyperglycemic values. For paralysis results, line separates values above baseline (defined as ‘1’ to show number of mice) with no paralysis (uninfected or cured).

Fig. 4. Anti-CD1d mAb reduces disease and enhances cytokines following virus infection in vivo

Male C57BL/6J WT or CD1d KO mice were infected with EMCV-D and followed as in Fig. 3. Results show individual animal data. A. Glucose tolerance test. B. Paralysis score. 50μg anti-CD1d mAb 1B1 or isotype control mAb, or 2μg α-galactosylceramide was given once / animal on day of infection, reducing diabetes (A.) and paralysis incidence of 6 week old mice (B.), measured at 7 days. Lines shown define hyperglycemia or values above baseline (defined as ‘1’ to show mice) of no paralysis (uninfected animals). The same groups of mice as shown in Fig. 4A,B were tested for serum cytokines. CD1d mAb could specifically induce elevated systemic Th1 cytokine levels. Results show individual animal day 7 serum p70 IL-12 (C.), IFN-γ (D.), and IFN-α (E.) ELISA data (lines indicate control uninfected and untreated animal baseline values).

Fig. 5. Decreased disease following treatment of virus infection with CD1d mAb Fab’₂ in vivo

Balb/c mice were infected with EMCV-D and followed as described previously (30,33). Results show individual animal Day 7 glucose tolerance test and paralysis score data analyzed as in Fig. 3, with lines indicating limits of normal values. 50μg anti-CD1d mAbs 3C11, 19G11, HB323 or it’s Fab’₂, or isotype control mAbs were given / animal on the day of infection, reducing diabetes (A.) and eliminating or greatly suppressing paralysis (B.) as in uninfected controls. C. Independent experiment Day 7 paralysis data, comparing anti-CD1d mAbs 1B1, HB323 and isotype control mAb or their corresponding Fab’₂. The sera from Fig. 5C were tested for serum cytokines. Intact CD1d mAb and Fab’₂ could induce elevated systemic Th1 cytokine levels. Results show individual animal Day 3 serum p70 IL-12 (D.), IFN-γ (E.), and IFN-α (F.) ELISA data (lines indicate control uninfected values). Similar results, although with less cytokines, were detected in serum from Day 7 (not shown).

Fig. 6. Treatment of established virus infection with α-Galcer

2μg α-Galcer or vehicle were given / animal before infection (Day -1) or on days 1 - 3 of EMCV-D infection of Balb/c mice as shown. Infected animals were monitored for disease as in Fig. 3, with lines indicating abnormal value limits. Results show individual animal glucose tolerance test results (A.) and paralysis scores (B.), measured on day 7 post-infection with % incidence of abnormal glucose results or paralysis. Normoglycemic values were defined as within 3 times the S.D. of the mean value of PBS injected uninfected controls.

Fig. 7. Protection following treatment of established virus infection with CD1d mAb

50μg anti-CD1d mAb 3C11 or isotype control mAb were given / animal on days 1, 2, or 3 of EMCV-D infection of Balb/c mice. Infected animals were monitored for disease and serum cytokines as shown in previous Figs. CD1d mAb could induce protection against EMCV-D mediated disease until Day 2 of infection, whereas α-Galcer exacerbated disease from Day 1 onwards (Fig. 7). CD1d mAb could induce protection against EMCV-D mediated disease and elevate systemic Th1 cytokine levels up to Day 2 post-infection. Results show individual paralysis results (line indicates lack of paralysis of control uninfected mice) and serum cytokines, measured on Day 5 post-infection (lines indicate median baseline values). p70 IL-12 (B.), IFN-γ (C.), and IFN-α (D.) ELISA data.