Neonatal exposure to a self-peptide-immunoglobulin chimera circumvents the use of adjuvant and confers resistance to autoimmune disease by a novel mechanism involving interleukin 4 lymph node deviation and interferon gamma-mediated splenic anergy

Abstract

Induction of neonatal T cell tolerance to soluble antigens requires the use of incomplete Freund's adjuvant (IFA). The side effects that could be associated with IFA and the ill-defined mechanism underlying neonatal tolerance are setbacks for this otherwise attractive strategy for prevention of T cell-mediated autoimmune diseases. Presumably, IFA contributes a slow antigen release and induction of cytokines influential in T cell differentiation. Immunoglobulins (Igs) have long half-lives and could induce cytokine secretion by binding to Fc receptors on target cells. Our hypothesis was that peptide delivery by Igs may circumvent the use of IFA and induce neonatal tolerance that could confer resistance to autoimmunity. To address this issue we used the proteolipid protein (PLP) sequence 139-151 (hereafter referred to as PLP1), which is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. PLP1 was expressed on an Ig, and the resulting Ig-PLP1 chimera when injected in saline into newborn mice confers resistance to EAE induction later in life. Mice injected with Ig-PLP1 at birth and challenged as adults with PLP1 developed T cell proliferation in the lymph node but not in the spleen, whereas control mice injected with Ig-W, the parental Ig not including PLP1, developed T cell responses in both lymphoid organs. The lymph node T cells from Ig-PLP1 recipient mice were deviated and produced interleukin (IL)-4 instead of IL-2, whereas the spleen cells, although nonproliferative, produced IL-2 but not interferon (IFN)-gamma. Exogenous IFN-gamma, as well as IL-12, restored splenic proliferation in an antigen specific manner. IL-12-rescued T cells continued to secrete IL-2 and regained the ability to produce IFN-gamma. In vivo, administration of anti-IL-4 antibody or IL-12 restored disease severity. Therefore, adjuvant-free induced neonatal tolerance prevents autoimmunity by an organ-specific regulation of T cells that involves both immune deviation and a new form of cytokine- dependent T cell anergy.

Figure 1

SJL/J mice injected with Ig–PLP1 at birth resist induction of EAE during adult life. Newborn mice (10 per group) were injected with 100 μg of affinity chromatography–purified Ig–PLP1 (▴) or Ig–W (○) in saline within 24 h of birth and were induced for EAE with free PLP1 peptide at 7 wk of age as described in Materials and Methods. Mice were scored daily for signs of paralysis for 100 d. A shows the daily mean clinical score of all mice, and B shows the daily mean score of only the surviving animals. Although all mice in both groups developed signs of paralysis, 40% of the mice that were injected with Ig–W at birth died of severe disease. Death did not occur in mice injected with Ig–PLP1 at birth. The mean maximal disease severity was 4.2 ± 0.9 in the mice recipient of Ig–W at birth and 2.7 ± 0.5 in the Ig–PLP1 group.

Figure 2

In vivo presentation of Ig–PLP1 by neonatal thymic and splenic APCs. Newborn mice (three per group) were injected with 100 μg Ig–PLP1 (▴) or Ig–W (○) in saline within 24 h of birth, and in vivo presentation of Ig–PLP1 was allowed for 2 d. The mice were then killed, and pooled thymic (A) and splenic (B) cells were irradiated and used as APCs for stimulation of the PLP1-specific T cell hybridoma 4E3 (28). IL-2 production in the supernatant, which was used as a measure of T cell activation, was determined using the IL-2–dependent HT-2 cell line as described in Materials and Methods. The indicated cpms represent the mean ± SD of triplicates.

Figure 3

Reduced splenic proliferative T cell response in mice injected with Ig–PLP1 at birth. Newborn mice (eight per group) were injected intraperitoneally within 24 h of birth with 100 μg Ig–PLP1 or Ig–W in saline. When the mice reached 7 wk of age they were immunized with 100 μg free PLP1 peptide in 200 μl CFA/PBS (vol/vol) subcutaneously in the foot pads and at the base of the limbs and tail. 10 d later the mice were killed, and the (A) lymph node (0.4 × 10⁶ cells/well) and (B) splenic (1 × 10⁶ cells/well) cells were in vitro stimulated for 4 d with 15 μg/ml of free PLP1 or PLP2, a negative control peptide corresponding to the encephalitogenic sequence 178–191 of PLP (26). 1 μCi/well of [³H]thymidine was added during the last 14.5 h of stimulation, and proliferation was measured using an Inotech β-counter and the trace 96 Inotech program. The indicated cpms represent the mean ± SD of triplicate wells for individually tested mice. The mean cpm ± SD of lymph node proliferative response of all Ig–PLP1 and Ig–W recipient mice was 34,812 ± 7,508 and 37,026 ± 10,333, respectively. The mean splenic proliferative response was 3,300 ± 3,400 for the Ig–PLP1 recipient group and 14,892 ± 4,769 for the Ig–W recipient group.

Figure 4

Neonatal injection of free PLP1 peptide in saline or IFA has a different effect than Ig–PLP1/saline on the proliferative T cell responses to a challenge with PLP1 in CFA. Three groups of newborn mice (seven mice per group) were injected at birth intraperitoneally with 100 μg PLP1 peptide in 100 μl saline (PLP1/Sln), 100 μg PLP1 peptide in 100 μl PBS/IFA (vol/vol) (PLP1/IFA), and 100 μg Ig–PLP1 in 100 μl saline (Ig–PLP1/Sln), and challenged with 100 μg PLP1 in CFA as in Fig. 3. 10 d later the mice were killed and lymph node (A) and splenic (B) proliferative responses were analyzed by [³H]thymidine incorporation as described in Fig. 3. A control group of mice that was not injected at birth (None) but immunized as adults with PLP1 in CFA was included for comparison purposes. The bars represent the mean cpm ± SD of seven individually tested mice.

Figure 5

Lymph node T cell deviation in mice recipient of Ig–PLP1 at birth. Newborn mice (eight per group) were injected intraperitoneally within 24 h of birth with 100 μg Ig–PLP1 or Ig–W in saline. When the mice reached 7 wk of age, they were immunized with 100 μg free PLP1 peptide in 200 μl CFA/PBS (vol/vol) subcutaneously in the foot pads and at the base of the limbs and tail. 10 d later the mice were killed, and the lymph node cells (0.4 × 10⁶ cells/well) were in vitro stimulated with free PLP1 or PLP2 (15 μg/ml) for 24 h. The production of IL-2 (A), IL-4 (B), and IFN-γ (C) was measured by ELISPOT as described in the Materials and Methods section using PharMingen anticytokine antibody pairs. The indicated values (spot forming units, SFU) represent the mean ± SD of eight individually tested mice.

Figure 6

Production of IL-2 but not IFN-γ by nonproliferative splenic T cells from mice injected with Ig–PLP1 on the day of birth. Splenic cells (10⁶ cells/well) from the mice described in Fig. 5 were in vitro stimulated with free PLP1 or PLP2 (15 μg/ml) for 24 h, and the production of IL-2 (A), IL-4 (B), and IFN-γ (C) in the supernatant was measured by ELISA using anticytokine antibody pairs from PharMingen according to the manufacturer's instructions. The indicated amounts of cytokine represent the mean ± SD of eight individually tested mice.

Figure 7

Cytokine-mediated restoration of splenic T cell proliferation in mice injected with Ig–PLP1 at birth. A group of five newborn mice was injected intraperitoneally with 100 μg of Ig–PLP1 and immunized with 100 μg PLP1 peptide in CFA at 7 wk of age, as in Fig. 3. 10 d later, the splenic cells (10⁶ cells/well) were in vitro stimulated with free PLP1 peptide (15 μg/ml) in the presence of 100 U/ml IFN-γ (A) or 10 U/ml IL-12 (B), and [³H]thymidine incorporation was measured as in Fig. 3. Cells from each mouse were stimulated with PLP1 peptide without addition of exogenous cytokines (dotted bars), with PLP1 peptide in the presence of cytokine (hatched bars), or with PLP2 peptide in the presence of cytokine (black bars). The indicated cpms for each mouse represent the mean ± SD of triplicate wells.

Figure 8

Restoration of EAE in Ig–PLP1 tolerized mice by administration of anti–IL-4 antibody. Newborn mice were injected intraperitoneally within 24 h of birth with 100 μg Ig–PLP1 in saline. When they reached 7 wk of age, a group of seven mice was injected intraperitoneally with 1 mg/mouse of affinity purified 11B11 anti–IL-4 antibody in 500 μl of PBS (□). A second group of five mice was injected with 1 mg/mouse of rat IgG in 500 μl PBS (⋄) to serve as control. On the next day all mice were induced for EAE with PLP1 peptide as described in Fig. 1. 5 d after disease induction, the mice were given a second injection of 1 mg/mouse intraperitoneally of 11B11 or rat IgG. The mice were scored daily for signs of paralysis. For comparison purposes the clinical scores of the mice described in Fig. 1 that were tolerized with Ig–PLP1 (▴) or Ig–W (○) at birth and induced for EAE with PLP1 peptide at 7 wk of age were included.

Figure 9

Restoration of EAE in Ig–PLP1-tolerized mice by administration of rIL-12. Newborn mice (seven per group) were injected intraperitoneally within 24 h of birth with 100 μg Ig–PLP1 in saline, and when the mice reached 7 wk of age they were induced for EAE with PLP1 peptide as described in Fig. 1. 4 h after disease induction the mice were injected intraperitoneally with 500 ng/mouse of rIL-12 (PharMingen). Additional intraperitoneal injections of rIL-12 (500 ng/mouse) were carried out on days 2, 4, and 7 after disease induction. These mice (□) were scored daily for signs of paralysis. For comparison purposes, the clinical scores of the mice described in Fig. 1 that were tolerized with Ig–PLP1 (▴) or Ig–W (○) at birth and induced for EAE with PLP1 peptide at 7 wk of age were included.

Figure 10

Restoration of splenic proliferation in Ig–PLP1 tolerized mice by administration of IL-12. Newborn mice (seven per group) were injected intraperitoneally within 24 h of birth with 100 μg Ig–PLP1 in saline and when the mice reached 7 wk of age they were immunized subcutaneously with 100 μg PLP1 peptide in CFA as described in Fig. 3. 4 h after immunization, the mice were injected intraperitoneally with 500 ng/mouse of rIL-12. Additional intraperitoneal injections of rIL-12 (500 ng/mouse) were carried out on days 2, 4, and 7 after immunization. On day 10 the mice were killed, and the spleen cells (10⁶/well) were stimulated with PLP1 peptide in the presence (cross-hatched bar) or absence (gray bar) of rIL-12 (10 U/well), and proliferation was measured as described in Materials and Methods. For comparison purposes, spleen cells from mice that did not receive rIL-12 in vivo were stimulated with PLP1 peptide in the presence (striped bar) or absence (white bar) of rIL-12 (10 U/well). Each bar represents the mean ± SD of seven individually tested mice.