Analysis of the cellular mechanism underlying inhibition of EAE after treatment with anti-NKG2A F(ab')2

Abstract

Autoimmune encephalomyelitis may be ameliorated experimentally by enhancing NK cell-mediated elimination of activated autoreactive T cells through a mutation that interrupts the interaction between Qa-1(b) and CD94/NKG2A. Here we evaluate the ability of an anti-NKG2A F(ab')(2) Ab to enhance elimination of autoreactive T cells and reduce experimental autoimmune encephalomyelitis (EAE). Anti-NKG2A F(ab')(2) treatment diminishes progression of both myelin oligodendrocyte glycoprotein (MOG)-induced EAE in intact C57BL/6 mice and after adoptive transfer of disease-causing T cells. Analyses of the underlying mechanism revealed that administration of anti-NKG2A F(ab')(2) Ab reduces CD4(+) T recall responses to MOG and skews the proportion of IL-17- and IFNgamma-producing CD4(+) T cells toward the protective IL-4- and IL-10-secreting CD4(+) T cell subpopulations. CD94/NKG2A-dependent inhibition of inflammatory damage to spinal cord is associated with decreased infiltration of T cells and reduced microglia activation in the central nervous system. Because anti-NKG2A F(ab')(2) treatment had no detectable effect on the numbers or activity of T and B lymphocytes and NK cells in peripheral lymphoid tissues, this anti-NKG2A-based approach may represent a safe and effective therapy for this CNS disorder.

Fig. 1.

Rationale for use of the 20d5 F(ab′)₂ fragment and determination of the optimal time window for Ab administration. (A) L cells infected with Qa-1 were used as target cells of IL-2–activated NK cells with or without Qdm peptide (30 μM). In some cases, cells were preincubated with 20d5 F(ab′)₂ or whole Ig for 1 h at 37 °C. Percentage of lysis is shown at the indicated E:T ratios. (B) EAE was induced in C57BL/6 mice as described in Methods. 20d5 F(ab′)₂ was given i.v. at days 0, 3, and 6. Development of EAE was monitored daily.

Fig. 2.

Anti-NKG2A F(ab′)₂ treatment ameliorates disease severity of EAE. (A) EAE was induced in C57BL/6 mice (n = 6) as described in Methods. Treatment with or without anti-NKG2A F(ab′)² or whole 20d5 rat IgG2a at days 13 and 15 is shown. Mice were monitored daily for EAE. (B) MOG-reactive CD4⁺ T cells (10⁶) were purified from EAE-experienced C57BL/6 mice and transferred into Rag2^−/− hosts followed by immunization s.c with 200 μg MOG in CFA at day 1 and injection i.p. with 200 ng pertussis toxin at days 1 and 2. EAE assessment was recorded daily. 20d5 F(ab′)₂ was administered at days 15 and 17. Data are shown as mean ± SEM (n = 6; *P < 0.05). (C) 2D2 transgenic CD4⁺ T cells (10⁴) were transferred into Rag2^−/− hosts (n = 6) and EAE was induced as described in B. 20d5 F(ab′)₂ was injected at days 15 and 16(*P <0.05).

Fig. 3.

20d5 F(ab′)₂ Ab treatment reduces cell infiltration and activation status of microglia and CD4⁺ T cells in spinal cord. EAE was induced in B6 mice and treated as described in Methods. (A) Mean cell counts in spinal cord were determined by hemocytometer and graphed with standard errors. (B) The absolute cell numbers of Qa-1^b-expressing CD4 cells and microglia were determined by flow cytometry by gating on CD45^hiCD4⁺ and CD45^medCD11b⁺ populations, respectively. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Correlation of Qa-1^b-expressing CD4 cells and microglia to clinical score in mice was measured at the day of sacrifice.

Fig. 4.

20d5 F(ab′)₂ Ab treatment decreases splenic CD4⁺ T cell recall responses. Splenocytes from B6-EAE mice (A) or Rag2^−/− mice transferred with MOG-reactive CD4 cells (B) were incubated with the indicated concentrations of MOG peptide in the presence of irradiated splenocytes from B6 mice. IL-2 secretion was measured by ELISA 48 h after culture. Data are shown as mean ± SEM (n = 4; *P < 0.05).

Fig. 5.

20d5 F(ab′)₂ Ab treatment alters CD4⁺ T cell cytokine profiles. EAE was induced in Rag2^−/− hosts by adoptive transfer of MOG-reactive CD4 cells. Intracellular levels of IFNγ (A) and IL-10, IL-4, and IL-17 (B) from spinal cord were assessed by FACS analyses as described in Methods. The percent of cytokine-secreting CD4 cells is shown with standard errors (n = 3–4; *P < 0.05).

Fig. 6.

20d5 F(ab′)₂ Ab treatment accumulates activated NK cells into spinal cord. (A) EAE was induced in B6 mice followed by treatment with anti-NKG2A F(ab′)₂ Ab. The numbers of spinal CD69-expressing and NKG2A⁺ NK cells were determined by FACS analyses on CD45^hiDX5⁺ cells (n = 3–4; *P < 0.05). (B) 2D2 CD4 cells were transferred into Rag2^−/− hosts and EAE was induced as described in Fig. 2C. The levels of different activation markers on spinal cord NK cells were determined by FACS analyses on CD45^hiNK1.1⁺ cells. The levels of NK activation markers were correlated with EAE scores at termination. (C) The percent of CRACC and Ly-9-expressing spinal cord CD4 and NK cells was determined on CD45^hiCD4⁺ and CD45^hiNK1.1⁺ populations, respectively. (D) EAE was induced in Rag2^−/− hosts by adoptive transfer of MOG-reactive CD4 cells. Spinal cord cells were plated on anti-NK1.1-coated plates and stimulated for 8 h with BD Golgiplug (BD Biosciences) for the last 7 h. The levels of intracellular perforin and IFNγ were determined on a CD45^hiDX5⁺ population. Data are shown as mean ± SEM (n = 4; *P < 0.05).