Figure 5. Effects of NK cells on the capacity of microglia and other CNS-derived APCs to promote myelin-reactive T cell proliferation and Th17 responses. (A) Proliferation of naive CD62Lhigh MOG35-55-specific CD4+ transgenic 2D2 T cells cultured with microglia, astrocytes (AS), mDCs, pDCs, CD11b+CD45+, or irradiated CD4− splenocytes from MOG-primed mice at the peak of EAE disease at an APC/T cell ratio of 5:1, in the presence or absence of 10 µg/ml MOG35-55 peptide. Mice were treated with control IgG or anti-NK1.1 before immunization. Results are expressed as the changes in counts per minute (CPM). Irradiated CD4− splenocytes stimulate 2D2 cells at 2/3 of the capacity of mDCs. (B) Release of IL-17 by T helper cell supernatant from A after 72 h. (C) Capacity of CNS-derived APCs to drive Th17 cell responses and to be affected by NK cells. Naive (CD25−CD62LhighCD44low) CD4+ T cells were sorted from lymph nodes and spleens of 2D2 transgenic mice. Subsequently, 2 × 105 cells of the indicated APC types and NK cells (all isolated from the CNS) were added to the culture. NK cells from the IL-2 complex–treated mice were injected into the brain. Il-17a transcripts were quantified by qRT-PCR. Data in A–C are representative of two independent experiments using cells from 15–20 mice/group. MG, microglia. Error bars represent the means ± SEM. P-values were determined by an ANOVA test. (D) Interactions between NK cells and microglia influence myelin-reactive Th17 cells in vivo and the importance of the NKG2A–Qa1 pathway. Naive (CD25−CD62LhighCD44low) CD4+ T cells were sorted from lymph nodes and spleens of 2D2 mice. Subsequently, 2 × 105 cells were injected i.v. into MOG/CFA/PT-primed RAG1−/−γc−/− mice. Simultaneously, the same numbers of microglia from Cx3cr1+/− (MG+/−) and Cx3cr1−/− (MG−/−) mice and NK cells from the IL-2 complex–treated mice were injected into the brain as previously described (Cardona et al., 2006b). STAT3 phosphorylation was quantified by FACS and compared in mice receiving CD4+ T cells alone or mice that also received microglia from Cx3cr1+/− or Cx3cr1−/− mice. RNA was isolated from 2D2 CD4+ cells and Rorα, Rorγ (also know as Rorc), and Il-17a transcripts were quantified by qRT-PCR. Data represent three experiments with 15–22 mice per group each. Error bars represent the means ± SEM. P-values wre determined by an ANOVA test. For C and D, + and − denote culture with or without NK cells.
Figure 5.

Effects of NK cells on the capacity of microglia and other CNS-derived APCs to promote myelin-reactive T cell proliferation and Th17 responses. (A) Proliferation of naive CD62Lhigh MOG35-55-specific CD4+ transgenic 2D2 T cells cultured with microglia, astrocytes (AS), mDCs, pDCs, CD11b+CD45+, or irradiated CD4− splenocytes from MOG-primed mice at the peak of EAE disease at an APC/T cell ratio of 5:1, in the presence or absence of 10 µg/ml MOG35-55 peptide. Mice were treated with control IgG or anti-NK1.1 before immunization. Results are expressed as the changes in counts per minute (CPM). Irradiated CD4− splenocytes stimulate 2D2 cells at 2/3 of the capacity of mDCs. (B) Release of IL-17 by T helper cell supernatant from A after 72 h. (C) Capacity of CNS-derived APCs to drive Th17 cell responses and to be affected by NK cells. Naive (CD25−CD62LhighCD44low) CD4+ T cells were sorted from lymph nodes and spleens of 2D2 transgenic mice. Subsequently, 2 × 105 cells of the indicated APC types and NK cells (all isolated from the CNS) were added to the culture. NK cells from the IL-2 complex–treated mice were injected into the brain. Il-17a transcripts were quantified by qRT-PCR. Data in A–C are representative of two independent experiments using cells from 15–20 mice/group. MG, microglia. Error bars represent the means ± SEM. P-values were determined by an ANOVA test. (D) Interactions between NK cells and microglia influence myelin-reactive Th17 cells in vivo and the importance of the NKG2A–Qa1 pathway. Naive (CD25−CD62LhighCD44low) CD4+ T cells were sorted from lymph nodes and spleens of 2D2 mice. Subsequently, 2 × 105 cells were injected i.v. into MOG/CFA/PT-primed RAG1−/−γc−/− mice. Simultaneously, the same numbers of microglia from Cx3cr1+/− (MG+/−) and Cx3cr1−/− (MG−/−) mice and NK cells from the IL-2 complex–treated mice were injected into the brain as previously described (Cardona et al., 2006b). STAT3 phosphorylation was quantified by FACS and compared in mice receiving CD4+ T cells alone or mice that also received microglia from Cx3cr1+/− or Cx3cr1−/− mice. RNA was isolated from 2D2 CD4+ cells and Rorα, Rorγ (also know as Rorc), and Il-17a transcripts were quantified by qRT-PCR. Data represent three experiments with 15–22 mice per group each. Error bars represent the means ± SEM. P-values wre determined by an ANOVA test. For C and D, + and − denote culture with or without NK cells.

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