The Transcription Factor NFATc1 Supports the Rejection of Heterotopic Heart Allografts

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  Figure 1. NFATc1 deficiency in T cells prevents acute rejection of allogeneic heterotopic heart transplants. (A) Syngeneic (C57BL/6, Syn) or fully allogenic (BALB/c, Allo) heart grafts were transplanted into wild-type (WT) C57BL6, Nfatc1flx/flx x mb1-cre, Nfatc1flx/flx x CD4-cre, or Nfatc1P2Δ recipient mice (n = 5–9 recipients per group). While Nfatc1flx/flx x mb1-cre mice bear NFATc1-deficient B cells, Nfatc1flx/flx x CD4-cre mice bear NFATc1-deficient CD4+ and CD8+ T cells. Nfatc1P2Δ mice carry an Nfatc1 P2 promoter deletion and, therefore, are deficient for P2-directed transcripts (23). The survival of heart function was monitored daily during the first 3 weeks after transplantation, then four times per week. (B) Representative western blot showing the efficient NFATc1 ablation in cytotoxic T cells (CTL) from Nfatc1flx/flx x CD4-cre (c1-ko) mice. Cytoplasmic (CP) and nuclear proteins (N) from CTLs of WT, Nfatc1flx/flx x CD4-cre (c1-ko) and Nfatc2−/− (c2-ko) mice were fractionated. For the generation of CTLs in vitro, splenic CD8+ T cells were stimulated by αCD3/CD28 antibodies (Abs) for 3 days, followed by culture in IL-2 containing medium for further 3 days. They were left un-induced…

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  Figure 2. Expression of NFATc1/αA in peripheral T cells leads to rejection of allogeneic heterotopic heart transplants. (A) Allogenic heart grafts (of BALB/c mice) were transplanted into five dlck-cre x Nfatc1flx/flx mice, or five dlck-cre x Nfatc1flx/flx x caNfatc1-STOPflx/flx mice expressing a constitutive active (ca) version of NFATc1/αA in peripheral T cells. (B) Immunoblots showing the expression of caNFATc1/αA in CD8+ T cells from two out of five dlck-cre x Nfatc1flx/flx x caNfatc1-STOPflx/flx mice. In lanes 1 and 6, cytosolic protein, in lanes 2, 3, and 7 nuclear protein from wild-type (WT) CD8+ T cells stimulated by αCD3/CD28 antibodies (Abs) in vitro for 2 days was fractionated. In lanes 4 and 8, cytosolic protein, and in lanes 5 and 9 nuclear protein of CD8+ T cells activated for 2 days from dlck-cre x Nfatc1flx/flx x caNfatc1-STOPflx/flx mice was fractionated. (C) Immunoblot showing NFATc1 expression in CD8+ T cells from five dlck-cre x Nfatc1flxl/flx mice. In lane 1, whole cell protein of WT CD8+ T cells stimulated by αCD3/CD28 Abs in vitro for 2 days was fractionated, in lanes 2–6, whole cell protein of activated CD8+ T cells from dlck-cre x Nfatc1flx/flx mice.

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  Figure 3. Graft-Infiltrating Cells (GICs) in transplanted hearts. (A) Representative hematoxylin–eosin stains of grafts from syngeneic and allogeneic wild-type (WT) recipients, and from Nfatc1flx/flx x CD4-cre recipients during the acute rejection phase (d5), and from Nfatc1flx/flx x CD4-cre recipients at d110. Bar, 300 µm. (B) Quantification of GICs in transplanted allogeneic hearts with (+) and without (−) cyclosporin A (CsA) treatment of animals. (C) Cytotoxicity of GICs. Luciferase assays were performed after cultivation of MOPC-luciferase target cells together with equal numbers of GICs isolated from WT or Nfatc1flx/flx xCD4-cre recipient mice during the acute rejection phase (d6, effector/target ratio 4/1, followed by 20 or 36 h incubation).

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  Figure 4. Cyclosporin A (CsA)-sensitive nuclear factors of activated T cell (NFAT) signaling is a major transcriptional pathway in Graft-Infiltrating Cells (GICs) from allogeneic heterotopic heart transplants. Syngeneic (C57BL/6, Syn) or fully allogeneic (BALB/c, Allo) heart grafts were transplanted into wild-type (WT) or NFATc1-deficient (Nfatc1flx/flxxCD4-cre) C57BL/6 recipients. A subgroup of WT recipients was treated daily with CsA (15 mg/kg, Allo CsA). GICs were purified during acute rejection phase (d5) and subjected to next-generation sequencing (NGS) assays. (A) RNA expression profiles (“heat map”) of T cell signature genes. GICs were purified from syngeneic recipients (lane 1), allogeneic recipients in the absence or presence of CsA (lanes 2 and 3, respectively), and from recipients bearing Nfatc1−/− T cells (lane 4). Their RNAs were isolated and subjected to NGS. (B) Ingenious Pathway Analyses. Expression data were normalized to the transcriptome of GICs in syngeneic transplantations. (C) RNA expression of genes encoding glycolytic enzymes. (D) Extracellular flux analyses of activated CD8+ Ts from WT and dlck-cre x Nfatc1flx/flx mice (left), and from WT and dlck-cre x…

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  Figure 5. The Cxcr3 gene is a direct NFATc1 target. (A) Representative immune histochemical stains of Cxcr3. Syngeneic (Syn) or allogeneic (Allo) heart grafts were transplanted into wild-type (WT) or cyclosporin A (CsA)-treated WT mice, or in recipient mice bearing NFATc1-deficient T cells (Nfatc1flx/flx x CD4-cre). Grafts and graft-infiltrating cells (GICs) were analyzed during the acute rejection phase on day 5. Bar, 500 µm. (B) Next-generation sequencing assays. Normalized RNA expression of Cxcr3 gene and of relevant ligand genes—Cxcl9, Cxcl10, and Cxcl11—in the transcriptome of GICs. RPKM, reads per kilobase per million reads. (C) Induction of Cxcr3 transcripts in CTL cells derived from WT, Nfatc1flx/flx x CD4-cre or Nfatc2−/− mice. Real-time PCR assays of RNAs were normalized to Actb expression. (D) Genomic organization of the Cxcr3 locus. The distribution of epigenetic modification histone mark H3K27Ac and the presence of DNaseI hypersensitive sites in non-expressing and cells expressing (as Th1 cells) is indicated. (E) Binding of NFATc1/A-Bio and NFATc2 (39) to the Cxcr3 locus in CTLs. ChIP seq assays using a bacterial artificial chromosome transgenic mice expressing NFATc1/A-Bio…