Deletion of Fanca or Fancd2 dysregulates Treg in mice

Abstract

Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca(-/-) or Fancd2(-/-) BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca(-/-) or Fancd2(-/-) mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25(+)Foxp3(+) Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25(+)Foxp3(+) Tregs of Fanca(-/-) or Fancd2(-/-) mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients.

Figure 1

FA Treg cells show lower suppression potential. (A) FA deficiency does not alter Treg cell number in native mice. Splenocytes from mice with the indicated genotypes were subjected to flow cytometry analysis for CD4, CD25, Foxp3 staining. CD4⁺ cells were gated for analysis of CD25⁺Foxp3⁺ cell portion. Representative flow graph (left) and quantification (right) are shown. Results are means plus or minus SD of 2 independent experiments (n = 6 per group). (B) FA Treg cells fail to suppress Teff cell proliferation in vitro. Carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled WT CD4⁺CD25⁻ Teff cells were cocultured with CD4⁺CD25⁺ cells freshly isolated from either WT or Fanca^−/−, Fancd2^−/− mice at a ratio of 2:1 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 5 days followed by flow cytometry analysis for CFSE retention. Data were analyzed by FlowJo software. Representative flow graph (left) and quantification (right) are shown. Results are means plus or minus SD of 3 independent experiments (n = 9 per group). (C) FA Treg cells are less suppressive in preventing GVHD in vivo. Lethally irradiated Balb/c recipients were transplanted with 5 × 10⁶ TCD BMCs from WT C57BL/6 animals alone, or with sorted WT Teff cells (CD4⁺CD25⁻, 5 × 10⁵) plus or minus equal numbers of sorted CD4⁺CD25⁺ Treg cells from either WT C57BL/6, Fanca^−/−. Survival of recipients was monitored by Kaplan-Meier curve method. Each group includes 7 to 10 mice. (D) No difference in cell number of donor-derived Treg cells. Splenocytes isolated from recipients described in (C) were subjected to Flow cytometric analysis for H-2^b, CD4, Foxp3 staining. Donor H-2^b+ cells were gated for CD4⁺Foxp3⁺ cell portion. Representative flow graph (left) and quantification (right) are shown.

Figure 2

FA deficiency affects Foxp3 transcriptional activity. (A) Relative expression levels of Foxp3 signature genes in WT and Fanca^−/− (upper) or Fancd2^−/− (lower) donor T cells. RNA extracted from H-2^b+CD4⁺ cells isolated from recipients transplanted with Treg cells from WT, Fanca^−/−, or Fancd2^−/− mice was used for real-time PCR analysis using primers specific for the indicated Foxp3 target genes. Samples were normalized to the level of GAPDH mRNA. (B) Change in expression levels of Foxp3 target genes in FA infiltrated donor T cells (H-2^b+CD4⁺). RNA extracted from H-2^b+CD4⁺ cells of the recipients described in (A) was used for real-time PCR analysis using primers specific for the indicated Foxp3 target genes. Each group includes 6 mice.

Figure 3

FA chimeras exhibit increased GVHD-inducing potential. (A) Survival of secondary BM transplanted chimeras. 5 × 10⁶ BMCs plus 5 × 10⁶ splenocytes from WT C57BL/6 mice (B6, H-2^b+, CD45.2⁺) or 10 × 10⁶ WBMCs plus 5 × 10⁶ splenocytes from Fanca^−/− (left) or Fancd2^−/− (right) mice (C57BL/6: B6, H-2^b+, CD45.2⁺) were transplanted to lethally irradiated Boy J recipients (C57BL/6: B6, H-2^b+, CD45.1⁺). Donor-derived chimera (CD45.2⁺) were assessed at 4 months after BMT. The recipients with greater than 95% donor-derived chimera were then subjected to 2nd BMT by 9 Gy irradiation and injecting 5 × 10⁶ BM cells along with 2 × 10⁶ T cells isolated from either B6 (syngeneic; H-2^b+, CD45.2⁺) or Balb/c (allogeneic; H-2^d+, CD45.2⁺) mice. Survival of the mice was monitored by Kaplan-Meier curve method. (B) Histopathologic examination of GVHD target organs. Tissue sections (skin, liver, lung, and small intestine) were stained with hematoxylin and eosin and examined by microscope. Asterisks show lymphocytic infiltrates; green arrows show tissue destruction; black arrows show focal alveolar hemorrhages in lung. The specimens shown are representative images of 5 mice in each group with similar histologic features. (C) GVHD clinical scores were determined as a measure of GVHD severity on days 14 and 21 after allogeneic BMT. Data are presented as means plus or minus SD of 2 independent experiments (n = 7 to 10 per group). (D) Weight loss of the recipients. Average weights are shown for mice described in (A). Data are presented as means plus or minus SD of 2 independent experiments (n = 7 to 10 per group).

Figure 4

FA T cell induces increased GVHD lethality. Fanca^−/− (A) and Fancd2^−/− (B) T cells induce higher GVHD lethality. 5 × 10⁶ of T cell deleted total BMCs (TCD) from WT mice plus 3 × 10⁶ T cells from either WT or FA mice were transplanted to lethally irradiated Balb/c recipients. Survival of recipients was monitored by Kaplan-Meier curve method. Each group includes 6 to 10 mice. (C) GVHD clinical scores were determined as a measure of GVHD severity in surviving animals on the indicated time points after allogeneic BMT. Data are presented as means plus or minus SD of 2 independent experiments. (D) Weight loss of the recipients. Average weights are shown for mice described in (A) and (B). Data are presented as means plus or minus SD of 2 independent experiments.

Figure 5

Exacerbated inflammation partially contributes to GVHD in FA. (A) Expression of GVHD effective cytokines in FA Treg-transplanted mice. Lethally irradiated Balb/c recipients were transplanted with 5 × 10⁶ TCD cells from WT C57Bl/6 animals alone, or with sorted WT Teff cells (CD4⁺CD25⁻, 5 × 10⁵) plus or minus equal numbers of sorted CD4⁺CD25⁺ Treg cells from either WT C57BL/6, Fanca^−/− or Fancd2^−/− mice. Donor T cells isolated from the small intestine of the Balb/c recipients transplanted with the indicated donor cells were stimulated with 50 ng/mL PMA and 2 μg/mL Ionomycin for 1 hour, followed by 3 hours incubation in the presence of 1 μg/mL Brefeldin A. Treated cells were stained H-2^b+ and CD4⁺ antibodies before the treatment with Cytofix/Cytoperm reagent. Cytokines were intracellularly stained with antibodies specific for TNF-α, IFN-γ, and IL-6 followed by flow cytometric analysis gated on the H-2^b+CD4⁺ cell compartment. Each group includes 6 to 10 mice. (B) Levels of TNF-α, IFN-γ, and IL-6 in sera of recipient mice described in (A). (C) Increased NF-κB transcription activity in infiltrated FA T cells. H-2^b+CD4⁺ T cells isolated from liver (left) and small intestine (right) of the recipients described in (A) were used for flow cytometric analysis for CD154. Representative flow graphs are shown. (D) p65 deletion partially reduces FA GVHD mortality. Treg cells from p65^f/fFanca^+/+ or p65^f/fFanca^−/− mice plus TCD + Teff cells were transplanted to lethally irradiated Balb/c recipients followed by DMSO or Tamoxifen treatment of 3 days. Survival of recipients was monitored by Kaplan-Meier curve method. Each group includes 6 to 9 mice.