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. 2016 Oct;186(10):2679-91.
doi: 10.1016/j.ajpath.2016.06.014. Epub 2016 Aug 18.

SAG/Rbx2-Dependent Neddylation Regulates T-Cell Responses

Nathan D Mathewson  1 , Hideaki Fujiwara  2 , Shin-Rong Wu  1 , Tomomi Toubai  2 , Katherine Oravecz-Wilson  2 , Yaping Sun  2 , Corinne Rossi  2 , Cynthia Zajac  2 , Yi Sun  3 , Pavan Reddy  4
Affiliations

SAG/Rbx2-Dependent Neddylation Regulates T-Cell Responses

Nathan D Mathewson et al. Am J Pathol. 2016 Oct.

Abstract

Neddylation is a crucial post-translational modification that depends on the E3 cullin ring ligase (CRL). The E2-adapter component of the CRL, sensitive to apoptosis gene (SAG), is critical for the function of CRL-mediated ubiquitination; thus, the deletion of SAG regulates neddylation. We examined the role of SAG-dependent neddylation in T-cell-mediated immunity using multiple approaches: a novel T-cell-specific, SAG genetic knockout (KO) and chemical inhibition with small-molecule MLN4924. The KO animals were viable and showed phenotypically normal mature T-cell development. However, in vitro stimulation of KO T cells revealed significantly decreased activation, proliferation, and T-effector cytokine release, compared with WT. Using in vivo clinically relevant models of allogeneic bone marrow transplantation also demonstrated reduced proliferation and effector cytokine secretion associated with markedly reduced graft-versus-host disease. Similar in vitro and in vivo results were observed with the small-molecule inhibitor of neddylation, MLN4924. Mechanistic studies demonstrated that SAG-mediated effects in T cells were concomitant with an increase in suppressor of cytokine signaling, but not NF-κB translocation. Our studies suggest that SAG is a novel molecular target that regulates T-cell responses and that inhibiting neddylation with the clinically available small-molecule MLN4924 may represent a novel strategy to mitigate T-cell-mediated immunopathologies, such as graft-versus-host disease.

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Figures

Figure 1
Figure 1
Neddylation inhibition in T cells. Analysis by Western blot of Cullin-1 protein in CD90.2+ splenic T cells. Loss of higher-molecular-weight band of Cullin-1 by treatment with 100 nmol/L (A) and 500 nmol/L (B) MLN4924 indicates level of inhibition of nedd8 attachment to cullin protein. α-Tubulin protein levels were analyzed as an equal loading control. C: CD90.2+ T-cell viability by annexin V staining after incubation with MLN4924 at indicated concentrations for 72 hours. One representative experiment each of three is shown. ∗∗∗P < 0.001 versus 0 h.
Figure 2
Figure 2
T-cell functions are attenuated by neddylation blockade. A: BALB/c T cells were cultured with irradiated (30 Gy) C57BL/6 bone marrow–derived dendritic cells (BMDCs) at 40:1 and 100:1 ratios (T cell/BMDC) in the presence or absence of MLN4924 for 72 hours. Where indicated, T cells were preincubated with MLN4924 at indicated dosage for 6 hours before plating. Enzyme-linked immunosorbent assay quantification of IL-2 (B), interferon (IFN)-γ (C), IL-4 (D), and IL-17 (E) release from T cells, cultured in the presence or absence of MLN4924 with allogeneic BMDCs as indicated in A for 72 hours. One representative experiment of three is shown in cells treated in triplicate. Analysis of T-cell stimulation with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28 in the presence or absence of vehicle or 500 nmol/L MLN4924 by 3[H] thymidine incorporation for the last 6 hours of 72-hour culture (F) or carboxyfluorescein diacetate succinimyl ester (CFSE) dilution for 48 hours with annexin V viability staining (G). G and H: Inhibition of neddylation by MLN4924 significantly reduces the proliferation of T cells when treated at time of plating or by treatment 24 hours after stimulation. H: Quantification of representative T cells after 48 hours total stimulation between T cells treated with vehicle or MLN4924 24 hours after stimulation. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus syngeneic control; †P < 0.05 versus unstimulated; ‡P < 0.05, ‡‡‡P < 0.001 versus vehicle control. Allo UnRx, Allo-BMT untreated; CPM, count per minute; DMSO, dimethyl sulfoxide; Pre-Inc. TC, preincubated T-cells; Syn, syngeneic.
Figure 3
Figure 3
Conditional knockout of SAG protein in T cells reduces allogeneic (Allo) T-cell proliferation. A: Breeding scheme of C57BL/6 LCK-Cre mice with C57BL/6 SAGfl/fl mice. F1 heterozygous generations were subsequently bred producing animals with [knockout (KO)] and without [wild type (WT)] the T-cell–specific deficiency of SAG. Mice were continually bred and used on the basis of confirmed genotype of Rnf7 deficiency by quantitative PCR (B) or SAG protein deficiency shown by immunoblot detection in peripheral splenic CD90.2+ T cells and in developing T-cell populations in the thymus in WT or KO animals, as indicated (C); CD8 single positive (SP), CD4 SP, CD4/CD8 double positive (DP), and CD4/CD8 double negative (DN). WT or KO T cells co-cultured with irradiated (30 Gy) syngeneic (Syn; C57BL/6 H-2b) or allogeneic (BALB/c H-2d) splenocytes for 96 hours measured by 3[H] thymidine incorporation during the last 6 hours of culture (D) or carboxyfluorescein diacetate succinimyl ester (CFSE) dilution with annexin V viability staining (E). ∗P < 0.05, ∗∗∗P < 0.001. CPM, count per minute.
Figure 4
Figure 4
SOCS proteins are degraded less in T cells with dysfunctional neddylation pathway. Protein analysis by Western blot of p65 isoform of NF-κB protein using nuclear (N) and cytosolic (C) fractions from wild-type (WT) and knockout (KO) T cells (A) or WT T cells cultured in the presence or absence of vehicle or 500 nmol/L MLN4924 (B), subjected to or withheld from concurrent stimulation with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28. Plots below Western blot images show densitometric and statistical analysis of p65 NF-κB presence in the nuclear fraction and cytosolic fraction. Lamin A/C and lactate dehydrogenase proteins were analyzed to demonstrate the presence of nuclear and cytosolic fractions, respectively. One representative experiment of three is shown. C: Immunocytochemistry analysis of p65 NF-κB (column 3) localization in bone marrow–derived dendritic cells cultured in the presence or absence of MLN4924 and stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28. Cell nuclei were stained using DAPI (column 1). Cytoplasmic actin was stained with phalloidin (column 2). Merged images of staining (column 4). One representative experiment of three is shown. D: Gene expression in WT and KO T cells identified in Affymetrix microarray analysis. The data set and top 24 enriched genes shown were obtained from two biological replicates. E: SOCS1 and SOCS3 gene expression in CD90.2+ T cells, stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28 where indicated, and treated with 500 nmol/L MLN4924 or vehicle. Flow cytometric analysis of SOCS1 protein (F) and SOCS3 protein levels (G) in WT and KO C57BL/6 T cells stimulated with irradiated (30 Gy) allogeneic BALB/c splenocytes for indicated time. One representative experiment of two is shown in groups treated in triplicate. H: Western blot analysis of SOCS1 protein levels in WT T cells treated with MLN4924 500 nmol/L and stimulated with 5 μg/mL α-CD3 and 2.5 μg/mL α-CD28 for 72 hours. One representative experiment of three is shown. ∗P < 0.05, ∗∗∗P < 0.001. Original magninfication, ×60 (C). SLCK, SAGfl/fl × Lck-Crel; Stim, stimulated; Unstim, unstimulated.
Figure 5
Figure 5
SAG deficiency mitigates T-cell functions in vivo and reduces graft-versus-host disease (GVHD). Flow cytometric analysis of splenic donor (H-2b) wild-type (WT) and knockout (KO) T cells shown on day 7 (A) and day 14 (B) after C57BL/6 → BALB/c (H-2d) allogeneic transplant. Similar levels of CD4+ and CD8+ were observed at both time points with a significant decrease in activated (CD44hi) and significant increase in naïve (CD62L+ CD44−) T cells. C: Significantly fewer SAG-deficient (KO) CD4+ T cells produced interferon (IFN)-γ, IL-4, and IL-17 on day 14 after allogeneic bone marrow transplantation (BMT); determined by flow cytometry gating and the total number of splenocytes isolated on analysis. Recipients of allogeneic transplant that received KO donor T cells exhibit significantly less severe GVHD (D) and improved survival (E), compared to recipients of WT donor allogeneic (allo)-T cells. Treatment of allo-BMT recipients that received WT donor T cells with 20 mg/kg MLN4924 sub-q resulted in less severe GVHD (F) and significantly improved survival (G). Animals treated with MLN4924 were treated for 5 days starting day −1 through day +3, relative to transplant on day 0. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control. SLCK, SAGfl/fl × Lck-Cre.
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