Ndfip1 mediates peripheral tolerance to self and INAUGURAL ARTICLE exogenous by inducing cell cycle exit in + responding CD4 T cells

John A. Altina, Stephen R. Daleya, Jason Howittb, Helen J. Rickardsa, Alison K. Batkina, Keisuke Horikawaa, Simon J. Prasada, Keats A. Nelmsa, Sharad Kumarc, Lawren C. Wud, Seong-Seng Tanb, Matthew C. Cooka,1, and Christopher C. Goodnowa,1,2

aJohn Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia; bFlorey Neurosciences Institute, University of Melbourne, Parkville, VIC 3010, Australia; cCentre for Cancer Biology, University of South Australia, Adelaide, South Australia 5000, Australia; and dDepartment of Immunology, Genentech, South San Francisco, CA 94080

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2013.

Contributed by Christopher C. Goodnow, December 17, 2013 (sent for review October 13, 2013) The NDFIP1 (neural precursor cell expressed, developmentally down- (11), rheumatoid arthritis (12), and multiple sclerosis (13). It regulated 4 family-interacting protein 1) adapter for the remains unclear which cellular mechanisms of tolerance are ITCH is genetically linked to human allergic and auto- disrupted by these genetic variants to result in allergic and immune disease, but the cellular mechanism by which these . enable foreign and self- to be tolerated is unresolved. Here, Ndfip1 and Itch were first revealed as important immune we use two unique mouse strains—an Ndfip1-YFP reporter and an regulators in mouse genetic studies. Homozygous inactivating Ndfip1-deficient strain—to show that Ndfip1 is progressively in- mutations in the Itchy strain cause dermatitis, lung mononuclear duced during T-cell differentiation and activation in vivo and that inflammation, lymphadenopathy with follicular hyperplasia, in- – its deficiency causes a cell-autonomous, Forkhead box P3-indepen- creased activated T cells (notably IL-4 producing Th2 cells), + dent failure of peripheral CD4 T-cell tolerance to self and exogenous expansion of B1b cells in the peritoneal cavity, and early death IMMUNOLOGY + antigen. In small cohorts of antigen-specific CD4 cells responding (5, 6, 14, 15). Although the murine pathology has often been in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit described as autoimmune because of its spontaneous de- cell cycle after one to five divisions and to abort Th2 effector differ- velopment, there is currently little direct evidence of T-cell au- toimmunity, and the predominant inflammation of skin and entiation, defining a step in peripheral tolerance that provides mucosal surfaces suggests an exaggerated response to innocuous insights into the phenomenon of T-cell anergy in vivo and is distinct environmental antigens. Indeed, elegant studies showed that Itch from the better understood process of Bcl2-interacting mediator Ndfip1 deficiency prevents high-zone tolerance in an experimental of cell death-mediated . deficiency precipitated model of respiratory exposure to an egg protein allergen (16). autoimmune pancreatic destruction and diabetes; however, this An almost identical skin and lung inflammatory syndrome occurs depended on a further accumulation of nontolerant anti-self T cells in mice inheriting a homozygous -trap insertion that greatly from strong stimulation by exogenous tolerogen. These findings reduces Ndfip1 mRNA and protein (2). Although much progress illuminate a peripheral tolerance checkpoint that aborts T-cell has been made elucidating diverse biochemical functions of Itch clonal expansion against allergens and autoantigens and demon- strate how hypersensitive responses to environmental antigens may Significance trigger . Advances in organ transplantation and treatment of allergy immunological tolerance | allergy | T lymphocyte | Interleukin-4 | and autoimmune disease hinge upon harnessing a physiologi- Aire (Autoimmune Regulator) cal switch that allows T cells to decide between proliferating extensively or actively becoming tolerant. The experiments n healthy individuals, mature T cells in peripheral lymphoid presented here illuminate a critical element of this natural switch, Itissues proliferate and acquire effector functions in response to Ndfip1 (neural precursor cell expressed, developmentally down- antigens from pathogenic microbes but remain tolerant to self- regulated protein 4 family-interacting protein 1), a partner pro- antigens and innocuous environmental antigens. Defects in this tein of ubiquitin ligases induced during the first several divisions phenomenon of “peripheral T-cell tolerance” are thought to after T cells encounter antigen. They define the cellular action of contribute to the burden of autoimmune and allergic disease, but Ndfip1 in vivo, acting within dividing helper T cells that have there is only a fragmented understanding of its cellular basis, its responded to innocuous foreign or self-antigen that should connection to specific genetic circuits, and the interconnection normally be tolerated, to force their exit from cell cycle before between autoimmunity and hypersensitivity to exogenous anti- they have divided so many times that they acquire tissue-dam- gens (1). This problem is exemplified by the genetic circuit aging effector functions. encoding Ndfip1 [neural precursor cell expressed, developmentally down-regulated protein 4 (NEDD4) family-interacting protein 1], Author contributions: J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., S.J.P., K.A.N., L.C.W., S.-S.T., M.C.C., and C.C.G. designed research; J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., S.J.P., K.A.N., a transmembrane protein localized to the Golgi and intracellular and M.C.C. performed research; J.H., S.K., and S.-S.T. contributed new reagents/analytic vesicles that recruits and activates the HECT-type E3 ubiquitin tools; J.A.A., S.R.D., J.H., H.J.R., A.K.B., K.H., K.A.N., L.C.W., S.-S.T., M.C.C., and C.C.G. ligase Itch (2–7). Human genetic studies have associated NDFIP1 analyzed data; and J.A.A., S.R.D., M.C.C., and C.C.G. wrote the paper. and ITCH with allergic and autoimmune diseases. Inherited ITCH The authors declare no conflict of interest. deficiency results in asthma-like chronic lung disease with non- Freely available online through the PNAS open access option. fibrotic lymphocytic pneumonitis (90% cases) and organ-specific 1M.C.C. and C.C.G. contributed equally to this work. autoimmunity (60% cases) variably involving the thyroid, liver, 2To whom correspondence should be addressed. E-mail: [email protected]. intestine, or pancreatic islets (8). Inherited NDFIP1 polymorphisms This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are associated with inflammatory bowel disease (9, 10), asthma 1073/pnas.1322739111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1322739111 PNAS | February 11, 2014 | vol. 111 | no. 6 | 2067–2074 Downloaded by guest on October 2, 2021 and Ndfip1 in many cell types (3, 17), the cellular basis for im- A BC mune dysregulation in their absence is unresolved, and their role C -58 nt in T-cell tolerance to self-antigens has yet to be examined. Exon 4 Exon 5 Exon 6 KO mutant Defects in several different cellular mechanisms for peripheral Wt: GTATG…….GG gt wild-type Mut: GTATG…….GG at -125 nt T-cell tolerance have been implicated in the inflammatory dis- -58 nt PY? ease caused by defects in the Itch-Ndfip1 genetic circuit. T-cell -125 nt PY2 Ndfip1 anergy is a mechanism defined initially in tissue culture that PY1 prevents initiation of T-cell proliferation when T cells are stim- D N ulated without a CD28 costimulus (18). Itch was required for 100 100 T-cell anergy in cultured cells rendered anergic by prolonged in 80 80 60 60 vitro treatment with ionomycin or harvested from TCR trans- 40 40 tubulin genic (Tg) mice 10 d after exposure to a high-tolerogenic dose of 20 20 foreign antigen. An intact Itch gene was correlated with di- 0 % surviving 0

% dermatitis-free 0 100 200 0 100 200 minished TCR signaling and proteolytic degradation of protein age (days) age (days) θ γ kru/kru +/+ , +/– kinase C (PKC)- , phospholipase C (PLC)- , JunB, and c-Jun Ndfip1(n=13)(n=13) Rag (n=13) kru/kru –/– proteins (16, 19). A role for Itch in nondegradative ubiquitina- Ndfip1((n=27)n=27) Rag (n=27) tion of the TCR CD3 ζ subunit to inhibit its phosphorylation and the activation of Zap-70 has also been shown (20). Likewise, Fig. 1. Immune-mediated lethal inflammatory syndrome in mice with Ndfip1 deficiency causes JunB accumulation (2) and allows T a truncating Ndfip1 splice site mutation. (A) Schematic showing the location of the Ndfip1kru mutation within the Ndfip1 exon 5 splice donor sequence cells to make IL-2 for a sustained period in vitro without the and the resulting two aberrant splice products. (B) Schematic showing the need for CD28 costimulation (21). Itch-deficient T cells also κ α topology of the Ndfip1 protein and position of the truncating mutations. (C) display exaggerated NF- B signaling in response to TNF- , be- Western blot of primary T-cell lysates from wild-type, Ndfip1kru/kru (mutant), − − cause Itch forms a complex that recruits Tnfaip3 to terminate and Ndfip1 / (KO) mice, probed with an antibody raised to a conserved N- NF-κB signaling (22). Itch has also been implicated in ubiquiti- terminal peptide of Ndfip1, and then stripped and reprobed with antibody nation and degradation of Bcl-10, an adaptor protein with an to tubulin to assess loading. (D) Dermatitis and survival of Ndfip1kru/kru mice essential role in TCR and CD28 signaling to NF-κB (23). TCR- with normal or null Rag1 , aged to 250 d or until moribund necessi- activated T cells that lack Itch or Ndfip1 form a diminished tating the animal to be killed. + percentage of Forkhead box P3 (Foxp3 ) induced T-regulatory cells (iTregs) when cultured with TGF (24, 25). Increased differentiation of Th2 effector cells is a prominent revealed a G-to-A substitution in the first nucleotide of the intron feature of Itch or Ndfip1 deficiency that is partly explained by following exon 5, disrupting the mRNA splice donor sequence ” their role in ubiquitination and degradation of JunB, an Il4 gene (Fig. 1A, allele denoted “Ndfip1kru ). Genotyping for this muta- factor preferentially expressed in Th2 cells (2, 14, tion showed perfect concordance between Ndfip1kru homozygos- 26). However, Tg mice that express equivalently high levels of ity and the lethal dermatitis phenotype in >100 affected offspring JunB in T cells do not exhibit a similar spontaneous accumula- (>800 meioses) generated by successively backcrossing to wild- tion of Th2 effector cells or inflammatory disease (26, 27). Ab- type C57BL/6 mice and intercrossing heterozygous progeny. normal Notch signaling within activated T cells might also Amplification and sequencing of cDNA from Ndfip1wt/wt ver- contribute (28–30) because the Itch ortholog in Drosophila, sus Ndfip1kru/kru splenocytes revealed the mutation abolished Suppressor of Deltex, was discovered as a negative regulator of normal splicing and resulted in two aberrantly spliced mRNA Notch signaling, and the Drosophila Ndfip1 ortholog has similar products, encoding frame shifts and premature stop codons genetic effects on this pathway (31, 32). Itch ubiquitinates and truncating the Ndfip1 protein either immediately preceding or terminates ligand-independent Notch signaling in endosomes, following the first of the three transmembrane domains (Fig. 1 A requiring an adaptor protein that may be Ndfip1 based on and B and Fig. S2 A and B). In transfected HEK293 cells, Ndfip1 Drosophila studies (31, 33, 34). protein was detected from only the aberrantly spliced cDNA Although there are many possible cellular explanations for retaining the first transmembrane sequence and from the wild- how allergy and autoimmunity may result from defects in the type cDNA (Fig. S2C). Western blotting of activated T-cell Itch-Ndfip1 genetic circuit, resolving these alternatives to place lysates from Ndfip1kru/kru mice with antibody to the Ndfip1 N the biochemical circuit in its correct cellular context awaits terminus (35) confirmed the absence of the ∼27-kDa wild-type a systematic comparison of the fates of mutant and wild-type T Ndfip1 protein and only trace amounts of ∼20-kDa protein cells responding to normally tolerogenic antigens in vivo. Here, corresponding to the aberrantly spliced mRNA retaining the first we perform this comparison and reveal that the critical function transmembrane sequence (Fig. 1C). This truncated residual for Ndfip1 in peripheral tolerance is as an induced, cell-auton- + protein included the N-terminal cytoplasmic PY motifs that bind omous brake against effector CD4 cell differentiation. and activate Itch and Nedd4 (3) but was evidently insufficient to suppress lethal inflammation. Results Because Ndfip1 is expressed widely and has been implicated Lethal Immune-Mediated Th2 Disease Caused by a Truncating in functions outside of the adaptive (36), we Mutation of Ndfip1. In a C57BL/6 × C57BL/10 mouse pedigree tested whether or not the dermatitis or premature lethality was suppressed by the selective elimination of T and B cells in segregating point mutations induced by ethylnitrosourea, off- − − spring exhibited a Mendelian recessive syndrome of spontaneous Rag1 / Ndfip1kru/kru mice. In contrast to lymphocyte-sufficient Ndfip1kru/kru littermate controls, dermatitis and mortality did not mast cell-rich dermatitis with median onset age 90 d, followed − − by weight loss and premature mortality at a median age of 160 d develop in Rag1 / Ndfip1kru/kru mice (Fig. 1D), demonstrating (Fig. 1 and Fig. S1 A and B). This was accompanied by lymph- that the spontaneous pathology absolutely requires T and/or adenopathy, splenomegaly, increased CD86 and CD23 activation B lymphocytes. markers on B cells, expansion of the activated/memory subset of + + + CD4 and CD8 T cells, greatly elevated serum IgE, and for- Ndfip1 Deficiency Causes Cell Autonomous Accumulation of CD4 + + + + mation of a prominent population of IL-4 and IFN-γ CD4 Effectors Not Corrected by Normal Foxp3 Cells. To determine cells (Fig. S1 C–E). The mutation was given the allele name which subsets of T or B cells are regulated by Ndfip1, 50:50 mixed “krusty” and mapped in a genome-wide scan of C57BL/6 × hematopoietic chimeras were constructed by transplanting bone C57BL/10 SNPs to a 18 region containing a strong marrow from Ndfip1kru/kru or wild-type donors, distinguished by − − candidate gene, Ndfip1, based on a similar phenotype in a strain CD45.1 or CD45.2 allotypic markers, into irradiated Rag1 / bearing a gene-trap insertion (2). Sequencing the Ndfip1 exons recipients. As observed in unmanipulated Ndfip1kru/kru mice, B cells

2068 | www.pnas.org/cgi/doi/10.1073/pnas.1322739111 Altin et al. Downloaded by guest on October 2, 2021 + + and CD8 T cells were both dramatically altered in chimeras CD4 and CD8 T cells was comparable to their thymic SP coun- kru/kru

reconstituted with only 50% Ndfip1 hematopoietic cells (Fig. terparts, whereas lower heterogeneous levels were observed in B INAUGURAL ARTICLE + – S3 A–C), but these effects were an indirect consequence of cells (Fig. 3B). Within splenic CD4 cells, CD44hi CD25 ef- + Ndfip1kru/kru in other lymphocytes because they occurred to an equal fector/memory and CD44int CD25 Treg fractions had ∼fivefold – + extent in the CD45.1 B and CD8 T cells bearing wild-type Ndfip1 increased YFP above CD44lo CD25 naïve CD4 T cells. These developing alongside Ndfip1kru/kru lymphocytes. By contrast, there results, measured at single-cell resolution, mirror the pattern of + was a direct, cell-autonomous requirement for Ndfip1 in CD4 T Ndfip1 mRNA expression observed in sorted T-cell subsets in the cells (Fig. 2). Ndfip1 deficiency resulted in greatly increased Immgen database (38). When splenocytes from Ndfip1-YFP Tg + + + frequencies of CD44hi IFN-γ and IL-4 cells among CD4 cells animals were cultured with plate-bound antibodies against CD3 in chimeras reconstituted with 100% Ndfip1kru/kru marrow. In mixed and CD28, YFP increased ∼30-fold above the level in resting + + chimeras bearing 50% Ndfip1kru/kru marrow, expansion of IL-4 CD4 T cells over the course of 2 d, with ∼2-fold induction by + + cells, CD44hi cells, and IFN-γ CD4 cells was limited to the 6hand∼10-fold induction by 24 h (Fig. 3C). Thus, Ndfip1 is + CD45.2 T cells lacking normal Ndfip1 and did not occur in the progressively induced by antigen- signaling as T cells + CD45.1 wild-type CD4+ cells that developed in the same ani- matureandthenbecomeactivated. + + mals. Analysis of Foxp3 CD4 cells in the mixed chimeras + showed that Ndfip1kru/kru neither increased nor decreased the Ndfip1 Aborts Antigen-Induced Division and Differentiation of CD4 + frequency of Foxp3 cells even under competitive reconstitution T Cells After Several Days. The findings above indicated that Ndfip1 + conditions where wild-type marrow was also present (Fig. 2). may regulate either the initial activation of naive CD4 cells by + Despite the presence of large numbers of wild-type Foxp3 cells innocuous foreign- or self-antigens or their subsequent clonal in these 50:50 mixed chimeras, accumulation of mutant CD44hi accumulation as effector cells. To resolve these alternatives, the + Th2 and Th1 CD4 effector cells was not corrected. Ndfip1kru mutation was crossed into the 3A9 TCR Tg strain (37, + 39), providing a source of naïve CD4 T cells recognizing hen Ndfip1-YFP Is Progressively Induced During T-Cell Maturation and egg lysozyme (HEL) peptide presented by the MHC class II + Activation. To understand the cell-autonomous requirement for protein I-Ak. Equal numbers of naïve HEL-specific CD4 T cells + Ndfip1 in preventing pathological accumulation of effector CD4 from wild-type 3A9 Tg donors (allotypically marked as hetero- T cells, Ndfip1-YFP Tg reporter mice were constructed by pro- zygous CD451/2) and Ndfip1kru/kru 3A9 Tg donors (allotypically nuclear injection of a mouse bacterial artificial chromosome marked as homozygous CD452/2) were mixed, labeled with car- (BAC) containing the Ndfip1 genomic with an eYFP cDNA boxyfluorescein diacetate succinimidyl ester (CFSE), and trans- 1/1 inserted at the Ndfip1 translation start site (Ndfip1-YFP Tg ferred into normal congenic B10.BR CD45 recipients. In these IMMUNOLOGY + + mice). In the , YFP fluorescence of CD4 CD8 double- experiments, normal Ndfip1 was present in all cells other than + positive (DP) cells was only marginally higher than control thy- the small subset of transferred CD452/2 CD4 TCR Tg cells. To mocytes from non-Tg mice, but fluorescence was ∼100-fold analyze the response to innocuous foreign antigen, the recipient + – – + higher in CD4 CD8 and CD4 CD8 single-positive (SP) cells, mice were injected i.p. with 100 μg of HEL protein in saline corresponding to the stage of positive selection by TCR-pMHC without adjuvant, and clonal expansion of the transferred T cells ligation (Fig. 3A). A further ∼fivefold higher YFP fluorescence monitored 3 or 6 d later using antibodies against the CD45 + + + was observed in the CD25 GITR subset of CD4 SP cells, allotypes and by CFSE fluorescence (Fig. 4A). After 3 d, donor T which comprise cells receiving a stronger TCR signal, including cells in control recipients that did not receive HEL persisted in + nascent Foxp3 regulatory T cells (Tregs) and cells undergoing equal frequencies and had not diluted CFSE, indicating that negative selection (37). In the spleen, YFP fluorescence in mature absence of Ndfip1 did not result in spontaneous activation of

Chimeras: 100% wt 50% kru 100% kru 100% 50% 100% wt wt kru CD45.1 cells: wt wt wt none wt wt wt CD45.2 cells: wt kru kru kru wt kru kru

Gated on: CD45.2 CD45.1 CD45.2 CD45.2

CD4+ cells CD4+++ cells CD4 cells CD4 cells

CD45.1

CD45.2

) CD45.1

CD45.2 CD45.2 100 100 100 100 + *** 80 80 80 80 5.25 5.42 25.1 28.3 hi 50 * 60 60 60 60 40 CD4

– 30 40 40 40 40 20 Fig. 2. Ndfip1 deficiency causes cell-autonomous

% of Max + 20 20 20 20 10 accumulation of activated/effector CD4 T cells that 0 0 0 0 % CD44 0 + 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 1 2 1 2 5. 5. 5. 5. is not corrected by the presence of wild-type Foxp3

(Foxp3 *** 105 105 105 105

) Tregs. Three groups of mixed bone marrow chi-

+ * 4 1.83 4 3.09 4 5.76 4 17.5 20 10 10 10 10 meras were generated by transplanting irradiated + 15 −/− 103 103 103 103 Rag1 mice: “100% kru,” transplanted with 100% CD4

IFNg 10 f kru/kru “ ” 102 102 102 102 Ndfip1 CD45.2 bone marrow; 50% kru, IFNg 5 0 0 0 0 transplanted with a 50:50 mixture of Ndfip1kru/kru %o 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 ( +/+ 105 105 105 105 CD45.2 bone marrow and Ndfip1 CD45.1 bone )

+ *** “ ” 4 4 4 4 marrow; 100% wt, transplanted with a 50:50 10 0.26 10 0.037 10 7.71 10 13.4 20 +/+ + 15 ** mixture of Ndfip1 CD45.2 bone marrow and 103 103 103 103 + +

-4 / IL-4 10 Ndfip1 CD45.1 bone marrow. Flow cytometry was 102 102 102 102 IL + 0 0 0 0 5 used to analyze lymph node CD4 cells, gated into + + 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 (% of0 CD4 1 either CD45.2 or CD45.1 cells, measuring cell sur- 105 105 105 105 ns

6.44 6.48 9.23 5.12 ) face expression of CD44, intracellular IFN-γ or IL-4

+ ns 104 104 104 104 25 + 20 after 3 h ex vivo phorbol myristate acetate (PMA)/ 103 103 103 103 15 ionomycin stimulation, and intracellular Foxp3. Rep-

Foxp3 2 2 2 2 10 10 10 10 10 resentative plots and quantitation in multiple animals 0 0 0 0 Foxp3 5 are shown. Lines link data points from the same re- 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 0 2 2 2 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 (% of CD4 cipient animal. *P < 0.05; **P < 0.01; ***P < 0.001 by CD44 Student t test.

Altin et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2069 Downloaded by guest on October 2, 2021 cellular flow cytometric staining at this time point revealed more ACthymocytes kru/kru +

100 JunB in Ndfip1 CD4 Tg cells than in coresident wild-type 100 105 5.69 86.5 6 hr kru/kru 80 cells and that most Ndfip1 cells produced high levels of IL-4 80 60 104 upon brief restimulation ex vivo, whereas few of the wild-type Tg 40 60 –

% of Max cells did (Fig. 4 C E). 20 103 CD4 40 0 The continued expansion and effector differentiation of 0102 103 104 105 + % of Max kru/kru 102 Ndfip1 20 CD4 T cells observed at day 6 of the response to 0.74 100 0 24 hr exogenous HEL “tolerogen” was not attributable to a deficiency 80 0 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 60 in transacting suppression by Tregs, because it occurred cell- CD8 Ndfip1-YFP 40 autonomously in recipient mice that had a normal T-cell reper- CD4+CD8– cells % of 20 Max toire. It was nevertheless possible that differences in Foxp3 0 + 100 0102 103 104 105 105 induction within the transferred mutant and wild-type CD4 T 100 80 48 hr cells might explain their divergent behavior. To test this possi- 104 80 60 bility, we repeated the cotransfer experiments with a parallel 3.2 60 2/2 103 40 group of recipients that received a mixture of CD45 Foxp3- GITR 40 1/1 % of Max % of Max 20 deficient 3A9 TCR Tg CD4 cells and CD45 wild-type Tg cells 102 20 0 2 3 4 5 0 010 10 10 10 (Fig. 5). Six days after transfer and administration of HEL in 91.2 Ndfip1-YFP kru/kru + 0 0102 103 104 105 saline, Ndfip1 CD4 T cells had expanded dramatically as 0102 103 104 105 unstimulated + CD25 Ndfip1-YFP before, but the expansion of Foxp3-deficient CD4 T cells was stimulated + indistinguishable from their cotransferred wild-type CD4 cells (Fig. 5A). Hence, Foxp3 is not required within tolerogen- + B splenocytes B220– cells responding CD4 T cells to curtail their proliferative response in 100 105 105 37.2 vivo, whereas Ndfip1 is essential. 80

104 104 Mitochondrial apoptosis regulated by the Bcl-2 and Bcl2- 60 interacting mediator of cell death (Bim) proteins is a well-

103 103

38.1 CD4 40 established pathway contributing to peripheral T-cell tolerance CD11b 50.4 % of Max 2 2 25.9 10 10 20

0 0

0 2 3 4 5 0102 103 104 105 0102 103 104 105 010 10 10 10 Ndfip1-YFP wt CD45 1/2 kru CD452/2 B220 CD8 AB10 + CD4 cells –HEL day 3 +HEL day 3 +HEL day 6 8

5 5 5 100 10 99.4 10 10 105 99 95 90.490 6 9.64 0.230.2 1.5 0.320.3 104 104 104 4 80 104 103 103 103 2 60 9.09.066 0.290.29 2.75 donor cells (%) 0 CD45.1 102 2 102 103 10 day: 3 3 6 0 0 0 CD25 40 HEL: – 2 3 4 5 2 3 4 5 2 3 4 5 + +

% of Max 010 10 10 10 010 10 10 10 010 10 10 10 102 77.5 9.31 20 CD45.2CD4 2 0 100 100 100 C 80 0 80 80 2 3 4 5 80 010 10 10 10 0102 103 104 105 60 CD44 Ndfip1-YFP 60 60 60

40 40 40 40

20 20 20 20 Fig. 3. Progressive induction of an Ndfip1 reporter gene during T-cell se- donor cells (%)

0 + 0 0 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0 lection and activation. Flow cytometric analysis of Tg Ndfip1 reporter 010 10 10 10 day: 3 3 6 CFSE IL-4 (Ndfip1-YFP Tg) mice bearing a BAC with eYFP integrated in place of the HEL: – + + + + + Ndfip1 translation initiation codon. (A) YFP fluorescence of CD4 CD8 , CD4 – – + + – – – + – + + CD8 ,CD4CD8 ,CD4 CD8 CD25 GITR ,andCD4 CD8 CD25 GITR thymo- 12/ 2/2 cyte populations from Ndfip1-YFP Tg mice, gated as indicated. (B)YFP D CD45 CD45 E 100 5 + + + + lo – + hi – 105 5.8 0 10 13 0 8000 fluorescence of CD4 , CD8 , B220 , CD4 CD44 CD25 , CD4 CD44 CD25 , 80 4 4 + int + 10 10 1/2 60 and CD4 CD44 CD25 splenocyte populations from Ndfip1-YFP Tg mice, 3 103 wt CD45 10 + 40 6000 2/2 2 102 gated as indicated. (C) YFP fluorescence of gated CD4 cells among sple- wt CD45 20 10 0 0 87 0.35 0 94 0 2 3 4 5 2 3 4 5 nocytes from Ndfip1-YFP Tg mice that were incubated with or without plate- 010 10 10 10 0102 103 104 105 010 10 10 10 4000 100 105 4 0.2 105 1.0 0.29 bound antibodies against CD3 and CD28 for the indicated periods. In all 80 4 10 104 JunB MFI g 60 IFNg 2000 1/2 3 3 cases, the shaded gray histogram shows the YFP fluorescence of cells from wt CD45 10 10 40

2 2/2 10 102 non-Tg control mice. kru CD45 20 0 93 2.9 0 28 70 0 0 2 3 4 5 2 3 4 5 0102 103 104 105 010 10 10 10 010 10 10 10 wt + wt wt + kru JunB IL-4 + + naïve CD4 T cells. Three days after HEL injection, however, Fig. 4. Ndfip1 is required in activated CD4 T cells to prevent effector ac- both mutant and wild-type donor T cells had increased in fre- cumulation and differentiation in response to exogenous antigen without + quency 8- to 10-fold and had undergone multiple rounds of cell adjuvant. (A) An equal mixture of HEL-specific CD4 T cells was prepared division, reported by CFSE dilution (Fig. 4 A and B). At this from 3A9 TCRHEL Tg mice that were Ndfip1kru/kru CD452/2 or wild-type CD451/2, initial stage of the response, there was little difference in the labeled with CFSE, and 2 × 106 total CD4+ cells injected into the circulation of + proliferation of mutant and wild-type TCR Tg CD4 cells, al- normal B10.BR CD451/1 recipient mice. A subset of the recipient mice was though the Ndfip1kru/kru T cells were ∼1.5-fold as frequent. given 100 μg of HEL antigen in saline i.p. at the time of cell transfer. Shown is A remarkable divergence between wild-type and Ndfip1kru/kru representative flow cytometric analysis of recipient spleen cells on the in- + + CD4 T cells developed by day 6 after exposure to HEL antigen dicated day after T-cell transfer. Upper graphs are gated on CD4 T cells and in saline, when the frequency of wild-type Tg donor cells had show the percentage derived from the two donors and the recipient. Lower decreased ∼fivefold compared with their peak at day 3, and the graphs show CFSE fluorescence of the respective donor and recipient cells. (B) Percentage of donor cells in multiple recipients analyzed as in A. Lines remaining cells retained appreciable CFSE (Fig. 4 A and B). In + link data points from the same recipient animal. (C) Percentage of donor T contrast, the Ndfip1kru/kru CD4 T cells had continued to increase ∼ cells expressing IL-4 measured by intracellular staining and gated as in A in frequency a further fourfold from day 3 and had diluted their after 3 h ex vivo PMA/ionomycin stimulation. (D) Intracellular staining for + CFSE label beyond the limit of detection. Consequently, at day JunB, IL-4, and IFN-γ on gated CD451/2 or CD452/2 donor CD4 cells at day 6 kru/kru + 6, the Ndfip1 CD4 progeny were ∼30- to 50-fold more after transfer and injection of HEL in saline. (E) Quantitation of the JunB MFI abundant than their cotransferred wild-type counterparts. Intra- of gated donor cells in multiple recipients analyzed as in D.

2070 | www.pnas.org/cgi/doi/10.1073/pnas.1322739111 Altin et al. Downloaded by guest on October 2, 2021 − − Aire / B10.BR mice with Ndfip1kru/kru bone marrow. Whereas ) AC1000 + 100 wt Aire-deficient recipients of wild-type marrow showed no signs INAUGURAL ARTICLE 80 100 ko of disease or histological evidence of pancreatitis, recipients of

+ 60 kru/kru – 10 Ndfip1 marrow lost weight and became moribund 30 40 d Ki-67 40 after transplant with extensive lymphocytic infiltration and auto- 1 fold expansion fold 20 immune destruction of the exocrine pancreatic cells (Fig. S4). This 0.1 0 (% among divided among CD4(% genetic cooperation mirrored that observed between Aire and

(relative to co-transferred wt) co-transferred to (relative ko ko ko ko kru kru bim bim Cblb, the latter encoding another ubiquitin ligase with a role in foxp3 peripheral tolerance (41). B –HEL +HEL To analyze peripheral tolerance to a defined self-antigen made kru kru Foxp3ko Bimko Ndfip1kru HEL CD452/2 CD452/2 CD452/2 CD452/2 in the pancreas, we crossed 3A9 TCR Tg mice with mice expressing HEL protein controlled by the rat gene promoter (insHEL Tg mice). Despite greatly increased thymic + production of CD4 T cells specific for the pancreatic islets, the majority of TCRHEL:insHEL double Tg mice with one or two wild-type Ndfip1 alleles were protected from diabetes (Fig. 6A).

Ki-67 This protection has previously been shown to be attributable to Aire-dependent thymic deletion (39) and Cblb-dependent pe- wt ripheral tolerance (43). By contrast, 100% of Ndfip1kru/kru double CD451/1 Tg animals developed diabetes by 60 d of age (Fig. 6A). Analysis of the thymi nevertheless showed no effect of the Ndfip1kru/kru CTV + mutation upon thymic deletion of 3A9 TCRHEL CD4 cells in Fig. 5. Ndfip1 mediates tolerance distinct from mitochondrial apoptosis or + insHEL Tg animals. Thus, the enhanced progression to diabetes Foxp3, by aborting the proliferative response of CD4 T cells. Cotransfer of caused by Ndfip1 deficiency likely reflected the fact that some HEL + allotypically distinguished 3A9 TCR Tg cells was performed as in Fig. 4 but TCRHEL CD4 cells escape thymic deletion and must be con- including parallel groups of recipients that received cell mixtures from wild- trolled by Ndfip1-mediated peripheral tolerance. type and Foxp3-deficient, or wild-type and Bim-deficient mice (all mice 3A9 The role of Ndfip1 in peripheral tolerance to pancreatic islets TCRHEL Tg), and using the cell division label CTV. (A) Quantitation of the fold- + kru/kru was pinpointed by transferring mature naïve CD4 T cells from expansion (relative to cotransferred wild-type cells) of wild-type, Ndfip1 , HEL kru/kru Foxp3-deficient or Bim-deficient CD4+ cells in recipient mice at day 6 fol- 3A9 TCR Tg wild-type or Ndfip1 mice into wild-type IMMUNOLOGY lowing injection of HEL in saline, compared with the cell inputs at day 0. (B) insHEL Tg recipients. This experimental design restricted Ndfip1 Representative Ki-67 and CTV data from CD451/2 recipients on day 6, gated deficiency to the small population of transferred cells, which on the cotransferred CD452/2 (red plots) or CD451/1 (blue plots) 3A9 TCRHEL were admixed into a normal, diverse repertoire of T cells in- + kru/kru + CD4+ T cells of the indicated genotypes. (C) Percentage of Ki-67 cells remaining cluding Tregs. Neither the wild-type nor the Ndfip1 CD4 T in cell cycle among donor CD4+ cells that had previously divided (CTVlo)in cells precipitated diabetes in insHEL Tg recipients unless 100 μg response to 100ug HEL in saline, measured in individual recipients as in B. of HEL in saline was also given at the time of T-cell transfer (Fig. 6B). Within 8 d, all of the mice that had received Ndfip1kru/kru T cells and exogenous HEL became diabetic, whereas none of the (1, 40). To test whether a defect in this pathway could replicate mice that received wild-type T cells and exogenous HEL became the failure of Ndfip1kru/kru cells to abort clonal expansion, we diabetic. Diabetes in exogenous HEL-exposed mice receiving included a parallel group of recipients that received a mixture of Ndfip1kru/kru T cells was accompanied by severe lymphocytic in- CD452/2 Bim-deficient 3A9 TCR Tg cells and CD451/1 wild-type filtration and destruction of most pancreatic islets (Fig. 6C). 3A9 Tg cells (Fig. 5). In contrast to the >50-fold greater ex- To test whether Ndfip1 deficiency affected the peripheral re- pansion of Ndfip1kru/kru cells, Bim-deficient cells exposed to sponse to islet-derived self-antigen alone, we tracked congeni- exogenous HEL in saline accumulated an average ∼eightfold cally marked mixtures of Ndfip1kru/kru and wild-type 3A9 higher than the wild-type internal controls (Fig. 5A). TCRHEL Tg T cells in insHEL Tg recipients. In insHEL Tg To compare how defects in Ndfip1 or Bim affected the pro- recipients not given exogenous HEL, a subset of Ndfip1kru/kru and + liferative state of the responding cells, we labeled the transferred wild-type donor CD4 T cells were stimulated to divide in re- T cells with cell trace violet (CTV) and measured division-linked sponse to low levels of self-HEL, but only the Ndfip1kru/kru di- CTV dilution together with expression of the cell cycle marker vided extensively based on analysis of CTV dilution on day 6, + + Ki-67 at day 6 of the response (Fig. 5 B and C). Analysis of CTV remained Ki-67 , and had differentiated into GATA-3 effector dilution indicated that, like their wild-type counterparts and also + cells (Fig. 6D). In control recipients that lacked the insHEL the Foxp3-deficient cells, most of the Bim-deficient CD4 cells transgene, neither mutant nor wild-type T cells were induced to + had responded to exogenous HEL tolerogen by dividing one divide or become GATA-3 . Thus, Ndfip1 is critical to abort + to five times. However, the resulting CTVlow progeny expressed CD4 T-cell proliferation and differentiation induced by pancreas- little Ki-67 on day 6, indicating that they had exited cell cycle by derived self-antigen. this time point. By contrast, the transferred Ndfip1kru/kru cells Although responding Ndfip1kru/kru T cells remained in cell + remained predominantly Ki-67 , revealing a failure to exit cell cycle after stimulation by self-antigen alone, their net expansion cycle (Fig. 5 B and C). These results establish that peripheral was only ∼threefold more than the cotransferred wild-type tolerance to innocuous exogenous antigen involves two geneti- counterparts (Fig. 6E). In insHEL Tg recipients that also re- + cally separate cellular mechanisms within responding CD4 T ceived 100 μg of exogenous HEL in saline, however, the cells: (i) Bim-dependent apoptosis; and (ii) Ndfip1-dependent Ndfip1kru/kru T cells accumulated to >10-fold higher frequencies exit from cell cycle that occurs even when apoptosis is defective. compared either to cotransferred wild-type T cells or to mutant T cells encountering self-antigen alone (Fig. 6 D and E). Because + Ndfip1 Prevents Autoimmune Disease by Aborting CD4 Cell limited amounts of HEL are made and presented to 3A9 TCR + Proliferation and Differentiation. Peripheral tolerance mechanisms Tg CD4 cells in the pancreas-draining nodes of insHEL Tg become critical backups when thymic tolerance fails (41); for recipients (44), we hypothesized that the amount of tolerogen example, because of inherited Aire mutations that disrupt thy- presented (from either self or foreign source) also governed the mic deletion of organ-specific T cells (39, 42). In B10.BR mice net accumulation of Ndfip1kru/kru T cells. We tested this hy- with intact peripheral tolerance mechanisms, Aire deficiency pothesis by including a recipient group that lacked the insHEL caused little clinical autoimmune disease on its own. We therefore transgene and was instead given a 1,000-fold lower dose of combined Aire and Ndfip1 deficiency by transplanting wild-type or exogenous HEL (0.1 μg) in saline (Fig. 6 D and E). Like the cells

Altin et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2071 Downloaded by guest on October 2, 2021 Ndfip1+/+ (n=14) insulitis: none Fig. 6. Ndfip1 deficiency disrupts peripheral T-cell toler- ABNdfip1kru/+ (n=20) T cells: wt wt kru kru Cmoderate ance to pancreatic islets. (A) Incidence of diabetes in a co- Ndfip1kru/kru(n=5) HEL PBS: - + - + 30 severe hort of mice with the indicated Ndfip1 genotypes that 100 HEL 100 25 were all doubly Tg for the 3A9 TCR transgene and an 80 80 20 insulin-promoter HEL transgene (insHEL Tg). (B) Daily blood diabetic 60 15 60 glucose readings in singly insHEL Tg mice injected i.v. with 6 + HEL 40 10 40 10 CD4 T cells from 3A9 TCR Tg donors that were ei- kru/kru 20 5 20 ther Ndfip1 or wild-type. Half of the recipients also % non-diabetic % μ

blood glucose (mM) received 100 g of HEL in saline i.p. at the time of T-cell 0 0 % pancreatic islets 0 01002003000 5 10 15 20 transfer. (C) Quantitation of insulitis in ≥10 individual islets age (days) days post cell transfer T cells: wild-type Ndfip1kru in individual recipients (denoted by columns) 3 wk after transfer of wild-type or Ndfip1kru/kru T cells together with 100ug HEL + DE–HEL insHEL + insHEL 0.1ug HEL HEL in saline. (D) A mixture of HEL-specific CD4 Tcells HEL kru/kru 2/2 100 from 3A9 TCR Tg mice that were Ndfip1 CD45 or wild-type CD451/2 was CTV-labeled and injected into the 1/1 10 circulation of non-Tg or insHEL Tg B10.BR CD45 recipient μ 10.9 77.6 99.5 28.3 mice. One group of insHEL Tg recipients was given 100 g 1 of HEL in saline i.p. at the time of cell transfer, whereas another group of non-Tg recipients received 0.1 μgofHEL

Ki-67 + donor cells 0.1 in saline i.p. The donor CD4 cellswereanalyzed6dlater (%, normalised) (%, in the spleen for CTV dilution and expression of Ki-67 and 7.26 15.9 89.7 8.24 0.01 GATA-3. Shown are representative plots of CTV fluores- CTV insHEL: ++ cence versus Ki-67 staining (upper plots) or GATA-3 stain- ing (lower plots) on the cotransferred kru CD452/2 CD4+ gHEL PBS: 100 0.1 cells (red) and wt CD451/2 CD4+ cells (blue). (E)Frequency kru Cd45 2/2 among CD4+ cells of wild-type and Ndfip1kru/kru donor cells wt Cd451/1 in individual recipient mice described in D, normalized to GATA-3 frequencies in a non-Tg recipient that received no HEL. Lines link data points from the same recipient animal.

exposed to tolerogenic HEL in other formats (insHEL and/or inflammatory disorder affecting particularly the skin, gastroin- + 100 μg of exogenous HEL), the Ndfip1kru/kru donor CD4 cells in testinal tract, and lungs, which could be recapitulated by selective recipients given the low dose of exogenous HEL differentiated Ndfip1 deficiency in the T-cell lineage and suppressed by skew- + + into GATA-3 effectors and remained Ki-67 , but their accu- ing the TCR repertoire to an innocuous ovalbumin OT-II TCR mulation was ∼10-fold less than in animals given the high dose. specificity (2, 21, 24, 25). In the earlier studies, mixed bone marrow chimera experiments found a higher fraction of Ndfip1- + + Discussion deficient CD4 and CD8 T cells were activated, but analysis was The experiments above illuminate cellular mechanisms for pe- limited because the mutant animals were on a heterogeneous ripheral T-cell tolerance, which remain to be harnessed for al- strain background derived variably from 129 and B6 strains, so lergy desensitization, transplantation tolerance, or autoimmune that complicated cell sorting and permeabilization was needed to disease therapy. As reviewed in ref. 1, many in vivo studies have distinguish mutant cells from the homogeneous B6-strain con- revealed that peripheral T-cell tolerance occurs by a burst of trols. The findings here extend those mixed chimera results using tolerogen-induced proliferation followed by Bim-mediated apo- matched congenic B6.CD45.2/CD45.1 strains, revealing that ac- + ptotic elimination. The results here reveal a separate mechanism, tivation of many CD8 cells and B cells is a reaction to Ndfip1 + + showing that Ndfip1 acts within individual CD4 T cells that deficiency in other hematopoietic cells, whereas CD4 cells have begun proliferating in response to innocuous self or foreign lacking Ndfip1 undergo cell-autonomous expansion into Th1 and antigens, forcing them to exit cell cycle after one to five divisions Th2 effector cells despite the presence of wild-type Tregs and + in vivo. Because CD4 cell differentiation into IL-4–producing other cells. + cells is a function of the number of times they have divided (45, Foxp3 Tregs that develop during negative selection in the 46), the failure of tolerogen-responding Ndfip1-deficient T cells thymus (nTregs) or during T-cell activation in the periphery + to exit cell cycle may explain their differentiation into GATA-3 (iTregs) mediate a non–cell-autonomous mechanism of periph- IL-4–producing Th2 effector cells. The failure to exit cycle eral tolerance, and previous studies established that Itch-or without Ndfip1 contrasted with the peripheral tolerance defect Ndfip1-deficient T cells are crippled in their differentiation into caused by Bim deficiency, where responding progeny still exited iTregs (24, 25). The experiments here showing that Ndfip1 mu- cycle after dividing but resisted elimination by apoptosis. These tation induced autoimmune and inflammatory disease in con- results identify an activation-induced partner of HECT ubiquitin genically matched mixed bone marrow chimeras and in cell + ligases, Ndfip1, to explain previous observations that Bim- transfer recipients despite numerous normal Foxp3 cells, and + deficient CD4 T cells are still able to cease dividing in re- failure of Ndfip1-deficient T cells to abort proliferation that was sponse to self-antigen in vivo despite their inability to undergo not recapitulated by Foxp3-deficient cells, excludes deficits in apoptosis (47). Our findings provide a cellular explanation for Foxp3-expressing cells or their function as a primary explanation. the association of NDFIP1 with both allergic and autoimmune The results here are consistent with Ndfip1 acting as a mech- disease (9–13), the co-occurrence of autoimmunity and asthma- anism to steadily “raise the stakes” for each to proceed like disease in humans with ITCH deficiency (8), and how cross- into successive rounds of division, thereby forcing cells that have reactive environmental antigens may help to trigger autoimmune received a suboptimal (tolerogenic) stimulus to abort ongoing disease (48, 49). As discussed below, Ndfip1-mediated exit from cell division several days into the response. Ndfip1-YFP Tg re- cycle may identify a critical in vivo counterpart of T-cell anergy porter mice revealed that TCR/CD28 stimulation causes a 30- in vitro. fold increase in YFP fluorescence occurring gradually over the The findings here address the unresolved role of Ndfip1 in course of 2 d. This result is consistent with the Ndfip1 induction peripheral T-cell tolerance. Previous studies have established previously reported for TCR/CD28-stimulated T cells: at the that Ndfip1 deficiency in mice causes a T-cell driven, lethal protein level in cultured unfractionated T cells (2) and at the

2072 | www.pnas.org/cgi/doi/10.1073/pnas.1322739111 Altin et al. Downloaded by guest on October 2, 2021 mRNA level in sorted naïve T cells cultured with TGF-β (25). to understand the integration of these diverse biochemical hi

The induction of Ndfip1-YFP in all CD44 activated/memory pathways in future studies. INAUGURAL ARTICLE T cells contrasts with models of T-cell anergy that would postulate A key finding from the experiments is that large numbers of selective induction of Ndfip1 by tolerizing stimuli. Instead, in- autoimmune effector T cells and autoimmune islet destruction duction of Ndfip1 in actively dividing T cells may progressively only developed when an intrinsic peripheral tolerance defect was + raise the signaling threshold needed to keep the cells in cycle, combined with a sufficiently large pool of organ-specific CD4 and in the absence of sufficient microbial costimuli, this may cells that had escaped thymic deletion and also a large exogenous force cell cycle exit. A feature of the “rising stakes” model of antigen trigger. The experiments highlight a third control Ndfip1-mediated peripheral tolerance is that it defers the de- mechanism—limiting amount of tolerogenic antigen stimulus— cision of whether to attack or disarm until after an initial pro- that has also frequently been observed as a key variable in pe- liferative response is made, providing more time for T cells ripheral T-cell tolerance (1). A high density of pMHC may to sample the environment without compromising their speed simply drive more rapid progress through successive cell cycles or of mobilization. + decrease apoptotic loss of daughter cells. One can envisage two Ndfip1-induced exit from cell cycle in CD4 cells responding situations in which self-reactive T cells that have evaded thymic to tolerogens may be an unexpected in vivo manifestation of T- deletion might be strongly stimulated: (i) when an exogenous cell clonal anergy. T-cell anergy has been best defined in T cells food, environmental, or microbial protein happens to contain stimulated in vitro through their TCR without CD28 cos- peptides of similar sequence to the self-antigen (48, 49); and (ii) timulation, and is mostly viewed as a mechanism blocking initi- where damage to an organ releases much more self-antigen for ation of T-cell IL-2 production and proliferation (18). Anergy presentation in the draining lymph node. Given the association has not been considered as a mechanism to trigger dividing T of NDFIP1 polymorphisms with a number of inflammatory dis- cells to exit cycle. T-cell responses to antigen in the absence of eases involving exogenous or self-antigens (9–13), it is not in- CD28 costimuli in vivo have nevertheless not recapitulated the in conceivable that the cellular defect in peripheral tolerance vitro models: instead, proliferation was initiated but followed by defined here could arise through polygenic inheritance patterns Bim-mediated apoptosis, although the remaining cells have been involving NDFIP1, HLA, and other T-cell regulatory genes. The shown to be anergic to reinitiation of proliferation or IL-2 pro- findings here imply that “high-zone tolerance” intervention with duction provided sufficient tolerogen was present (1, 47). Itch exogenous peptides or whole antigens, as practiced in some allergy- has been shown to be required in T cells for in vitro and in vivo desensitization regimes, may in fact exacerbate disease in indi- T-cell anergy, as measured by reinitiation of cytokine production + viduals where there is an intrinsic failure of tolerogen-reactive + and cell proliferation (16, 19). Ndfip1-deficient CD4 cells CD4 cells to exit cycle and abort effector differentiation. IMMUNOLOGY stimulated in culture with antibodies to CD3 in the absence of CD28 have recently been shown to make more IL-2 for an ex- Materials and Methods − − − − − − tended period (21). This difference could nevertheless be sec- Mice. The 3A9 TCR Tg, insHEL Tg, Aire / ,Bim / , and Foxp3 / mice were as ondary to a failure to exit cell cycle without Ndfip1, because IL-2 described previously (37, 39, 61). Mice were either on the C57BL/6 back- production normally increases on a per cell basis as a function of ground, the congenic C57BL/6.SJL-PtprcCD45.1 background, or the congenic the number of TCR-induced cell divisions (45, 46), and the IL-2 B10.BR and B10.BR.SJL-PtprcCD45.1 strains, as indicated. All procedures measurements did not control for cell division history. Decreased were approved by the Australian National University Animal Ethics and production of IL-2 without apparent loss of proliferative re- Experimentation Committee. sponse during in vitro restimulation has been observed in anergic + DO11 TCR Tg CD4 T cells that have exited cycle in response to Ndfip1 cDNA Sequencing and Western Blotting. TRIzol-isolated RNA from tolerogenic self antigen but failed to undergo apoptosis because splenocytes was reverse transcribed with oligo-dT, amplified using primers of Bim deficiency (47). The extent to which Ndfip1 forces cell flanking the Ndfip1 coding region, separated by agarose gel electrophoresis, cycle exit by down-regulation of IL-2 synthesis or by independent cloned into pMXs-IRES-GFP retroviral vector, and sequenced on an ABI3730 + – effects on TCR-induced cell proliferation will require cell capillary sequencer. Naïve T cells (CD4 CD44loCD62LhiCD25 ) were sorted division-based analysis with Il2/Ndfip1 double-deficient T cells from wild-type, Ndfip1kru/kru, and Ndfip1null/null mice, stimulated 48 h with in future studies. 5 μg/mL plate-bound anti-CD3 (145-2C11) and 2 μg/mL soluble anti-CD28 Another future question raised by the findings here is which (37.51), and lysed in 1× SDS sample buffer with sonication and boiling, and biochemical targets of Ndfip1 result in exit from cell cycle in lysates were resolved on a polyacrylamide gel. Proteins were blotted to + CD4 T cells stimulated by self- or foreign-antigen in the ab- PVDF membrane, blocked with 5% (wt/vol) milk, probed with rat mono- sence of adjuvant. Induction of Ndfip1 in actively dividing T cells clonal antibody against an N-terminal peptide of mouse Ndfip1 (35), and may impose a sustained and elevated TCR-CD28 costimulatory developed with HRP-conjugated anti-rat IgG (Millipore) and Enhanced Chem- requirement by downregulating TCR-ζ (20), PKC-θ,PLC-γ, iluminescence Substrate (Perkin-Elmer). JunB and c-Jun proteins (16, 19), Bcl-10, and NF-κB (22, 23). This is supported by the demonstration that Ndfip1-deficient T Flow Cytometry. In vitro stimulation and staining for cell surface and in- cells make more IL-2 than wild-type T cells even when CD28 is tracellular proteins and CFSE dilution was performed as previously described genetically ablated from both (21). However, normal T cells (61). CTV labeling was done at room temperature as described (62) with abort their proliferation to tolerogenic stimuli in vivo even when slight modifications as described in SI Materials and Methods. – CD28 signals have been received (50 56). The presence of ad- × ditional cytokines is also normally required to promote sustained Bone Marrow Chimeras. RAG1-deficient mice were X-irradiated with 1 500 cGy, and wild-type or Aire-deficient mice were given 2 × 500 cGy 4 h apart, rounds of T-cell division and effector differentiation, notably × 6 α β – before i.v. injection of 2 10 T cell-depleted bone marrow cells per mouse. IL-12, IFN- and - , IL-1, and IL-4 (55, 57 59). These cyto- Recipients were maintained on polymixin B and neomycin for 6 wk. kines are typically produced extrinsically to the responding T cells in response to infection, adjuvants, or cell damage, although T-Cell Responses to HEL Protein in Vivo. CD45 and CFSE/CTV-labeled splenocyte autocrine or paracrine sources arise if the T cells divide enough – × 6 3A9 + γ suspensions containing 0.5 2 10 TCR -expressing CD4 T cells were times to differentiate into effector cells that produce IFN- or transferred by i.v. injection to B10.BR-congenic recipients. Where indicated, IL-4. Ndfip1 may suppress the potential for autocrine production μ + recipients received 0.1 or 100 g of HEL in PBS by i.p. injection on the same of IL-2 (21) or IL-4 in actively dividing CD4 cells by degrading day. For cellular analysis, spleens were collected on days 3 or 6 for flow c-Jun and JunB (2, 14), and by inhibiting Notch (31, 32). Ndfip1 + cytometry. For analysis of pathology, recipients were monitored daily for deficiency may also allow tolerogen-stimulated CD4 T cells to hyperglycemia using a blood glucose meter (Medisense Optium), and at remain in cycle by crippling the TGFβ signaling pathway (24, 25), 3 wk posttransfer, pancreata were formalin-fixed, paraffin-embedded, and which normally delivers an important anti-proliferative signal for stained with hematoxylin/eosin, and the extent of lymphocytic infiltration of T cells (60). The findings here provide the in vivo cellular context individual islets was scored blinded to the sample identity.

Altin et al. PNAS | February 11, 2014 | vol. 111 | no. 6 | 2073 Downloaded by guest on October 2, 2021 ACKNOWLEDGMENTS. We thank the Genotyping Team of the Australian work was supported by National Institutes of Health Grants AI100627 and Phenomics Facility (APF) for expert mapping, sequencing, and genotyp- AI054523, Wellcome Trust Grant 082030/B/07/Z, and National Health and ing; the staff of the APF for husbandry; Debbie Howard for expert Medical Research Council Grants 585490, 1016953, 427620, 1009190, technical assistance; and Dr. Ian Parish for advice on the manuscript. This and 1002863.

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