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

Abstract

The NDFIP1 (neural precursor cell expressed, developmentally down-regulated protein 4 family-interacting protein 1) adapter for the ubiquitin ligase ITCH is genetically linked to human allergic and autoimmune disease, but the cellular mechanism by which these proteins enable foreign and self-antigens to be tolerated is unresolved. Here, we use two unique mouse strains--an Ndfip1-YFP reporter and an Ndfip1-deficient strain--to show that Ndfip1 is progressively induced during T-cell differentiation and activation in vivo and that its deficiency causes a cell-autonomous, Forkhead box P3-independent failure of peripheral CD4(+) T-cell tolerance to self and exogenous antigen. In small cohorts of antigen-specific CD4(+) cells responding in vivo, Ndfip1 was necessary for tolerogen-reactive T cells to exit cell cycle after one to five divisions and to abort Th2 effector differentiation, defining a step in peripheral tolerance that provides insights into the phenomenon of T-cell anergy in vivo and is distinct from the better understood process of Bcl2-interacting mediator of cell death-mediated apoptosis. Ndfip1 deficiency precipitated autoimmune pancreatic destruction and diabetes; however, this depended on a further accumulation of nontolerant anti-self T cells from strong stimulation by exogenous tolerogen. These findings illuminate a peripheral tolerance checkpoint that aborts T-cell clonal expansion against allergens and autoantigens and demonstrate how hypersensitive responses to environmental antigens may trigger autoimmunity.

Keywords: Aire (Autoimmune Regulator); Interleukin-4; T lymphocyte; allergy; immunological tolerance.

Fig. 1.

Immune-mediated lethal inflammatory syndrome in mice with a truncating Ndfip1 splice site mutation. (A) Schematic showing the location of the Ndfip1^kru mutation within the Ndfip1 exon 5 splice donor sequence and the resulting two aberrant splice products. (B) Schematic showing the topology of the Ndfip1 protein and position of the truncating mutations. (C) Western blot of primary T-cell lysates from wild-type, Ndfip1^kru/kru (mutant), and Ndfip1^−/− (KO) mice, probed with an antibody raised to a conserved N-terminal peptide of Ndfip1, and then stripped and reprobed with antibody to tubulin to assess loading. (D) Dermatitis and survival of Ndfip1^kru/kru mice with normal or null Rag1 genes, aged to 250 d or until moribund necessitating the animal to be killed.

Fig. 2.

Ndfip1 deficiency causes cell-autonomous accumulation of activated/effector CD4⁺ T cells that is not corrected by the presence of wild-type Foxp3⁺ Tregs. Three groups of mixed bone marrow chimeras were generated by transplanting irradiated Rag1^−/− mice: “100% kru,” transplanted with 100% Ndfip1^kru/kru CD45.2 bone marrow; “50% kru,” transplanted with a 50:50 mixture of Ndfip1^kru/kru CD45.2 bone marrow and Ndfip1^+/+CD45.1 bone marrow; “100% wt,” transplanted with a 50:50 mixture of Ndfip1^+/+CD45.2 bone marrow and Ndfip1^+/+CD45.1 bone marrow. Flow cytometry was used to analyze lymph node CD4⁺ cells, gated into either CD45.2⁺ or CD45.1⁺ cells, measuring cell surface expression of CD44, intracellular IFN-γ or IL-4 after 3 h ex vivo phorbol myristate acetate (PMA)/ionomycin stimulation, and intracellular Foxp3. Representative plots and quantitation in multiple animals are shown. Lines link data points from the same recipient animal. *P < 0.05; **P < 0.01; ***P < 0.001 by Student t test.

Fig. 3.

Progressive induction of an Ndfip1 reporter gene during T-cell selection and activation. Flow cytometric analysis of Tg Ndfip1 reporter (Ndfip1-YFP Tg) mice bearing a BAC with eYFP integrated in place of the Ndfip1 translation initiation codon. (A) YFP fluorescence of CD4⁺CD8⁺, CD4⁺CD8^–, CD4^–CD8⁺, CD4⁺CD8^–CD25^–GITR^–, and CD4⁺CD8^–CD25⁺GITR⁺ thymocyte populations from Ndfip1-YFP Tg mice, gated as indicated. (B) YFP fluorescence of CD4⁺, CD8⁺, B220⁺, CD4⁺CD44^loCD25^–, CD4⁺CD44^hiCD25^–, and CD4⁺CD44^intCD25⁺ splenocyte populations from Ndfip1-YFP Tg mice, gated as indicated. (C) YFP fluorescence of gated CD4⁺ cells among splenocytes from Ndfip1-YFP Tg mice that were incubated with or without plate-bound antibodies against CD3 and CD28 for the indicated periods. In all cases, the shaded gray histogram shows the YFP fluorescence of cells from non-Tg control mice.

Fig. 4.

Ndfip1 is required in activated CD4⁺ T cells to prevent effector accumulation and differentiation in response to exogenous antigen without adjuvant. (A) An equal mixture of HEL-specific CD4⁺ T cells was prepared from 3A9 TCR^HEL Tg mice that were Ndfip1^kru/kru CD45^2/2 or wild-type CD45^1/2, labeled with CFSE, and 2 × 10⁶ total CD4⁺ cells injected into the circulation of normal B10.BR CD45^1/1 recipient mice. A subset of the recipient mice was given 100 μg of HEL antigen in saline i.p. at the time of cell transfer. Shown is representative flow cytometric analysis of recipient spleen cells on the indicated day after T-cell transfer. Upper graphs are gated on CD4⁺ T cells and show the percentage derived from the two donors and the recipient. Lower graphs show CFSE fluorescence of the respective donor and recipient cells. (B) Percentage of donor cells in multiple recipients analyzed as in A. Lines link data points from the same recipient animal. (C) Percentage of donor T cells expressing IL-4 measured by intracellular staining and gated as in A after 3 h ex vivo PMA/ionomycin stimulation. (D) Intracellular staining for JunB, IL-4, and IFN-γ on gated CD45^1/2 or CD45^2/2 donor CD4⁺ cells at day 6 after transfer and injection of HEL in saline. (E) Quantitation of the JunB MFI of gated donor cells in multiple recipients analyzed as in D.

Fig. 5.

Ndfip1 mediates tolerance distinct from mitochondrial apoptosis or Foxp3, by aborting the proliferative response of CD4⁺ T cells. Cotransfer of allotypically distinguished 3A9 TCR^HEL Tg cells was performed as in Fig. 4 but including parallel groups of recipients that received cell mixtures from wild-type and Foxp3-deficient, or wild-type and Bim-deficient mice (all mice 3A9 TCR^HEL Tg), and using the cell division label CTV. (A) Quantitation of the fold-expansion (relative to cotransferred wild-type cells) of wild-type, Ndfip1^kru/kru, Foxp3-deficient or Bim-deficient CD4⁺ cells in recipient mice at day 6 following injection of HEL in saline, compared with the cell inputs at day 0. (B) Representative Ki-67 and CTV data from CD45^1/2 recipients on day 6, gated on the cotransferred CD45^2/2 (red plots) or CD45^1/1 (blue plots) 3A9 TCR^HEL CD4+ T cells of the indicated genotypes. (C) Percentage of Ki-67⁺ cells remaining in cell cycle among donor CD4⁺ cells that had previously divided (CTV^lo) in response to 100ug HEL in saline, measured in individual recipients as in B.

Fig. 6.

Ndfip1 deficiency disrupts peripheral T-cell tolerance to pancreatic islets. (A) Incidence of diabetes in a cohort of mice with the indicated Ndfip1 genotypes that were all doubly Tg for the 3A9 TCR^HEL transgene and an insulin-promoter HEL transgene (insHEL Tg). (B) Daily blood glucose readings in singly insHEL Tg mice injected i.v. with 10⁶ CD4⁺ T cells from 3A9 TCR^HEL Tg donors that were either Ndfip1^kru/kru or wild-type. Half of the recipients also received 100 μg of HEL in saline i.p. at the time of T-cell transfer. (C) Quantitation of insulitis in ≥10 individual islets in individual recipients (denoted by columns) 3 wk after transfer of wild-type or Ndfip1^kru/kru T cells together with HEL in saline. (D) A mixture of HEL-specific CD4⁺ T cells from 3A9 TCR^HEL Tg mice that were Ndfip1^kru/kru CD45^2/2 or wild-type CD45^1/2 was CTV-labeled and injected into the circulation of non-Tg or insHEL Tg B10.BR CD45^1/1 recipient mice. One group of insHEL Tg recipients was given 100 μg of HEL in saline i.p. at the time of cell transfer, whereas another group of non-Tg recipients received 0.1 μg of HEL in saline i.p. The donor CD4⁺ cells were analyzed 6 d later in the spleen for CTV dilution and expression of Ki-67 and GATA-3. Shown are representative plots of CTV fluorescence versus Ki-67 staining (upper plots) or GATA-3 staining (lower plots) on the cotransferred kru CD45^2/2 CD4+ cells (red) and wt CD45^1/2 CD4+ cells (blue). (E) Frequency among CD4⁺ cells of wild-type and Ndfip1^kru/kru donor cells in individual recipient mice described in D, normalized to frequencies in a non-Tg recipient that received no HEL. Lines link data points from the same recipient animal.