Histone Deacetylase 7 mediates tissue-specific autoimmunity via control of innate effector function in invariant Natural Killer T Cells
- Gladstone Institute of Virology and Immunology, United States
- University of California, San Francisco, United States
- Buck Institute for Research on Aging, United States
Figures
Figure 1 with 3 supplements
A Gain-of-Function HDAC7 Mutant, HDAC7-ΔP, Arrests Thymic iNKT Development.
(A) Representative flow cytometric plots of iNKT cells and conventional αβ T-cells, identified by staining with TCRβ and CD1D tetramer, empty or loaded with αGalCer as indicated, in thymocytes from …
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Figure 1—source data 1
Multi-sheet Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.
- https://doi.org/10.7554/eLife.32109.007
Figure 1—figure supplement 1
Supporting Data on iNKT Phenotype of HDAC7-ΔP Transgenic Mice.
(A) Representative flow scatter plots showing expression of CD44 vs. TCRβ (left), CD4 vs. CD8α (center), and CD8α vs. CD8β (right) in intra-epithelial lymphocytes isolated from small intestines of …
Figure 1—figure supplement 2
Supporting Data on the iNKT Phenotype of WT: HDAC7-ΔP Mixed Hematopoietic Chimeras.
(A) Log2 fold ratio of HDAC7-ΔP-derived (CD45.2) to WT-derived (CD45.1) cells at the indicated thymocyte stages. A composite DN1-4 engraftment ratio (Lin-CD4-CD8-) was calculated per mouse to …
Figure 1—figure supplement 3
Full Gating Strategy for Detection and Staging of iNKT Cells in HDAC7-ΔP Transgenic Mice and WT: HDAC7-ΔP Mixed Hematopoietic Chimeras.
(A) Full gating for representative panels from thymus shown in Figure 1A and C. Bottom row of plots is as shown in Figure 1A and C. From left to right, top row shows gating for leukocytes by FSC …
Figure 2 with 2 supplements
Deletion of HDAC7 in thymocytes Reduces iNKT Numbers and Expands an Innate-Memory CD8 Population.
(A) Representative flow plots showing CD4/CD8 expression (left), loaded and empty CD1D tetramer reactivity (center), and CD44/NK1.1 expression of magnetically enriched iNKT cells (right) from thymus …
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Figure 2—source data 1
Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.
- https://doi.org/10.7554/eLife.32109.011
Figure 2—figure supplement 1
Supporting Data on T Cell Phenotypes of Hdac7-KO Mice.
(A) Representative flow scatter plots showing analysis of WT (top) and HDAC7-ΔP TG (bottom) thymocytes for the frequency of γδ T cells (second column), and their Vδ6.3-positive (third column) and …
Figure 2—figure supplement 2
Supporting Data on Memory Markers and Cytokine Production in WT: Hdac7-KO Mixed Hematopoietic Chimeras.
(A) Bar chart showing log2 ratios of Hdac7-KO/WT cells present in WT: Hdac7-KO hematopoietic chimeras at the indicated thymic developmental stages. (B) Representative flow plots showing gating for …
Figure 3 with 1 supplement
HDAC7-ΔP Blocks Innate Effector Development in iNKT Cells and Converts Them to Naive-Like T-cells.
(A, B) Representative flow cytometric plots showing TCRβ vs. PBS-57 tetramer staining (A), and CD44 vs. NK1.1 staining of iNKT cells (B) in thymus (top) and spleen (bottom) of littermate mice with …
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Figure 3—source data 1
Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.
- https://doi.org/10.7554/eLife.32109.014
Figure 3—figure supplement 1
Supporting Data on iNKT Phenotype of Vα14/Jα18 X HDAC7-ΔP Transgenic Mice.
(A, B) Restoration of iNKT cells (Tet + TCRβ+) in Hdac7-KO mice by expression of the Vα14-Jα18 TCR transgene (A, top row), as well as representative CD44/NK1.1 staging for each genotype (bottom …
Figure 4
Nuclear HDAC7 Retention Restricts PLZF Expression and Mirrors PLZF-Associated T Cell Phenotypes.
(A, B) Representative flow cytometric plots (A) and total quantification (B) of PLZF expression in TCRβ+ cells from thymus, spleen, and liver. (C) PLZF expression in mature CD4 SP (CD4+ CD8- TCRβ+) …
Figure 5 with 1 supplement
HDAC7 Regulates a Cassette of Genes in Glycolipid-Reactive Cells That is Highly Relevant to Innate Effector Function, Inflammation, Autoimmunity, and Autoimmune Liver Disease.
(A) Scatter charts showing gene expression changes in Cd1d/αGalCer-reactive Vα14 Tg (X axis) and Vα14 X HDAC7-ΔP Tg (Y axis) thymocytes (left) or CD4 splenocytes (right) vs naïve CD4SP thymocytes or …
Figure 5—figure supplement 1
HDAC7 Regulates a Cassette of Genes in Glycolipid-Reactive Cells That is Highly Relevant to Innate Effector Function, Inflammation, Autoimmunity, and Autoimmune Liver Disease.
Related to Figure 5. (A) Table showing top putative upstream regulators (Column 1) of the genes from Figure 5A that were suppressed by HDAC7-ΔP, based on analysis with Ingenuity Pathway Analysis …
Figure 6
The intersection of HDAC7-regulated genes in iNKT development and GWAS hits for IBD and PSC highlights key signaling pathways.
(A) Venn diagram showing enumeration of genes that are GWAS risk loci for PSC and IBD from (Liu et al., 2013; Jostins et al., 2012), and/or also regulated by HDAC7 during NKT development according …
Figure 7 with 1 supplement
HDAC7 Can Physically Bind and Functionally Antagonize PLZF Transcriptional Activity.
(A) Immuno-blots showing co-immunoprecipitation with endogenous HDAC7 of PLZF from PLZF-transgenic thymocytes. (B) Immunoblot showing Co-immunoprecipitation of HA-tagged full-length PLZF from …
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Figure 7—source data 1
Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically (Figure 7D,E,F).
- https://doi.org/10.7554/eLife.32109.021
Figure 7—figure supplement 1
Diagram of GAL4-PLZF, HDAC7, and GAL4 reporter constructs employed in experiments shown Figure 7F in main text.
The GAL4 DBD-PLZF fusion and HDAC7 constructs shown were co-transfected into HEK293T cells, together with the 5XGAL4 site-containing pGL2 Promoter luciferase reporter shown at right.
Figure 8 with 1 supplement
Loss of iNKT Cells in HDAC7ΔP Mice Contributes to Tissue-Specific Autoimmunity.
(A) Schematic of mixed BM chimeras used to monitor HDAC7-ΔP-mediated autoimmunity time course and severity. Lethally irradiated CD45.1 BoyJ recipients were reconstituted (6 × 106 cells) with a 1:5 …
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Figure 8—source data 1
Microsoft Excel workbook containing numerical data matrices for all figure panels (on separate sheets) in which individual data points are not represented graphically.
- https://doi.org/10.7554/eLife.32109.024
Figure 8—figure supplement 1
Supporting Data on restoration of iNKTs in HDAC7-ΔP mixed chimeras with Vα14-Jα18 TG bone marrow and autoimmune disease course.
(A) Representative flow scatters showing CD45.1 vs. CD45.2 expression in spleens and livers of WT CD45.1/.2 heterozygote recipients, three days after retro-orbital transfer of 5 × 106 CD45.2 iNKT …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | Histone Deacetylase 7 (HDAC7) | NA | HDAC7 | Coding sequence used for HDAC7 expression constructs and the HDAC7-ΔP transgene. |
| Gene (Homo sapiens) | Promyelocytic Leukemia, Zinc Finger (PLZF) | NA | ZBTB16 | Coding sequence used for PLZF expression constructs. |
| Strain, strain background (Mus musculus) | C57/BL6 | Jackson Laboratories | Stock Number: 000664 | |
| Strain, strain background (Mus musculus) | Boyj (B6.SJL-Ptprca Pepcb/BoyJ) | Jackson Laboratories; PMID: 11698303 | Stock Number: 002014 | B6 strain congenic for Cd45.1 |
| Genetic reagent (Mus musculus) | Vα14/Jα18 Transgenic, Tg(Cd4-TcraDN32D3)1Aben | Jackson Laboratories; PMID: 18031695 | MGI:4880641 | Vα14/Jα18 Transgenic from Bendelac laboratory |
| Genetic reagent (Mus musculus) | Lck-Cre Transgenic, Tg(Lck-cre)548Jxm | Jackson Laboratories; PMID: 8618846 | MGI: 2176199 | Cre strain for thymicHdac7 deletion |
| Genetic reagent (Mus musculus) | Hdac7flox/flox, Hdac7tm2Eno | Eric Olson, UTSW; PMID: 16873063 | MGI: 1891835 | HDAC7 floxed allele |
| Genetic reagent (Mus musculus) | Hdac7-/+, Hdac7tm1Eno | Eric Olson, UTSW; PMID: 16873063 | MGI: 1891835 | HDAC7 null allele |
| Genetic reagent (Mus musculus) | Lck-PLZF transgenic, C57BL/6-Tg(Cd4-Zbtb16)1797Aben/J | Jackson Laboratories; PMID: 18703361 | MGI:4881493 | PLZF Transgenic strain from Bendelac Laboratory |
| Genetic reagent (Mus musculus) | HDAC7-ΔP Transgenic | Our laboratory; PMID: 23103766 | NA | Transgenic expression of HDAC7-ΔP under control of Lcl proximal promoter/CD2 LCR in C57BL/6 |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 FL | this paper | NA | Expression construct for FL FLAG-tagged human HDAC7 |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 1–497 | this paper | NA | Expression construct for FLAG-tagged human HDAC7 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 65–497 | this paper | NA | Expression construct for FLAG-tagged human HDAC7 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 120–497 | this paper | NA | Expression construct for FLAG-tagged human HDAC7 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 1–220 | this paper | NA | Expression construct for FLAG-tagged human HDAC7 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) FLAG-HDAC7 1–180 | this paper | NA | Expression construct for FLAG-tagged human HDAC7 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF FL | this paper | NA | Expression construct for FL HA-tagged human PLZF |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 1–318 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 1–405 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 105–460 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 280–673 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 320–673 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 395–673 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) HA-PLZF 455–460 | this paper | NA | Expression construct for HA-tagged human PLZF truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) GAL4-PLZF FL | this paper | NA | Expression construct for GAL4 DNA-binding domain (1-142) fused to full-length PLZF |
| Recombinant DNA reagent | pCDNA 3.1(+) GAL4-PLZF 455–460 | this paper | NA | Expression construct for GAL4 DNA-binding domain (1-142) fused to PLZF1 - 460 truncation mutant |
| Recombinant DNA reagent | pCDNA 3.1(+) GAL4-PLZF 455–460 | this paper | NA | Expression construct for GAL4 DNA-binding domain (1-142) fused to PLZF1 - 318 truncation mutant |
| Software, algorithm | Ingenuity Pathway Analysis | Qiagen | RRID:SCR_008653 | Tool for pathway mapping and other gene ontology analysis from RNAseq transcript abundance data. |
| Software, algorithm | Bowtie 2.0 | Johns Hopkins University; PMID: 22388286 | RRID:SCR_005476 | Tool for aligning raw sequence reads to genome |
| Software, algorithm | SeqMonk | Babraham Institute | RRID:SCR_001913 | Tool for calculating RNA transcript abundances from Bowtie-mapped sequence reads (.SAM files) |
Additional files
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Supplementary file 1
Excel spreadsheet containing SeqMonk Normalized expression values for all present RNAs in our 18 samples (six genotypes, three biological replicates each, as defined in Materials and methods), with means for each genotype (Columns A-Z), summary statistics for key comparisons (mean, log2 mean/mean, and T-test, Columns AA-AK), and aligned data from relevant published studies (Columns AL-AT).
Additional notes and PMIDs for gene-specific published findings for disease-associated GWAS loci are provided in Columns AY-BA.
- https://doi.org/10.7554/eLife.32109.025
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Supplementary file 2
Full table of Ingenuity Pathway Analysis overrepresented pathways for the comparison of genes expressed in CD4 SP cells for Vα14Jα18 TG X HDAC7-ΔP TG mice vs Vα14Jα18 TG littermates in spleen and thymus.
- https://doi.org/10.7554/eLife.32109.026
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Supplementary file 3
Full table of Ingenuity Pathway Analysis predicted upstream regulators and their targets for the comparison of genes expressed in CD4 SP cells for Vα14Jα18 TG X HDAC7-ΔP TG mice vs Vα14Jα18 TG littermates in spleen and thymus.
- https://doi.org/10.7554/eLife.32109.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.32109.028
