Research ArticleTransplantation

Zbtb7a induction in alveolar macrophages is implicated in anti-HLA–mediated lung allograft rejection

See allHide authors and affiliations

Science Translational Medicine  12 Jul 2017:
Vol. 9, Issue 398, eaal1243
DOI: 10.1126/scitranslmed.aal1243
  • Fig. 1. Profile of lung transcriptome after MHC class I ligation by specific Ab.

    (A) Heat map presentation of differentially expressed genes after intrabronchial administration of H-2Kb or isotype control Abs into C57BL/6 mice. Three mice per group per day were analyzed and plotted. Probe intensity was normalized by z score, and red and blue indicate higher and lower expression levels, respectively. Data have been deposited into the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GEO ID: GSE71426). (B) Scatterplot highlighting 23 selected genes differentially expressed between H-2Kb and isotype control groups. Mean signal intensities were transformed into log2, and quantile normalization was applied. Differential expression was determined by two-way analysis of variance (ANOVA) test, and expression level exceeding 1.5-fold (P < 0.05 and FDR q < 0.1) was deemed significant. The central solid line represents line of equality, and genes expressed >1.5-fold demarcated by dashed lines are annotated. Red, up-regulated; blue, down-regulated. (C) Quantitative polymerase chain reaction (qPCR) validation of Zbtb7a expression was conducted in a parallel experiment. Three mice per time point were analyzed for Zbtb7a expression and normalized with that of Actb. Zbtb7a expression in untreated C57BL/6 lungs was set as baseline expression. Data are presented as means ± SEM, multiple t test was applied with multiple comparisons by Holm-Sidak method, and *P < 0.05 is considered significant.

  • Fig. 2. Disruption of Zbtb7a expression in lungs prevents anti-MHC–induced lung-restricted autoimmunity and OAD.

    (A) Schematic representation of siRNA lentivirus transduction and subsequent Ab challenge. Mice were intrabronchially inoculated with lentivirus 3 days before intrabronchial Ab regimen. (B) Repression in the Zbtb7a expression was quantified by qPCR. n = 3. (C) Representative detection of Zbtb7a expression in lung lysates by Western blot analysis. (D) Representative Masson’s trichrome staining on day 45 after Ab administration. Fibrotic areas with deposition of collagenous ECM are stained blue. Scale bars, 100 μm. (E) Histopathologic score for a data point represents average of five fields from each mouse. Median lines are drawn. (F) Titers for serum anti-Kα1T and Col V were evaluated by enzyme-linked immunosorbent assay (ELISA) from eight mice, and mean ± SEM lines are drawn. (G) Development of T cell autoreactivity was analyzed by enzyme-linked immunospot (ELISPOT) on day 45. The data are presented as spot-forming cell (SFC) per million cells. n = 8 per group. Results are presented as means ± SEM, multiple t tests were applied with correction for Holm-Sidak method. P values are indicated. *P < 0.05 is considered significant.

  • Fig. 3. Early induction of ZBTB7A in lung transplant recipients who developed de novo DSA and BOS.

    BAL cell ZBTB7A expression was evaluated by qPCR after normalization with ACTB, CD68, and PPARG. ΔCt values at 3 months after transplantation were set as baseline for respective lung transplant recipients (table S2), and relative expression for subsequent time points was calculated as 2−ΔΔCt. (A) ZBTB7A induction in a cohort of 21 BOS+ lung transplant recipients and time-matched 21 lung transplant recipients who remained BOS-free, (B) kinetic study at 6 ± 1 and 12 ± 1 posttransplant months on 12 lung transplant recipients who later developed BOS, and (C) 11 lung transplant recipients who remained stable were evaluated. (D) Ten lung transplant recipients who developed de novo DSA (MFI > 2000) and received Ab-directed therapies were stratified as pre-DSA (MFI < 2000), DSA-peaked (highest MFI observed during follow-up of individual patient), DSA-reduced (2000 < MFI < peak), and DSA-resolved (MFI < 2000). (E) Serial samples from eight stable lung transplant recipients (DSA and not treated by Ab-directed therapy) were evaluated. (F) Role of DSA and autoAb (anti-Kα1T and anti–Col V) in ZBTB7A induction evaluated in lung transplant recipients DSA, autoAb (n = 11); DSA, autoAb+ (either anti-Kα1T or anti–Col V; n = 5); DSA+, autoAb (n = 10); DSA+, one autoAb (either anti-Kα1T or anti–Col V; n = 11), and DSA+, two autoAbs (anti-Kα1T and anti–Col V; n = 19). Two-tailed Mann-Whitney U test was applied, and P values are indicated. *P < 0.05 is considered significant.

  • Fig. 4. Induced Zbtb7a in lungs is localized to AMs.

    (A) Representative reverse transcription PCR analysis on total RNA isolated from AMs (lanes 1, 4, 7, and 10), non-AM BAL cells (lanes 2, 5, 8, and 11), and alveolar respiratory epithelial cells (REC; lanes 3, 6, 9, and 12) on day 7 from siRNA lentivirus–transduced and Ab-treated mice (n = 6). bp, base pair. (B) Freshly isolated human AMs (from stable lung transplant recipients; DSA, autoAb, and with no documented rejection) and mouse AMs (from C57BL/6) were cultured at 1 × 106 cells per well (33). Surface MHC class I was ligated by anti–H-2Kb and anti-HLA, respectively, in mouse and human AM cultures, and MHC class II on mouse AMs was ligated by anti–I-A/I-E. Isotype-matched controls for MHC Abs and ligation of a non-MHC molecule (keratin) were also evaluated. Zbtb7a expression was normalized to that of Actb and presented as relative expression compared to no Ab–treated AMs. n = 5. Data are presented as means ± SEM, two-tailed unpaired t tests are applied, *P < 0.05 is considered significant, and P values are indicated. (C) BAL cells from five BOS+ lung transplant recipients (P-1 to P-5) and four BOS-free lung transplant recipients (P-6 to P-9) with stable lung function were fractionated into AM and non-AM cells. The lung biopsy specimens were obtained on the same day of BAL fluid collection. Fold induction in ZBTB7A expression compared to the peripheral blood leukocytes for individual lung transplant recipient is plotted as heat map.

  • Fig. 5. Induction of lung-restricted autoimmunity and OAD by anti-MHC ligation of AM is Zbtb7a-dependent.

    (A) Expression of lineage markers and H-2Kd transgene in huCD68-Kd Tg C57BL/6 AMs. BAL cells were pregated for CD45+, CD19, and CD3 leukocytes. (B) Representative trichrome-stained images showing fibrotic scar and occluded bronchiole in anti–H-2Kd–induced OAD on day 30 and (C) histopathology assessment of the OAD lesion. n = 6. Scale bar, 100 μm. (D) Development of de novo Ab titers and (E) stimulation of TH17 cells specific for Kα1T and Col V were evaluated on day 30. n = 6 to 7. (F) Representative surface expression of MHC (class I and II) and costimulatory molecules in Zbtb7a-sufficient or Zbtb7a-deficient AMs after anti–H-2Kd administration was analyzed on day 30. n = 3. Data were analyzed by multiple t tests, and statistical significance (*P < 0.05) was determined using the Holm-Sidak method.

  • Fig. 6. Zbtb7a expression is essential in elicitation of AM-directed humoral and cellular alloimmunity.

    Allogeneic reconstitution of AM was achieved by intrabronchial transfer of 4 × 105 huCD68-Kd Tg C57BL/6 AM into C57BL/6 recipients. (A) Development of H-2Kd–specific Abs in BAL fluid was measured by flow cytometry at 15 weeks. Representative anti–H-2Kd titers (in table S5) are presented as overlaid histograms (n = 5 to 7), and individual MFI values are indicated. (B) ELISPOT assay measured alloreactive (H-2Kd–specific) IL-17–producing cells per million lung leukocytes (mean ± SEM) at 15 weeks. One-way ANOVA was applied to test statistical significance (*).

  • Fig. 7. Zbtb7a deficiency in donor AMs reduces donor-derived exosome production.

    (A) Quantitation of total exosomes isolated from cell-free BAL fluid after adoptive transfer of allogeneic AMs from Fig. 6. Data from three mice per AM transfer group were individually analyzed and are presented as means ± SEM. One-way ANOVA was applied with Brown-Forsythe test that compares exosomes from the AM transfer groups with those from C57BL/6 as the baseline expression. Significance (P values) of differences is indicated. (B) Representative flow cytometric analysis on BAL exosomes from AM transfer groups (n = 3 to 4) was performed by anti-CD63–coated Exo-Flow magnetic beads. The AM-derived exosomes (Siglec-F+) were analyzed for frequency of donor or recipient origin as defined by H-2Kd and H-2Kb staining (summary in table S6). Constitution of the BAL exosomes (mean) derived from AMs, non-AM cells, and donor AMs (alloexosomes) is also shown. SSC-A, side scatter area; FSC-A, forward scatter area; APC, allophycocyanin.

  • Fig. 8. Zbtb7a deficiency impairs antigen presentation by AMs.

    Adoptively transferred Zbtb7a-sufficient or Zbtb7a-deficient huCD68-Kd Tg AMs in Fig. 6 were antigen-pulsed (with L-OVA or L-BSA delivering 5 μg of protein or 1 μg of OVA 323-339 peptide) in vivo at 15 weeks after transfer, harvested at 6 hours after pulsing, and transferred (1 × 105) with 5 × 106 CFSE-labeled naïve OT-II T cells into C57BL/6 recipients. (A) Representative plot of OT-II T cell proliferation in lungs and spleen measured by CFSE dilution at 72 hours after AM and T cell transfer. Cells were pregated for CFSE+ and CD3+. n = 5. SSC-H, side scatter height. (B) Summary of the CFSE dilution. Mean ± SEM is plotted for T cell proliferation in the lungs. n = 5.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/398/eaal1243/DC1

    Materials and Methods

    Fig. S1. Ab ligation of MHC class I increases lung-infiltrating B cells and neutrophils.

    Fig. S2. Zbtb7a knockdown in lungs reduces anti-MHC–induced pulmonary inflammation.

    Fig. S3. Zbtb7a knockdown reduces development of anti-MHC–induced iBALT in lungs.

    Fig. S4. Zbtb7a deficiency does not diminish endocytosis by AMs.

    Fig. S5. Zbtb7a deficiency in AM alters molecular responses to anti-MHC ligation.

    Fig. S6. Exogenous alloAb induces Zbtb7a expression in the accepted lung allograft after mouse orthotopic lung transplantation.

    Fig. S7. Lymphocytes are dispensable in anti-MHC–induced Zbtb7a expression but are required for OAD development.

    Table S1. Differential gene expression in lungs after Ab ligation of MHC class I.

    Table S2. Clinical characteristics of the lung transplant recipients.

    Table S3. ZBTB7A expression in lung transplant recipients.

    Table S4. Summary of Zbtb7a expression in response to exogenous Abs after mouse orthotopic lung transplantation.

    Table S5. Summary of the allogeneic AM transfer protocol.

    Table S6. Summary of the AM-derived alloexosome production.

    Table S7. Primary data.

    References (71, 72)

  • Supplementary Material for:

    Zbtb7a induction in alveolar macrophages is implicated in anti-HLA–mediated lung allograft rejection

    Deepak K. Nayak,* Fangyu Zhou, Min Xu, Jing Huang, Moriya Tsuji, Jinsheng Yu, Ramsey Hachem, Andrew E. Gelman, Ross M. Bremner, Michael A. Smith, Thalachallour Mohanakumar*

    *Corresponding author. Email: tm.kumar{at}dignityhealth.org (T.M.); deepak.nayak{at}dignityhealth.org (D.K.N.)

    Published 12 July 2017, Sci. Transl. Med. 9, eaal1243 (2017)
    DOI: 10.1126/scitranslmed.aal1243

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Ab ligation of MHC class I increases lung-infiltrating B cells and neutrophils.
    • Fig. S2. Zbtb7a knockdown in lungs reduces anti-MHC–induced pulmonary inflammation.
    • Fig. S3. Zbtb7a knockdown reduces development of anti-MHC–induced iBALT in lungs.
    • Fig. S4. Zbtb7a deficiency does not diminish endocytosis by AMs.
    • Fig. S5. Zbtb7a deficiency in AM alters molecular responses to anti-MHC ligation.
    • Fig. S6. Exogenous alloAb induces Zbtb7a expression in the accepted lung allograft after mouse orthotopic lung transplantation.
    • Fig. S7. Lymphocytes are dispensable in anti-MHC–induced Zbtb7a expression but are required for OAD development.
    • Table S1. Differential gene expression in lungs after Ab ligation of MHC class I.
    • Table S2. Clinical characteristics of the lung transplant recipients.
    • Table S3. ZBTB7A expression in lung transplant recipients.
    • Table S4. Summary of Zbtb7a expression in response to exogenous Abs after mouse orthotopic lung transplantation.
    • Table S5. Summary of the allogeneic AM transfer protocol.
    • Table S6. Summary of the AM-derived alloexosome production.
    • References (71, 72)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S7 (Microsoft Excel format). Primary data.