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. 2022 Oct 3;219(10):e20212554.
doi: 10.1084/jem.20212554. Epub 2022 Aug 23.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection

J Luke Postoak  1 , Wenqiang Song  1 , Guan Yang  1 , Xingyi Guo  2 , Shiyun Xiao  3 , Cherie E Saffold  1 , Jianhua Zhang  4   5 , Sebastian Joyce  1   6 , Nancy R Manley  3 , Lan Wu  1 , Luc Van Kaer  1
Affiliations

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection

J Luke Postoak et al. J Exp Med. .

Abstract

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

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Conflict of interest statement

Disclosures: L. Van Kaer reported “I am a member of the scientific advisory board of Isu Abxis Co., Ltd. (South Korea), for which I get financial compensation. The company focuses on the development of antibody-mediated treatment for cancer and rare diseases. The work at the company is not directly related to this publication.” No other disclosures were reported.

Figures

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Graphical abstract
Figure S1.
Figure S1.
Vps34TEC phenotype. (A) Flow cytometric analysis of TECs (CD45−EpCAM+) and thymocytes (CD45+, EpCAM−) for tdTomato expression isolated from 1-wk-old Foxn1-Cre−Ai14+ or Foxn1-Cre+Ai14+ mice. (B) Dorsal skin tissue sections from 8-wk-old Vps34f/f or Vps34TEC mice stained with H&E (scale bar = 100 μm). (C) Gross images of thymic lobes isolated from 1-wk-old Vps34f/f or Vps34TEC mice. (D) Flow cytometric analysis of CD4−CD8−Lin (CD3, B220, NK1.1)− DN thymocytes including DNI (CD44+CD25−), DNII (CD44+CD25+), DNIII (CD44−CD25+), and DNIV (CD44−CD25−) isolated from 6- to 8-wk-old Vps34f/f and Vps34TEC mice. The graph represents the relative frequency of each indicated DN thymocyte population. (E) Thymus tissue sections from 7- to 10-d-old Vps34f/f or Vps34TEC mice stained with H&E (scale bar = 1,000 μm), anti-keratin 5 (K5; green) and DAPI (blue), or UEA-1 lectin (red) and DAPI (blue; scale bar = 500 μm). (F and G) Flow cytometric analysis of (F) total TECs and (G) TEC subsets, including mTECs (UEA-1+Ly-51−) and cTECs (UEA-1−Ly-51+) at 10 d of age. (H) mTEChi (MHC IIhiCD80hi) and mTEClo (MHC IIloCD80lo) frequency among mTECs. (I) Sca-1 and Itga-6 expression by UEA-1lo TECs. The graph represents Sca-1+Itga6+ cells among UEA-1lo TECs. Data are representative of at least two independent experiments with at least two mice per group. Each graphed data point represents a biological replicate. Data signify the mean ± SD. *, P < 0.05; ***, P < 0.001; ns, not significant by unpaired t test.
Figure 1.
Figure 1.
Vps34 deficiency in TECs causes thymic hypoplasia, decreased thymopoiesis, and T cell lymphopenia. (A) Thoracic cavity at 1 wk (top, scale bar = 0.5 cm) and 4 wk (bottom, scale bar = 0.4 cm) of age in Vps34f/f or Vps34TEC mice. (B) Total thymocyte cellularity at the indicated ages in Vps34f/f or Vps34TEC mice. (C) Flow cytometric analysis of CD4 and CD8 DP thymocytes at the indicated ages in Vps34f/f or Vps34TEC mice. The graph represents the frequency of DP thymocytes among all thymocytes. Data for A–C obtained from two or three independent experiments (n = 3–10 mice per genotype). (D) CD4 and CD8 expression profiles of T cells isolated from spleen (top) and mesenteric lymph nodes (mLN; bottom) in 7-wk-old Vps34f/f or Vps34TEC mice. Graphs represent total CD4+ T cells (CD4+CD3+) and CD8+ T cells (CD8+CD3+) in the spleen (top) and mLN (bottom). (E) CD44 and CD62L expression profiles of T cells isolated from the mLN of 7-wk-old Vps34f/f or Vps34TEC mice. Graphs represent the frequency of CD62LhiCD44lo cells among total CD4+ T cells (top panels) or total CD8+ T cells (bottom panels). Data for D and E obtained from two independent experiments (n = 5–6 mice per genotype). Data signify the mean ± SD where each data point represents a biological replicate. *, P < 0.05; ***, P < 0.001; ns, not significant by unpaired t test.
Figure 2.
Figure 2.
TEC-specific Vps34 deficiency modulates TEC autophagy, cellularity, and morphology. (A) Schematic of LC3-EGFP/RFP reporter. (B) Flow cytometric analysis of TECs (CD45−EpCAM+) for LC3-EGFP/RFP reporter expression in nontransgenic control (left), Vps34f/f (middle), and Vps34TEC (right) mice at 3 d of age. The graph represents percent EGFPlo TECs, cTECs (UEA-1− TECs), and mTECs (UEA-1+ TECs) among corresponding RFP+ TEC populations. Data are representative of four (for all TECs) and two (for cTEC and mTEC) independent experiments. (C) Thymus tissue sections from 3-d-old Vps34f/f or Vps34TEC mice were stained with H&E (scale bar = 800 μm), anti-keratin 5 (K5; green) and DAPI (blue), or UEA-1 lectin (red) and DAPI (blue; scale bar = 500 μm). Data are representative of three independent experiments with three biological replicates. (D and E) Flow cytometric analysis of total TECs (D) and TEC subsets (E), including mTECs (UEA-1+Ly-51−) and cTECs (UEA-1−Ly-51+) at 3 d of age. Graphs represent frequency (left) and total number (right) of TECs or TEC subsets. (F) Ki67 expression among cTECs (MHC IIhiUEA-1− TECs) and mTECs (MHC IIhiUEA-1+ TECs). The graph represents frequency of Ki67+ cells among TEC subsets. Data for D–F obtained from two independent experiments with four to seven biological replicates. Data signify the mean ± SD where each data point represents a biological replicate in all graphs. **, P < 0.01; ***, P < 0.001; ns, not significant by unpaired t test. PE, phosphatidylethanolamine.
Figure 3.
Figure 3.
Postnatal deletion of Vps34 in thymic stromal cells decreases cellularity and TEC homeostasis in a transplant model. (A) Schematic of the experimental design. Thymic lobes isolated from newborn Rosa26-CreERT2; Vps34f/f, or Vps34f/f mice were transplanted under the kidney capsule of 6–8-wk-old C57BL/6 recipient mice. Recipients were treated with tamoxifen or vehicle by oral gavage 3 wk after grafting and were analyzed 3 wk after the last tamoxifen treatment. (B) Examples of kidneys transplanted with a thymus lobe from vehicle- or tamoxifen-treated recipient mice (scale bar = 0.5 cm). The graph represents total cellularity from transplanted thymi. (C) Flow cytometric analysis of TECs isolated from vehicle- or tamoxifen-treated mice. The graph represents total TEC cellularity (CD45−EpCAM+ cells) from transplanted thymi. Data signify the mean ± SD. Data are representative of four independent experiments where each data point represents a biological replicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by unpaired t test.
Figure S2.
Figure S2.
DN thymocytes, iNKT cells, MAIT cells, and splenic T cells in neonatal Vps34TEC mice. (A) Flow cytometric analysis of CD4−CD8−Lin (CD3, B220, NK1.1)− DN thymocytes including DNI (CD44+CD25−), DNII (CD44+CD25+), DNIII (CD44−CD25+), DNIV (CD44−CD25−), and ETPs (CD44+CD25−cKit+) isolated from 3-d-old Vps34f/f and Vps34TEC mice. The graph represents total numbers of the indicated thymocyte subset per thymic lobe. Data obtained from two independent experiments (n = 3–4 biological replicates per genotype). (B and C) Splenocytes from 3-d-old Vps34f/f or Vps34TEC mice were analyzed by flow cytometry. Flow cytometric analysis of (B) CD4 and CD8 expression, and (C) Foxp3-RFP expression among CD4+ T cells (CD3+CD4+). Graphs represent frequency and total numbers of the indicated splenic subsets. Data obtained from three independent experiments (n = 5–7 mice per genotype). (D) Flow cytometric analysis of iNKT cells (PBS57/CD1d-tetramer+CD3+) isolated from the thymus of the indicated mouse strains at the indicated ages. (E) Flow cytometric analysis of MAIT cells (5-OP-RU/MR1-tetramer+CD3+) isolated from 3-d-old Vps34f/f or Vps34TEC mice. As a control, cells were stained with 6-FP/MR1-tetramers (top). Data obtained from two independent experiments with at least three biological replicates. Data signify the mean ± SD where each graphed data point represents a biological replicate. **, P < 0.01; ***, P < 0.001; ns, not significant by unpaired t test.
Figure 4.
Figure 4.
Defective CD4+ T cell development in neonatal Vps34TEC mice. Thymocytes from 3-d-old Vps34f/f or Vps34TEC mice were analyzed by flow cytometry. (A) CD4 and CD8 expression. Graphs represent frequency and total numbers of indicated thymocyte subsets. Data obtained from three independent experiments (n = 8–10 mice per genotype). (B and C) CD69 and TCRβ expression (top panel) on thymocytes was used to gate on the indicated developmental thymocyte stages, and the percentage of cells within each stage is depicted in the graph at the top. Cells within stages II–IV were then analyzed for CD4 and CD8 surface expression. Graphs represent the frequency of DP, CD4 SP, and CD8 SP thymocytes within each specified developmental stage (B) and CD24 and TCRβ expression (C). The graph represents the frequency of mature thymocytes (TCRβ+CD24lo) among the CD4 SP or CD8 SP subsets. Data for B and C obtained from two independent experiments (n = 4–5 mice per genotype). (D) Foxp3-RFP and CD25 expression of cells within the CD4 SP CD3+ gate. The graph represents the frequency of thymic regulatory T cells (Foxp3-RFP+CD25+) among CD4 SP CD3+ thymocytes. Data obtained from three independent experiments (n = 5–7 mice per genotype). Data signify the mean ± SD where each data point represents a biological replicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by unpaired t test.
Figure 5.
Figure 5.
Vps34 in TECs is critical for positive selection of MHC class II–restricted but not MHC class I–restricted transgenic TCRs. Six separate TCR transgenes were introgressed into the Vps34TEC strain and analyzed for positive selection compared with TCR transgenic Vps34f/f controls (see Table S1 for the specificities of these TCRs). (A–G) Flow cytometric analysis of thymocytes for CD4 and CD8 expression from the following TCR transgenic lines: OTI (A), P14 (B), OTII (D), LLO56 (E), LLO118 (F), and Marilyn (females only; G). Representative flow plots or histograms show clonotypic TCR expression profiles of Vps34f/f (blue line), Vps34TEC (red line), and control staining (shaded gray) of the indicated cell populations. (C) Total cell number (top) and frequency (bottom) of CD8 SP thymocytes in the indicated MHC class I–restricted TCR transgenic lines. (H) Total cell number (top) and frequency (bottom) of CD4 SP thymocytes in the indicated MHC class II–restricted TCR transgenic lines. (I) Surface CD5 expression on DP thymocytes (CD4+CD8+) from the indicated mouse strains. Data signify the mean ± SD. Data are representative of at least two independent experiments with three to six biological replicates per genotype. Each data point represents a biological replicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by unpaired t test. FMO, fluorescence minus one.
Figure S3.
Figure S3.
Splenic phenotype of T cells in TCR transgenic mice. (A) DP thymocyte frequency of 1-wk-old Vps34f/f or Vps34TEC mice transgenic for the indicated MHC class I–restricted (left) or MHC class II–restricted (right) TCRs. (B and C) Flow cytometric analysis of splenocytes for CD4 and CD8 expression from the following MHC class I–restricted TCR transgenic lines: (B) OTI and (C) P14. (D) Frequency (left) and total cell number (right) of CD8 SP cells in the indicated TCR lines. (E–H) Flow cytometric analysis of splenocytes for CD4 and CD8 expression from the following MHC class II–restricted TCR transgenic lines: (E) OTII, (F) LLO56, (G) LLO118, and (H) Marilyn (females only). (I) Frequency (left) and total cell number (right) of CD4 SP in the indicated transgenic TCR lines. (J) Marilyn thymocyte development in male Vps34TEC and Vps34f/f mice. Data signify the mean ± SD. Data are representative of at least two independent experiments (n = 3–6 mice per genotype) where each data point represents a biological replicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant by unpaired t test.
Figure 6.
Figure 6.
TCRβ chains display altered gene usage and repertoire sharing in CD4 SP thymocytes of neonatal Vps34TEC mice. The somatically rearranged TCRβ CDR3 sequences were amplified from genomic DNA isolated from CD4 SP TCRβ+ thymocytes flow sorted from 4- or 6-d-old Vps34f/f or Vps34TEC mice (n = 3/genotype). (A) Numbers of productive TCR sequences. (B) Simpson clonality indexes of TCR repertoire diversity. (C) Distribution of CDR3 lengths (χ2 test). (D) Distribution of TCR Vβ gene usage. (E) Heatmap of Morisita repertoire overlap across all samples. The graph represents the Morisita overlap index for each individual comparison organized by group. Data signify the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by unpaired t test unless otherwise indicated.
Figure S4.
Figure S4.
Vps34 deficiency attenuates the incidence and severity of active EAE. EAE was induced by active immunization with MOG35–55 peptide. (A) Daily clinical score (ANOVA). (B) Disease onset (Kaplan–Meier curves by log-rank test). (C) Peak disease score (unpaired t test). Results are accumulated from three independent experiments with a combined total of 15 mice per group. The data shown are the average ± SEM. *, P < 0.05; **, P < 0.01; dpi, days postimmunization.
Figure 7.
Figure 7.
cTECs present increased abundance of CLIP-bound I-Ab complexes in the absence of Vps34. (A–C) cTECs (CD45−EpCAM+Ly-51+) isolated from the indicated strains (isotype and FMO [fluorescence minus one] staining are from Vps34f/f isolated cells) were analyzed for (A) MHC class I (H-2Kb), (B) MHC class II (H-2I-A/I-E), or (C) CLIP-bound I-Ab expression by flow cytometry. Data are representative of at least two independent experiments (n = 4–7 per genotype). (D) Protein isolated from control and Vps34-deleted C9 cells were analyzed by Western blot for markers of autophagy and vesicle trafficking. β-Tubulin was used as loading control. The graph represents the relative densities of pre-pro-cathepsin L, pro-cathepsin L, and mature cathepsin L bands (CatL) between sgLacZ and sgVps34. Data are representative of two independent experiments. (E) Light micrograph of live control (sgLacZ) and Vps34-deleted (sgVps34) C9 cells (top, scale bar = 100 μm). Thymus tissue sections from 3-d-old Vps34f/f or Vps34TEC mice stained with H&E (bottom, scale bar = 75 μm). Arrows highlight areas of significant vacuolization. The graph represents the number of vacuolated areas per medullary islet. Data are from one randomly selected medullary islet from three independent biological replicates of each genotype. (F) sgVps34 and sgLacZ cells were stained with Lysosensor green probe and then analyzed by flow cytometry. The graph represents relative mean fluorescence intensity (MFI) of Lysosensor green. Data are representative of two independent experiments. All data signify the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; nd, none detected by unpaired t test. Source data are available for this figure: SourceData F7.
Figure S5.
Figure S5.
Vps34 regulates CD4+ T cell positive selection in a canonical autophagy-independent mechanism. (A and B) Flow cytometric analysis of (A) total TECs (CD45−EpCAM+) and (B) TEC subsets, including mTECs (UEA-1+Ly-51−) and cTECs (UEA-1−Ly-51+) on postnatal day 10. Graphs represent frequency (left) and total number (right) of TECs or TEC subsets. Data obtained from two independent experiments (n = 4 mice per genotype). (C–E) Thymocytes were analyzed for CD4 and CD8 expression by flow cytometry. (C) Analysis of 10-d-old Atg5f/f or Atg5TEC mice. Graphs represent frequency and total numbers of the indicated thymocyte subsets. Data obtained from two independent experiments (n = 5–6 mice per genotype). (D) B6 or Atg5TEC mice 5 wk after lethal irradiation following engraftment with OTII TCR transgenic bone marrow. Graphs represent frequency and total numbers of OTII cells (CD4 SP TCR Vα2hi). Data obtained from two independent experiments (n = 7 mice per group). (E) OTII TCR transgene was introgressed into the Atg5TEC strain and offspring were analyzed for positive selection compared with OTII TCR transgenic Atg5f/f controls. Data obtained from two independent experiments (n = 4 mice per genotype). Graphs represent frequency and total numbers of OTII cells (CD4 SP TCR Vα2hi). Data signify the mean ± SD where each data point represents a biological replicate. ns, not significant by unpaired t test.
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