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. 2021 Apr 5;218(4):e20201904.
doi: 10.1084/jem.20201904.

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection

Izumi Ohigashi  1 , Melina Frantzeskakis  2 , Alison Jacques  2 , Sayumi Fujimori  1 , Aya Ushio  2 , Fusano Yamashita  1 , Naozumi Ishimaru  3 , Da Yin  4 , Margaret Cam  4 , Michael C Kelly  5 , Parirokh Awasthi  6 , Kensuke Takada  7 , Yousuke Takahama  2
Affiliations

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection

Izumi Ohigashi et al. J Exp Med. .

Abstract

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.

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Conflict of interest statement

Disclosures: The authors declare no competing interests exist.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
The thymoproteasome affects V(D)J sequences of TCRα and TCRβ chains in polyclonal CD8+ T cells. (A) Numbers (means and SEMs, n = 4 in two independent measurements) of TCR sequences assigned to in-frame V(D)J-rearranged TCRs in 106 CD8+ T cells from four individual β5t+/− (Het) mice and four individual β5t−/− (KO) mice. (B) Numbers (means and SEMs, n = 4) of unique TCR reads (left) and Shannon-Weaver diversity indexes of TCR repertoire diversity (right) in CD8+ T cells from Het mice and KO mice. (C) Frequencies (means and SEMs) of CDR3 length in CD8+ T cells from Het mice and KO mice. Statistically significant increases and decreases of frequencies in KO cells are highlighted by red and blue asterisks, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired t test). (D) Circos plots showing the use of V-J combinations in CD8+ T cells from Het mice and KO mice. Statistically significant difference in frequencies between Het (blue) and KO (red) groups is highlighted by letters in blue or red. (E) Venn diagrams showing unique and overlapped numbers of TCR full-length sequences shared in all four Het mice (1,361 TCRα genes and 354 TCRβ genes) and all four KO mice (3,489 TCRα genes and 1,797 TCRβ genes). (F) Quantitative RT-PCR analysis of mRNA expression levels (means and SEMs, n = 7 in seven independent measurements) of indicated TCR full-length sequences relative to TCR-Cα or TCR-Cβ levels in CD4+CD8+ thymocytes isolated from Het mice. Statistically significant increases and decreases of mRNA levels in KO cells are highlighted by red and blue asterisks, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired t test with Welch’s correction for unequal variances).
Figure S1.
Figure S1.
The use of TCRα and TCRβ V regions detected in CD8+ T cells from β5t-deficient mice. (A and B) Frequencies (means and SEMs) of the use of TCRα V regions (A) and TCRβ V regions (B) detected in CD8+ T cells from β5t+/− (Het) mice and β5t−/− (KO) mice. Statistically significant increases and decreases of frequencies in KO cells are highlighted by red and blue asterisks, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by unpaired t test).
Figure 2.
Figure 2.
The thymoproteasome shapes the TCR repertoire of positively selected thymocytes in the thymic cortex before migration to the thymic medulla. (A) Flow cytometric analysis of thymocytes from TCRα-KO mice, TCRα#1-Tg TCRα-KO mice, and TCRα#2-Tg TCRα-KO mice at 6 wk old. Total viable thymocyte numbers (means and SEMs, n = 5–7 in three independent measurements) are listed. Shown are CD8 and CD4 profiles of total viable thymocytes (left), intracellular (IC) TCRβ histograms in CD4+CD8+ thymocytes (middle), and surface Vα8.3 expression in CD4+CD8+ surface TCRβhigh thymocytes (right). Background fluorescence histograms are also shown (shaded). Numbers in plots indicate the frequency of cells within indicated area. (B–D) Flow cytometric analysis of thymocytes from bone marrow (BM) chimera mice. BM cells from TCRα#1-Tg TCRα-KO or TCRα#2-Tg TCRα-KO mice (CD45.1−CD45.2+) were transferred into lethally irradiated β5t-sufficient (control [Ctrl]) or β5t-deficient (KO) mice (CD45.1+CD45.2−). (B) Representative profiles of reconstituted CD45.1−CD45.2+ thymocytes were gated as indicated and analyzed for cell-surface expression of indicated molecules. Numbers in plots indicate the frequency of cells within the indicated area. (C and D) Frequencies (means and SEMs, n = 8 in seven independent measurements) of indicated thymocyte subpopulations in CD45.1−CD45.2+ thymocytes derived from TCRα#1-Tg TCRα-KO (C) and TCRα#2-Tg TCRα-KO (D) BM cells are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by paired t test).
Figure S2.
Figure S2.
Flow cytometric analysis of spleen cells from indicated bone marrow chimera mice. Total viable cell numbers (means and SEMs, n = 5–6 in four independent measurements) are listed. Representative profiles of reconstituted CD45.1−CD45.2+ spleen cells were gated as indicated and analyzed for cell-surface expression of indicated molecules. Numbers in plots indicate frequency of cells within indicated area. Ctrl, control.
Figure S3.
Figure S3.
Flow cytometric profiles of CD62L, CD69, CCR7, Vα8.3, and TCRβ expressed by thymocyte subpopulations from bone marrow chimera mice.(A) Flow cytometric analysis of CD62L, CD69, and CCR7 expressed by CD4−CD8+ TCRβhigh thymocytes from indicated bone marrow chimera mice and by CD4−CD8+ TCRβhigh and CD4−CD8+ TCRβnegative/low thymocytes from B6 mice. Numbers in histograms indicate mean fluorescence intensity. (B) Flow cytometric analysis of Vα8.3 and TCRβ expressed by CD4−CD8+ CD62Lhigh thymocytes from indicated bone marrow chimera mice and by CD4−CD8+ CD62Lhigh and total thymocytes from B6 mice. Background fluorescence histograms are shown in gray. Representative results of n = 4 in two independent measurements are shown. Ctrl, control.
Figure 3.
Figure 3.
Single-cell RNA sequencing analysis of thymocyte subpopulations. (A) t-Distributed stochastic neighbor embedding plots of indicated thymocyte subpopulations isolated from β5t-Het I-Aβ-KO and β5t-KO I-Aβ-KO mice at 6 wk old (n = 2 in two independent measurements). I-Aβ-KO mice, lacking cell-surface MHC-II expression, were used to specifically detect MHC-I–dependent selection and development of CD4+CD8+ (DP) immature thymocytes into CD4−CD8+ mature thymocytes. (B and C) Feature plots for indicated genes encoding intracellular molecules (B) and cell-surface molecules (C).
Figure S4.
Figure S4.
CD5 and CD8 expression in CD4+CD8+ CD69+CCR7− thymocytes. Mean fluorescence intensity (MFI; means and SEMs, n = 4 in two independent measurements) of cell-surface CD5 and CD8 expression in CD4+CD8+ CD69+CCR7− Vα8.3+ thymocytes from TCRα#1-transgenic, TCRα-deficient mice and TCRα#2-transgenic, TCRα-deficient mice (by unpaired t test).
Figure 4.
Figure 4.
The thymoproteasome optimizes CD8+ T cell production in the absence of the thymic medulla. (A) Flow cytometric analysis of Liberase-digested thymic cells from 2-wk-old relB-deficient mice. Dot plots show EpCAM and CD45 expression in total thymic cells (left) and UEA1 reactivity and Ly51 expression in CD45−EpCAM+-gated epithelial cells (right). Numbers in dot plots indicate the frequency of cells within the indicated area. (B) Cell number (means and SEMs, n = 9 in four independent measurements) of CD45−EpCAM+UEA1−Ly51+ cTECs and CD45−EpCAM+ UEA1+Ly51− mTECs. ***, P < 0.001 (by unpaired t test with Welch’s correction). (C) Immunofluorescence analysis of thymic sections from 2-wk-old relB-KO mice. β5t (green), UEA1 reactivity (blue), and Aire (red). Representative data from three independent experiments are shown. Scale bars, 100 µm. (D) Hematoxylin and eosin–stained liver sections from indicated mice. Representative results of at least three independent experiments. Scale bar, 100 µm. (E) Inflammation grades in indicated tissues from control mice and relB-KO mice. (F) Flow cytometric analysis of thymocytes from relB-KO mice and relB/β5t-double KO (DKO) mice at 1 wk old. Cell number (means and SEMs, n = 10 in seven independent measurements) of indicated thymocyte populations. **, P < 0.01 (by unpaired t test with Welch’s correction). Histograms for TCRβ expression in propidium iodide (PI)− viable cells and dot plots for CD8 and CD4 expression in PI− TCRβhigh cells are also shown. Numbers indicate the frequency of cells within the indicated area. wo, wk old.
Figure S5.
Figure S5.
Thymocyte profiles and tissue inflammation grades in β5t-deficient and relB-deficient mice. (A–C) Flow cytometric analysis of thymocytes from indicated mice at 1 wk old (A) and 2 wk old (B and C). Cell number (means and SEMs, n = 9–17 in five to seven independent measurements) of indicated thymocyte populations. ***, P < 0.001 (by unpaired t test with Welch’s correction). Histograms for TCRβ expression in PI− viable cells and dot plots for CD8 and CD4 expression in PI− TCRβhigh cells are also shown. Numbers indicate the frequency of cells within the indicated area. (D) Inflammation grades in indicated tissues from relB/β5t-double KO (DKO) mice. wo, wk old.
Figure 5.
Figure 5.
The thymoproteasome optimizes CD8+ T cell production in the absence of additional antigen-presenting cells. (A) MHC-I expression in indicated cell populations from 2-wk-old B6 mice. Histograms show the expression of H-2Kb (blue) and background fluorescence (shaded) in cTECs, mTECs, PDGFRβ+CD45−EpCAM− fibroblasts, PDGFRβ−CD45−EpCAM− other nonhematopoietic cells, CD11c+ DCs, CD19+ B cells, TCRβhigh thymocytes, and CD11c− CD19−TCRβnegative-low other hematopoietic cells. Numbers in histograms indicate median fluorescence intensity (MFI) for H-2Kb (blue) and background (black). Plots (means and SEMs, n = 3–4 in three independent measurements) show net MFI values for H-2Kb and H-2Db. (B–D) dGuo-treated fetal thymuses from control mice, β5t-KO mice, relB-KO mice, and double KO (DKO) mice were reconstituted with β2m-KO fetal thymocytes cultured for 7 d. (B) Net MFI values (means and SEMs, n = 3–16 in at least two independent measurements) for H-2Kb (top) and H-2Db (bottom) in indicated cell populations are shown. Heterozygous or homozygous β5t-Venus knock-in knock-out allele was included, so that Venus+ TECs represented β5t-expressing cTECs. (C and D) Contour plots for CD8β and CD4 expression in PI− TCRβhigh thymocytes from indicated fetal thymus organ cultures are shown. Numbers in plots indicate the frequency of cells within the indicated area. Cell numbers (means and SEMs; n = 8–12 in C, n = 11–16 in D in four independent measurements) of indicated thymocyte populations measured in four independent experiments are plotted. *, P < 0.05 (by unpaired t test with Welch’s correction).
Figure 6.
Figure 6.
Contribution of negative selection in thymoproteasome-dependent production of CD8+ T cells. (A) Flow cytometric analysis of viable thymocytes from indicated mice at 4–10 wk old. Total viable cell numbers (means and SEMs, n = 8–9 in seven independent measurements) are listed. The frequency of cells within the indicated area is also shown. (B and C) Generation of indicated thymocyte subpopulations in Bcl2-Tg–negative mice (B) and Bcl2-Tg–positive mice (C) is shown as the frequency of cell numbers relative to the numbers of CD4+CD8+ CD69−CCR7− immature thymocytes. (D and E) Differences (D) and fold changes (E) in cell numbers of indicated thymocyte subpopulations between Bcl2-Tg–positive and –negative mice are shown. **, P < 0.01; ***, P < 0.001 (by paired t test).
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