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Specialized proteasome subunits have an essential role in the thymic selection of CD8+ T cells

Nature Immunology volume 17, pages 938–945 (2016)Cite this article

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Abstract

The cells that stimulate positive selection express specialized proteasome β-subunits different from those expressed by all other cells, including those involved in negative selection. Mice that lack all four specialized proteasome β-subunits, and therefore express only constitutive proteasomes in all cells, had a profound defect in the generation of CD8+ T cells. While a defect in positive selection would reflect an inability to generate the appropriate positively selecting peptides, a block at negative selection would point to the potential need to switch peptides between positive selection and negative selection to avoid the two processes' often cancelling each other out. We found that the block in T cell development occurred around the checkpoints of positive selection and, unexpectedly, negative selection as well.

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Figure 1: Severe defect in mature polyclonal CD8+ T cells in 4KO mice.
Figure 2: Defect in positive selection of OT-I or P14 CD8+ T cells in 4KO thymi.
Figure 3: Cells of the CD8+ lineage express CD69 and TCRβ in 4KO mice.
Figure 4: 4KO thymocytes receive positive selection signals independently of MHC class II.
Figure 5: Expression of CCR7 and markers of commitment to the CD8+ lineage on 4KO thymocytes.
Figure 6: 4KO thymocytes are lost during negative selection.
Figure 7: 'Rescue' of the development of CD8+ T cells in 4KO thymus by Bcl2l11−/− thymocytes.

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Acknowledgements

We thank D. Littman (New York University School of Medicine) for Runx3dYFP/YFP mice, and A. Singer and E. Huseby for discussions. Core resources of the University of Massachusetts were used (supported by the Diabetes Endocrinology Research Center (DK32520)). Supported by the US National Institutes of Health (AI20248 and AI110374 to K.L.R., and T32CA130807-02 to E.Z.K.), the University of Massachusetts Diabetes and Endocrine Research Center (DK32520 for K.R.) and Japan Society for the Promotion of Science KAKENHI (21000012 to K.T., and 25221102 to S.M.).

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Authors and Affiliations

  1. Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Eleanor Z Kincaid & Kenneth L Rock

  2. Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Shigeo Murata

  3. Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan

    Keiji Tanaka

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  1. Eleanor Z Kincaid
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Contributions

E.Z.K. designed and did experiments, analyzed data and wrote the paper; S.M. and K.T. generated the β5t-defidient mice and discussed results and conclusions; and K.L.R. designed experiments, supervised the experiments and wrote the paper.

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Correspondence to Kenneth L Rock.

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Integrated supplementary information

Supplementary Figure 1 Additional staining and gating of 4KO thymi.

(a) Quantitation of total thymocytes in wild-type and 4KO animals. (n = 8 mice per genotype, male and female) (b) Flow cytometry of WT and 4KO thymi. Numbers in quadrants indicate percent CD4+CD8α+ (uppser left) or CD4−CD8α− (upper right) cells among total thymocytes. (c,d) Quantitation of CD4−CD8α− (c) or CD4+CD8α+ (d) thymocytes in wild-type and 4KO mice, as in b. (e) Quantitation of CD19+B220+ cells in wild-type thymi, wild-type inguinal lymph nodes (LN), 4KO thymi, or 4KO inguinal lymph nodes. (n = 6 mice per genotype, male and female) (f) Quantitation of CD4−CD8α+TCRβhi and CD4−CD8β+TCRβhi thymocytes in wild-type (n = 9, male and female) and 4KO (n = 8, male and female) mice. (g) Flow cytometry of CD8α vs CD8β on CD4−CD8α+TCRβhi thymocytes in wild-type and 4KO mice. (h,i) Geometric mean fluorescence intensity of CD5 on CD4−CD8+TCRβhi (h) and CD4+CD8−TCRβhi (i) thymocytes. Each symbol (a,c,d,e,f,h,i) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P=0.0079 ** P<0.0001 (Student’s t-test). Data are representative of two experiments (b,g) or are pooled from two experiments (a,c,d,e,f,h,i)

Supplementary Figure 2 TCR Vβ analysis.

(a,b) Frequency of cells staining positive for each indicated Vβ among CD4+ (a) or CD8+ (b) splenocytes. (n = 8 mice per genotype, male and female). Bars indicate the mean (± s.d) * P=0.0101 ** P=0.0019 *** P=0.0008 **** P=0.0006 ***** P<0.0001 (Student’s t-test). There is no significant difference between wild-type and 4KO by Two-way ANOVA. Data are pooled from two experiments.

Supplementary Figure 3 Normal development of CD8+ T cells in mice heterozygous for deficiency in H2-Kb and H2-Db despite a decrease in MHC class I on cTECs similar to that in 4KO mice.

(a) Geometric mean fluorescence intensity of H2-Kb on wild-type (n=8, male and female), H2-Kb+/ –H2-Db+/– (n=4, male and female), and 4KO (n=4, male and female) splenocytes. (b) Quantitation of CD4−CD8+TCRβhi thymocytes in wild-type and H2-Kb+/ –H2-Db+/– mice. (c) Flow cytometry of wild-type CD45−CD326+ adherent thymocytes. Numbers in quadrants indicate percent CD249+UEA-1− (cTEC)(upper right) or CD249−UEA-1+ (mTEC)(lower left) cells among CD45−CD326+ adherent thymocytes. (d,e) Geometric mean fluorescence intensity of H2-Kb on wild-type (n=6, male and female), H2-Kb+/ –H2-Db+/– (n=3, male and female), and 4KO (n=5, male and female) mice on CD45−CD249+CD326+ (d) or CD45−CD249+CD326+ (e) cells, presented relative to that in wild-type mice, set as 100%. Each symbol (a,b,d,e) represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P<0.05 (One-way ANOVA with Dunnett’s multiple comparison post-test). Data are representative of three experiments (c), or are pooled from four (a,b) or three (d,e) experiments.

Supplementary Figure 4 4KO thymocytes receive positive selection signals independently of MHC class II.

(a) Quantification of CD69+TCRβint thymocytes in wild-type and 4KO fetal thymic organ cultures in the presence of monoclonal antibody to MHC class II (500 μg/ml). (b) Quantification of wild-type and 4KO CD4+CD8−TCRβhi thymocytes in the presence of control monoclonal antibody (LTF-2) or monoclonal antibody to MHC class II (M5/114) (each at a concentration of 500 μg/ml). (n = 20 lobes (WT + LTF-2), n = 21 lobes (WT + M5/114), n = 21 lobes (4KO + LTF-2) or n = 17 lobes (4KO + M5/114)). Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P=0.0058 and ** P=0.0012 (Student’s t-test). Data are pooled from five experiments.

Supplementary Figure 5 Alternative gating of wild-type and 4KO thymocytes with CD69 and TCRβ.

(a) Flow cytometry of wild-type and 4KO thymocytes (n = 9 mice per genotype, male and female). Numbers in outlined areas indicate percent CD69−TCRβ −cells (bottom left), CD69+TCRβint cells (top middle), CD69+TCRβ hi cells (top right), or CD69− TCRβ hi cells (bottom right) among total thymocytes. Numbers in quadrants indicate percent of cells in that quadrant and the various populations (above plots) gated as on the far left. (b,c,d) Quantitation of CD4+CD8+(b), CD4+CD8− (c) and CD4−CD8+ (d) thymocytes in wild-type (open bars) and 4KO (closed bars) mice gated as in a. Bars indicate the mean (± s.d) Data are representative of three experiments (a), or are pooled from three (b,c,d) experiments.

Supplementary Figure 6 Effects of Bim deficiency on CD4+CD8+ double-positive and CD4SP thymocytes are similar in wild-type and 4KO thymi.

(a,b) Quantitation of CD4+CD8+(a) and CD4+CD8-TCRβhi (b) thymocytes in WT→WT (n = 22), Bcl2l11−/− →WT (n = 16), WT→4KO (n = 16), or Bcl2l11−/− →4KO (n = 16) chimeras. (c) Fold increase in CD4+CD8−TCRβhi thymocytes in Bcl211−/−→WT vs WT→WT chimeras (WT), and Bcl211−/−→4KO vs. WT→4KO chimeras (4KO). (Flow cytometry shown in Fig 4) Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.d.). * P=0.0289 (Student’s t-test). Data are pooled from 5 experiments.

Supplementary Figure 7 Peptide switching allows many thymocytes to survive negative selection.

In wild-type animals, thymocytes are positively selected on peptides produced by thymoproteasomes in cTECs. Many positively selected thymocytes subsequently avoid deletion because they are negatively selected on largely different peptides, produced by constitutive, immuno or mixed proteasomes in mTECs and DCs, and consequently don’t encounter their high affinity ligands. In the 4KO mice, there is no peptide switching, and most CD8 lineage cells die during negative selection.

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Kincaid, E., Murata, S., Tanaka, K. et al. Specialized proteasome subunits have an essential role in the thymic selection of CD8+ T cells. Nat Immunol 17, 938–945 (2016). https://doi.org/10.1038/ni.3480

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