N-glycosylation bidirectionally extends the boundaries of thymocyte positive selection by decoupling Lck from Ca2+ signaling (original) (raw)

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Acknowledgements

We thank B. Andersen for _K14_-Cre mice and K.H. Khachikyan for helping with experiments. We thank the members of M.D.'s lab for proofreading the manuscript. Supported by the US National Institutes of Health through the National Institute of Allergy and Infectious Disease (R01AI053331 to M.D.) and the National Heart, Lung, and Blood Institute (F30HL108451 to H.M.).

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Authors and Affiliations

  1. Department of Neurology, University of California, Irvine, Irvine, California, USA
    Raymond W Zhou, Ani Grigorian, Amanda Hong, David Chen, Araz Arakelyan & Michael Demetriou
  2. Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California, USA
    Raymond W Zhou, Haik Mkhikian & Michael Demetriou
  3. Institute for Immunology, University of California, Irvine, Irvine, California, USA
    Raymond W Zhou, Haik Mkhikian, Ani Grigorian & Michael Demetriou

Authors

  1. Raymond W Zhou
  2. Haik Mkhikian
  3. Ani Grigorian
  4. Amanda Hong
  5. David Chen
  6. Araz Arakelyan
  7. Michael Demetriou

Contributions

R.W.Z. performed all experiments with the assistance of H.M., A.H., D.C., A.G. and A.A. M.D. wrote the paper with assistance from R.W.Z.

Corresponding author

Correspondence toMichael Demetriou.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 N-glycan branching regulates T cell development.

(a) The Golgi N-glycan GlcNAc branching pathway. The N-acetylglucosaminyltransferases Mgat1, 2, 4, and 5 act sequentially to generate N-acetylglucosamine (GlcNAc) branched N-glycans on glycoproteins transiting the Golgi. These are variably extended with galactose to generate N-acetyllactosamine units. Galectins bind N-acetyllactosamine, with avidity increasing with the number of N-acetyllactosamine units (i.e., branching). Loss of Mgat1 blocks all branching and results in only high-mannose type N-glycans. The plant lectin L-PHA binds tri- and tetra-antennary GlcNAc branched N-glycans and serves as a marker for Mgat1 deleted cells. GalT3, galactosyltransferase3; iGnT, β1,3 N-acetylglucosaminyl transferase; MII/MIIx, mannosidase II/IIx. (b) Flow cytometric analysis of ConA binding on wildtype thymic subpopulations, normalized to CD4SP thymocytes. NS, not significant, *P<0.05. P-values by unpaired t-test (2-tailed) with Welch’s and Bonferroni corrections. Data are one experiment representative of three (b; mean ± s.e.m. of three technical replicates) independent experiments.

Supplementary Figure 2 Characterization of Mgat1-deficient mice.

(a) Histology of the spleen. (b) Quantification of DN, DP, CD4, CD8 SP thymocyte and splenic CD4 and CD8 cells. Each symbol represents one mouse; small horizontal lines indicate the mean (n=10 mice). (c) Flow cytometric analysis of surface CD24 expression on thymocytes. (d) Flow cytometric analysis of 7-AAD staining of total thymocytes after one day culture at rest. Error bars indicate s.e.m. of three technical replicates. (e) Pictures of mice (left) revealing lack of gross skin defects and flow cytometric analysis of skin epithelial cells (right) from Mgat1 f/f and Mgat1 _f/f_K14-Cre+ mouse confirming deletion of Mgat1 in Mgat1 _f/f_K14-Cre+ mouse. Numbers indicate the mean percent cells of indicated areas. (f) Percentage of L-PHA− (left) and Annexin V+ (right) ex vivo splenic T cells after 56 days doxycycline treatment provided in the drinking water; error bars indicate s.e.m. of three technical replicates. NS, not significant, *P<0.05, ***P<0.001. P-values by unpaired t-test (1-tailed) with Welch’s corrections. Data are one experiment representative of three (a, c), or two (d, e) independent experiment, or pooled from 10 (b) independent experiments.

Supplementary Figure 3 Characterization of Mgat2-deficient mice.

(a) Total number (left) of thymocytes (n=15 mice) and splenocytes (n=7 mice) from 4-8 weeks old mice; quantification (right) of thymic and splenic subpopulations (n=6 mice). Each symbol represents one mouse; small horizontal lines indicate the mean. (b) Flow cytometric analysis of CD4 and CD8 expression (left) and L-PHA binding (right) of thymocytes and splenocytes. Numbers indicate the mean percent cells of indicated areas. (c) Flow cytometric analysis of Annexin V binding on ex vivo thymocytes; DP thymocytes were gated for analysis. NS, not significant, *P<0.05, **P<0.01, ***P<0.001. P-values by unpaired t-test (1-tailed) with Welch’s corrections. Data are pooled from 15 (a) independent experiments, and one experiment representative of six (b; mean of three technical replicates) or three (c; mean ± s.e.m of three technical replicates) experiments.

Supplementary Figure 4 Characterization of Mgat1-deficient mice and non-TCR induced death.

(a) Flow cytometric analysis of CD4 and CD8 expression (left) and L-PHA binding of thymocytes and splenocytes (right). Numbers indicate the mean percent cells of indicated areas. Note that although Mgat1 is expected to be deleted at the DP stage in Mgat1 f/f CD4-Cre+ mice, loss of cell surface branching is delayed until the SP stage due to the time required for membrane turnover of cell surface glycoproteins. (b) Flow cytometric analysis of cell surface expression of IL-7R on thymic subpopulations. (c-e) Annexin V+ DP thymocytes following one day of culture at rest in the presence of dexamethasone (c), TNF (d), and α-Fas (e); gated on DP thymocytes for analysis. Data for each genotype are normalized to its own non-treated control. Relative Annexin V+ cells (%) = % Annexin V+treated – % Annexin+non-treated. Data are one representative experiment of four (a, mean of three technical replicates) or two (b-e; mean ± s.e.m. of three technical replicates) independent experiments.

Supplementary Figure 5 Characterization of cell death.

(a-e) Flow cytometric analysis of Annexin V and L-PHA binding on gated DP thymocytes cultured at rest (a, c), with and without z-VAD-FMK for one day (b), or with and without 40 mM GlcNAc, 10 mM Uridine, and 10 μM Kifunensine for two days (d, e). (f, g) Flow cytometric analysis of L-PHA and Annexin V binding on thymocyte without (f) and with ConA binding (g); DP thymocytes were gated for analysis. (h) Immunoblot of total Bcl-xL, Bax, Bim(EL), and Mcl-1 in ex vivo thymocytes. (i) Flow cytometric analysis of cell surface expression of CD69 and TCRβ in total thymocytes. Numbers indicate the mean percent cells of indicated areas. (j) Annexin V+ DP thymocytes after one day of culture with and without PMA and Ionomycin. NS, not significant, *P<0.05, **P<0.01, ***P<0.001. P-values by unpaired (a-c, j) or paired (d, e, g) t-test (1-tailed) with Welch’s and Bonferroni (d, e) corrections. Data are one experiment representative of three (a, b, f-h, j; mean (± s.e.m.) of three technical replicates), two (c-e; mean ± s.e.m. of three technical replicates) or four (i) independent experiments.

Supplementary Figure 6 Characterization of CD4, CD8 expression and cell death in Mgat1- and Mgat2-deficient thymocytes.

(a) Real-time RT-PCR analysis of Cd4 and Cd8α mRNA expression in total thymocytes; results are presented relative to Actb expression. (b) Immunoblot of total CD4 and CD8α of ex vivo thymocytes. (c) Flow cytometric analysis of CD4 and CD8α on gated Annexin V+ and Annexin V- DP thymocytes from Mgat1 _f/f_Lck-Cre+ mouse after one day culture at rest. (d, e) Flow cytometric analysis of surface CD4 and CD8α expression on ex vivo Mgat2 _f/f_Lck-Cre+ and Mgat5 −/− DP (d, e) and SP (d) thymocytes; gated on L-PHAlo cells for Mgat2 _f/f_Lck-Cre+ mice for analysis; data were normalized to control and each symbol represents one mouse, small horizontal lines indicate the mean (d). (f) Nur77+ DP thymocytes following one day stimulation by plate-bound α-CD3ɛ plus α-CD28. (g, i, j) Flow cytometric analysis of Annexin V binding on gated DP thymocytes after one day culture in the presence of PMA and Ionomycin (g), Ionomycin (i), or stimulated by α-CD3ɛ (10 μg/ml) plus α-CD28 (50 μg/ml) with cyclosporine A (j). (h) Annexin V+ ex vivo thymocytes, gated on T3.70+CD8+ cells for analysis. NS, not significant, *P<0.05, **P<0.01, ***P<0.001. P-values by unpaired t-test (1-tailed) with Welch’s corrections. Data are one experiment representative of three (a-c, g, h), or two (e, f, i, j) independent experiments (mean ± s.e.m. of three technical replicates).

Supplementary Figure 7 A model for N-glycan branching control of positive selection.

(a) A model for the interaction of branching with pMHC-TCR affinity in controlling positive selection. N-glycosylation provides a novel sliding scale that dynamically expands the range for positive selection in two directions by differentially controlling both the lower and upper limits of affinity from which pMHC-TCR interactions positively select T cells. (b) In response to low affinity pMHC, branching promotes positive selection by enhancing cell surface retention of the CD4 and CD8 co-receptors, which stabilize binding of low-affinity pMHC to TCR and enhance recruitment of Lck to TCR for low level signaling. Reduced branching disrupts binding of glycoproteins to galectins, leading to CD4/CD8 endocytosis, non-responsiveness to low affinity pMHC and death by neglect. In response to high affinity pMHC, high branching limits TCR clustering and downstream signaling to promote positive selection. Reduced branching disrupts galectin-TCR interactions, thereby promoting TCR clustering/signaling, and Ca2+ flux in response to high affinity pMHC, therefore enhancing negative selection.

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Zhou, R., Mkhikian, H., Grigorian, A. et al. N-glycosylation bidirectionally extends the boundaries of thymocyte positive selection by decoupling Lck from Ca2+ signaling.Nat Immunol 15, 1038–1045 (2014). https://doi.org/10.1038/ni.3007

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