Research ArticleIMMUNOLOGICAL MEMORY

Enzymatic synthesis of core 2 O-glycans governs the tissue-trafficking potential of memory CD8+ T cells

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Science Immunology  13 Oct 2017:
Vol. 2, Issue 16, eaan6049
DOI: 10.1126/sciimmunol.aan6049
  • Fig. 1 Memory CD8+ T cells traffic into VacV-infected skin independent of antigen specificity.

    (A) Naïve B6 mice were infected with LCMV. On day 60 after LCMV infection, LCMV-immune or naïve age-matched controls were infected with VacV on the left ear skin by scarification. On day 3 after infection with VacV, trafficking of CD8+ T cells into the skin was quantified. (B and C) LCMV-specific CD8+ T cells (H2-Db-GP33–41 and H2-Db-GP276–284) were identified in the VacV-infected skin and spleen. Core 2 O-glycosylated CD43 was measured with the 1B11 antibody. (D and E) WT or Gcnt1−/− B6 mice were infected with LCMV. On day 60 after LCMV infection, mice were infected with VacV on the left ear skin. Trafficking of GP33-specific (D) and GP276-specific (E) CD8+ T cells was quantified on day 3 after infection. n.s., not significant. (F) Naïve P14 CD8+ T cells were transferred into naïve B6 mice and infected with LCMV-Armstrong. On day 75 after infection, LCMV-immune mice were challenged with VacV-GP33 on the left ear skin and VacV-OVA on the right ear skin. Trafficking of P14 CD8+ T cells into the skin was quantified on day 3 after VacV infection. (G) Quantification of (F). (H) Same as (F), except VacV was quantified from naïve or LCMV-immune mice on day 4 after infection of the skin.

  • Fig. 2 Core 2 O-glycan synthesis is stimulated by IL-15 signaling.

    (A) Memory P14 CD8+ T cells were purified and stimulated with IL-15 (250 ng/ml) or IL-15 + 5 mM BADG for 3 days. Binding of E- and P-selectin chimeric proteins was quantified. (B) Same as (A), except after IL-15 stimulation, memory P14 CD8+ T cells were treated with neuraminidase before analyzing E- and P-selectin binding. (C) The synthesis of complex core 2 O-glycans requires the collective action of a number of glycosyltransferases. (D) Memory P14 CD8+ T cells were purified and stimulated in vitro with IL-15 (500 ng/ml) for 3 days. Changes in gene expression of Gcnt1, B3gnt3, B4galt5, St3gal4, Fut7, and St3gal1 were determined by quantitative polymerase chain reaction. (E) St3gal1 caps the core 1 O-glycan substrate with α2,3-linked sialic acid, which prevents core 2 O-glycan synthesis (by Gcnt1) and can be detected with the lectin MAL II. PNA binds to core 1 O-glycans only if they do not contain an α2,3-linked sialic acid. (F) Memory P14 CD8+ T cells were stimulated as in (A), and binding of MAL II, PNA, or the 1B11 antibody was analyzed. (G) LCMV-immune mice (90 days after infection) were given control IgG or IL-15–neutralizing antibody for 10 days. PBL, peripheral blood lymphocyte. The frequency, E-selectin binding (H), and P-selectin binding (I) of the memory P14 CD8+ T cell population in the circulation were quantified. (J) Mice from (G) to (I) were infected with VacV, and memory P14 CD8+ T cells were quantified in the skin on day 3 after VacV infection.

  • Fig. 3 Core 2 O-glycan synthesis in memory CD8+ T cell subsets.

    (A) Memory P14 CD8+ T cells were analyzed 75 days after LCMV infection for expression of CD62L and KLRG1. (B to E) Memory P14 CD8+ T cell subsets from (A) were analyzed for expression of core 2 O-glycans (1B11) (B), CD122 (C), E-selectin binding (D), and P-selectin binding (E). MFI, mean fluorescence intensity. (F) 1B11 cells from each subset as shown in (A) were sorted by fluorescence-activated cell sorting and cultured in vitro for 3 days in media alone or supplemented with IL-15 (50 or 250 ng/ml). (G) Memory CD8+ T cell subsets were sorted, and 5 × 105 cells were transferred into B6 mice and challenged with CpG intranasally. Recruitment into the challenged lung and binding of E- and P-selectins were analyzed at 60 hours after challenge. (H to J) Quantification of data shown in (G).

  • Fig. 4 Memory CD8+ T cells that become terminally differentiated by multiple antigen encounters lose core 2 O-glycan synthesis activity.

    (A) Expression of CD62L and KLRG1 on primary and tertiary memory P14 CD8+ T cells 90 days after LCMV infection and (B) binding to E- and P-selectins. (C and D) Quantification of (B). (E) Expression of CD122 and CD132. (F and G) Quantification of (E). (H) Expression of total core 1 O-glycans (Jacalin) and unsialylated core 1 O-glycans (PNA). (I and J) Quantification of (H). (K) Primary and tertiary memory P14 CD8+ T cells were purified and stimulated with the indicated concentration of IL-15 for 15 min, and phosphorylation of STAT5 (Y694) was analyzed by immunoblot. (L) Primary and tertiary memory CD8+ T cells were purified and cultured with IL-15 for 3 days. Binding of MAL II, PNA, and the 1B11 antibody was analyzed. (M) Same as (L), except the glycosylation of PSGL-1 was analyzed by immunoblot. (N) Same as (L), except binding to E-selectin was analyzed by flow cytometry. (O) Quantification of (N) from four independent experiments. (P and Q) Same as (N) and (O), except for P-selectin.

  • Fig. 5 Primary memory CD8+ T cells traffic into the inflamed lung mucosa better than tertiary memory.

    (A) Primary (Thy1.1/1.2) and tertiary (Thy1.1/1.1) memory P14 CD8+ T cells were purified, and equal numbers of both were transferred into naïve B6 (Thy1.2/1.2) mice. (B) Mice from (A) were challenged with saline or CpG, and recruitment to the lung was analyzed 60 hours after challenge. (C and D) Quantification of (B). (E) Expression of core 2 O-glycans (1B11) from (B). (F) Cells from (B) were analyzed for binding to E- and P-selectins. (G and H) Quantification of (F).

  • Fig. 6 Primary memory CD8+ T cells traffic into VacV-infected skin and provide protective immunity against viral infection.

    (A) Frequencies of primary and tertiary memory P14 CD8+ T cells in individual B6 mice. (B) Mice containing equal frequencies of either primary or tertiary memory CD8+ T cells were infected with VacV-OVA on the left ear skin, and recruitment of memory GP33-specific P14 CD8+ T cells was analyzed on day 3 after infection. (C) Quantification of (B). (D) Expression of core 2 O-glycans (1B11) on primary and tertiary memory P14 CD8+ T cells in the blood and VacV-infected skin. (E) Binding to E- and P-selectins on P14 CD8+ T cells isolated from the skin. (F and G) Quantification of (E). (H) Primary and tertiary memory P14 CD8+ T cells were purified from spleens, 2 × 106 cells were transferred into naïve B6 mice, and the left ear skin was infected with VacV-GP33. Recruitment of memory P14 CD8+ T cells into the infected and control skin was analyzed on day 3 after infection. (I) Same as (H), except viral load was measured by standard plaque assay on day 4 after infection.

  • Fig. 7 Inflationary KLRG1+ TEM MCMV-specific CD8+ T cells lose core 2 O-glycan synthesis.

    (A) Naïve B6 mice were infected with MCMV. On day 120 after infection, the frequency of MCMV-specific CD8+ T cells was quantified by MCMV-specific major histocompatibility complex class I tetramers. (B and C) Expression of CD62L and KLRG1 on MCMV-specific CD8+ T cells from (A). (D) Core 2 O-glycan expression (1B11) from (A). (E) CD8+ T cells of MCMV-infected mice were purified and stimulated in vitro with IL-15 (250 ng/ml) for 3 days. Expression of core 2 O-glycans (1B11) was analyzed. (F) MCMV-infected mice were infected with VacV on the left ear skin. Frequency of M57- and M38-specific CD8+ T in the blood and skin on day 5 after VacV infection was quantified. (G) Quantification of trafficking efficiency from (F). (H) Same as (F). Expression of KLRG1 on conventional (M45-specific) and inflationary (M38-specific) CD8+ T cells from the blood and skin after VacV infection. (I) Quantification of (H).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaan6049/DC1

    Materials and Methods

    Fig. S1. Trafficking of memory CD8+ T cells into VacV-infected skin.

    Fig. S2. CD8+ T cells require Gcnt1 to synthesize core 2 O-glycans for binding to E- and P-selectins.

    Fig. S3. Trafficking of memory CD8+ T cells into VacV-infected skin requires interactions with E- and P-selectins.

    Fig. S4. IL-15 regulates E- and P-selectin binding during VacV infection.

    Fig. S5. TCM CD8+ T cells synthesize core 2 O-glycans and maintain expression of CD62L.

    Fig. S6. Phenotype of terminally differentiated tertiary memory CD8+ T cells using repetitive LCMV-Armstrong infection.

    Fig. S7. Primary memory CD8+ T cells are recruited into the skin better than tertiary memory CD8+ T cells during contact hypersensitivity.

    Table S1. Primer pairs for gene expression analysis in Fig. 2 and fig. S4.

    Table S2. Raw data sets and statistical analyses.

  • Supplementary Materials

    Supplementary Material for:

    Enzymatic synthesis of core 2 O-glycans governs the tissue-trafficking potential of memory CD8+ T cells

    Jossef F. Osborn, Jana L. Mooster, Samuel J. Hobbs, Michael W. Munks, Conrad Barry, John T. Harty, Ann B. Hill, Jeffrey C. Nolz*

    *Corresponding author. Email: nolz{at}ohsu.edu

    Published 13 October 2017, Sci. Immunol. 2, eaan6049 (2017)
    DOI: 10.1126/sciimmunol.aan6049

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Trafficking of memory CD8+ T cells into VacV-infected skin.
    • Fig. S2. CD8+ T cells require Gcnt1 to synthesize core 2 O-glycans for binding to E- and P-selectins.
    • Fig. S3. Trafficking of memory CD8+ T cells into VacV-infected skin requires interactions with E- and P-selectins.
    • Fig. S4. IL-15 regulates E- and P-selectin binding during VacV infection.
    • Fig. S5. TCM CD8+ T cells synthesize core 2 O-glycans and maintain expression of CD62L.
    • Fig. S6. Phenotype of terminally differentiated tertiary memory CD8+ T cells using repetitive LCMV-Armstrong infection.
    • Fig. S7. Primary memory CD8+ T cells are recruited into the skin better than tertiary memory CD8+ T cells during contact hypersensitivity.
    • Table S1. Primer pairs for gene expression analysis in Fig. 2 and fig. S4.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data sets and statistical analyses..

    Files in this Data Supplement:

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