Research ArticleIMMUNOMETABOLISM

Clonal expansion of vaccine-elicited T cells is independent of aerobic glycolysis

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Science Immunology  07 Sep 2018:
Vol. 3, Issue 27, eaas9822
DOI: 10.1126/sciimmunol.aas9822
  • Fig. 1 IL-27 is required for the CD8+ T cell response to adjuvanted subunit vaccination but not to infectious challenge.

    (A and B) Tetramer staining on splenic CD8+ T cells from control (unimmunized), C57BL/6J, and IL-27R−/− mice immunized with poly (I:C)/αCD40/OVA or Pam3Cys/αCD40/OVA or infected with 5 × 106 PFU of VV-WR at 7 dpi. ns, not significant. (C) Tetramer-positive T cells were analyzed by flow cytometry for CD122 (IL-2 receptor β-chain) expression 5 days after immunization with poly (I:C)/αCD40/OVA. Data indicate means ± SEM, n ≥ 3 mice per group, representative of five experiments.

  • Fig. 2 IL-15 is required for the generation of the primary CD8+ T cell response to vaccination but not to infectious challenge.

    (A and B) The abundance of splenic antigen-specific T cells was determined for WT or IL-15−/− mice 7 days after vaccination with poly(I:C)/αCD40/OVA or Pam3Cys/αCD40/OVA. The percentage of CD8+ T cells that were tetramer-positive (A) and the total number of tetramer-positive T cells (B) are shown for cells responding to immunization, VV-WR, or LM-OVA. (C to E) Similarly, tetramer staining was performed in mice responding to specified single adjuvants plus OVA 7 dpi. Data indicate means ± SEM, n ≥ 3 mice per group, representative of four (A and B) and three (C, D, and E) experiments. Tet, tetramer; Flag, flagellin.

  • Fig. 3 Tbet and Eomes are required for maximal CD8+ T cell responses to adjuvanted subunit vaccination.

    (A to C) Congenically marked OT-1 T cells from WT and IL-27R−/− backgrounds were cotransferred into C57BL/6J mice and harvested for flow cytometric analysis 1, 2, and 3 days after vaccination. (A) OT-1 cotransfer method schematic. (B) Flow plots show the gating strategy for WT and IL-27R−/− OT-1 cells and representative Tbet and Eomes staining. (C) The MFI of Tbet and Eomes at day 3 after immunization in WT/IL-27R−/− OT-1 cotransfers and in single transfers of WT OT-1 cells transferred into WT mice or IL-15−/− mice. (D) Tetramer staining was performed on CD8+ T cells in control (unimmunized), C57BL/6J (WT), Tbet KO, or Eomes KO (Eomesfl/fl × dLck-Cre) mice 7 dpi. Data shown are means ± SEM, n ≥ 3 mice per group, representative of three (for Tbet−/−) and four (for Eomesfl/fl × dLck-Cre) experiments.

  • Fig. 4 IL-27 and IL-15 influence IRF4 expression, a required transcription factor for the T cell responses to subunit vaccination.

    (A and B) C57BL/6J mice received 5 × 103 purified OT-1 T cells 1 day before vaccination. Total numbers of WT and IL-27R−/− OT-1 cells were analyzed at 3 and 6 days after vaccination (A), and the ratio of WT to IL-27R−/− OT-1 cells was determined for each time point (B). (C and D) Endogenous CD8+ T cells were analyzed for Kb-SIINFEKL tetramer staining 7 days after immunizing C57BL/6J mice (C), and the ratio of tetramer MFI:CD3ε MFI was determined for each of four mice per group (D). (E) C57BL/6J and IRF4 cKO (Irf4fl/fl × CD8α-Cre) mice were challenged with influenza virus and tetramer-stained 7 dpi. Representative plots show distribution of low and high tetramer staining. Graphs show MFI of tetramer on tetramer-positive events (left) and the ratio of tetramer-high versus tetramer-low events (right) in WT and IRF4 cKO mice. (F) C57BL/6J and IRF4 cKO mice were vaccinated and tetramer-stained 7 dpi. Representative plots show the percentage of tetramer staining cells out of total CD8+ T cells. The graph below shows means ± SEM, n = 3 to 4 mice per group. (G) IRF4 staining on tetramer-positive cells from WT, IL-15−/−, and IL-27R−/− mice 3 days after immunization. Data shown are means ± SEM, n = 3 mice per group, representative of three (A and B), five (C and D), two (E and F), and four (G) experiments.

  • Fig. 5 Vaccine-induced T cell responses display a metabolic program characterized by mitochondrial function, not aerobic glycolysis.

    (A) Seven days after immunization or infection with LM-OVA, relative RNA expression was determined for antigen-specific T cells sorted from C57BL/6J mice. Differentially expressed genes (adjusted P value false discovery rate (FDR) < 0.1 and fold change of 1.1 or greater) were filtered for Gene Ontology Consortium (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway association with electron transport chain, oxidative phosphorylation (Oxphos), tricarboxylic acid cycle and metabolic process, or glycolysis. Gene expression in T cells responding to LM challenge or combined adjuvant vaccine immunization T cells was determined by Affymetrix gene arrays (as described in Materials and Methods) using Transcriptome Analysis Console Software (Thermo Fisher Scientific). All differentially expressed genes (significant between vaccine- and LM-OVA–responding T cells) were next determined with a fold change cutoff at ±1.1. (B) Proteomics analyses performed on OT-1 T cells purified 5 dpi, showing proteins important to glycolysis and the Krebs cycle in vaccine-treated cells versus LM-OVA. (C) Glut-1 expression and 2-NBDG uptake on antigen-specific endogenous CD8+ T cells were tetramer-stained 5 dpi as a percentage of CD44 naïve CD8+ T cell MFI. Data are combined from two experiments, where n = 6 to 7 mice per group. (D) Representative flow plots and average gMFI of pAkt, mTor, cMyc, p70pS6K, and 4EBP1 on OT-1 T cells 5 days after vaccination (blue), infection (magenta), or in CD44 endogenous CD8+ T cells (gray). Data shown are means ± SEM, representative of ≥3 experiments. (E and F) Metabolic flux was performed on splenic OT-1 T cells purified 5 dpi with LM-OVA, or αCD40/poly(I:C)/OVA, respectively. (E) ECAR was measured at baseline, and after injection with glucose, oligomycin, and 2-DG, at indicated time points, from which baseline aerobic glycolysis and glycolytic capacity were determined. Data shown are combined from four separate experiments, n = 4 per group, where each n value equals the average of all the biological replicates from one of the four experiments. The total numbers of mice included were 22 for LM-OVA and 26 for vaccine. (F) OCR was measured at baseline and after injection of oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin and rotenone (A/R). From this, baseline ATP production and SRC were determined. Data shown are combined from three separate experiments, n = 3 per group, where each n value equals the average of all the biological replicates from one of the three experiments. Ten mice (LM-OVA) or 14 (vaccine) mice were included. Ratio paired t tests were used to determine significance for (E) and (F). (G) OT-1 T cells were purified 5 dpi and analyzed by TEM. Representative TEM images are shown. Scale bar, 0.5 μm. Cell surface area and the total number of mitochondria were quantified from 27 cells per group. The surface area of each mitochondrion from each cell was also quantified.

  • Fig. 6 Vaccine-induced augmentation of SRC is dependent on IL-27 and IL-15.

    WT OT-1 T cells were adoptively transferred into C57BL/6J or IL-15−/− recipients (WT, IL-15−/−), or IL-27R−/− OT-1 T cells were transferred into C57BL/6J recipients (IL-27R−/−) and vaccinated 1 day later. Alternatively, WT OT-1 T cells were transferred into C57BL/6J recipients and infected 1 day later (LM-OVA). OCR and ECAR were measured ex vivo on purified OT-1 cells 5 dpi (A and B). Ant/Rot, antimycin and rotenone. Baseline ATP production, SRC, aerobic glycolysis, and glycolytic capacity were determined as in Fig. 5. RNA-seq on WT and IL-27R−/− OT-1 T cells responding to subunit vaccination was performed as described in Materials and Methods (C). The resulting gene list was filtered for all differentially expressed genes (adjusted P value FDR < 0.1) with a fold change of 1.15 or greater and that are associated with the GO metabolic pathways metabolic process, trixarboxylic acid cycle, and/or electron transport chain (GO:0008152, GO:0006099, and GO:0022900). Heat map was generated using Morpheus (Broad Institute). For OCR, data are combined from two experiments, where n = 6 mice per group (all vaccination groups) and n = 3 mice (infection), and values are means ± SEM. For ECAR, data are representative from a total of three experiments each, where n = 7 (WT versus IL-27R−/−) or n = 3 (WT versus IL-15−/−).

  • Fig. 7 Glycolytic blockade reduces T cell response to infection but not to subunit vaccination.

    WT C57BL/6J mice were either immunized, infected with 1 × 107 PFU of VV-OVA, or infected with 2 × 103 CFU of LM-OVA. Starting 1 day later, mice were injected intraperitoneally (IP) with PBS or 2-DG daily. (A) Experimental schematic. (B) Spleens were harvested at 7 dpi, and tetramer staining was performed for Kb-TSYKFESV (B8R) or Kb-SIINFEKL (OVA) tetramer-positive cells in VV-WR–infected and vaccinated mice (left) or Kb-SIINFEKL tetramer-positive cells in LM-OVA–infected or vaccinated mice (right). Shown are means ± SEM from a representative experiment (VV) or two experiments combined (LM-OVA) of ≥3 experiments each, where n = 4 to 5 mice per group. The relative percentages of SLEC and MPEC subsets were assessed in tetramer-positive events (C), and the ratios of the number of SLECs to the number of MPECs were determined (D). Splenocytes were also stained 7 dpi for granzyme B and for intracellular cytokines after 5 hours of stimulation with SIINFEKL peptide (E). (F) Percentage of IFN-γ+TNFα+IL-2+ within all CD8+ T cells, making any one or more of these cytokines (left). Relative quantity of IFN-γ, TNFα, or granzyme B produced by CD8+ T cells, making the respective effector proteins normalized to percentage of max for LM or vaccine, respectively, for each protein (right). Percentages shown (C and E) are the averages from four to five mice per group.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/27/eaas9822/DC1

    Methods

    Fig. S1. In response to subunit vaccination, the formation of T cell memory is substantially reduced in an IL-15−/− host.

    Fig. S2. IL-15 is required to support survival of vaccine-elicited T cells, not for their initial expansion to antigenic challenge.

    Fig. S3. Vaccine-elicited T cells express levels of IRF4 substantially higher than in T cells responding to viral challenge.

    Fig. S4. Vaccine- and infection-elicited T cells express both overlapping and unique gene sets when compared with naïve T cells.

    Fig. S5. Vaccine-elicited T cells express predominantly a central memory phenotype as compared with T cells responding to infection.

    Fig. S6. Global gene expression differences and representative gene sets in WT versus IL-27R−/− T cells responding to subunit vaccination.

    Fig. S7. Tbet expression in vaccine-elicited T cells from WT mice is elevated compared with WT T cells responding to virus infection.

    Table S1. Raw data.

    References (6674)

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. In response to subunit vaccination, the formation of T cell memory is substantially reduced in an IL-15−/− host.
    • Fig. S2. IL-15 is required to support survival of vaccine-elicited T cells, not for their initial expansion to antigenic challenge.
    • Fig. S3. Vaccine-elicited T cells express levels of IRF4 substantially higher than in T cells responding to viral challenge.
    • Fig. S4. Vaccine- and infection-elicited T cells express both overlapping and unique gene sets when compared with naïve T cells.
    • Fig. S5. Vaccine-elicited T cells express predominantly a central memory phenotype as compared with T cells responding to infection.
    • Fig. S6. Global gene expression differences and representative gene sets in WT versus IL-27R−/− T cells responding to subunit vaccination.
    • Fig. S7. Tbet expression in vaccine-elicited T cells from WT mice is elevated compared with WT T cells responding to virus infection.
    • References (6674)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data.

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