Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells - PubMed (original) (raw)

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

Roberto Tinoco et al. J Immunol. 2018.

Abstract

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus-specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7-/- effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7-/- CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells.

PubMed Disclaimer

Figures

FIGURE 1.

P-selectin expression and P-selectin binding are increased during influenza viral infection in the lung. (A) WT mice were infected with PR8-OVAII i.n. and their lungs isolated at the indicated time points. Representative sections shown are at original magnification ×20 and black arrows indicate P-selectin staining (brown). (B) Inflammation score and P-selectin staining in lungs. (C) Number of P-selectin binding (Psel+) and nonbinding (Psel−) OT-II T cells at 8 dpi. (D) P-selectin binding by dividing cells in dLN at 6 dpi, gated on CD4+CD45.1+ cells. (E) Cytokine production by Psel− and Psel+ OT-II T cells in dLN at 8 dpi, gated on CD4+CD45.1+Psel− or CD4+CD45.1+Psel−. Data are representative of three independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05.

FIGURE 2.

Fucosyltransferase activity is upregulated and maintained on subsets of effector and memory CD4+ T cells. (A) P-selectin binding in OT-II CD4+ T cells in the indicated organs during PR8-OVAII influenza viral infection. (B) Representative FACS plots showing P-selectin binding and IL-7Rα expression in the indicated tissues during influenza viral infection, gated on CD4+CD45.1+ cells. Data are representative of three independent experiments with ≥5 mice per group. Error bars indicate SEM.

FIGURE 3.

Fucosyltransferase-deficient CD4+ T cells can respond during primary influenza infection. (A) WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. CD4+ T cell frequencies in lung, dLN, and spleen are shown at the indicated time points postinfection. The absolute numbers of WT and Fut4/7−/− OT-II T cells are shown at 8 dpi (B) and 21 dpi (C). WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of three independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05, **p < 0.005, and ***p < 0.001.

FIGURE 4.

CD4+ T cells deficient in fucosyltransferase activity can home to tissues and proliferate like WT T cells during primary influenza viral infection. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. (A) WT and Fut4/7−/− OT-II effector T cell infiltration in the indicated tissues 4 dpi and (B) 6 dpi. (C) BrdU incorporation in WT and Fut4/7−/− T cells at 8 dpi and representative FACS plots (D). WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of two independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as ***p < 0.001.

FIGURE 5.

Fucosyltransferase-deficient CD4+ T cells had increased apoptosis and similar cytokine production. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. and tissues analyzed at 8 dpi (A–E). (A) Frequencies of Caspase3+ cells in lung. (B) Frequencies of Caspase3+ and propidium iodide+ cells in dLN and spleen. (C) OT-II T cell positioning in lung vasculature and parenchyma after in vivo Ab labeling. Cytokine production in the lung (D) and dLN and spleen (E). Data are representative of two (A and B) or three (C–E) independent experiments with ≥5 mice per group. WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05 and **p < 0.005.

FIGURE 6.

Fucosyltransferase-deficient CD4+ T cells cannot engage P-selectin and have reduced T-bet and NKG2A/C/E expression. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. (A) Frequencies of NKG2A/C/E and P-selectin binding at 8 dpi. (B) Frequencies of T-bet and P-selectin binding at 8 dpi. WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of two independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as **p < 0.005 and ***p < 0.001.

FIGURE 7.

Defective secondary expansion of fucosyltransferase-deficient memory CD4+ T cells during secondary influenza infection. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. (A) Frequencies of WT and Fut4/7−/− OT-II expansion in lung, dLN, and spleen during primary influenza infection at the indicated time points. (B) PR8-OVAII immune mice were reinfected with ×31-OVAII. Frequencies of WT and Fut4/7−/− memory T cell expansion postinfection in lung, dLN, and spleen at the indicated time points. WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of three independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05 and **p < 0.005.

FIGURE 8.

CD4+ T cells require fucosyltransferase activity to generate tissue-resident memory cells. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred into WT mice and infected with PR8-OVAII i.n. to generate memory T cells and tissues analyzed at 30 dpi. (A) Memory T cell subsets were quantified based on TCM (CD62L+), TEM (CD62L−), and TRM (CD62L−CD69+CXCR3+) phenotypes and representative FACS plots are shown. (B) Frequencies of memory T cells expressing LFA-1, β1-integrin, and α1-integrin. WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of three independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05, **p < 0.005, and ***p < 0.001.

FIGURE 9.

Fut4/7−/− CD4+ T cells fail to accumulate after secondary infection. WT and Fut4/7−/− OT-II CD4+ T cells were cotransferred in WT mice and infected with PR8-OVAII i.n. to generate memory T cells. (A) Memory T cells were sorted from the spleen at 30 dpi and cotransferred at 1:1 ratio in naive WT mice that were subsequently infected with PR8-OVAII. (B) Frequencies of WT or Fut4/7−/− OT-II T cells at 9 and 20 dpi in the indicated tissues. (C) Absolute numbers of WT and Fut4/7−/− OT-II T cells in lung, dLN, and spleen at the indicated time points. (D) BrdU incorporation in dLN at 6 dpi. WT cells were gated by CD4+CD45.1+ and Fut4/7−/− cells by CD4+Thy1.1+ staining. Data are representative of two independent experiments with ≥5 mice per group. Error bars indicate SEM. A two-tailed unpaired Student t test statistical analysis was used to compare outcomes. Significance was set to p < 0.05 and represented as *p < 0.05, **p < 0.005, and ***p < 0.001.

References

1. Kreijtz J. H., Fouchier R. A., Rimmelzwaan G. F. 2011. Immune responses to influenza virus infection. Virus Res. 162: 19–30. -PubMed
1. Strutt T. M., McKinstry K. K., Marshall N. B., Vong A. M., Dutton R. W., Swain S. L. 2013. Multipronged CD4(+) T-cell effector and memory responses cooperate to provide potent immunity against respiratory virus. Immunol. Rev. 255: 149–164. -PMC -PubMed
1. McEver R. P. 2015. Selectins: initiators of leucocyte adhesion and signalling at the vascular wall. Cardiovasc. Res. 107: 331–339. -PMC -PubMed
1. Carlow D. A., Gossens K., Naus S., Veerman K. M., Seo W., Ziltener H. J. 2009. PSGL-1 function in immunity and steady state homeostasis. Immunol. Rev. 230: 75–96. -PubMed
1. Lord G. M., Rao R. M., Choe H., Sullivan B. M., Lichtman A. H., Luscinskas F. W., Glimcher L. H. 2005. T-bet is required for optimal proinflammatory CD4+ T-cell trafficking. Blood 106: 3432–3439. -PMC -PubMed

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells - PubMed (original) (raw)