Research ArticleIMMUNOLOGICAL MEMORY

Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids

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Science Immunology  17 Mar 2017:
Vol. 2, Issue 9, eaaj1996
DOI: 10.1126/sciimmunol.aaj1996
  • Fig. 1 LFA-1–ICAM-1 interactions are required for CD8+ T cell motility in the liver.

    (A) Two hours before the transfer of 7 × 106 in vitro activated OT-I T cells, mice (WT or mT/mG) were treated with blocking antibodies (Abs) to ICAM-1. Four hours after cell transfer, mice were prepared for intravital imaging and imaged by two-photon microscopy using a standard galvanometer scanner to acquire a 50-μm-deep Z-stack about every 30 s. Representative images from time-lapse imaging of mT/mG mice either with or without anti–ICAM-1 are shown. Scale bar, 30 μm. (B) Movement parameters of OT-I cells after anti–ICAM-1 treatment; data pooled from four experiments and analyzed using LMMs with experiment and mouse as random effects and speed, meandering index, or arrest as the fixed effects. Means and SD are shown. (C) In vitro activated Itgal−/− OT-I T cells (7 × 106) [labeled with CellTrace Violet (CTV)] and 7 × 106 in vitro activated GFP+ WT OT-I+ T cells were cotransferred to WT recipient mice. Mice were imaged as in (A); the image shows a representative frame from a time-lapse movie showing tracks of the Itgal−/− (yellow) and WT T cells (white); scale bar, 50 μm. (D) Movement parameters of Itgal−/− and WT cells in the livers of naive recipient mice, as described in (C); data are pooled from three mice in two independent experiments and analyzed as in (B). (E) In vitro activated Itgal−/− OT-I CD8+ T cells (2 × 106) (labeled with CTV) and 2 × 106 in vitro activated GFP+ WT OT-I CD8+ T cells were cotransferred to WT recipient mice. Twenty-four hours later, the blood, lymph nodes (LNs), spleen, liver, and lungs were harvested, and the proportion of WT and Itgal−/− cells in each organ was determined by flow cytometry (representative plots all from the same mouse shown). (F) Summary data for the proportions of WT and Itgal−/− cells in organs harvested from five mice in one of two similar independent experiments, analyzed by one-sample t test (compared with the input proportions of WT and Itgal−/− cells). Means and SD are presented. ***P < 0.001.

  • Fig. 2 Itgal−/− cells do not efficiently protect against sporozoite challenge.

    (A) Itgal−/− or littermate WT OT-I T cells (2 × 106) were transferred to C57BL/6 mice 1 day before mice were challenged with 5 × 103 P. berghei CS5M sporozoites. Twenty-four hours after challenge, livers were harvested from the recipient mice and controls, and the parasite load was assessed by quantitative reverse transcription polymerase chain reaction. Data are from one of two similar experiments with five to seven mice per group, assessed by one-way ANOVA with Tukey’s post-test for multiple comparisons. Means and SD of log-transformed data are presented. rRNA, ribosomal RNA; A.U., arbitrary units. *P < 0.05, ****P < 0.0001. (B) EL4 target cell killing after incubation with in vitro activated Itgal−/− or littermate WT OT-I T cells. Data are expressed as the number of live-pulsed target cells recovered compared with the number of live-unpulsed target cells after 6 hours. Means and SD are based on three technical replicates, from one of two experiments, and P value is the probability that the median inhibitory concentration values are different (extra sum-of-squares F test). (C) Mice were infected with 1.5 × 105 P. berghei CS5M-GFP sporozoites; 15 hours later, the mice received either 7 × 106 Itgal−/− or littermate WT OT-I T cells labeled with CellTrace Violet; 20 hours after infection, the mice were prepared for imaging, and a 40-μm Z-slice of each parasite was taken. Pie charts show the proportion of parasites with 0, 1, and ≥2 T cells in contact analyzed by χ2 test, whereas (D) shows the number of T cells per parasite for each condition analyzed by Mann-Whitey U test. Data are from three mice receiving Itgal−/− cells and four mice receiving WT OT-I cells. Bars show means and SD. **P < 0.01.

  • Fig. 3 Memory CD8+ T cells display patrolling behavior in the liver.

    GFP+ OT-I cells (2 × 104) were transferred to C57BL/6 mice before immunization with 5 × 104 P. berghei CS5M sporozoites. One week (A) and 4 weeks (B) after immunization, mice were prepared for intravital imaging, and the livers were imaged by two-photon microscopy using a resonant scanner to collect time-lapse moves of a single Z-slice at ~3 frames/s; images are representative time points with T cell tracks shown in white; scale bar, 50 μm. (C) Mean speed versus polarity of T cells in the liver, 1 week (green points) and 4 weeks (gray points) after immunization. (D) Proportion of cells exhibiting different T cell migration behaviors 1 and 4 weeks after immunization. Analysis was performed by using a χ2 test. ****P < 0.0001. (E) Mean speed (i) and arrest coefficients (ii) of OT-I GFP T cells in the liver 4 weeks after immunization (analysis based on 50-μm Z-stacks at 1 frame/30 s). Mice received 50 μg of anti-ICAM or isotype control antibodies 3 hours before imaging. Analysis was performed by one-tailed Mann-Whitney U test because the direction of the expected effect was already known from previous experiments. Data are pooled from six mice in each experimental group; medians and interquartile ranges are presented. *P < 0.05.

  • Fig. 4 LFA-1 is highly expressed on a subset of liver memory CD8+ T cells.

    CD45.1+ OT-I cells (2 × 104) were transferred to C57BL/6 mice before immunization with 5 × 104 P. berghei CS5M sporozoites. Organs were harvested and cells were prepared for flow cytometry analysis 1, 2, and 4 weeks after immunization. (A) Representative flow cytometry plots from a single mouse at each time point, showing the expression of CD11a (ITGAL) on CD45.1+ CD8+ OT-I T cells in the spleen, lymph nodes, and liver at the indicated time points. Values indicate the percentage of cells that are CD11ahi. (B) Summary data pooled from two independent experiments showing the proportion of CD45.1+ CD8+ OT-I cells that are CD11ahi. Data were analyzed using an LMM including the experiment and mouse as random effects and organ and time point as fixed effects. Bars show means and SD. ***P < 0.001, ****P < 0.0001.

  • Fig. 5 CD11ahi memory CD8+ T cells in the liver have a TRM phenotype.

    CD45.1+ naïve OT-I cells (2 × 104) were transferred to C57BL/6 mice before immunization with 5 × 104 P. berghei CS5M sporozoites. (A) Representative flow cytometry plots of the expression of CD69, KLRG1, CD103, and CXCR3 by CD45.1+ CD8+ OT-I T cells expressing intermediate and high levels of ITGAL (CD11aint and CD11ahi) in the liver 4 weeks after immunization. (B) Summary plots showing the proportion of cells expressing the indicated phenotypes 4 weeks after immunization in the liver. Data are pooled from four independent experiments for CD69 and KLRG1, a single experiment for CD103, and two independent experiments for CXCR3; data were analyzed by LMMs including mouse as a random effect and CD11a subset as a fixed effect. Bars show means and SD. *P < 0.05, ***P < 0.001. (C) Representative flow cytometry plots showing the coexpression of CD69 and CD11a in the indicated organs (from a single animal) 4 weeks after immunization. (D) Summary of the proportion of OT-I cells in the indicated organs that are CD11ahi CD69+. Data are pooled from three independent experiments and analyzed using an LMM including mouse as a random effect and the organ as the fixed effect. Bars show means and SD. **P < 0.01.

  • Fig. 6 LFA-1 is required for residence of Plasmodium-specific TRM cells in the liver.

    (A) Naïve CD45.1+ WT OT-I cells (2 × 104) were cotransferred with 2 × 104 naïve GFP+ Itgal−/− OT-I cells to C57BL/6 1 day before immunization with 5 × 104 P. berghei CS5M sporozoites; at 1 and 4 weeks after immunization, organs were harvested, and the number and phenotype of transferred cells were determined by flow cytometry. (B) Representative plots from a single mouse at each time point showing the expansion of the different OT-I+ populations in the spleen and liver 1 and 4 weeks after immunization. (C) Summary data showing the overall ratio of Itgal−/− (KO) to WT OT-I cells in the spleen and liver of mice (i) 1 week and (ii) 4 weeks after immunization. (D) Representative flow cytometry plots showing the TRM phenotype of WT and Itgal−/− OT-I cells in the spleen and liver of a single animal 4 weeks after immunization. (E) Summary data showing the percentage of WT and Itgal−/− OT-1 cells that are TRM in (i) the spleen and (ii) livers 4 weeks after immunization. (F) Summary data showing the overall ratio of Itgal−/− (KO) to WT OT-I cells that are (i) TRM and (ii) non-TRM. (G) Summary data of the overall ratio of Itgal−/− (KO) to WT OT-I cells in different organs of mice analyzed 4 weeks after immunization; bars show means and SD. All data are pooled from nine mice in two independent experiments. (C) and (F) were analyzed using LMMs with mouse as a random effect and organ as the fixed effect. (E) was analyzed similarly to (C) but with genotype as the fixed effect. (G) was analyzed similarly to (C) but with experiment included as a random effect. **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 Itgal−/− mice do not form liver TRM cells after LCMV infection.

    Itgal−/− and littermate mice were infected with LCMV Armstrong at 2 × 105 PFU per mouse. (A) One week after infection, the percentage of CD8+ T cells in the blood that was NP396-specific was measured by flow cytometry; data are means and SD analyzed by a two-tailed Student’s t test. Four weeks after infection, the NP396-specific immune response was measured in the spleen and liver by flow cytometry, with (B) representative flow cytometry plots from individual mice and (C) summary data presented. We further determined the proportion of antigen-specific cells that had the TRM phenotype (CD69+ KLRG1lo) by flow cytometry for each organ and genotype, with (D) representative flow cytometry plots from individual mice and (E) summary data presented. Data in (B) to (E) were analyzed using LMMs including mouse as a random effect and organ and genotype as fixed effects; pairwise P values derived from the models are given. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/9/eaaj1996/DC1

    Materials and Methods

    Fig. S1. CD44, VCAM-1, and β2-microglobulin are not required for CD8+ T cell motility in the liver.

    Fig. S2. Phenotype of ITGAL-C77F mice.

    Fig. S3. Similar phenotypes of WT and Itgal−/− cells after in vitro priming.

    Fig. S4. Expression of CD11a on different populations of OT-I T cells.

    Fig. S5. CD11ahi cells in the liver and lung are CD69+, KLRG1, CD103, and IVAb+ after sporozoite immunization.

    Fig. S6. LCMV infection induces populations of CD11hi cells in the liver and lung that are phenotypically similar to those seen after sporozoite immunization.

    Fig. S7. NKT cells have a similar CD11ahi CD69+ KLRG1 phenotype to liver TRM cells.

    Fig. S8. LFA-1 is required for the retention of cells in the liver and lung in the steady state.

    Fig. S9. LFA-1 is required for the formation of TRM cells in the liver in the steady state.

    Movie S1. Anti–ICAM-1 inhibits effector T cell migration in the hepatic sinusoids.

    Movie S2. LFA-1–deficient cells display impaired motility in the liver.

    Movie S3. Migration of OT-I cells in the livers of mice 1 week after sporozoite immunization.

    Movie S4. Migration of OT-I cells in the livers of mice 4 weeks after sporozoite immunization.

  • Supplementary Materials

    Supplementary Material for:

    Up-regulation of LFA-1 allows liver-resident memory T cells to patrol and remain in the hepatic sinusoids

    H. A. McNamara, Y. Cai, M. V. Wagle, Y. Sontani, C. M. Roots, L. A. Miosge, J. H. O'Connor, H. J. Sutton, V. V. Ganusov, W. R. Heath, P. Bertolino, C. G. Goodnow, I. A. Parish, A. Enders, I. A. Cockburn*

    *Corresponding author. Email: ian.cockburn{at}anu.edu.au

    Published 17 March 2017, Sci. Immunol. 2, eaaj1996 (2017)
    DOI: 10.1126/sciimmunol.aaj1996

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. CD44, VCAM-1, and β2-microglobulin are not required for CD8+ T cell motility in the liver.
    • Fig. S2. Phenotype of ITGAL-C77F mice.
    • Fig. S3. Similar phenotypes of WT and Itgal-/- cells after in vitro priming.
    • Fig. S4. Expression of CD11a on different populations of OT-I T cells.
    • Fig. S5. CD11ahi cells in the liver and lung are CD69+, KLRG1-, CD103-, and IVAb+ after sporozoite immunization.
    • Fig. S6. LCMV infection induces populations of CD11hi cells in the liver and lung that are phenotypically similar to those seen after sporozoite immunization.
    • Fig. S7. NKT cells have a similar CD11ahi CD69+ KLRG1- phenotype to liver TRM cells.
    • Fig S8. LFA-1 is required for the retention of cells in the liver and lung in the steady state.
    • Fig. S9. LFA-1 is required for the formation of TRM cells in the liver in the steady state.
    • Legends for movies S1 to S4

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Anti–ICAM-1 inhibits effector T cell migration in the hepatic sinusoids.
    • Movie S2 (.mov format). LFA-1–deficient cells display impaired motility in the liver.
    • Movie S3 (.mov format). Migration of OT-I cells in the livers of mice 1 week after sporozoite immunization.
    • Movie S4 (.mov format). Migration of OT-I cells in the livers of mice 4 weeks after sporozoite immunization.

    Files in this Data Supplement: