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

LFA-1 helps T cells feel at home in the liver

Liver-resident T cells are critical to defense against infections such as malaria and hepatitis B virus. To protect the hepatic sinusoids, these cells must enter circulation yet still remain in the liver. McNamara et al. now report that liver-resident CD8+ T cells up-regulate LFA-1 and that LFA-1–ICAM-1 (intercellular adhesion molecule–1) interactions are critical for T cell patrolling of the hepatic sinusoids. Moreover, in the absence of LFA-1, CD8 T cells do not form liver-resident memory populations, even after infection with either Plasmodium or lymphocytic choriomeningitis virus. Thus, LFA-1 expression acts as a sort of invisible fence, allowing the liver-resident memory T cells free reign within the boundaries of the liver.

Abstract

Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: How can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103—a key integrin for T cell residence in epithelial tissues—we investigated other candidate adhesion molecules. Using intravital imaging, we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent on LFA-1–ICAM-1 (intercellular adhesion molecule–1) interactions. Liver-resident CD8+ T cells up-regulate LFA-1 compared with effector memory cells, presumably to facilitate this behavior. Last, we found that LFA-1–deficient CD8+ T cells failed to form substantial liver-resident memory populations after Plasmodium immunization or lymphocytic choriomeningitis virus infection. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.

INTRODUCTION

CD8+ T cells play critical roles in protection against infectious diseases and cancers. One problem that T cells must overcome is that the number of infected cells can represent a tiny fraction of cells in the body. To achieve efficient tissue surveillance, specialized populations of CD8+ T cells that patrol different niches develop after priming. The first populations to be defined, on the basis of the expression of CD62L and CCR7, were central and effector memory CD8+ T cells (1). More recently, populations of tissue-resident memory (TRM) cells have been identified in numerous tissues—especially barrier tissues—such as the skin, lung, gut, and female reproductive tract (26). Such cells are strictly defined by their inability to recirculate from their tissue of residence, though they are frequently identified by the expression of CD69 and the integrin CD103 (2, 3, 6). Intravital imaging studies have revealed that CD8+ TRM cells in the skin are largely sessile cells that may act as sentinels against invading pathogens (7). This is consistent with the finding that these cells function as the first line of defense in peripheral tissues, able to recruit other cells to the immune response (8).

Given that the liver is the target organ of important pathogens, including hepatitis B virus, hepatitis C virus, and Plasmodium, several recent studies have begun to characterize TRM cells in this organ (5, 9, 10). Although the liver was previously thought of as a “graveyard” for CD8+ T cells, it is now clear that it harbors large numbers of memory CD8+ T cells capable of protecting against pathogen challenge (11, 12). We have recently shown that the formation of robust CD69+ CD8+ T cell populations is essential for effective protection against Plasmodium (malaria) liver stages (9). On the basis of parabiosis studies, these CD69+ cells in the liver have been defined as a resident population that does not recirculate within 2 to 3 months (5, 9). Liver TRM cells also share with epithelial TRM cells a common gene expression signature dependent on the expression of the transcription factors Hobit and Blimp1 (9, 10). However, liver CD8+ T cells have some distinct features compared with epithelial TRM cells: Most liver TRM cells are present in the circulation, as revealed by in vivo antibody labeling experiments (5, 9). Intravital imaging has further shown that liver TRM cells display motile patrolling behavior in the hepatic sinusoids (9).

A critical question therefore is: What are the molecular interactions that retain CD8+ T cells in the liver and facilitate their patrolling behavior? Liver TRM cells do not express high levels of CD103 (9, 10), an integrin that is required for TRM cells to be retained in many epithelial tissues (13). On the other hand, a variety of other adhesion molecules have been implicated in the migration of CD8+ T cells to the liver. The initial trapping of CD8+ T cells in the liver appears to be mediated by interactions between CD8+ T cells and platelets bound to the endothelium via CD44, rather than selectin-mediated rolling interactions (14). Some studies have suggested that intercellular adhesion molecule–1 (ICAM-1) is required for the retention of naive and activated CD8+ T cells in the liver but only in the presence of antigen (15, 16). Although the ICAM-1 ligand LFA-1 has been found to be critical for natural killer T (NKT) cell retention in the liver (17, 18), this canonical adhesion molecule has been regarded as dispensable for the intrahepatic retention of activated CD8+ T cells (14, 17).

Here, we investigated the roles of a range of adhesion molecules in the intrahepatic migration of effector and memory CD8+ T cells using intravital imaging. We found that, unexpectedly, ICAM-1–LFA-1 interactions are indeed important for the movement of activated CD8+ T cells in the liver. Further analysis revealed that LFA-1 is highly expressed specifically on liver TRM cells and that its absence results in their inability to establish residence in the liver. Our data thus reveal an unexpected role for the adhesion molecule LFA-1, rather than CD103, in the retention of liver TRM cells and highlight the distinct adhesion molecule requirements for memory T cells that patrol vascular, rather than barrier, tissues.

RESULTS

Activated CD8+ T cells use LFA-1–ICAM-1 interactions to patrol the liver

Previous studies have shown that CD8+ T cells migrate in the hepatic sinusoids with a characteristic patrolling behavior that facilitates their ability to scan the liver and find pathogens, such as Plasmodium and hepatitis B virus (14, 19). We used antibody blockade to investigate the roles of ICAM-1, VCAM-1 (vascular cell adhesion molecule–1), and CD44 on the patrolling behavior of transferred in vitro activated CD8+ T cells. These molecules have been proposed to have roles in the intrahepatic accumulation of CD8+ T cells, though with the exception of CD44, their roles in migratory behavior within the liver have not been studied (14, 16). We also transferred cells to β2-microglobulin–deficient recipients to examine the role of major histocompatibility complex class I interactions, which have also been suggested to be important for CD8+ T cell adhesion in the liver (20). We subsequently examined the migration of the activated CD8+ T cells in the liver by time-lapse multiphoton microscopy (movie S1). Unexpectedly, only ICAM-1 blockade had any effect on CD8+ T cell movement, with cells in treated mice moving more slowly and spending more time arrested than cells in control mice (Fig. 1, A and B), suggesting that ICAM-1 and its ligands might be important for CD8+ T cell patrolling of the liver. In contrast, antibodies to other adhesion molecules as well as rat immunoglobulin G2b (IgG2b) isotype control antibodies had no effect on migration of CD8+ T cells (fig. S1). The reduction in T cell speed seen in anti–ICAM-1–treated mice is consistent with previous in vitro studies that show that ICAM-1 is required for the crawling motility of lymphocytes as well as their adhesion (21).

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.

Given that ICAM-1 is highly expressed on the surface of liver sinusoidal endothelial cells and hepatocytes (14, 20), we hypothesized that T cells were crawling on these surfaces using the integrin LFA-1 that is highly expressed on CD8+ T cells. LFA-1 is composed of integrin α-L (ITGAL; CD11a) combined with integrin β-2 (CD18). To further investigate a possible role of LFA-1 in T cell migration in the liver, we used a mouse line carrying a Cys77Phe mutation caused by a G>T change in Exon 3 of the Itgal gene (Chromosome 7, position 127299608). This mutation causes a complete lack of ITGAL on the cell surface (fig. S2A); accordingly, we designate them Itgal−/− for simplicity. It is likely that Cys77 forms disulfide bonds and that the mutation destabilizes the integrin structure. These mice were identified from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen for immune phenotypes in the blood (22) because they have an elevated proportion of NKT cells in the blood (fig. S2B). This may be explained by the fact that LFA-1 has been shown to be important for the intrahepatic retention of NKT cells (18). Closer analysis of our Itgal−/− mice revealed that they also have elevated CD8+ T cells in the blood, particularly CD44hi-activated CD8+ T cells (fig. S2B).

To investigate the effect of loss of ITGAL on CD8+ T cell migration in the liver, we activated Itgal−/− OT-I cells in vitro and cotransferred them to mice with similarly activated but differentially labeled wild-type (WT) OT-I cells. Migration in the liver was then assessed by multiphoton microscopy (movie S2 and Fig. 1C). In agreement with our ICAM-1 blockade data, Itgal−/− cells did not display patrolling behavior; rather, they spent large amounts of time arrested and moved with slower average speeds than WT cells (Fig. 1D). The Itgal−/− and WT cells showed similar expression of activation markers and other β2 integrins, suggesting that the reduced migration was not due to inadequate priming of Itgal−/− cells, resulting in the expression of different adhesion molecules (fig. S3). We further used flow cytometry to quantify the accumulation of cells in the liver and other organs in mice that received equal numbers of WT and Itgal−/− cells. In the liver and lungs, Itgal−/− cells constituted <20% of the cells recovered from these mice; in contrast, Itgal−/− cells formed the major proportion of cells recovered from the spleen, blood, and lymph nodes (Fig. 1, E and F). One hypothesis is that the spleen and lymph nodes act as a sink for the Itgal−/− cells; however, our intravital imaging data (Fig. 1, B and C) and our finding that there are also elevated numbers of Itgal−/− cells circulating in the blood suggest that this is not the case. Rather, our data suggest that LFA-1 is important for the retention of activated CD8+ T cells in the liver and lungs.

LFA-1 is required for efficient CD8+ T cell–mediated protection against malaria

We next wanted to determine whether the lack of patrolling cells in the liver would affect protection against the Plasmodium parasite. Although CD8+ T cells are capable of killing parasites in the liver (19), it is not known whether the cells conferring protection are those circulating in the blood or those migrating in the sinusoids. Given the results of the previous experiment, in which the Itgal−/− cells are enriched in the blood and WT cells are enriched in the liver, we were able to test this by transferring activated Itgal−/− or WT OT-I cells to mice before challenge with Plasmodium berghei CS5M parasites that express the SIINFEKL epitope within the immunodominant circumsporozoite (CS) protein (23). We found that the parasite burden was significantly greater in mice that received the Itgal−/− cells than in those that received WT cells (Fig. 2A). Although a component of this impaired protection by Itgal−/− cells may be attributed to a defect in cytotoxicity (Fig. 2B), we also observed by intravital microscopy that WT OT-I cells were better able to associate with and cluster around P. berghei CS5M parasite than Itgal−/− OT-I cells (Fig. 2, C and D). Thus, our data are consistent with the hypothesis that LFA-1–mediated patrolling of the liver is required for efficient immune surveillance, though we cannot exclude the possibility that LFA-1 is also important for CD8+ T cell killing of infected cells.

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.

Memory T cells in the liver adopt a characteristic patrolling behavior in the hepatic sinusoids

We next wanted to determine whether the migratory behavior that we observed in in vitro activated cells reflects that of in vivo primed cells. Accordingly, we immunized mice that had received naïve green fluorescent protein–expressing (GFP+) OT-I cells with P. berghei CS5M sporozoites. The livers of mice were then imaged at effector (1 week) and memory (4 weeks) time points after immunization (Fig. 3, A and B, and movies S3 and S4). At effector time points, the behavior of donor T cells in the liver was different from that observed with in vitro activated cells. There were many rounded-up cells and cells moving with the blood flow. To capture these rapidly moving cells, we modified our imaging protocol and made high frame-rate movies (3 frames/s) using the resonant scanner on our microscope. Mathematically, we were then able to distinguish three migratory phenotypes among the GFP+ cells using the parameters of cell speed and polarity (Fig. 3C). We designated the first population as “flowing” cells; these cells moved rapidly (speed, >25 μm/min; median, 208 μm/min) in the blood though sometimes they briefly arrested on the walls of the sinusoids. Among the nonflowing cells moving at <25 μm/s, we observed a population of “rounded” cells, which we formally defined as having a polarity of <1.5; these cells were often arrested but sometimes detached (median speed, 5.8 μm/min). Last, we were able to observe some “patrolling” cells that we defined by their higher polarity (>1.5). Overall, these cells moved similarly to in vitro activated cells, though they had a higher median speed (9.5 μm/s). At the memory time point, a notably different picture was seen, with a substantial increase in the number of patrolling cells that increased from <5% of cells 1 week after immunization to >50% of the total cells at 4 weeks after immunization (Fig. 3D). We further found that ICAM-1 blockade reduced the speed and increased the arrest coefficient of memory CD8+ T cells in the liver (Fig. 3E), suggesting that these cells, like in vitro activated effectors, use LFA-1–ICAM-1 interactions for their patrolling behavior.

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.

Liver-resident memory CD8+ T cells express exceptionally high levels of LFA-1

Given that LFA-1 was found to be important for migration of in vitro activated CD8+ T cells in the liver, we speculated that the formation of a patrolling memory population in the liver might be associated with an increase in the expression of LFA-1 (ITGAL; CD11a) on CD8+ T cells. We examined the phenotypes of OT-I cells in the spleen, lymph node, and livers of mice at 1, 2, and 4 weeks after immunization with P. berghei CS5M sporozoites (Fig. 4A). Naïve cells expressed low levels of LFA-1 that increased upon priming (fig. S4). CD11a was expressed at intermediate levels on activated CD8+ T cells in the spleen at all time points; however, at 2 and 4 weeks after immunization, we observed a distinct population of CD11ahi cells in the liver (Fig. 4, A and B, and fig. S4). This population did not appear to be present in the lymph nodes and spleen, which is consistent with our previous data (Fig. 1, E and F), suggesting that LFA-1 is dispensable for homing to these organs.

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.

We were further interested in knowing the broader phenotype of the CD11ahi population, particularly whether they might correspond to liver TRM CD8+ T cells. We found that, indeed, CD11ahi cells were almost exclusively CD69+ KLRG1 CXCR3+ (Fig. 5, A and B), a typical profile for liver TRM cells (9, 10). We also found that the CD11ahi cells in the liver did not express high levels of CD103 (Fig. 5B), whereas intravenous injection of fluorescein isothiocyanate–labeled anti-CD8a antibody revealed these cells to be in the circulation (fig. S5A), which is also in agreement with previous descriptions of liver TRM cells (5, 9, 10). To determine whether this CD11ahi population is unique to the liver or may be seen in other tissues, we examined the spleen, lymph node, blood, skin, and lungs of mice immunized with sporozoites. Although we were unable to detect antigen-specific cells in the skin of sporozoite immunized mice, we were able to detect CD11ahi CD69+ cells in the lungs of immunized mice (Fig. 5, C and D). However, these lung CD11ahi cells were unlikely to be TRM cells because lung TRM cells express CD103 and are not labeled by intravenously injected antibody (IVAb) (6, 24), whereas the CD11ahi cells that we observed were CD103 and IVAb+ (fig. S5).

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.

To determine whether the elevated expression of LFA-1 on liver CD8+ T cells was specific to Plasmodium immunization, we also examined CD11a expression on NP396 Tetramer+ cells after intraperitoneal infection with lymphocytic choriomeningitis virus (LCMV) Armstrong. Similar to our results with sporozoites, we identified a CD11ahi population in the liver that corresponded to previously defined liver TRM populations, as they are CD69+ CD103 KLRG1 (fig. S6, A to C). We also identified a similar population of CD11ahi CD69+ KLRG1 cells in the lung after LCMV infection (fig. S6D). Although a modest proportion (~20%) of Tetramer+ cells were IVAb in this case, we did not detect any appreciable CD103 expression (fig. S6D), which is in agreement with previous observations that show that intraperitoneal LCMV infection does not induce lung TRM cells (25, 26). Independently, a distinct population of CD11ahi cells in the liver has been observed previously after vesicular stomatitis virus infection (27), though this observation was not followed up. Last, we analyzed NKT cells as the other liver-resident lymphocyte subset and found that NKT cells also have an identical CD11ahi CD69+ KLRG1 phenotype to liver TRM cells (fig. S7).

LFA-1 is required for the retention of TRM cells in the liver

To determine whether LFA-1 is required for the intrahepatic retention of CD8+ T cells, we first made mixed bone marrow chimeras containing CD45.2+ Itgal−/− cells mixed with CD45.1+ WT cells as well as control chimeras reconstituted with equal numbers of CD45.2+ and CD45.1+ WT cells. The contributions of Itgal−/− cells to the CD8+ T cell population were analyzed in the spleen, lung, and liver (fig. S8A). Itgal−/− CD8+ T cells migrated normally to the spleen but did not accumulate efficiently in either the lung or the liver, though the overall defect was only just significant in the liver (fig. S8, B and C). Specific analysis of the contribution of Itgal−/− cells to the CD69+ and CD69 liver cell populations (fig. S9A) revealed that the intrahepatic CD69+ subset came almost entirely from the WT cells (fig. S9, B and C), indicating that ITGAL is required for its residency. In contrast, when we examined CD69 CD8+ T cells in the liver, we found that these were not dependent on ITGAL for retention in the liver (fig. S9, B and C). A similar analysis of the lung CD69+ and CD69 cell populations revealed that, in contrast to the liver, ITGAL is critical for the accumulation of both these populations in the lung (fig. S9, D to F).

To determine whether LFA-1 is required for the formation of antigen-specific liver TRM cell populations after immunization, we cotransferred GFP+ Itgal−/− and WT CD45.1+ OT-I cells to CD45.2+ mice before immunization with P. berghei CS5M sporozoites (Fig. 6A). At 1 week after infection, we observed that the WT cells outnumbered the Itgal−/− cells in both the liver and spleen by about 30:1 (Fig. 6B), a result consistent with the priming defect previously reported for Itgal−/− cells (28). However, whereas this ratio remained constant at 4 weeks after immunization in the spleen, the Itgal−/− cells were now outnumbered by about 60:1 in the liver, suggesting that these LFA-1–deficient cells have a specific defect in forming memory populations in this organ (Fig. 6C).

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.

Examination of the phenotypes of the cells revealed that the difference between the spleen and liver was largely driven by the relative proportions of CD69+ KLRG1 (TRM) cells (Fig. 6D). This population typically made up ~30% of the WT OT-I cells in the liver but accounted for only ~10% of the Itgal−/− cells in the same mouse (Fig. 6E). These cells may not be lost altogether but may enter the general circulation, because the situation was reversed in the spleen, with a higher proportion of TRM phenotype cells among the Itgal−/− OT-I cells compared with the WT OT-I cells (Fig. 6E). As a result of this, the ratio of WT to Itgal−/− OT-I TRM cells was 120:1 in the livers of immunized mice but was only 8:1 in the spleens (Fig. 6F). Nonetheless, the ratio of Itgal−/− to WT non-TRM cells was still significantly lower in the liver compared with the spleen, suggesting that activated non-TRM cells may still use ITGAL to accumulate in the liver.

We also investigated the ratio of Itgal−/− to WT OT-I cells in other organs in this experiment (Fig. 6G). In agreement with our bone marrow chimera data, lung OT-I cells had an even stronger requirement for ITGAL than liver OT-I cells, with Itgal−/− cells being outnumbered by about 120:1 by WT cells. In contrast, Itgal−/− cells were only outnumbered by about 8:1 by WT OT-I cells in the lymph nodes. This suggests that some Itgal−/− cells may accumulate in lymph nodes, though the proportion of OT-I cells in the lymph nodes was very small (fig. S5).

Last, we were concerned that our data might be biased by the poor priming of the LFA-1–deficient cells by Plasmodium, especially in a competitive environment. Accordingly, we infected Itgal−/− mice and their WT littermates with LCMV Armstrong and measured the formation of antigen-specific T cell responses using NP396 tetramers. In this infection model, there was no detectable defect in priming in the Itgal−/− mice because similar percentages of Tetramer+ cells were seen in the blood 1 week after immunization (Fig. 7A). Moreover, 4 weeks after immunization, there were similar proportions and numbers of Tetramer+ cells between the Itgal−/− and littermate mice in both the spleen and liver (Fig. 7, B and C). However, whereas robust populations of antigen-specific CD69+ KLRG1 TRM cells were formed in livers of littermate control mice, few were seen in Itgal−/− mice (Fig. 7, D and E). Similar to our results with Plasmodium immunization, there was a ~2-fold increase in the average numbers of CD69+ KLRG1 cells in the spleens of Itgal−/− compared with littermates, suggesting that these cells may accumulate in secondary lymphoid organs if they are unable to be retained in the liver, though this did not reach statistical significance when correction was made for multiple comparisons. Collectively, these data from our bone marrow chimeras and two distinct models of infection suggest that LFA-1 is critically required for the retention of TRM cells in the liver.

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.

DISCUSSION

Several recent papers have identified populations of tissue-resident CD69+ antigen-specific memory cells in the liver that patrol the sinusoids (5, 9, 10). A critical question then is: How can an intravascular population in the circulation also be tissue-resident? Here, we show a critical role for ITGAL—and, by extension, the integrin LFA-1—in the migration of activated CD8+ T cells in the liver. We further show that LFA-1 is required for the retention of TRM cells in the liver. It is possible that these cells might occasionally exit the liver if they were to enter the hepatic venules, though it may be that these cells rapidly recirculate back to the liver because of the elevated expression of ICAM-1 in these tissues and the slow blood flow within this organ (14, 20). Overall, though, our data support the hypothesis that it is the expression of high levels of LFA-1 on the surface of liver TRM cells that allows them to remain in the liver and patrol the hepatic sinusoids.

Our data further suggest that TRM populations in different tissues have different adhesion molecule requirements for tissue retention. In agreement with previous reports, we do not detect high levels of the integrin CD103 on the surface of our CD11ahi liver TRM cells (5, 10). CD103 is highly expressed on epithelial TRM cells, such as those found in the skin and the gut, and has been shown to be required for T cell residence in the skin (2, 3, 13). Its expression appears to be induced, even in vitro, by transforming growth factor–β (TGF-β) signaling (29). In vivo, the level of CD103 expression increases progressively during skin TRM development as they migrate to the epidermis (30). However, although CD103 may be critical for the residence of TRM cell populations in various epithelia, it is presumably dispensable for TRM populations that may remain exposed to the circulation. TGF-β signaling has previously been shown to down-regulate LFA-1 expression, suggesting that these integrins may be reciprocally regulated (31, 32).

Despite ICAM-1–LFA-1 interactions being considered canonical in the leukocyte adhesion cascade, their role in liver residence has been unclear. Whereas ICAM-1 has been suggested to have a role in the retention of naïve and activated cells in the liver (16, 20, 33), antibody blockade with α–LFA-1 antibodies was not found to inhibit the accumulation of activated CD8+ T cells in the liver (14). Moreover, LFA-1–deficient mice do not have abnormal numbers of conventional T cells in the liver (17). Our data resolve many of these contradictory observations. As with previous studies, we did not see any effect of antibody blockade on retention of activated effector cells to the liver, only a defect in migration. Our data also explain why previous studies missed a role of LFA-1 in CD8+ T cell migration to the liver, because LFA-1 is only absolutely required for the retention of the TRM phenotype cells, whereas other CD8+ T cells that are nonresident can transiently associate with the liver in the absence of LFA-1. Thus, previous studies of bulk CD8+ T cell populations were unable to detect the role of LFA-1 in this recently identified cell type.

Although our studies have focused on the role of LFA-1 in CD8+ T cell accumulation in the liver, we also find that this integrin is important for CD8+ T cell retention in the lung. We identified a population of cells in the lung that was CD11ahi, which, like the corresponding population in the liver, were CD69+ and KLRG1. However, our CD11ahi CD69+ cells are unlikely to be lung TRM cells because lung TRM cells are typically IVAb and CD103+ (6, 24). The absence of lung TRM cells is not unexpected because Plasmodium does not infect the lung, whereas LCMV only forms significant lung TRM populations after intratracheal infection (25). In contrast to the liver, both CD69+ and CD69 CD8+ T cells in the lung had a strong requirement for LFA-1 to be retained. This again highlights the different adhesion molecule requirements among diverse populations of CD8+ T cells in different organs. Previous studies have shown that LFA-1 is required for the retention of early effector cells in the lung (34), whereas another study has shown that LFA-1 is required for memory cell entry to the lung airways (35). Our results extend these previous results in showing an important role for LFA-1 for the retention of vascular CD8+ T cell populations (as opposed to just those in the airways) at memory time points. Whereas liver sinusoids are characterized by slow blood flow, the flow is faster and cells experience more sheer stress in the lung (36). Thus, LFA-1 may be universally required among CD8+ T cell populations in the lung to allow even transient associations with the endothelium.

In summary, we have found a previously unappreciated role for LFA-1 in the retention of CD8+ T cells in the liver and for the movement of these T cells within hepatic sinusoids. This motility is what appears to be important for the efficient surveillance of the liver and the identification of infected cells (9). Crucially, because of the potential ability of the liver TRM cells to enter the circulation directly, the mechanism of retention in the liver appears different from that described for epithelial or mucosal TRM cells. Overall, our data suggest that the nature of tissue-resident CD8+ T cell populations may be even more diverse and complex than has previously been suggested.

MATERIALS AND METHODS

Details of standard immunological methods used in this study are given in Supplementary Materials and Methods.

Study design

The initial aim of the study was to determine the molecules involved in CD8+ T cell migration in the liver. Accordingly, experiments wherein we blocked candidate adhesion molecules and measured motility by intravital imaging were performed (Fig. 1). Because the n for these experiments was determined by the number of cells, each mouse was considered an experimental repeat. No randomization or blinding was performed in these experiments; however, the hypothesis that blocking these molecules would affect migration was specified in advance, though the direction of the effect was not predicted. We therefore analyzed these experiments using a linear mixed model (LMM) approach, which accounts for variation between mice and experiments as random effects in addition to our fixed effects (movement parameters). These experiments suggested a role for LFA-1 in accumulation and migration, which we tested by cotransferring LFA-1–deficient and WT cells to mice and measuring migration and accumulation by intravital microscopy and flow cytometry. In these experiments, the hypothesis was specified in advance. Randomization and blinding could not be performed in these experiments because both the experimental and control groups were contained in the same mouse.

Having identified LFA-1 as a likely candidate for T cell migration and residence in the liver, we performed a series of controlled laboratory experiments to investigate this further (Figs. 2 to 7). In these experiments, at least four mice per group were used in each individual experiment where the n was determined by the number of mice. Data presented were typically pooled from multiple experiments and were therefore usually analyzed using LMMs, which account for variation due to random effects (individual mice and experiments). Data from all mice studied are presented in the figures, and no outliers were excluded. In these experiments, the hypotheses were specified in advance. No randomization or blinding was performed; however, in many experiments, control and experimental groups (cells) were contained within the same animal.

Mice

C57BL/6.J mice, Rag1−/− mice, OT-I mice (37), B2m−/− mice (38), and uGFP mice (39) were bred in-house at the Australian National University (ANU). mT/mG mice (40) were purchased from the Jackson Laboratory and maintained at the ANU. ITGAL-C77F (Itgal−/−) mice were identified in our ongoing ENU mutagenesis screens (22). ITGAL-C77F mice were maintained on a C57BL/6.J background and crossed to an OT-I uGFP background, as required. All animal procedures were approved by the Animal Experimentation Ethics Committee of the ANU (protocol numbers A2013/12, A2014/62, and A2015/76).

Immunizations

Mice were immunized intravenously with 5 × 104 P. berghei CS5M sporozoites expressing mCherry (23) dissected by hand from the salivary glands of Anopheles stephensi mosquitoes. Mice were then treated with 0.6 mg of choloroquine intraperitoneally daily for 10 days to prevent the development of blood stage infection. LCMV Armstrong was delivered intraperitoneally at a dose of 2 × 105 plaque-forming units (PFU) per mouse.

Antibody blockade

The following antibodies were injected intravenously 2 hours before transfer of activated OT-I CD8+T cells: anti–ICAM-1 [clone YN1/1.7.4; BioXCell; 50 μg per mouse (18)], anti–VCAM-1 [clone 429; BioLegend; 50 μg per mouse (16)], anti-CD44 [clone KM81, blocking CD44 binding to hyaluronan; Cedarlane; 20 μg per mouse (14)], and rat IgG2b isotype control (clone LTF2; BioXCell; 50 μg per mouse).

Multiphoton microscopy

Mice were prepared for multiphoton microscopy essentially as described in Supplementary Materials and Methods (41). Imaging was performed using a FluoView FVMPE-RS multiphoton microscope system (Olympus) equipped with an XLPLN25XWMP2 objective (25×; numerical aperture, 1.05; water immersion; 2-mm working distance). Images were acquired using FV30 software (Olympus) and exported to Imaris (Bitplane) for downstream processing, as described in Supplementary Materials and Methods. Movies were annotated and prepared for display using Adobe After Effects (Adobe).

Statistical analysis

Details of statistical analysis for each experiment are given in the relevant figure legend. χ2 tests, t tests, Mann-Whitney U tests, and one-way analyses of variance (ANOVAs) were performed using Prism 6 (GraphPad). Where data were pooled from multiple experiments, multilevel analyses were performed using an LMM in GenStat (VSNi). LMMs are regression analysis models containing both fixed and random effects, with fixed effects being the variable/treatment under examination, whereas random effects are unintended factors that may influence the variable being measured. If significance was found from running an LMM, pairwise comparisons of the least significant differences of means were undertaken to determine at which level interactions were occurring. Statistical significance was assumed if the P value was <0.05 for a tested difference (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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.

REFERENCES AND NOTES

Acknowledgments: We thank M. Devoy, H. Vohra, and C. Gillespie of the Imaging and Cytometry Facility at the John Curtin School of Medical Research for assistance with flow cytometry and multiphoton microscopy. We also thank T. Neeman of the ANU Statistical Consulting Unit for assistance with statistical analysis of the data and D. Godfrey (University of Melbourne) for reagents and advice in the initial identification of the Itgal mutant mice. Funding: This work was supported by start-up funds from the ANU (to I.A.C.) and grants from the Perpetual Foundation (to I.A.C., FR2014/1152), the Ian Potter Foundation (to I.A.C., grant number 32616), the Ramaciotti Foundation (to C.G.G. and A.E.), the NIH (to C.G.G., grant number U19 AI100627), and the National Health and Medical Research Council (to A.E., grant number GNT1035858). Author contributions: Study conception and design: H.A.M., P.B., W.R.H., I.A.P., C.G.G., A.E., and I.A.C. Acquisition of data: H.A.M., Y.C., M.V.W., Y.S., C.M.R., L.A.M., J.H.O., H.J.S., I.A.P., and I.A.C. Analysis and interpretation of data: H.A.M., V.V.G., L.A.M., J.H.O., A.E., and I.A.C. Drafting of manuscript: H.A.M. and I.A.C. Critical revision: W.R.H., P.B., I.A.P., and A.E. Competing interests: The authors declare that they have no competing interests.
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