2.1 Mice
All mice used in this study are from the C57BL/6 (B6) background and at 6-8 weeks of age. C57BL/6 WT mice are purchased from the National Laboratory Animal Center (Taipei, Taiwan). CD45.1/45.2+ congenic and NK-deficient (NFIL3 KO) mice will be kindly provided by Dr. Chien-Kuo Lee and Dr. Tak Mak (The Campbell Family Institute for Breast Cancer Research), respectively. All mice are bred and kept in specific pathogen–free (SPF) facility at College of Medicine, National Taiwan University.
Procedures and the use of the animals are reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
2.2 Hydrodynamics-based transfection
pAAV/HBV plasmid DNA are purified by using Endotoxin-Free Maxi plasmid kit.
C57BL/6 mice (male, 6-8 weeks old) will be warmed under pet lamp. Ten micrograms of plasmid DNA/DPBS in a volume equivalent to 8% of the mouse body weight will be injected via a tail vein in 5s. Facial vein blood will be collected weekly with lancet. Serum levels of HBsAg will be determined by using Roche's cobas e 411 Immunoassay Analyzer.
The cutoff value for determining HBsAg positivity is a signal-to-cutoff (S/CO) ratio of ≥ 1 according to manufacturer’s instructions.
2.3 Anti-ASGM1 treatment in vivo
The lyophilized anti-Asialo GM1 antisera (Wako, JP) was dissolved with distilled water according to the manufacturer’s instructions. Mice (male, 6-8 weeks old) were intraperitoneal injected 20 μl antisera solution one day before hydrodynamically transfection and twice a week for 8-10 weeks. The intrahepatic immune cells population was determined by flow cytometry.
2.4 Isolation of intrahepatic lymphocytes
Mice are anesthetized by administrated 10 µl Zoletil/rompun premix intravenous and then perfused by 10ml DPBS. Suspend the cells in HBSS buffer by mincing the perfused liver with a plunger and passed through a 70 µm strainer. Hepatocytes and large cell clumps are removed by 50 g centrifugation 3 min twice. The supernatant containing intrahepatic leukocytes (IHLs) is pelleted by 300 g centrifugation at 4℃ for 10 min.
Resuspend the cells to 40%/70% percoll (GE) gradient centrifugation, 1200 g, 20min.
Interface cells are washed by HBSS once.
2.5 Analysis of intrahepatic lymphocytes and splenocytes
Single cell suspensions were firstly stained with FC receptor blocker (BD Biosciences, San Jose, CA, USA) for 10 min on ice and then subjected to surface marker-specific antibody staining for 60 min. Cells were labeled with polyclonal antibody specific for ASGM1 and monoclonal antibodies specific for CD3ε(17A2), CD8α (53-6.7), CD25 (pc61.5), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD103 (M290), CD107a (1D4B), CD127 (SB/199), CD137 (17B5), CXCR3(CXCR3-173), ICOS (7E.17G9), KLRG1 (2F1), LFA-1 (H155-78), NK1.1 (PK126), PD-1 (29F-1A12) or RANKL (IK22/5) from BioLegend, San Diego, CA. After washed twice, the cells were resuspended in FACS buffer (2% FBS in PBS) and analyzed by flow cytometry (Canto II, BD Biosciences, San Jose, CA, USA). Regarding cell sorting, single cell suspensions were stained with FC receptor blocker and then subjected to anti-ASGM1, anti-CD44 or anti-LFA-1 antibody single-staining for 60 minutes. Cells were sorted under indicated criteria using the FACS Aria IIIu (BD Biosciences, San Jose,
2.6 Immunohistochemistry (IHC)
Perfused livers were fixed in 10% formalin embedded in paraffin. Intrahepatic HBcAg was detected by IHC staining with rabbit anti-HBc and Envision System, horseradish peroxidase (DAB) (Dako, Glostrup, Denmark). Hematoxylin was used to stain liver section nuclei.
2.7 Adoptive transfer
CD45.1/45.2+ congenic mice were intravenous administrated of 1 μg KN-62. 30 minutes later, intrahepatic leukocytes were harvested and stained. A FACSAria cell sorter ((BD Biosciences, San Jose, CA) was used to purify ASGM1+ and ASGM1- CD8 T cells. Purity of sorted cells was more than 90%, as verified by flow cytometry.
CD45.2+ C57BL/6 mice received intravenously 8x104 donor cells.
2.8 Gene expression profile analysis
3 x 104 ASGM1+ and ASGM1- CD8 T cells from the liver of naive NFIL3 KO mice were sorted on a FACSAria III (BD Biosciences, San Jose, CA). For each sample, we extracted total RNA using Trizol (Invitrogen, Waltham, MA). After amplification and conversion to cDNA, samples were hybridized to an Affymetrix Transcriptome Array (Mouse Clariom D) (Affymetrix Inc. Santa Clara, CA) at NCFPB High-throughput Genome Analysis Core Facility. Image analysis was done using Affymetrix Transcriptome Analysis Suite (TAS) (Affymetrix Inc. Santa Clara, CA).
2.9 Enzyme-linked immunospot assay (ELISPOT)
Intrahepatic leukocytes cells or splenocytes were cultured and assayed for the frequency of antigen-specific IFN-γ-secreting cells using a Murine IFN-γ Single-Color Enzymatic ELISPOT Assay kit (Cellular Technology Limited, Shaker Heights, OH).
Briefly, 1 × 105 mononuclear cells were cultured with HBcAg18-27 and HBcAg93-100
peptides (50 μg/ml, Mission Biotech, Taipei, TW) in 200 μl CTL-Test Medium and incubated for 18 h at 37°C. Spot-forming cells were revealed by biotin-conjugated
detection Ab with alkaline phosphatase-conjugated streptavidin and substrates, BCIP and NBT.
2.10 Quantitative PCR analysis
For quantitative PCR, total RNA was isolated from indicated cells using Trizol (Invitrogen, Waltham, MA) and cDNA was synthesized with iScript (Bio-Rad, Hercules, CA) according to the manual. Quantitative amplification was performed with SYBR Premix ExTaq II (TaKaRa, Shiga, Japen) in an Thermo PIKOREAL 96 system (Thermo Fisher Scientific, Vantaa, Finland) with the primers: (Inpp4b: 5’- CAGTAGCAAGGATGGAGAGGCA-3’ (forward), 5’- TTGCTCGGTTCACCACTT CTCC-3’ (reverse); Itgae: 5’- GAAGTGGAACGGAG GGTCCTTT-3’ (forward), 5’- GTCTGGAACTCGTAGGTGACCT-3’ (reverse); Eomes: 5’- CCACTGGATGAG GCAGGAGATT-3’ (forward), 5’- GTCCTCTGTCACTTCCACGATG-3’ (reverse);
Ly6C2: 5’- GCGCCTCTGATGGATTCTGCAT-.
2.11 Materials
NucleoBond Xtra EF Macherey-Nagel Düren DE
Fluorescent-conjugated antibodies BD Biosciences Pharmingen San Diego, USA
MojoSort CD3 T cell isolation kit BioLegend San Diego, CA, USA
Fixation/Permeabilization kit Invitrogen, Carlsbad, CA, USA
ImmnoSPOT kit Cellular Technology, OH, USA
2.12 Statistical analysis
Statistical analysis of experimental groups is performed by unpaired Student’s t-tests using GraphPad Prism 6.0 software (GraphPad Software, San Diego USA). P<0.05 was considered significant.
Chapter 3 Results
3.1 Anti-AsialoGM1(ASGM1) treatment resulted in lack of sustained HBsAg
clearance from serum.
To study the role of NK cells in HBV clearance, anti-ASGM1 was intraperitoneal (i.p.) injected to BALB/c or C57BL/6 mice to deplete NK cells. The percentages of NK cells in the liver were markedly decreased in mice treated with the antisera (Figure 1a, e).
After hydrodynamic injection (HDI) of a replication competent HBV DNA, the anti-ASGM1 treated mice were remained HBsAg seropositive until the end-point and led to significant higher serum-HBsAg titer in anti-ASGM1 treated mice than isotype-control treated mice (Figure 1b, f). The appearance of anti-HBs antibodies in the serum was delayed in mice treated with anti-ASGM1 comparing to isotype control (Figure 1c, g).
Similarly, the immunohistochemistry analysis revealed that the staining for HBcAg remained detectable in the livers of anti-ASGM1-treated BALB/c and C57BL/6 mice on Day 42 post-transfection. However, the staining was much lower in untreated mice (Figure 1e, h). Taken together, our results indicated that anti-ASGM1 treatment
significantly impaired the ability of HBV clearance in both BALB/c and C57BL/6 mice, suggesting that some ASGM1+ immune cells contribute in clearing virus.
To further investigate the role of NK cells, we employed NFIL3 KO mice which are known to have the deficiency of NK development. The percentages of NK cells in the liver decreased in NFIL3 KO mice comparing to wild-type C57BL/6 mice (Figure 2a).
Unexpectedly, the average HBsAg level in the sera of NFIL3 KO mice was comparable to WT mice after HDI with HBV DNA (Figure 2b). There was no significant difference of the ability of producing anti-HBs antibodies (Figure 2c), and the expressing of HBcAg in the liver six weeks after HDI (Figure 2d) between NFIL3 KO mice and the WT mice.
Our data showed that NFIL3 KO mice were functional competent in HBV clearance, suggesting that NK cells might be not the key effector cells whose depletion causing the impairment of HBV clearance when the mice were treated with anti-ASGM1 antisera.
3.3 The non-NK ASGM1 positive immune cells mediated HBV clearance.
Since anti-ASGM1 treatment significantly abolished the HBV clearance in WT mice, while the loss of conventional NK cells in NFIL3 KO mice had the normal ability to clear virus, it raised a question that is there a cell type which can be depleted by anti-ASGM1 treatment and is crucial in HBV clearance in NFIL3 KO mice? We treated NFIL3 KO
the ability of HBV clearance in NFIL3 KO mice. As WT mice, the HBsAg level was much higher in anti-ASGM1 treated mice than in isotype control treated mice (Figure 3a) after HDI. None of anti-ASGM1 treated mice was found anti-HBsAg antibodies in the serum, while a significantly increasing of the anti-HBs antibodies in the sera after HDI in control mice (Figure 3b). The expressing of HBcAg in the liver were still detectable in anti-ASGM1 treated mice six weeks after HDI (Figure 3c). Our data indicated that there are the non-NK cells which can be depleted by anti-ASGM1 treatment and is critical in HBV clearance.
3.4 The majority of intrahepatic ASGM1 positive immune cells in NFIL3 KO mice
were CD8 T cells.
To further analyze the possible cells which can be depleted by anti-ASGM1 treatment and is critical in HBV clearance, we sorted the intrahepatic ASGM1+ cells from WT or NFIL3 KO mice and analyzed the immune phenotype of these cells. The percentage of NK1.1+CD3- cells were significantly higher in sorted cells than in total intrahepatic leukocytes from WT mice (Figure 4a), consistent with our knowledge that conventional NK cells expresses large amount of ASGM1. Our data showed that the
intrahepatic leukocytes as well from both WT mice and NFIL3 KO mice (Figure 4a, b).
Furthermore, the percentage of CD8+ cells among CD3+ cells were significantly higher in sorted cells than in total cells in both mice (Figure 4c, d). In addition, anti-ASGM1 treatment depleted the intrahepatic HBcAg specific CD8 T cells in NFIL3 KO mice (Figure 5). These data pointed out that the cell critical in HBV clearance in NFIL3 KO
mice might be CD8 T cells.
3.5 The intrahepatic ASGM1 positive CD8 T cells in HBV carrier mice expressed
CD44 and LFA-1.
CD8 T cells were considered as one of the key effector immune cells in HBV clearance. CD8 T depleted chimpanzee and CD8 KO mice were showed to lost the ability to against HBV (Thimme et al. 2003). The inactivation of PD1+ CD8 T cells from HBV carrier mice can be reversed by PD1 blockage treatment (Tzeng, H.T., et al., 2012).
Chronic HBV carrier mice regained the ability to clear HBV after treated with anti-PD1 antibodies. However, only partial of CD8 T cells express ASGM1. It raised a question that are the CD8 T cells which express ASGM1 and are critical in HBV clearance a sub-population of CD8 T cells. Therefore, we tested a panel of phenotypic markers of CD8 T
cells, most of them expressed CD44 and LFA-1 (Figure 6, 7a) instead of naïve markers CD62L and CD127. The expression of other phenotypic markers (such as CD25, CD103, CD107, CD137, ICOS, RANKL and Tim-3 as shown in Figure 6) were low or absent.
Among these markers, the expression patterns of LFA-1 and CXCR3 were significantly different between ASGM1+ and ASGM1- CD8 T cells (Figure 6b). Meanwhile, we noticed that almost all ASGM1+ CD8 T cells expressed LFA-1 and can be divided into two group, LFA-1int and LFA-1hi. Regarding LFA-1hi were reported to be one of the markers of a distinct liver resident CD8 T cells which are CD69 positive as well, we analyzed the expression of CD69 and LFA-1 among ASGM1 positive or negative CD8 T cells.
3.6 The intrahepatic ASGM1, CD44 and LFA-1 triple positive CD8 T cells were a
distinct sub-population.
Figure 7a demonstrate that among intrahepatic ASGM1-positive CD8 T cells, most
of them expressed CD44, CD69, and LFA-1. By comparing the CD44, LFA-1 and ASGM1 expression patterns of CD8 T cells, we found that the intersection occupied almost all the LFA-1 set and the majority of ASGM1 set, implicating that this triple-positive CD8 T cells belonged to a distinct sub-population (Figure 7b). To further verify
One day after injecting anti-ASGM1 antibodies, the CD44+LFAhi CD8 T cells of both WT or NFIL3 KO mice were depleted (Figure 7c). Collectively, our data showed that ASGM1, CD44 and LFA-1 triple positive CD8 T cells was not only a distinct sub-population, but occupied most of the phenotypically liver resident CD8 T cells.
3.7 Intrahepatic ASGM1+ CD8 T cells had distinct transcriptional profile from
ASGM1-CD8 T cells and showed similarity to core gene signature of Trm cells.
We next sought to determine whether differences between ASGM1+ and ASGM1- CD8 T cells in not only the phenotypic marker, but also the transcriptional signature. Both cells were respectively sorted for gene expression microarray analysis. 1,129 expressed genes showed a fold change of 2 or greater (Figure 8a), indicated that ASGM1+ CD8 T cells were distinct from ASGM1- ones in transcriptional level. The differentially expressed genes included those encoding chemokine signaling pathway, T cell receptor signaling pathway, integrin-mediated cell adhesion, focal adhesion and XPodNet protein-protein interactions (Figure 8b). This raised the question that ASGM1+ CD8 T cells might have unique adhesive behavers in the liver. We found that many genes represented in the core gene signature of liver Trm cells (Mackay LK et al. 2016, Fernandez-Ruiz et al. 2016) were similarly up- or down-regulated in liver ASGM1+ CD8 T cells (Figure 8c).
We confirmed several of these findings by qPCR (Figure 8d), suggesting that these cells are liver resident cells.
3.8 Intrahepatic ASGM1+ CD8 T cells homed to and persisted in the liver after
transplantation.
Recent evidence indicates that large numbers of resident CD8 T cells are harbored within liver for protection against pathogen challenges (Keating R, et al. 2007).
Nevertheless, whether liver resident CD8 T cells contribute the major role among total CD8 T cells in host protection against HBV is still unclear. Our results indicate that the major intrahepatic ASGM1-positive immune cells in NFIL3-KO mice highly co-expressed LFA-1, which is recently reported as a phenotypic marker of liver-resident T cells (Beura et al. 2018; McNamara et al. 2017). To further address the liver resident role of intrahepatic ASGM1+ CD8 T cells, we compared the expression of LFA-1 between intrahepatic and splenic CD8 T cells first. As showed in Figure 9a, LFA-1 was highly expressed by intrahepatic ASGM1+ CD8 T cells but not by splenic T cells. Moreover, only ASGM1+ CD8 cells sorted from liver but not spleen homed to the livers of recipients after adoptive transferring, and these liver-homing donor cells persisted for more than
phenotype were also depleted after anti-ASGM1 Ab treatment (Figure 9 d). Collectively, ASGM1-positive CD8 T cells not only phenotypically but also functionally showed liver residency.
3.9 Blocking of ASGPR1 dampened the liver homing of ASGM1+ CD8 T cells.
The liver resident CD8 T cells express high level of ASGM1. Meanwhile, NK cells, which are known to express high level ASGM1, are especially enriched in the liver. Those implied that the expression of ASGM1 might have role in adhesion and there might be some ASGM1 receptors enriched in the liver. Trm of intestinal intraepithelial leukocytes (IEL) are known to express CD103. Neither CD103 positive nor negative IEL expressed certain ASGM1 in naive mice, indicating that the expression of ASGM1 was not universal to the tissue resident CD8 T cells in different organs.
Chapter 4 Discussion
4.1 Contribution of this work.
We showed that HBV clearance of mice was abolished by α-ASGM1 treatment on both BALB/c, C57BL/6 and NFIL3 KO mice, while NFIL3 KO mice without α-ASGM1 treatment were immune competent against HBV as wild type mice. This result suggested there is a non-cNK ASGM1+ cell critical in eliminating HBV. We further characterize a distinct CD44+LFAhi CD8 T cells highly expressing ASGM1 among intrahepatic leukocytes and can be depleted by α-ASGM1 treatment. Next, we compare the gene expression profile of this distinct subset of CD8 T cells with core gene signature of TRM, and employed adoptively transferring to explore that intrahepatic ASGM1+ CD8 T cells were phenotypically and functionally liver-resident. Finally, we test the possibility that ASGPR might be the hepatic receptor of ASGM1+ cells with ASGPR1 blocking antibody.
4.2 Deficient of conventional NK cells is not enough to impair the ability of HBV
clearance.
A study consisted with our data to demonstrate that anti-ASGM1 treatment abolished the HBV elimination. However, they further showed that the ability of HBV clearance was impaired in NFIL3 KO mice (Zheng et al., 2016). We notice that the kinetic of the serum HBsAg in their NFIL3 KO mice work was different from the anti-ASGM1
ASGM1 treatment. Nevertheless, HBV clearance in NFIL3 KO mice looks slightly different from wild type mice. Our results didn’t rule out the possibility that NK cells can help to enhance the quantity of HBV-specific CD8 T cells. Instead, we strengthen the important role of CD8 T cells since the depletion the liver-resident CD8 T led to the abolish of HBV clearance in NFIL3 KO mice.
4.3 The role of distinct CD8 T subset in the immune response needs to be explore.
Current antiviral therapies such as reverse transcriptase inhibitors-nucleotide analogue suppress viral load but fail to achieve HBV cure. Combination of antiviral and immune modulatory therapies is thought to cure hepatitis B virus infection. There were several clinical trials of immune checkpoint blockade about activating HBV specific T cells. However, the safety and efficacy of anti-PD-1 therapy for HBV clearance remain concerned (Johnson et al. 2016; El-Khoueiry et al 2017; Gane et al. 2019). New approaches are needed to help HBV specific T cells to eliminate HBV.
Anti-ASGM1–mediated NK cell depletion was thought to be a powerful tool to analyze in vivo functions of NK cells, nevertheless, the expression of ASGM1 is found on not only NK cells, but a subpopulation of NKT, CD8 T and other hematopoietic cells
that about 10%~30% naïve CD8 T cells (Kosaka et al. 2007) expressed ASGM1, but from where these T cells come and what is the biological role of them are remain undetermined.
It has been also documented that most of the LCMV-specific CD8 T cells expressed ASGM1 (Slifka et al. 2000), while what these cells are and whether they are functionally different from ASGM1 negative T cells remain unknowns as well. Herein we provided the first evidence that ASGM1 is expressed by a distinct effector CD8 T cells expressing CD44 and LFA-1. It has been documented that activated T cells expressing specific activation profile have their own distinct character (Wei et al. 2017). The authors defined five distinct MC38 tumor-infiltrating T cell clusters phenotypically, only two of them were CD44, CD69 double positive. Different clusters showed unique immune response under the treatment of immune-checkpoint blockade, implied that CD44 and CD69 might not merely represent the activation of T cell. Soon after activation, T cells expressed CD44 which binds to hyaluronan (HA) for T cells rolling and extravasation. Though that CD44 was considered a memory marker as well, it has been documented that the engagement of CD44 reduces the formation of memory CD8 T cells (Lee-Sayer et al.
2018). The role of CD69, a C-type lectin, have remained elusive for a long time though that it is rapidly expressed after T cell activation.
4.4 TRM or liver resident CD8 T will be the potential target of immune therapy.
TRM cells are thought to reside in peripheral tissues and is crucial for protective immunity in peripheral tissue. TRM is a functional term raised from the study of CD8 T cells from infected skins. Conventional TRM highly expressed CD44, CD49a, CD103, and CD69. The former three binds Hyaluronic acid, type IV collagen and E-cadherin, respectively. The last inhibits S1PR1 and further stop the egress of the cells to blood.
Those molecules coordinately help cells to reside in peripheral tissues. However, accumulating evidences pointed out that liver-resident CD8 T cells are atypical TRM since they don’t express CD49a and CD103 (Fernandez-Ruiz et al. 2016; McNamara et al. 2017). Accordingly, the study employed CD69, CD103 double positive markers as the premise of human liver TRM without other validations became no significance (Pallett et al. 2017). Thus, other molecule must take place to provide enough power for residency and be considered as a reliable liver-resident marker..
it has been demonstrated that effector CD8 T cells which arrested within liver sinusoids contributed to recognize hepatocellular antigens in HBV mouse model
it has been demonstrated that effector CD8 T cells which arrested within liver sinusoids contributed to recognize hepatocellular antigens in HBV mouse model