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Keywords:

  • Human liver;
  • Tetramer;
  • Phenotype;
  • CTL

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The frequency and phenotype of human antiviral memory CD8+ T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8+ T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature ‘effector memory’ phenotype of antigen-specific CD8+ T cells within the liver. An enrichment of an activated subset of NKT cells (Vα 24/Vβ 11) was also observed, a finding which may be relevant to the regulation of the antiviral populations.

Abbreviations:
Tetramer:

HLA class I peptide tetrameric complex

MCMV:

Murine CMV

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

CD8+ T lymphocytes play a critical role in the initial control of virus infections and in some instances play an ongoing role in the post-acute or memory phase 1. Studies using HLA class I peptide tetrameric complexes (tetramers) have greatly expanded our knowledge of the frequencies, the phenotype and, to some extent, the function of antiviral CD8+ T lymphocytes. However, in man, these studies have largely been restricted to blood for logistical reasons. In some cases, such as persistent viral hepatitis, the intrahepatic populations have been analyzed in a limited number of cases 2, 3. However, the interpretation of these results is difficult since no data exists on the relative distribution and phenotype of virus-specific T cell populations within an uninfected liver. In murine studies, the nonlymphoid tissue compartment appears to be important, since about 50% of memory CD8+ T cells mayexist in these areas and the phenotype appears to be skewed towards an effector memory subset 4, 5.

Memory CD8+ T cells have been examined in great detail, and a spectrum of these cells appears to exist. Broadly, CD8+ T cells may maintain, or revert to, the expression of markers associated with lymph node homing (CD62L, CCR7), described as central memory T cells, with less immediate protective capacity, but strong proliferative capacity 6. Conversely, effector memory T cells lack these markers 6 and, in murine studies, appear to have an important protective role, particularly within the tissue compartment 7, 8. In man, the more ‘mature’ effector memory cells lose certain cell surface markers (CD27, CD28) as they acquire intracellular perforin 9, 10. These cells may re-express CD45RA (an isoform of CD45), which has been traditionally associated with naïve cells. The biological relevance of this expression is unknown, although it has been linked with ‘terminally-differentiated’ cells 11, 12, albeit contentiously 13, 14.

Across the antiviral memory T cell spectrum there are wide variations in phenotype. HCV-specific CD8+ T cells are CD27/28 high and perforin low, whilst at the opposite extreme, CMV-specific CD8+ T cells express high levels of perforin and are generally CD27/28 low, and EBV-specific CD8+ T cells lie in between 10. One hypothesis to explain this difference is that during HCV infection, more mature effector cells may be trapped in the liver, perhaps undergoing cell death within that compartment. To further investigate this and to define the nature of memory cells found in human tissue as opposed to blood/lymph nodes under normal conditions, we investigated a unique series of liver/blood paired samples from donors without liver inflammation.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 Relative percentage of virus-specific CD8+ T cells within the liver

Initially, the frequencies of tetramer-positive T cells in blood and liver were compared in a series of donors (Table 1). Relative enrichment of tetramer-positive CD8+ T cells within the liver compared to blood (greater than twofold difference as a percentage of total lymphocytes) was seen in six of nine responses for the CMV- or EBV-specific T cells (Fig. 1, upper panels, Fig. 2A). The enrichment of tetramer-positive cells within the liver compared to the blood lymphocytes was very marked in some cases, with relative increases of up to 45-fold. However, the overall effect was variable with mean enrichment factors of 2.2 for CMV-specific cells, 4.5 for EBV-specific cells and 3.2 for both kinds of cells combined.

The chemokine receptors CXCR3 and CCR5 have been shown to be important in selective T cell recruitment to HCV-infected livers 15. To determine whether these chemokine receptors may be important in the recruitment to the liver seen for some of the CMV- and EBV-specific CD8+ T cells, we analyzed several of the tetramer-specific populations for these markers. The level of CXCR3 expression was low (0–1.14%; n=4) as was expression of CCR5 (0–3.54%; n=4) on the liver lymphocytes analyzed.

Table 1. Relevant HLA types and tetramer-specific responses
PatientRelevant HLA typesCMV responseEBV response
1A2+
2A2, B7+ (B7)
3B7+ (B7)
4A2, B8+ (B8)
5B7+
6A2++
7A2, B8+ (A2, B8)
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Figure 1.  Example of liver and peripheral lymphocyte tetramer stains using the HLA-B7 CMV (patient 2) and HLA-B8 EBV (patient 7) tetramers (top panel). Example of antibody stains (CD45RO and CD27) used in this study (bottom panel).

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2.2 Phenotypic and functional analysis of virus-specific CD8+ T cells

The phenotype of the virus-specific T cell populations was analyzed by using surface stains for markers of maturation. Compared to the total CD8 population, CMV-specific T cells in blood had a more ‘mature’ or ‘late-differentiated’ phenotype: CD27/CD28 low, CCR7 low, with relatively high expression of CD45RO (and reciprocally lower expression of CD45RA) and CD95. As previously described in blood, EBV-specific T cells had a higher expression of CD27 and CD45RA than the CMV-specific T cells (Fig. 2B–H). In the liver, relatively little change in CD27 and CD28 expression was seen in the tetramer-positive populations (Fig. 2B–D), which largely reflects the fact that the tetramer-positive cells in blood are generally CD27/CD28 low. This contrasts with the overall CD8+ T cell population (which is typically CD28/ CD27 double or single positive in blood), a significant enrichment of CD27/28 double-negative (i.e. mature or late-differentiated) CD8+ T cells was readily seen in the liver (Fig. 2B).

Similarly, CCR7 expression was low on all tetramer-positive populations in blood and liver, but the CCR7-negative (effector memory) population was enriched in the liver amongst the total CD8+ T cell population. Also, high CD95 expression was seen on tetramer-positive lymphocytes in blood and liver, but was significantly enriched in the liver within the total CD8+ population. Therefore, the overall liver population is enriched for CD95+/CCR7/CD28/CD27 T cells, as typified by late effector memory antiviral T cells.

Analysis of the effector function of virus-specific T cell populations, as assessed by intracellular IFN-γ  production, was also attempted. However, the cells did not respond efficiently to the PHA and ionomycin treatment as a positive control to warrant analysis, which was most likely linked to an aspect of long-term cell storage. In an attempt to overcome this technical problem, we used the murine CMV (MCMV) model to analyze tetramer-specific T cell function in the liver, as previously described 16. Using this model, we observed a strong IFN-γ  production in the liver lymphocyte populations analyzed, which was comparable to that in the spleen (Fig. 3).

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Figure 2. (A) Percentage of CMV (◊) and EBV (▪) tetramer-specific lymphocytes within cells obtained from liver (L) and PBMC (P). Percentage of (B) CD27/CD28 double-negative cells, and (C) CD27+, (D) CD28+, (E) CD45RA+, (F) CD45RO+, (G) CD95+ and (H) CCR7+ expressed on CD8+ lymphocytes (▵), combined CMV and EBV tetramer-specific lymphocytes (•), CMV tetramer-specific, and EBV tetramer-specific lymphocytes from liver or PBMC. The frequencies were compared between results for each group from liver and PBMC using a paired t test, and the means of each group are indicated with a horizontal bar. Significant differences (p<0.05) between groups are indicated with their p values.

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Figure 3. Peptide-induced IFN-γ  expression of tetramer-specific (MCMVpp89) CD8+ T cells within the spleen and liver of a latently infected mouse, using an intracellular cytokine stain after peptide stimulation. The proportion of the tetramer-positive cells producing IFN-γ  (top right of quadrant) is indicated as a percentage.

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2.3 Potential regulation of virus-specific CD8+ T cells within the liver

NKT 17 and regulatory T cells 18 have both been shown to regulate T cell proliferation and differentiation. Since the liver is known to be a regulatory environment, we analyzed the percentage of these populations within the corresponding blood and liver lymphocytes. A significant enrichment of a particular subset of NKT cells (Vα 24+/Vβ 11+) was seen within the liver compared to blood (0.088% vs. 0.015%; Fig. 4A, B). The NKT cells within the liver also expressed a higher amount of CD69, indicating that these cells are more strongly activated (Fig. 4A, C). In contrast, the percentage of regulatory T cells (CD4+/CD25+) within the liver was lower (Fig. 5A), despite a similar percentage of CD25+ cells within the CD4+ populations (Fig. 5C). The decrease in frequency was due to the overall frequency of CD4+ cells being lower in the liver than in the blood (Fig. 5B).

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Figure 4. (A) Example of liver and peripheral NKT cell (Vα 24/Vβ 11) antibody stains (top panel) and of CD69 antibody stains of the NKT cell populations (bottom panel). Percentage of cells within the lymphocyte population obtained from the liver (L) and PBMC (P) expressing Vα 24/Vβ 11 (B) or expressing CD69 within the Vα 24/Vβ 11 population (C). The frequencies were compared between results for each group from liver and PBMC using a paired t test, and the means of each group are indicated with a horizontal bar. Significant differences (p<0.05) between groups are indicated with their p values.

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thumbnail image

Figure 5. Percentage of cells within the lymphocyte population, obtained from the liver (L) and PBMC (P), that are CD4/CD25 double positive (A) or express CD4 (B) within the lymphocyte population or that express CD25 within the CD4+ population (C). The frequencies were compared between results for each group from liver and PBMC using a paired t test, and the means of each group are indicated with horizontal bars. Significant differences (p<0.05) between groups are indicated with their p values.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Analysis of CMV- and EBV-specific CD8+ T cells isolated from blood and the liver sinusoid revealed that significant frequencies of virus-specific CD8+ T cells can be found in the liver in the absence of inflammation, although these frequencies appear to be variable between individuals. The localization of cells does not appear to be closely linked to the expression of the chemokine receptors CXCR3 and/or CCR5. This compares to a recent study during the memory phase of an MCMV infection, which showed a relative enrichment of MCMV-specific CD8+ T cells within the liver by a factor of 1.5 16, compared to mean enrichments of 2.2 for CMV and 4.5 for EBV in our study. We estimate that at any time point, the total number of CMV- or EBV-specific CD8+ T cells in a normal human liver is in the order of 1×106 for each single specificity. Based on the murine model, the finding of such mature effector cells in a parenchymal organ is unlikely to be liver-specific, although the localization of such cells to the liver is of particular importance with reference to viral hepatitis and T cell regulation. Although CMV reactivation may occur in the liver, studies in the mouse suggest that this is a rare, sporadic event 19. Given the observed presence of effector cells of diverse specification in the liver infiltrates, this suggests that the localization of such cells is not dependent on specific viral reactivation in this site, but rather reflects a particular homing pattern of a CD8+ T cell type 20.

Most strikingly, there was a significant shift in the tetramer-positive populations in terms of CD45 isoform expression. The tetramer-positive cells expressing CD45RO were significantly enriched in the liver as compared to the blood fraction. This effect was seen in both the CMV- and EBV-specific populations, but was most marked in the latter population. Reciprocal changes were seen in the expression of CD45RA. Similar changes within the total CD8+ T cell populations were also seen, although interpretation of this is slightly different from the tetramer-positive population, as this population includes both naïve and antigen-experienced cells. The tetramer-positive population contains only expanded memory populations, and it is of some significance that a functional difference (i.e. homing) is seen between the CD45RA+/CD45RO and reciprocal CD45RA/CD45RO+ subsets, since only in vitro effects have been shown to date, and even these have been contentious 1114, 21.

If effector memory subsets were to be defined by the relocation of such CD8+ T cells to peripheral organs, then CD45RA+ cells would appear to be less common amongst effector memory cells, arguing against the expression of this marker as a consequence of differentiation. A potential caveat is that such cells may die very rapidly on entering the liver and thus would not appear in our analysis.

Activation of virus-specific T cells was not analyzed in this study. Previous studies using identical samples have shown that a significant percentage of CD8+ T cells express CD69 in the liver compartment 22. It is not clear what drives this activation, and it appears from the data that it is not antigen-specific, since even large populations such as those specific for CMV comprise only a fraction of the total CD8+ T cells detected. This activation is likely to be functionally important and must be borne in mind during analysis of activation in the context of disease. It is possible that activation of innate subsets enriched in the liver, possibly in the context of gut-derived bacterial products, releases cytokines that can lead to activation of ‘bystander’ CD8+ T cells. One such subset consists of Vα 24/Vβ 11 double-positive NKT cells, which we find strongly enriched in the normal human liver. NKT cells have been shown to play an important role in the regulation of immunity to pathogens within this environment 23, 24. Previous studies on the Vα 24/Vβ 11 subset of NKT cells analyzed patients during persistent HCV infection and showed a comparatively high (up to 0.37%) percentage of these cells within the liver 25, 26. The NKT cell subset was also more highly activated (>90%) in the HCV-infected livers as compared to an average of 50% seen in the ‘normal’ liver. This correlates with another study analyzing the general NKT cell population in the liver-infiltrating lymphocytes of HCV-positive and -negative individuals 27. Hence, this specific subset of NKT cells may have an important role in HCV infection. It is noteworthy that compared to HCV-specific T cell infiltrates within inflamed HCV-infected livers 2, 3, the CMV- and EBV-specific subsets are still relatively large and could potentially contribute to immunopathology through non-specific activation pathways.

The liver has also been proposed as a site of immune ‘tolerance’ 28, and several cell populations have been proposed to play a role in this. In particular, regulatory T cells are known to suppress CD8+ T cell activation in vivo29. The decreased percentage of CD4+/CD25+ T cells seen in the liver argues against a specific regulatory T cell effect in the liver due to increased local activity. However, further functional studies, particularly by using novel regulatory T cell markers, are required to test these ideas. One other feature relevant to the regulation of CD8+ T cells in the liver was the relatively high expression of Fas (CD95) on CD8+ T cells as compared to that in blood. It is possible that cell death through the Fas/Fas ligand pathway contributes to the elevation of such activated cells in the liver 30.

In conclusion, using a unique source of human tissue-derived lymphocytes, we have shown that virus-specific T cells enter the normal human liver from the blood and can be found at significant frequencies even in the absence of obvious inflammation. The mechanisms leading to the accumulation and/or activation of such cells are not fully understood, but markers associated with CD45RO as opposed to the CD45RA subset of antigen-experienced CD8+ T cells should be examined in future studies. The role of activated NKT cells in homeostasis of normal human antiviral CTL demands further analysis.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Cell samples

Mononuclear cells were obtained from blood (PBMC) and livers (LMC) of organ donors and stored in liquid nitrogen as previously described 22. The liver-derived cells were previously found to represent the liver sinusoidal lymphocyte population 22.

4.2 Tetramers and antibodies

Peptide-HLA class I tetramers were constructed as described 31, using peptide epitopes derived from EBV (HLA A2-restricted peptide GLCTLVAML, HLA B8-restricted peptide RAKFKQLL) and CMV (HLA A2-restricted peptide NLVPMVATV, HLA B7-restricted peptide TPRVTGGGAM), and were used with a PerCP-conjugated anti-CD8 mAb to label cells in conjunction with FITC- or APC-conjugated mAb specific for CD27, CD28, CD45RA, CD45RO, and CD95 (Becton Dickinson). The unconjugated anti-CCR7 antibody (Becton Dickinson) was combined with an APC-conjugated secondary antibody (Dako). The following mAb were also used: anti-CD4-APC, anti-CD25-FITC, anti-CD69-APC (Becton Dickinson), anti-Vα 24-PE and anti-Vβ 11-FITC (Serotec).

4.3 Flow cytometric staining and analysis

Immunostaining and fluorescence-activated cell sorter (FACS) analysis were performed as described 26. Briefly, 1×106 cells were incubated with an excess of tetramer for 20 min at 37°C. Cells were washed with PBS containing 2% FCS. Antibodies were then added in excess for 20 min at 4°C, and cells were washed as before. Individual samples were stained 3–4 times during phenotypic analyses, resulting in multiple data points for the calculation of tetramer-specific cell frequencies. Data were collected on at least 5×104 cells using a FACSCalibur and analyzed using CellQuest software (both Becton Dickinson).

4.4 Murine IFN-γ  secretion

Murine infection with MCMV, MCMVpp89 tetramer staining, and IFN-γ  staining were performed as described 16. Lymphocytes were prepared from liver and spleen of mice at 1 year post-infection.

4.5 Statistical analysis

Statistical analysis was performed using GraphPad Prism Version 3.0a. Data were compared using a paired t test with a significant value of p<0.05.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

We are grateful to the Wellcome Trust, NHMRC (Australia) and the Lions Medical Research Foundation for financial support.

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