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Features of immune senescence in liver
transplant recipients with established grafts
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Liver Transplantation May 2010
William Gelson 1, Matthew Hoare 1, Sarah Vowler 2, Arun Shankar 1, Paul Gibbs 3, Arne N. Akbar 4, Graeme J. M. Alexander 1
1Department of Medicine, University of Cambridge, Cambridge, UK
2Centre for Applied Medical Statistics, University of Cambridge, Cambridge, UK
3Department of Surgery, University of Cambridge, Cambridge, UK
4Department of Immunology, University College London, London, UK
email: Graeme J. M. Alexander (gja1000@doctors.org.uk)
"We believe this is the first study of immune senescence in the field of solid organ transplantation......Outside the transplant setting, cardiac disease, hematological malignancy, cerebrovascular disease, and infections are known to be associated with shortened telomere length in PBMCs.....The field of immune senescence consists mainly of cross-sectional studies. Longitudinal studies that examine telomere dynamics over time have not yet been reported, although no doubt many are underway.[40] Factors thought to cause accelerated immune senescence include chronic viral infections (CMV, EBV, and human immunodeficiency virus), smoking, obesity, stress, diabetes mellitus, and sarcoidosis.[3][4][41-44] It is not possible to ascertain whether these are causal associations......Liver transplant recipients with established allografts had shorter lymphocyte telomeres (in both CD4+ and CD8+ T cell subsets) when compared with the healthy control group with similar demographics.....these observations suggest first that liver transplant recipients with established grafts have fewer immature lymphocytes and second that their immature lymphocytes are more aged than healthy controls. This is likely to decrease the potency of immune responses to novel antigen.....The presence of HCC at engraftment and skin malignancy after transplantation were associated independently with shortened lymphocyte telomeres....Previous CMV infection (as determined by positive for anti-CMV antibody at sampling) was found to be associated with decreased expression of CD8+ CD127+, CD8+ CD28+, CD8+ CD27+, CD4+ CD28+, and CD4+ CD27+ and increased expression of CD8+ CD57+, but not with telomere length.....Lymphocyte telomere length was associated negatively with age in all lymphocyte subsets studied, consistent with the fact that telomeres shorten with age. Lymphocyte markers of maturity (CD45RO, CD57, and KLRG1) were associated positively with age, while lymphocyte markers of immaturity (CD28, CD27 and CD127) were associated negatively with age. Lymphocyte telomere length was associated positively with markers of lymphocyte maturity and associated negatively with markers of immaturity in cases, consistent with the concept of lymphocyte telomere loss with the proliferation associated with antigen experience.[2] Telomere length was also significantly shorter in mature than immature CD4+ and CD8+ T cells."
ABSTRACT
Immune senescence is the normal process whereby the human immune system ages, but becomes less effective. We investigated whether liver transplant recipients have features of immune senescence. Lymphocytes from 97 liver transplant recipients with established grafts and 41 age-matched and sex-matched controls were subjected to an 8-color flow cytometry assay that measured expression of killer cell lectin-like receptor subfamily G member 1, cluster of differentiation 127 (CD127), CD45RO, CD27, CD28, CD4, CD8, and CD57. Lymphocyte telomere length was assessed by flow-fluorescence in situ hybridization. Cases were compared with controls for each marker of immune senescence using a Mann-Whitney U test. For liver transplant recipients, linear regression analyses identified associations between markers of immune senescence and clinical or demographic characteristics. Lymphocytes from liver transplant recipients expressed more phenotypic markers of maturity than did lymphocytes from controls. Lymphocyte telomeres were shorter in liver transplant recipients than in controls. Age, hepatocellular carcinoma at transplantation, and skin malignancy developing after transplantation were associated independently with shortened lymphocyte telomeres. Increasing age and previous cytomegalovirus infection were associated independently with phenotypic markers of lymphocyte maturity. Thus, lymphocytes from liver transplant recipients are older biologically than lymphocytes from age-matched and sex-matched controls. Hepatocellular carcinoma at transplantation, subsequent skin malignancy, and previous cytomegalovirus infection are associated with lymphocyte senescence in liver transplant recipients.
Article Text
The peripheral T lymphocyte pool is maintained by a combination of antigenic stimulation and other, less well-defined homeostatic mechanisms.[1] Peripheral T lymphocytes turn over constantly. This may be part of a slow homeostatic process, with bursts of activity when a target antigen is experienced, or through chronic antigenic stimulation, for example in the presence of chronic viral infection.[2-4] The result of constant turnover is immune senescence, characterized by a population of exhausted lymphocytes with a mature cell surface phenotype that demonstrate replicative senescence.[5][6]
All healthy cells, including T lymphocytes, have a finite lifespan. Human cluster of differentiation 4-positive (CD4+) lymphocytes sustain around 33 population doublings in culture; CD8+ cells sustain fewer, around 23.[7][8] As T cells reach replicative senescence, they stop dividing due to the development of cell-cycle arrest,[9] at which point the cell becomes resistant to apoptosis and there are significant changes in immune function.[10] In animals, aged naïve T cells produce less interleukin-2 and more interferon- than their young counterparts and express fewer membrane activation markers (CD25, CD62L, and CD154) when stimulated in vitro.[11-13] The ability of aged naïve T cells to provide B cell help also wanes.[14] Further, aged memory T cells derived from aged naïve T cells fail to proliferate or provide help in vitro.[3]
Telomeres are formed by a repeated hexameric sequence of nucleotides (TTAGGG),[15] which are found at the ends of chromosomes and shorten by 50-100 base pairs (bp) in most somatic cells (including lymphocytes) at each cellular division.[16] When telomere length becomes critically short, the cell becomes senescent.[17] Telomere length thus provides a surrogate in vivo marker for the assessment of immune senescence in different cell populations, including lymphocytes.
Many previous studies in nontransplant populations have demonstrated that cardiac disease, malignancy, cerebrovascular disease, and infections are associated closely with shortened telomeres in peripheral blood mononuclear cells (PBMCs); furthermore, prospective studies in healthy elderly populations reveal that shortened telomeres identify a cohort with a subsequent increase in morbidity and mortality.[16][18-21] These observations suggest first that immune senescence carries an increased risk and second that cardiac disease, malignancy, cerebrovascular disease, and infections are disorders associated with the process of immune senescence.
There is a marked increase in the prevalence of cardiac disease, malignancy, cerebrovascular disease, and infections in patients with established liver grafts, eventually affecting a majority of cases and which in the past have been attributed to agents used to suppress immune responses.[22-31] However, an alternative (and not exclusive) hypothesis is that liver transplant recipients develop premature immune senescence, which is associated with an increased risk of cardiac disease, malignancy, cerebrovascular disease, and infection, perhaps consequent to chronic alloantigenic stimulation.[16] To our knowledge, immune senescence has not been studied in the context of transplantation.
Based on these observations, we investigated key features of immune senescence (lymphocyte telomere length and lymphocyte phenotypic markers) in liver transplant recipients with established liver allografts. The relation between immune senescence and both clinical features of alloimmunity and complications of transplantation were also investigated.
Abbreviations:
ALD, alcohol-related liver disease; AIH, autoimmune hepatitis; APC, allophycocyanin; bp, base pair; CD, cluster of differentiation; CI, confidence interval; CMV, cytomegalovirus; Cy5, cyanine 5; EBV, Epstein-Barr virus; FISH, fluorescent in situ hybridization; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; KLRG1, killer cell lectin-like receptor subfamily G member 1; mfi, mean fluorescence intensity; PBC, primary biliary cirrhosis; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PE, phycoerythrin; PSC, primary sclerosing cholangitis.
DISCUSSION
Lymphocyte telomere length was associated negatively with age in all lymphocyte subsets studied, consistent with the fact that telomeres shorten with age. Lymphocyte markers of maturity (CD45RO, CD57, and KLRG1) were associated positively with age, while lymphocyte markers of immaturity (CD28, CD27 and CD127) were associated negatively with age. Lymphocyte telomere length was associated positively with markers of lymphocyte maturity and associated negatively with markers of immaturity in cases, consistent with the concept of lymphocyte telomere loss with the proliferation associated with antigen experience.[2] Telomere length was also significantly shorter in mature than immature CD4+ and CD8+ T cells.
We believe this is the first study of immune senescence in the field of solid organ transplantation. Liver transplant recipients with established allografts had shorter lymphocyte telomeres (in both CD4+ and CD8+ T cell subsets) when compared with the healthy control group with similar demographics. Lymphocyte telomere length was similar in liver transplant recipients and healthy controls when analysis was restricted to aged lymphocytes (CD4+ CD45RO+ and CD8+ CD57+ T cells). In addition to having lymphocytes with shorter telomeres, liver transplant recipients also had more mature CD4+ T cells (CD45RO+) and fewer immature CD4+ and CD8+ T cells (CD27+, CD28+, and CD127+). Taken together, these observations suggest first that liver transplant recipients with established grafts have fewer immature lymphocytes and second that their immature lymphocytes are more aged than healthy controls. This is likely to decrease the potency of immune responses to novel antigen.[2]
Several studies demonstrate expansion and telomere shortening of antigen-specific cells to chronic viral antigens.[3][4][35] However, antigen-naïve lymphocytes were also more aged in liver transplant recipients than healthy controls. It seems improbable that this is due to chronic antigenic exposure, and alternative explanations must be sought; either non-antigen dependent lymphocyte turnover is responsible,[1] or there is a preferential antigen-dependent selection/survival of naïve cells with long telomeres. We have not pursued these hypotheses in this study.
The presence of HCC at engraftment and skin malignancy after transplantation were associated independently with shortened lymphocyte telomeres. HCC and skin malignancy have not been studied in relation to lymphocyte or PBMC telomere length. However, patients were found to have shorter telomeres than age-matched controls in all groups in a study of 92 patients with head and neck cancer, 135 with bladder cancer, 54 with lung cancer, and 32 with renal cell carcinoma.[36] The differences were 0.9 kb, 0.2 kb, 0.4 kb, and 0.2 kb, respectively. Given that PBMC telomere loss is between 15 and 55 bp/year in adults,[37] this equates to between 6 and 26 years of additional aging (taking a mean value of 35 bp/year). In our patient group, the difference was smaller, at about 6 years of additional aging for HCC and 4 years for cancer that developed following liver transplantation. However, the patients in the present series were quite different. None of the patients with HCC at transplantation was known to have recurrent HCC at sampling and all were at least 3 years from definitive treatment by liver transplantation. Skin malignancy in our study included squamous cell carcinoma, basal cell carcinoma, and one melanoma. Only one patient had an active skin malignancy at the time of lymphocyte sampling (head and neck squamous cell carcinoma).
Previous CMV infection (as determined by positive for anti-CMV antibody at sampling) was found to be associated with decreased expression of CD8+ CD127+, CD8+ CD28+, CD8+ CD27+, CD4+ CD28+, and CD4+ CD27+ and increased expression of CD8+ CD57+, but not with telomere length. There is a recognized association of low CD28+, CD27+, and CD127+ expression and high CD57+ expression with previous CMV infection.[4][38][39] KLRG1 may be up-regulated by CMV infection,[4] but this study did not confirm that observation.
Normal liver biochemistry was associated with an immature CD8+ phenotype (CD8+ CD127+, CD8+ CD28+, and CD8+ CD57-). Normal liver biochemistry may identify a group that includes those with operational tolerance; expression of these markers may, in some, therefore relate to clinical tolerance. A study of immune suppression withdrawal would be required to investigate this further. Another possibility is that patients with more naïve lymphocytes may respond to the complications of transplantation more effectively than those who have an exhausted immune system, or conversely that those patients with healthy grafts have had fewer complications and therefore have not worn out their immune system. A longitudinal study would be required to assess this phenomenon further and is underway. This will allow an assessment of the utility of these biomarkers in risk stratification.
Outside the transplant setting, cardiac disease, hematological malignancy, cerebrovascular disease, and infections are known to be associated with shortened telomere length in PBMCs.[16][18-21] Such associations were not found in this study. However, this series was small and based largely on healthy patients attending a liver transplant clinic for a routine appointment. A larger study is required to investigate these associations further in the field of liver transplantation.
The hallmarks of immune senescence are replicative senescence (characterized by short telomeres, altered immune function, and poor proliferative ability in long-term cell culture), the expansion of lymphocyte populations with a mature, antigen-experienced cell-surface phenotype and a diminished T cell receptor repertoire.[10][16] In vitro studies of proliferative capacity and immune function and assessment of T cell receptor repertoire would help to further develop our understanding of immune senescence in liver transplantation.
The field of immune senescence consists mainly of cross-sectional studies. Longitudinal studies that examine telomere dynamics over time have not yet been reported, although no doubt many are underway.[40] Factors thought to cause accelerated immune senescence include chronic viral infections (CMV, EBV, and human immunodeficiency virus), smoking, obesity, stress, diabetes mellitus, and sarcoidosis.[3][4][41-44] It is not possible to ascertain whether these are causal associations.
This study has shown, for the first time, an association of solid organ transplantation with features of immune senescence (short lymphocyte telomeres and mature cell surface phenotype). Because immune senescence may predispose to diseases that are common in liver transplant recipients, namely cardiovascular disease, malignancy, and infections, this may be an important finding.
If there was a causal relationship between liver transplantation and immune senescence, one might expect a correlation between the number of transplants performed and features of immune senescence. This correlation was found only for telomere length in CD8+ CD57+ cells; thus, such a relationship might not exist. However, only 13 of 97 recipients received more than 1 graft which may be too few for analytical purposes; the hypothesis assumes that lymphocyte telomeres shorten but never lengthen, which may not be correct.
Many factors may contribute to immune senescence in liver transplantation, including chronic liver disease before and after engraftment, alloimmune responses, infection, and immune suppression. We have embarked upon a longitudinal study comparing features of immune senescence before and after engraftment to investigate a causal relationship between these factors and immune senescence.
RESULTS
The Relation Between Age with Telomere Length and Lymphocyte Surface Phenotype in Cases and Controls
Age was associated inversely and significantly with telomere length in both cases and controls in all lymphocyte subsets studied (Fig. 2 and Table 2). There was correlation between age and cell-surface markers of cellular senescence, which was significant for all lymphocyte subsets studied in the cases. Further, there was close correlation between lymphocyte telomere length and cell-surface markers of cellular senescence in cases but not controls. These observations confirm that age was associated with increased lymphocyte maturation, which was most marked in cases; thus lymphocyte telomeres shortened with increasing age, the proportion of lymphocytes with a mature lymphocyte phenotype increased (KLRG1, CD57, and CD45RO), and the proportion of lymphocytes with an immature phenotype decreased (CD27, CD28, and CD127).
Comparison of Liver Transplant Recipients with Healthy Controls
There was no evidence of an age (P = 0.93) or sex (P = 0.18) difference between cases and controls. Table 3 shows a comparison of telomere length in different lymphocyte subsets in liver transplant recipients (n = 97) with healthy controls (n = 41). Telomere length was shorter in liver transplant recipients than controls in all T cell subsets. These differences were significant in lymphocytes overall (P = 0.004), CD4+ T cells (P = 0.01), CD8+ T cells (P = 0.01), less mature T cells (CD4+ CD45RO- [P = 0.004] and CD8+ CD57- [P = 0.003]). Using a regression line for all data in Fig. 2, the differences were interpreted in terms of absolute age, indicating a difference between liver transplant recipients and controls of between 4 and 5 years of additional immune aging. The observation that the difference was more marked in immature than mature T cell subsets (and was significant in immature but not mature T cell subsets) was surprising but noteworthy.
To assess whether accelerated immune senescence is an ongoing process in liver transplant recipients, telomere length for CD4+ cells were plotted against age for cases and controls (Fig. 2). The slopes and intercepts of the regression lines were compared with an analysis of covariance test. The slopes were not significantly different (P = 0.95); the intercepts were significantly different (P < 0.001).
Table 4 shows a comparison of lymphocyte phenotypic markers of maturity in liver transplant recipients (n = 97) with healthy controls (n = 41). For all markers, there was a higher proportion of lymphocytes with a mature phenotype (CD4+ KLRG1+, CD4+ CD45RO+, CD8+ KLRG1+ and CD8+ CD57+) in liver transplant recipients than controls and a lower proportion of lymphocytes with an immature phenotype (CD4+ CD27+, CD4+ CD28+, CD4+ CD127+, CD8+ CD27+, CD8+ CD28+, and CD8+ CD127+) in liver transplant recipients than controls. These differences were significant for CD4+ CD45RO+ (P = 0.004), CD4+ CD27+ (P = 0.003), CD4+ CD28+ (P = 0.02), CD4+ CD127+ (P = 0.04), CD8+ CD27+ (P = 0.03), CD8+ CD28+ (P = 0.045), and CD8+ CD127+ (P = 0.006) and were not significant for CD4+ KLRG1+ (P = 0.09), CD8+ KLRG1+ (P = 0.81), and CD8+ CD57+ (P = 0.35) lymphocytes.
Taken together, these observations suggest that liver transplant recipients have more lymphocytes with a cell-surface phenotype of increased aging than controls and that immature lymphocytes from liver transplant recipients are biologically older than those from controls. The fact that regression line slopes for cases and controls were similar, but that the intercepts were not, suggests that cases and controls were aging at a similar rate, but that cases had a lower baseline age.
Relation Between Clinical Characteristics and Features of Immune Senescence in Liver Transplant Recipients
With each of the experimental variables as outcomes, simple linear regression was used to screen for associated clinical and demographic characteristics (P < 0.10). These characteristics were taken through into multiple linear regressions, with experimental variables as outcomes. Table 5 summarizes the clinical characteristics that were associated independently with experimental variables.
Associations with Telomere Length in Liver Transplant Recipients
Age was associated with telomere length in all lymphocyte subsets. The average coefficient for age and telomere length over all lymphocyte subsets was -0.50 (standard deviation [SD] = 0.10); thus, as a liver transplant recipient with an established graft gets 1 year older, their telomere length shortens by 0.5 mfi.
HCC at transplantation was associated negatively with telomere length in all lymphocyte subsets studied, except for CD4+ CD45RO- T cells, where there was no association. The average coefficient for HCC at transplantation and telomere length was -12.40 (SD = 2.91); telomere length in patients with HCC who underwent transplantation would be expected to be 12.40 mfi shorter than those transplanted without an HCC (which equates to about 6.2 years of additional aging).
Skin malignancy subsequent to transplantation was associated negatively with telomere length in lymphocytes, CD4+ CD45RO+ T cells, CD8+ CD57+ T cells, and CD8+ CD57- T cells. The average coefficient for skin malignancy after transplantation and telomere length was -7.35 (SD = 0.43); telomere length in patients with skin malignancy after transplantation would be expected to be 7.35 mfi shorter than those without skin malignancy after transplantation (which equates to around 3.7 years of additional aging).
There were no consistent independent associations between telomere length and: sex; pediatric or adult recipient at engraftment; number of transplants; underlying disease etiology; CMV status; having normal liver biochemistry at sampling; calcineurin inhibitor or sirolimus-based immune suppression; number of prescribed immune-suppressive agents at sampling; number of infective episodes; number of treated episodes of acute rejection; and history of malignancy (other than skin) and precancerous lesions. A relation to cardiovascular or cerebrovascular events could not be addressed because the events were rare in this series.
Associations with Lymphocyte Cell-Surface Phenotype in Liver Transplant Recipients
Age was associated consistently and independently with markers of lymphocyte surface phenotype. The associations were negative for markers of immaturity (CD28, CD27, and CD127) and positive for markers of maturity (CD45RO, CD57, and KLRG1). The coefficient varied in magnitude from 0.26 and 0.59, equating to changes in cell surface expression of 0.26% and 0.59% per year.
Evidence of previous exposure to CMV was associated independently and negatively with expression of CD8+ CD127+, CD8+ CD28+, CD8+ CD27+, CD4+ CD28+, and CD4+ CD27+ and associated positively with CD8+ CD57+ expression. The coefficient was greater in CD8+ cells than CD4+ cells, accounting for up to 29.95% reduction in CD8+ CD27+ cells.
Normal liver biochemistry was associated independently and positively with expression of CD8+ CD127+ and CD8+ CD28+ and negatively with CD8+ CD57+ expression. The greatest coefficient was for CD8+ CD57+ cells at -16.81.
There were no consistent independent associations between lymphocyte cell surface phenotype and: sex; pediatric or adult recipient at engraftment; number of transplants; underlying disease etiology; the presence of HCC at transplantation; having normal liver biochemistry at sampling; calcineurin inhibitor or sirolimus-based immune suppression; number of prescribed immune suppressive agents at sampling; number of infective episodes; number of treated episodes of acute rejection; and history of malignancy and precancerous lesions.
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