Immunological characteristics of the elderly allograft recipient

Abstract

The increasing number of elderly people with a demand for organ transplantation poses an important medical challenge. The effect of aging on the immune system concerns wide modifications with a considerable influence on transplant outcomes. Aging causes significant changes in immune cells repertoire. Thymic involution impairs the production of new naïve cells. Immune remodeling induces important alterations in the activity of immunological molecules. Therefore, clinical implications in elderly transplant recipients should consider appropriate organ allocation with adequate individualization of immunosuppression.

Introduction

The growing phenomenon of global population aging, especially in the developed countries, presents a new challenge for the increasing need for organ transplantation. Survival benefits of solid-organ transplantation are unquestionable. More than 2 million life-years have been saved by solid-organ transplants in the last 25-year period only in the United States [1].

Chronological age alone is not considered as a contraindication against transplantation. Nevertheless, physicians who take care of patients prepared for transplantation and after transplantation should bear in mind that older patients have a higher risk of post-transplant infections, malignancy and renal dysfunction, which is exacerbated by immunosuppressant drugs [2]. On the other hand, the risk of rejection is lower in the elderly due to reduced immunological activity which occurs with aging [3]. The senescence of immunological system seems to be mostly associated with chronological aging but also with an increased risk of opportunistic infections, mainly CMV. Chronic diseases, including systemic lupus erythematosus, coronary artery disease, rheumatoid arthritis, also exhibit features of acute immune remodeling. For the above-mentioned reasons, the understanding of immunosenescence and immune remodeling is very important for adequate individualization of immunosuppression after transplantation.

The innate immune system is far older in comparison with the adaptive immune system but newer in its recognition as a significant factor in organ transplantation [4]. As T cells are necessary for the rejection, much more attention has been paid on cells of the adaptive immune system. It is now recognized that up-regulation of proinflammatory mediators in the allograft is present before the T cell response. Such early inflammation associated with innate response and connected with tissue injury is independent of the adaptive immune system [5], [6], [7], [8]. Innate immune system is a major barrier against infections, and infections are indeed the first cause of death in elderly renal transplant recipients. Mechanisms of the innate immune system are changed with advanced age [9], [10]. Aging is associated with hyperinflammation that may exacerbate chronic damage such as atherosclerosis. Proinflammatory cytokines among others IL-6, Il-1B, TNF-alpha, C-reactive protein, increase with aging [11], [12]. Chemokines and adhesion molecules have their role in homing of lymphocytes and graft rejection. The role of vascular endothelium is also important in regulation of inflammatory but also postinflammatory fiber reactive processes in transplantation [13], [14], [15].

Expression but also activity of Toll-like receptors may be dysregulated with aging which contributes to the increased morbidity and mortality from infectious diseases found in the elderly [12], [16].

In older patients there is an alteration of the receptor-driven functions of neutrophils, such as apoptosis, chemotaxis and superoxide anion production which cause a decrease in signaling elicited by specific receptors [17]. The role of natural killer (NK) cells in both allograft rejection and tolerance is significant. Recently it was shown that NK cell status may predict morbidity and mortality in older people, emphasizing the importance of innate immunity [18]. Natural killer cells seem to be important in transplantation due to their ability to differentiate allogeneic major histocompatibility complex (MHC) antigens but also their potent cytolytic effector mechanisms. NK cells participate in the immune response against solid organ grafts which suggests that their role in rejection and tolerance might be significant [19]. NK cells, being innate immune lymphocytes, participate also in inspection against transformed cells, viruses and other pathogens which plays an essential role in elderly patients concerning risk of infections and cancers. Senescence has a differential effect on distinct NK cells’ biological functions [20]. Upon stimulation with IL-2 a decrease proliferative response of NK cells was observed.

It should be also mentioned that components of the innate immune system called complement participate in the graft response of the elderly, too [21]. The complement system has an ability to participate in non-specific inflammation and membrane injury but also antigen-specific immune stimulation. In renal transplantation, the complement cascade plays role in both non-immune- and immune-mediated damage [22]. Complement component C4 copy number variation in human longevity was noticed lately by a German group during investigation of more than 3000 individuals [23] Table.

Homeostatic proliferation is a common physiological process triggered by lymphopenia to maintain a stable level of T cells [24]. Transplantation is a rare model in which lymphopenia is intentionally induced for its immunosuppressive effect.

Calendar aging is associated with thymic involution accompanied by changed tissue architecture and reduced tissue mass together with a decrease in CD3 + T cell production [3], [4], [5]. Involution of thymic tissue is the cause of T cell aging in healthy people, which is related to reduced numbers of circulating naïve T cells. It corresponds with an increased differentiation and proliferation paths leading to the creation of memory T cells. At the age of about 50 years immature thymocytes represent only about 10%, while in 70-year-olds thymic activity is almost absent [25], [26]. The contact with antigens throughout life causes the reduction in the size of naïve cells and the expansion of memory cells, which results in moving the naïve compartment to memory compartment with aging. Peripheral homeostatic proliferation of T cells prevents lymphopenia which appears in the elderly as a result of thymic involution [26].

The progressively inadequate thymic output of naive T-cells seems to be one of more important mechanism of senescence of the immune system. When the thymic output of new naïve T cells does not restore equilibrium, a peripheral proliferation of surviving T cells in secondary lymphoid organs, known as homeostatic proliferation (HP) takes over with all the consequences that this brings about [27], [28], [29]. This mechanism is also important for memory T cells which have high impact on rejection and the induction of tolerance [30]. Alloreactive memory T cells may be generated in transplant recipients that have not earlier been exposed to alloantigen through mechanisms such as cross-reactivity and homeostatic proliferation.

Flow cytometry has been applied to examine how different populations of peripheral blood leukocytes purified from healthy volunteers changed with age. A significant decline in the percentage of naïve T cells and CD8 + T cells and an increase in the percentage of effectors memory cells, CD4 + foxp3 + T cells and NK cells [31] have been noticed in aging.

Age-related alteration of immunity may reveal changes in the repertoire of T cells and B cells which are capable to respond to antigenic challenges. Absolute numbers and proportions of lymphocyte subpopulations are altered but also the repertoire of antigen receptor genes expressed by lymphocytes is subject to modifications. A diversified repertoire makes adaptive immunity possible and offers an adequately varied selection of specificities against immunological events. Such a variety may be achieved by genomic rearrangements, combining variable diversity and joining gene segments to build antigen binding regions of immunoglobulin chains and T cell receptor chains.

In early life stages positive and negative selection to remove receptors that have high affinity for self-antigen occurs in the bone marrow or in the thymus. In consequence, mature naïve lymphocytes have functional receptors that are not characterized by high affinity for autoantigen. Additionally, memory lymphocyte repertoires reveal individual antigen response history.

In older population the immune system is distinguished by the repertoire narrowing and reduced efficiency. It manifests itself as a decreased ability of responding to immunological challenge and inappropriate immune activation. The former is tantamount to poor responses to vaccination and increased vulnerability to infection, while the latter signifies a tendency for autoantibodies formation and enhanced release of inflammatory mediators. The hypothesis suggests a failure in positive and negative selection [32].

Immunological studies of aging document changes in the proportions and absolute numbers of different lymphocyte subsets. A reduction in naïve lymphocyte production together with an increased proportion of clonally expanded memory cells have been noticed. This may decrease the ability to respond to new antigenic challenges.

The immunological T cell aging correlates with age-related deterioration of cellular immunity, which results in decreased vaccination effectiveness, enhanced susceptibility to infections and a higher risk of the appearance of tumors [33]. Increased numbers of differentiated CD4 + T cells have also been connected with the prevalence and severity of atherosclerotic disorders [34].

Aging is associated with a progressive decline in immune functions. A decrease in T-cell functions predominates in the aging process. Calendar aging has an impact on major signaling pathways of T cell apoptosis [35]. Shortening of telomeric DNA, as described for T cells with advancing age, is one of independent factors contributing to increased apoptosis [36].

Immunosenescence has also an impact on B cell population and humoral response. It is thought that naïve B cells decrease in comparison with memory cells. The production of antibodies is reduced but also shortened duration of protective immunity following immunization has been noticed [37], [38], [39]. It has been suggested that aging in mice results in a decline of the earliest stages of B cell development [40].

The function of B cells might be also decreased by defects in immunoglobulin class switch recombination, activation induced cytidine deaminase, and diminished E47 expression (transcription factor necessary for activation-induced cytidine deaminase, which is one of molecular mechanisms for the reduced activity of B cells in aged mice) [39]. These defects have been observed both in mice and humans. Studies have also shown that suppressed tristetraprolin activity contributes to a decreased E47 mRNA stability in aged B lymphocytes [39].

The evolutionarily conserved function of immunity system named autophagy is also known to decline with age [41]. In this process intracellular material is degraded within the lysosome and the macromolecular constituents are recycled. A dysfunction of the autophagy machinery leads to the accumulation of abnormal proteins or damaged organelles and is associated with diabetes, autoimmunity, cancer and infections [42]. Autophagy influences T cell survival after TCR activation and may destabilize the immunological synapse. Autophagy removes mitochondria and the endoplasmatic reticulum influencing central tolerance by thymic naïve T cell repertoire selection [43], [44], [45]. Autophagy is also required for the development and survival of B1 cells and in absence of autophagy plasma cells secrete an increased amount of immunoglobulins [46], [47].

The gradual biological process which occurs throughout life and describes mechanisms of damage and repair of the immune system was interestingly called immune remodeling [48]. Alterations may be marked by reduced levels of antigen-specific immune responses among people aged 65 years or older in contrast to younger people. Immune remodeling is a positive adaptation of aging to support healthy survival beyond reproductive performance. Acute, more drastic remodeling may occur excessively among young patients with chronic diseases. Such acute remodeling causes the risk of premature exhaustion in context of the immune repertoire.

Immunosenescence as a term describing faulty adaptive immune responses observed in elderly people was introduced as early as in the 1950s [49]. The decreased activity of humoral and/or cell-mediated responses was thought to result from physiological weakening that occurs during chronological or calendar aging.

Immunological response against foreign antigens depends on the diversity of the T-cell receptor (TCR) repertoire. The contact with antigen stimulates specific T cells to clonal proliferation. After antigen elimination some T cells remain as memory cells but most undergo apoptosis.

TCR diversity is maintained up to the age of 65 years and is strongly reduced later due to clonal proliferation of T cells [26]. The alteration of the TCR repertoire helps to explain reduced antigen-specific responses among the elderly.

Recurring antigenic exposure has an essential role in the expansion of non-dividing T cells [50].

Senescent T cells are memory cells and may be identified by a deficiency in the expression of CD28. The molecule CD28 is the major costimulatory receptor present in CD4 + and CD8 + cells and is required to sustain TCR-driven activation [51]. CD28^null T cells either are senescent or approach the end-stages of senescence due to transcriptional inactivation [28], [29], [30], [31]. CD28^null T cells are resistant to apoptosis, which explains why they accumulate throughout life [32], [33]. Ligation of the TCR– CD3 complex on these cells induces reduced phosphorylation and attenuates TCR signal transduction [52].

Senescent and pre-senescent T cells have partial or no capacity for cell division due to telomere erosion, therefore they are generally long-lived and functionally active [53].

Senescence of T cells may lead to the appearance of long-lived natural killer (NK)-like T cells [54]. Such NK-like senescent T cells express untypically higher densities of receptors that are usually found on natural killer cells, called the killer cell immunoglobulin-like receptors (KIR), which are the most diverse natural killer receptors (NKR). There are not only dissimilarities in the number but also patterns of expression of KIRs and NKRs on senescent T cells [55], [56]. KIRs comprise a family of surface stimulatory or inhibitory receptors. They are specific for allelic forms of human leukocyte antigen (HLA) class I molecules [38], [39]. NKR + T cells are fundamentally different from the so-called natural killer T (NKT) cells. Both NKR + T cells and NKT cells have oligoclonal TCRs. In simple terms, NKT cells express neither CD4 + nor CD8 + but NKR + T cells are either CD4 + or CD8 + cells expressing various NKR [40], [41]. The accumulation of NKR + T cells in vivo is a physiological adaptation of aging to retain immune homeostasis. With thymus degeneration the accumulation of these clonally proliferative TCR cells, which may be resistant to apoptosis, might be a way to preserve T-cell numbers and avoid clinical lymphopenia in older people [32], [42].

Biological features of the exhausted immune system may appear earlier than they gradually occur in calendar aging. Patients with chronic immune-mediated diseases reveal the TCR repertoire perturbations as well. Systemic lupus erythematosus, coronary artery disease, rheumatoid arthritis and cytomegalovirus infection among others are examples of chronic diseases which demonstrate features of acute immune remodeling [48].

It should be emphasized that some of the above-mentioned diseases may lead to chronic renal insufficiency and the necessity of renal replacement therapy. Therefore, such patients may be renal transplant recipients. Additionally, these people may suffer from coronary artery disease, which may exacerbate perturbations. It indicates that immunosenescence may be present more often in transplant patients already in younger age groups.

Young patients suffering from chronic inflammatory, vascular, infectious diseases demonstrate oligoclonal expansion of T cells. These clonal T cells may be CD28^null and NKR +. Severe clinical disease manifestations of chronic diseases are associated with the presence of oligoclonal senescent CD28^null NKR + T cells [43], [44]. Moreover, chronic diseases may lead to telomere shortening, reduced mitotic capacity, decreased thymic output and the reduction of TCR diversity, which are typical of immunosenescence [48].

Cytomegalovirus (CMV) infection is usually acquired early in life. Around 10% of the total circulating T cells become CMV-specific and remain present throughout life (usually less than 0.1% of T cells are specific for the other pathogen). Cytomegalovirus is still the main cause of opportunistic infections in the first year after kidney transplantation [57]. Latent CMV infection may be present in 60%–90% of all kidney transplant patients [58]. Without routine preventive therapy, symptomatic CMV infection occurs in about 20%–60% of cases, typically within the first three months [59]. The CMV-antigen-specific memory T cells are characterized by the lack of CD28 molecule [9], [48].

Lately it has been noticed that CMV infection reduces relative telomere length (RTL) of CD8 + T cells in ESRD patients. An increased T cell differentiation has also been observed with the increasing percentage of CD28^null CD4 + and CD8 + memory T cells. These CD28^null T cells have shorter telomeres in comparison with CD28 + T cells. It has been concluded that CMV infection increases T cell differentiation as observed in premature T cell aging [60].

Premature T-cell aging resulting in a defective T-cell immunity may be also induced by uremia [50], [51]. Faulty T-cell-mediated immunity as the consequence of a loss in renal function has such clinical implications as a poor vaccination response, an increased susceptibility to infections and an increased prevalence of virus-associated cancers among others [52], [53], [54].

The retention of uremic toxins and cytokines in end-stage renal disease (ESRD) generates oxidative stress and inflammation which influence premature T-cell aging [61]. Younger patients aged 25–45 years with ESRD resemble immunologically older healthy volunteers aged 60–80 years. Younger patients with ESRD had a significant loss of naïve T cells and a relative increase in memory T cells [62]. Aging of the T-cell system was associated with a progressive decrease in newly formed T cells from the thymus named thymic output. A significant decrease in telomere length in ESRD patients additionally confirmed immunological aging. Thymic output can be described by evaluating the content of T-cell receptor excision circles (TRECs) [61]. TRECs are circular DNA episomes formed during rearrangement of TCR genes in the thymus.

The impact of uremia on cells from the lymphoid and myeloid cell lineage is diverse [63]. Cells belonging to the lymphoid cell lineage (T, B and NK cells, plasmacytoid dendritic cells — pDC) are generally diminished in number and function, which might be caused by the combination of the proinflammatory uremic milieu and intensified oxidative stress. The number of cells belonging to the myeloid cell lineage (granulocytes, monocytes, macrophages, myeloid dendritic cells — mDC) is higher or the same as in a healthy person. These cells participate in the creation of proinflammatory and increased oxidative stress environment. The characteristics for the immune aging CD4 + CD28^null T cells are expanded.

Recently thymic output together with CD31 + naïve T-cell numbers and the relative telomere length (RTL) with T-cell function determined by measuring cytokine production were analyzed before and after kidney transplantation (3, 6, 12 months) [64]. Post-transplantation memory T-cell numbers were reduced but restored to pretransplant values 1 year after renal transplantation. The relative telomere length has not changed. TREC content and CD31 + naïve T-cell numbers were stable after renal transplantation. The T-cell function did not improve after transplantation. The findings have shown that uremia is associated with T cell aging, which is not reversible after transplantation.

There are also interesting observations that the expression of the KLOTHO gene is downregulated in patients with ESRD because of methylation of the promoter region stimulated by oxidative stress [65]. KLOTHO was named an anti-aging gene because KLOTHO knockout mice have a characteristic premature aged phenotype. It shows that chronic inflammation together with increased oxidative stress may augment the induction of epigenetic changes, connected with aging of the immune system.

The age-related homeostatic proliferation might have the opposite effect and may accelerate rejection, but on the other hand to understand the role of the mechanism in the elderly may have important implications for tolerance induction or transplant survival [24]. The homeostatic proliferation becomes the source of new T cells in the elderly after thymus regression. These cells acquire a memory phenotype.

Chronic rejection may be transformed to acute rejection by T cells that have gone through homeostatic proliferation in lymphopenic environment. Such cells consistently cause reliable rejection. The findings have significant implications in the context of tolerance induction or graft-prolonging protocols [24].

Depleting antibodies in the elderly may have an additional effect on homeostatic proliferation which may be dangerous in case of infection [66], [67].

In the strategy of transplanting old kidneys to old recipients two kinds of weaknesses are combined. Old organs suffer from atherosclerotic damage, diminished number of viable glomeruli, lower potential for repair, and therefore are more sensitive to ischemic-reperfusion injury. Tissue injury induces the response that stimulates immune recognition and vicious cycle of further injuries, leading to higher immunogenicity of older grafts.

The fusion of dysregulated immune response with stronger immunogenicity of older grafts had clinical consequences observed in the elderly where the features of more potent early immune response were revealed with more acute rejections registered in whole senior cohort [68]. On the other hand, death with functioning graft is the main cause of graft loss in the elderly with the significant contribution – besides cardiovascular disease – of infections and neoplasias.

Owing to the lack of organs for transplantation, expanded criteria donors (ECD) have been introduced to expand the donor pool. The rule “old for old” is applied in clinical practice to optimize the allocation system and utilize older organs. A transplantation of an older kidney to an older recipient may be sufficient and most appropriate, especially taking into account the decreased alloresponse of an older recipient, even despite increased immunogenicity of older organs.

Despite the efforts made to expand the pool of kidney donors, the number of patients on the kidney transplant waiting list is continuously growing. Living kidney donation seems to be a large potential alternative for recipients, even the one from older living donors. The outcomes of living kidney donation from older donors (aged ≥ 60 years) have been analyzed lately using the UNOS database from 1994 to 2012 [69].

The transplant recipients who received older LD kidneys had lower graft and overall survival in comparison with the recipients who received a younger LD kidney. The recipients with an older LD kidney had better graft and overall survival in comparison with ECD transplant recipients.

Considering elderly recipients, the first implication should concern the chance of an old recipient to receive a kidney from a young deceased donor, which is very small. Therefore, living donation even from older donors may be a better solution than waiting. Regarding fewer chronic rejections and more deaths caused by infections compared to younger patients, the second implication draws attention to the significance of over-immunosuppression avoidance.

Age-connected changes in the immune system described as immunosenescence, including immune remodeling, should be taken into account in the immunosuppressive management of the elderly. The real challenge and task for clinical studies is to elaborate an adequate immunosuppressive protocol for elderly recipients receiving kidney from old donors. It should be sufficient to control alloresponse in the early period with diminished exposure to calcineurin inhibitors, allowing curing the ischemic-reperfusion injury, compensated with an induction of anti-interleukin-2 receptor antibodies and mycophenolate mofetil. After six months the protocol could be individualized based on the clinical course and immune monitoring.