Interventions to restore appropriate immune function in the elderly

One of the issues which is often overlooked is the considerable amount of energy required to undertake an immune response. Naïve T cells which are normally in a close to quiescent state must shift to activation by changing their metabolism after encountering antigen. Successful activation is followed by an increased uptake of nutrients, increased mitochondrial oxidative phosphorylation and the cells metabolism becomes predominantly glycolytic. This shift presages the demand for energy and cell components precursors required for proliferation [16]. For many older individuals the necessary energy to make a successful immune response may be lacking because of either an inadequate diet or the change in the permeability of the gut to specific elements. Insufficient energy to deliver an immune response which is adequate may be due to a number of simple reasons. For example one previous study indicated a significant association between inadequate calorie intake and being underweight in older people with deficient or poorly functioning dentition [17]. Studies in which the diet of older individuals was supplemented, with an energy source and trace elements revealed that treated individuals could produce a greater response to influenza [18‐21] and pneumococcal vaccination [22, 23]. Changes in the permeability of the gut alter the uptake of trace elements such as zinc. The critical role of zinc for the immune system comes from studies on zinc deficient individuals who may have thymic atrophy, lymphopenia and recurrent infection [24] however studies on zinc supplementation of the diet of older individuals has failed to provide a conclusive result which would be beneficial to vaccine practitioners [25]. The immunomodulatory role and the potential immune enhancer property of vitamin D have been highlighted recently. Studies which have looked at ability of vitamin D supplements to improve the effectiveness of vaccines provide conflicting information [26].

The fungus Streptomyces hygroscopicus found in a soil sample from Easter Island was shown to produces a macrolide compound named rapamycin which was subsequently found to inhibit proliferation of mammalian cells and to possess immunosuppressive properties. These properties led to it being approved by the Food and Drug Administration in the United States for cancer therapy such as renal cell carcinoma. The normal oral dose for cancer patients would be in the region of 10 mg a day. Later experiments in mouse models suggested that at lower doses rapamycin could improve response to influenza antigen possibly by inhibiting the formation of germinal centres thus reducing the production of high affinity antibodies but permitting the production of lower affinity antibody and increasing the presence of influenza-specific IgM antibodies [27]. These results prompted trials in humans where recipients received 0.5 mg daily, or 5 or 20 mg weekly or placebo. After a six-week course and a two-week gap the recipients were vaccinated with the trivalent influenza vaccine. Those in the treatment group showed considerable and significant improvement in their immune response when compared to those in the placebo group [28].

One approach to restoring an effective immune response to a vaccine antigen in older individuals and to ensure adequate levels of protection has been to introduce an adjuvant in the vaccine composition [29]. The classic adjuvant containing aluminium salts has more recently been replaced by oil in water emulsions which have been used with great effect to promote a response to a zoster subunit vaccine for example. However success with adjuvants is a double edged sword. In one study of those who received the adjuvanted herpes zoster subunit vaccine 81.5% showed injection site reactions and 66.1% showed systemic reactions [30]. The mechanisms of action of adjuvants are not entirely clear yet. It is thought that they may in part act as depots of antigen prolonging it presence in the periphery, or interact with pattern recognition elements on accessory cells, or stimulate a local inflammatory response so bringing more cells to the area [29]. Whilst adjuvants have been shown to be effective in improving vaccine related responses in older adults their use may be limited to vaccines which are given infrequently. For example it may not be wise to vaccinate an older individual every year for 30 years (from age 65 to 95) with an influenza vaccine supplemented with adjuvant. Used in such a way this may drive all specific clones of responding cells to reach their replicative limit sooner which may prove counterproductive.

Increasing the amount of antigen within the dose of vaccine has been also shown to improve the response in older individuals, but not in a directly correlative manner. So, giving for example ten times the antigen dose will not produce a ten-fold increase in the immune response to the corresponding vaccine antigen. Vaccines with increased amounts of antigen include the influenza vaccine with four times the amount of antigen than the standard-dose vaccine (60 μg instead of 15 μg of haemagglutinin antigen for each of the three influenza A/H1N1, A/H3N2 and B strains) and Zostavax a live attenuated varicella zoster virus vaccine administered only to seniors, which contains more than 14 times the number of plaque forming units than the standard varicella vaccine recommended for children. This latter vaccine boosts immunity to the varicella zoster virus in older individuals and significantly reduces the incidence of herpes zoster and post-herpetic neuralgia [31].

One of the issues which is often considered to be a major problem in older people is latent virus infections. Control of these viruses is present throughout life but as we age this control begins to wain as seen with those harbouring the varicella zoster virus (human herpes virus 3 or HHV-3) who become susceptible to increased bouts of shingles the disease associated with herpes zoster and an increase in the prevalence of post herpetic neuralgia with age. Control of cytomegalovirus (CMV or HHV-5) has also been shown to decline with age with the appearance of detectable virus in the urine of older people [32]. Studies using the adoptive transfer of CMV specific CD8 T cells showed that overt disease could be controlled in individuals post-transplant [33, 34]. Whilst this treatment is still at an early phase and unlikely to be offered to older individuals in the near future more recent work suggests that virus specific T cells could be generated from autochthonous stem cells [35].

Heterochronic parabiosis; the surgical joining of two animals of different ages such that they have a common blood circulation has been used successfully in the past to identify means of ameliorating age related changes in the cardiac system [36] and in cognitive function [37]. Experiments to modify the immune system of the older partner of the parabiont by an extrinsic factor from the younger parabiont revealed that whilst pre-T cells from either parabiont could seed the opposing thymus and undergo thymopoiesis there was no reversal of thymic atrophy suggesting that the defect was in the thymic environment [38, 39]. Immune rejuvenation by a serum based factor is therefore ruled out as an option but the idea of rejuvenating the peripheral immune system by the adoptive transfer of blood from younger to older individuals has a long history. In the early 1920’s Prof A.A. Bogdanov tested the hypothesis that the transfusion of blood from young into older individuals may rejuvenate the latter. He experimented on himself, accepting transfused blood on up to 11 occasions, before the 12th transfusion killed him in 1928 [40].

Half a litre of blood (500 ml) is the amount normally donated for transfusion and this amount of blood will contain approximately 7.5 × 10⁸ T cells. In individuals aged 20 to mid 30’s approximately 50% or more of these cells will be naïve and the rest memory cells. It would seem feasible to collect this amount of blood and extract the leukocytes on a regular basis and then store them in ampoules in liquid nitrogen until the individual becomes much older. The memory cells will have a repertoire dependent on the pathogens and antigens already encountered and so should be slightly more restricted in terms of its diversity than the naïve T cell population. The latter should contain recent thymic emigrants and so have a diverse receptor repertoire.

In older individuals age related changes in both the peripheral T and B pool of cells include the presence of holes in the antigen specific receptor repertoire which consequently is diminished and the presence of lymphocytes at or close to their limit of replication. Re-transfusion of leukocytes at a later stage of life could provide a means of combatting both of these problems. There are of course issues around how many cells to transfuse back into a recipient, whether this number would integrate easily into the indigenous population and whether the receptors for specific pathogens would be present in the transfused lymphocytes. Preliminary answers to these questions have been provided by animal work [41], but more work is needed on human volunteers to determine these parameters. Despite this, several companies have grown up offering a service for individuals to collect their leukocytes early in their lifespan and store them in lymphocytes in liquid nitrogen for a prolonged period so that they can be reinfused at later date.

Interleukin 7 (IL-7) is a type 1 short chain cytokine produced by the stromal cells of primary and secondary lymphoid organs which binds to a cell surface receptor composed of an IL-7 specific alpha chain (CD127) and a common gamma chain (CD132). The latter is termed common since it is used as one of the receptor chains for cytokines IL-2, 4, 9, 15 and 21 [42]. Expression of CD127 and CD132 together are found at several stages of the T cell development pathway from early progenitors to memory cells. At the early intrathymic stages of the T cell pathway the IL-7:IL-7R interaction is responsible for ensuring cell survival [43] may be responsible for generating T cell receptor diversity [44] and is associated with clonal expansion of mature thymocytes prior to their export to the naïve T cell pool [45]. In peripheral T cells the IL-7 ligand receptor interaction is mainly associated with clonal maintenance through ensuring cell survival and proliferation in both the naïve and memory T cell pools [46]. The potential for IL-7 to act as an immune rejuvenating agent came from initial studies by Bhatia et al. [47] on young mice treated for prolonged periods with anti-IL-7 antibody. These mice showed severe thymic atrophy and a decline in thymic cellularity similar to that seen in older animals. Treatment of old mice with IL-7 could reverse age-related atrophy of the thymus, leading to a restoration of thymic output and an improvement in peripheral T cell function [43, 48]. One striking feature of using IL-7 to reverse thymic atrophy was the importance of the quantity of IL-7 present in the thymus as shown by experiments in which three lines of transgenic mice were generated in which the IL-7 gene was placed under the control of an lcx promoter. Each line produced different amounts of IL-7 and the line which produced the most (termed TgB) showed lower levels of thymocytes numbers when compared with wild type controls and were found to have a bottleneck in differentiation at the early stage of T cell development [49]. Later work suggest that high levels of IL-7 antagonise Notch signalling and as such impact on the choice of T versus B cell lineage by stem cells in the thymus [50]. Rejuvenation of immunity in old female rhesus macaques treated with recombinant simian IL-7 prior to vaccination with influenza showed an increase in thymic output and higher haemagglutinin titres in treated animals compared to saline treated controls [51]. The first clinical trial using IL-7 examined the therapeutic effects of IL-7 administered to humans with metastatic cancer [52]. A more recent placebo controlled double blind phase IIa trial in patients with lymphopaenic metastatic breast cancer [53], some of whom received recombinant IL-7 as part of their treatment regimen reported encouraging results with IL-7 inducing a significant increase in T cell numbers when delivered before chemotherapy. In another study on HIV infected patients who were also receiving antiviral therapy the data suggest that repeated cycles of recombinant human IL-7 were tolerated quite well by the recipients and sustained T cell restoration was seen in the majority of the participants of the study [53]. To date no trial has reported on the treatment of healthy older individuals who have received IL-7 therapy.

Early work on rodents showed that surgical castration could lead to reversal of the age associated atrophy seen in the thymus [54, 55] although this has been reported to be transitory [56]. Chemical castration using luteinizing hormone releasing hormone agonists have been shown to be effective in inducing thymic regrowth in old rodents [57] and in old primates [58] through a mechanism which may involve the upregulation of Delta-like 4 a Notch ligand crucial for T cell differentiation [59]. Experiments in humans suggest that this might be an attractive approach to improve immunity in older males [60, 61], however the side effects associated with the treatment may mitigate against this choice of approach.

Keratinocyte Growth Factor (KGF) is a member of the fibroblast growth factor family, a family which contains secreted proteins which signal to receptor tyrosine kinases and intracellular non-signalling proteins which act as co-factors for voltage gated calcium channels [62]. KGF is the 7th member of this family and a secreted signalling molecule which interacts with its receptor expressed on thymic epithelial cells and induces the proliferation of thymic epithelial cell progenitors and mature epithelial cells in fetal thymic organ cultures [63]. Early experiments in murine system suggested that the subcutaneous administration of KGF to mice for 3 consecutive days at 5 mg/Kg per day enhanced thymopoeisis for almost 2 months. Moreover the treatment restored thymopoeisis in old mice [64]. These results prompted experiments in primates which were reconstituted with CD34⁺ peripheral blood progenitor cells after irradiation. Animals receiving treatment with KGF showed increased frequencies of naïve T cells in their lymph nodes compared with controls and more cells recognised as recent thymic emigrants. In addition, KGF treated animals preserved their thymic architecture up to at least 12 months after the reconstitution [65].

Palifermin is a recombinant form of KGF of bacterial origin which has been used to treat patients receiving chemotherapy or radiation therapy to stimulate the growth of cells in the mucosal barrier. The positive non-human primate results prompted experiments using Palifermin on patients with HIV-1 infection who were receiving antiretroviral therapy. The hypothesis was that poor CD4 levels in these patients was due to reduced thymic function and treatment with Palifermin should improve the blood profile of CD4 cells in these patients. However a randomised double blind placebo controlled trial in HIV infected patients receiving ART [66] revealed no significant change in either the numbers of recent thymic emigrants, naïve cell numbers or thymic size. The dose range used in these studies was from 20 to 60μg/Kg which is considerable less than the 5 mg/Kg given to the mice in the original model and may be the reason why no change was seen alternatively it is possible that the structure of the thymus was such that it was irreparable within the time frame of the experiment.