Dose response kinetics of CD8 lymphocytes from young animals transfused into old animals and challenged with influenza

Transfusion of autologous leukocytes after prolonged storage has been proposed as a means of rejuvenating the immune system of older individuals. The rationale for this approach is that age related immune decline is associated with a diminished pool of naïve T cells following atrophy of the thymus and reduction in thymic output. The presence of high levels of naïve T cells within the blood of young individuals could provide a boost to the immune system of an older “self” through a rejuvenation of the naïve T cell pool. However what remains unresolved is whether the cells could be incorporated effectively into the T cell pool of the host and whether effectors could be generated. Using CD45 congenic mice in our experiments we show that the transfusion of young donor cells into older congenic host animals leads to their successful incorporation into the peripheral T cell pool. When the recipients were challenged with influenza virus, specific effector CD8 cells were generated which were of both host and donor origin. We found no relationship between the number of responder cells of donor origin at the time of assay and the number of cells injected.     

Major features of immunesenescence,including reduced thymic output,are ameliorated by high levels of physical activity in adulthood

It is widely accepted that aging is accompanied by remodelling of the immune system including thymic atrophy and increased frequency of senescent T cells, leading to immune compromise. However, physical activity, which influences immunity but declines dramatically with age, is not considered in this literature. We assessed immune profiles in 125 adults (55–79 years) who had maintained a high level of physical activity (cycling) for much of their adult lives, 75 age‐matched older adults and 55 young adults not involved in regular exercise. The frequency of naïve T cells and recent thymic emigrants (RTE) were both higher in cyclists compared with inactive elders, and RTE frequency in cyclists was no different to young adults. Compared with their less active counterparts, the cyclists had significantly higher serum levels of the thymoprotective cytokine IL‐7 and lower IL‐6, which promotes thymic atrophy. Cyclists also showed additional evidence of reduced immunesenescence, namely lower Th17 polarization and higher B regulatory cell frequency than inactive elders. Physical activity did not protect against all aspects of immunesenescence: CD28^?veCD57^+ve senescent CD8 T‐cell frequency did not differ between cyclists and inactive elders. We conclude that many features of immunesenescence may be driven by reduced physical activity with age.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Interleukin‐21 administration to aged mice rejuvenates their peripheral T‐cell pool by triggering de novo thymopoiesis

The vaccination efficacy in the elderly is significantly reduced compared to younger populations due to thymic involution and age‐related intrinsic changes affecting their naïve T‐cell compartment. Interleukin (IL)‐21 was recently shown to display thymostimulatory properties. Therefore, we hypothesized that its administration to ageing hosts may improve T‐cell output and thus restore a competent peripheral T‐cell compartment. Indeed, an increase in the production of recent thymic emigrants (RTEs) attributable to intrathymic expansion of early thymic progenitors (ETPs), double‐negative (DN), and double‐positive (DP) thymocytes as well as thymic epithelial cell (TEC) was observed in recombinant (r)IL‐21‐treated aged mice. In sharp contrast, no alterations in the frequency of bone marrow (BM)‐derived progenitors were detected following rIL‐21 administration. Enhanced production of naïve T cells improved the T‐cell receptor (TCR) repertoire diversity and re‐established a pool of T cells exhibiting higher levels of miR‐181a and diminished amounts of the TCR‐inhibiting phosphatases SHP‐2 and DUSP5/6. As a result, stimulation of T cells derived from rIL‐21‐treated aged mice displayed enhanced activation of Lck, ZAP‐70, and ERK, which ultimately boosted their IL‐2 production, CD25 expression, and proliferation capabilities in comparison with T cells derived from control aged mice. Consequently, aged rIL‐21‐treated mice vaccinated using a tyrosinase‐related protein 2 (Trp2)‐derived peptide exhibited a substantial delay in B16 tumor growth and improved survival. The results of this study highlight the immunorestorative function of rIL‐21 paving its use as a strategy for the re‐establishment of effective immunity in the elderly.     

Signal transduction via the T cell antigen receptor in naïve and effector/memory T cells

T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.     

Induction of protective immunity by primed B-1 cells in Toxoplasma gondii -infected B cell-deficient mice

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.     

Optimal window of caloric restriction onset limits its beneficial impact on T‐cell senescence in primates

We have recently shown in non‐human primates that caloric restriction (CR) initiated during adulthood can delay T‐cell aging and preserve naïve CD8 and CD4 T cells into advanced age. An important question is whether CR can be initiated at any time in life, and whether age at the time of onset would modulate the beneficial effects of CR. In the current study, we evaluated the impact of CR started before puberty or during advanced age on T‐cell senescence and compared it to the effects of CR started in early adulthood. Our data demonstrate that the beneficial effects of adult‐onset CR on T‐cell aging were lost by both early and late CR onset. In fact, some of our results suggest that inappropriate initiation of CR may be harmful to the maintenance of T‐cell function. This suggests that there may be an optimal window during adulthood where CR can delay immune senescence and improve correlates of immunity in primates.     

Strong homeostatic TCR signals induce formation of self‐tolerant virtual memory CD8 T cells

Virtual memory T cells are foreign antigen‐inexperienced T cells that have acquired memory‐like phenotype and constitute 10–20% of all peripheral CD8⁺ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen‐experienced memory T cells are incompletely understood. By analyzing T‐cell receptor repertoires and using retrogenic monoclonal T‐cell populations, we demonstrate that the virtual memory T‐cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self‐reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T‐cell compartment via modulating the self‐reactivity of individual T cells. Although virtual memory T cells descend from the highly self‐reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self‐reactivity in polyclonal T cells for the generation of functional T‐cell diversity.     

Virtual memory cells make a major contribution to the response of aged influenza-naïve mice to influenza virus infection

Background

A diverse repertoire of naïve T cells is thought to be essential for a robust response to new infections. However, a key aspect of aging of the T cell compartment is a decline in numbers and diversity of peripheral naïve T cells. We have hypothesized that the age-related decline in naïve T cells forces the immune system to respond to new infections using cross-reactive memory T cells generated to previous infections that dominate the aged peripheral T cell repertoire.

Results

Here we confirm that the CD8 T cell response of aged, influenza-naïve mice to primary infection with influenza virus is dominated by T cells that derive from the memory T cell pool. These cells exhibit the phenotypic characteristics of virtual memory cells rather than true memory cells. Furthermore, we find that the repertoire of responding CD8 T cells is constrained compared with that of young mice, and differs significantly between individual aged mice. After infection, these virtual memory CD8 T cells effectively develop into granzyme-producing effector cells, and clear virus with kinetics comparable to naïve CD8 T cells from young mice.

Conclusions

The response of aged, influenza-naive mice to a new influenza infection is mediated largely by memory CD8 T cells. However, unexpectedly, they have the phenotype of VM cells. In response to de novo influenza virus infection, the VM cells develop into granzyme-producing effector cells and clear virus with comparable kinetics to young CD8 T cells.

     

Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4⁺ and CD8⁺ T cells, microarray analysis was performed pre‐ and post‐TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T‐cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4⁺ and CD8⁺ naïve T cells. Evaluation of these differences provides in‐depth insight into potential functional and phenotypical differences among these populations.     

Nasal peptide vaccination elicits CD8 responses and reduces viral burden after challenge with virulent murine cytomegalovirus

Infection of BALB/c mice with murine cytomegalovirus (MCMV) leads to CD8 cell responses to an immunodominant epitope YPHFMPTNL. We presented this epitope as a nasal peptide vaccine in combination with cholera toxin adjuvant, and evaluated immune responses and protection from MCMV challenge. Vaccination of naïve mice generated elevated numbers of peptide‐specific interferon‐7‐secreting splenocytes (median 80/million, range 60 to 490), compared to control mice (median 2/million, range —4.5 to 8; P=0.008, Mann‐Whitney test). Twelve days after challenge with virulent MCMV, vaccinated mice had a 1.1 log₁₀ reduction in salivary gland viral titer compared to unvaccinated controls (5.36±0.24 vs. 6.42±0.12, mean±SD log₁₀ plaque‐forming‐units; P<0.001, t‐test). Mice with chronic MCMV infection had consistent responses to the peptide (183±24/million interferon‐γ‐secreting splenocytes). Nasal peptide vaccination during chronic infection boosted peptide‐specific responses in two of four mice to >900/million interferon‐γ‐secreting splenocytes. Nasal peptide vaccination was immunogenic in naïve and MCMV‐infected mice, and reduced viral burden in naïve mice after virulent MCMV challenge. The nasal route may be useful for peptide presentation by novel human vaccines.     

Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function

Background

As individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences.

Results

We examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition.

Conclusions

Our data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

     

Analysis of naïve and memory CD4 and CD8 T cell populations in breast cancer patients receiving a HER2/<Emphasis Type="Italic">neu</Emphasis> peptide (E75) and GM-CSF vaccine

We are conducting clinical trials of the E75 peptide as a vaccine in breast cancer (BrCa) patients. We assessed T cell subpopulations in BrCa patients before and after E75 vaccination and compared them to healthy controls. We obtained 17 samples of blood from ten healthy individuals and samples from 22 BrCa patients prior to vaccination. We also obtained pre- and post-vaccination samples of blood from seven BrCa patients who received the E75/GM-CSF vaccine. CD4, CD8, CD45RA, CD45RO, and CCR7 antibodies were used to analyze the CD4+ and CD8+ T cells by four-color flow cytometry. Compared to healthy individuals, BrCa patients have significantly more memory and less naïve T cells and more effector-memory CD8+ and less effector CD4+ T cells. Phenotypic differences in defined circulating CD4+ and CD8+ T cell subpopulations suggest remnants of an active immune response to tumor distinguished by a predominant memory T cell response and by untapped recruitment of naïve helper and cytotoxic T cells. E75 vaccination induced recruitment of both CD4+ and CD8+ naïve T cells while memory response remained stable. Additionally, vaccination induced global activation of all T cells, with specific enhancement of effector CD4+ T cells. E75 vaccination causes activation of both memory and naïve CD4+ and CD8+ T cells, while recruiting additional naïve CD4+ and CD8+ T cells to the overall immune response.     

T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment

The following study was undertaken to better understand the mechanisms that relate the homeostatic set point of the peripheral T cell population to energy availability in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+T cells notably declined after one week of malnourishment, a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Overall levels of IL-7 were similar in malnourished and control mice. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to quickly adjust the total number of T cells during malnutrition.     

Orally Administered Bisphenol A Disturbed Antigen Specific Immunoresponses in the Naïve Condition

Bisphenol A [2,2-bis(4-hydoxyphenyl)propane; BPA] is an endocrine disrupter widely used in polycarbonate plastics and epoxy resins. We investigated the effects of orally administered BPA on antigen-specific responses of the naïve immune system.

BPA was orally administered to T cell receptor transgenic mice, and the antigen-specific responses of immune cells were investigated. Administered BPA moderately reduced interleukin (IL)-2, 4, and interferon (IFN)-γ secretion and increases in IgA and IgG2a production.

Additionally, it was found that orally administered BPA increased antigen-specific IFN-γ production of T cells and modified whole antigen presenting cells (APCs) to suppress antigen-specific cytokine production from T cells.

These findings suggest that BPA can augment the Th1-type responses of naïve immune systems, though the bioavailability of orally administered BPA was low in our experiments.     

Impact of sex steroid ablation on viral, tumour and vaccine responses in aged mice

Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.