Generation of both cross-reactive and virus-specific T-cell populations after immunization with serologically distinct influenza A viruses.

Specificity of cytotoxic T-cell function was investigated for a range of different influenza viruses. T cells from mice immunized with A or B strain influenza viruses, or with vaccinia virus, showed reciprocal exclusion of cytotoxicity. Extensive cross-reactivity was, however, found for lymphocyte populations from mice infected with a variety of serologically distinct influenza A viruses, though serum antibodies did not cross-react when tested in a radioimmunoassay using comparable target cells as immunoadsorbents. This apparent lack of T-cell specificity was recognized for immune spleen cells generated after intraperitoneal inoculation of high titers of virus, and for mediastinal lymph node populations from mice with pneumonia due to infection with much less virus. The phenomenon could not be explained on the basis of exposure to the chicken host component, which is common to A and B strain viruses. However, not all of the virus-immune T-cell clones are cross-reactive. Competitive-inhibition experiments indicate that a considerable proportion of the lymphocyte response is restricted to the immunizing virus. Even so, the less specific component is significant. Also, exposure to one type A virus was found to prime for an enhanced cell-mediated immunity response after challenge with a second, serologically different A strain virus.

Induction of virus-specific modifications recognized by cytotoxic T cells is not altered by prior substitution of target cells with trinitrophenol.

Cytotoxic thymus-derived lymphocytes generated after interaction with trinitrophenyl (TNP)-substituted or virus-infected cells only lyse H-2 compatible target cells modified with the component used to immunize (TNP or virus). Prior saturation of TNP-reactive sites inhibits neither the infectivity of influenza A viruses, nor the capacity of infected cells to develop antigenic changes recognized by influenza-immune T cells. The two antigens are distinct entities on the cell membrane and do not obviously compete to form interactions with H-2 molecules.

Shortened telomeres in the expanded CD28-CD8+ cell subset in HIV disease implicate replicative senescence in HIV pathogenesis.

OBJECTIVE: To test the hypothesis that the expanded population of non-proliferative CD28-CD8+ T cells in HIV disease have shortened telomeres, thereby providing evidence that increased rounds of CD8+ cell division occur during HIV disease, possibly leading to replicative senescence and exhaustion of CD8+ T-cell responses. DESIGN: CD8+ cells play a central role in control of HIV infection. In late HIV disease, an expanded population of CD28-CD8+ cells with reduced proliferative potential has been documented. A similar population of CD28-CD8+ cells has been identified in ageing humans, where telomere length measurements have suggested that these cells have reached the irreversible state of replicative senescence. METHODS: CD8+ cells from HIV-infected and control subjects were sorted by flow cytometry into CD28+ and CD28- fractions. Telomere lengths were determined as mean terminal restriction fragment (TRF) lengths by Southern hybridization. RESULTS: The TRF lengths of sorted CD28-CD8+ cells in HIV-infected subjects ranged between 5 and 7 kilobases (kb) and were significantly shorter than TRF lengths of CD28-CD8+ cells in uninfected subjects (P = 0.003). The TRF length in CD28-CD8+ cells from HIV-infected subjects was the same as that observed for centenarian peripheral blood mononuclear cells and is compatible with a state of replicative senescence. CONCLUSIONS: The shortened telomeres in the CD28-CD8+ cells in HIV-infected subjects and the poor proliferative potential of these cells identifies CD8+ cell replicative senescence as a newly described feature of HIV disease. Our results provide a mechanism for the loss of CD8+ cell control of viral replication that accompanies advanced HIV disease. Replicative senescence may contribute to exhaustion of the T-cell response as a result of chronic HIV disease. Whether this phenomenon occurs in other chronic viral infections is unknown.

Telomere shortening in T cells correlates with Alzheimer's disease status.

Telomeres, the repeated sequences that cap chromosome ends, undergo shortening with each cell division, and therefore serve as markers of a cell's replicative history. In vivo, clonal expansion of T cells during immune responses to both foreign and autoantigens is associated with telomere shortening. To investigate possible immune alterations in Alzheimer's disease (AD) that might impact current vaccine-based therapeutic strategies, we analyzed telomere lengths in immune cell populations from AD patients. Our data show a significant telomere shortening in PBMC from AD versus controls (P=0.04). Importantly, telomere length of T cells, but not of B cells or monocytes, correlated with AD disease status, measured by Mini Mental Status Exam (MMSE) scores (P=0.025). T cell telomere length also inversely correlated with serum levels of the proinflammatory cytokine TNFalpha (a clinical marker of disease status), with the proportion of CD8+ T cells lacking expression of the CD28 costimulatory molecule, and with apoptosis. These findings suggest an immune involvement in AD pathogenesis.

Telomeres and HIV disease.

Telomere measurement, envisioned as a novel approach to elucidate T-cell dynamics in HIV disease, failed to reveal any consistent pattern in CD4+ T cells. By contrast, significant telomere shortening, as well as other hallmarks suggestive of replicative senescence, was observed within the CD8+ T-cell subset. Telomere studies have thus provided unanticipated insight into a novel facet of memory CD8+ T lymphocyte dynamics that may explain the exhaustion of the protective antiviral immune response. Strategies aimed at manipulating replicative senescence, therefore, offer unique approaches to immune reconstitution.

Transgenic mice expressing a truncated Peromyscus leucopus TNF-alpha gene manifest an arthritis resembling ankylosing spondylitis.

Several studies have implicated tumor necrosis factor-alpha (TNF-alpha) in autoimmune diseases, such as rheumatoid arthritis (RA). To elucidate further the role of TNF-alpha in inflammatory arthritis, we generated transgenic mice harboring a truncated Peromyscus leucopus TNF-alpha (Pe-TNF) gene. An arthritic phenotype closely resembling human ankylosing spondylitis was observed only in transgenic lines expressing the Pe-TNF transgene at the mRNA level. We characterized the arthritic phenotype in detail by radiographic and histologic techniques. It consisted of severe axial skeletal kyphosis and ankylosis, accompanied by an inflammatory and fibrotic process at the end plates and enthesis. Peripheral joint lesions were absent in mice expressing the P. leucopus TNF-alpha gene, in contrast to the RA-like phenotype described in transgenic mice expressing a truncated human TNF-alpha gene. The Pe-TNF transgenic mouse model provides a unique opportunity to explore potential mechanisms whereby TNF-alpha may initiate an autoimmune arthritis resembling ankylosing spondylitis.

Long-term immunological memory against viruses.

This review addresses a novel facet of human T cell biology that constitutes a fundamental problem for long-term maintenance of immunological memory against viruses. The finite proliferative capacity of human T lymphocytes is sufficiently great to accommodate the waves of clonal expansion associated with primary and even secondary immune responses. However, long-term memory to viruses that establish latency and to repeatedly encountered viruses such as influenza may be severely impaired by "replicative senescence", a genetically programmed process affecting most somatic cell types of human origin. Consistent with this idea, memory CD8+ T cells with hallmarks of replicative senescence have been identified in vivo. Such cells may contribute to compromised viral immunity and response to vaccines, and furthermore, their very presence may negatively influence homeostatic mechanisms that control the size of the memory T cell pool in elderly persons.

The accumulation of non-replicative, non-functional, senescent T cells with age is avoided in calorically restricted mice by an enhancement of T cell apoptosis.

Peripheral blood lymphocytes of elderly humans show an increased percentage of T cells with characteristics of replicative senescence. Similarly, the overall decrease in T cell proliferation in aged mice reflects a progressively increasing proportion of non-functional cells rather than a uniform decline in function by all cells. The improved immune function of calorically restricted (CR) animals is, paradoxically, accompanied by a relative lymphopenia. To test whether the reduction in lymphocyte number in the CR mice might reflect more efficient elimination of T cells, we measured apoptosis in young, old and CR old mice. T cell apoptosis induced by irradiation, Staurosporine, anti-CD3, and heat shock was reduced by 62, 42, 32, and 30%, respectively, in old compared with young mice. Caloric restriction normalized apoptosis in T cells from aged mice. Enhanced elimination of non-functional T cells in CR mice may be, at least in part, responsible for their improved immune functional status relative to non-CR mice of the same age.

Calorie restriction inhibits the age-related dysregulation of the cytokines TNF-alpha and IL-6 in C3B10RF1 mice.

TNF-alpha and IL-6 are generally increased in the sera of aged humans and mice. The dysregulation of these cytokines may be critical in autoreactivity and immune dysfunction. In earlier studies we demonstrated that production of TNF-alpha and IL-6 following in vitro stimulation of peritoneal macrophages by LPS was reduced in old compared to young mice, and that dietary caloric restriction (CR) had no effect on the induction of TNF-alpha in this system. In the present study we examined the effects of age and calorie restriction on the constitutive production of both TNF-alpha and IL-6. Serum levels of both cytokines were significantly higher in old versus young mice. However, in old mice subjected to long term CR the serum levels were comparable to those of young mice. The potential involvement of normalization of TNF-alpha and IL-6 levels in the life extension effect of CR are discussed.

The in vitro senescence of human T lymphocytes: failure to divide is not associated with a loss of cytolytic activity or memory T cell phenotype.

Normal human T lymphocytes, activated in vitro and cultured in the continuous presence of the growth factor interleukin 2 (IL2), have a limited proliferative potential. Senescent T cell cultures will not proliferate, even if restimulated by the original allogeneic stimulator cells. However, we have now observed that such restimulation induces an increase in the percentage of cells expressing the 55 kDa chain of the IL2 receptor (IL2R alpha, CD25) without any associated increase in cell number. A younger culture, which showed a comparable increase in CD25, underwent two population doublings in the same time period after restimulation. The senescent cultures, (primarily of the CD8+, cytotoxic/suppressor, phenotype), were also found to be highly potent and specific effector cells in a 51chromium release assay for cytolytic activity. Furthermore, senescent cultures maintain the surface phenotype of memory T cells. These findings demonstrate that while senescent T cells are unable to proliferate in response to restimulation or to IL2, they are able to recognize the foreign stimulator cells and to initiate an otherwise normal T cell response. Our results lend support to the hypothesis that in vitro senescence is not associated with a generalized decline in functional activity in a differentiated cell type, but with a specific event which limits cell division. Thus, the long term T lymphocyte culture system will be useful for studying the mechanism by which proliferation is blocked in these, apparently, post-mitotic cells.

T cells and aging (update february 1999).

Deterioration of the immune system with aging ("immunosenescence") is believed to contribute to morbidity and mortality in man due to the greater incidence of infection, as well as possibly autoimmune phenomena and cancer in the aged. Dysregulation of T cell function is thought to play a critical part in these processes. Factors contributing to T cell immunosenescence may include a) stem cell defects, b) thymus involution, c) defects in antigen presenting cells (APC), d) aging of resting immune cells, e) disrupted activation pathways in immune cells, f) replicative senescence of clonally expanding cells. This review aims to consider the current state of knowledge on the scientific basis for and potential clinical relevance of those factors in immunosenescence.

Loss of CD28 expression on T lymphocytes: a marker of replicative senescence.

The CD28 molecule, a disulfide-linked homodimer expressed on peripheral T cells and thymocytes, mediates an essential costimulatory signal following engagement of the T cell receptor (TCR). Increased proportions of CD28- T cells have been observed during aging and in situations of chronic immune stimulation, but the origin and functional characteristics of these cells have been unclear. T cells which reach replicative senescence in culture after multiple rounds of cell division have shortened telomeres, respond poorly to stress stimuli, and no longer express CD28, suggesting that CD28- T cells observed in vivo may be the progeny of memory cells which have been repeatedly stimulated. This review describes the features of T cell replicative senescence, presents several possible mechanisms for the generation of senescent T cells in vivo, and proposes that replicative senescence may explain immune exhaustion.

Strong HLA-DR expression in T cell cultures after activation is necessary for IL-2-dependent proliferation.

We have examined the appearance of DR antigens on human T cells activated by PHA, using a monoclonal anti-DR framework antibody and a large panel of human HLA typing sera. Strong DR expression within the culture by day 8 was associated with the ability of cells to grow for long periods in IL-2 containing medium, whereas weak or absent DR expression was predictive of poor in vitro growth. All the cells responded equivalently to initial stimulation with PHA. These data support the hypothesis proposed by Moretta et al. to explain blocking of IL-2-dependent proliferation by an anti-DR antibody--that DR molecules may be involved in the transmission of signals by IL-2.

Insights on immunological aging derived from the T lymphocyte cellular senescence model.

A human T cell culture system has been developed to study cellular senescence in a manner analogous to that used for fibroblasts. Using this system, several markers of replicative senescence have been identified. Evidence is provided to show that new insights into the immune deficiency of aging as well as HIV infection have emerged from the in vitro model. The culture model is now being exploited to dissect the basic mechanisms underlying the senescence program in T cells.

Neonatal T cells as a model system to study the possible in vitro senescence of lymphocytes.

In this study long-term neonatal T-lymphocyte cultures were initiated from cord blood following alloantigenic stimulation. Growth curves and population doublings were measured for replicate cultures, functional and phenotypic analyses performed, and cells were cloned. Thus, newborn T cells were shown to constitute a potentially excellent model for the analysis of possible in vitro senescence of immunologically relevant cells. Certain problems of the system centering around "crisis" periods and reproducibility, were additionally explored.

Resistance to apoptosis in human CD8+ T cells that reach replicative senescence after multiple rounds of antigen-specific proliferation.

We have established an in vitro culture model of cellular aging in which antigen-specific T cells are stimulated repeatedly to divide until they reach the irreversible state of growth arrest known as "replicative senescence." T lymphocytes that reach replicative senescence in culture show complete loss of CD28 expression, shortened telomeres, undetectable telomerase, and reduced ability to produce heat shock proteins. We now document that in response to treatment with apoptotic stimuli, senescent CD8+ T-cell cultures show reduced apoptosis and diminished caspase 3 activity compared with quiescent early passage cultures from the same donor. Our results suggest that the progressive accumulation of T cells showing many of the hallmarks of replicative senescence during aging, chronic infection, and autoimmune disease may, in part, reflect the diminished capacity of such cells to undergo normal programmed cell death.

Human T lymphocytes possess a limited in vitro life span.

The T lymphocyte offers certain theoretical advantages over other available cell types for the study of aging. Immunosenescence is a well-established part of, and may be directly relevant to, mammalian aging, and the T lymphocyte is well-characterized as to function, cell-surface antigen make-up, and other factors. However, prior efforts at studying in vitro aging of T cells have been hampered by poor reproducibility in doubling potential and the occurrence of a peculiar type of crisis. We have improved the culture conditions for long-term in vitro propagation of normal human T lymphocytes so that previously described variability between identically manipulated cultures and the crisis period have been eliminated. Analysis of the growth patterns of 109 individual cultures revealed a limited proliferative life span, with the number of cumulative population doublings corresponding to that reported for adult human fibroblasts. This accord between the in vitro life spans of two vastly different cell types lends further support to the concept of the Hayflick Limit as a general biological phenomenon.

CD28 expression in T cell aging and human longevity.

Functional decrements of the immune system have a major contribution to aging and age-related diseases. Here, we further characterize the decline in proportion of CD28-positive T cells previously identified in centenarians. Cohorts of 97 centenarians, 40 subjects aged 70-90 (ELD group), and 40 young adults (under age 40) were phenotyped for T cell surface expression of CD28, CD4, and CD8 antigens. The significant decline in T cells expressing CD28 (p < 10(-4) for comparisons between adults and either ELD or centenarians) affects preferentially the CD8+ subset of T cells. This decline accounts largely for the age-related diminution of T cell responsiveness to mitogenic signals. CD28 expression is modulated in T cell cultures in a growth-related fashion and this modulation is dampened in cultures from centenarians. We propose that the decrease in CD28 expression reflects a compensatory adaptation of the immune system during aging in the face of chronic stimulation.

Immune effects of hormone replacement therapy in post-menopausal women.

Hormone replacement therapy (HRT) confers many health benefits to post-menopausal women. Despite links between estrogen and immune function prior to menopause, the immune status of women receiving HRT has not been rigorously investigated. This case-control study uses clinical laboratory assessment, flow cytometry, and functional assays to measure immune function. Participants included 27 post-menopausal women taking estrogen/progestin combinations, and 22 post-menopausal women not receiving HRT. Compared to the (-)HRT group, the (+)HRT group had more B-cells (p<0.05), higher mitogen-induced T-cell proliferation (p<0.05), and higher levels of induced TNF-alpha (p<0.05). There was a trend towards a lower proportion of CD4+ T-cells expressing the activation marker CD25+ (p<0.10). These findings represent a reversal of immune alterations associated with normal aging, suggesting that preservation or improvement of immune function may be associated with the use of HRT.

Decline in CD28+ T cells in centenarians and in long-term T cell cultures: a possible cause for both in vivo and in vitro immunosenescence.

The dramatic decline in immune function with age, especially in T cell proliferative activity, has been documented extensively in experimental animal models and in clinical studies of the elderly. A similar proliferative decline is also seen in long-term T lymphocyte cultures used to study in vitro cellular senescence. We have compared the peripheral blood T lymphocytes of centenarians and younger controls for the cell surface expression of CD28, a costimulatory molecule that is required for optimal activation and proliferation following engagement of the T cell receptor. Our analysis shows a significant decrease (p < 0.001) in the percentage of T cells expressing CD28 in the elderly cohort, with values ranging from 44% to 90%, as compared to the mean control value of 91%. The decline in the percentage of CD28+ T cells correlates with a reduction in the CD4/CD8 ratio (r2 = 0.695, p < 0.0001). Concommitantly, experiments using an in vitro T cell culture system showed a progressive loss of CD28 expression with culture "age." The concordance of proliferative decline and loss of CD28 in the centenarians and in the in vitro cultures suggest that a Hayflick phenomenon may operate in vivo leading to immunosenescence.