The immune system in extreme longevity.

Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of the immune function, but is rather the result of a remodeling where some functions are reduced, others remain unchanged or even increased. In addition, it appears that the ancestral/innate compartment of the immune system is relatively preserved during aging in comparison to the more recent and sophisticated adaptive compartment that exhibit more profound modifications. The T-cell branch displays an age-dependent decline of the absolute number of total T-cells (CD3+), involving both CD4+ and CD8+ subsets, accompanied by an increase of NK cells with well-preserved cytotoxic function and by a reduction of B-cells. One of the main characteristics of the immune system during aging is a progressive, age-dependent decline of the virgin T-cells (CD95-), which is particularly profound at the level of the CD8+ subpopulation of the oldest old subjects. The progressive exhaustion of this important T-cell subpopulation dedicated primarily to the defense against new antigenic challenges (viral, neoplastic, bacterial ones), could be a consequence of both the thymic involution and the lifelong chronic antigenic stimulation. The immune function of the elderly, is therefore weakened by the exhaustion of CD95- virgin cells that are replaced by large clonal expansions of CD28- T-cells. The origin of CD28- cells has not been completely clarified yet, but it is assumed that they represent cells in the phase of replicative senescence characterized by shortening telomers and reduced proliferative capacity. A major characteristic of the immune system during aging is the up-regulation of the inflammatory responses which appears to be detrimental for longevity. In this regard, we have recently observed a progressive age-dependent increase of type 1(IL-2, IFN-gamma, TNF-alpha) and type 2 (IL-4, IL-6, IL-10) positive CD8+ T-cells; in particular, type 1 cytokine-positive cells significantly increased, with age, in all CD8+ subsets particularly among effector/cytotoxic and memory cells. A major force able to drive a chronic pro-inflammatory state during aging may be represented by persistent viral infections by EBV and CMV. Therefore, we have determined the frequency and the absolute number of viral antigen-specific CD8+ T-cells in subjects older than 85 years, who were serologically positive for CMV or EBV. In the majority of these subjects we detected the presence of T lymphocytes positive for epitopes of CMV or EBV. In all subjects the absolute number of CMV-positive CD8+ cells outnumbered that of EBV-positive ones. In addition, the majority of CMV+ T cells were included within the CD28- subpopulation, while EBV+ T cells belonged mainly to the CD28+ subset. These data indicate that the chronic antigenic stimulation induced by persistent viral infections during aging bring about important modifications among CD8+ subsets, which are particularly evident in the presence of CMV persistence. The age-dependent expansions of CD8+CD28- T-cells, mostly positive for pro-inflammatory cytokines and including the majority of CMV-epitope-specific cells, underlines the importance of chronic antigenic stimulation in the pathogenesis of the main immunological alterations of aging and may favour the appearance of several pathologies (arteriosclerosis, dementia, osteoporosis, cancer) all of which share an inflammatory pathogenesis.

Cytotoxic chemotherapy preceding apheresis of peripheral blood progenitor cells can affect the early reconstitution phase of naive T cells after autologous transplantation.

Transient T cell immunodeficiency is a common complication following hematopoietic stem cell transplantation. In breast cancer patients transplanted with autologous peripheral blood progenitor cells (PBPC) harvested after cytotoxic treatment with either cyclophosphamide or epirubicin plus paclitaxel, we evaluated T cells infused in grafts and in peripheral blood during the early reconstitution phase. We found that PBPC grafts harvested after treatment with epirubicin plus paclitaxel contained substantially larger numbers of T cells with less altered composition than after cyclophosphamide. Three months after high-dose cytotoxic chemotherapy, the numbers and the kinetics of circulating naive T cells, but not of memory and CD28- T cells, correlated positively with the number of naive T cells infused PBPC grafts. Finally, retrospective analysis of two cohorts of patients transplanted in different clinical settings with PBPC grafts harvested following cyclophosphamide or epirubicin plus paclitaxel showed apparently different susceptibilities to develop endogenous varicella zoster virus reactivation in the first year after high-dose cytotoxic chemotherapy. On the whole, these data indicate that number and composition of T cells in PBPC grafts vary according to the former cytotoxic therapy, and suggest that autologous transfer of T cells may accelerate the early T cell reconstitution phase and possibly ameliorate immune competence in patients rendered lymphopenic by high-dose chemotherapy.

A stochastic model for CD8(+)T cell dynamics in human immunosenescence: implications for survival and longevity.

We propose here a stochastic model for the CD 8(+)T lymphocyte dynamics on the long time-scale of the human lifespan. Our purpose has been to test the hypothesis, recently proposed on the basis of our experimental data (Fagnoni et al., 2000), that the depletion of virgin CD8(+)T lymphocytes can be considered a reliable biomarker related to the risk of death. This hypothesis is embedded in a more general theory of immunosenescence according to which the accumulation of antigen experienced (AE) T cells and the concomitant exhaustion of antigen non-experienced (ANE) T cells with age, mostly due to the chronic lifelong exposure to antigens, is a major characteristic of the remodeling of the human immune system with age. In our model we considered a deterministic balance of ANE and AE T cell concentrations plus a stochastic forcing, which describes the chronic antigenic stress fluctuations, assuming a mean genetically determined capability of individuals to respond to antigens. The major results of our model is the validation of the above-mentioned hypothesis, since the model is capable of fitting the experimental data concerning the changes of ANE T cell concentration over age, and at the same time to reproduce survival curves similar to the demographic ones. Furthermore, the stochastic process results in being responsible for the peculiar shape of the survival curves.

Circulating CD33+ large mononuclear cells contain three distinct populations with phenotype of putative antigen-presenting cells including myeloid dendritic cells and CD14+ monocytes with their CD16+ subset.

BACKGROUND: In peripheral blood, myeloid markers identify a heterogeneous mixture of cells in transit from the bone marrow to peripheral tissues. Similarly, HLA-class II DR expression usually identifies mononuclear cells with the potential for developing antigen-presenting activity. We gathered putative antigen presenting cells bearing myeloid markers (My-APC) to study their composition by cell surface phenotype. METHODS: To gather and dissect My-APC phenotype while excluding lymphocytes and granulocytes, we developed a strategy based on staining red cell-lysed peripheral blood and gating cells bearing myeloid markers and physical parameters of large mononuclear cells. RESULTS: Phenotypic analysis within the My-APC gate showed three distinct populations. The largest fraction was constituted by CD14+ monocytes that extended into the other two populations, each expressing gradually lower levels of CD14 surface antigen along with increasing levels of CD16 and CD2, respectively. The CD16 and CD2 expression patterns extended from CD16+CD14+ or CD2+CD14+ double- positive intermediate cells toward each single positive subset, but they were reciprocally exclusive. Interestingly, CD2+CD14- cells within the My-APC gate were equivalent to myeloid dendritic cell precursors (pre-DC) defined previously by the absence of lineage markers and expression of HLA-DR and myeloid markers. Phenotypic analysis of each population revealed differences in the expression of costimulatory molecules and CD62L. CONCLUSIONS: This novel analytical approach allowed us to distinguish circulating My-APC in three subsets and to identify relationships between monocytes and other related myeloid populations including DC.

Immunotherapy: on the edge between experimental and clinical oncology.

Cancer immunotherapy is still largely confined to the laboratory bench and experimental animal models. Yet the field is rapidly moving forward and some immunological tools are now entering into clinical use. The first and perhaps best example of such progress is given by bioengineered humanized monoclonal antibodies of which some have been already approved for therapy in B-cell lymphoma and breast cancer. Unexpectedly, another remarkable form of immunotherapy has turned out to derive from T-cell adoptive therapy associated with allogeneic bone marrow transplantation. Its benefits render such an approach the first choice therapy for a large number of hematological malignancies and it is now being adapted also for treatment of advanced solid tumors. Finally, harnessing the immune system against the autologous tumor remains the most ambitious but still distant design for immunotherapy. Recent technical advances and a better understanding of the immune system in cancer patients should concur in defining the best strategy for active immunotherapy in clinical oncology.

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients.

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.

Shortage of circulating naive CD8(+) T cells provides new insights on immunodeficiency in aging.

Clinical observations indicate that elderly people are prone to severe, often lethal infectious diseases induced by novel pathogens. Since the ability to mount primary immune responses relies on the availability of naive T cells, the circulating naive T-cell reservoir was evaluated throughout the human life span. Naive T cells were identified as CD95(-) T lymphocytes for their phenotypic and functional features. Indeed, the lack of CD95 marker is sufficient to identify a population of naive T cells, as defined by coincidence with previously characterized CD45RA(+) CD62L(+) T cells. Naive CD95(-) T cells, as expected, require a costimulatory signal, such as CD28, to optimally proliferate after anti-CD3 stimulation. Cytofluorimetric analysis of circulating T lymphocytes from 120 healthy subjects ranging in age from 18 to 105 years revealed that naive T cells decreased sharply with age. The younger subjects had a naive T-lymphocyte count of 825 +/- 48 cells/microL, and the centenarians had a naive T-lymphocyte count of 177 +/- 28 cells/microL. Surprisingly, the naive T-cell count was lower in CD8(+) than in CD4(+) subsets at any age, and the oldest individuals were almost completely depleted of circulating naive CD8(+) T cells (13 +/- 4 cells/microL). Concomitantly, a progressive expansion of CD28(-) T cells occurs with age, which can be interpreted as a compensatory mechanism. These data provide new insights into age-related T-cell-mediated immunodeficiency and reveal some analogies of T-cell dynamics between advanced aging and human immunodeficiency virus (HIV) infection. In conclusion, the exhaustion of the naive CD8(+) T-cell reservoir, which has never been reported before, suggests that this T-cell pool is a major target of the aging process and may define a parameter possibly related to the life span of humans. (Blood. 2000;95:2860-2868)

Biomarkers of immunosenescence within an evolutionary perspective: the challenge of heterogeneity and the role of antigenic load.

Under an evolutionary perspective, antigens can be considered nothing else than chronic stressors that constituted the major selective pressure for immune system emergence and evolution. In this review, recent data are discussed under the hypothesis that human immunosenescence is the consequence of the continuous attrition caused by chronic antigenic overload/stress. The advantage of this theoretical approach is that a unifying hypothesis is proposed, which tries to fill in the current gap between the conceptualizations concerning the mechanisms which counteract aging and favor longevity in invertebrates and vertebrates. The hypothesis is that the immune system is, at a higher level of biological organization and complexity, the counterpart of the anti-stress response network identified in invertebrates as the major determinant of survival. We argue that some of the most important characteristics of immunosenescence, i.e. the accumulation and the clonal expansion of memory and effector T cells, the reduction/exhaustion of naive T cells, and the shrinkage of T cell repertoire, are compatible with this assumption. Thus, immunosenescence can be envisaged as a global reduction of the "immunological space." Concomitantly, immunosenescence results in the progressive generation of cellular mosaicism which is the consequence of the heterogeneous replicative histories and telomere shortening of T and B cell subsets, as well as hemopoietic stem cells. Most of the parameters affected by immunosenescence appear to be under genetic control, and future research on biomarkers should address this point. On the whole, immunosenescence can be taken as a proof that the beneficial effects of the immune system, devoted to the neutralization of dangerous/harmful agents early in life and in adulthood, turn to be detrimental late in life, in a period largely not foreseen by evolution. This perspective fits with basic assumptions of evolutionary theories of aging, such as antagonistic pleiotropy.

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells.

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.

T lymphocyte proliferative capability to defined stimuli and costimulatory CD28 pathway is not impaired in healthy centenarians.

It is generally assumed that T cell proliferation is impaired in aged individuals. We report data on the proliferative capability of peripheral blood mononuclear cells (PBMC) and T lymphocytes from 40 healthy people of different ages, (19-107 years), including 14 centenarians, to defined mitogenic stimuli. We observed no age-related proliferative impairment both in PBMC and in purified T cells stimulated by anti-CD3 mAb or phorbol myristate acetate (PMA). Furthermore, T cells stimulated by anti-CD3 mAb or PMA and costimulated by CD28 mAb did not proliferate differently among young, middle aged subjects and centenarians. Thus, short term T cell proliferation is not affected even at extreme age when well defined stimuli are used on cells deriving from carefully selected healthy subjects.

Dendritic cells that process and present nominal antigens to naive T lymphocytes are derived from CD2+ precursors.

Dendritic cells (DC) are potent APC that, in mature form, can be distinguished from other mononuclear cells on the basis of their distinct morphology, absence of lineage markers, and dense expression of MHC and costimulatory molecules. While comparing different DC preparation methods, we observed that DC derived from cultured PBMC that had been depleted of CD2+ cells before culture were functionally distinct from DC derived from PBMC that had not been depleted of CD2+ cells. Thus, both types of DC stimulated allogeneic T cells to proliferate in the MLR, but only DC derived from CD2+ precursors could sensitize naive T cells to soluble Ags such as keyhole limpet hemocyanin and HIV gp160 glycoprotein. Subsequent studies confirmed the existence of CD2+ and CD2- DC precursor populations among HLA-DRbright, lineage-negative PBMC. Immediately after their isolation, these populations were morphologically similar to one another by light and electron microscopy, and neither had substantial Ag-presenting activity. After culture for 24 to 48 h with supernatant from PHA-activated PBMC, both populations developed dendrites, formed clusters with T cells, and stimulated allogeneic T cell responses in the MLR as well as autologous T cell responses to tetanus toxoid, a recall Ag. However, CD2+ DC precursors alone gave rise to APC that presented soluble Ags to naive CD4+ T cells, a property that could be inhibited by Abs to CD4, CD11a, and CD28 on T cells or CD86 on DC. The expression of CD54 and CD86 on CD2+ DC precursors was increased markedly after their culture and differentiation, while the expression of these molecules on CD2- DC precursors was not remarkably changed. These findings reveal the existence of two functionally distinct populations of DC, each derived from a phenotypically distinct precursor present in monocyte-depleted peripheral blood.

Expansion of cytotoxic CD8+ CD28- T cells in healthy ageing people, including centenarians.

Ageing is associated with complex remodelling in the phenotypic and functional profiles of T lymphocytes. We investigated whether expression of CD28 antigen on T cells is conserved throughout adulthood and ageing in humans. For this purpose we analysed T cells obtained from peripheral blood of 102 healthy people of ages ranging from 20 to 105 years. We found an age-related increase of CD28- T cells in percentage and absolute number, predominantly among CD8+ T cells. CD28- T cells from aged donors analysed by flow cytometry appeared as resting cells (not expressing CD25, CD38, CD69, CD71, DR), bearing markers of cytotoxic activity (CD 11b and CD 57) and with a phenotype compatible with 'memory' cells (up-regulated CD2 and CD11a; CD62L absent). At the functional level, freshly isolated purified CD28- CD8+ T cells showed high anti-CD3 redirected cytotoxic activity against Fc-bearing P815 cells. The same activity tested on freshly isolated bulk T lymphocytes was significantly augmented with age. We found a positive correlation between age, number of CD8+ CD28- T cells and anti-CD3 redirected cytotoxicity by freshly isolated T cells. These data suggest that an activation of unknown nature within the cytotoxic arm of the immune system occurs with age. We speculate that these cytotoxic T lymphocytes (CTL) in vivo may constitute armed effector cells for immediate killing of targets bearing peptides from pathogens of intracellular origin.

B4B, a novel growth-arrest gene, is expressed by a subset of progenitor/pre-B lymphocytes negative for cytoplasmic mu-chain.

We subjected PBMC of normal adults to density fractionation to enrich for an immunoblast fraction that would include early immune lineage precursors. Differential display PCR experiments identified one transcript that is expressed specifically in this immunoblast fraction. This cDNA, designated B4B, encodes a novel gene product containing four putative transmembrane-spanning domains. B4B+ cells, detected with anti-B4B Ig, were found at very low frequency in PBMC (0.01%) and were enriched significantly in intermediate density fractions (0.1-1.0%). B4B+ cells were shown to be CD19+CD45+HLA-DR+ and negative for CD20, cytoplasmic mu-chain, CD3, CD16, CD56, CD34, and CD68 (monocyte), consistent with a progenitor/pre-B lymphocyte subset that does not express cytoplasmic mu-chain and thus may lack productive Ig rearrangement. This phenotypic description of the B4B+ subset agrees with our finding that the frequency of B4B+ cells was greatly increased in bone marrow (3-10%) as compared with PBMC (0.01%). The B4B polypeptide sequence exhibits significant homology to only one known protein, PMP-22/gas-3, a Schwann cell-specific protein that induces cell growth arrest. Transient expression of B4B specifically inhibited cellular proliferation by more than 50%. Based on its antiproliferative effect and pattern of expression restricted to a subpopulation of immature B cells, the B4B gene product may be involved in the elimination of B cells before productive VDJC rearrangement of Ig loci or, alternatively, in the growth arrest of transformed progenitor B cells.

Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells.

In this pilot study, we investigated the ability of autologous dendritic cells pulsed ex vivo with tumor-specific idiotype protein to stimulate host antitumor immunity when infused as a vaccine. Four patients with follicular B-cell lymphoma received a series of three or four infusions of antigen-pulsed dendritic cells followed, in each instance, by subcutaneous injections of soluble antigen two weeks later. All patients developed measurable antitumor cellular immune responses. In addition, clinical responses have been measured with one patient experiencing complete tumor regression, a second patient having partial tumor regression, and a third patient resolving all evidence of disease as detected by a sensitive tumor-specific molecular analysis.

Inhibition of antigen-presenting cell function by alendronate in vitro.

Bisphosphonates are potent inhibitors of bone resorption in vivo and are emerging as important and widely used drugs for the treatment of a variety of abnormal bone resorptive processes. In the current study we investigated the in vitro effects of 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), a recently developed, extremely potent bisphosphonate, on the immune functions of human peripheral blood mononuclear cells (PBMCs). PBMC proliferation induced by lectins, alloantigens, and a nominal antigen (tetanus toxoid) was inhibited in a dose-dependent manner by alendronate. Pretreatment of monocytes, but not T cells, with the compound at concentrations ranging from 10(-4) to 10(-8) M was inhibitory, indicating that alendronate acts selectively on antigen-presenting cells (APCs). Alendronate did not affect the viability of monocytes or T cells or the expression of cell surface molecules known to play critical roles in antigen presentation. Alendronate exhibited dose-dependent inhibition of the production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) by activated monocytes. The inhibitory effect of 10(-6) M alendronate on PBMC proliferation was reversed by 10 U/ml recombinant rIL-1 beta, whereas other cytokines such as IL-6, TNF-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) had no effect. Thus, alendronate acts on monocytes to inhibit their antigen-presenting/accessory cell functions through a mechanism that can be overcome by exogenous IL-1. The inhibitory effect of this agent on cytokine production may contribute to its inhibitory effect on bone resorption.

Role of B70/B7-2 in CD4+ T-cell immune responses induced by dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T-cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti-CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC-dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T-cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation.

Cellular and molecular basis of human gamma delta T cell activation. Role of accessory molecules in alloactivation.

Although gamma delta T cell receptor-bearing lymphocytes (gamma delta T cells) constitute a significant minority of circulating and tissue-associated T lymphocytes, the mechanism responsible for the activation of these cells is unknown. To address this question, resting gamma delta TCR+, CD3+, CD4-, CD8- cells isolated from the blood of healthy volunteers were cultured with allogeneic dendritic cells (DC) or monocytes, and their proliferative response measured. DC alone induced gamma delta T cells to proliferate, with a peak response on the sixth day of culture. Pretreatment of DC with an anti-HLA-DR mAb, but not anti-HLA class I or anti-CD1 mAbs, inhibited the response of gamma delta T cells. Antibodies to gamma delta T cell receptor, CD2, CD3, or CD11a were also inhibitory, whereas antibodies to alpha beta T cell receptor, CD4, CD5, and CD8 had no effect. Although only 40-60% of freshly isolated gamma delta T cells expressed CD28, mAbs directed against CD28 or its ligand, CD80, were markedly inhibitory. Moreover, removal of CD28+ cells from the gamma delta T cell population nearly abrogated the response to DC. These results demonstrate that resting gamma delta T cells recognize and respond to MHC class II determinants on allogeneic DC in a manner that is highly dependent on the CD28 activation pathway as well as molecules such as CD2 and CD11a that mediate cell-to-cell adhesion.

Identification of a human OX-40 ligand, a costimulator of CD4+ T cells with homology to tumor necrosis factor.

The human OX-40 cell surface antigen is a CD4+ T cell activation marker that acts as a costimulatory receptor and is a member of the nerve growth factor receptor/tumor necrosis factor (TNF) receptor family. Using a soluble form of the receptor, the extracellular region fused with human immunoglobulin Fc, we expression cloned the human OX-40 ligand cDNA from a library derived from an activated B lymphoblastoid cell line MSAB. The encoded protein is identified as gp34, a type II transmembrane antigen previously known to be expressed only by human T cell lymphotropic virus 1-infected cells. We describe gp34 as a new member of the TNF family, and find that the recombinant ligand expressed in COS cells costimulates phorbol myristate acetate, phytohemagglutinin, and anti-CD3-induced CD4+ T cell proliferation.

Complex alteration of thyroid function in healthy centenarians.

Several changes in thyroid function have been described in the elderly and largely attributed to concomitant nonthyroidal illness. The extent to which aging per se contributes to these changes remains to be elucidated, and scanty data are available in extremely old subjects. The present study was designed to focus on thyroid function during physiological aging, taking advantage of two groups of selected aged individuals: group A of healthy centenarians (n = 41; age range, 100-110 yr) and group B including healthy elderly subjects selected by the criteria of the EURAGE SENIEUR protocol (n = 33; age range, 65-80 yr). Control groups included 98 healthy normal adult subjects (group C; age range, 20-64 yr) and 52 patients with miscellaneous nonthyroidal illness (group D; age range, 28-82 yr). Our previous report of a low prevalence of thyroid autoantibodies in centenarians was confirmed and extended by the finding of a similar low autoantibody prevalence in the highly selected healthy elderly population of group B. Subclinical primary hypothyroidism was found in 3 (7.3%) centenarians, and their data were excluded from further statistical evaluation. No significant difference was found in the median serum free T4 levels of groups A-C. Median (and range) serum free T3 (FT3) was lower in centenarians [3.67 pmol/L (2.3-5.5)] than in group B [5.22 pmol/L (3.4-6.1)] and group C [5.38 pmol/L (2.9-8.4); P < 0.0001 vs. both groups]. Similarly, the median serum TSH level of centenarians [0.97 mU/L (< 0.09 to 2.28)] was lower than those in groups B [1.17 mU/L (0.53-2.74)] and C [1.7 mU/L (0.4-4.8); P < 0.0001 vs. both groups]; moreover, serum TSH was also significantly (P < 0.01) lower in group B than in group C. Both serum FT3 and TSH concentrations showed a significant inverse correlation (r = -0.634; P < 0.0001 and r = -0.377; P < 0.0001, respectively) with age. Median serum FT3 in centenarians was lower than that in group D patients [4.61 pmol/L (2.15-6.6); P < 0.0001]. In contrast, median serum rT3 in centenarians [0.40 nmol/L (0.20-0.77)], although higher than those in groups B [0.24 nmol/L (0.15-0.37); P < 0.0001] and C [0.22 nmol/L (0.05-0.46); P < 0.0001], was significantly lower than that in group D [0.60 nmol/L (0.13-2.08); P < 0.0001]. In conclusion, thyroid function appears to be well preserved until the eighth decade of life if healthy subjects are studied, whereas a reduction of serum FT3 is observed in extreme aging.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphocyte subsets and natural killer cell activity in healthy old people and centenarians.

The contribution of the immune system to healthy aging and longevity is still an open question. For this reason, several immune parameters (T, B, and natural killer [NK] cell subsets; non-major histocompatibility complex [MHC]-restricted cytotoxic activities, ie, natural and redirected killing [RDK] activities) were studied in a total of 138 healthy subjects of different ages, from 4 to 106 years of age, including 26 centenarians. The major age-related modifications were the following: (1) a decrease in the absolute number of T lymphocytes (CD3+), involving both CD4+ and CD8+ subsets, accompanied by a marked concomitant increase in the number of activated T cells (CD3+, HLA-DR+); (2) a marked decrease in the number of B lymphocytes (CD19+); and (3) an increase in the number of cells with markers of NK activity and of T lymphocytes able to mediate non-MHC-restricted cytotoxicity. These modifications linearly progressed with age and centenarians followed the trend, suggesting that their immune system did not escape the aging process. However, other immunohematologic parameters (number of red blood cells, platelets, and leukocytes) and important immune functions, such as cytotoxic activities (NK and RDK cell activities), were well preserved throughout life until the last decades of life. Unexpectedly, in apparently healthy middle-aged subjects, a decrease of cytotoxic activities was observed in comparison with those of both young controls and centenarians. In conclusions, our data suggest that in centenarians some immune responses are kept at a high level of efficiency, likely contributing to their successful aging. However, this selected group of people does not escape the aging process, as shown by the progressive derangement of a variety of immune parameters.