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R B Effros

Publications and source records attributed to R B Effros.

At least 37 records · Page 2Linked to original sources
PubMed1997

Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion?

Extensive in vitro research on fibroblasts has defined numerous genetic and phenotypic changes associated with replicative senescence. Identification of T-cell replicative senescence as a feature of human immunodeficiency virus (HIV) disease and ageing suggests this phenomenon merits more careful consideration by immunologists, especially with regard to chronic infection, memory and adoptive immunotherapy.

Allergy and Immunology↗
PubMed1996

Mechanism of telomerase induction during T cell activation.

The progressive shortening of the ends of chromosomes (telomeres) during cell division may serve as a mitotic clock for replicative senescence. Telomerase, a ribonucleoprotein which synthesizes telomeric DNA and maintains telomere length, is absent from most normal somatic cells but is expressed in immortal cells. Low levels of telomerase activity have been detected in peripheral blood mononuclear cells (PBMC) and hematopoietic cells and an increase in telomerase activity during T cell activation has recently been reported. In this study, we show that the increase in telomerase activity during T cell activation was transient and did not prevent the loss of telomeres in long-term T cell cultures. Analysis of the mechanism of telomerase induction showed that the increase in telomerase activity was accompanied by an increase in the levels of hTR, the RNA component of human telomerase. Moreover, telomerase induction occurred in the presence of aphidicolin, indicating that DNA synthesis was not required. Increased telomerase expression was observed when PBMC were activated with phorbol myristate acetate (PMA) and ionomycin, indicating that it was independent of early transmembrane signals. It was, however, linked to the T cell signal transduction pathway, as inhibiting protein kinase C with bisindolylmaleimide prevented the increase in telomerase activity.

Adult↗
PubMed1996

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.

CD28 Antigens↗
PubMed1994

Stress response of senescent T lymphocytes: reduced hsp70 is independent of the proliferative block.

Senescent human T lymphocyte cultures are unable to undergo proliferation, but show no difference from early passage cells in cytotoxic function or surface antigenic profile. A second feature of senescent T cells is the dramatic reduction in hsp70 production in response to heat shock. This decline is associated with a decrease in binding of nuclear extracts to the consensus heat shock element. Interestingly, the progressive decline in the heat shock response of cultured T cells correlates with the percent proliferative life span completed rather than with the actual proliferative activity at the time of heat shock. This suggests that for senescent T cells the reduced ability to respond to heat shock by producing hsp70, although possibly lying at the level of transcriptional control, may nevertheless be unrelated to the reduced DNA synthesis or the diminished proliferative activity also manifested by these cells.

Cell Division↗
PubMed1993

In vitro cellular aging in T-lymphocyte cultures: analysis of DNA content and cell size.

Like other normal diploid mammalian cells, human T-lymphocytes display a limited in vitro lifespan. Long-term cultures of normal adult peripheral blood T cells, activated in vitro and passaged in the presence of interleukin-2 undergo 23 +/- 7 cumulative population doublings. Cell cycle analysis revealed that in senescent T cell cultures, restimulation with the original antigen resulted in 16-22% of the cells entering S phase, compared to 60% entering cycle in young cultures. In addition, within 1 week of restimulation, the senescent cultures do not increase in cell number, but 93% of the cells return to the G1/G0 DNA content, a proportion typically seen in quiescent (nonrestimulated) cultures. Coculture of early- and late-passage cells at two different ratios excluded a putative inhibitory factor produced by the old cells and similarly eliminated a possible stimulatory product in early cultures. Flow cytometry measure of forward angle light scatter revealed no difference in cell size between early- and late-passage cells, in contrast to the findings with senescent fibroblasts. Thus, while increasing cell size may contribute to the senescent phenotype of fibroblast cultures, it is not a factor in the senescence of human T-lymphocytes, and it is therefore doubtful that alterations in cell size are fundamental to in vitro cellular aging.

Cell Cycle↗
PubMed1993

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.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Influences of dietary restriction on immunity to influenza in aged mice.

Our previous studies of the immune response of aged mice inoculated with influenza A virus revealed age-related decreases in antigen-specific cytotoxic T-lymphocyte function (CTL), T-cell proliferation, IL-2 production, antigen presentation, and antibody production. Because dietary restriction (DR) of rodents has been shown to extend maximum life span, delay the onset of tumors, and improve many immunologic parameters in aged animals, we tested the effect of such a regimen on the immune response to the influenza virus. We report that DR significantly inhibited the age-related decline in antigen presentation and T-cell proliferation. It also reduced the decline in antibody production to the virus. This is the first demonstration of improved immunity to an actual infectious agent resulting from DR. The improvement appears to be on a number of levels and to reflect more than one operative mechanism.

Aging↗
PubMed1991

Influence of age and caloric restriction on macrophage IL-6 and TNF production.

Aging is correlated with a plethora of immunological changes resulting in both decreased immunity to exogenous antigens and increased autoreactivity. Immunosenescence is generally believed to primarily affect the B- and T-cell compartments, leaving macrophage (M phi) function relatively intact. However, several recent reports show diminished presentation of certain antigens and decline in interleukin 1 (IL-1) production by aged M phi s suggesting the need for more extensive assessment of M phi function. In the present study we demonstrate that aged murine M phi s show diminished IL-6 and TNF production. In addition, we show that dietary caloric restriction, a regimen which extends life span and improves many immunological functions, has no effect on the declining TNF production.

Age Factors↗
PubMed1989

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.

Cell Survival↗
PubMed1987

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.

Cell Division↗
PubMed1987

Immunization of normal human splenocytes in vitro to produce human monoclonal antibodies to cellular antigens.

We have obtained human monoclonal antibodies to polymorphic cell surface determinants by immunizing normal human splenecytomy in vitro to allogeneic cells. Splenocytes from young patients undergoing splenectomy secondary to traumatic injury are separated into T and B lymphocyte populations. The T lymphocytes are irradiated with 1500 rad to selectively inactivate T suppressors. Responder T and B cells are then recombined at a 1:1 ratio. Maximal IgM and IgG production is obtained when pokeweed mitogen and irradiated stimulator cells are added to the cultures. Stimulator cell specific antibody levels peak at day 7 of in vitro immunization, and fall thereafter. Fusion of the immunized splenocytes to a human fusion partner as early as day 4 results in hybridomas secreting antibodies to cellular antigens. Transformation by EBV, expansion and fusion of the immunized cell line also yield hybridomas secreting stimulator cell specific antibody.

Antibodies, Monoclonal↗
PubMed1986

A human-human hybridoma secreting anti-HLA class II antibody.

In this report, we describe the production and characterization of the first human-human hybridoma secreting antibody to HLA Class II determinants. The hybridoma (GMEC101), which has been stable in tissue culture for greater than 20 mo, secretes 10 to 50 micrograms/ml of IgM-kappa antibody. This antibody binds to a wide range of human cell lines, but not to the HLA-A,B,C, and DR-negative K562 cell line. Functionally, GMEC101 strongly inhibits a unidirectional mixed lymphocyte reaction (MLR) at the level of the stimulator cell. Neither the cellular ELISA binding nor the MLR inhibition is lost after a triple platelet absorption (which removes Class I but not Class II activity). Because the binding and MLR blocking show no correlation with the known DR or DQ specificities, we suggest that GMEC101 may be detecting a novel HLA Class II determinant.

Antibody Specificity↗
PubMed1985

Detection of antibodies to cell surface antigens by a simplified cellular ELISA (CELISA).

An improved method for screening human hybridoma antibodies to cell surface antigens is described. The following modifications have been developed: rapid expansion of desired screening cell types by EBV transformation; use of only 5 X 10(4) cells/well; elimination of the need for glutaraldehyde fixation; elimination of the requirement for PLL to attach cells to microplates; preparation of a large number of plates which can be stored at 4 degrees for 3 months; Protein A-peroxidase ELISA assay yielding excellent replicates, low background "noise", and high OD readings for positive wells. The techniques we have developed should greatly simplify and shorten the assay procedures for detecting human antibodies to a variety of cell surface antigens.

Antibodies, Monoclonal↗
PubMed1985

Production of a human monoclonal antibody to HLA by human-human hybridoma technology. A preliminary report.

An Epstein-Barr-virus-transformed lymphoblastoid cell line (ECEBV) was derived from a multiply transfused renal dialysis patient. ECEBV was shown to secrete specific antibody in a cellular enzyme-linked immunosorbent assay (CELISA) and was hybridized with the mutagenized human fusion partner G M1500 resistant to 6-thioguanine and ouabain. Hybridomas surviving hypoxanthine-aminopterin-thymidine (HAT) and ouabain selection were cloned by limiting dilution. The hybridomas continue to secrete antibody which reacts with some human cells but not with others after 14 months in culture. None reacts with K562 (no HLA-A, -B, -C or -DR) or with Daudi (no HLA-A, -B, or -C). This is a preliminary report of the production of a human monoclonal antibody to HLA. Application of this technique could result in the large-scale production of human monoclonal antibodies for HLA typing, the production of anti-idiotype antibodies for use in transplant patients to prevent acute rejection, and for the study of the structure and function of HLA in man.

Antibodies, Monoclonal↗
PubMed1985

Ye-1, a monoclonal antibody that cross-reacts with HLA-B27 lymphoblastoid cell lines and an arthritis causing bacteria.

A monoclonal antibody, Ye-1, was generated by immunizing BALB/c mice with Yersinia enterocolitica. This antibody also reacted with all of 12 B27 positive lymphoblastoid cell lines, but only four of 31 B27 negative ones. Three of the four reactive B27 negative cell lines were B7 positive. A B27 positive cell line which has lost the B27 expression because of experimentally-induced mutation became unreactive with the Ye-1. These findings support the possibility that there is cross-reactivity between HLA-B27 antigens and Y. enterocolitica.

Animals↗
PubMed1984

The effect of age on the antigen-presenting mechanism in limiting dilution precursor cell frequency analysis.

We have utilized limiting dilution analysis (LDA)2 to compare the intrinsic precursor cytotoxic T lymphocyte (pCTL) frequency for influenza-plus-self in young and old C57BL/6 mice. Under conditions of excess interleukin 2 (IL-2) and antigen presenting cells (APC) derived from spleens of mice matched in age to those being tested, we found more than a twofold difference in pCTL frequency between young and old animals. However, there was no difference in pCTL frequency between the two age groups if antigen was presented to the old responder cells on spleen cells derived from young mice. The apparent decrease in pCTL frequency in old mice by standard LDA may in fact be due to a defect in the antigen processing and/or presentation mechanism of old spleen cells. We conclude that the age-associated defective CTL activity previously reported by us and by others may be due at least in part to a defect in the antigen presentation mechanism of aging mice.

Aging↗
PubMed1983

The immune response of aged mice to influenza: diminished T-cell proliferation, interleukin 2 production and cytotoxicity.

In view of the fact that T lymphocytes play a key role in the recovery from viral infections, and that many immune functions involving T cells show a decline in aged individuals, we examined the ability of old mice to mount a T-cell response to influenza virus. Both primary and secondary cytotoxic T-lymphocyte (CTL) responses following in vivo immunization with influenza virus were minimal in old mice at the time point of peak response in young mice. Analysis of CTL activity generated in an in vitro microculture system revealed both a diminished proliferative response and a decrease in Interleukin 2 (IL-2) production in "old" compared to "young" cultures following stimulation with virus. Furthermore, addition of exogenous IL-2 at the initiation of the culture period augmented both subtype-specific and A-strain cross-reactive CTL activity of old spleen cells. Finally, exogenous IL-2 increased the lower proliferative response of the old cultures. These findings, if also true for elderly humans, might have broad clinical relevance to the problem of suitable prophylactic approaches to influenza infection in the elderly.

Aging↗