Arnika Rehbein

Human T‐cell clones in long‐term culture as a model of immunosenescence

Summary: We have consistently observed that like other normal somatic tissue cells, human T lymphocytes manifest a finite proliferative capacity in culture in vitro. When measured in population doublings (PD), this averages about 35 PD for T‐cell clones (TCC) derived from mature peripheral T cells of young adults and about 20 PD more for TCC derived from T‐cell precursors in their bone marrow. We believe that alterations in surface marker phenotypes and corresponding functional changes observed in these human TCC as they progress through their finite lifespans in vitro can provide valuable information on processes of T‐cell immunosenescence in vivo. They may also provide a model system for studying ways of modulating the ageing process to delay or prevent immunosenescence in the elderly and the chronically infected or possibly to accelerate immunosenescence in organ transplantation.

Decreased proliferative capacity and increased susceptibility to activation-induced cell death in late-passage human CD4+ TCR2+ cultured T cell clones.

The growth characteristics in vitro of interleukin 2 (IL 2)-dependent human CD4+ alpha beta-T cell receptor-positive helper T cell clones (TCC) were studied in relation to alterations in surface phenotype, cytokine responsiveness, and susceptibility to activation-induced cell death (AICD). TCC derived from peripheral blood T cells had finite lifespans averaging 33 population doublings (PD) with a recorded maximum lifespan of 80 PD (n = 208). First analyses of the TCC were undertaken at ca. 25 PD, at which time all cells of all TCC expressed high intensity CD45RO and low intensity CD45RA, as well as high intensity CD95 (fas) and MHC class II antigens. The expression of these molecules remained elevated throughout the proliferative lifespan of the clones, but for those TCC which were initially CD28+ (the majority), the density of expression of the latter was diminished in most late-passage clones. Concomitant with this, late-passage cells showed reduced responsiveness to CD28-mediated costimulation by CHO transfectants expressing human CD80 compared to early-passage cells. Additionally, the level of expression of IL 2R gamma c and IL 7R chains was commonly reduced, as was the response to IL 2 and IL 7. Despite unchanged levels of fas expression on TCC with time, late-passage cells were more susceptible to AICD than early, passage cells. These observations further document functional and phenotypic alterations in long-term cultured human T helper cells, which may be considered as biomarkers of immunosenescence. This may contribute to an improved understanding of the mechanisms underlying depressed T cell function in old age.

In vitro senescence models for human T lymphocytes.

Immunosenescence is an age-associated dysregulation of immune function which may contribute to the increased susceptibility of the elderly to infectious disease. Although age-associated changes are measurable in the innate immune system, it is the adaptive arm of the immune system which is particularly susceptible to the deleterious effects of ageing, especially the T cell compartment. In this review, the characteristics of longitudinal ageing in cultured monoclonal human T cell populations will be summarized. It will be argued that parallels between this in vitro model and T cell senescence in vivo suggest the use of such models to screen for interventions ameliorating immunosenescence in vivo.

Relative Roles of Natural Killer- and T Cell-Mediated Anti-Leukemia Effects in Chronic Myelogenous Leukemia Patients Treated with Interferon-α

Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.

Extrathymic T cell differentiation in vitro from human CD34+ stem cells

Although it is well established that T cells are derived from CD34+ stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in vitro from CD34+ cells in the absence of any thymic influence. We now present a limiting dilution cloning culture system that supports the differentiation of highly purified human CD34+ cells to CD3+ T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum‐free medium supplemented with a cocktail of cytokines including flt‐3 ligand, interleukin‐3 (IL‐3), stem cell factor (SCF), and IL‐2. CD4+ T cell clones capable of mitogen‐stimulated proliferation and response to IL‐2, and expressing a varied TCR‐Vβ repertoire were obtained under these conditions. This culture system therefore supports human T lymphopoiesis in the absence of any thymic influence and may prove useful for the evaluation of extrathymic T cell differentiation in vitro. J. Leukoc. Biol. 64: 733–739; 1998.

Cellular immune responses to autologous chronic myelogenous leukaemia cells in vitro

Abstract Using a modification of the autologous mixed lymphocyte/tumour cell culture (MLTC), it is demonstrated here that lymphocytes from chronic-phase myelogenous leukaemia (CML) patients (n = 58), but not from their HLA-identical siblings, proliferated upon coculture with autologous tumour cells. However, in most cases, the level of proliferation measured was low (stimulation index <3, n = 37). This was most likely related to the amount of interleukin-10 (IL-10) released into the culture medium by the CML cells, because addition of neutralizing anti-IL-10 serum to MLTC markedly enhanced proliferative responses. In addition, supplementation of media with IL-1α further enhanced proliferative responses and a combination of anti-IL-10 serum and IL-1α was more effective than either agent alone. Only HLA-DR-matched CML cells, but not HLA-DR-mismatched CML cells or matched or mismatched PBMC restimulated proliferation of IL-2-dependent T cell lines derived from MLTC supplemented with IL-1α and anti-IL-10 serum. The responding cells under these conditions were predominantly CD4+ and secreted IL-2, and interferon γ; some secreted IL-4, but none secreted IL-10. These data therefore suggest the existence of an HLA-DR-restricted DTH/Th1-type of tumour-specific immunity in CML patients, which may be down-regulated in vitro by excessive secretion of IL-10 together with depressed secretion of IL-1.

Cytokine modulation of TH1/TH2 phenotype differentiation in directly alloresponsive CD4+ human T cells.

Organ graft rejection is caused by the recognition of allogeneic MHC molecules by recipient T cells by two different pathways. The indirect pathway of alloreactivity requires the presentation of MHC peptides from the graft by autologous APC, as with conventional antigen. The direct pathway, on the other hand, requires the recognition of foreign MHC on foreign cells. The regulatory mechanisms for this component of alloreactivity have not been extensively studied. We show here that the T cell response activated by alloantigens in the direct pathway is similarly constrained and modulated by cytokines, as has been shown for classic antigen presentation. Thus, the inclusion of IL-2 or TGF-beta in MLC performed with purified responder T cells resulted in outgrowth of cells secreting IL-2 and IFN-gamma, whereas addition of IL-4, IL-10, or anti-TGF-beta encouraged outgrowth of cells secreting IL-4 and IL-10. T cells alloactivated via the direct pathway and then cloned in IL-2 alone secreted IL-4 and IL-10 as well as IFN-gamma and IL-2 (Th0 phenotype). Established clones remained susceptible to cytokine modulation, such that IL-4 and IL-10 decreased their secretion of IL-2 and IFN-gamma, whereas TGF-beta suppressed IL-4 and IL-10 secretion. The first alterations of Th0 toward Th1 or Th2 phenotypes could already be observed after only a very brief exposure to cytokines of 48 hr, followed by extended culture with IL-2 alone. These results confirm that human T cells with Th1 and Th2 phenotypes, recognizing alloantigen via the direct pathway, derive from the same IL-2-secreting precursor and can be manipulated by cytokines in an analogous fashion to conventional antigen-reactive cells. These findings may have implications for manipulating the direct pathway of alloantigen recognition in human organ transplantation.

Partial correction of defective generation of lymphokine-activated killer cells in patients with chronic myelogenous leukaemia after in vivo treatment with interferon-α (wellferon)

SummaryPatients with chronic myelogenous leukaemia (CML) in untreated chronic phase are deficient in their ability to generate lymphokine-activated killer (LAK) cells from peripheral blood mononuclear cells although they posses essentially normal levels of CD16+ and Leu19+ lymphocytes, which do not seem to be actively suppressed by tumour cells. Attempts to enhance LAK cell generation in these patients are reported here. Combining the lymphokines interleukins-2, with -4 and -5 (IL-2, IL-4, IL-5), was not successful; in fact, IL-4 depressed LAK cell induction in both normal donors and CML patients. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also failed to enhance cytotoxicity of normal donors or patients, and indomethacin was similarly without effect. The only agent found to enhance LAK cell induction by IL-2 in normal donors was interferon-γ (but not IFN-α) and even this modest effect was not seen with the cells of CML patients. Increasing concentrations of IL-2 and/or culture duration also failed to improve LAK cell generation by patients. The only improvement in LAK cell generation was observed in CML patients treated for one or more months with IFN-α, where a steady increase of LAK activity with time after initiation of therapy was noted. These results show that the blockade of LAK cell induction in chronic-phase myelogenous leukaemia patients is difficult to lift pharmacologically in vitro but possibly susceptible to biological response modifiers in vivo.

Restimulation properties of cloned alloactivated lymphocytes: Detection of a novel type of HLA-D/DR region determinant and allelic subtypes for HLA-Dw3 using cloned reagents

Lymphocytes alloactivated by Dw3 homozygous typing cells were cloned by the method of limiting dilution and cultured for prolonged periods using T-cell growth factor and irradiated pooled leukocytes (as feeder cells). Restimulation specificity of two clones functioning as primed lymphocyte typing reagents was investigated in panel and family studies. Cells from one of the clones (12-2) were always specifically stimulated by HLA-Dw3 antigens shared between the original priming cells and the stimulating panel cells. In an informative family KOH, however, cells from this clone seemed to detect a split in the Dw3 cluster. Cells from the other clone (12-8) failed to respond to Dw3 antigens as expressed by the original priming cells or by panel stimulating cells; rather, specificity of restimulation seemed to be associated with the expression of Dw4 antigens. Family segregation analysis did not support this conclusion however, since stimulating products segregated with one of the three Dw3-bearing haplotypes and with none of three Dw4-bearing haplotypes. This suggested that 12-8 cells may be responsive to antigens different from those detected by homozygous typing cells.

Age-related loss of function of alloactivated interleukin 2-propagated human primed lymphocyte typing clones.

Human lymphocytes alloactivated in vitro were cloned by limiting dilution in the presence of filler cells and interleukin 2 (IL 2)‐containing supernatants of phytohaemagglutinin‐stimulated lymphocytes. Clones with allospecific proliferative reactivity (PLT clones), measured by tritiated thymidine (3H‐TdR) incorporation, were selected for extensive IL 2‐dependent expansion. The cloned lines had finite lifespans, ranging from an estimated minimum of 28 to > 65 doublings. Function as PLT reagents, however, was retained in all cases for only an estimated 30 cell doublings. This apparent cessation of function was not caused by loss of the ability to metabolize thymidine, since lines continuing to grow for > 30 doublings still incorporated 3H‐TdR in the presence of IL 2. An altered requirement for stimulating antigen (number of stimulating cells), or altered response kinetics, did not contribute to loss of PLT function. Exogenous IL 2 added during restimulation to responders previously ‘rested’ overnight without IL 2 did not restore the response. Thus, under present experimental conditions, functional lifespans of cloned PLT reagents appear fixed at ˜ 30 cell doublings.