Measurement of polyclonal and antigen-specific cytotoxic T cell function.

Measurement of in vitro cytotoxic function of human T cells can be accomplished by polyclonal stimulation of T cell effectors using anti-CD3 antibody, which stimulates all cytolytic effector cells, or with a specific stimulating antigen. Accordingly, two sets of assays of cytolytic T cell function are described in this unit, one for measuring anti-CD3-mediated cytotoxicity and the other for measuring antigen-specific cytotoxicity. Although the calcein release assay (CARE-LASS) described here is for use with antigen-activated cytotoxic T lymphocytes (CTL) as well as natural killer (NK) or lymphokine-activated killer (LAK) cells, minor changes in the protocols that address polyclonal T cell activation are described that make them suitable for use with calcein-labeled target cells.

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.

Identification of differentially expressed genes in young and senescent T cells.

The need to analyze changes in gene expression patterns occuring in senescent cells has stimulated the search for proper methods to identify the actual differences between young and senescent cells. In studies of aging, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) has already been applied successfully by several groups. Altered gene expression in young and senescent fibroblast cultures (1), human mammary epithelial cells (2), and rat brain cells (3, 4) has been detected with this technique.

Prerequisites for the immunotherapy of cancer.

Tumours express proteins not commonly found in normal cells, or over-express certain proteins. These may in some cases serve as target antigens for immunological attack. It is therefore essential to improve our understanding of the nature of these target epitopes and the cells which recognize them, in order to develop immunotherapy as a realistic treatment for cancer. A small group of around 40 investigators recently came together at the Heinrich Fabri Institute of the University of Tübingen to discuss the identification of human tumour antigens and the exploitation of this knowledge for effective immunotherapy.

T cell immunosenescence in vitro and in vivo.

The term "immunosenescence" refers to an age-associated dysregulation of immune function which contributes to the increased susceptibility of the elderly to infectious disease. Although there are age-associated changes measurable in the innate immune system (Pawelec et al., 1998c), it is the adaptive, particularly T cell, system which is most susceptible to the deleterious effects of aging. In this minireview, characteristics of aging in long-term human T cell cultures will be summarized, and the parallels between the in vitro model and in vivo immunosenescence will be documented. The use of culture models to screen for ways of manipulating immunosenescence in vitro may provide a basis for intervention to ameliorate immunosenescence in vivo.

Tumour-specific MHC-class-II-restricted responses after in vitro sensitization to synthetic peptides corresponding to gp100 and Annexin II eluted from melanoma cells.

In a search for potentially tumour-specific MHC-class-II-restricted antigens, the immunogenicity of endogenous peptides that had been eluted from HLA-DR molecules of the human melanoma cell line FM3 (HLA-DRB1*02x, DRB1*0401) was tested in vitro. Two 16-mers representing gp100 positions 44-59, and annexin II positions 208-223 bound well to isolated DRB1*0401 molecules and are discussed here. HLA-DR-matched normal donors' T cells were cultured with peptide-pulsed artificial antigen-presenting cells (CHO cells cotransfected with genes for HLA-DRB1*0401 and CD80 and coexpressing high levels of both human molecules). Specific sensitization was achieved against both peptides, as measured in assays of autocrine proliferation and interleukin-2 secretion. Moreover, responses to native autologous melanoma cells but not to autologous B cells were also observed. In view of the expression of fas by the activated T cells and of fas ligand by the melanoma cells, blockade of potential fas/ fas-ligand interactions was undertaken using monoclonal antibodies (mAb). The antagonistic fas-specific mAb M3, but not the fas agonist M33, caused a markedly enhanced T cell response to FM3 cells. These results demonstrate that synthetic peptide antigens are able to sensitize T cells in vitro for effective MHC-class-II-restricted recognition of melanoma cells.

The T cell in the ageing individual.

Aged persons frequently manifest declining parameters of those immune functions which protect the young against disease. Longitudinal studies are beginning to show that number, type and function of T cells may be associated with longevity, morbidity and mortality in free-living elderly humans. Multi-faceted alterations in the ability of T cells from old donors to respond to stimulation are being dissected, and pathways which are compromized in the elderly compared to the young are being defined. Successful immune responses depend upon waves of rapid and extensive clonal expansion to combat primary infection, followed by death of most T cells, survival of memory cells and their later reactivation and further expansion. This implies that the finite replicative potential of T cells might impose a critical limiting factor on the maintenance of immune responses in the face of thymic involution and drastically reduced capacity to generate new naive T cells. This type of 'clonal exhaustion' can readily be studied in vitro using human T cell clones and the findings can be applied to the in vivo situation. Understanding the processes of replicative senescence in such in vitro models may shed light not only on some of the underlying mechanisms of immunosenescence but also in situations of chronic antigenic stimulation in vivo. Moreover, it might begin to indicate how the system could be manipulated on the one hand to prevent or reverse T cell senescence without nullifying the control mechanisms of tumor suppression, and on the other hand, to reconstitute possibly faulty suppression, for example in autoimmune disease.

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

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.

Interleukin 10 protects activated human T lymphocytes against growth factor withdrawal-induced cell death but only anti-fas antibody can prevent activation-induced cell death.

Interleukin 10 (IL-10) is a pleiotropic T cell-derived cytokine best known for its negative regulatory effects on T cell immunity. It inhibits responses indirectly by downregulating expression of major histocompatibility complex (MHC) molecules and co-stimulatory molecules such as CD80 on antigen presenting cells as well as directly via its effects on responding cells. On the other hand, IL-10 has been shown to protect activated T cells against apoptosis caused by withdrawal of the major growth factor, IL-2, and allow proliferation of T cells in the absence of IL-2. However, we show here that this IL-10-dependent, IL-2-independent proliferative response is short-lived, and that IL-10-responsive T cells cannot multiply in its presence. Moreover, inclusion of exogenous IL-10 in clonal cultures propagated with IL-2 results in suppression of their growth. These findings, together with the observation that IL-10 fails to protect T cells against activation-induced cell death (a fas/fas-ligand-dependent phenomenon blocked only by certain antagonistic anti-fas reagents), suggest that the negative regulatory effects of IL-10 outweigh the upregulated proliferation observed on some T cell clones (TCC) in the absence of IL-2.

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.

Long-term culture of monoclonal human T lymphocytes: models for immunosenescence?

Human monoclonal T lymphocyte populations maintained in long-term culture by intermittent reactivation via the antigen receptor and supplied with exogenous interleukin 2 manifest finite proliferative lifespans. T lymphocytes cloned from mature peripheral T cells of adult donors were constantly lost from the time point of their first isolation up to an estimated maximum of 80 population doublings (PD) for the longest lived. T lymphocytes cloned from T cell progenitors in bone marrow, on the other hand, survived for a maximum of ca. 100 PD. One facet of the functional capacity of cells derived from these two different sources was assessed by measuring their autocrine proliferation after mitogenic stimulation. For a majority of T cell clones (TCC), autocrine proliferative capacity decreased as a function of culture age, becoming absent by 50 PD for adult-derived-TCC and by 70 PD for bone marrow-derived TCC, thereby clearly occurring prior to the end of the proliferative life spans of the clones. Limiting dilution frequency analysis showed that the number of autocrine proliferative precursors within these monoclonal populations declined with age, paralleling loss of autocrine proliferative capacity in the 'bulk' clones. Of a variety of surface structures monitored during culture ageing of TCC, the density of expression of the coreceptor molecule CD28 was found to correlate with decreasing autocrine proliferative capacity in two-thirds of the clones. Thus, at least for a fraction of monoclonal human T lymphocytes, decreasing autocrine proliferative capacity, a measure of clonal expansion, may correlate with decreasing numbers of CD28 molecules expressed on the surface and therefore presumably with the strength of costimulatory signal delivered via this important coreceptor.

Role of three quantitatively dominant endogenous peptides from HLA-DRB1*0401 molecules in class II specific alloreactivity.

Alloreactivity remains an important barrier to organ transplantation and is caused by T cell recognition of foreign histocompatibility antigens (HAg) in two ways: (1) indirect recognition, in which processed HAg peptides are presented by self MHC like any other foreign antigen, and (2) direct recognition, where the foreign MHC itself is recognized in contravention of the T cell recognition rule of self restriction. Whereas the role of endogenous peptides in direct MHC class I specific recognition is now established, their role in class II specific direct alloreactivity remains controversial, since no defined endogenous peptide has been shown to be required for alloreactivity. That mutations resulting in defective antigen processing impair class II specific allostimulation, however, suggests that the endogenous pathway is important for class II as well as class I alloreactivity. We attempted to establish the importance of endogenous peptides for alloreactivity by identifying common sequences of peptides bound by DR molecules of an HLA-DRB1*0401 homozygous B cell line. Peptides corresponding to three of these (calreticulin, HLA class I and an unidentified molecule) were used to restimulate established allospecific HLA-Dw4 reactive T cell clones, as well as to sensitize allogeneic T cells de novo in vitro. Xenogeneic chinese hamster ovary (CHO) cells coexpressing the relevant DR allele together with CD80 were used as antigen presenting cells. The role of CD80 could be determined on these cells because (1) they are xenogeneic and (2) they do not express B7 family members bound by CTLA-4Ig.(ABSTRACT TRUNCATED AT 250 WORDS)

Requirements for stimulation or anergy induction in alloreactive human T cell clones.

The "two signal" concept for T cell activation is widely accepted. Signal 1 is commonly delivered via the antigen receptor, and signal 2 via accessory interactions. Delivery of both signals results in activation, signal 1 alone in induction of hyporesponsiveness. The nature of signal 1 in alloreactivity is not completely clear; most evidence suggests that a complex of foreign major histocompatibility complex molecules and their bound peptides is recognized. Interactions between B7 (CD80) ligand and CD28/CTLA-4 receptors are currently considered the most important sources of signal 2. Xenogeneic cells transfected with human genes provide useful stimulators for dissecting signals 1 and 2 in alloreactivity. We show here that the majority of DR-specific alloreactive T cell clones (TCC) fails to recognize Chinese hamster ovary (CHO) cells transfected with human DR, whether or not these are cotransfected with genes for CD80 or LFA-3. Stimulation was not observed even in the presence of a pool of peptides isolated by low pH release from B cell line (BCL)-derived DR molecules, or in the presence of synthetic peptides corresponding to the sequences of the three most commonly identified endogenous peptides. Lack of recognition was observed both in failure to stimulate proliferation and in failure to induce anergy. However, one TCC was identified which responded weakly to DR+ CHO cells, and for this clone, the presence of either CD80 or LFA-3 strongly enhanced proliferative responses. Anergy was not induced, even in the absence of CD80. Immobilized HLA-DR molecules purified from a BCL also failed to stimulate proliferation, but unlike the CHO transfectants, they did induce anergy. Stimulation with BCL also induced anergy if CD80-dependent interactions were blocked with soluble CTLA-4-Ig receptor. These results are consistent with the model that DR molecules expressed in the absence of appropriate peptide are simply not recognized by most alloreactive T cells, whereas DR molecules containing appropriate bound peptide are recognized as signal 1 and induce anergy. CTLA-4-Ig blocking confirms that CD80-dependent interactions can be important in preventing anergy induction, but that they are not always necessary is illustrated by the existence of a single clone which recognized DR molecules on CHO transfectants, giving very weak proliferation without CD80, and nonetheless no anergy induction.

Extrathymic development and function of human T-lymphocytes from bone marrow cells in vitro.

To enrich low-density human bone marrow (BM) cells for putative progenitors of T-lymphocytes, CD7+ CD3- cells were sorted (purity was estimated at > 99.9%) and cultured under limiting dilution conditions with irradiated allogeneic stimulator cells, interleukin (IL) 2, and PHA. Clonal populations were available for analysis from Day 25 onward. By this time, all clones (n = 54) expressed CD3 and alpha/beta-T cell receptor (TCR2). Fifty percent of the clones were CD4+ and 50% were CD8+, with no double positives, whereas almost all clones obtained under identical conditions from peripheral blood (PB) cells were CD4+. All clones were capable of autocrine proliferation, which was blocked by CD25 or CD71 mAb. Most or all clones tested (n = 15) responded to IL 4 and IL 7 as well as IL 2, but not to IL 3 or GM-CSF and only two responded to IL 9. Most clones accumulated mRNA for GM-CSF, IL 2, IL 3, IL 4, IL 5 and also IL 9, but 6 of 11 were negative for IFN-gamma mRNA, and all were negative for IL 6 mRNA. Sixty-two percent of CD4+ and 85% of CD8+ clones (total 70% of all clones) mediated lectin-dependent cell lysis; but whereas 35% of CD4+ and 65% of CD8+ clones (total 46% of all clones) lysed K562 natural killer (NK)-susceptible targets, only 24% of CD4+ and 5% of CD8+ clones (total 17% of all clones) killed lymphokine-activated killer (LAK)-susceptible Daudi cells. Only three clones lysed allogeneic LCL targets and none lysed autologous targets. Furthermore, none of the clones proliferated when stimulated by autologous cells, neither did they suppress proliferative responses of autologous cells. These results suggest that CD3- cells from the bone marrow can acquire functional cytotoxic and proliferative programs extrathymically during in vitro culture with IL 2, mitogens and allogeneic cells, but do not manifest autoreactivity in the three test systems, cytotoxicity, suppression, or autocrine proliferation.

Evolution of a CD3+CD4+ alpha/beta T-cell receptor+ mature T-cell clone from CD3-CD7+ sorted human bone marrow cells.

In order to study extrathymic differentiation in vitro, CD7+CD3- lymphocytes were sorted from normal human bone marrow and cultured under conditions of limiting dilution together with irradiated pooled allogeneic peripheral blood mononuclear cells (PBMC) and phytohemagglutinin (PHA) in the presence of 1000 U/ml of interleukin-2 (IL-2). One clone was obtained that failed to react with monoclonal antibody (mAb) TCR delta 1 (TCR1, gamma/delta-specific) or WT31 (TCR2, alpha/beta-specific). From day 35 through day 74 in culture, the surface phenotype of this clone evolved into CD3+, CD4+, CD8-, TCR2+, TCR1-, and was further characterized as CD2+, CD45RO+, CD16-, and CD56-. The presence of mRNA for TCR alpha and beta but not gamma and delta chains was confirmed by Northern blotting. Accessory cell-dependent autocrine proliferative responses to PHA (most likely driven by IL-2) were initially absent, but became measurable at the same time as the TCR was acquired. However, in the absence of PHA, the clone failed to respond to a panel of homozygous B-cell lines representing the majority of MHC class II alleles. Autoreactivity was also not demonstrable. Cytotoxicity was limited to MHC unrestricted "natural killer (NK)-like" lysis of K562 target cells, with no autocytotoxicity detected. The NK-like lysis diminished over time in parallel with the acquisition of surface TCR. The cloned cells were not suppressive for mature lymphocyte proliferation. After stimulation, the cells secreted tumor necrosis factor alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF) detected by immunoassays, and T-cell growth factors, most likely IL-2, as detected by bioassays. Polymerase chain-reaction methods demonstrated the presence of mRNA for IL-2, IL-3, IL-4, IL-9, interferon-gamma, and GM-CSF in these cells after stimulation with PHA and B-LCL. These results suggest that cells with the phenotype and some functional characteristics of mature T lymphocytes can evolve extrathymically in vitro from T-cell precursors sorted from normal human bone marrow.

The effects of the antifungal azoles itraconazole, fluconazole, ketoconazole and miconazole on cytokine gene expression in human lymphoid cells.

The antifungal azole drugs itraconazole (itra; R51,211), fluconazole (flu; UK-49,858), ketoconazole (keto) and micronazole (mico) have been investigated for their suppressive influence on the gene expression of the immunoregulatory cytokines IL2, IL4, IL9, GM-CSF, TNF-alpha, IFN-gamma, as well as both chains of the IL2 receptor in human PBMC and of the cytokines in the human keratinocyte cell line HaCat 17.5. The results obtained in Northern blot analysis were compared with the effects of the established immunosuppressant drug CSA and the new immunosuppressive drug FK 506, as well as the cytokine TGF-beta, which is also immunosuppressive. While 1 microgram/ml CSA and 0.1 microgram/ml FK 506 completely suppressed PHA-stimulated accumulation of mRNA for IL2, IL4, IL9, GM-CSF, TNF-alpha and IFN-gamma in PBMC, flu, keto and TGF-beta failed to inhibit any (except TNF-alpha blocked by TGF-beta). Itra and mico did suppress accumulation of mRNA, but unlike CSA and FK 506, only at high doses (10 micrograms/ml) and after extended incubation (24 h). None of the drugs nor TGF-beta suppressed the expression of the IL2R-alpha and IL2R-beta genes or TNF-alpha-stimulated cytokine gene expression in keratinocytes. Itra and mico, 1 mg/ml (achievable serum level), caused only slight inhibition of the cytokines in PBMC after 6 and 24 h of incubation. These results demonstrate that the mode of action of the azoles is different from CSA and FK 506.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of liposomal interleukin-2 in vitro.

Preclinical in vitro assessment of highly purified natural human interleukin-2 (IL-2) packed in egg lecithin liposomes was performed in short- and long-term T-cell cloning and propagation systems, and in experiments testing induction of lymphokine-activated killer (LAK) cells. Liposomal IL-2 (lip-IL-2) was essentially as active as free natural or recombinant IL-2 for cloning and culture of both helper and cytotoxic alloreactive T cells. However, lip-IL-2 was found to be markedly inferior to free natural or recombinant IL-2 for the induction of LAK cells from normal donors. Nevertheless, lip-IL-2 was able to maintain LAK cytotoxicity of populations preactivated with free IL-2. These results suggest that lip-IL-2 can interact with activated T cells and LAK cells in the same way as free IL-2, but that it is much less efficient at activating LAK-cell precursors.