CD8+ T-cell clones in old mice

Summary: Most old mice and human beings contain large clones of CD8⁺αβ TCR⁺ T cells. In mice clones bearing Vβ7 appear more frequently in animals infected with mouse hepatitis virus than in uninfected animals. This property is controlled by some non MHC gene in the animals. The frequency of old mice containing such clones is affected by the origin of the animals. Although the clones are relatively anergic to acute stimuli in vitro, they can divide in vivo since in old animals they divide and turnover with about the same kinetics as other, non-clonally expanded CD8⁺T cells. Moreover the clones expand slowly but inexorably after transfer into recipient animals. These data suggest that the CDS⁺αβ TCR clones arise because they are specific for some exogenous or auto antigen to which the cells are continuously exposed in vivo.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Effector CD4+ and CD8+ T-cell mechanisms in the control of respiratory virus infections

Summary: Human NK cells express several specialized inhibitory receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA CLASS I alleles leads to NK-mediated target cell lysis. Receptors specific for groups of HLA-C (p58), HLA-B (p70) and HLA-A (p140) alleles belong to the Ig superfamily with two or three Ig-like domains in their extracellular portion, and a long cytoplasmic tail containing ITIM motifs and associated with a non-polar transmembrane portion. In contrast, the CD94/NKG2-A receptor complex is composed of type II proteins with a C-type lectin domain which displays a more broad specificity for different class I alleles. Recently, activatory forms of the HLA-C-specific receptors have been identified in some donors. They are virtually identical to the inhibitory forms in their extracellular portions, but display a short cytoplasmic tail lacking ITIM motifs associated with a Lys-containing transmembrane portion (p50). A subset of activated T-lymphocytes. primarily CD8+ and oligoclonal or monoclonal in nature, express NK-type class I-specific receptors. These receptors exert an inhibitory activity on T-cell receptor-mediated functions and may provide an important mechanism of down-regulation of T-cell responses.     

Induction of CD8+ CTL Recognizing Mycobacterial Peptides

Mycobacterium tuberculosis is the single, most important cause of morbidity attributable to a single infectious organism. CD8⁺ T cells play an important role in anti-tuberculous immune responses in both mice and humans. Data concerning the identity of mycobacterial antigens recognized by CD8⁺ T cells is limited; consequently, few CTL epitopes have been characterized. The authors identified allele-specific (H-2^{b and d}) MHC class I binding motifs in six prominent M. tuberculosis protein antigens (the 19 and 38 kDa lipoglycoproteins and the 10, 16, 65 and 70 kDa stress proteins). These predicted epitopes were tested for MHC binding as well as their ability to elicit peptide-specific CTL following in vivo priming. The authors were able to identify eight previously undescribed mycobacterial CTL epitopes by using spleen cells from peptide-immunized mice. In addition, CTL specific for at least one of these epitopes also recognized the naturally processed epitope presented on transfected EL4 target cells. These mycobacteria-derived CTL epitopes could be important for future analysis of the involvement of CD8⁺ T cells in M. tuberculosis infection, pathogenesis and vaccine development.     

CD4+ and CD8+ Distribution Profile in Individuals Infected by Schistosoma mansoni

Rationale: Patients with chronic Schistosoma mansoni infection show lower anti‐soluble egg antigen (SEA) proliferation responses and higher responses to soluble worm antigen preparation (SWAP). Objective: To compare the activation status and proliferation response of peripheral blood mononuclear cells (PBMC) of infected (XTO) and egg‐negative individuals (NI) living in the same endemic area. Methods: XTO (n = 51) and NI individuals from the same geographical area (n = 37) and healthy blood donors (n = 22) were evaluated before and after stimulation with SEA and SWAP. The expression of activation markers (CD4⁺ HLADR⁺, CD8^high+HLA‐DR⁺ and CD8⁺ CD28⁺) and proliferation assay was assessed by flow cytometry. Findings: PBMC from infected patients showed lower frequency of CD4⁺ but no change in CD8⁺ T cells when compared with the healthy donor group. The ratio CD4⁺/CD8⁺ was 1.3, 0.6 and 0.5 in healthy donors, infected and non‐infected individuals, respectively. The HLA‐DR⁺ expression on CD8⁺ was higher in PBMC from infected and non‐infected individuals than from healthy donors, but similar in both total lymphocytes and CD4⁺ populations. No intergroup proliferation response differences were observed in CD4⁺ and CD8⁺ PBMC unstimulated and stimulated with SEA and SWAP. The SEA but not SWAP‐stimulated cells showed a decrease in the expression of phosphorylated extracellular signal‐regulated kinase (ERK1/2). Conclusions: XTO and NI individuals living in the same area presented a smaller per cent of CD4⁺ and a higher per cent of CD8⁺ cells. The activation by either CD8^high+HLA‐DR⁺ or CD8^high+HLA‐DR⁺/CD8⁺ was enhanced and decreased in XTO and NI by CD8⁺ CD28⁺ and CD8⁺ CD28⁺/CD8⁺ when compared with healthy donor. ERK phosphorylation was attenuated in XTO and NI individuals when stimulated with SEA but not SWAP.     

Expression of CD28 on CD8+ and CD4+ Lymphocytes During HIV Infection

CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, costimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4⁺ and CD8⁺ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4⁺ and CD8⁺ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8⁺CD28⁻ cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.     

Programming, demarcating, and manipulating CD8+ T-cell memory

Summary: In response to infection, antigen‐specific CD8⁺ T cells undergo massive expansion in numbers, acquire effector mechanisms, and disseminate throughout the body. The expansion phase is followed by a contraction (death) phase, where 90–95% of antigen‐specific CD8⁺ T cells are eliminated. The remaining antigen‐specific CD8⁺ T cells form the initial memory pool, which can be stably maintained for life. In this review, we discuss evidence that early events after infection ‘program’ CD8⁺ T cells to expand, contract, and generate memory in a fashion that is largely insensitive to the duration of infection or antigen display. Recent data demonstrate, despite numerical stability, that memory CD8⁺ T‐cell populations undergo phenotypic and functional changes with time after immunization. However, the early suggestion that specific markers can be used to identify memory CD8⁺ T cells has not been supported by recent studies. Thus, we argue that specific functional characteristics, such as the ability to persist and undergo vigorous secondary expansion leading to elevated memory cell numbers, remain the best markers of ‘good’ memory cells. Finally, we discuss experimental approaches to manipulate and accelerate generation of CD8⁺ T cells with memory characteristics, and how these systems can inform both basic and applied immunology.     

Selective Killing of CD4+ and CD8+ Cells with Immunotoxins Containing Saporin

Saporin, a ribosome-inactivating protein from the seeds of Saponaria officinalis, was covalently linked to an anti-CD4 monoclonal antibody. The resulting immunotoxin at 10(-9)M concentration was toxic to CD4+ lymphocytes without affecting other cells. Selective elimination of CD4+ and CD8+ cells was also obtained with murine monoclonal anti-CD4 and anti-CD8 antibodies and an immunotoxin consisting of saporin linked to an anti-mouse IgG antibody.     

Induction of Interleukin-2 Production in CD4+ and CD8+ T-Cell Subsets after Activation via CD3 and CD2

The activation signals necessary for interleukin-2 (IL-2) receptor induction, IL-2 production, and DNA synthesis in resting T cells were investigated. IL-2 receptors were induced after activation via CD2 or CD3 alone, while IL-2 production in both CD4+ and CD8+ T cells required activation via both CD3 and CD2. The sequence of activation signals via CD3 and CD2 was shown to be important since DNA synthesis was induced when the primary activation signal was delivered via CD3, and the CD2 signal within 8 h. In contrast, no DNA synthesis was demonstrated when the primary activation signal was delivered via CD2 and the CD3 signal later. Ciclosporin A (CyA) inhibited T-cell DNA synthesis after activation via CD2 and CD3. The inhibition seemed to be due to the prevention of IL-2 synthesis.     

CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice

During T-cell development the transition in the thymus of CD4-CD8- double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T-cell receptor (TCR)-beta-chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B-17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.     

Activation of Resting, Pure CD4+, and CD8+ Cells via CD3

We studied the requirements for secondary activation signals in pure CD4+ and CD8+ T cells after stimulation with anti-CD3 antibodies. Stimulation of CD4+ or CD8+ cells with anti-CD3 monoclonal antibodies (MoAb) bound to polystyrene monosized particles never resulted in a proliferative response. However, DNA synthesis was observed when recombinant interleukin 2 (IL-2) or other secondary signals, such as those provided by phorbol myristate acetate (PMA) or autologous accessory cells (AC), were also added. These secondary signals were not in themselves capable of inducing DNA synthesis in the absence of particle-bound anti-CD3. We also found that the signals provided by AC may be dependent on the activation state of these cells. Thus, the effects of accessory cells were enhanced by a factor present in fetal calf serum (FCS), most likely endotoxin or lipopolysaccharide (LPS), which alone, however, were not able to activate T cells, even in the presence of particle-bound anti-CD3. Recombinant IL-1 over a broad dose range was unable to replace PMA or activated AC after stimulation with particle-bound anti-CD3. Purified CD4+ and CD8+ T cells behaved identically in all the experiments, indicating that the basic mechanisms for activation in the two T-cell subsets are identical.     

Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes

Background CD4⁺ and CD8⁺ T-lymphocytes are suggested to differentially affect airway inflammation in asthma. Agents which increase intracellular cAMP levels, such as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affect cytokine expression. Differences in the PDE isoenzyme profile between CD4⁺ and CD8⁺ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CD4⁺ and CD8⁺ T-lymphocytes. Methods CD4⁺ and CD8⁺ T-lymphocytes were purified (>98%) from peripheral blood mononuclear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4⁺ and CD8+ T-lymphocyte homogenates, PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0.5μM cyclic nucleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I, II and V activities were detected. About 20% of total eAMP hydrolysing capacity at 0.5 μM cAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE I-IV. The PDE isoenzyme pattern was not different between CD4⁺ and CDS⁺ T-lymphocytes. Moreover, representative inhibitors of PDE HI and IV activity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4^h and CD8⁺ T-lymphocytes. Conclusion Normal CD4⁺ and CD8⁺ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.     

CD4+CD25+ regulatory cells in acquired MHC tolerance

Summary: Tolerance to self-antigens is an ongoing process that begins centrally during T-cell maturation in the thymus and continues throughout the cell's life in the periphery by a network of regulated restraints. Remaining self-reactive T-cells that escape intrathymic deletion may be silenced within the peripheral immune system by specialized regulatory CD4⁺ cells. By analogy, regulatory CD4⁺ cells that control immunity to "acquired self" should arise in circumstances where the immune system acquires tolerance to foreign MHC, such as the tolerance that develops following the exposure to foreign MHC antigens during the neonatal period. We have used this classic model of neonatal tolerance to examine the role of regulatory CD4⁺ cells in acquired tolerance to disparate class I and class II MHC. Adoptive transfer of unfractionated but not CD4⁺-depleted spleen cells from neonatal tolerant mice into SCID recipients inhibited skin graft rejection by immunocompetent CD8⁺ T cells. Using 5-bromo-2'-deoxyuridine incorporation, standard cytotoxic T-lymphocyte assays, short-term interferon-γ ELISPOT, and intracellular FACS analysis to study CD8⁺ T-cell effector function, we demonstrated that neonatal tolerant mice contain CD4⁺CD25⁺ cells that suppress the development of anti-donor CD8⁺ T-cell responses in vitro . We conclude that regulatory CD4⁺CD25⁺ cells initiate and/or maintain tolerance by preventing the development of CD8⁺ T-cell alloreactivity.     

Linomide Enhances Apoptosis in CD4+CD8+ Thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4⁺CD8⁺ thymocytes, were reduced in number by 75%, mature CD4⁺CD8^? or CD4^?CD8⁺ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4⁺CD8^? and CD4^?CD8⁺) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4⁺CD8⁺ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4⁺CD8⁺. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4⁺CD8⁺ subset.     

The role of CD8+ T-cell replicative senescence in human aging

Summary: The strict limit in proliferative potential of normal human somatic cells – a process known as replicative senescence – is highly relevant to the immune system, because clonal expansion is fundamental to adaptive immunity. CD8⁺ T cells that undergo extensive rounds of antigen‐driven proliferation in cell culture invariably reach the end stage of replicative senescence, characterized by irreversible cell‐cycle arrest and a critically short telomere length. Cultures of senescent CD8⁺ T cells also show resistance to apoptosis, permanent loss of CD28 expression, altered cytokine profiles, reduced ability to respond to stress, and various functional changes. Cells with similar characteristics accumulate during normal aging as well as in younger persons infected with human immunodeficiency virus, suggesting that the process of replicative senescence is not an artifact of cell culture but is also occurring in vivo. Interestingly, in elderly persons, the presence of high proportions of CD8⁺ T cells with characteristics of replicative senescence is correlated with reduced antibody responses to vaccines as well as with osteoporotic fractures. CD8⁺CD28^– T cells also accumulate in patients with certain types of cancer. The emerging picture is that senescent CD8⁺ T cells may modulate both immune and non‐immune functions, contributing not only to reduced anti‐viral immunity but also to diverse age‐related pathologies.     

Requirements for Phytohaemagglutinin Activation of Resting Pure CD4+ and CD8+ T Cells

We have utilized a new method for obtaining highly purified cells using positive selection by immunomagnetic separation to study the conditions required for phytohaemagglutinin (PHA) activation of pure T4 and T8 cells. In the presence of accessory cells (AC), a comparable proliferative response was obtained in the two subsets. In the absence of AC, PHA induced low levels of interleukin 2 (IL-2) receptor expression as well as responsiveness to IL-2 in both T4 and T8 cells. If AC or 12- O -tetradecanoyl-phorbol-13-acctate (TPA) were also present, IL-2 production and DNA synthesis were seen in both subsets. A short preincubation with PHA primed' T fells for subsequent responsiveness to IL-2 or TPA, while preincubation with TPA did not induce response to PHA. Thus, PHA alone is sufficient for the first step of T cell activation lending to IL-2 receptor expression. The second step leading to IL-2 production, is dependent on direct interaction with AC in the presence of PHA. While T8 cells are dependent on help by T4 cells for proliferation to occur during stimulation with antigen, in PHA stimulation the requirements for activation and proliferation seem to he identical for T4 and T8 cells.     

Restricted and Individual Usage of T-cell Receptor β-Gene Variables in Nickel-Induced CD4+ and CD8+ Cells

Nickel allergy is manifested as contact allergic eczema elicited by delayed-type hypersensitivity, the reaction being mediated by T lymphocytes. We examined the T-cell receptor (TCR) β-chain variable gene segment (Vβ) use of nickel-induced CD4⁺ and CD8⁺ T cells in the peripheral blood of nickel-sensitive and non-sensitized subjects. The results show that each patient had an individual Vβ repertoire overexpressed, these being in CD4⁺ cells Vβ10 and Vβ13 (in subject A); Vβ1, Vβ2, Vβ13 and Vβ21 (subject B); Vβ1 and Vβ10 (subject C); Vβ9 and Vβ19 (subject D). Thus, no single Vβ gene dominated in a majority of the CD4⁺ samples. The Vβ genes overexpressed in patient CD8⁺ nickel-induced T cells were Vβ1 (in subject A), Vβ1 (subject B), Vβ1 and Vβ2 (subject C) and Vβ7 (subject D), domination of Vβ1 being seen in most of the CD8⁺ samples (75%). No specific overexpression of any Vβ genes in the nickel-allergic subjects was found in comparison with the non-sensitized subjects. In conclusion, an individual pattern of restricted Vβ genes was induced with nickel in CD4⁺ and CD8⁺ T cells in each nickel allergy patient.     

JNK and p38 MAP kinases in CD4+ and CD8+ T cells

Summary: The c‐Jun aminoterminal kinase (JNK) and p38 mitogen‐activated protein (MAP) kinase signaling pathways have been associated with cell death, differentiation and proliferation. CD4⁺ and CD8⁺ T cells have different effector functions after antigen stimulation and control specific aspects of the immune response. The studies carried out in our group indicate that the role of JNK and p38 MAP kinases in CD4⁺ T cells is different from their role in CD8⁺ T cells. Moreover, these two pathways are not redundant in either T cell population. We have also shown that p38 MAP kinase regulates early stages of T cell development in the thymus. It is therefore important to consider the specific function of these kinases in each T cell population when pharmacological inhibitors of JNK and p38 MAP kinases are used for therapeutic purposes to control the immune response.