Chicken CD4, CD8alphabeta, and CD8alphaalpha T cell co-receptor molecules

New knowledge has recently been obtained about the evolutionary conservation of CD4, CD8alphaalpha, and CD8alphabeta T cell receptor (TCR) co-receptor molecules between chicken and mammals. This conservation extends from biochemical structure and tissue distribution to function. Panels of monoclonal antibodies and polyclonal antisera against different epitopes of chicken CD8 and CD4 molecules have proven their value in several recent studies. Chicken CD8 allotypes and homozygous strains carrying these allotypes have been established and these strains provide excellent models for further studies. The extensive polymorphism of CD8alpha in chickens has not been observed in any other species, suggesting that CD8alpha and CD8beta have evolved under different selective pressure in the chicken. A large peripheral blood CD4+CD8+ T cell population in chicken resembles that observed in some human individuals but the inheritance of peripheral blood CD4CD8alphaalpha T cells in the chicken is a unique observation, which suggests the presence of a single gene responsible for CD8alpha, but not CD8beta, specific expression. Despite these unique findings in chicken, the data on CD4, CD8alphaalpha, and CD8alphabeta molecules show that they have evolved before the divergence of mammalian and avian branches from their reptilian ancestors.     

Soluble CD4, CD8, and HLA molecules in commercial immunoglobulin preparations

CD4 and CD8 exist in membrane-bound and soluble forms. These antigens are the physiological ligands for molecules of HLA classes I and II, which we have found in soluble form in commercial immunoglobulin preparations. We measured concentrations of soluble CD4 and CD8, as well as soluble HLA-ABC and HLA-RQP in sixteen immunoglobulin preparations by enzyme immunosorbent assays. Only two preparations contained detectable CD8, and nine had soluble CD4 in varying amounts. There was great variation in concentrations of soluble HLA molecules. It is possible that the immunomodulating effects of immunoglobulin therapy in disorders with immune aetiology are due, at least partly, to these contaminating molecules.     

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.     

Cytokines and soluble CD4 and CD8 molecules in rheumatoid arthritis: relationship to systematic vasculitis and microvascular capillaroscopic abnormalities

The head and neck cancer patient often presents with both protein malnutrition and trace element deficiencies. Zinc has been found to be deficient in many head and neck cancer patients. In this study, pretreatment zinc status and nutritional status (measured by the Prognostic Nutritional Index [PNI]) were correlated with clinical outcomes in 47 patients. The patients were followed-up for a median of 52 mo from the time of enrollment. Our results showed that the tumor size and overall stage correlated significantly to zinc status whereas no such correlation was seen with PNI, alcohol intake, or smoking in our subjects. The results also showed that impaired zinc status was associated with an increased number of treatment morbidities, unplanned hospitalizations, and treatment delays (P < 0.05). Nutritional status was not associated with any studied outcome variable. The disease-free interval was highest for the group which had both zinc-sufficient and nutrition-sufficient status. Although our data do not prove conclusively, they do suggest that impaired zinc status at presentation may contribute to treatment morbidity, and that for an optimal mean disease-free interval, a sufficient zinc and nutritional status is required.     

Tissue distribution, regulation and intracellular localization of murine CD1 molecules

CD1 molecules are MHC-unlinked class Ib molecules consisting of classical (human CD 1a-c) and non-classical subsets (human CD1d and murine CD1). The characterization of non-classical subsets of CD1 is limited due to the lack of reagents. In this study, we have generated two new anti-mouse CD1 monoclonal antibodies, 3H3 and 5C6, by immunization of hamsters with purified CD1 protein. These antibodies recognize CD1-transfected cells and have no reactivity to cells isolated from CD1-/- mice. Both antibodies precipitate the 52 kDa heavy chain and 12 kDa beta2m from thymocytes and splenocytes by radio-immunoprecipitation. Deglycosylation of CD1 reduces molecular mass of the heavy chain by 7.5 kDa, which can be detected by 3H3 but not 5C6. 3H3 and 5C6 detect surface CD1 expression on cells from the thymus, spleen, lymph node and bone marrow, but not on intestinal epithelial cells. Developmentally, CD1 is expressed on thymocytes prior to TCR rearrangement and remains constant throughout thymic development. CD1 is expressed early in the fetal liver (day 14) and remains expressed in hepatocytes postnatally. These data support evidence of a role for CD1 in the selection and/or expansion of NK1- T cells of both thymic origin and extrathymic origin. Unlike classical class I molecules, murine CD1 levels are not affected by IFN-gamma, but like human CD1b can be up-regulated by IL-4 and GM-CSF although only moderately. Similar to human CD1b, murine CD1 is found by immunofluorescence microscopy on the cell surface, and in various intracellular vesicles, including early and late endosomes. Localization in endocytic compartments indicates that murine CD1 may be capable of binding endocytosed antigens.     

The expression of CD4 and CD8 molecules conditions the behavior of V beta + murine thymocytes upon superantigenic challenge

We investigated the capacity of the Staphylococcal enterotoxin (SE) B, a superantigen (SAg) specific for TCR V beta domain, to modulate V beta 8+ thymocytes selection in adult mice. Thymocytes were collected at various time intervals after SEB injection (10 and 100 micrograms) and V beta 8+ modulation was analysed by three color flow cytometry. SEB failed to affect V beta 8+ thymocytes comprised in the less mature compartments, namely, CD4+8+ and CD4-CD8-, whereas it selectively affected V beta 8+CD4+8+ (downward modulation) and V beta 8+CD4-8+ thymocytes (upward modulation). The different response to SEB challenge between CD4+8- and CD4-8+ thymocytes appeared dependent on the CD4/MHC class II interaction, as V beta 8+CD4-8+ thymocytes carrying a transgenic CD4 molecule capable of interacting with MHC class II showed the same response of V beta 8+CD4+8- thymocytes. At variance with thymocytes, however, V beta 8+CD4+8- and V beta 8+CD4-8+ splenic T lymphocytes responded to SAg challenge in identical manner (upward modulation) highlighting the importance of maturation status and/or microenvironment in SAg response. V beta 8+ thymocytes remaining in the thymus were assessed for their capacity to respond to a SAg challenge. Thus, thymocytes were obtained at various time intervals after SEB injection and cultured in the presence of SEB or SEA, a Sag specific for V beta 10 as control. A reduced mitotic response to SEB but not to SEA was noticed irrespective of the number of V beta 8+ responding cells present in culture. It is concluded that SAgs affect TCR specific thymocytes by conditioning their redistribution and inducing an anergic status.     

The proliferative response of HIV+ T-cells (CD4+ and CD8+) are severely suppressed via CD28 coactivation

Several studies have suggested that regulation of expression of the costimulatory molecule CD28 on the T-cell surface may play an important role in AIDS pathogenesis. In a study of T-cells from HIV+ donors, we find that activation with anti-CD3 plus anti-CD28 results in a mitogenic response which was approximately 86% suppressed for both CD4+ and CD8+ T-cells when compared to normal control cells. With PMA costimulation (instead of anti-CD28), the anti-CD3 response was suppressed much less, by 64 and 61%, respectively. With Con A as opposed to CD3 stimulation, the degree of suppression was less with either coactivator but still more severe with CD28 than with PMA coactivation. It has been reported that the CD28 subset of CD8+ T-cells is diminished in HIV+ individuals and could account for these results. It is possible as well that the CD28 costimulatory pathway in the CD4+ T-cells particularly is altered due to intervention by the HIV. While our data do not differentiate between these two possibilities, it show that the immune status is compromised in the HIV+ individual not only in terms of number of CD4+ T-cells, but in their activation response as well.     

Co-capping studies reveal CD8/TCR interactions after capping CD8 beta polypeptides and intracellular associations of CD8 with p56(lck)

A reaction-diffusion model was developed to predict the fate of nitric oxide (NO) released by cells of the immune system. The model was used to analyze data obtained previously using macrophages attached to microcarrier beads suspended in a stirred vessel. Activated macrophages synthesize NO, which is oxidized in the culture medium by molecular oxygen and superoxide (O2-, also released by the cells), yielding mainly nitrite (NO2-) and nitrate (NO3-) as the respective end products. In the analysis the reactor was divided into a "stagnant film" with position-dependent concentrations adjacent to a representative carrier bead and a well-mixed bulk solution. It was found that the concentration of NO was relatively uniform in the film. In contrast, essentially all of the O2- was calculated to be consumed within approximately 2 microm of the cell surfaces, due to its reaction with NO to yield peroxynitrite. The decomposition of peroxynitrite caused its concentration to fall to nearly zero over a distance of approximately 30 microm from the cells. Although the film regions (which had an effective thickness of 63 microm) comprised just 2% of the reactor volume and were predicted to account for only 6% of the NO2- formation under control conditions, they were calculated to be responsible for 99% of the NO3- formation. Superoxide dismutase in the medium (at 3.2 microM) was predicted to lower the ratio of NO3- to NO2- formation rates from near unity to <0.5, in reasonable agreement with the data. The NO3-/NO2- ratio was predicted to vary exponentially with the ratio of O2- to NO release rates from the cells. Recently reported reactions involving CO2 and bicarbonate were found to have important effects on the concentrations of peroxynitrite and nitrous anhydride, two of the compounds that have been implicated in NO cytotoxicity and mutagenesis.     

IL-10 impacts autoimmune diabetes via a CD8+ T cell pathway circumventing the requirement for CD4+ T and B lymphocytes

IL-10 is essential for an early phase of diabetes in nonobese diabetic (NOD) mice, but later becomes protective against its development. The mechanism by which IL-10 mediates the pathway to diabetes in these mice is unknown. Herein, we dissected the cellular and costimulation requirements for diabetes in transgenic (tg) NOD mice that expressed IL-10 in their pancreatic islets (IL-10-NOD mice). We found that IL-10 alone did not cause diabetes because the offspring (IL-10-NOD-scid mice) from back-crosses of IL-10-NOD mice with NOD-scid mice had no diabetes. Moreover, these IL-10-NOD-scid mice were free of lymphocytic infiltration. Treatment of IL-10-NOD mice with depleting anti-CD4 mAb or control mAb had no effect on diabetes. Surprisingly, depletion of CD8+ T cells by treatment with the corresponding mAb inhibited diabetes without attenuating insulitis, demonstrating a critical role for CD8+ T cells in the disease process. Interestingly, B cell-deficient IL-10-NOD mice readily developed diabetes with kinetics and incidence similar to those observed in wild-type mice, demonstrating that B lymphocytes as APCs were not required in the disease process. Administration of anti-CD40 ligand (CD40L) mAb did not prevent disease, indicating that CD40/CD40L costimulation is not required for diabetes in IL-10-NOD mice. Immunization of IL-10-NOD mice with CFA or heat-shock protein 65, known to block diabetes in NOD mice, had no effect on their diabetes. We demonstrate that IL-10 contributes early to the pathology of diabetes via a CD8+ T cell pathway, eliminating the requirement for B lymphocytes and CD40-CD40L costimulation. Our findings provide a mechanism for the participation of IL-10 in the early development of diabetes.     

CD8+ T cells in intracellular bacterial infections of mice

In the normal course of an immune response, both CD4+ and CD8+ T cells respond to each of the bacterial pathogens we have discussed and both responses may be required for the most potent immunity to infection. In this discussion, we have focused on the ability of these organisms to prime CD8+ T-cell responses in vivo and the ability of CD8+ T cells as sole mediators of acquired immunity, to protect against infection. It is clear that the vacuolar location of bacterial pathogens such as Salmonella or Mycobacteria does not prevent in vivo priming of CD8+ T-cell responses to these pathogens. However, vacuolar localization may affect the potency of CD8+ T-cell responses under experimental conditions that assess the capacity of CD8+ T cells as the sole mediators of acquired immunity. In the case of cytoplasmic L. monocytogenes, clear evidence exists that antigen-specific CD8+ T cells, in the absence of immune CD4+ T cells, can provide substantial acquired immunity to naive mice. Similar clear experimental results with Salmonella and Mycobacteria are lacking. Such results would provide stronger support for vaccines that elicit CD8+ T-cell responses to these vacuolar pathogens. Although our discussion has focused on only three specific organisms, we suggest that detection of an in vivo CD8+ T-cell response to a bacterial antigen does not ensure that the response will be protective against infection in a vaccine setting. In the case of Salmonella and Mycobacteria, this issue remains unresolved.     

Human immunodeficiency virus type 2 envelope glycoprotein binds to CD8 as well as to CD4 molecules on human T cells

We report here that human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (gp105), but not HIV-1 gp120, can bind to CD8 molecules as well as to CD4 molecules on human T cells. This phenomenon may lead to differences in the life cycles of HIV-1 and HIV-2, and it may be related to the differences in disease manifestations of HIV-1 and HIV-2 infection, including longer survival of HIV-2-infected patients.     

CD3/TCR complex-associated lymphocyte activation gene-3 molecules inhibit CD3/TCR signaling

The lymphocyte activation gene-3 (LAG-3) molecule is a T cell activation Ag closely related to CD4 at the gene and protein levels. We investigated whether LAG-3 itself may down-regulate the immune response by interfering with TCR signaling. The binding of Ab to the LAG-3 molecule followed by cross-linking (XL) inhibits cell proliferation and cytokine secretion in response to CD3XL on activated T cells. LAG-3XL-induced down-regulation is associated with functional unresponsiveness, as well as with high CD25 expression levels and reversion by exogenous IL-2. It is also associated with a down-modulation of CD3/TCR complex expression. At the biochemical level, LAG-3XL inhibits calcium response to CD3 stimulation. This inhibition is observed with different LAG-3- and CD3-specific mAbs on condition that the two receptors are cross-linked together. Finally, the capping of CD3 was shown to induce cocapping of LAG-3 molecules. Together, these results show that CD3/TCR complex-associated LAG-3 molecules can play an active role in negatively regulating the CD3/TCR activation pathway. They ultimately suggest that LAG-3 is an inhibitory receptor in activated T lymphocytes.     

Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.     

Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

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.