Murine CD8 lymphocyte expansion in vitro by artificial antigen-presenting cells expressing CD137L (4-1BBL) is superior to CD28, and CD137L expressed on neuroblastoma expands CD8 tumour-reactive effector cells in vivo

The ability to expand tumour-infiltrating lymphocytes in vitro has been greatly enhanced by the use of antigen-independent mechanisms of immune cell costimulation. We have produced human, using the K562 cell line, and murine, using YAC-1 cells, artificial antigen presenting cells (aAPC) and demonstrate that these cell types stimulate murine lymphocyte populations in distinct ways. Using aAPC that have been transfected with CD137L (4-1BBL) and CD32 (FcRgammaII), as a means to bind anti-CD3 and anti-CD28 antibody, we found that CD4 cells preferentially expanded in vitro with K562 aAPC, while CD8 cells expanded with both K562 and YAC-1 aAPC. Co-stimulation mediated by CD137L on aAPC was superior to that mediated by anti-CD28 antibody. This was seen in both long and short-term expansion assays, and by the rapid induction of a CD8+ DX5+ population. DX5 serves, under these in vitro conditions, as a general marker for lymphocyte activation. In vivo, the superiority of CD137L was demonstrated by the induction of T helper 1 effectors seen in freshly isolated splenocytes from mice immunized with CD137L-expressing neuroblastoma tumour vaccines. The ability to stimulate a strong CD8 CTL response in vivo correlated with the induction of a DX5+ cell population in splenocytes with a memory-effector phenotype. The presence of this unique DX5+ cell population, phenotypically distinct with regards to CD69 and CD62L expression from DX5+ cells induced by aAPC in vitro, may be associated with the ability of CD137L to induce strong anti-tumour immunity.     

Dietary n-3 polyunsaturated fatty acids modulate purified murine T-cell subset activation

Studies in humans and murine disease models have clearly shown dietary fish oil to possess anti-inflammatory properties, apparently mediated by the n-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). To determine the mechanisms by which dietary EPA and DHA modulate mouse T-cell activation, female C57BL/6 mice were fed diets containing either 2% safflower oil (SAF), 2% fish oil (FO), or a 2% purified EPA/DHA ethyl ester mixture for 14 days. Splenic CD4 T cells ( approximately 90% purity) or CD8 T cells ( approximately 85% purity) were incubated with agonists which act at the plasma membrane receptor level [anti(alpha)-CD3/anti(alpha)-CD28], the intracellular level (PMA/Ionomycin), or at both the receptor and intracellular levels (alphaCD3/PMA). CD4 T cells stimulated with alphaCD3/alphaCD28 or PMA/Ionomycin proliferated and produced principally IL-2 (i.e. a Th1 phenotype), whereas the proliferation of CD4 T cells stimulated with alphaCD3/PMA was apparently driven principally by IL-4 (i.e. a Th2 phenotype). The IL-4 driven proliferation of putative Th2 CD4 cells was enhanced by dietary n-3 fatty acids (P = 0.02). Conversely, IL-2 production by alphaCD3/alpha CD28-stimulated CD4 T cells was reduced in FO-fed animals (P < 0.0001). The alphaCD3/alphaCD28-stimulated CD8 cells cultured from FO-fed animals exhibited a significant decrease (P < 0.05) in proliferation. There were no dietary effects seen in alphaCD3/PMA-stimulated CD8 cells, which produced both IL-2 and IL-4, or in PMA/Ionomycin-stimulated CD8 cells, which produced principally IL-2. These data suggest that dietary n-3 fatty acids down-regulated IL-2 driven CD4 and CD8 activation, while up-regulating the activation of the Th2 CD4 T-cell subset. Thus, the anti-inflammatory effects of n-3 fatty acids may result in both the direct suppression of IL-2-induced Th1 cell activation and the indirect suppression of Th1 cells by the enhanced cross-regulatory function of Th2 cells.     

Mesenchymal stem cells control alloreactive CD8+CD28− T cells

CD28/B7 co-stimulation blockade with belatacept prevents alloreactivity in kidney transplant patients. However, cells lacking CD28 are not susceptible to belatacept treatment. As CD8⁺CD28⁻ T-cells have cytotoxic and pathogenic properties, we investigated whether mesenchymal stem cells (MSC) are effective in controlling these cells. In mixed lymphocyte reactions (MLR), MSC and belatacept inhibited peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent manner. MSC at MSC/effector cell ratios of 1:160 and 1:2·5 reduced proliferation by 38·8 and 92·2%, respectively. Belatacept concentrations of 0·1 μg/ml and 10 μg/ml suppressed proliferation by 20·7 and 80·6%, respectively. Both treatments in combination did not inhibit each other''s function. Allostimulated CD8⁺CD28⁻ T cells were able to proliferate and expressed the cytolytic and cytotoxic effector molecules granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. While belatacept did not affect the proliferation of CD8⁺CD28⁻ T cells, MSC reduced the percentage of CD28⁻ T cells in the proliferating CD8⁺ T cell fraction by 45·9% (P = 0·009). CD8⁺CD28⁻ T cells as effector cells in MLR in the presence of CD4⁺ T cell help gained CD28 expression, an effect independent of MSC. In contrast, allostimulated CD28⁺ T cells did not lose CD28 expression in MLR–MSC co-culture, suggesting that MSC control pre-existing CD28⁻ T cells and not newly induced CD28⁻ T cells. In conclusion, alloreactive CD8⁺CD28⁻ T cells that remain unaffected by belatacept treatment are inhibited by MSC. This study indicates the potential of an MSC–belatacept combination therapy to control alloreactivity.     

Role of increased CD8/CD28(null) T cells and alternative co-stimulatory molecules in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease; it is a leading cause of death and existing treatments have no proven disease-modifying effect. The mechanisms underlying this resistance are largely unknown, but suggest the presence of some self-maintaining pathogenic process, possibly initiated by cigarette smoking, that prevents the normal resolution of inflammation. We have previously reported increased production of proinflammatory cytokines and granzyme b by CD8(+) T cells in COPD; costimulatory receptor/ligand interactions required include CD80:86/CD28, B7-1/CTLA4, 4-1BB/1BBL and OX40/OX40L. We hypothesized that a dysregulated expression/function of these molecules may play a role in inflammatory/autoimmune components of COPD. We analysed T cell co-stimulatory molecules in blood from 34 controls, 15 smokers and 48 COPD subjects. We assessed the potential functional relevance of CD8/CD28(null) cells in COPD by measuring their production of proinflammatory cytokines, co-stimulatory molecules, granzyme and perforin. A smoke-exposed murine model was applied to investigate the relative expression of CD8/CD28(null) T cells in blood, lung tissue and airway. CD8/CD28(null) cells were increased in both current- and ex-smoker COPD groups; these cells expressed significantly more interferon (IFN)-γ, OX40, 4-1BB, CTLA4, granzyme and perforin when stimulated than CD8/CD28(+) T cells. There were no changes in CD4/CD28(null) T cells. In mice exposed to cigarette smoke for 12 weeks, CD8/CD28(null) T cells were significantly increased in the airway with a trend for an increase in lung tissue and blood. Increased production of proinflammatory cytokines and expression of alternative co-stimulatory molecules by CD8/CD28(null) T cells may play a role in inflammatory or autoimmune responses in COPD and identify therapeutic targets.     

CD8beta/CD28 expression defines functionally distinct populations of peripheral blood T lymphocytes

Peripheral blood CD8+ T lymphocytes generally express the CD8 coreceptor as an alphabeta heterodimer. On these cells, the CD8beta chain is present either at high (CD8betahigh) or low density (CD8betalow). CD8betahigh cells are CD28+, whereas CD8betalow cells are CD28+ or CD28-. Therefore, three subpopulations of CD8+ T cells can be described: (i) CD8betahighCD28+ (ii) CD8betalowCD28+, and (iii) CD8betalowCD28- cells. Phenotypic and functional characterization of these CD8+ T cell subsets revealed significant differences. CD8betahighCD28+ cells predominantly express CD45RA. In contrast, CD8betalowCD28+ cells frequently express CD45R0 and the activating NK receptor CD161. CD8betalowCD28- cells frequently revert to the CD45RA phenotype. In addition, these cells express CD16, CD56, CD94, and the killer-inhibitory receptors NKB1 and CD158a. Intracellular IL-2 was frequently detected in CD8betahighCD28+ cells and CD8betalowCD28+ cells, but not CD8betalowCD28- cells. CD8betalowCD28+ cells and CD8betalowCD28- cells frequently stained positive for IFN-gamma. In addition, these cells contain intracellular perforin and granzyme A. Expression of Fas (CD95) as well as susceptibility to apoptosis is markedly increased in CD8betalowCD28+ and CD8betalowCD28- cells as compared to CD8betahighCD28+ cells. In vitro activation of peripheral blood lymphocytes triggered expansion of CD8betahighCD28+ cells as well as a development into CD8betalowCD28+ and CD8betalowCD28- cells. Similarly, activation of CD8betahighCD28+ cord blood cells resulted in the appearance of CD8betalowCD28+ and CD8betalowCD28- cells. These data suggest that CD8betahighCD28+ cells can differentiate into CD8betalowCD28+ and CD8betalowCD28- cells upon TCR stimulation. Therefore, the CD8beta/CD28 subsets in peripheral blood may reflect distinct stages of post-thymic CD8+T cell development.     

Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice

Previously, we and others have demonstrated that CD4-deficient mice have a normal number of T cells and B cells with a significant population of CD4^-8^-TcRαβ⁺ T cells. Surprisingly, however, these mice lacking CD4 show in vivo immunoglobulin isotype class switching from IgM to IgG in response to sheep erythrocytes and vesicular stomatitis virus. In this study we have depleted various subpopulations of T cells in vivo and shown that the population of CD4^-8^-TcRαβ⁺ T cells is responsible for providing “help” in the antibody response of CD4-deficient mice to vesicular stomatitis virus infection. We have used antigen-specific proliferation assays and blocking studies with class I and II major histocompatibility complex (MHC)-specific purified antibodies to show that these cells are class II MHC-restricted in responses against the T cell-dependent antigen keyhole limpet hemocyanin (KLH). Finally, phenotypic analysis of the CD4^- CD8^-thymocytes in CD4-deficient mice shows that these cells have a more mature phenotype than the CD4^-8^- thymocytes in wild type mice. These results indicate that CD4 is not absolutely necessary for positive selection or effector function of class II MHC-restricted helper T cells.     

Presentation of high antigen‐dose by splenic B220lo B cells fosters a feedback loop between T helper type 2 memory and antibody isotype switching

Effective humoral immunity ensues when antigen presentation by B cells culminates in productive cooperation with T lymphocytes. This collaboration, however, remains ill‐defined because naive antigen‐specific B cells are rare and difficult to track in vivo. Herein, we used a defined transfer model to examine how B lymphocytes, as antigen‐presenting cells, shape the development of T‐cell memory suitable for generation of relevant antibody responses. Specifically, we examined how B cells presenting different doses of antigen during the initial priming phase shape the development of CD4 T‐cell memory and its influence on humoral immunity. The findings indicate that B cells presenting low dose of antigen favour the development of T helper type 1 (Th1) type memory, while those presenting a high antigen dose yielded better Th2 memory cells. The memory Th2 cells supported the production of antibodies by effector B cells and promoted isotype switching to IgG1. Moreover, among the B‐cell subsets tested for induction of Th2 memory, the splenic but not peritoneal B220^lo cells were most effective in sustaining Th2 memory development as well as immunoglobulin isotype switching, and this function involved a tight control by programmed death 1–programmed death ligand 2 interactions.     

GITR/GITRL: more than an effector T cell co-stimulatory system

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNFR superfamily, expressed in several cells and tissues including T lymphocytes, NK cells and antigen-presenting cells (APC). GITR activation, upon interaction with its ligand (GITRL), functions as a co-activating signal. GITRL is mainly expressed on APC and GITR/GITRL interaction is important for the development of immune response. This review summarizes recent results about the GITR/GITRL system, focusing on the interplay between APC, effector and regulatory T cells.     

Enhanced apoptosis of T cells in common variable immunodeficiency (CVID): role of defective CD28 co-stimulation

CVID is a primary immune disorder in which hypogammaglobulinaemia may be associated with a number of T cell defects including lymphopenia, anergy, impaired lymphocyte proliferation and deficient cytokine secretion. In this study we show that T cells of CVID subjects, in comparison with control T cells, undergo spontaneous apoptosis in culture and markedly accelerated apoptosis after gamma-irradiation. Although costimulation of the CD28 receptor following engagement of the TCR/CD3 receptor normally provides a second signal necessary for IL-2 secretion, CD28 costimulation in CVID does not significantly increase IL-2 production, nor does this combination of activators enhance the survival of irradiated CVID T cells, as it does for cultured normal T cells. Addition of IL-2 enhances CVID T cell survival, suggesting that the IL-2 signalling pathways are normal. CVID T cells have similar expression of Bcl-2 to control T cells. CD3 stimulation up-regulates T cell expression of bcl-xL mRNA for normal T cells, but anti-CD28 does not augment bcl-xL expression for CVID subjects with accelerated apoptosis. Defects of the CD28 receptor pathway, leading to cytokine deprivation and dysregulation of bcl-xL, could lead to poor T cell viability and some of the cellular defects observed in CVID.     

The life (and death) of CD4+CD28null T cells in inflammatory diseases

Inflammation contributes to the development and perpetuation of several disorders and T lymphocytes orchestrate the inflammatory immune response. Although the role of T cells in inflammation is widely recognized, specific therapies that tackle inflammatory networks in disease are yet to be developed. CD4⁺CD28^null T cells are a unique subset of helper T lymphocytes that recently shot back into the limelight as potential catalysts of inflammation in several inflammatory disorders such as autoimmunity, atherosclerosis and chronic viral infections. In contrast to conventional helper T cells, CD4⁺CD28^null T cells have an inbuilt ability to release inflammatory cytokines and cytotoxic molecules that can damage tissues and amplify inflammatory pathways. It comes as no surprise that patients who have high numbers of these cells have more severe disease and poor prognosis. In this review, I provide an overview on the latest advances in the biology of CD4⁺CD28^null T cells. Understanding the complex functions and dynamics of CD4⁺CD28^null T cells may open new avenues for therapeutic intervention to prevent progression of inflammatory diseases.     

ICOS controls Foxp3+ regulatory T‐cell expansion,maintenance and IL‐10 production during helminth infection

Foxp3⁺ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3⁺ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3⁺ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3⁺ Treg‐cell responses. Infection of ICOS^?/? mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3⁺ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3⁺ Treg‐cell apoptosis, a Foxp3⁺ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios^?Foxp3⁺ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3⁺ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4⁺ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3⁺ Treg‐cell expansion and function during helminth infections.     

IL-2 induces in vivo suppression by CD4(+)CD25(+)Foxp3(+) regulatory T cells

Interleukin-2 (IL-2) treatment is currently used to enhance T cell-mediated immune responses against tumors or in viral infections. At the same time, IL-2 is essential for the peripheral homeostasis of CD4(+)CD25(+)Foxp3(+ )regulatory T cells (Treg). In our study, we show that IL-2 is also an important activator of Treg suppressive activity in vivo. IL-2 treatment induces Treg expansion as well as IL-10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL-2 via gene-gun vaccination using IL-2 encoding DNA plasmids (pIL-2) inhibited naive antigen-specific T cell proliferation as well as a Th1-induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL-2-treated mice and the suppression depends on the synergistic action of IL-10 and TGF-beta. These data highlight that during therapeutic treatment with IL-2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.     

Relative efficiency of porcine and human cytotoxic T-lymphocyte antigen 4 immunoglobulin in inhibiting human CD4+ T-cell responses co-stimulated by porcine and human B7 molecules

α1,3-Galactosyltransferase gene-knockout pigs transgenic for porcine cytotoxic T-lymphocyte antigen 4 immunoglobulin (pCTLA4-Ig) have been produced to reduce T-cell-mediated rejection following xenotransplantation. The level of soluble pCTLA4-Ig in their blood was greatly in excess of the therapeutic level in patients, rendering the pigs immune-incompetent. Soluble pCTLA4-Ig produced by these transgenic pigs was evaluated for binding to porcine and human (h) B7 molecules, and for its inhibitory effect on allogeneic and xenogeneic human T-cell responses. Porcine CTLA4-Ig-expressing peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (AECs) were evaluated for their direct inhibitory effect on hCD4+ T-cell responses. Soluble pCTLA4-Ig and purified hCTLA4-Ig showed similar binding to pB7 molecules, but pCTLA4-Ig showed significantly less binding to hB7 molecules. The pCTLA4-Ig and hCTLA4-Ig inhibited the response of hCD4+ T cells to pAECs equally, but pCTLA4-Ig was less successful in inhibiting the human allogeneic response. The hCD4+ T-cell response to PBMCs from pCTLA4-Ig pigs was significantly lower than that of non-pCTLA4-Ig pigs. Although pCTLA4-Ig was detected in the cytoplasm of pCTLA4-Ig-expressing pAECs, only a minimal level of soluble pCTLA4-Ig was detected in the supernatant during culture, and pCTLA4-Ig-expressing pAECs did not inhibit the xenogeneic direct human T-cell response. High-level tissue-specific production of pCTLA4-Ig may be required for sufficient immunosuppression for organ or cell (e.g., islets) transplantation.     

Human double-negative (CD4-CD8-) T cells bearing alpha beta T cell receptor possess both helper and cytotoxic activities.

Expression of CD4 or CD8 on the cell surface is an important guide for discriminating the immunological functions of T cells. However, a minor T cell subset, which lacks both CD4 and CD8 molecules but bears the usual form of T cell receptor (TCR) alpha beta (CD4-CD8-TCR alpha beta+ T cells), has recently been found not only in mice but also in humans, and its role in immune response is now of considerable interest. In order to clarify the characteristics of this newly defined T cell subpopulation, we established five IL-2-dependent CD4-CD8-TCR alpha beta+ T cell clones from the peripheral blood of a healthy individual, and examined their various biological functions. It was found that all clones not only helped B cells in immunoglobulin production, but also exerted major histocompatibility complex-unrestricted cytotoxicity. Although their CD3/TCR complexes were functionally competent, the cytotoxicity seemed to be mediated via unknown molecules other than the CD3/TCR complex, as evidenced by the failure of CD3 MoAb to inhibit the cytotoxic activity. Our present findings showed that CD4-CD8-TCR alpha beta+ T cells possess potential bifunction, i.e. helper and cytotoxic activities. Their roles in the pathogenesis of immunodeficiency are discussed.     

Estrogen enhances the functions of CD4(+)CD25(+)Foxp3(+) regulatory T cells that suppress osteoclast differentiation and bone resorption in vitro

Cross-talk has been shown to occur between the immune system and bone metabolism pathways. In the present study, we investigated the impact of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells on osteoclastogenesis and bone resorption. Treg cells that were isolated and purified from peripheral blood mononuclear cells (PBMCs) of healthy adults inhibited both the differentiation of osteoclasts (OCs) from human embryo bone marrow cells (BMCs) and the pit formation in a dose-dependent manner. In cell cocultures, the production levels of both interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) were proportionally upregulated as the ratio of Treg cells to BMCs was increased, and the inhibition of OC differentiation and bone resorption by Treg cells was completely reversed by anti-IL-10 and anti-TGF-β1 antibodies. Treatment of BMC and Treg cell cocultures with 17β-estradiol (E2) at concentrations between 10⁻⁷ and 10⁻⁹ mol/l suppressed OC differentiation and bone resorption more efficiently than it did in cultures of BMCs alone; this enhanced suppression occurred via the stimulation of Treg cell IL-10 and TGF-β1 expression. These data suggest that Treg cells suppress OC differentiation and bone resorption by secreting IL-10 and TGF-β1. E2 enhances the suppressive effects of Treg cells on OC differentiation and bone resorption by stimulating IL-10 and TGF-β1 secretion from these cells. Therefore, Treg cell-derived IL-10 and TGF-β1 are likely involved in the regulation of E2 on bone metabolism and represent potential therapeutic targets for the treatment of postmenopausal osteoporosis (PMO).     

Circumvention of regulatory CD4(+) T cell activity during cross-priming strongly enhances T cell-mediated immunity

Immunization with purified antigens is a safe and practical vaccination strategy but is generally unable to induce sustained CD8(+) T cell-mediated protection against intracellular pathogens. Most efforts to improve the CD8(+) T cell immunogenicity of these vaccines have focused on co-administration of adjuvant to support cross-presentation and dendritic cell maturation. In addition, it has been shown that CD4(+) T cell help during the priming phase contributes to the generation of protective CD8(+) memory T cells. In this report we demonstrate that the depletion of CD4(+) T cells paradoxically enhances long-lasting CD8-mediated protective immunity upon protein vaccination. Functional and genetic in vivo inactivation experiments attribute this enhancement primarily to MHC class II-restricted CD4(+) regulatory T cells (Treg), which appear to physiologically suppress the differentiation process towards long-living effector memory T cells. Since, in functional terms, this suppression by Treg largely exceeds the positive effects of conventional CD4(+) T cell help, even the absence of all CD4(+) T cells or lack of MHC class II-mediated interactions on priming dendritic cells result in enhanced CD8(+) T cell immunogenicity. These findings have important implications for the improvement of vaccines against intracellular pathogens or tumors, especially in patients with highly active Treg.     

Allergen-Specific increase in interleukin (IL)-4 and IL-5 secretion from peripheral blood mononuclear cells during birch-pollen immunotherapy

The mechanisms behind the effects of immunotherapy (IT) with birch-pollen extract are largely unknown. In this pilot study, we measured the cytokine secretion in vitro from peripheral blood mononuclear cells (PBMC) obtained from birch-pollen-allergic patients undergoing IT treatment ( n =4) or placebo administration (n=4), collected before treatment, 1 and 4 weeks after start of treatment, and during and just after the pollen season (12–14 weeks after start of treatment). The PBMC were stimulated with birch-pollen extract in vitro for 7 days, followed by restimulation with the mitogens phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) for 24 h, to enhance the production of cytokines. The supernatants were analyzed with ELISA and radioimmunoassay for interferon-gamma (IFN-y), interleukin (IL)-4, and IL-5. In the therapy group, we noted an increased secretion of IL-4 and IL-5 from PBMC collected at 4 weeks after the start of treatment (IL-4: 29±21 pg/ml [day 0] to 3741448 pg/ml [week 4], mean± SD; IL-5: 95+48 pg/ml to 11471697 pg/ml). No increase was seen in the placebo group. During the pollen season, we noted a trend toward increased IL-4 and IL-5 secretion in both groups. We conclude that the temporary increase in serum IgE observed in many IT studies may be a consequence of increased IL-4 production due to the allergen exposure.