Interleukin-18 regulates T helper 1 or 2 immune responses of human cord blood CD4+ V alpha 24+V beta 11+ natural killer T cells

Natural killer T (NKT) cells, exhibiting both T-cell and NK-cell markers, are known to regulate immune responses by secreting T-helper (Th) 1 and Th2 cytokines. We analyzed NKT cells in cord blood (CB) for phenotypical and functional characteristics and regulatory mechanisms that control Th1 and Th2 determination. Human CB V alpha 24+V beta 11+ NKT cells were predominantly the CD4+ single positive (SP) phenotype (approximately 96%), in contrast to adult peripheral blood V alpha 24+V beta 11+ NKT cells which are composed of a dominant population of the CD4-CD8-double negative (DN) phenotype and a minor population of the CD4+ SP phenotype. The CB CD4+ V alpha 24+ NKT cells, following stimulation with the primary culture, gained the capacity to secrete interferon (IFN)-gamma, a Th1 cytokine, and interleukin (IL)-13, a Th2 cytokine. The combination of IL-18 and IL-12 induced IFN-gamma production in CB CD4+ V alpha 24+ NKT cells, while IL-18 in combination with IL-2 induced IL-13 production in these cells. Thus, IL-18 regulates the determination of the Th1 or Th2 immune response by human CD4+ V alpha 24+ NKT cells through different cytokine combinations.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

CD4+ T-cell clones from autoimmune thyroid tissue cannot be classified according to their lymphokine production

In order to define whether CD4+ T cells from autoimmune and non-autoimmune thyroid tissue could be classified according to their mediator production, lymphokine production was studied in 63 thyroid-derived CD4+ T-cell clones from four patients with Graves' disease, one with Hashimoto's thyroiditis, and one with non-toxic goitre (9-12 clones per patient). The production of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), lymphotoxin (LT), interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) was assessed at the mRNA level by slot-blot analysis in unstimulated clones as well as after activation with monoclonal anti-CD3 (OKT3) and IL-2. No lymphokine production was found in unstimulated clones, whereas 56% of the clones produced all six lymphokines simultaneously after stimulation. In the remaining 44% usually not more than one lymphokine was missing from the complete panel. Lymphokine mRNA concentrations varied between different clones and different patients, but, in this small sample, not between the diseases from which the clones were originated. There was a significant correlation between IL-6, LT, and IL-2 mRNA levels and T-cell helper function, which was estimated by the stimulation of thyroid microsomal autoantibody production using autologous peripheral B cells. TGF-beta and IFN-gamma mRNA expression was unrelated to T-cell help. The results demonstrate that intrathyroid T cells from autoimmune and non-autoimmune thyroid disorders cannot be classified according to their lymphokine production, unlike some results with in vitro-induced mouse T-cell clones, where two populations, Th1 and Th2, have been described. Single T cells are capable of producing a whole panel of lymphokines and thus are capable of triggering a multitude of different processes.     

"Bystander polarization" of CD4+ T cells: activation with high-dose IL-2 renders naive T cells responsive to IL-12 and/or IL-18 in the absence of TCR ligation

Responsiveness of CD4+ T cells to the IFN-gamma-inducing cytokines IL-12 and IL-18 is generally thought to be acquired only after stimulation via the TCR. We report herein that stimulation of naive CD4+ T cells with high-dose IL-2 (1000 U/ml) renders these cells responsive to IL-12 and/or IL-18 without a requirement for TCR ligation. Naive CD4+CD62L+ Tcells from normal C57BL/6 mice or from DO11.10/Rag2(-/- )OVA-specific TCR-transgenic mice secreted substantial amounts of IFN-gamma when stimulated concurrently with high-dose IL-2 plus IL-12 or IL-18. mRNA encoding both chains of the IL-12 and the IL-18 receptors was expressed by CD4+ T cells after stimulation with high-dose IL-2. Furthermore, anti-CD3-induced IL-12/IL-18 responsiveness was fully abrogated in the presence of cyclosporin A whereas IL-2-induced IL-12/IL-18 responsiveness was not, reminiscent of the previously reported IL-12+IL-18 innate pathway of T cell activation. Lastly, after stimulation with IL-2+IL-12, naive CD4+ T cells from DO11.10/Rag2(-/- )mice exhibited polarization towards a Th1 phenotype (high IFN-gamma but no IL-4) during secondary stimulation with immobilized anti-CD3. We have coined the term "bystander polarization" to describe this phenomenon and we speculate that bystander polarization of naive CD4+ T cells may occur in vivo during strong antigen-specific immune responses.     

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-gamma expression.

Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-gamma (IFN-gamma) (Th2- and Th1-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P = 0.0075 and P = 0.0004, respectively) compared with non-atopic controls, whereas IFN-gamma production was not significantly different. In contrast to allergen, the prototypic Th1-type antigen M. tuberculosis PPD induced an excess of IFN-gamma over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-gamma or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.     

Immunization with alpha-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis.

We have compared the immune responses of mice immunized either with alpha-galactosylceramide (alpha-GalCer), capable of eliciting a CD1-metiated stimulation of V alpha14+ NK T cells, or with lipoarabinomannan (LAM), a glycophospholipid derived from mycobacteria which is known to be presented by CD1b in humans. Within 24 h, alpha-GalCer induces a burst of IFN-gamma secretion in vivo, and recall with antigen in vitro leads to the synthesis of IL-4 and IL-10 in addition to IFN-gamma. Associated with this in vivo cytokine release is a polyclonal activation of splenic B and T cells. CD1-reactive NK T lymphocytes mediate these events, because none of them are observed in alpha-GalCer-immunized CD1-/- mice. LAM immunization fails to promote similar early responses in vivo. Repeated exposure of mice to alpha-GalCer induces splenic T cells to secrete IL-4 and IL-10 but dramatically reduced levels of IFN-gamma. Such a bias in the cytokine balance triggered by NK T cells stimulated with multiple doses of alpha-GalCer suggests that this compound might be useful in the induction of Th2 immune responses and the prevention of chronic inflammatory conditions mediated by Th1 cytokines.     

Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway

Recent studies have implicated cytokines associated with CD4+ T lymphocytes of both T helper (Th)1 and Th2 subsets in resistance to experimental blood stage malaria. As the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to Th1/Th2 cytokine and immunoglobulin isotype profiles and to the development of a protective immune response to malaria in NIH mice infected with Plasmodium chabaudi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin (IL)-4 and up-regulation of interferon (IFN)-gamma responses by P. chabaudi-specific T cells and by reduction of P. chabaudi-specific immunoglobulin G1 (IgG1). The shift towards a Th1 cytokine pattern corresponded with efficient control of acute parasitaemia but an inability to resolve chronic infection. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies raised IFN-gamma production over that seen with CD86 blockade alone, with augmentation of this Th1-associated cytokine reducing levels of peak primary parasitaemia. These results demonstrate that IL-4 production by T cells in P. chabaudi-infected NIH mice is dependent upon CD86/CD28 interaction and that IL-4 and IFN-gamma contribute significantly, at different times of infection, to host resistance to blood stage malaria. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-gamma-producing T cells during P. chabaudi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. This study indicates a role for B7/CD28 costimulation in modulating the CD4+ T-cell response during malaria, and further suggests involvement of this pathway in other infectious and autoimmune diseases in which the Th cell immune response is also skewed.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

NKT cell cytokine imbalance in murine diabetes mellitus.

A minor subset of murine MHC class I-restricted T cells which express both the alphabeta form of the T cell receptor and a NK lineage marker, termed NKT cells, is capable of secreting significant amounts of Interleukin-4 and Interferon-y upon activation. As such NKT cells may play a role in development of Th1 and Th2 cells during T cell ontogeny or expansion of T cells expressing a dominant cytokine pattern in the effector phase. We have studied the role of NKT cells in a murine model of disease multidose streptozotocin induced diabetes mellitus (MDSDM). In MDSDM thymic and splenic NKT cells are present at normal levels but have greatly reduced capacity to secrete Interleukin-4 upon stimulation with anti-TCR antibody compared to control mice; conversely, Interferon-y secretion is maintained. By analysis of cytokine RNA production we found that treatment of several strains of mice with streptozotocin changes the peripheral helper T cell phenotype elicited after immunization with Keyhole Limpet Hemocyanin from a mixed Th1- and Th2-type cytokine pattern (characterized by IFN-gamma and IL-4 and IL-5 expressions, respectively) to predominately Th1-type. Furthermore, susceptibility to MDSDM is significantly enhanced when NKT cells are selectively eliminated in vivo by administration of depleting anti-CD122 antibody TMbeta-1. In addition, antibody depletion of NKT cells from non-obese diabetic mice significantly accelerates onset of disease. Collectively these data support a model for development of murine diabetes mellitus in which NKT cell cytokine expression influences the development of Th1-type diabetogenic T cells.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice.

Accumulating evidence suggests that Th1 T cells play a pivotal role in the development of autoimmune diabetes. Conversely, promoting a Th2 response inhibits disease progression. However, it has not been determined whether Th2 cells are regulatory T cells that fail at the time of diabetes development in naive non-diabetic NOD mice. Therefore, in order to evaluate cytokine secretion by spleen and islet infiltrating T cells in NOD mice at different stages of the autoimmune process, we developed an ELISPOT assay that detects IL-2, IL-4, and interferon-gamma (IFN-gamma) secretion in vitro at the single-cell level. We showed that, whatever the age considered, IFN-gamma is predominantly secreted, and that no IL-4-secreting cells are detected in the islets of male and female NOD mice. Spleen cells from 8-week-old female NOD mice, which include regulatory suppressor T cells, do not secrete IL-4, either upon presentation of islet cell antigens in vitro, or after transfer in vivo, but do secrete IFN-gamma. IFN-gamma secretion by T cells from diabetic mice results from CD4 but not CD8 T cells in transfer experiments into NOD/severe combined immunodeficient (SCID) recipients. These results suggest that (i) detection of regulatory CD4 T cells in NOD mice is not paralleled by a Th2 response; (ii) beta cell destruction does not depend on a switch from a Th2 to a Th1-type response; and (iii) CD8 T cells do not participate in induction of diabetes by secreting IFN-gamma.     

Human peripheral blood CD4+ and CD8+ T cells express Th1-like cytokine mRNA and proteins following in vitro stimulation with heat-inactivated Brucella abortus.

Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.     

Recombinant adenovirus-transduced human dendritic cells engineered to secrete interleukin-10 (IL-10) suppress Th1-type responses while selectively activating IL-10-producing CD4+ T cells

Recombinant adenoviruses (rAd) are efficient tools for genetic modification of human dendritic cells (DC) in vitro. Infection of DCs by rAd encoding beta-galactosidase (betagal) results in partial maturation of DCs, as witnessed by the upregulation of major histocompatibility complex and costimulatory molecules. Accordingly, these DCs are more potent stimulators of Th1-type proliferative responses. We now demonstrate that infection of immature DCs with rAd encoding human interleukin (IL)-10 results in the secretion by the DCs of large amounts of IL-10, while not affecting expression of activation markers indicative of partial DC maturation. In contrast to rAd-betagal-infected DCs, rAdIL-10-infected DCs are very poor stimulators of monoclonal and polyclonal Th1-type responses. Instead, stimulation of nonpolarized CD4+ T-cell cultures with rAdIL-10-infected DCs selectively activates and expands an IL-10-producing CD4+ T-cell subset capable of suppressing Th1 responses in vitro. Our data argue that rAd-infected human DCs genetically engineered to produce IL-10 may be exploited for the modulation of harmful Th1-type responses in transplantation and autoimmune diseases.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Intracellular IFN-gamma production and IL-12 serum levels in latent autoimmune diabetes of adults (LADA) and in type 2 diabetes.

Th1 cytokines, such as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), and Th1-inducing cytokines, such as IL-12, are involved in the pathogenesis of various organ-specific autoimmune diseases, including autoimmune diabetes. In this study, we investigated intracellular IFN-gamma release by T lymphocytes and IL-12 serum levels in 48 type 2 and 36 latent autoimmune diabetes of adults (LADA) diabetics and 25 control subjects in an attempt to evaluate their role in the pathogenesis of these clinical entities. Ionomycin (ION) and phorbol-12-myristate-13-acetate (PMA)-activated peripheral blood mononuclear cells (PBMCs) were stained with anti-CD4-FITC or anti-CD8-FITC and anti-IFN-gamma phycoerythrin (PE) monoclonal antibodies (mAbs) and analyzed by flow cytometry. IL-12 serum levels were determined by enzyme-linked immunosorbent assay (ELISA). In all study groups, IFN-gamma content of CD4(+) and CD8(+) lymphocytes was significantly upregulated by stimulation. Furthermore, it was observed that CD4(+) and CD8(+) lymphocytes from type 2 diabetics produced significantly lower levels of IFN-gamma compared with LADA patients and controls. However, the percentages of CD4(+)/IFN-gamma(+) and CD8(+)/IFN-gamma(+) cells from type 2 diabetics were significantly higher compared with controls. The flow cytometric picture of intracellular IFN-gamma release in LADA patients did not differ from that observed in controls. However, IL-12 serum levels in type 2 and LADA diabetics were lower than in controls. Because Th1 cytokines have been associated with the pathogenesis of autoimmune diabetes, these results preclude Th1 involvement in the autoimmune phenomena observed in LADA patients. In contrast, the low IFN-gamma levels observed in type 2 diabetics in combination with the low IL-12 serum levels might be a contributing factor in the frequently observed chronic complications in these patients.