IL-4-producing NK T cells are biased towards IFN-γ production by IL-12. Influence of the microenvironment on the functional capacities of NK T cells

NK T cells are an unusual T lymphocyte subset capable of promptly producing several cytokines after stimulation, in particular IL-4, thus suggesting their influence in Th2 lineage commitment. In this study we demonstrate that, according to the cytokines present in the micro environment, NK T lymphocytes can preferentially produce either IL-4 or IFN-γ. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4⁻ CD8⁻ TCRα β⁺ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-γ in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-γ is strikingly enhanced when IL-12 is added either during their expansion or the anti-CD3 stimulation, while IL-4 secretion is always absent. A similar effect of IL-12 on IFN-γ production was observed when NK T lymphocytes were obtained after expansion with IL-7. It is noteworthy that whatever cytokines are used for their expansion, IL-12 stimulation, in the absence of TCR/CD3 cross-linking, promotes consistent IFN-γ secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant up-regulation of the capacity of NK T cells to produce IFN-γ after anti-CD3 mAb injection when mice were previously treated with IL-12. In conclusion, we provide evidence that the functional capacities of NK T cells, which ultimately will determine their physiological roles, are strikingly dependent on the cytokines present in their microenvironment.     

Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate

Type 2 cytokines are thought to have a protective role in psoriasis vulgaris by dampening the activity of T helper 1 (Th1) lymphocytes. The aim of the present study was to determine the effect of monomethylfumarate (MMF), the most active metabolite of the new anti-psoriatic drug Fumaderm®, on the production of cytokines and the development of Th subsets. MMF was found to enhance interleukin (IL)-4 and IL-5 production by CD2/CD8 monoclonal antibody-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Maximal effects of MMF were found at a concentration of 200 μM and resulted in tenfold enhanced levels of IL-4 and IL-5 production. MMF did not affect the levels of IL-2 production, interferon (IFN)-γ production or proliferative T cell responses in these cultures. Similar effects of MMF were observed in cultures of purified peripheral blood T cells indicating that this compound can act directly on T cells. MMF did not influence cytokine production by purified CD4⁺CD45RA⁺ (unprimed) T cells, but greatly enhanced IL-4 and IL-5 production without affecting IFN-γ production by purified CD4⁺CD45R0⁺ (primed) T cells. Furthermore, MMF also augmented IL-4 and IL-5 production in established Th1/Th0 clones that were stimulated with CD2/CD28 monoclonal antibody. Finally, when PBMC were challenged with Mycobacterium tuberculosis that typically induces Th1 recall responses with strong IFN-γ secretion, MMF again appeared to induce high levels of IL-4 and IL-5 secretion while IFN-γ production was unaffected. These results may be relevant for the development of therapeutic regimens designed to correct inappropriate Th1 subset development in immunopathologic conditions.     

Mechanism of enhanced antigen presentation by B cells activated with anti-μ plus interferon-γ: Role of B7-2 in the activation of naive and memory CD4+ T cells

B cells activated with anti-γ antibody plus interferon (IFN)-γ exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-μ, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-γ further augmented the expression of B7-2 on anti-μ-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44^low naive CD4⁺ T cells, whereas CD45RB^low memory CD4⁺ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4⁺ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-μ plus IFN-γ. These results suggest that IFN-γ plays an important role in recruitment of naive T cells for an immune response.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin-12 is required for priming thymocytes to produce both Th1 cytokines and interleukin-10

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin-1 (TSST-1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)-2, in the absence or presence of exogenous IL-4, interferon (IFN)-γ or IL-12. The resulting CD4⁺ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL-2, IL-4, IL-5, IL-10, IFN-γ and tumor necrosis factor (TNF)-β in response to stimulation with phorbol 12-myristate 13-acetate (PMA)+anti-CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4⁺ T cell clones generated from human thymocytes produced high concentrations of IL-2, IL-4 and IL-5, but no IFN-γ, TNF-β or IL-10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)-3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN-γ. However, upon addition of exogenous IL-12, the resulting CD4⁺ thymocyte clones produced TNF-β, IFN-γ, and IL-10 in addition to IL-4 and IL-5. These results suggest that CD4⁺ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL-4 and IL-5, whereas the ability to produce both Th1 cytokines and IL-10 is acquired only after priming with IL-12.     

Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand

IL- 12 is the prominent inducer of Th1 responses in humans and in the mouse. CD40 ligand (CD40L) plays important roles in regulation of immune responses, including T cell-dependent activation of B cells and cytokine production by monocytes and dendritic cells. The present study examined the influences of IL-12 on the CD40L expression of activated human CD4⁺ T cells. IL-12 enhanced CD40L expression on CD4⁺ T cells stimulated with immobilized anti-CD3 in the complete absence of accessory cells, whereas IL-4 and IL-10 decreased it. Exogenous interferon-gamma (IFN-γ) did not increase CD40L expression on immobilized anti-CD3 stimulated CD4⁺ T cells at any time up to 168 h of culture. The IL-12-induced enhancement of CD40L expression on anti-CD3 activated CD4⁺ T cells was not influenced in the presence of a metalloproteinase inhibitor KB8301, which up-regulated CD40L expression by preventing the processing of membrane-bound CD40L, or B cells, which down-regulated CD40L expression by receptor-mediated endocytosis. These results indicate that IL-12 enhances the CD40L expression of activated CD4⁺ T cells independently of the IFN-γ production. The data thus suggest that Th1 responses induced by IL-12 might play an important role in the regulation of humoral immune responses through up-regulated CD40L expression.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture

The lymphocyte activation gene 3 (LAG-3) is a CD4 homolog with binding affinity to MHC class II molecules. It is thought that LAG-3 exerts a bimodal function, such that co-ligation of LAG-3 and CD3 could deliver an inhibitory signal in conventional T cells, whereas, on regulatory T cells, LAG-3 expression could promote their inhibitory function. In this study, we investigated the role of LAG-3 expression on CD4⁺ T cells in patients with long bone fracture. We found that LAG-3⁺ cells represented approximately 13% of peripheral blood CD4⁺ T cells on average. Compared to LAG-3^? CD4⁺ T cells, LAG-3⁺ CD4⁺ T cells presented significantly higher Foxp3 and CTLA-4 expression. Directly ex vivo or with TCR stimulation, LAG-3⁺ CD4⁺ T cells expressed significantly higher levels of IL-10 and TGF-β than LAG-3^? CD4⁺ T cells. Interestingly, blocking the LAG-3-MHC class II interaction actually increased the IL-10 expression by LAG-3⁺ CD4⁺ T cells. The frequency of LAG-3⁺ CD4⁺ T cell was positively correlated with restoration of healthy bone function in long bone fracture patients. These results together suggested that LAG-3 is a marker of CD4⁺ T cells with regulatory function; at the same time, LAG-3 might have limited the full suppressive potential of Treg cells.     

Epigenetic Modification Mediates the Increase of LAG-3<Superscript>+</Superscript> T Cells in Chronic Osteomyelitis

Immune suppression plays critical roles in the development of chronic osteomyelitis, and the mechanisms underlying the development of immune suppression in chronic osteomyelitis have attracted much attention. LAG-3 is an important suppressor of T cell activation, but the role of LAG-3 in the immune regulation of chronic osteomyelitis is currently unknown. We sought to demonstrate if LAG-3 plays crucial roles in chronic osteomyelitis progression and has effects on immune suppression and exhausting of T cells, and what is the mechanism underlying LAG-3 deregulation in chronic osteomyelitis. We examined the expression of LAG-3 in the T cells of peripheral blood of 50 healthy controls and 50 patients with chronic osteomyelitis by flow cytometry. Clinical data were analyzed to determine the correlation between inflammation index and LAG-3 expression. Moreover, we isolated the CD4⁺ T cells from healthy controls and chronic osteomyelitis patients to compare cell proliferation and IFN-γ production. Chromatin immunoprecipitation assays were utilized to analyze the epigenetic modification on LAG-3 expression in T cells. We found that LAG-3 was significantly increased in the T cells of peripheral blood from chronic osteomyelitis patients. Subsequently, clinical data analysis suggested that the higher expression of LAG-3 was associated with severer inflammation situation. Consistently, LAG-3⁺CD4⁺ T cells exhibited impaired cell proliferation and IFN-γ secretion. Deregulation of histone methylation mediated the increase of LAG-3⁺ T cells during chronic osteomyelitis. Taken together, our study demonstrates the increase of LAG-3⁺ T cells and its immune regulatory roles in chronic osteomyelitis progression, suggesting new mechanisms and potential therapeutic targets for chronic osteomyelitis.     

Differential effects of interleukin-12 on the development of naive mouse CD4+ T cells

The influence of interleukin (IL)-12 and IL-4 on the differentiation of naive CD4⁺ T cells was studied in an accessory cell-free in vitro system. Dense CD4⁺ T cells were purified from unimmunized mice and activated using immobilized anti-CD3 monoclonal antibodies (mAb) in the presence of IL-4, IL-12, or a combination of both cytokines, and restimulated after 6 days by re-exposure to anti-CD3-coated culture wells. T cells initially activated in the presence of IL-4 produced substantial amounts of IL-4 and trace amounts of interferon (IFN)-γ after restimulation at day 6 with plate-bound anti-CD3 mAb. By contrast, T cells primed in the presence of IL-12 produced high levels of IFN-γ and only minimal amounts of IL-4, thus indicating that IL-12 and IL-4 by acting directly on stimulated naive CD4⁺ T cells support the development of T_H1 and T_H2 cells, respectively. When naive CD4⁺ T cells were stimulated in the presence of IL-12 together with IL-4 in comparable concentrations, the effect of IL-12 on T_H1 differentiation was largely inhibited by IL-4. On the other hand, IL-12 exerted no inhibitory effect on IL-4-induced T_H2 differentiation but rather enhanced the production of IL-4 after restimulation of the respective T cells. Decreasing amounts of IL-4 in combination with a high level of IL-12 led to an increasing production of IFN-γ by the emerging T cells and, simultaneously, to a relatively high production of IL-4. These data were confirmed by time-course experiments which revealed that the delayed addition of IL-4 to IL-12-primed T cell cultures resulted in a gradual restoration of IFN-γ production whereas in parallel the secretion of IL-4 was not reduced over a wide period of delay (6–72 h). These results, therefore, demonstrate that (a) IL-4 dominates the effect of IL-12, (b) IL-12 promotes the development of T_H1 cells; however, in the presence of IL-12 and relatively high levels of IL-4 also the development of T_H2-like cells is slightly but significantly enhanced by IL-12, and (c) high amounts of IL-12 in combination with relatively low levels of IL-4 give rise to a T cell population that upon rechallenge exhibited a cytokine profile resembling that of T_H0 cells.     

The origin of regulatory from the effector cells in LAG-3-marked Th1 immunity against severe influenza virus infection

In severe respiratory virus infections, including influenza, an exaggerated host immune response has been linked to the severe disease and death. Control of the overwhelming immune response is thus essential. Efforts with broad-spectrum immunosuppressive agents such as steroids are disappointing. A better understanding of host immune response using animal experimental system is required to avoid undesired outcome of experimental manipulation. Following severe influenza virus infection in influenza hemagglutinin antigen-specific transgenic mouse experimental model, step-wise evolving cells from a pool of naïve hemagglutinin-specific CD4⁺ T cells were studied for phenotypic, genomic, and functional characterization in vivo. Naïve CD4⁺ T cells respond with Th1 commitment in the absolute majority. They first develop into LAG-3^MedIFN-γ-secreting Th1 effectors and then evolve into LAG-3^HighIFN-γ-not-secreting regulators with increasing LAG-3 expression upon continuous activation and cell division. The LAG-3^MedIFN-γ-secreting effectors contribute to inflammation, boost inflammatory response of cognate antigen-specific CD8⁺ T cells, and aggravate the disease despite facilitated virus clearance. In contrast, LAG-3^High regulators do not contribute to inflammation, suppress CD8⁺ T cell inflammatory response, alleviate lung pathology, and ameliorate the disease with preserved virus clearance. Moderated CD8⁺ T cells retain proliferative capacity, and persist beyond virus clearance. Such moderation is distinct from Foxp-3⁺ regulator-mediated suppression, which suppresses proliferative and inflammatory responses of the CD8⁺ T cells and impairs virus clearance with inflammation alleviation. Origin of regulatory from the effector cells of LAG-3-marked Th1 immunity alleviates lung inflammation without impairment of virus eradication.     

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.     

T lymphocytes from the normal human peritoneum contain high frequencies of Th2-type CD8+ T cells

The majority of peritoneal T lymphocytes have been shown to be CD8⁺ and to co-express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)-4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8⁺ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)-γ and exhibited high alloantigen-specific cytolytic activity, some clones secreted IL-4 and IL-5 but no detectable IFN-γ. These Th2-type CD8⁺ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8⁺ T cell may occur in different immune compartments.     

Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell-mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8⁺ T cells producing interferon (IFN)-γ which mediate the response and CD4⁺ T cells producing interleukin (IL)-4 and IL-10 which negatively regulate the magnitude and duration of the response. Since CD4⁺ T cell development to either IFN-γ- (Th1) or IL-4/IL-10- (Th2)-producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4⁺ T cell induction in a Th1-promoting environment would not only alter the CD4⁺ T cell cytokine-producing phenotype but also the course of the CHS response. Administration of the Th1-promoting cytokine IL-12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten-sensitized mice depleted of CD8⁺ T cells, treatment with IL-12 induced effector CD4⁺ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed-type hypersensitivity than CHS. Hapten-primed CD4⁺ T cells from IL-12 treated, sensitized mice produced IFN-γ, but not IL-4 in response to T cell receptor-mediated stimulation. Use of neutralizing anti-IFN-γ antibody indicated that IL-12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN-γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4⁺ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.     

Distinct modulation by interferon-gamma (IFN-γ) of CD23 expression on B and T lymphocytes of atopic subjects

The low-affinity IgE receptor (FcεRII/CD23) plays a role in IgE production. Cytokines participating in IgE synthesis also modulate CD23 expression on lymphocytes, but whether this modulation is different in atopic subjects remains unclear. We studied CD23 expression on B and T lymphocytes in 10 asthmatic patients with Dermatophagoides pteronyssinus hypersensitivity and 10 healthy non-atopic subjects. Studies were performed by flow cytometry, in phytohaemagglutinin (PHA) or IL-4-stimulated mononuclear cell cultures, alone or in the presence of IFN-γ. Soluble CD23 (sCD23) released in the culture supernatants was measured by enzyme-linked immunoassay. Both PHA and IL-4 induced the expression of CD23 on lymphocytes of atopic and non-atopic subjects. Whereas PHA increased both the percentage and mean fluorescence intensity of CD23⁺ B and T cells, IL-4 alone did not increase the percentage of CD23⁺ T cells. The effects of IFN-γ were different in both groups, since it was able to reduce the percentage of PHA-stimulated CD23⁺ T cells only in non-atopic individuals. In non-atopic subjects more than atopic, levels of sCD23 were increased in the supernatants of PHA and IL-4 cultures. These results show that the modulation of CD23 expression is different on B and T cells, and that IFN-γ acts differently in atopic and non-atopic individuals.     

Compartmentalization of T cell responses following respiratory infection with Bordetella pertussis: hyporesponsiveness of lung T cells is associated with modulated expression of the co-stimulatory molecule CD28

We have used a murine respiratory challenge model to examine the local T cell responses in the lung during infection with Bordetella pertussis. T cells from lung parenchyma and airways of naive and infected mice were refractory to both antigen and mitogen stimulation in the presence of lung macrophages. Furthermore irradiated mononuclear cells from the lungs suppressed antigen and mitogen-induced proliferation, but not IFN-γ production, by splenic T cells. Removal of macrophages and stimulation of purified lung T cells in the presence of irradiated splenic antigen-presenting cells fully restored the response to mitogen. However, T cells purified from the lung during the acute phase of infection with B. pertussisfailed to proliferate or produce detectable levels of IL-2, IL-4, IL-5 or IFN-γ in response to purified bacterial antigens. In contrast, splenic T cells from these animals produced high levels of IL- 2 and IFN-γ and proliferated strongly to a range of bacterial components. Phenotypic analy sis of bronchoalveolar lavage cells during the course of infection revealed transient infiltra tion of neutrophils, followed by macrophages, CD4+ T cells and smaller numbers of CD8+ T cells and γ δ+ T cells. Cell surface expression of B7 on infiltrating macrophages and CTLA-4 on T cells did not change significantly during infection. However, expression of the CD28 co- stimulatory molecule was profoundly reduced on lung T cells during the acute phase of infection. In contrast, lung T cells from mice primed by B. pertussisinfection or vaccination were resistant to CD28 down-regulation. These results suggest compartmentalization of T cell responses between the lung and the periphery during B. pertussisinfection and that B. pertussismay have immunomodulatory properties on local T cell populations in the lungs of naive mice.     

CD40 ligand and IFN-γ synergistically restore IL-12 production in HIV-infected patients

IL-12 production in HIV-infected (HIV⁺) individuals is severely impaired after stimulation by bacterial products or T cell-dependent stimuli. Because CD40-CD40 ligand (CD40L) interactions are the major mechanism involved in the T cell-dependent activation of antigen-presenting cells, we investigated whether this pathway was functional in HIV⁺ donors. CD40 expression was increased on freshly isolated monocytes from HIV⁺ individuals compared to HIV⁻ donors. However, equivalent CD40 expression was obtained in the two groups after cytokine stimulation. Since CD40 expression was intact in HIV⁺ donors' cells, we determined whether IL-12 production could be restored by providing exogenous T cell-dependent stimuli, CD40L and IFN-γ, at the time of bacterial stimulation. IL-12 production was not altered by CD40L alone, was increased by IFN-γ, and was synergistically restored to normal values by IFN-γ + CD40L. This combination was more efficient for enhancing IL-12 production than granulocyte-macrophage colony-stimulating factor + CD40L or neutralizing anti-IL-10 antibody + CD40L. CD40L did not affect IL-10 production, whereas IFN-γ significantly decreased it. This study demonstrates that the defect in IL-12 production by leukocytes from HIV⁺ donors can be overcome in vitro if the interacting cells are provided with the right T cell-dependent co-stimuli.