IL-18 enhances IL-4 production by ligand-activated NKT lymphocytes: a pro-Th2 effect of IL-18 exerted through NKT cells.

NKT cells are a remarkably versatile population whose functional capacities are determined by cytokines present in their microenvironment. In this study, we provide evidence for a new immunoregulatory effect of the proinflammatory cytokine IL-18 on NKT cells. We found that IL-18, mainly known for its involvement in NK cell activation and in Th 1 immune responses, substantially enhanced IL-4 production as well as the percentage of IL-4(+) cells among NKT lymphocytes activated by their specific ligand alpha-galactosylceramide (alpha-GalCer). The effect of IL-18 on IL-4 production by activated NKT cells took place both in vivo and in vitro and was not affected by IL-12 which increased IFN-gamma secretion in the same conditions. We show that NKT cells are the main targets for IL-18-induced IL-4 production since it occurred neither in NKT-deficient mice nor after stimulation of Th2 lymphocytes. Finally, we provide evidence that the IL-4 promptly generated by NKT cells in response to IL-18 plus alpha-galactosylceramide in vivo can effectively contribute to the adaptive Th2 immune response by up-regulating the early activation marker CD69 on B cells. Our data support the notion that, in contrast to the exclusive IFN-gamma inducer IL-12, IL-18 acts in a more subtle manner as a costimulatory factor in both pro-Th1 and pro-Th2 responses depending on the nature of the stimulation and the target cells.

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes.

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.

A distinct IL-18-induced pathway to fully activate NK T lymphocytes independently from TCR engagement.

NK T lymphocytes are characterized by their ability to promptly generate IL-4 and IFN-gamma upon TCR engagement. Here, we demonstrate that these cells can also be fully activated in the absence of TCR cross-linking in response to the proinflammatory cytokine IL-18 associated with IL-12. NK T cells stimulated with IL-18 plus IL-12 proliferated, killed Fas+ target cells, and produced high levels of IFN-gamma without IL-4. In these conditions, IFN-gamma production was at least 10-fold higher than that upon TCR cross-linking. Interestingly, a 2-h pretreatment with IL-12 plus IL-18 sufficed to maintain the high IFN-gamma-producing potential during subsequent stimulation with anti-TCR mAbs or with the specific Ag alpha-galactosylceramide. Similar effects were observed in vivo, because splenic CD4+ NK T cells from MHC class II-deficient mice secreted IFN-gamma without further stimulation when removed 2 h after a single injection of IL-12 plus IL-18. In conclusion, our evidence for activation of NK T lymphocytes in response to IL-18 plus IL-12 in the absence of TCR engagement together with the maintenance of preferential IFN-gamma vs IL-4 production upon subsequent exposure to specific Ags is consistent with the active participation of this cell population in innate as well as acquired cellular immune responses.

IL-4-producing NK T cells are biased towards IFN-gamma 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 microenvironment, NK T lymphocytes can preferentially produce either IL-4 or IFN-gamma. In agreement with our previous reports showing that their IL-4-producing capacity is strikingly dependent on IL-7, CD4-CD8-TCRalphabeta+ NK T lymphocytes, obtained after expansion with IL-1 plus granulocyte-macrophage colony-stimulating factor, produced almost undetectable amounts of IL-4 or IFN-gamma in response to TCR/CD3 cross-linking. However, the capacity of these T cells to produce IFN-gamma 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-gamma 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-gamma secretion by NK T cells without detectable IL-4 production. Experiments in vivo demonstrated a significant upregulation of the capacity of NK T cells to produce IFN-gamma 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.

Natural killer T cells: a potent cytokine-producing cell population.

Natural killer (NK) T cells constitute a subset of TCRalphabeta+ T lymphocytes characterized by natural killer surface receptor expression as well as by a restricted TCR repertoire. This population is capable both of secreting several cytokines, especially IL-4 and IFN-gamma, shortly after stimulation and of killing target cells via Fas/FasL interactions. The cytokines present in the microenvironment of NK T cells play a pivotal role in their potential immunoregulatory functions which are exerted through their ability to induce apoptosis and to modulate the development of Th1 or Th2 cells. Recent reports concerning the physiological roles of the NK T cell population will be discussed in this review.

Requirement of IL-7 for IL-4-producing potential of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes.

IL-7 plays an important role in the growth and differentiation of T cells. We have previously reported that IL-7 induces preferential expansion of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes which express a skewed V beta repertoire and are potent IL-4 producers. In this report, we provide evidence that IL-1 in combination with granulocyte macrophage colony stimulating factor can also expand this population. Yet, these cells do not share the functional characteristics of those obtained in the presence of IL-7, i.e. cytotoxic activity and high IL-4 production. These functional capacities can be acquired by adding IL-7. In conclusion, our findings demonstrate that the capacity of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes to produce IL-4 as well as to kill target cells is IL-7 dependent.

NK1.1+ T cells from IL-7-deficient mice have a normal distribution and selection but exhibit impaired cytokine production.

Particular subsets of T cells expressing the NK1.1 antigen have been proposed to play an immune regulatory role by their fast and strong production of cytokines, in particular IL-4. We sought to determine factors driving the functional differentiation of NK1.1+ T cells. Since NK1.1+ T cells are exquisitely sensitive to IL-7 stimulation, we analyzed the development, selection and IL-4 production of NK1.1+ T cells in IL-7-deficient mice (IL-7-/-m mice). Besides a sharp reduction of all T cell subsets, NK1.1+ T cells develop at normal relative frequencies in IL-7-/- mice. They also undergo a normal selection process, as revealed by the biased V beta TCR repertoire identical to the one in IL-7+/+ mice. However, NK1.1+ T cells from IL-7-/- mice were found to be impaired in IL-4 and IFN-gamma production in in vitro and in vivo models. In addition, IL-7 was able to restore IL-4 production by NK1.1+ thymocytes from IL-7-/- mice. Finally, IL-7 but not IL-2 or IL-4 was able to maintain and increase IL-4 production by NK1.1+ thymocytes from normal mice. These data suggest that the functional maturation of NK1.1+ T cells requires a cytokine-driven differentiation process, in which IL-7 plays a major role.

IL-7 reverses NK1+ T cell-defective IL-4 production in the non-obese diabetic mouse.

Converging data suggest an important role for IL-7 in T lymphocyte maturation as illustrated by the severe T lymphopenia observed in IL-7-deficient mice. We recently reported that IL-7 preferentially promotes the in vitro expansion of a discrete MHC class I-dependent lymphocyte subset comprising both CD4+ and CD4-CD8- TCR alpha beta + cells bearing several NK cells markers such NK1.1 and Ly-49. These T cells, designated as NK1+ T cells, have the unique property among thymocytes of producing large amounts of IL-4 upon primary stimulation via the TCR. We have further demonstrated that thymic NK1+ T cells of non-obese diabetic (NOD) mice, a spontaneous model of autoimmune type I diabetes, are markedly deficient in maturation both quantitatively and functionally (IL-4 production). In the present experiments, the addition of exogenous IL-7 completely restored IL-4 production by anti-TCR alpha beta-stimulated mature (HSA-CD8-) thymocytes in NOD mice. A short 2 h preincubation with IL-7 was sufficient to restore both the expression of IL-4 mRNA and IL-4 production capacity. This was related to a direct effect on NK1+ thymocytes since: (i) the effect of IL-7 was restricted to the non-mainstream MEL-14- 3G11- TCR alpha beta + subset which mostly concentrates the IL-4-producing capacity and (ii) IL-7 did not restore IL-4 production in class I-deficient mice which lack the NK1+ T cell subset. Importantly, this activity of IL-7 on NK1+ T cells was also demonstrated in non-autoimmune strains of mice. These results were extended in vivo by showing that the IL-7 treatment significantly increased the anti-CD3 triggered IL-4 production by NK1+ T spleen cells. These findings confirm the role of IL-7 in NK1+ T cell maturation and suggest that the NK1+ T cell defect in NOD mice could be related to insufficient intrathymic IL-7 bioavailability.

In vitro T cell unresponsiveness following low-dose injection of anti-CD3 MoAb.

Anti-CD3 MoAb treatment is widely used as an immunosuppressive therapy. In the present study we examined the in vitro T cell response in mice having received 24 h before a single i.v. injection of 10 microgram of anti-CD3 MoAb. We found that splenocytes from these mice displayed a dramatically decreased proliferative response to the T cell mitogens concanavalin A (Con A), anti-CD3, phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) + calcium ionophore, while the effect of lipopolysaccharide (LPS) was not impaired. T cell suppression persisted for about 10 days after anti-CD3 injection, returning to normal within 15 days. The F(ab')2 fragment of anti-CD3 had no such effect, indicating the requirement for in vivo activation. At the dose used, anti-CD3 resulted neither in T cell depletion nor in down-modulation of the CD3/T cell receptor (TCR) complex. The low proliferation was also not explained by apoptosis, following secondary challenge with Con A. Splenocytes from anti-CD3-injected mice were highly responsive to IL-2, but generated little or no IL-2, IL-3, IL-4 and interferon-gamma (IFN-gamma) when exposed to Con A. Normal cytokine production could not be restored by the addition of optimal doses of IL-2 during Con A stimulation. Transforming growth factor-beta (TGF-beta) was the only cytokine whose mRNA expression was not modified in stimulated splenocytes from anti-CD3-injected mice. Furthermore, anti-TGF-beta antibodies increased Con A-induced T cell proliferation, but not cytokine production.

Prolactin receptor expression in lymphocytes from patients with hyperprolactinemia or acromegaly.

Previous reports demonstrated that prolactin receptors (PRL-R) are widely expressed on cells of the immune system. We analyzed a possible regulation of PRL-R expression on human mononucleated blood cells by prolactin (PRL) itself. PRL-R expression was analyzed by immunofluorescence on T and B lymphocytes and monocytes from peripheral blood mononucleated cells (PBMC) of patients with hyperprolactinemia or acromegaly compared with sex- and age-matched control subjects. The frequency of PRL-R positive cells and the intensity of PRL-R expression was only modified among the CD8+ T cell population of hyperprolactinemic patients with macroadenoma. No correlation was reported between PRL-R expression and circulating PRL levels. The percentage of PRL-R+ cells on B or T lymphocytes and monocytes as well as the capacity of PBMC to proliferate in response to T cell mitogens were not significantly different in bromocriptine-treated compared with untreated patients. These findings suggest that factors other than pituitary PRL play the major role in regulating PRL-R expression on cells of the immune system.

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response.

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.

MHC class I-selected CD4-CD8-TCR alpha beta+ T cells: an important source of IL-4.

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial antigen priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well-defined. Here, a novel source of IL-4, the class I-selected CD4-CD8-TCR alpha beta+ T cells, potentially implicated in the development of specific Th2 immune cells, is reviewed.

Prolactin receptors and the immune system.

The existence of a physiological immunoneuroendocrine network clearly contributing to homeostasis has now been demonstrated. In this context, nervous, endocrine, and immune systems communicate with each other, using common mediators and respective receptors. An interesting aspect of this network involves the interactions between prolactin (PRL) and the immune system. Prolactin plays a significant role in regulation of the humoral and cellular immune responses in physiological as well as pathological situations, such as autoimmune diseases. This role is exerted via the existence of specific receptors on cells on the immune system. Recently, using monoclonal antibodies (mAbs) raised against the extracellular domain of the rat liver PRL-receptor (PRL-R), we demonstrated by immunochemistry and molecular biology the presence of functional PRL receptors on thymic epithelial cells, one of the major components of the thymic microenvironment, which significantly influences early events in T-cell differentiation. Furthermore, using analytical fluocytometry, we showed that human and murine lymphoid cells also expressed PRL receptors. In both of the primary lymphoid organs, namely the thymus and bone marrow, more than 80% of cells expressed this receptor. In the periphery, all B cells and macrophages and 70% of T cells, with similar percentages of CD4+ and CD8+ cells, were PRL-R+. The density of receptors was lower on T cells than on B cells and macrophages, but this density was significantly enhanced following stimulation by T cell mitogens. These data, together with the demonstration of PRL production by thymocytes led to the hypothesis that, in addition to the classical endocrine pathway, autocrine and paracrine PRL/PRL-R interactions may exist in both central and peripheral lymphoid organs, and involve lymphocytes and microenvironmental cells.

Expression of short and long forms of prolactin receptor in murine lymphoid tissues.

Two different forms of the prolactin receptor, differing in the length of their cytoplasmic domains, have been characterized in many tissues of rodents. To better understand whether short and long forms of PRLR are involved in the immune effects of PRL, we evaluated the distribution of these different forms in the thymus, spleen, lymph nodes and bone marrow from rats and mice. Total RNA was used for cDNA synthesis which was amplified by PCR, using oligonucleotides specific to the different forms of the prolactin receptor. We detected transcripts encoding both forms of prolactin receptor in all lymphoid tissues examined in mouse and rat. Finally we studied the transcript encoding prolactin itself in these rodent tissues; a clear signal was only found in rat thymus. The ubiquitous presence of both forms of receptor transcripts in different lymphoid tissues points to an important role of the PRLR and suggests that such forms of the receptor may be involved in differential functions in lymphocytes.

Skewed V beta TCR repertoire of CD8+ T cells in murine Trypanosoma cruzi infection.

We have followed CD4 and CD8 TCR V beta repertoires during the acute phase of Trypanosoma cruzi infection in a resistant mouse strain (C57BL/6). No major changes were found in the V beta TCR distributions analyzed (covering roughly 40% of the TCR repertoire) in peripheral CD4 T lymphocytes, confirming the polyclonal nature of CD4 T cell responses. In contrast, in most animals, an over-representation of V beta 5 and V beta 14 TCR families was disclosed in the CD8 T cell compartment, superimposed on a predominantly polyclonal response. The preferential expansion of V beta 5+CD8+ T cells was also observed after infection of sensitive (C3H/HeJ, BALB/c) mouse strains. These observations suggest the existence of CD8 T cell-directed superantigenic activities associated with parasites.

Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection.

Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcR alpha beta and TcR gamma delta cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4lowCD8- and CD4-CD8low cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection.

Studies on the thymus in Chagas' disease. II. Thymocyte subset fluctuations in Trypanosoma cruzi-infected mice: relationship to stress.

Changes in thymic T-cell subsets in mice acutely infected with Trypanosoma cruzi have been studied in both C3H/HeJ and C57BL/6 mice. The significant decrease in thymocyte number, observed in both mouse strains on day 14 post-infection correlated with a drastic decrease in CD4+CD8+ cell number, whereas the number of CD4-CD8-, CD4+CD8- and CD4-CD8+ cells remained essentially unchanged. The important increase in CD3hi cell frequency confirmed that resistant thymocytes during Chagas' disease development were mostly medullary thymocytes, whereas the thymic cortex was largely depleted, as previously observed on thymus sections. This involution of the thymus could have been due to the increase of circulating glucocorticoid levels observed after infection. However, similar cell modifications were found in infected adrenalectomized mice whose serum corticosterone levels were only slightly augmented. Thus, the thymic alterations appear not to be linked to stress responses, at least those dependent on high levels of circulating glucocorticoids.

Studies on the thymus in Chagas' disease. I. Changes in the thymic microenvironment in mice acutely infected with Trypanosoma cruzi.

Previous observations demonstrated severe thymocyte depletion in mice undergoing acute Chagas' disease. These data led us to investigate the status of the thymic microenvironment in these animals. Young adult C57BL/6 and C3H/HeJ mice were infected i.p. with 10(5) blood-derived trypomastigote forms of Trypanosoma cruzi (CL strain) and killed 7-14 days after infection. Sera were then analyzed for thymic hormone (thymulin) levels, and frozen thymus sections were studied by immunohistochemistry for the expression of functional antigens (thymulin and Ia), the distribution of distinct thymic epithelial cell subsets and extracellular matrix components. Infected mice exhibited a transient decrease in thymulin production and those with severe thymic atrophy showed a denser Ia-bearing cellular network. In addition, an abnormal localization of the TR5 and CK18 antigens restricted to the medullary and cortical TEC subsets, respectively, was observed. Furthermore, an increase in the basement membrane proteins was detected within thymic lobules. We suggest that the thymic microenvironment is also affected during T. cruzi infection, extending the concept that the thymus should be regarded as a target in Chagas' disease.