Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses.

CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4+ CD25+ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4+ CD25+ T cells from their CD4+ CD25- counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4+ CD25+ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo. Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.

Natural regulatory CD4 T cells expressing CD25.

In normal mice, a subpopulation of CD4 T cells constitutively express CD25. These cells behave as regulatory T cells in autoimmune and inflammatory reactions, in tolerance to superantigens, and in peripheral T-cell homeostasis. They are unable to produce interleukin (IL)-2, and are dependent on IL-2 for growth in vitro and in vivo. CD4 CD25(+) T cells spontaneously secrete IL-10, which is involved in some of their regulatory functions. They are resistant to apoptosis, but can be tolerized by anergy.

In vivo elimination of viral superantigen-activated CD4+ T cells: apoptosis occurs at a distance from the activation site.

Local injection of mouse mammary tumor virus (MMTV) induces a local immune response, with activation of viral superantigen (vSAG)-specific T cell subsets followed by their clonal deletion. We investigated the fate of vSAG-reactive T cells following footpad injection of MMTV(SW) to mice. Activated T cells accumulated in draining lymph nodes. However, we demonstrated that apoptosis did not occur at the activation site, on the contrary of what has been shown after bacterial SAG activation. Although activated T cells were already shown to have the capacity to migrate to the gut, the fate of gut homing cells remains unclear. We demonstrate that the number of vSAG-specific T cells activated in the periphery was increasing in the follicles of gut-associated lymphoid organs, together with the number of apoptotic cell clusters. These results strongly suggested that gut-associated lymphoid tissue was the specific graveyard for apoptotic vSAG-activated CD4 T cells.

Induction and inhibition of CD40-CD40 ligand interactions: a new strategy underlying host-virus relationships.

Interaction between CD40 and the CD40 ligand (CD40L) is required for mouse mammary tumor virus (MMTV) propagation. We found that Fas was expressed on B cells and CD40L on a small subset of viral superantigen-cognate T cells 12 h after MMTV(SW) infection. CD40L and Fas were down-regulated after 24 h. All CD4 T cells then became resistant to anti-CD3-induced CD40L induction in vitro for 2 wk. Initiation of CD40L expression and its rapid shut-off was associated with IL-12 production and was controlled by IFN-gamma and shedding of soluble CD40. These results suggest that a rapid, transient CD40-CD40L interaction involving a small number of cells is sufficient for MMTV propagation. Modulation of CD40L expression may be a major mechanism regulating the balance between viral propagation and host defenses, allowing mutual survival.

Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency.

We recently characterized a CD4+ T cell population expressing the IL-2R alpha chain (CD25), producing IL-10 and resisting clonal deletion induced by viral superantigen (vSAG) encoded by mouse mammary tumor virus [MMTV(SW)]. We now report that these apoptosis-resistant cells are generated in the thymus but not from the immature CD4+ CD8+ thymocytes. They migrate from the thymus and are found in the periphery from at least the 10th day of life, after which they expand with the same kinetics in normal and MMTV(SW)-infected mice. Their strong capacity for expansion in the periphery makes this population insensitive to thymectomy in adulthood. CD4+ CD25+ cells were totally dependent on exogenous IL-2 for growth in vitro and in vivo, and were missing in IL-2 knockout (KO) mice. The absence of this population and/or an inability to produce IL-10 may be the missing link between IL-2R alpha KO, IL-2 KO and IL-10 KO mice, which all die of inflammatory bowel disease.

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.

Changes in 1,25-(OH)2D3 synthesis and its receptor expression in spleen cell subpopulations of mice infected with LPBM5 retrovirus.

This study examines the influence of chronic retroviral infection of mice with a LPBM5 virus mixture on the paracrine system involving immune cells and 1,25-(OH)2D3 in the spleen. Plasma ionized calcium, 25-(OH)D and 1,25-(OH)2D of infected mice were unchanged. In contrast, the specific binding of 1,25-(OH)2D3 to spleen cytosol and the number of monocyte/macrophages expressing 1,25-(OH)2D3 receptors (VDR) were markedly increased. The retroviral infection also influenced the local production of 1,25-(OH)2D3 in the spleen. It did not alter this production in monocyte/macrophages but increased that in isolated T cells. Isolated B cells in control mice did not produce 1,25-(OH)2D3, but they increased the ability of isolated T cells to produce this metabolite during coculture incubations. Infection altered this cell interaction as 1,25-(OH)2D3 production in infected T cells decreased when these cells were cocultured with infected B cells. Thus, chronic retroviral infection alters both the local vitamin D metabolism and VDR expression by immune cells in mice. These findings suggest close local interactions between 1,25-(OH)2D3 and immune system activation during retroviral infection.

Massive mammary gland infection and pregnancy-dependent mammary tumor development in mice infected neonatally with mouse mammary tumor virus (SW) but not in mice infected as adults, despite a dramatic local response.

Mouse mammary tumor virus (MMTV) (SW) caused a high incidence (65%) of pregnancy-dependent adenocarcinomas in BALB/c(SW) mice infected as newborns by suckling their mothers' milk. These tumors were type B adenocarcinomas which developed early, at about 1 year of age. Uninfected breeding females and those infected at an age of 8 weeks by injection of virus had the same low incidence of malignant tumors (13%), and the tumors developed later (at approx. 23-24 months). The low incidence of tumors in adult-infected mice was correlated with partial infection of the mammary glands, and delayed transmission of MMTV(SW) to the offspring. Although the virus was rapidly disseminated in both types of infection, the responses of neonatally infected and adult-infected mice to MMTV(SW) infection and viral superantigen (vSAG) presentation were different. Activation by and presentation of the vSAG was impaired in mice infected neonatally, and tolerance induction by clonal deletion was delayed. Local activation was dramatic in mice infected as adults and clonal deletion followed rapidly. Although interaction between B and T cells is needed for completion of the virus life cycle and viral amplification, the strong local immune response to MMTV(SW) in adult-infected mice limits mammary gland infection, and protects them against mammary tumor development.

T cell deletion induced by chronic infection with mouse mammary tumor virus spares a CD25-positive, IL-10-producing T cell population with infectious capacity.

We found that T cells recognizing viral superantigen (vSAG) can be subdivided into two distinct functional subsets based on IL-2R alpha (CD25) expression. CD4+Vbeta6+CD25- and CD4+Vbeta6+CD25+ T cells were sensitive to vSAG activation. When obtained from BALB/c(SW) mice, both subsets were infected and capable to induce the tolerance process when transferred into noninfected recipients. However, in contrast to CD4+Vbeta6+CD25- cells, which were gradually deleted in MMTV(SW)-infected mice, the pool of CD4+Vbeta6+CD25+ lymphocytes was constant even at the end of the deletion process, and maintained a limited reactivity to vSAG-induced activation. The constant number of Vbeta6+CD25+ observed in infected mice could not be explained by their rapid turnover (deletion and renewal), as their proliferative rate measured by BrdU incorporation was similar in infected and naive mice, as well as in virus-nonspecific (Vbeta8.2+) cells. Neither was the Vbeta6+CD25+ subset dependent on vSAG activation since it was also present in MMTV-free mice and was not generated from Vbeta6+CD25- cells upon in vivo vSAG stimulation. Vbeta6+CD25+ T cells constitutively expressed IL-4 and IL-10 mRNA. IL-10 has been shown to be associated with viral, bacterial, and parasitic infections. This permanent CD25+ subpopulation may play a role in the control of viral infection and tolerance induction via vSAG recognition and IL-10 production.

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.

Mouse mammary tumor virus production by thymic epithelial cells in vivo.

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV-encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV-induced negative selection by showing that thymic epithelial cells do express MMTV-encoded proteins.

In vivo T cell response to viral superantigen. Selective migration rather than proliferation.

Superantigens induce T cell activation and proliferation in vitro, and some also induce cell activation in vivo. MMTV(SW) is an infectious mouse mammary tumor virus (MMTV) encoding a superantigen with the same Vbeta specificity as MIs-1a (Mtv-7), which induces a strong local response in vivo. injection of MMTV(SW) into mouse footpads leads to accumulation of superantigen-reactive T cells (Vbeta6+CD4+) and B cells in the draining lymph nodes (LN). We investigated the kinetics of this cell accumulation by measuring cell activation (blastogenesis, CD25 and CD69 expression), cell migration (using syngenic FITC-labeled CD4+ cells and L-selectin detection), and cell proliferation (using in vivo labeling with bromodeoxyuridine). Specific T cells selectively migrated to the draining LN. Accumulating Vbeta6+CD4+ T cells were large CD69+ cells, but remained CD25 negative and showed down-regulated L-selectin expression. Their DNA synthesis rate, studied by pulse labeling and continuous administration of bromodeoxyuridine, was increased, but remained too low to explain the draining LN hyperplasia. These data show that the local T cell response to MMTV(SW) mainly consists of selective migration followed by local activation of reactive T cells, and that cell proliferation is only a minor component of the response. By contrast, the optimal dose of staphylococcal enterotoxin B that, nevertheless, leads to a lower reactive T cell accumulation in the draining LN induces a very high proliferation rate.

Resistance to murine AIDS in offspring of mice infected with LP-BM5. Role of CD8 T cells.

The murine-acquired immunodeficiency syndrome (MAIDS) is caused by a mixture of murine leukemia viruses (LP-BM5 MuLV). The influence of perinatal contact with retroviruses or their Ags on the response to infection was tested by infecting with LP-BM5 (MuLV) the adult offspring of mice with MAIDS. These offspring were resistant to disease after virus challenge. Most of them were free of defective viral DNA, and even those with molecular evidence of infection had lymphoid cells with a lower infectious capacity to cause MAIDS in naive recipients. No ecotropic, xenotropic, or mink cell focus-forming (MCF) virus expression was found at the age of 5 wk, which is the time of LP-BM5 (MuLV) challenge. However, at 22 wk of age, one-half of the offspring from MAIDS mothers had ecotropic virus-expressing cells in their spleens. At the time of suckling, offspring from infected mothers had enhanced percentages of B cells and CD4 and CD8 T cells in the spleen, possibly followed by a slight persistent splenomegaly. These results suggest that immune reactivity, rather than tolerance to the virus, is responsible for resistance to disease after challenge. The offspring of MAIDS mice could clear the virus after challenge. This clearance was mediated by CD8 T cells, as continuous CD8 T cell depletion initiated at the time of viral challenge abrogated the resistance of these mice to MAIDS.

Neonatal impaired response to viral superantigen encoded by MMTV(SW) and Mtv-7.

MMTV(SW) is an exogenous mouse mammary tumor virus that codes for a superantigen sharing the same V beta specificity as Mtv-7 (Mis-1a). Neonatal mice infected by suckling-infected milk show a deletion of the CD4+ V beta 6+ T cell subset within 8 weeks. In contrast, adult mice infected by injection of the virus in the footpad have a much faster deletion, which occurs within 2 weeks. In the present work, we investigated possible mechanisms for the different kinetics of deletion in the adult and newborn mice. To find out if the route of infection could be responsible for this discrepancy, we infected 5-day-old and adult mice by injection in the footpad. Our results demonstrate that the route of infection is not responsible for the delayed kinetics of reactive T cell deletion since newborn mice injected with the virus show similar kinetics to neonates infected by maternal milk. To exclude differences in viral spreading between the two models, we used a PCR assay to detect proviral DNA. Spreading of the virus was shown to occur at a similar rate or even more rapidly in neonates than in adults. We also compared the activation induced by MMTV(SW) or Mis-1a spleen cells in the draining lymph node in neonatal and adult mice and showed that a poor local activation is induced in neonates compared with adults. In vitro, neonatal T cell reactivity to anti-V beta 6 antibody was also impaired. Thus, the delay in clonal deletion could be linked to impaired expression, presentation and/or response to the viral superantigen. Our results suggest that the initial response to MMTV(SW) could be of importance for the kinetics of reactive T cell deletion.

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.

Non-exclusive Fas control and age dependence of viral superantigen-induced clonal deletion in lupus-prone mice.

To investigate the role of Fas in the induction of tolerance by viral superantigen (SAG), we infected MRL-+/+ and MRL-lpr (Fas mutant) mice with mouse mammary tumor virus (MMTV) (SW), a virus encoding an SAG with the same specificity as endogenous Mtv-7-SAG. In normal mice, this infection has two distinct consequences on specific V beta 6+CD4+ T cells, consisting of activation followed by clonal deletion. MMTV (SW)-SAG-induced activation in vivo was identical in MRL-+/+ and MRL-lpr mice. In contrast, clonal deletion showed age-dependent impairment. Early infection (5 weeks) led to identical clonal deletion of specific T cells in blood lymphocytes from MRL-+/+ and MRL-lpr mice, although clonal deletion was slightly impaired in the MRL-lpr lymph nodes. Late infection (10 weeks) of MRL-lpr mice led to markedly delayed and reduced clonal deletion. V beta 6+CD4+ T cells which escaped clonal deletion in aging MRL-lpr mice were not anergized by interaction with SAG. These results show that peripheral clonal deletion induced by viral SAG in adult mice is controlled by Fas, but not exclusively so.

Endogenous granulocyte-macrophage colony-stimulating factor is involved in IL-1- and IL-7-induced murine thymocyte proliferation.

We have reported previously that IL-1 induces murine thymocyte proliferation in the absence of artificial comitogens, provided that the cells are cultured at high densities. In the present study, we show that, in these conditions, TdR uptake in response to IL-1 is diminished significantly by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) Abs. Indeed, a substantial production of this growth factor occurs when thymocytes are cultured in the presence of IL-1. Maximal GM-CSF levels are attained within 3 days of culture, and mRNA expression is detected after a 48-h stimulation. Both GM-CSF production and IL-1-induced thymocyte proliferation are decreased considerably by the depletion of I-A+ Mac-1+ accessory cells. Yet, addition of exogenous GM-CSF to accessory cell-depleted thymocytes does not restore the proliferative response to IL-1 alone, suggesting the implication of another accessory cell-derived mediator. Our data design IL-7 as the endogenous factor required in our culture system because: 1) GM-CSF can reverse the decrease in the proliferation after accessory cell depletion when IL-7 is provided together with IL-1, and 2) the proliferative response to IL-1 plus IL-7 is diminished as much by neutralization of GM-CSF by its specific Abs as by accessory cell removal (approximately 30%). Finally, the cells responding to IL-1 + IL-7 were identified as mature CD4-CD8-TCR+ thymocytes by the use of bromodeoxyuridine (BrdUrd), suggesting that the GM-CSF produced by thymic accessory cells in response to IL-1 participates in IL-7-dependent, intrathymic expansion of the CD4-CD8-TCR+ compartment.

Interleukin 7 induces preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4+8- murine thymocytes positively selected by class I molecules.

We analyzed the phenotype and V beta-T cell receptor (TCR) repertoire, together with interleukin 7 receptor (IL-7R) expression in unfractionated thymocytes stimulated in vitro with IL-7. This culture system results in a specific proliferation of mature thymocytes belonging to the CD3+CD4-, CD4+8-, and CD4-8+ subsets. IL-7 induced a preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4-8- thymocytes. This phenomenon is not observed in beta 2-microglobulin-deficient mice, showing that a fraction of CD4+8- thymocytes, enriched in V beta 8.2+ cells, is selected by class I molecules in normal mice, as are a large proportion of CD4-8- alpha beta TCR+ thymocytes. Our findings also establish that IL-7 plays a major role in the expansion of rare thymocyte subsets, which could exert important functions in inflammatory and immune responses.

MTS-32 monoclonal antibody defines CD4+8- thymocyte subsets that differ in their maturation level, lymphokine secretion, and selection patterns.

We have previously described MTS-32 as identifying an Ag on both thymic stromal cells and thymocytes. In contrast with CD4+8+ and CD4-8+ thymocytes, of which the vast majority are MTS-32+, a notable subset of CD4+8- thymocytes is MTS-32-. Here we show that with regard to heat-stable Ags, Qa-2, and CD69 expression CD4+8- MTS-32- thymocytes are phenotypically enriched in mature cells when compared with their MTS-32+ counterparts. Moreover, sorted CD4+8- MTS-32+ thymocytes are unable to respond to anti-CD3 cross-linking, whereas MTS-32- CD4+8- thymocytes respond to the same stimulus by producing IL-4, IL-5, IL-10, IFN-gamma, and trace amounts of IL-2. In addition, MTS-32- CD4+8- and CD4-8- TCR-alpha beta+ thymocytes differ in their TCR V beta repertoire on a Mls-1a selecting background. We therefore suggest that the MTS-32 ligand is involved in signals consecutive with TCR recognition in the thymus, i.e., selection, activation, and lymphokine production.

A kinetic study on the deletion of thymic, peripheral, and gut-associated V beta 6+ T cells in an Mls-1b BALB/c colony infected with an exogenous mouse mammary tumor virus.

Mls-1a expression results in the deletion of T cells bearing V beta 6 chains of the TCR. However, V beta 6+ T cells are also deleted in Mls-1b BALB/c mice that have been infected with an exogenous mouse mammary tumor virus (swiss mice) via maternal milk intake, and whose open reading frame region is markedly similar to that of the provirus Mtv-7. In this report we describe the kinetics of V beta 6+ T cell deletion in the thymus, spleen, lymph nodes, and gut-associated lymphoid populations of these BALB/c mice from the early weeks of life to 6 mo of age. Deletion of V beta 6+ T cells within the CD4+ T cell population was more obvious in the thymus than in the spleen at 8 wk of age. However, the earliest incidence of deletion was observed in the gut intraepithelial lymphocyte population at 5 wk of age. Furthermore Mtv-7 (SW) transcripts were only found in the gut in the first wk of life, whereafter they could be detected in the thymus, spleen, and lymph nodes. This report suggests that after entering the intestinal tract of host mice, mouse mammary tumor virus (swiss mice) is subsequently transferred to the thymus and peripheral lymphoid organs resulting in the deletion of CD4+V beta 6+ T cells in that order.