Overexpression of the antiapoptotic protein A1 promotes the survival of double positive thymocytes awaiting positive selection.

As it has been shown for Mcl-1, Bcl-xl and Bcl-2, proteins of the Bcl-2 family play a crucial role during T-cell development in the thymus. We here show that the expression of the antiapoptotic gene A1 is specifically enhanced at the DN3/DN4 transition and in DP thymocytes that have been positively selected suggesting that A1 expression might be considered as a transcriptional signature of thymocytes that have received pre-TCR or TCR survival signal. Furthermore, we observed that A1-a overexpression in recombination activation gene 1-deficient mice transgenic for the major histocompatibillity complex class I-restricted F5 TCR enhances cell survival of DP thymocytes and permits accumulation of DP cells awaiting positive selection. However, A1-a overexpression has no effect on negative selection. Therefore, our results suggest that A1 plays a specialized role in allowing survival of DP thymocytes and that its role can be distinguished from that of Mcl-1, Bcl-xl and Bcl-2.

HIV type 1 abrogates TAP-mediated transport of antigenic peptides presented by MHC class I. Transporter associated with antigen presentation.

Downregulation of MHC class I expression following human immunodeficiency virus 1 (HIV-1) infection is thought to play an important role in viral escape from immune recognition by cytotoxic T-lymphocytes (CTLs). Since exogenous addition of HIV-1-derived peptides restores susceptibility of HIV-1-infected cells to CTL-mediated lysis, we tested whether endogenous peptide loading is impaired in these cells. Our results show that in HIV-1-infected cells the ability of the transporter associated with antigen presentation (TAP) to translocate antigenic peptides from the cytosol to the lumen of the ER for presentation on MHC class I molecules is abolished. These data suggest that interference with the supply of antigenic peptides to the MHC class I pathway provides an additional mechanism by which HIV-1 evades the CTL-mediated immune response.

Blimp-1 over-expression abrogates IL-4- and CD40-mediated suppression of terminal B cell differentiation but arrests isotype switching.

Following stimulation, primary B cells either directly undergo terminal differentiation to IgM-secreting plasma cells or enter the memory pathway characterized by affinity maturation and isotype switching. Which of the various fates is adopted by B cells is determined by the strength and duration of the antigenic signal, the availability and quality of T cell help and additional signals derived from the germinal center milieu. High rate secretion is correlated with endogenous Blimp-1 levels and can be caused by ectopic expression of Blimp-1. Using cultures of resting primary mouse B cells stimulated in vitro in various combinations with IL-4, anti-mu F(ab')2 or anti-CD40 in the absence or presence of lipopolysaccharide, we show that IgM secretion and the expression of Blimp-1 is either not induced or even suppressed by B cell receptors (BCR) or CD40 ligation and by IL-4. Additional treatment with IL-2 and IL-5 induces Blimp-1 expression and facilitates IgM and IgG1 secretion, which can also be achieved by retroviral transduction of Blimp-1. On the other hand, the drastic increase in membrane IgG1(+) cells with time in cultures treated with IL-4 is greatly diminished in cells forced to express Blimp-1. We conclude that suppression of Blimp-1 by antigen-BCR interaction and T helper cell-dependent CD40 and IL-4 signaling are necessary to facilitate entrance into the memory pathway and to prevent terminal differentiation.

Control of T cell hyperactivation in IL-2-deficient mice by CD4(+)CD25(-) and CD4(+)CD25(+) T cells: evidence for two distinct regulatory mechanisms.

In IL-2-deficient mice, antigen-activated CD4 T cells accumulate and cause lethal immune pathology. Wild-type cells of hematopoietic origin present in the same animal are able to prevent this hyperactivation of T cells, but the mechanisms and cells controlling the IL-2-deficient cells are unknown. Here we show that IL-2(-) CD4 cells with an ovalbumin-specific transgenic TCR (IL-2(-) OVAtg) undergo both clonal expansion and clonal contraction when transferred to euthymic recipients and challenged with antigen, but continuously expand in athymic hosts. Cotransfer of wild-type CD4 T cells prevents the accumulation of IL-2-deficient cells. On the residual IL-2(-) TCRtg cells CD69 and CD25 are up-regulated, suggesting that activation per se is not suppressed and that the cells had received an IL-2 signal. Since IL-2 is able to restore the defective antigen-induced cell death (AICD) of IL-2-deficient T cells in vitro, paracrine IL-2 provided by the wild-type CD4 cells may thus be able to allow clonal contraction of IL-2-deficient cells also in vivo. Interestingly however, regulatory CD4(+)CD25(+) cells also efficiently contain the clone size of antigen-stimulated IL-2-deficient T cells. Since CD4(+)CD25(+) cells do not produce IL-2, this suggests a mechanism of suppression distinct from paracrine IL-2 delivery. In keeping with this, the residual IL-2(-) TCRtg cells recovered after cotransfer of regulatory CD4(+)CD25(+) cells do not show increased CD25 or CD69 expression, suggesting that they had not received paracrine IL-2 and that clonal containment occurred at the level of initial activation rather than clonal contraction by AICD. IL-2 deficiency therefore may upset T cell homeostasis by two distinct mechanisms: the failure to program expanding T cells for apoptosis, and the failure to generate functional CD4(+)CD25(+) regulatory cells.

C/EBPbeta enhances IL-4 but impairs IL-2 and IFN-gamma induction in T cells.

C/EBP transcription factors have been described to control the activity of the human IL-4 promoter. The C/EBP binding sites within the IL-4 promoter overlap with composite NF-AT and AP-1 binding motifs. We show here that similar binding sites are part of the murine IL-4 promoter. Retroviral overexpression of C/EBPbeta in murine EL-4 thymoma cells led to a strong induction of endogenous IL-4 and a reduction in IL-2 and IFN-gamma expression. Similarily, in primary murine T cells C/EBPbeta induction resulted in an increase in IL-4 levels, whereas in human Jurkat T cells a decrease in IL-2 RNA was detected. Like AP-1, C/EBP factors belong to the large class of basic leucine zipper proteins. However, unlike AP-1, C/EBPbeta does not act in synergy with NF-AT in the induction of the murine IL-4 promoter. Instead, both factors compete in their binding to the P4/Pu-bD site, one of the most important sequence elements of the IL-4 promoter. Whereas NF-AT factors require high levels of free Ca2+ and calcineurin activity for induction, C/EBP induction in T cells is Ca2+/calcineurin independent. These observations suggest that various induction conditions lead to the activation of transcription factors, inducing IL-4 promoter activity at specific developmental stages of T cells.

Primary immune response in skin and skin-associated lymphoid tissue of interleukin-4 transgenic mice.

The interleukin-4 transgenic mice investigated here exhibit a ubiquitous expression of interleukin-4 in all organs, including the skin. In this study, the induction phase of oxazolone-induced local primary contact hypersensitivity and croton oil-induced irritant contact dermatitis in transgenic and wild-type mice was analysed. Compared to wild-type mice, the transgenic mice showed a decreased activation of the skin-draining lymph nodes but a strong hyperreactivity in the skin after topical sensitisation. In contrast to this, both the transgenic and the wild-type mice developed a strong and comparable inflammatory skin reaction after topical irritation. A striking increased expression level of tumour necrosis factor-alpha and macrophage inflammatory protein-2 genes were found in the skin of the transgenic mice during primary local contact hypersensitivity, while both the transgenic and the wild-type mice developed comparable expression levels of these cytokines during irritant contact dermatitis. Compared to wild-type mice, a strongly enhanced expression level of interleukin-6 transcripts derived from epidermal antigen presenting cells were detected in the skin of IL-4 transgenic mice, whereas in the skin-draining lymph nodes of transgenic mice significantly lower levels were detected. We conclude that the migration of epidermal antigen-presenting cells towards the skin-draining lymph nodes is reduced in transgenic mice, which could be due to the different cytokine balance in these mice strains. The atypical irritant-like reaction observed in transgenic mice after topical sensitisation is a phenomenon comparable to atopic diseases and therefore this transgenic strain might be a helpful model for investigating the immunopathophysiological features of these diseases.

Reversal of Blimp-1-mediated apoptosis by A1, a member of the Bcl-2 family.

Blimp-1 (B lymphocyte-induced maturation protein 1) is strongly expressed during the late stages of B cell differentiation to immunoglobulin-secreting plasma cells. Overexpression of Blimp-1 in B lymphoma cells has been reported to induce either growth arrest and cell death or Ig secretion and terminal differentiation, depending on the developmental stage of the recipient lymphomas. By using a retroviral expression system we show that Blimp-1-transduced immature WEHI 231 murine B lymphoma cells produce J chain, increased levels of the secretory form of micro heavy chain mRNA and secrete IgM for a short period of time. Concomitantly, they exhibit altered ratios of c-myc/mad4 mRNA levels, a reduction in the expression of the anti-apoptotic bcl-2 family member A1 and a distinct growth disadvantage, followed by cell death. Reintroduction of A1 by retroviral transduction greatly extends the life span of Blimp-1-expressing WEHI 231 cells which continue to secrete IgM. These data suggest that levels of A1 may determine the checkpoint between death and survival of Blimp-1-expressing B cells at different stages of differentiation.

A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death.

Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. We show here that cross-linking CD40 stimulates the expression of A1, a member of the anti-apoptotic Bcl-2 family, in primary murine B lymphocytes. CD40-dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line. We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein. A1-transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor. In contrast, constitutive expression of A1 did not abrogate anti-IgM-induced c-myc down-regulation. Consistent with this, A1 did not release anti-IgM-induced cell cycle arrest. Our data indicate that CD40-stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti-IgM antibodies and suggests a protective role for A1 in antigen receptor-mediated apoptosis in B cells.

Impaired thymopoietic potential of immature CD3(-)CD4(+)CD8(-) T cell precursors from SIV-infected rhesus monkeys.

Immature thymocyte subpopulations were examined for their capacity to differentiate in a newly developed xenogeneic monkey-mouse fetal thymus organ culture (FTOC) system. We provide evidence for impaired precursor function of CD3(-)CD4(+)CD8(-) thymocytes after in vivo infection with SIVmac251 as indicated by a reduced cell number per FTOC and a lower percentage of thymocytes with more mature phenotypes. Addition of recombinant SIV glycoprotein 120 (rgp120) also resulted in a dose-dependent impairment of T cell maturation in FTOC. The data suggest that in patients infected with HIV, T cell maturation and thus replenishment of peripheral pools may be compromised as a result of intrathymic infection or circulating viral gp120.

Inhibition of T cell/B cell interaction by B-CLL cells.

The course of disease in patients suffering from chronic lymphocytic leukemia (CLL) is determined by a profound dysregulation of the immune system. The resulting immune suppression is the main cause of death in those patients. In the present study we addressed the question of whether leukemic B cells (B-CLL) are able to suppress regular T cell/B cell interaction. Activated CD4+ T cell clones induce expression of the early activation antigen CD23 on B lymphocytes in vitro. Under conditions used, this B cell activation event was dependent upon direct T cell contact. Addition of certain bystander B-CLL cells or normal B lymphocytes resulted in a cell number-dependent inhibition of B cell induction. This seems to reflect the competition of B-CLL cells for a cell contact-mediated T cell helper signal. By using CD40 ligand transfected fibroblasts as a substitute for T cell help, we show that the same B-CLL cells also suppress CD40 ligand-mediated B cell activation. B-CLL cells differ in their ability to inhibit CD40 ligand-mediated B cell activation. Some B-CLL cases (eight out of 14) are unable to compete for the T cell or CD40 ligand-mediated signal, even though they can functionally interact with CD40 ligand and thereby get activated themselves. In addition, these results indicate that the observed inhibition is not a result of cell crowding by merely reducing the chance of specific B cell/T cell interactions. Collectively, these data indicate that B-CLL cells are able to inhibit the interaction of activated T lymphocytes with normal B lymphocytes in vitro. Perturbed T cell/B cell interaction may represent an important mechanism underlying the various defects of the specific immune system observed in patients suffering from B-CLL.

In vitro skewing of TCR transgenic CD4+ T cells from interleukin-2 deficient mice towards Th1 and Th2 in the absence of exogenous interleukin-2.

Previous experiments from several groups have indicated that in vitro priming for Th2 cells rigorously requires IL-4 but also depends on IL-2 [1-3]. On the other hand, IL-2 deficient mice characteristically have highly increased serum levels of the Th2-dependent isotypes IgG1 and IgE [4]. The overproduction of these isotypes is lost in IL-2 x IL-4 double deficient animals [5]. To readdress the question of a need for IL-2 for Th2 skewing in vitro we used T cells from IL-2-/- mice also transgenic for the DO11.10 TCR which is specific for OVA + IAd [6]. CD4+ cells from these mice were primed in vitro on IL-2-/- dendritic cells in the presence of OVA peptide and IL-4, IL-12 and IL-15, respectively. Following restimulation, cytokine production was analysed by intracellular staining with anti IL-4 and anti IFNgamma antibodies and flow cytometry. The data show that IL-4 primes IL-2-/- T cells for IL-4 production even in the absence of exogenous IL-2, while IL-12, as expected, polarises towards IFNgamma production. The ability to be primed for IL-4 production in the absence of IL-2 was also exhibited by naive CD4+CD62LlowTCR transgenic IL-2-/- cells and thus was not restricted to the CD44high CD62Llow cells which make up a high proportion of CD4+ cells in IL-2 deficient mice. We conclude that IL-2 is not absolutely required for in vitro skewing of naive T cells towards Th2.

The interleukin 2 receptor alpha chain/CD25 promoter is a target for nuclear factor of activated T cells.

The expression of the murine interleukin (IL)-2 receptor alpha chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions -585 and -650 located upstream of the proximal CD25 promoter. Immediately 3' from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1-like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp-mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp-deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor alpha chain promoters, both of which are regulated by NF-AT factors.

Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice.

NF-ATp and NF-ATc are the most prominent nuclear NF-AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF-ATp in vivo we have inactivated the NF-ATp gene by gene targeting in mice. We show that NF-ATp deficiency leads to the accumulation of peripheral T cells with a "preactivated" phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen-reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF-ATp-deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF-ATp-deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+/ IgD+ B lymphocytes. Contrary to the T lymphocytes from IL-2-deficient mice which develop a phenotype similar to the NF-ATp-/- mice, NF-ATp-/- T cells do not show obvious defects in Fas-mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF-ATp.

IL-2 receptor alpha-chain expression is independently regulated in primary and secondary lymphoid organs.

The IL-2R is composed of three chains: IL-2R alpha, IL-2R beta, and IL-2R gamma. In mice, IL-2Ra is critical and determines IL-2 binding to the tripartite IL-2R complex. To extend our previous studies, which demonstrated that IL-2 regulates IL-2R alpha expression in vitro, we have analyzed expression in IL-2-deficient mice in vivo. As in control animals, CD4- CD8- thymocytes and bone marrow-derived B220+ pre-B cells were IL-2R alpha positive. In contrast, activated lymph node and splenic CD4 T cells (CD4+ CD69+) were found to be IL-2R alpha negative, whereas approximately 20% of the same cell populations from the MLR/lpr strain, which also accumulate large numbers of CD4-activated T cells in the presence of intact IL-2, retained expression. A similar pattern of IL-2R alpha expression was found among splenic CD8 cells from IL-2(-/-) and IL-2(+/-) animals. These findings demonstrate that in primary lymphoid organs, IL-2 is not directly involved in IL-2R alpha expression. However, at the level of mature lymphocytes, and more specifically CD4 T cells, IL-2 remains in vivo, as in vitro, the most critical cytokine controlling both IL-2R alpha expression and sensitivity to IL-2.

Cyclosporin A interferes with the inducible degradation of NF-kappa B inhibitors, but not with the processing of p105/NF-kappa B1 in T cells.

The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.

Low-grade B cell lymphomas of mucosa-associated lymphoid tissue (MALT-type) require CD40-mediated signaling and Th2-type cytokines for in vitro growth and differentiation.

To investigate the mechanisms of T cell dependence underlying the development of extranodal mucosa-associated lymphoid tissue (MALT)-type B cell lymphomas, the activation, proliferation, and differentiation of lymphoma B cells were studied using ligand binding to the CD40 membrane receptor. The activation and proliferative response of all investigated low-grade MALT-type lymphomas (n = 6) was strongly dependent on anti-CD40-mediated signals and was complemented by cytokines produced by T helper cells of the Th2 type (interleukin-4 (IL-4) and/or IL-10). Th1 cytokines (IL-2 and/or interferon-gamma) bad little effect. Low-grade, but less so high-grade, MALT-type lymphoma B cells were induced to secrete large amounts of tumor immunoglobulin in response to IL-10. In contrast, high-grade MALT-type lymphomas (n = 5) proliferated in response to both Th2- and Th1-type cytokines and CD40 stimulation, whereas Burkitt lymphomas (n = 3) could not be rescued from apoptosis by CD40 stimulation with or without cytokines. These results suggest that CD40 signaling in combination with Th2 cytokines are essential for the development and progression of low-grade MALT-type B cell lymphoma. We conclude that T cells, which activate B cells in a CD40-dependent fashion, may contribute to lymphoma pathogenesis.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules.

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1-infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1-infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VV-expressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1-specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.

Constitutive expression of interleukin (IL)-4 in vivo causes autoimmune-type disorders in mice.

The transgenic (tg) expression of interleukin (IL)-4 under the control of a major histocompatibility complex (MHC) class I promoter leads to B cell hyperactivity in mice, characterized by increased B cell surface MHC class II and CD23 expression, elevated responsiveness of the B cells to polyclonal ex vivo stimulation, and increased immunoglobulin (Ig)G1 and IgE serum levels. Tg mice develop anemia, glomerulonephritis with complement and immune deposition in the glomeruli, and show increased production of autoantibodies. Treatment of IL-4 tg mice with anti-IL-4 neutralizing antibodies protected the mice from disease development, showing that IL-4 was responsible for the observed disorders. Deletion of superantigen responsive autoreactive T cells in the IL-4 tg mice was normal and treatment of mutant mice with deleting anti-CD4 antibodies failed to ablate the onset of autoimmune-like disease, suggesting that CD4+ T cells were not the primary cause of the disorders. Furthermore, the deletion of B cells reacting against MHC class I molecules was also normal in the IL-4 tg mice. Therefore the most likely explanation for the increased production of autoantibodies and the autoimmunelike disorders is that IL-4 acts directly on autoreactive B cells by expanding them in a polyclonal manner. Taken together our results show that inappropriate multi-organ expression of IL-4 in vivo leads to autoimmune-type disease in mice.

Systemic autoimmune disease as a consequence of defective lymphocyte death.

A major group of systemic autoimmune diseases is associated with abnormal lymphoproliferation, as a result of defects in the termination of lymphocyte activation and growth. Recent progress has been made in understanding the causes and consequences of these abnormalities. At the molecular level, the defects in CD95 and its ligand are only the most obvious reasons for the breakdown of 'clonal contraction' which in fact requires the participation of multiple gene products, including the IL-2-IL-2-receptor system, to set up a functional apoptotic machinery.