Blimp-1 is a master regulator of terminal B cell differentiation and plays a pivotal role in various developmental processes. In addition to full length Blimp-1, a Blimp-1 mRNA lacking exon 7 (Blimp-1Δ7) has been described to occur in murine B cells. The activity and function of the mutant mRNA-encoded protein (Blimp-1Δ7), lacking three crucial zinc fingers necessary for DNA interaction, is completely unknown. Since isoforms of other prdm family proteins affect each other's functions, we wondered whether Blimp-1Δ7 still plays a role in B cells, independent of direct DNA binding. In this study, we found that Blimp-1Δ7 is preferentially expressed in naïve CD19+ B cells. A fraction of Blimp-1Δ7 migrates to the nucleus, colocalizes with HDAC2 and is found at sites of repressed chromatin, although it does not bind to the Blimp-1 DNA consensus site. Unexpectedly, Blimp-1 and Blimp-1Δ7 homodimerize as well as heterodimerize with each other. Ectopic expression of Blimp-1Δ7 in WEHI 231 cells, a Blimp-1-negative murine lymphoma line, leads to cessation of proliferation and enhancement of apoptosis. Importantly, LPS-induced differentiation is suppressed in the presence of Blimp-1Δ7. This is in agreement with our finding that Blimp-1Δ7 interferes with endogenous Blimp-1 expression. Thus, our data suggest an auto-regulatory mechanism of Blimp-1 activation.

Present address: Laboratory of Molecular and Experimental Pathology, Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, P.R. China. yPresent address: Department of Genetics, University of Erlangen, D-91058 Erlangen,Germany. zShared senior authorship. Correspondence to: Thomas Hu« nig, Institute for Virology and Immunobiology, University of Wu« rzburg,Verbacher Stra e 7, D-97078 Wu« rzburg,Germany E-mail: huenig@vim.uni-wuerzburg.de

In multiple sclerosis, CD8 T-cells are thought play a key pathogenetic role, but mechanistic evidence from rodent models is limited. Here, we have tested the encephalitogenic potential of CD8 T-cells specific for the model antigen ovalbumin (OVA) sequestered in oligodendrocytes as a cytosolic molecule. We show that in these 'ODC-OVA' mice, the neo-self antigen remains invisible to CD4 cells expressing the OVA-specific OT-II receptor. In contrast, OVA is accessible to naïve CD8 T-cells expressing the OT-I T-cell receptor, during the first 10 days of life, resulting in antigen release into the periphery. Introduction of OT-I as a second transgene leads to fulminant demyelinating experimental autoimmune encephalomyelitis with multiple sclerosis-like lesions, affecting cerebellum, brainstem, optic nerve and spinal cord. OVA-transgenic oligodendrocytes activate naïve OT-I cells in vitro, and both major histocompatibility complex class I expression and the OT-I response are further up-regulated by interferon-gamma (IFN-gamma). Release of IFN-gamma into the circulation of ODC-OVA/OT-I double transgenic mice precedes disease manifestation, and pathogenicity of OT-I cells transferred into ODC-OVA mice is largely IFN-gamma dependent. In conclusion, naïve CD8 T-cells gaining access to an 'immune-privileged' organ can initiate autoimmunity via an IFN-gamma-assisted amplification loop even if the self-antigen in question is not spontaneously released for presentation by professional antigen presenting cells.

We have used the 5′ flanking sequence of the myelin basic protein gene known to include the core promoter and a strong oligodendrocyte (ODC)‐specific enhancer to target expression of the well‐studied model antigen ovalbumin (OVA) to ODC in transgenic mice. OVA protein was detected in a tissue‐ and cell‐specific manner in these "ODC‐OVA" mice. Without immunization, CD4 T cells and B cells remained ignorant of the neo‐self antigen expressed in the central nervous system (CNS), as indicated by unimpaired development and lack of activation of OVA/IAb‐specific TCR transgenic T cells in these mice, and the ability to mount normal OVA‐specific recall and antibody responses. Upon immunization with OVA in complete Freund's adjuvant, about half of the transgenic mice developed neurological symptoms characteristic of experimental autoimmune encephalomyelitis (EAE). Mononuclear infiltrates in the brain and spinal cord contained both macrophages and T cells, similar to classical models of EAE induced by immunization with CNS antigens in adjuvant. The wealth of immunological reagents available to study and manipulate the OVA‐specific response should make this new model useful for the investigation of components and mechanisms involved in CNS‐specific autoimmunity.

Abstract The transcription factor C/EBPβ transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPβ also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPβ represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPβ-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPβ. Whereas sumoylation does not affect the C/EBPβ-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPβ and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPβ.

The transcriptional repressor Blimp-1 regulates terminal differentiation of B-lymphocytes and myeloid cells. We now show that Blimp-1 is also expressed in human and murine primary T lymphocytes. Blimp-1 expression is highest in freshly isolated primary T cells with an antigen experienced phenotype. Th2 and CD4 + CD25 + cells exhibited higher levels of Blimp-1 mRNA than Th1 cells. However, ectopic expression of Blimp-1 by retroviral transduction neither altered the frequency of IFN-γ or IL-4 producing cells nor did it induce suppressor activity. In non-polarized cells, retroviral transduction of Blimp-1 led to a marked reduction in IL-2 secretion, to an inability to proliferate and to reduced viability. Our data suggest that Blimp-1 is physiologically expressed in T lymphocytes during late stages of differentiation, induces down regulation of IL-2 production and a shortened life span and might thus contribute to a limitation of T cell immune responses.

Terminal differentiation of B cells into antibody secreting plasma cells is an essential step for eliciting a successful humoral immune response. The two transcription factors B lymphocyte-induced maturation protein-1 (Blimp-1) and X-box-binding protein-1 (XBP-1) are indispensable for this process. Hereby, XBP-1 activation depends on Blimp-1. However, it is not known if Blimp-1 alone, in the absence of differentiation signals, can induce XBP-1 processing. Here we show that ectopic expression of Blimp-1 is sufficient to induce an unfolded protein response (UPR), as evidenced by the generation of the processed form of XBP-1 and upregulation of the classical UPR target BIP, in both the B cell lymphoma cell line WEHI 231 and in mouse primary splenic B cells. Interestingly, the amino terminal part of Blimp-1 comprising amino acids 1-751 was sufficient to induce the above effects while the carboxy terminal part comprising amino acids 465-856 had no effect. Taken together our results identify Blimp-1 as the upstream molecule, capable of triggering the UPR in B cells resulting in XBP-1 processing, which is an important step during plasma cell generation.

Dendritic cells (DC) are unique antigen‐presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo . Microbial stimulation induces early IL‐2 production by mouse DC. Previous reports demonstrated that IL‐2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC‐derived IL‐2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone morrow‐derived DC results in early IL‐2 production unless IL‐4 is introduced in DC cultures. Here we show that IL‐2 produced by LPS‐activated DC is dispensable for cognate T cell responses since IL‐2 loss of function DC elicit OVA‐specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL‐2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL‐2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.

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.

Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4+ T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit α enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.

A decade ago, it was discovered unexpectedly that mice defective in IL-2 signalling do not suffer from impaired T-cell development or immune responsiveness, but rather from hyperactivation of the immune system leading to the autoimmune-inflammatory "IL-2 deficiency syndrome." Here we discuss the mechanisms unravelled so far which are likely to contribute to this immunopathological disorder. Definite evidence has been obtained for a defect in the generation and/or the maintenance of "regulatory" suppressor T-cells, and for a cell autonomous defect in the susceptibility of activated CD4 T-cells to apoptosis, which normally serves to terminate immune responses by clonal contraction. In addition, an as-yet unconfirmed defect in the negative selection of autoreactive CD4 T-cells is being discussed. In conclusion, it has become clear that while redundant as a T-cell growth factor, IL-2 serves a unique role as a negative regulator of the immune system with an essential task in maintaining self tolerance.

Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymph okine expression, in particular the synthesis of IL-5. Using primary murine CD4(+) T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit a enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.

A decade after the first description of IL-2-deficient mice, the redundancy of IL-2 as a T cell growth factor is well accepted and the focus of research has shifted to the unexpected multiorgan autoimmunity and inflammation observed in mice lacking components of the IL-2/IL-2R system. So far, a set of defects at the levels of repertoire selection, the generation of suppressive regulatory T cells, T cell homing and clonal contraction via activation induced cell death (AICD) have been documented. We propose that these individual defects jointly contribute to the severe disturbance of T cell homeostasis and self-tolerance underlying the immunopathology of the IL-2 deficiency syndrome.

Hematopoietic progenitor kinase 1 (HPK1) is a member of germinal center kinases that is predominantly expressed in hematopoietic cells and transiently activated by T-cell receptor (TCR) triggering. We show here that HPK1 supports apoptosis of T cells. When HPK1 was overexpressed in murine CD4(+) T cells, a substantial increase was observed in spontaneous and TCR/CD3-mediated apoptosis as well as in Fas ligand (FasL) expression. In H2O2-treated EL-4 thymoma cells, which show an increase in reactive oxygen species (ROS) and apoptosis, overexpression of HPK1 enhanced ROS-mediated apoptosis, whereas expression of HPK1 antisense (AS) RNA impaired apoptosis. HPK1 expression also led to a sustained increase in c-Jun N-terminal kinase (JNK) activity, suggesting that JNK activation contributes to the HPK1-mediated apoptosis in H2O2-treated EL-4 cells. Under the same conditions, a rapid cleavage of HPK1 was observed, and overexpression of N- and C-terminal cleavage products in CD4(+) T cells resulted in, similar to full-length HPK1, an increase in apoptosis. In agreement with published data, we show that the C-terminal portion of HPK1 suppresses IkappaBalpha degradation, thereby inhibiting nuclear factor (NF)-kappaB activation. These findings suggest that by inhibiting the antiapoptotic action of NF-kappaB and inducing the proapoptotic activity of JNK, OHPK1 supports apoptosis in T cells.

Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.

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.

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.

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-2deficient 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-2deficient 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.