Ingolf Berberich

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. Consistant 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.

IL-2 and autoimmune disease.

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.

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression

T cell–specific NFAT2 deletion results in reduced CXCR5+ follicular regulatory T cells, leading to uncontrolled germinal center responses and humoral autoimmunity.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-κB control

RelB is an unusual member of the Rel/NF-κB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IκBs, the cytosolic NF-κB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IκBs. However, RelB degradation differs from that of IκBs since it is not induced by TNFα but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelBDM, site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-κB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-κB activity.

Mitochondria-Dependent Caspase-9 Activation Is Necessary for Antigen Receptor-Mediated Effector Caspase Activation and Apoptosis in WEHI 231 Lymphoma Cells

Engagement of the B cell Ag receptor (BCR) on immature B cells leads to growth arrest followed by apoptosis. Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis. Previously, we have shown that cross-linking CD40 on B cells stimulates the expression of A1, an antiapoptotic member of the Bcl-2 family, and that transduction of the murine B lymphoma line WEHI 231, a model for immature B cells, with A1 protected the cells against BCR-induced apoptosis. Here we demonstrate that A1 strongly interferes with activation of caspase-7, the major effector caspase activated after BCR cross-linking on WEHI 231 lymphoma cells. The pathway leading to activation of the effector caspase cascade including caspase-7 is unclear. Using retrovirally transduced WEHI 231 cell populations, we show that a catalytically inactive mutant of caspase-7 is cleaved almost as efficiently as the wild-type form, arguing against autocatalysis as the sole activating process. In contrast, overexpression of catalytically inactive caspase-9 strongly interferes with caspase-7 processing, poly(ADP-ribose) polymerase cleavage, and DNA laddering, suggesting a role for caspase-9 and hence for the mitochondrial pathway. The importance of the mitochondrial/caspase-9 pathway for BCR-triggered apoptosis is highlighted by our finding that both A1 and the mutant caspase-9 attenuate BCR-induced apoptosis. Thus, our data suggest that the BCR-mediated apoptotic signal in immature B cells spreads via a mitochondrial/caspase-9 pathway.

The Stability and Anti-apoptotic Function of A1 Are Controlled by Its C Terminus

Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transfered could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis.

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 μ 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.

C/EBPβ enhances IL‐4 but impairs IL‐2 and IFN‐γ 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/EBPβ in murine EL‐4 thymoma cells led to a strong induction of endogenous IL‐4 and a reduction in IL‐2 and IFN‐γ expression. Similarily, in primary murine T cells C/EBPβ 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/EBPβ 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.

SUMOylation Interferes with CCAAT/Enhancer-Binding Protein β-Mediated c-myc Repression, but Not IL-4 Activation in T Cells

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 complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant α-chain paired with β-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like α-galactosylceramide (α-GalCer). iNKT cells can be visualized with α-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-γ secretion after α-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes α-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to α-GalCer presented by rat or mouse CD1d and efficiently bound α-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to α-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent α-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Vα14) α-chains and wild-type or mutated BV8S2 (Vβ8.2) β-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.