Hajime Karasuyama

Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.

Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response

A poorly immunogenic murine colon cancer was used to investigate mechanisms of antitumor immunity. Injection of tumor cells engineered by gene transfection to secrete IL-2 stimulated an MHC class I-restricted cytolytic T lymphocyte (CTL) response against the parental tumor. The tumor cells secreting IL-2 produced an antitumor response in vivo, even in the absence of CD4+ T cells. Animals immunized with the engineered cells were protected against subsequent challenge with the parental tumor cell line. Similar findings were demonstrated for other tumor types. Thus, provision of a helper lymphokine in a paracrine fashion induced a tumor-specific immune response involving activation of endogenous CTLs and other immune effector cells. These findings demonstrate that the failure of an effective antitumor immune response may be primarily due to a helper arm deficiency of the immune system rather than a paucity of tumor-specific cytotoxic effector cells. Furthermore, they outline a novel strategy for augmenting tumor immunity.

Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome

Hyper-immunoglobulin E syndrome (HIES) is a compound primary immunodeficiency characterized by a highly elevated serum IgE, recurrent staphylococcal skin abscesses and cyst-forming pneumonia, with disproportionately milder inflammatory responses, referred to as cold abscesses, and skeletal abnormalities. Although some cases of familial HIES with autosomal dominant or recessive inheritance have been reported, most cases of HIES are sporadic, and their pathogenesis has remained mysterious for a long time. Here we show that dominant-negative mutations in the human signal transducer and activator of transcription 3 (STAT3) gene result in the classical multisystem HIES. We found that eight out of fifteen unrelated non-familial HIES patients had heterozygous STAT3 mutations, but their parents and siblings did not have the mutant STAT3 alleles, suggesting that these were de novo mutations. Five different mutations were found, all of which were located in the STAT3 DNA-binding domain. The patients’ peripheral blood cells showed defective responses to cytokines, including interleukin (IL)-6 and IL-10, and the DNA-binding ability of STAT3 in these cells was greatly diminished. All five mutants were non-functional by themselves and showed dominant-negative effects when co-expressed with wild-type STAT3. These results highlight the multiple roles played by STAT3 in humans, and underline the critical involvement of multiple cytokine pathways in the pathogenesis of HIES.

Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo

Cell lines and clones were established from PB76‐positive mouse fetal liver at day 13 and 14 of gestation, which proliferated with division times of a day in serum‐substituted cultures under the stimulatory influence of adherent stromal cells and the cytokine IL‐7 for periods longer than half a year. These lines expressed varying levels of the B lymphocyte lineage related markers PB76, B220, BP‐1, VpreB and lambda 5, but no surface Ig or MHC class II molecules. All clones expressed PB76, VpreB and lambda 5 in a high percentage of cells, while B220 and/or BP‐1 expression was low or undetectable in some. A cell line, and several clones established from it, all had kappa and lambda light chain genes in germ‐line configuration. Either one or both of their H‐chain‐gene containing chromosomes carried a DH to JH. These pre B cell lines and clones could be induced to VH to DH and VL to JL rearrangements. This resulted in the development of varying percentages of sIg‐positive surface, MHC class II negative, LPS‐reactive B cells within 2–3 days, in the absence of contacts with stromal cells and/or IL‐7. When injected into SCID mice, the cultured pre B cells populated the spleen of these mice to 5% with surface Ig‐, MHC class II‐positive LPS‐reactive cells for greater than 25 weeks. The long‐term in vitro proliferative capacity of these DH‐JH rearranged pre B cell clones makes them major candidates for committed stem cells of the B lineage.

The surrogate light chain in B-cell development

The proteins encoded by the VpreB and lambda 5 genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners-the classical heavy (H) chain, the DHJHC mu-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Fritz Melchers and colleagues review the structures of the VpreB and lambda 5 genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.

Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development

The nature of signals that govern the development of immunoglobulin heavy chain-dependent B cells is largely unknown. Using mice deficient for the B cell-expressed Src-family protein tyrosine kinases (SFKs) Blk, Fyn and Lyn, we show an essential role of these kinases in pre-B cell receptor (pre-BCR)– mediated NF-κB activation and B cell development. This signaling defect is SFK specific, as a deficiency in Syk, which controls pre-B cell development, does not affect NF-κB induction. Impaired NF-κB induction was overcome by the activation of protein kinase C (PKC)-λ, thus suggesting the involvement of PKC-λ in pre-BCR–mediated SFK-dependent activation of NF-κB. Our data show the existence of a functionally distinct SFK signaling module responsible for pre-BCR–mediated NF-κB activation and B cell development.

The expression of Vpre-B/λ5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice

Precursor B (pre-B) cells in bone marrow of normal and B cell-deficient mutant mice were analyzed for the expression of Vpre-B/lambda 5 surrogate light chain (SL). The surface expression of SL is confined to the early stages (pro-B and pre-B-I) of pre-B cell development and becomes undetectable once mu heavy chain (microH) is produced. The cell-cycle analysis revealed that cytoplasmic microH+ large cells (large pre-B-II), approximately 30% of which coexpressed SL in the cytoplasm, were most actively cycling, whereas cytoplasmic microH+ small cells (small pre-B-II) were SL- and not in cycle. The analysis of pre-B cells in B cell-deficient mice suggests that the large pre-B-II stage is a critical step for the selection and amplification of cells carrying functionally rearranged microH genes.

Thymic stromal lymphopoietin–elicited basophil responses promote eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a food allergy–associated inflammatory disease characterized by esophageal eosinophilia. Current management strategies for EoE are nonspecific, and thus there is a need to identify specific immunological pathways that could be targeted to treat this disease. EoE is associated with polymorphisms in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation, but how TSLP might contribute to EoE disease pathogenesis has been unclear. Here, we describe a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils, as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, we observed elevated TSLP expression and exaggerated basophil responses in esophageal biopsies, and a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, these data suggest that the TSLP-basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat this disease.

Mouse and human neutrophils induce anaphylaxis

Anaphylaxis is a life-threatening hyperacute immediate hypersensitivity reaction. Classically, it depends on IgE, FcεRI, mast cells, and histamine. However, anaphylaxis can also be induced by IgG antibodies, and an IgG1-induced passive type of systemic anaphylaxis has been reported to depend on basophils. In addition, it was found that neither mast cells nor basophils were required in mouse models of active systemic anaphylaxis. Therefore, we investigated what antibodies, receptors, and cells are involved in active systemic anaphylaxis in mice. We found that IgG antibodies, FcγRIIIA and FcγRIV, platelet-activating factor, neutrophils, and, to a lesser extent, basophils were involved. Neutrophil activation could be monitored in vivo during anaphylaxis. Neutrophil depletion inhibited active, and also passive, systemic anaphylaxis. Importantly, mouse and human neutrophils each restored anaphylaxis in anaphylaxis-resistant mice, demonstrating that neutrophils are sufficient to induce anaphylaxis in mice and suggesting that neutrophils can contribute to anaphylaxis in humans. Our results therefore reveal an unexpected role for IgG, IgG receptors, and neutrophils in anaphylaxis in mice. These molecules and cells could be potential new targets for the development of anaphylaxis therapeutics if the same mechanism is responsible for anaphylaxis in humans.

Positive and negative selection events during B lymphopoiesis

Early in B-cell development, large numbers of cells have to be generated, each of which expresses only one type of B-cell receptor (i.e. Ig) on its surface. This is achieved by the surface expression of a pre-B cell receptor containing a mu heavy chain/surrogate light chain which differentially provides signals for two responses of precursor B cells at this stage of development. On the one hand, it signals inhibition of further rearrangements of variable heavy chain to diverse-joining heavy chain loci to achieve allelic exclusion at the heavy-chain locus. On the other hand, it signals proliferative expansion by factors between 20 and 100. Later in B-cell development, tolerance to autoantigens must be established and maintained. Tolerance is achieved by developmental arrest and induction of secondary light-chain gene rearrangements in those IgM+ immature B cells that are reactive to autoantigens presented in the primary B-cell generating organs. Even later in development, when mature surface (s)IgM+/sIgD+ B cells encounter autoantigens presented to them in the periphery, either deletion or anergy of the autoantigen-reactive cells occurs. Anergic cells have a sIg-dependent, sIg-proximal defect in signaling and are short-lived. Anergy can be broken in vitro by polyclonal activation via ligation of CD40 in the presence of IL-4. A small part of the remaining immature B cells not reactive to autoantigens are selected to become mature, antigen-reactive sIgM+/sIgD+ B cells. Molecules which might guide such positive selection of B cells still remain to be identified.