Hidefumi Kojima

Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes

To study the contribution of putative perforin-independent mechanism in the antigen-specific target destruction by cytotoxic T lymphocytes CD8+ CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-/-) embryonic stem cells into blastocysts of RAG-2(-/-) mice. When tested on normal concanavalin A blasts, these perforin-deficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas-mediated pathway in antigen-specific cytolysis.

Mechanics of single kinesin molecules measured by optical trapping nanometry.

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.

Abnormal B lymphocyte development and autoimmunity in hypoxia-inducible factor 1α-deficient chimeric mice

Immune cells are exposed to low oxygen tensions as they develop and migrate between blood and different tissues, but the mechanisms by which lymphocytes adapt to hypoxia are poorly understood. Studies reported here of hypoxia-inducible factor 1α (HIF-1α) in lymphocyte development and functions suggest that it has a critical role in regulation of these processes. HIF-1α deficiency in Hif1α−/− → Rag2−/− chimeric mice results in dramatic and cell lineage-specific defects, which include appearance of abnormal peritoneal B-1-like lymphocytes, with high expression of B220 (CD45) receptor-associated protein tyrosine phosphatase and autoimmunity (accumulation of anti-dsDNA antibodies and rheumatoid factor in serum, deposits of IgG and IgM in kidney and proteinuria) as well as distortions of maturation of B-2 lymphocytes in bone marrow.

Cutting edge: hypoxia-inducible factor 1alpha and its activation-inducible short isoform I.1 negatively regulate functions of CD4+ and CD8+ T lymphocytes.

To evaluate the role of hypoxia-inducible factor 1α (HIF-1α) and its TCR activation-inducible short isoform I.1 in T cell functions, we genetically engineered unique mice with: 1) knockout of I.1 isoform of HIF-1α; 2) T cell-targeted HIF-1α knockdown; and 3) chimeric mice with HIF-1α gene deletion in T and B lymphocytes. In all three types of mice, the HIF-1α-deficient T lymphocytes, which were TCR-activated in vitro, produced more proinflammatory cytokines compared with HIF-1α-expressing control T cells. Surprisingly, deletion of the I.1 isoform, which represents <30% of total HIF-1α mRNA in activated T cells, was sufficient to markedly enhance TCR-triggered cytokine secretion. These data suggest that HIF-1α not only plays a critical role in oxygen homeostasis but also may serve as a negative regulator of T cells.

Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity

The acquisition of cytotoxic effector function by CD8+ T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8+ cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.

TACI attenuates antibody production costimulated by BAFF-R and CD40.

B cell activating factor of the TNF family (BAFF), plays critical roles in B cell survival, activation, differentiation, and antibody (Ab) production. BAFF binds to three receptors: BAFF‐R, transmembrane activator and calcium‐modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen. While BAFF‐R is the primary receptor for B cell costimulation by BAFF, TACI is reported to serve as a positive or negative regulator for B cell responses depending on conditions. To determine the real role of TACI in B cell responses, we examined the functional relationship between TACI and BAFF‐R in Ab production from human peripheral blood B cells using agonistic mAb. BAFF‐R and CD40 enhanced IgG secretion and B cell proliferation, which were inhibited by TACI. Although TACI induced mild B cell apoptosis, its extent did not correlate with that of TACI‐mediated inhibition of IgG secretion. In addition, TACI inhibited B‐lymphocyte‐induced maturation protein‐1 expression, IgG secretion from previously IgG‐negative selected B cells, and activation‐induced cytidine deaminase expression enhanced by BAFF‐R and CD40. Importantly, BAFF‐R and CD40 enhanced B cell responsiveness to TACI‐mediated suppression. Thus, BAFF may attenuate T cell‐independent and ‐dependent B cell responses by TACI.

Notch2 Signaling Is Required for Potent Antitumor Immunity In Vivo

CD8+ T cells play a central role in cancer immunosurveillance, and the efficient induction of CTLs against tumor Ags is required for successful immunotherapy for cancer patients. Notch signaling directly regulates the transcription of effector molecules in CTLs. However, it remains unclear whether Notch signaling in CD8+ T cells is required for antitumor CTL responses and whether modulation of Notch signaling can augment antitumor CTL responses. In this study, we demonstrate that signaling by Notch2 but not Notch1 in CD8+ T cells is required for antitumor CTL responses. Notch2flox/flox mice crossed with E8I-cre transgenic (N2F/F-E8I) mice, in which the Notch2 gene is absent only in CD8+ T cells, die earlier than control mice after inoculation with OVA-expressing EG7 thymoma cells. In contrast, Notch1flox/flox mice crossed with E8I-cre transgenic mice inoculated with EG7 cells die comparable to control mice, indicating that Notch2 is crucial for exerting antitumor CTL responses. Injection of anti-Notch2 agonistic Ab or delta-like 1-overexpressing dendritic cells augmented the antitumor response in C57BL/6 mice inoculated with EG7 cells. These findings indicate that Notch2 signaling in CD8+ T cells is required for generating potent antitumor CTLs, thus providing a crucial target for augmenting tumor immune responses.

Cytotoxic T Lymphocytes: The Newly Identified Fas (CD95)-Mediated Killing Mechanism and a Novel Aspect of Their Biological Functions

Publisher Summary This chapter summarizes the recently revealed cytotoxic mechanisms operating in the antigen-specific target cell killing by cytotoxic T lymphocytes (CTLs) and discusses the possible immune regulatory role of CTLs in antibody responses. The revelation of the two distinct pathways of killing necessitates a thorough reevaluation of a significant portion of the knowledge on specific target lysis by CTLs. Interpretation of an observation of CTL-mediated killing in conventional experimental situations in which the two killing systems could have been operating is not simple. Biochemical events leading to cell death are understood. In particular, the information on Fas-mediated cell death is extremely limited. Signal transduction in the Fas pathway is substantiated by the search for interacting proteins, as recently reported for the tumor necrosis factor receptor system. Cytotoxic factors, such as adenosine triphosphate, may have direct effects on target cell killing in a perforin- and Fas-independent manner, or they may serve as cofactors that work in concert with perforin and/or Fas. The highly specific and selective nature of CTL-mediated killing despite the nonselectivity of the effect of these molecules indicates the existence of a strict control mechanism that focuses the effector molecules solely on the specific target cells. Elucidation of such mechanisms is essential to understanding the physiological role of CTLs. A true understanding of the biolopal significance of the redundancy of killing mechanisms requires extensive studies on animals with genetic defects in one of the two systems. It is of interest to know whether the Fas pathway alone is sufficient to eliminate infection caused by intracellular pathogens, because CD4+ T cells can complement the lack of CD8+ CTLs in β2-microglobulin-deficient mice to clear the lymphocytic choriomeningitis virus infection, and the Fas pathway may play a role in eliminating infection by listeria monocytogenes in the absence of perforin.

Identification of a glioma antigen, GARC‐1, using cytotoxic T lymphocytes induced by HSV cancer vaccine

Despite several ongoing clinical trials of immunotherapies against glioma, few glioma‐specific antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified. We recently demonstrated that intratumoral inoculation with herpes simplex virus (HSV) as a cancer vaccine activates tumor‐specific CTLs. To identify glioma antigens recognized by CTLs, we used the HSV cancer vaccine to vaccinate mice harboring a syngeneic mouse glioma cell line, GL261. From the splenocytes of the immunized mice, we generated an H‐2Db‐restricted CTL line, GCL‐1, that was specific for GL261. Then, a cDNA expression library generated from GL261 was screened with GCL‐1, and a new gene encoding glioma antigen, GARC‐1, was isolated. Sequence analysis revealed that the GARC‐1 gene isolated from GL261 had a point mutation causing an amino acid change (Asp to Asn at position 81). T‐cell epitope analysis revealed that the mutated peptide GARC‐177–85 (AALLNKLYA) but not the wild‐type peptide (AALLDKLYA), was recognized by GCL‐1. These results suggest that HSV cancer vaccination may be a useful method for inducing tumor‐specific CTLs and identifying tumor antigens. Furthermore, this GL261/GARC‐1 murine glioma model may be useful for the development of immunotherapy for brain tumors.

Differentiation Stage-Specific Requirement in Hypoxia-Inducible Factor-1α–Regulated Glycolytic Pathway during Murine B Cell Development in Bone Marrow

Hypoxia-inducible factor (HIF)-1α plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1α gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1α–enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1α–deficient B220+ bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3, was greatly reduced in HIF-1α–deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory chain-related genes and TCA cycle-related genes in HIF-1α–deficient cells than in wild-type cells. In agreement with these findings, HIF-1α–deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1α–enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1α plays an important role in B cell development.