Nobukata Shinohara

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.

DNA immunization via intramuscular and intradermal routes using a gene gun provides different magnitudes and durations on immune response.

We investigated the antibody (Ab) and cytotoxic T lymphocyte (CTL) responses to gene gun (GG)-mediated DNA immunization via the intramuscular (i.m.) and intradermal (i.d.) routes. BALB/c mice were immunized five times at weekly intervals with plasmid DNA encoding enhanced green fluorescent protein (EGFP). EGFP production was rapidly detected in the target tissues after injection via either delivery route. There were significant differences in the magnitude and duration of the Ab and CTL responses according to the route employed. Intradermal injection elicited higher Ab and CTL responses to EGFP than i.m. injection 1 week after the last immunization. However, both immune responses were reduced rapidly 5 weeks after the last immunization via i.d. injection. In contrast, in mice injected via the i.m. routes, Ab and CTL responses 5 weeks after the last immunization remained at levels similar to those detected after 1 week. All mice generated a predominantly IgG1 Ab response via either route. These findings suggest that a combination of these two routes of DNA immunization would provide optimal conditions for induction of a broad immune response, and this information is expected to be very important for future applications of DNA vaccination.

Ectopic expression of a T-box transcription factor, eomesodermin, renders CD4+ Th cells cytotoxic by activating both perforin- and FasL-pathways

During viral infection, CD8(+) cytotoxic T lymphocytes (CTL) play a central role to eliminate viruses by destructing virus-infected cells utilizing two cytolytic pathways, i.e., perforin/granzyme pathway and FasL-Fas pathway. It has been shown that effector functions of CTL are critically controlled by two T-box transcription factors, T-bet and eomesodermin (Eomes), although their precise activities in constructing CTL functions are not fully understood. To investigate the functional potency and activities of Eomes, the effects of ectopic expression of Eomes in two terminally differentiated murine CD4(+) Th lines, on their effector functions were analyzed. The results showed that in Eomes-transfected Th hybridoma, cell surface FasL expression upon Con A stimulation was markedly enhanced, although perforin expression was not induced. In normal, non-transformed Th2 cells, introduction of Eomes elicited perforin expression, and also augmented FasL up-regulation. Interestingly, cyotlytic activity of Eomes-transfectant was more efficient than that of perforin-transfected Th2 cells which expressed high levels of perforin and granzyme B mRNA, indicating that Eomes may play additional roles other than preparation of these cytolytic effector molecules. In contrast, stimulation-induced CD154 up-regulation, one of the typical helper T cell characteristics, was repressed in Eomes-transfectant. Collectively, these results suggest that Eomes may not only be involved in perforin/granzyme expression but also play various functions, including FasL up-regulation, to develop the characteristics of CD8(+) CTL. These studies have also suggested that introduction of Eomes alone was sufficient to convert the functions of fully differentiated Th cells toward those of CTL.

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.

Full reconstitution of hematopoietic system by murine umbilical cord blood.

Background. Murine umbilical cord blood cells (UCBCs) were studied for their ability to reconstitute the hematopoietic system. Methods. On average, 150 &mgr;L of cord blood per fetus containing 1.2 to 2×104 nucleated cells were collected from day 18.5 fetal umbilical cord, and 3 to 6×103 cells per fetus were obtained after separation by gradient centrifugation. Results. Although lineage marker−, c-Kit+, and Sca-1+ cells were detectable among UCBCs, cells designated to be in the side population (SP) by Hoechst 33342 staining were hardly detectable within this population; the frequency of cells of this phenotype was less than 1 of 105. Instead, the lineage marker−, c-Kit+, and Sca-1− population contained a considerable number of SP cells. Nevertheless, UCBCs obtained from fetuses of green fluorescent protein-transgenic mice successfully reconstituted the blood cells of lethally irradiated recipients. Fluorescent cells could be readily detected in every blood cell lineage and among immature cell populations. Furthermore, fluorescent SP cells sorted from the recipient bone marrow cells could also reconstitute the blood cells in the secondary recipients, indicating that UCBCs also replenished bone marrow stem cells. Conclusion. Murine UCBC could fully reconstitute the hematopoietic system of lethally irradiated recipients including hematopoietic stem cells in bone marrow.

Cross-Positive Selection of Thymocytes Expressing a Single TCR by Multiple Major Histocompatibility Complex Molecules of Both Classes: Implications for CD4+ versus CD8+ Lineage Commitment

This study has investigated the cross-reactivity upon thymic selection of thymocytes expressing transgenic TCR derived from a murine CD8+ CTL clone. The Idhigh+ cells in this transgenic mouse had been previously shown to mature through positive selection by class I MHC, Dq or Lq molecule. By investigating on various strains, we found that the transgenic TCR cross-reacts with three different MHCs, resulting in positive or negative selection. Interestingly, in the TCR-transgenic mice of H-2q background, mature Idhigh+ T cells appeared among both CD4+ and CD8+ subsets in periphery, even in the absence of RAG-2 gene. When examined on β2-microglobulin−/− background, CD4+, but not CD8+, Idhigh+ T cells developed, suggesting that maturation of CD8+ and CD4+ Idhigh+ cells was MHC class I (Dq/Lq) and class II (I-Aq) dependent, respectively. These results indicated that this TCR-transgenic mouse of H-2q background contains both classes of selecting MHC ligands for the transgenic TCR simultaneously. Further genetic analyses altering the gene dosage and combinations of selecting MHCs suggested novel asymmetric effects of class I and class II MHC on the positive selection of thymocytes. Implications of these observations in CD4+/CD8+ lineage commitment are discussed.

Cell-Mediated Fas-Based Lysis of Dendritic Cells Which Are Apparently Resistant to Anti-Fas Antibody

In this report, the controversy concerning the sensitivity of dendritic cells (DCs) to Fas‐dependent induction of apoptosis was examined using murine DCs. Although DCs could not be lysed when exposed to an anti‐Fas antibody, Jo2, the observed resistance turned out to reflect their lack of the expression of FcγR necessary for crosslinking the antibody, rather than their intrinsic resistance. Thus, at least a fraction of DCs was sensitive to Jo2 in the presence of FcγR‐expressing by‐standers. Consistently, a significant fraction of DCs was sensitive to Fas‐dependent lysis mediated by T cells including the antigen‐specific killing by CD4+ T cells. Both immature (class II MHClow) and mature (class II MHChigh) DCs were sensitive to the Fas‐based induction of apoptosis.

Effective blocking of natural cytotoxicity of young rabbit serum on murine thymocytes by high concentration of glucose in complement-dependent cytotoxicity method

By an accidental observation, the natural cytotoxicity of 21-day-old rabbit sera to murine thymocytes was found to be almost completely inhibitable by 4.5 mg/ml glucose. This enabled us to use unabsorbed young rabbit serum as a source of complement for antibody-mediated cytotoxic treatment of murine thymocytes. This glucose-sensitive natural cytotoxic activity was recovered in the immunoglobulin fraction purified on an affinity column, and was dependent on complement activity. The unabsorbed 21-day-old rabbit sera had much higher complement activity than those from 14-day-old rabbits and commercially available low background complement. Indeed, complete depletion of CD4+ cells from murine thymocytes and splenocytes was achieved using unabsorbed 21-day rabbit serum as a source of complement and a monoclonal antibody GK1.5 which has been claimed to be inefficient in complement-dependent killing. Owing to the increase in complement titer and circulating blood volume 14-21 days after birth, the use of 21-day-old rabbit sera assures a much more abundant supply of complement for the treatment of murine lymphocytes.

Cognate interaction plays a key role in the surveillance of autoreactive B cells in induced mixed bone marrow chimerism in BXSB lupus mice

The effects of bone marrow transplantation (BMT) as a treatment for and/or preventive measure against autoimmune diseases in mice were investigated extensively. The reconstitution of the hematopoietic system with a mixture of autologous and heterologous bone marrow cells was reported to suppress the development of autoimmune diseases. However, the pathological mechanism through which mixed chimerism results in the suppression of disease development is still unknown. We have previously reported that the induction of fully major histocompatibility complex (MHC)-mismatched allogeneic mixed chimerism can prevent the disease development in BXSB mice. Interestingly, serum anti-dsDNA IgM antibody (anti-DNA IgM) levels were not significantly decreased in these chimeric mice, though other symptoms of autoimmune disease were ameliorated. In this study, we showed that self-reactive anti-DNA IgM production was mainly attributable to genetically normal B cells from the donor rather than genetically deficient B cells from the host. Host-type B cells responded normally to foreign antigens and produced the appropriate antibodies. BMT from fully MHC-matched or haplo-identical donors could suppress the production of anti-DNA antibodies. Our present study suggests the existence of a surveillance system dependent on the recognition of MHC molecules on B cells.

Significant involvement of nuclear factor-κB-inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotypehigh+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8low+ idiotypehigh+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.