Akemi Kawasaki

Perforin-secreting killer cell infiltration and expression of a 65-kD heat-shock protein in aortic tissue of patients with Takayasu's arteritis.

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of vascular cell injury in Takayasus arteritis. To clarify the immunological mechanisms involved, we examined the expression of a cytolytic factor, perforin in infiltrating cells of aortic tissue samples from seven patients with Takayasus arteritis. We also examined the expression of a 65-kD heat-shock protein (HSP-65), human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 in the aortic tissue. Immunohistochemical studies showed that the infiltrating cells mainly consisted of gamma delta T lymphocytes, natural killer cells, macrophages, cytotoxic T lymphocytes and T helper cells, and that perforin was expressed in gamma delta T lymphocytes, natural killer cells, and cytotoxic T lymphocytes. In situ hybridization analysis also revealed expression of perforin mRNA in the infiltrating cells. Immunoelectron microscopic studies demonstrated that the infiltrating cells released massive amounts of perforin directly onto the surface of arterial vascular cells. We also found that expression of HSP-65, human leukocyte antigen classes I and II, and intercellular adhesion molecule-1 was strongly induced in the aortic tissue and might facilitate the recognition, adhesion and cytotoxicity of the infiltrating killer lymphocytes. These findings provide the first direct evidence that the infiltrating cells in the aortic tissue mainly consist of killer cells, and strongly suggest that these killer cells, especially gamma delta T lymphocytes, may recognize HSP-65 and play a critical role in the vascular cell injury of Takayasus arteritis by releasing perforin.

Role of Perforin in Lymphocyte-Mediated Cytolysis

Publisher Summary This chapter discusses structure and expression of perforin. Perforin is a primary candidate as mediator of the cellular cytotoxicity exhibited by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The granule exocytosis model of lymphocyte-mediated cytolysis, wherein perforin is expected to play a central role, has been proposed on the basis of a great deal of circumstantial evidence. The central role of perforin and universality of the granule exocytosis model have been challenged based on evidence that some CTLs apparently lacking perforin expression are found and that some CTLs and lymphokine-activated killer cells lysed certain target cells without apparent granule exocytosis. The physiological relevance of the perforin-dependent and -independent pathways of lymphocyte-mediated cytolysis, mainly based on recent observations is discussed. The propriety of the perforin/granule exocytosis model has been challenged by extensive studies using various T cell clones, including classical CD8+ CTLs and recently characterized CD4+ helper/killer T cells. Perforin expression in primary CTLs in peritoneal exudate lymphocytes (PELS) after intraperitoneal immunization with allogeneic spleen cells is examined.

Microanatomical localization of PD-1 in human tonsils

PD-1 is an immunoinhibitory receptor, which belongs structurally to the CD28 family. PD-1-deficient mice show breakdown of peripheral tolerance and manifest multiple autoimmune symptoms. We previously described expression of PD-1 on activated T and B lymphocytes and myeloid cells. However, little is known about the microanatomical distribution of PD-1 in lymphoid organs. In this study, we performed immunohistochemistry using monoclonal antibodies against human PD-1. In human tonsils, PD-1 was expressed on most of T cells and a small subset of centrocytes in the light zone of germinal centers (GCs), where clonal selection of centrocytes takes place. These results suggest that PD-1 may play an important role in GC reaction.

Blastocyst MHC, a Putative Murine Homologue of HLA-G, Protects TAP-Deficient Tumor Cells from Natural Killer Cell-Mediated Rejection In Vivo

Blastocyst MHC is a recently identified mouse MHC class Ib gene, which is selectively expressed in blastocyst and placenta, and may be the mouse homolog of HLA-G gene the products of which have been implicated in protection of fetal trophoblasts from maternal NK cells and evasion of some tumor cells from NK cell attack. In this study, we identified two blastocyst MHC gene transcripts encoding a full-length α-chain (bc1) and an alternatively spliced form lacking the α2 domain (bc2), which may be homologous to HLA-G1 and HLA-G2, respectively. Both placenta and a teratocarcinoma cell line predominantly expressed the bc2 transcript. When these cDNAs were expressed in TAP-deficient RMA-S or TAP-sufficient RMA cells, only bc1 protein was expressed on the surface of RMA cells, but both bc1 and bc2 proteins were retained in the cytoplasm of RMA-S cells. Significantly, the RMA-S cells expressing either bc1 or bc2 were protected from lysis by NK cells in vitro. This protection was at least partly mediated by up-regulation of Qa-1b expression on the surface of RMA-S cells, which engaged the CD94/NKG2A inhibitory receptor on NK cells. More importantly, the bc1- or bc2-expressing RMA-S cells were significantly protected from NK cell-mediated rejection in vivo. These results suggested a role for blastocyst MHC in protecting TAP-deficient trophoblasts and tumor cells from NK cell attack in vivo.

Mouse CD94 Participates in Qa-1-Mediated Self Recognition by NK Cells and Delivers Inhibitory Signals Independent of Ly-49

Inhibitory receptors expressed on NK cells recognize MHC class I molecules and transduce negative signals to prevent the lysis of healthy autologous cells. The lectin-like CD94/NKG2 heterodimer has been studied extensively as a human inhibitory receptor. In contrast, in mice, another lectin-like receptor, Ly-49, was the only known inhibitory receptor until the recent discovery of CD94/NKG2 homologues in mice. Here we describe the expression and function of mouse CD94 analyzed by a newly established mAb. CD94 was detected on essentially all NK and NK T cells as well as small fractions of T cells in all mouse strains tested. Two distinct populations were identified among NK and NK T cells, CD94bright and CD94dull cells, independent of Ly-49 expression. The anti-CD94 mAb completely abrogated the inhibition of target killing mediated by NK recognition of Qa-1/Qdm peptide on target cells. Importantly, CD94bright but not CD94dull cells were found to be functional in the Qa-1/Qdm-mediated inhibition. In the presence of the mAb, activated NK cells showed substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic and “missing self” target cells. These results suggest that mouse CD94 participates in the protection of self cells from NK cytotoxicity through the Qa-1 recognition, independent of inhibitory receptors for classical MHC class I such as Ly-49.

Relationship of large and small CD3- CD56+ lymphocytes mediating NK-associated activities.

We have defined a population of CD3‐, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non–major histocompatibility complex (MHC)–restricted lysis, and have been equated with CD3‐ large granular lymphocytes (LGLs). In the present study we extended the observation that CD3‐, CD56+ SLs can mediate NK‐ and antibody‐dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3‐, CD56+ SLs in comparison to CD3‐ CD56+ LGLs. Our results show that CD3‐ SLs, similar to CD3‐ LGLs, exhibited activated killing in response to interleukin‐2 (IL‐2). In addition, after IL‐2 activation, the CD3‐ SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3‐ LGLs. Similar to CD3‐ LGLs, CD3‐ SLs could be directly activated by IL‐2 alone to secrete significant quantities of interferon‐γ (IFN‐γ) and to express IL‐2 receptor (IL‐2R) p55. Examination of serine esterases and pore‐forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3‐ LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL‐2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3‐, CD56+ LGLs. These CD3‐, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.

Functional characterization of mouse CD94 by using a novel monoclonal antibody

Cytotoxic activity of NK cells is regulated in delicate balance between activating signals and inhibitory signals mediated by functionally different receptor families, so-called activating receptors and inhibitory receptors. With the aim of identifying novel NK receptors, we generated mAbs by immunizing activated NK cells and selected a novel mAb designated Yuri3. Biochemical and molecular analysis indicated that Yuri3 recognizes mouse CD94. Here we explored the expression of mouse CD94 and evaluated its function on primary NK cells by using the mAb. CD94 was expressed on essentially all NK and NKT cells as well as some subsets of T cells. Two distinct populations were observed in NK and NKT cells, CD94bright and CD94dull cells, and the expression of CD94 was independent of the expression of Ly-49 family members. Addition of the anti- CD94 mAb abrogated the inhibition of the target cell lysis mediated by the NK recognition of Qa-1. Interestingly, CD94bright but not CD94dull population was responsible for Qa-1 recognition. Substantial cytotoxicity against autologous target cells as well as enhanced cytotoxicity against allogeneic target cells was observed in the presence of the mAb, suggesting that CD94 plays an important role in self protection from NK cytotoxicity and its inhibitory function is independent of inhibitory receptors for classical MHC class 1 such as Ly-49 family members.