Yoichi Shinkai

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.

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.

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.

Function and Control of Recombination‐Activating Gene Activitya

The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.

PROBING IMMUNE FUNCTIONS IN RAG-DEFICIENT MICE

Inactivation of recombination activating gene (RAG)-1 or RAG-2 in mice results in the inability of developing lymphocytes to initiate V(D)J recombination, leading to the arrest of lymphocyte differentiation at a very early stage. Introduction of functionally assembled antigen-receptor genes or other potentially relevant genes into the RAG-deficient background can bypass the V(D)J recombination block and promote differentiation of the lymphocytes of RAG-deficient mice to various stages. This approach offers new means for analyzing the control of lymphocyte differentiation. In addition, generation of somatic chimeric mice by injecting mutant embryonic stem cells into the RAG-2-deficient blastocysts has also provided a powerful new method for assaying the potential roles of genes or regulatory elements in lymphocyte development or function.

Molecular cloning and chromosomal assignment of a human perforin (PFP) gene.

Human perforin cDNA was isolated and the complete nucleotide sequence of the gene determined. The deduced amino acid sequence of human perforin showed 68.4% similarity to that of mouse perforin. RNA blot analysis of the human perforin gene revealed that the gene product is expressed preferentially in killer-type cells among cell lines tested, and in large granular lymphocytes among the peripheral blood mononuclear cells. In situ hybridization analysis with a human perforin cDNA probe revealed that the human perforin (PFP) gene is located on chromosome17q11-21.

Activities Involved in V(D)J Recombination

The assembly of Immunoglobulin (Ig) and T cell receptor (TCR) variable region gene segments (V, D, J) is a complex reaction that likely involves numerous components. In a simplified view, the recombination mechanism involves recognition of conserved heptamer-spacer-nonamer recombination sequences (RS) that flank each germline V, D, or J segment, Introduction of double stranded breaks between the elements to be joined and the flanking RS elements, potential loss and/or addition of nucleotides at the coding junctions, polymerization and ligation activities to complete the joining process (Fig.1; Alt and Baltimore 1982; for review see Blackwell and Alt 1989; Lieber 1991). The RS sequences that flank V, D, and J segments are sufficient to target the site-specific activities of the V(D)J recombination system to the adjacent “coding” sequences (Akira et al. 1987; Hesse et al. 1989). A notable aspect of this recombination reaction is the asymmetric processing of the coding and RS joins; the latter rarely involve nucleotide deletion and/or addition (Lieber 1991). The relative orientation of the sequences in the chromosome determines the fate of the reaction products. If the two coding gene sequences are in “opposite” transcriptional orientation the reaction will lead to inversion of the segment of DNA between the coding and RS joins with retention of all products in the chromosome. If the two sequences are in the same transcriptional orientation, the coding joins will be retained in the chromosome while the RS joins will be deleted as a circle (Okasaki and Sakano 1988; Toda et al. 1988; Fig.l). However, linear deletion products have also been observed to accumulate in thymus providing more direct evidence for the occurrence of double stranded breaks during this recombination process (Roth et al. 1992).

Mouse immunoglobulin allotypes: multiple differences between the nucleic acid sequences of the IgE a and IgE b alleles

To clarify the allotypic difference of the IgE antibody molecule, we determined the complete nucleotide sequence of the genes encoding the constant portion of mouse IgE of a (BALB/c) as well as b (B10.A) allotypes. A comparison of the sequences revealed that there were 12 single-base changes: 2 single-base changes in CH1 and CH2, 3 in CH3, and 7 in CH4. Five of them were silent changes, but seven resulted in amino acid substitutions. Although the silent changes are scattered through CH1 to CH4, the nonsilent substitutions were found only in CH3 (two substitutions) and CH4 (five). The allotypic determinant(s) that conventional antisera detect most likely reflects an amino acid difference(s) in CH3 and/or CH4.