Interleukin 2 receptor signaling regulates the perforin gene through signal transducer and activator of transcription (Stat)5 activation of two enhancers.

Optimal T cell differentiation into effector cells with specialized functions requires the participation of cytokine receptor signals. In T helper cells, this process is controlled by chromatin changes and distal and proximal regulatory elements as well as specific transcription factors. Analogous events during cytotoxic T lymphocyte (CTL) differentiation remain to be identified. This process is known, however, to be crucially regulated by interleukin (IL)-2 receptor (R) signals. It is accompanied by the induction of perforin expression via a mechanism that does not entail proximal regulatory elements. In this report, transgenically expressed human perforin gene locus DNAs demonstrate that IL-2R signals target two IL-2-dependent enhancers approximately 15 and 1 kilobase upstream of the promoter. The most distal enhancer may also respond to TCR signals. In transient transfections, both enhancers required two identically spaced Stat-like elements for their activation, which was abolished by expression of a dominant negative signal transducer and activator of transcription (Stat)5 molecule, whereas a constitutively active Stat5 molecule bypassed the requirement for IL-2R signals. These results provide a molecular explanation for the activation of the perforin gene during CTL differentiation and complement the analysis of animals deficient in the activation of the IL-2R Stat signaling pathway by establishing perforin as a target gene.

Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis.

This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose-dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL-mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell-mediated cytolysis.

Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences.

We have cloned the human perforin (P1) gene and sequenced 6.2-kb genomic DNA, containing 1.4-kb 5'-flanking region, the 5' untranslated region, the complete coding region and the beginning of the 3' untranslated region. The P1 gene including at least 95-bp 3' untranslated region is organized in only three exons: the first exon (97 bp) contains all but four nucleotides of the 5' untranslated region and was determined by primer extension and S1 nuclease mapping. This exon is separated by 1.7 kb from the second exon containing the remaining (4 bp) 5' untranslated region, the leader peptide and the N-terminal region of P1 up to--but not including--the C9 homologous region. The third exon is separated by a 1.2-kb intron and contains the remainder of the molecule, including at least 90 bp of the 3' untranslated region. This simple gene organization differs from that of the more complicated C9 gene. Because of the unusual intron in the 5' untranslated sequence the transcription initiation (cap) site is located almost 1.8 kb upstream of the ATG start signal. The more immediate 5' flanking sequence contains a CCAAT and GC box but lacks other known promoter elements. Instead, we find three different sequence repeats. One of them, a hexanucleotide sequence with the consensus GCCCTG of unknown significance occurs 19 times within a stretch of 240 bp. Further upstream we localized sequences homologous to the following enhancer and promoter elements: c-fos proto-oncogene, IFN-gamma and phorbol ester response elements, five cAMP response elements, and three motifs corresponding to general inducer elements. In addition, a sequence conserved in the 5'-flanking region of several T cell genes was identified. The 5' flanking regions of P1. CCP1 (granzyme B) and CCP2 (granzyme C) (kindly provided by Dr. Bleackley) contain as only significant homology cAMP response elements. These findings are consistent with a tight control and regulation of P1, which appears to be distinct from that of granzymes.

Structure and function of the murine perforin promoter and upstream region. Reciprocal gene activation or silencing in perforin positive and negative cells.

Gene expression of the cytolytic protein perforin is restricted to and tightly regulated in cytolytic lymphocytes. To begin to understand the molecular basis of perforin gene transcription, we cloned and analyzed 5.1 kb of the genuine murine perforin promoter and upstream region. The murine perforin promoter is located approximately 2.1 kb upstream of the translation start codon in the genomic DNA due to an intron in the 5' untranslated sequence. Although the sequenced murine promoter and upstream region was found to be quite homologous to that of the human gene, most of the interspecies conserved sequences lacked obvious consensus to known regulatory elements. Functional analysis of this region, however, indicated that it contains regulatory elements that may determine the cell-type-specific expression of this killer protein. After transient transfection into several cell lines, the perforin promoter and upstream region was used to drive the expression of the chloramphenicol acetyltransferase (CAT) reporter gene. High levels of CAT activities, exceeding 110 times the expression of a promoterless reporter gene construct, were expressed in CTL. In contrast, in perforin-negative cell types the perforin promoter and upstream region mediated barely detectable transcription of the CAT gene. Analysis of the immediate proximal perforin promoter, -120 to +2, revealed that it was ubiquitously active and that it expressed in all cells tested 20- to 50-fold higher CAT activity than the promoterless reporter gene construct. The cell-type restricted transcriptional activity of the perforin promoter and upstream region, however, was controlled by at least four negative and positive cis-acting upstream regions that spread over the entire 5 kb of the cloned DNA and acted reciprocally in different cells. Thus, in perforin-negative cells, the transcriptional activity of the immediate proximal perforin promoter was dominantly suppressed by several upstream negative regulatory elements, whereas in perforin-positive cells, the promoter activity was enhanced more than fivefold by several upstream regulatory elements.

Non-killer cell-specific transcription factors silence the perforin promoter.

Perforin is a pore-forming killer protein exclusively expressed by cells functionally defined as cytolytic lymphocytes. Previous reporter gene investigations have indicated that the cell-type-specific expression of the perforin gene is determined by the promoter and upstream region but the respective cis- and trans-acting molecules remain to be identified. In this investigation, we differentially display DNA-protein interactions of perforin-positive vs perforin-negative cell types and functionally test their biological relevance. Our results provide molecular evidence for the transcriptional repression of the perforin gene in non-killer cells by two novel regulatory elements. One of the respective transcription factors that are exclusively expressed by non-killer cells appears to be an Ets family member. Thus, the development of cells expressing perforin, and perhaps cytolytic lymphocytes in general, may involve a shutdown of the genes for these proteins.

Transgenic control of perforin gene expression. Functional evidence for two separate control regions.

Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murine perforin 5' flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Tissues not yet reported to contain perforin-expressing lymphocytes were identified. Transgene expression occurred in all cells that physiologically are able to express perforin, i.e., in T cells and NK cells, and in some T cells that normally may express little or no perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8+, as well as on CD4+ T cells. Also targeted were Thy-1.2+ gamma delta T cells, but not Thy1.2- gamma delta T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4-CD8-) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5' flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or NK cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation.

A central role of perforin in cytolysis?

Studies on the gene structure, on the transcript, and on perforin protein are reviewed, including intracellular trafficking. Perforin transcription is tightly regulated and specific for CTL and NK. Two independent pathways for perforin induction exist, only one of them being IL-2 independent. Perforin expression in vitro and in vivo correlates with the functional expression of cytotoxicity in viral infection, transplant and tumor rejection, and in autoimmunity. Perforin together with granzymes is localized in cytolytic granules. However, the trafficking of those two proteins is quite different. Since the properties of perforin containing granules encompass the characteristics of secretory granules and of lyzosomes, the term granulosomes is used to describe this unique organelle. Evidence is reviewed to refute the concept that the homologous restriction factors of complement also restrict the lysis of homologous cells by perforin.

Perforin expression in human peripheral blood mononuclear cells. Definition of an IL-2-independent pathway of perforin induction in CD8+ T cells.

Perforin gene expression upon in vitro stimulation was studied at the mRNA level in normal human PBMC and in subpopulations. Freshly isolated PBMC express low levels of perforin mRNA. Increased perforin expression is rapidly induced by the calcium ionophore A23187 and by rIL-2. Phorbolesters (PMA), by comparison, are poor inducers of perforin RNA. Perforin induction by Ca-ionophore, unlike granzyme 2 and IL-2 induction, did not synergize with phorbolesters in PBMC or in purified T cells. Instead, perforin mRNA induction by A23187 in purified T cells requires the presence of adherent cells. Ca-ionophore plus adherent cell-induced perforin occurred in CD8+ T cells and was abolished by depletion of CD8+ T cells but not by depletion of CD4+ T cells. Adherent cells alone did not express perforin under any condition. Perforin mRNA induction by both A23187 and by rIL-2 is independent of de novo protein synthesis. The half-life of perforin mRNA induced by either stimulus is approximately 100 min. Cyclosporin A completely abrogates perforin induction by A23187 but only slightly inhibits the effect of rIL-2 on perforin mRNA expression. These data show that A23187 activates perforin gene expression in CD8+ cells by an IL-2-independent pathway and that the molecular mechanism of perforin expression may be different from the one induced by IL-2. Granzyme 2 (human leukocyte protease-HLP, homologous to murine granzyme B) mRNA expression was studied in comparison to perforin. Granzyme 2 in contrast to perforin responds to the synergistic action of phorbolester and Ca-ionophore in PBMC. In addition, the kinetics of the induction of granzyme and perforin mRNA, by various signals are different. Our data suggest that situations in vivo may exist that allow perforin expression in CD8+ cells in the absence of cytokines by a combination of Ca signals and accessory receptor ligation. The same signals may not be sufficient for granzyme 2 expression in any T cell subpopulation.

Potent inhibition of CTLA-4 expression by an anti-CTLA-4 ribozyme.

Blockading the negative-regulatory CTLA-4 receptor has emerged as a powerful strategy with clinical potential to enhance T-cell responses. Some experimental tumors, for example, are rejected when anti-CTLA-4 antibodies are administered in vivo. The concise target cells and downstream events, however, remain to be defined. The development of gene transfer reagents that inhibit CTLA-4 may facilitate such investigations and may expand the therapeutic range. This communication describes an anti-CTLA-4 hairpin ribozyme that specifically abrogates CTLA-4 expression after gene transfer into a murine T-cell model. The analysis of multiple and independently derived clones and bulk cultures showed that CTLA-4 induction was inhibited > 90% at the RNA level and that it was undetectable at the protein level, with and without selective pressure. This potent inhibition required the catalytic function of the ribozyme. The anti-CTLA-4 ribozyme may be an alternative tool with which to continue the functional and therapeutical exploration of CTLA-4.

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells.

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.

Structure and function of human perforin.

Perforin (P1) is a cytolytic protein with similarity to complement component C9. P1 has been described as a unique component of murine cytolytic T-cell and rat natural killer cell granules Previous studies indicated that human granules and P1 differed from murine granules and P1 in that they appeared to be cytolytically less active and lacked the haemolytic activity characteristic of P1. It has been suggested that P1, like C9, is under the control of the homologous restriction factor. Here we determine the primary structure of human P1, re-examine its functional properties, and address the question of homologous restriction.

Perforin and granzyme B as markers for acute rejection in heart transplantation.

Histological analysis of endomyocardial biopsies (EMB) is regarded as the most satisfactory technique for monitoring crisis of rejection in heart transplanted patients. In this study, 42 biopsies from 14 patients who underwent heart transplantation were examined. Three patients did not present any rejection crisis at the date of the biopsy analysis, six were examined during an early rejection crisis (day 7-70 post-graft), and five were examined during a late rejection crisis (day 74-960 post-graft). Since granzyme B and perforin are proteins associated with cell lysis histological grading and cell phenotype analysis, in situ hybridization using granzyme B and perforin [35S]RNA probes was performed on 30 EMB to characterize the cytolytic activation of heart infiltrating cells. Our data suggest that granzyme B and perforin could be used as predictive markers for acute rejection in patients with early rejection crisis. Their detection might be an indication to administrate corticoids to resolve an acute rejection crisis. In contrast, their absence in patients with late rejection crisis appears as a good prognostic factor for the outcome of rejection and raises the question of the necessity to treat such patients with additional corticoid treatment.