Resistance of cytolytic lymphocytes to perforin-mediated killing. Murine cytotoxic T lymphocytes and human natural killer cells do not contain functional soluble homologous restriction factor or other specific soluble protective factors

CTL and NK cells produce a cytolytic pore-forming protein (perforin, cytolysin) localized in their cytoplasmic granules. These cytotoxic cells are resistant to killing mediated by other lymphocytes and by purified perforin. A membrane factor, known as homologous restriction factor (HRF), has been suggested to confer protection to different cell types against both C- and perforin-mediated lysis. The granules of human large granular lymphocytes have been reported to contain, in addition to perforin, a soluble HRF activity that can be eluted from anion-exchange columns at 115 mM NaCl. Here, we report that a soluble HRF activity is absent in the granules or the cytosol of murine CTL and human NK cells. Our data indicate that the inhibition attributed to HRF could be explained by exogenous EDTA added during granule fractionation. EDTA was shown to bind to Mono Q and to elute at 90 to 120 mM NaCl. A second perforin-inhibitory activity was also eluted from such a column. However, it was present in preparations obtained not only from CTL and NK cells, but also from some perforin-susceptible tumor cell lines, indicating that it has nonrestricted distribution and suggesting that it is probably irrelevant to the perforin-protection mechanism. Our results argue against a role for soluble granule HRF or other soluble factors in mediating resistance of cytotoxic lymphocytes against perforin-mediated lysis.     

Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.     

Perforin mRNA in primary peritoneal exudate cytotoxic T lymphocytes

Considerable evidence indicates that cloned CTL cell lines kill target cells by releasing toxic granules that contain a cytolytic protein, called perforin, and several serine esterases (granzymes A to F). However, primary CTL, such as the highly cytolytic peritoneal exudate lymphocyte (PEL) cell population, have been found by a hemolytic assay to have no perforin, or perhaps only borderline levels of that protein, suggesting that these cells use a different lytic mechanism. To determine whether or not primary CTL express the perforin gene, we have here compared mRNA from PEL CTL and from a cloned CTL cell line, 2C, by Northern blot analysis using a perforin cDNA probe. CD8+ PEL CTL contain approximately 30% of the amount of perforin message present in 2C. Moreover, depletion of CD8+ T cells from the total peritoneal exudate cell population removes both cytolytic activity and perforin message. We have previously shown that PEL CTL elicit the same changes in target cells as cloned CTL cell lines and are resistant to lysis by the toxic granules purified from these cells lines. Taken together these results are consistent with the view that primary CTL, as well as long term cloned CTL cell lines, exercise their cytolytic activity by means of perforin.     

Expression and function of synaptotagmin VII in CTLs

The Ca(2+) sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca(2+)-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII(+/+) controls, Syt VII(-/-) animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII(-/-) mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII(+/+) or Syt VII(-/-) CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.     

Involvement of granule proteins in T-cell-mediated cytolysis

Cytolytic T lymphocytes (CTL) and large granular lymphocytes contain dense cytoplasmic granules which, when isolated, are lytic for a variety of target cells. Granule proteins are released from the effector cell upon target cell interaction, further suggesting that they play a role in the cytolytic mechanism. Major proteins in CTL granules are a family of serine esterases (granzymes) and a pore-forming protein called perforin (cytolysin). Despite structural similarities between functionally conserved regions of perforin and the ninth component of complement (C9), these two lytic molecules are clearly distinct in their mode of target cell recognition. Perforin, unlike C9, is not dependent on a protein receptor molecule but binds to the target cell membrane via phosphorylcholine in a Ca2(+)-dependent manner. Here, we discuss the stimulus-secretion model for T-cell-mediated cytotoxicity with respect to our current understanding of perforin and the granzyme proteases.     

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

Inhibition of lymphocyte mediated cytotoxicity by perforin antisense oligonucleotides.

The granule/perforin exocytosis model of CTL mediated cytolysis proposes that CTL, upon recognition of the specific targets, release the cytolytic, pore-forming protein perforin into the intercellular space which then mediates the cytotoxic effect. However, direct evidence for the involvement of perforin is still lacking, and indeed, recent results even seem incompatible with the model. To determine directly the role of perforin in CTL cytotoxicity, perforin antisense oligonucleotides were exogenously added during the stimulation of mouse spleen derived T cells and human peripheral blood lymphocytes (PBL), respectively. Perforin protein expression in lymphocytes was reduced by up to 65%, and cytotoxicity of stimulated T cells by as much as 69% (5.7-fold). These results provide the first experimental evidence for a crucial role of perforin in lymphocyte mediated cytotoxicity.     

Inhibition of the lytic activity of perforin by lipoproteins

Cytoplasmic granules isolated from cytolytic T lymphocytes (CTL) lyse red blood cells or tumor cell lines in a nonspecific manner. The activity of highly purified granules was inhibited by human or rabbit serum at dilutions as high as 1/10,000. The main inhibitory activity of human serum was isolated by chromatography and was determined to be high density lipoprotein (HDL). HDL not only inhibited at a concentration of 70 ng/ml the lytic activity of isolated granules, but also of the purified, pore-forming protein perforin present in the granules. Purified low density lipoprotein was equally active. Because the CTL granule activity was inhibited by pure egg lecithin vesicles at a concentration equivalent to the phospholipid content of lipoproteins, the lipid portion of lipoproteins is the likely candidate for granule inactivation. Lipoproteins also decreased in a dose-dependent manner the cytotoxic activity of intact cytolytic T cells. However, cytotoxicity was not completely suppressed, and only in the case of CTL exhibiting low efficiency in killing their targets. It is proposed that lipoproteins inactivate perforin and may thereby inhibit a possible lysis of innocent bystander cells.     

Differential susceptibility of type III erythrocytes of paroxysmal nocturnal hemoglobinuria to lysis mediated by complement and perforin

Previous reports have suggested that a 65 kDa membrane protein, termed homologous restriction factor (HRF), in addition to protecting erythrocytes (E) against lysis by homologous complement (C), may also be involved in protecting cytolytic lymphocytes against lysis mediated by a pore-forming protein (PFP/perforin), one of their own lytic mediators. Here, we used HRF-deficient type III E of patients with paroxysmal nocturnal hemoglobinuria (PNH) to study their susceptibility to lysis mediated by homologous C and perforin, and compared it with lysis of HRF-bearing control or PNH type I E. We show that type III E of PNH patients are indeed more susceptible to lysis mediated by homologous C than control or type I E, but they are as susceptible to perforin-mediated lysis as type I E. In addition, all human E (type I or III) tested here are equally susceptible to lysis mediated by either human (homologous) or murine (heterologous) perforin. By immunoblot analysis, we confirm that type III E, in contrast to type I E, were deficient in the 65 kDa HRF. These results support the notion that homologous species restriction is seen in the C- but not in the lymphocyte perforin-system and argue against an active participation of HRF in protecting cells from perforin-mediated lysis.     

Differential requirement for IFN-gamma in CTL maturation in acute murine graft-versus-host disease

Although IFN-gamma is the archetypal Th1 cytokine, its role in CTL maturation is uncertain. We used an in vivo mouse model of CTL development, parent-into-F(1) acute graft-vs-host disease (AGVHD), to evaluate this issue. In AGVHD, transfer of naive parental T cells into F(1) hosts stimulates the development of allospecific CTL effectors that eliminate host lymphocytes, particularly B cells. Complete elimination of IFN-gamma, using IFN-gamma-deficient donors and administering anti-IFN-gamma mAb, suppressed B cell elimination, down-regulated TNF-alpha production, and enhanced Th2 cytokine production, but did not allow the B cell expansion characteristic of chronic GVHD (CGVHD). Because complete CTL inhibition results in full-blown CGVHD that is IFN-gamma independent, these observations indicate that IFN-gamma elimination only partially blocks CTL development. IFN-gamma elimination did not inhibit donor T cell engraftment or activation in the AGVHD model, but almost completely blocked Fas/Fas ligand (FasL) gene expression, protein up-regulation, and Fas/FasL-mediated CTL killing. In contrast, IFN-gamma elimination only partially inhibited perforin gene expression and perforin-mediated CTL activity. The contributions of IFN-gamma to CTL development were indirect, because IFN-gamma receptor-deficient donor cells differentiated normally into allospecific CTLs. Consistent with the view that the Fas/FasL and perforin pathways each mediate CTL killing in AGVHD, the absence of both perforin and IFN-gamma (perforin knockout donor cells and anti-IFN-gamma mAb) converted AGVHD to CGVHD. Thus, both IFN-gamma-dependent induction of Fas/FasL and IFN-gamma-independent induction of perforin contribute to CTL-mediated elimination of host B cells in AGVHD. Suppression of both pathways is required for typical CGVHD development.     

In vivo expression of perforin by CD8+ lymphocytes in autoimmune disease. Studies on spontaneous and adoptively transferred diabetes in nonobese diabetic mice

A potent cytolytic pore-forming protein (perforin or cytolysin) has previously been found to be associated with the cytoplasmic granules of CTL and NK cells. Inasmuch as all previous studies on perforin have been conducted with cultured CTL and NK cell lines, it is not clear whether perforin may play a role in the cytotoxicity mediated by CTL that have been primed in vivo. In this study, we investigated the presence of perforin in pancreata from nonobese diabetic (NOD) mice, which have been studied as a model of autoimmune, insulin-dependent (type I) diabetes mellitus. Whereas adult NOD mice spontaneously develop diabetes, it is possible to induce diabetes in young, irradiated NOD mice by adoptive transfer of splenocytes obtained from diabetic donors. By means of immunohistochemical analysis, we were able to detect perforin Ag in a small subpopulation of CD8+/Thy-1+/asialo GM1-/CD4- lymphocytes in the pancreatic islets of animals undergoing both spontaneous and adoptive transfer-mediated insulitis. Perforin+/CD8+ lymphocytes were found in small clusters and were observed to display the morphology of large granular lymphocytes. These observations show for the first time the presence of perforin-containing CD8+ lymphocytes in tissues of animals undergoing autoimmune disease.     

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.     

Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.     

Cellular localization of perforin 1 in murine cloned cytotoxic T lymphocytes

The localization of perforin 1 (P1) in cytotoxic cells was studied by immuno-electron microscopy by using a monospecific rabbit antiserum against highly purified mouse P1 and protein A gold as a second ligand. P1 was found in specific granules of cloned cytotoxic T lymphocytes (CTL). Within the granules, P1 antigen was localized in the fine granular matrix, whereas the vesicular compartment remained free of gold particles. The amount of P1 antigen detectable by immuno-electron microscopy varied between different CTL clones. CTL with NK-like activity had the highest level of P1 antigen. A cytotoxicity loss CTL mutant had no detectable P1 antigen, suggesting an important role of P1 during cell-mediated cytolysis. P1 antigen was undetectable also in bone marrow macrophages, indicating a different cytolytic mechanism of these cells.     

Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.     

Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These include the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.     

IL-4 diminishes perforin-mediated and increases Fas ligand-mediated cytotoxicity In vivo

CTL have evolved two major mechanisms for target cell killing: one mediated by perforin/granzyme secretion and the other by Fas/Fas ligand (L) interaction. Although cytokines are integral to the development of naive CTL into cytolytic effectors, the role of cytokines on mechanisms of CTL killing is just emerging. In this study, we evaluate the effects of IL-4 in Fas(CD95)/FasL(CD95L)-mediated killing of Fas-overexpressing target cells. Recombinant vaccinia viruses (vv) were constructed to express respiratory syncytial virus M2 Ag alone (vvM2) or coexpress M2 and IL-4 (vvM2/IL-4). MHC-matched Fas-overexpressing target cells (L1210Fas+) were used to measure both perforin- and FasL-mediated killing pathways. In contrast to Fas-deficient (L1210Fas-) target cells, effectors from vvM2/IL-4-immunized mice were able to lyse L1210Fas+ target cells with similar magnitude as vvM2-infected mice. Addition of EGTA/Mg2+ revealed that effectors from vvM2/IL-4-infected mice primarily lyse targets by a Ca2+-independent Fas/FasL pathway. Analysis of FasL expression by flow cytometry showed that IL-4 increased cell surface FasL expression on CD4+ and CD8+ splenocytes, with peak expression on day 4 after infection. These data demonstrate that IL-4 increases FasL expression on T cells, resulting in a shift of the mechanism of CTL killing from a dominant perforin-mediated cytolytic pathway to a dominant FasL-mediated cytolytic pathway.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells

Cytolytic lymphocytes (CTLs) are characterized by their inclusion of cytoplasmic granules containing effector molecules such as perforin and the serine proteases. Here we describe the cDNA cloning and functional characterization of two related isoforms of a cytolytic granule protein designated TIA-1. Sequence analysis reveals that the 40 kd TIA-1 isoform (rp40-TIA-1) is structurally related to the poly(A)-binding proteins, possessing three RNA-binding domains and a carboxy-terminal, glutamine-rich auxiliary domain. The 15 kd TIA-1 isoform, the major species present in cytolytic granules, appears to be derived from the carboxy-terminal auxiliary domain of rp40-TIA-1 by proteolytic processing. Both natural and recombinant TIA-1 were found to induce DNA fragmentation in digitonin permeabilized thymocytes, suggesting that these molecules may be the granule components responsible for inducing apoptosis in CTL targets.