cell-mediated cytotoxicity: contact and secreted factors

Cell-mediated cytotoxicity: contact and secreted factors

Sergei Apasov, Frank Redegeld and Michail Sitkovsky

National Institutes of Health, Bethesda, USA

The list of cells with cytotoxic potential now may include small resting

T cells, but the exact nature of ‘lethal hit delivery’ by cytotoxic T lymphocytes remains elusive. Cell-mediated cytotoxicity by cytotoxic T

lymphocytes is a complex, multistep process which seems likely to be mediated by several different pathways. Recent experimental evidence

for the functioning of a novel cytotoxic mechanism through a target cell’s surface receptor illustrates and emphasizes the necessity to study

the interactions of cytotoxic T lymphocytes and target cells as a whole.

Progress is evident in the description of molecular requirements for

triggering cytotoxicity, cellLcell contacts and the regulation of the effector responses of cytotoxic T lymphocytes by extracellular, intracellular and

granular proteins. Extracellular Ca 2+-dependent secretion of perforin and protease may explain several aspects of cellular cytotoxicity, whereas the apoptosis-mediating cell surface Fas protein is now implicated in

Ca2+-independent cytotoxicity.

Current Opinion in Immunology 1993, 5:40+410

Introduction

Cytotoxic T lymphocytes (CTIs) are believed to function via their ability to kill target cells that bear antigen and to secrete different lymphokines, including interferon (IFN)-y. These responses of CTIs, which are initiated and propagated during their initial interactions with target cells, include the following: ‘productive’ conjugate formation; the recognition of antigen by the T-cell receptor (TCR) of CTIs; transmembrane triggering; ‘lethal hit’ delivery to the target cell; and activation of cytoplasmic processes (e.g. granule exocytosis) and nuclear events in the CTL as well as in the target cell.

The latest developments in these closely interrelated fields of cellular cytotoxicity studies bring us closer to settling some of the most hotly debated issues and these are discussed below.

Surface contact requirements in cytotoxic

response and adhesion/co-receptor molecules

in CTL-target cell interactions

On the basis of in vitro studies it is believed that CTIs make contact with most surrounding cells. Although there is a predominance of non-lethal engagements even with the antigen-bearing target cell [ 11, only the antigen- bearing target cells are able to trigger the lethal hit via TCR crosslinking.

Important insights into the role of CD8+ CTIs in vivo are expected from studies of mice deficient in CD8 + cells or @-microglobulin ( p2m-/- ) obtained using the gene ‘knock-out’ approach. Studies of p2rn-/- mice [2] con- firm the important role of the MHC class I antigen in the generation of CD8+ CTIs. However, the P2rn-/- mice should be used with caution in studies of the in vivo role of CD8+ CTIs, as normal numbers of highly lytic CD8+ CTIs directed against MHC class I molecules are detected in &rn-/- mice after intraperitoneal immunization with tumor cells [3].

The mechanism of target cell recognition and triggering of specific cytotoxicity in CD8+ CTIs based on the presentation of peptide in association with MHC class I molecules and recognition by TCR is further conlirmed. Such recognition is reflected in the ability of CTIs to kill each other if synthetic peptides recognized by CTLs are added [ 41, providing a convenient and simple method for a rapid visual assay of the specificity of CTIs by screen- ing for CTL-CTL self-killing induced by synthetic peptides [51.

The critical determinant of CTL activation by antigenic peptides seems to be occupancy of highly localized receptors (TCR and CD8) over a contiguous region of the cell surface as a large number of small artificial antigen- presenting particles were not able to mimic the triggering of CTIs by large particles (4 to 5 pm diameter) [6*]. This may reflect the existence of ‘safeguards’ against CTL ‘mis- firing’ due to the binding of subcellular fragments and points to the need for the high local concentration of

Abbreviations f&m-f& microglobulin; CsA-cyciosporin A; CTL-cytotoxic T lymphocyte; IFN-interferon; IL-interleukin;

mA&monoclonal antibody; NK-natural killer; W-CAMP-dependent protein kinase; TCR-T-cell receptor.

404 @ Current Biology Ltd ISSN 0952-7915

Cell-mediated cytotoxicity Apasov, Redegeld and Sitkovsky 405

second messengers. The CD8 molecules are considered as especially important both as adhesion proteins and as co-receptors by enhancing the contact interactions of CTLs with target cells that express low levels of MHC class I molecules or CTIS that have low affinity TCRs for the antigenic peptide [7,8]. Indeed, interactions between both CD8 and class I MHC molecules, and lymphocyte function-associated antigen-l and intercellular adhesion molecules are triggered through the activated TCR and involve the cytoskeleton [9].

The in uiuo role of CTL adhesion molecules in CTL-target cell interactions, the migration of CTLs and their arrival near target cells is now better understood; a correlation has been reported between the protective activity of CTIs in viva and levels of expression of the adhesion molecules CD44 and very late activation antigen-4 [ 10’1. In addition, Tanaka et al. [ ll*l suggest a novel role for cytokines bound to proteoglycan and associated with the endothelium (e.g. macrophage inflammatory protein-la) in augmenting the integrin-mediated adhesion of CD8+ CT&

Recently, the co-stimulatory molecule CD28 was implicated in the activation of cytotoxicity mediated by small resting T lymphocytes [12*-l, suggesting the presence of pre-existing CTLs or rapidly activatable CTL precursors (‘paramedics’ of cellular immune responses?), which re- quire stimulation with anti-CD3 monoclonal antibody (mAb) (or with anti-CD2 mAb) and co-stimulation by CD28-B7 interactions for their cytotoxicity.

Biochemistry of CT1 triggering by CTL-target

cell interactions

Intracellular signals generated by TCR crosslinking by antigen-MHC complexes on target cells are propagated by such early participants as tyrosine protein kinases and by serine/threonine kinases (e.g. protein kinase C) [13-161. However, the tyrosine phosphorylation of TCR CD3 chains may be not absolutely required for CTL triggering since the &chains of CD3 are not phosphorylated i:aj, but are nevertheless able to redirect cytotoxicity

The ‘late’ transmembrane signaling events and ‘off signals (which allow CTLs to stop the lethal hit delivery and/or to disengage from target cells have been the subject of studies due to their decisive role in the ex- ecution of the effector function. The CAMP-dependent protein kinase (PKA), previously shown to function as a down-regulating ‘off signal in the regulation of both granule exocytosis and of cytotoxicity [ 191, has been dim rectly implicated in the propagation of TCR signals for IFN-)I gene expression and secretion by CTIs. This indicates that PKA is involved in both ‘off and ‘on’ signaling and thus has a dual role in CTL responses [ 201. Thus, the enhancement of CTL cytotoxicity triggered by TCRs and of granule exocytosis, and simultaneous inhibition of the production of IFN-)I were observed after pretreatment of CTLs with the Ca catalytic subunit of PKA encoded

by antisense mRNA oligos [20]. A similar effect was observed when the inhibitor of serine/threonine phosphatases (okadaic acid) was used [ 211. These are unusual effects as to our knowledge this is the first demonstration that it is possible to enhance CTL cytotoxicity and granule exocytosis induced by target cells by interfering with the activity of individual enzymes (PKA [ 201 or PP2A protein phosphatase [21]).

It was discovered recently [ 221 that immunosuppressants cyclosporin A (CsA) and FK506 have the ability to in terfere in T-cell transcriptional events by blocking the activity of the Ca* +/‘c~llodulin~dependelit phosphata,se calcineurin (the major calmodulin-binding protein in 1’ lymphocytes [23]). This provides direct evidence of the involvement of this protein phosphatase in the regulation of TCR-triggered cytotoxicity, which is independent of protein synthesis, granule exocytosis and IFN-y pro duction, which is dependent on protein synthesis, as these CTL responses were previously shown to be inhih ited by CsA [ 1,241,

CTL-induced changes in the target cell and the

contribution of target cells to their own death

There are additional indications that target cell intracel~ lular processes (e.g. DNA fragmentation as a part of the apoptotic process of target cell death [ 251) contribute to CTL-induced death either by triggering a programed cell death pathway (the ‘internal disintegration‘ model [lOj ) or by counteracting the effects of the lethal hit leading to the exhaustion of target cells due to the depletion of intracellular ATP [ 271. As yet another example of the ac tive role of target cells in interactions with CTLs [2x] the Na+,/Ca2+ exchanger is postulated t(_) play ;I role in the resistance of target cells to perf(~rin~‘c)~ol~sin-indu~~ti (1’ tolysis, which determines whether a target cell will sun?\,e the damage to the plasma membrane. The induction of stress (e.g. heat shock) induces target cell resistance to the CTL lytic hit [29] leading to speculation that ill IYIY~ starvation of tumor cells (severe stress condition) ma) result in their resistance to CTL attack.

Judging by the number of studies of CTI. induced DN.\ degradation many investigators still consider this to bc an indispensible event in CTL-induced target cell deatll even though evidence that contradicts this vie\v is ;I< cumulating [30,31]. Thus, the addition of inhibitors ot DNA topoisomerase I and II [30] inhibited DNA frag!- mentation in the target cell in a dose-dependent nlan ner without affecting CTL-mediated cytotoxicity Clearly, in this case the nuclear disintegration component is not required for the disruption of the target cell membranes and for the subsequent death of the target cell. It has also been shown that CTIs can induce both apopto sis and necrosis in target cells [32], and that zinc (an inhibitor of nuclease activity) can block DNA fragmentation without blocking target-cell lysis. Finally. using virall!, transformed murine fibroblast clones as targets for CTIs [ 311, clones were found that were susceptible to lysis b!.

406 lymphocyte activation and effector functions

CTLs and exhibited all manifestations of cell death, but failed to show genome digestion. This was shown to correlate with a deficiency in Ca2+ -dependent DNase I like endonuclease activity,

The characterization of Ca2+/Mg2+-dependent endonuclease activity, which could be involved in nuclear DNA degradation after the attack by CTIs resulted in the finding that it is identical to or has the same properties as DNase I [ 331. It is suggested that this enzyme gains access to the nucleus after the breakdown of the endoplasmic reticulum and nuclear membrane [33].

The number of granule proteins that have interesting en- zymatic activities in vitro is growing with the demonstration that the polyadenylate-binding protein TIA-1 induces DNA fragmentation in permeabilized target cells and, therefore, could be involved in the induction of programed cell death in target cells after attack by CTLs [34]. The finding that three cytolytic granule proteases have fragmentin activity [35] indicates that CTLs may have specialized ‘weapons’ for any particular target cell that cause unique patterns of DNA fragmentation in different target cells. Thus, the susceptibility of any individual target cell to lysis by a given CTL may be determined by the relative abundance of the particular fragmenting protease (to which the given target cell is especially sensitive).

Resolution of the two opposing views on the role of CTL-triggered DNA fragmentation in target cell death may come from conllicting results on the role of polytADPrybose) polymerase (PADPRT) in target cell death. An attractive model of target cell death due to CTL- induced DNA fragmentation, which can activate PADPRT and may, in turn, exhaust the intracellular pools of NAD and ATP, was given support using both inhibitors of PADPRT and PADPRT-deficient target cell mutants [ 281. Considered together with the results of Ucker [3I] it seems that the relative contribution of different C&induced and potentially lethal processes in target cells is dependent on the particular biochemical make-up of a given target cell.

Mechanisms of CTL-mediated ‘lethal hit’ triggering

The differences in molecular requirements between CTL cytotoxicity, perform exocytosis and pore formation [l] have yet to be resolved in direct experiments, but extensive efforts have been undertaken to distinguish between two major alternatives for CD8+ CTL-mediated target cell lysis: killing due to the triggering of target cell surface receptor by CTIs mediated signaling for target cell disintegration [1,36], and target cell death triggered by com- promising the integrity of the plasma membrane due to pore formation by perform released by CTL and natural killer (NK) cells [37]. The consensus is, however, that more than one mechanism may account for CTL-mediated cytotoxicity and a recent study of the cytotoxicity of different human CD4+ and CD8+ CTL clones strength-

ened the view that activated CTLs utilize several different cytotoxic mechanisms [ 381. Similarities and differences between CD4+ and CD8+ CTIs were the subject of several recent studies and it was shown that the normal T-cell response against HIV viral proteins (e.g. gp120 and gpl6O) could be mediated not only by CD8+ CTIs, but also by CD4+ CTIs [39]. Lysis by CD4+ CTLs is characterized by the same time course, morphological changes and DNA degradation in the target cell [40] as described for CD8+ CTIs. However, in contrast to CD8+ CTIs, the CD4+ CTIs deliver lethal hit relatively quickly and some CD4+ CTIs are able to kill bystander cells that do not bear antigen suggesting the release of a lytic activation-induced cytotoxic factor that has a short range and is quick acting [40]. The molecular nature of the CD4+ CTL-derived cytotoxic factor is not known and the cell surface form of tumor necrosis factor-cl was recently ruled out as the lytic intermediate for at least some CD4 + CTLs [ 391. The dominant and widely accepted model of cytolytic granule exocytosis was further supported by studies of perform and perforingranzyme transfectants [41--l. Direct experimental support was recently provided for the receptor triggered disintegration model by implica- tion of the Fas protein in Ca2+ -independent target cell lysis [42*-l.

Granule exocytosis model

According to the current view of the granule exocytosis model (P Henkart, personal communication) [41*,43] a polarized secretory event in the CTL is triggered by its surface receptors resulting in the generation of second messengers, cytoplasmic polarization and the transloca- tion of the bulk of the secretory granules toward the synapse-like junctional cleft formed between the CTL and target cell. After exocytosis the high local concentration of granular components in this cleft enables them to diffuse into the target cell cytoplasm if plasma membrane permeabilization takes place due to the perform cytolysin. Such permeabilization can cause sufficient ionic imbalance to lead to the death of the target cell, but can also provide access for other granule-derived mediators. Significant support for this model and the explanation of the apoptotic character of target cell death is provided by the recent demonstration (which is predictable, and expected in view of similar previous experiments using purified proteases and perform [44,45]) by Shiver and colleagues [41**] that serine protease granzyme A causes the breakdown of target cell DNA when secreted along with perform during membrane-triggered degranulation of double-transfected RBL cells. The wide acceptance of granule exocytosis model is reflected in the extensive efforts to further characterize both previously described and novel components of granules. An immunohistochemical study of CTL granules showed they are related to Iysosomes, which have many small internal vesicles and a dense core [46]. With

Ceil-mediated cytotoxicity Apasov, Redegeld and Sitkovsky 407

respect to the distribution of perform and granzymes they appear to be a homogeneous population. A recently described major component of CTL granules is the calcium-binding protein calreticulin, which was shown to co-localize with perform in the cytolytic granules, and is secreted upon TCR stimulation [47]. Calreticulin was suggested to act in a chaperon-like manner, protecting perform from inactivation by keeping it in its metastable, globular conformation.

Perforin expression and properties

Attempts to correlate the expression of perforin with CTL activity in viva are divided into the experiments designed to demonstrate release of perforin related to cytotoxicity and the tissue distribution of perforin-expressing CTIs. Indeed, the proportion of cells containing per forin among the alloreactive CTLs is greatly diminished after in vitro stimulation of the TCRCD3 complex by anti-perform mAb, indicating the release of the per for-in after CTL activation [48]. A correlation has been described between the cytolytic activity exhibited by NK cells and CD8+ CTIs and between the expression of perform [49]. It has been shown that perform can lyse oligodendrocytes that synthesize the myelin sheets enfolding segments of neighboring axons. The damage of oligodendrocytes is implicated in the mechanism of several diseases. Increases in the level of Ca2+ induced by per-for-in are invoked to explain the mechanism of de- myelination due to CTL attack [ 501.

Per-for-in has been detected in infiltrating CD8+ T cells during virus infection, autoimmunity, transplant rejection, tumor rejection [43] and in the synovial fluid of patients with rheumatoid arthritis [ 511. Perform and granzyme A are currently being explored as predictors and markers for rejection in cardiac transplantation [ 521.

While these results are interpreted as support for the role of perforin in the qtotoxicity of CTIs in vivo it is still possible that the detection of perform in vivo is not related to the cytotoxic event but reflects the presence of activated CTIs. Indeed, it seems that an upstream silencer is responsible for the regulation of perform expression and that this silencer is specifically inactivated during the activation of CTLs and NK cells [53]. The existence of two independent pathways is suggested for control of perform expression [48]: first, an increase in the level of intracellular Ca2+ induces a rapid increase in perform expression and upregulates interleukin (IL)-2 receptor expression; second, the presence of IL-2 and of up-regu- lated IL-2 receptor will result in additional enhancement of Ca2+ -independent perform expression.

Expression and functional role of granular proteases

Although the most compelling data with granzyme A and perforin double transfected RBL cells implicate pro-

teases in DNA fragmentation of perforin-permeabilized target cells [41], it is also possible that the proteases of CTIs could be involved in the processing of other granular components [ 54,551, interactions with nuclear proteins in target cells [ 561, pore formation ([57,58]; P Henkart,personal communication), DNA degradation [45] and the arrest of the cellular proliferation of target cells without their death [ 591. Indeed, the inactivation of lymphocyte granule proteases by specific protease inhibitors resulted in the reduction of lysis by granule extracts and perforin [ 571. Similarly, diminished cytotoxicin of CTLs toward target cells was observed when the expression of granzyme A was inhibited by transfection of a CTL clone with antisense mRNA for granzyme A [ 581. In addition, the protease-dependent pathway is implicated in both target cell DNA degradation and plasma membrane damage by experiments where the protease in hibitor was delivered into the cytoplasm by hypotonic shock (H Nakajima, P Henkart, abstract, FASEB meet- ing, Denver, 1993). However, all these results have to be reconciled with data obtained when no cotransfection of proteases was needed for lysis of target cells by cells transfected with perform [4I-,44,45].

A tantalizing clue on possible substrates for granule-lo cdted proteases was provided by observations by Paster- nack and colleagues [ 561 who found that granzyme A can selectively bind and cleave the nuclear protein nucleolin, suggesting that such binding may occur in vivo and nuclear proteins could be the natural targets of the granular enzymes.

Several recent reports implicate granule proteases in CTL activity in zdvo. The expression of the family of granzyme molecules and perforin in murine Thy-l dendritic epidermal T cells is suggested by Tschopp and colleagues [ 601 to reflect the involvement of yF cytotoxic epidermal T cells in immune surveillance. The use of protease inhibitory dipeptides was also fruitful in implicating CTLs in the rejection of grafts bearing multiple MHC-encoded antigenic disparities [GI]. In addition, Granzyme A has been suggested as a marker for rejection in cardiac transplantation and rheumatoid arthritis [ 511,

The list of experimental data not explained by the granule exocytosis model includes: the failure to detect pores in target cells [ 361 and to inhibit the activity of CTLs with antibodies to perform; the blocking of granule exocytosis by the pretreatment of CTLs with agents that induce CAMP without abolishment of cytotoxicity [ 191; and the observation of cytotoxicity in the absence of Ca2+ (when no granule exocytosis and the polymerization of perforin are possible [ 1,621. More recent observations not explained by the granule exocytosis model include the failure to detect perform in some CD4+ CTLs clones [631, and the inability to mobilize intracellular Ca2+ in some TCR j-chain transfectants that are still able to kill target cells [64]. In addition, a study of CTL development using reverse-transcribed PCR to evaluate granzyme expression showed that the time course of only granzyme B expression correlated with development of cytotoxic activity [ 651.

408 Lymphocvte activation and effector functions

Fas protein is implicated in Ca*+-independent

CT1 cytotoxicity

An important breakthrough was made recently by Gol- stein and colleagues [42**] in experiments that implicate Fas (or Fasassociated) target cell surface molecule(s) in target cell death triggered by CTIs in the absence of Ca2+. Their report is the first experimental demonstration of the role of a particular target cell surface molecule in the transmission of cell-mediated cytotoxicity due to contact interactions.

The next important question is how CTIs signal Fas molecules on the target cell and what is the nature of the Fas ligand (CTL surface associated molecule or CTL-derived soluble ligand?). An attractive possibility is to assume that Fas on target cells is bound and/or co- valently modified by some non-specific small molecular weight ligand (e.g. extracellular ATP [66,67] which is specifically accumulated by a TCR-triggered CTL) [68].

The extracellular ATP model of CTL-mediated

cytotoxicity

Extracellular ATP was considered as an intermediate in lysis by CTLs as it is able to lyse tumor target cells, whereas CTIs themselves are resistant to lysis, and CTIs can accumulate extracellular ATP after triggering via their TCRs [6671]. In addition, ATP can trigger apoptosis (including DNA fragmentation) and programed cell death in thymocytes and in other susceptible tumor cells [70,71] by two independent mechanisms, one of which is apoptosis. ATP-induced DNA fragmentation does not involve mitochondrial DNA [69] indicating that this is a specific nuclear event.

Studies of cytotoxicity mediated by CTIs and extracellular ATP (dependent on pH and Mg2 +, respectively) were designed to discriminate between the possibilities of extracellular ATP being a ‘hit’ molecule (ATP*- molecular species acting via lethal hit alone) or a ‘messenger’ (M~ATP~- molecular species acting in concert with e.g., ecto-kinases). The results suggest that the role of ectok- nases in addition to the role of receptors for ATP should be more carefully explored [66],

Nitric oxide as a lytic molecule

Recently a novel biological messenger, nitric oxide, was implicated in the many cellular responses including me- diation of macrophage actions [ 721. No convincing experiments have been reported suggesting a role for this mediator in CTL function and attempts in our laboratory to implicate it in cytolysis by using an inhibitor of nitric oxide synthase failed (F Redegold, P Smith and MV Sitkovsky, unpublished data).

Conclusion

It seems clear from this review, that while molecular pa- rameters of cytotoxic cellular interactions are increasingly well delined, the major direction of research is concerned with the exact role of individual effector molecules, be it regulatory enzymes (protein kinases, phosphatases) or effector ‘cytotoxic’ molecules (proteases, DNases, pore- forming proteins). It is expected that current efforts to determine the contribution of perform molecules in cellular cytotoxicity by in vivo gene ‘knockout’ approach will shed light not only on the exact role of cytolytic granules but also on the complex interplay and possible redundancy of different cytotoxic mechanisms in viva.

Acknowledgements

The authors wish to thank Dr. Pierre He&art for critical reading of the manuscript and Shirley Starnes for expert preparation of the manuscript.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: . . . 1.

2.

3.

4.

5.

6. .

of special interest of outstanding interest

SITKOVXY MV: Mechanistic, Functional and Immunophar- macological Implications of Biochemical Studies of Anti- gen Receptor-triggered Cytolytic T-lymphocyte Activation. Immunol Rev 1988, 103:127-160.

SPRICCS MK, KOLLER BH, SATO T, MOFNSSEY PJ, FANSLOW WC, SMITHIES 0, VOICE RF, WIDMER MB, MALISZEWSKI CR: Pa-Microglobulin-, CD8+ T-cell-deficient Mice Survive Inoculation with High Doses of Vaccinia Virus and Ex- hibit Altered IgG Responses. Proc Nat1 Acad Sci USA 1992, 89:60706074.

APASOV S, SITKOVSKY M: Highly Lytic CDS+, @TCR Cyto- toxic T Cells with MHC Class I Antigen Directed Cytotoxi- city in the BamicrogIobuIin, MHC Class I Deficient Mice. Proc Natl Acud Sci USA 1333, in press.

WALDEN PR, EISEN HN: Cognate Peptides Induce Self-destruc- tion of CDS+ Lymphocytes. Proc Nat1 Acad Sci USA 1990, 879015-9019.

BURROWS SR, SUHKBIER A, KHANNA R, MOSS DJ: Rapid Viiuai Assay of Cytotoxic T-cell Specificity Utilizing Synthetic Peptide Induced T-cell-T-cell wing. Immunology 1992, 76:174-175.

ME~CHER MF: Surface Contact Requirements for Acti- vation of Cytotoxic T Lymphocytes. J Immunol 1992, 149~2402-2405.

The elegant use of artificial antigen-presenting particles allowed the clar- ification of the requirements for cell-cell surface contact in CTL triggering via TCR.

7. O’ROURKE AM, MESCHER MF: Cytotoxic T-lymphocyte Activa- tion Involves a Cascade of SignaiIing and Adhesion Events. Nature 1992, 358:253-255.

8. O’ROIJRKE Ahl, RICERS J, MESCHER MF: Activated CD8 Binding to CIass I Protein Mediated by the T-cell Receptor Results in SignaIiing. Nature 1990, 346:187-189.

9. O’ROURKE AM, APGAR JR, KANE KP, MART-Z E, MESCHER MF: Cytoskeletal Function in CDS- and T CeU Receptor-medi-

Cell-mediated cytotoxicity Apasov, Redegeld and Sitkovsky 409

ated Interaction of Cytotoxic T Lymphocytes with Class I Protein. J Exp Med 1991, 173241-249.

10. RODRIGUES M, NUSSENZWEIG RS, ROMERO P, ZAVAIA F: The in . Vfuo Cytotoxlc Activity of CD8+ T CeU Clones Correlates

with Their Levels of Expression of Adhesion Molecules. J Exp Med 1992, 175:895-905.

The correlation between the expression of adhesion molecules and in viva antiparasite activity of otherwise fully cytolytic CD8+ CT& points to the clinical outcome determining the importance of adhesion molecules in CTL migration through tissues.

11. TANAKA Y, ADAMS DH, HUBSCHER S, HIRANO H, SIEBENUST U, . SHAW S: T-cell Adhesion Induced by Proteoglycan-imrnobi-

lized Cytokine MIP-IS. Nature 1993, 361:7’%35. Implicates cytokines with glycosaminoglycan-binding sites in migration of CD8+ CTLS in viva.

12. AZUMA M, CAYABYAE% M, BUCK D, PHILLIPS JH, LEER LL: CD28 . . Interaction with B7 Costimulates primary Allogeneic Pro-

liferative Responses and Cytotoxicity Mediated by SmaU, Resting T Lymphocytes. J E@ Med 1992, 175:353-360.

The discoveT of the ability of resting T cells to deliver a lethal hit after co-stimulation via CD28 opens new possibilities in studies of cytotoxic cells and widens the physiological role of resting T cells.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

SUGIYAMA H, APA?OV S, TRENN G, SITKOVSKY M: Identification of Protein Kinases and Protein Phosphatases Involved in CTL Elfector Functions. In Cytotoxic Celk Edited by Sitkovsky M, He&art P. Boston-Base-Berlin: Birhauser; 1993: in press,

NISHIOKA WK, WELCH RM: Inhibition of Cytotoxic T Lymphocyte-induced Target Cell DNA Fragmentation, hut Not Lysis, by Inhibitors of DNA Topoisomerases I and II. J Exp Med 1992, 175~23-27.

31.

O’SHEA JJ, MCVICAR DW, KUHNS DB, ORTALDO JR: A Role for Protein Tyrosine Kinase Activity in Natural Cytotoxi- city as Well as Antibody-dependent Cellular Cytotoxicity. J Immunol 1992, l&:2497-2502.

LICKER DS, OBERMII~R PS, ECKHART W, APGAR JR, BEHGL:R NA,

MYERS J: Genome Digestion is a Dispensable Consequence of Physiological CeU Death Mediated by Cytotoxic T Lym- phocytes. Mel Cell Biol 1992, 12:3060-3069.

32.

EINSPAHR KJ, ABRAHAM P.T, BINSTADT BA, UEHARA Y, LEIBSON PJ: Tyroslne Phosphorylation Provides an Early and Requisite Signal for the Activation of Natural Killer Cell Cytotoxic Function. Proc Nat1 Acad Sci USA 1991, 88:627+6283.

ZYCHUNSKV A, ZHENG LM, LIL! C~C, YOUUG JDE: Cytolytic Lym- phocytes Induce Both Apoptosis and Necrosis in Target Cells. J Immunol 1991, 146:393-400.

33.

BERREBI G, TAKAYAMA H, SITKOVSKY MV: Antigen-receptor Interaction Requirement for Conjugate Formation and Lethal-hit Triggering by Cytotoxic T Lymphocytes can be Ionophores. Proc Nat1 Acad Sci USA 1987, &1:1364-1368

PEI%CH MC, POLZAR B, STEPHArr II, CRUvll”lX~\ T. XIAcl1OULI~

HR, MANNHEU HG, TSCHOPP J: Characterization of the En- dogenous Deoxyribonuclease Involved in Nuclear DNA Degradation During Apoptosis (Programmed CeU Death). EMBO J 1993, 12:371-377.

34.

BAUER A, MCCONKEY DJ, HOWARD FD, CLAYTON LK, NOVICK D, KOYA.SU S, REINHERZ EL: DilTerential Signal Transduction via T-cell Receptor CD3<2, CD3<--7\, and CD3q2 Isoforms. Proc Nat1 Acud Sci USA 1991, 88:3842-3846.

THAN Q, STRELIU M, SAII‘O II, SCHI.OSS,%~ SF, ANDERSON P: A Polyadenylate Binding Protein Localized to the Granules of Cytolytic Lymphocytes Induces DNA Fragmentation in Target Cells. Cell 1991, 67:629639.

35.

ROMEO C, SEED B: Cellular Immaturity to HIV Activated by CD4 Fused to Cell or Fc Receptor Polypeptides. Cell 1991, 64:1037-1046.

SHI I., K&v C-M, POwT% JC, AEBEKXXI) R. ~RIIFUHER<; At I. Purification of Three Cytotoxic Lymphocyte Granule Ser- ine Proteases that Induce Apoptosis Through Distinct Substrate and Target CeU Interactions. ./ E.I;I, ,Iled 1992. 176:1521&1529.

36. TAKAYAMA H, TRENN G, SITKOVSKY MV: Locus of Inhibitory Action of cAMP-dependent Protein Kinase in the Anti- gen Receptor-triggered Cytotoxic T Lymphocyte Activation Pathway. J Biol Cbem 1988, 263:2330-2336.

BERKE G: Lymphocyte-triggered Internal Target Disinregra- tion. Immunol To&y 1991, 12~396399.

SLJGNAMA H, CHEN P, HUNTER M, TAFFS R, SITKOVSKY M: The Dual Role of the CAMP-dependent Protein Kinase CCL Subunit in T-cell Receptor-triggered T-lymphocyte Effec- tor Functions. J Biol Chem 1992, 267~2525625263.

TAFFS RE, REDEGELO FA, SITKOVSKY Mv: Modulation of Cy- tolytic T Lymphocyte Functions by an Inhibitor of Ser- lne/Threonine Phosphatase, Okadaic Acid. J Immunoll991, 147~722-728.

LILT AY, MISKO~~KY EP, STANHOPE PE, SILICIANO RF Production of Transmembrane and Secreted Forms of Tumor Necrosis Factor (TNF)-a by HIV-l-specific CD4+ Cytolytic T Lym- phocyte Clones. J Immunol 1992, 148:378%3798.

FLANAGAN WM, CORTHESY B, BRAM RJ, CRABTREE GR: Nuclear Association of a T-cell Transcription Factor Blocked by FK- 506 and Cyclosporin A. Nature 1991, 352:803-807.

37.

38.

39.

40.

41. . .

ORTAUX) JR, WINKLER~PICKE~ RT, NAGA\HIMA K, YA(;IT-\ tt.

OKUMURA K: Direct Evidence for Release of Pore-forming Protein During NK Cellular Lysis. .I Leukocyte Biol 1992, 52:483488.

S.n?-rti MJ, NOKIHISA Y, ORTALDO JR: Multiple Cytolytic Merh anisms Displayed by Activated Human Peripheral Blood T CeU Subsets. J Immunol 1992, 148:55-62.

OZDEM~RL~ M, EL-KHATRI M, BA.STL+NI L, AKDENK H, KI-CHKO~

V, Jr1 S-T: The Cytotoxic Process of CD4 Thl Clones. ,I Immunol 1992, 149:1883-1895.

KINCAID RL, TAKAYAMA H, BULINGSLEY ML, SITKO~~KY MV: Dif- ferential Expression of Calmodulin-binding Proteins in B, T Lymphocytes and Thymocytes. Nature 1987, 330:176178.

SHIVER JW, Su L, HENKART PA: Cytotoxicity with Target DNA Breakdown by Rat Basophilic Leukemia Cells Expressing Both Cytolysin and Granzyme A. Cell 1992, 71:315-322.

TRENN G, TAFFS R, HOHMAN R, KINCA~D R, SHEVACH EM, The demonstration of DNA breakdom in target cells during cytolysin- SITKOV~KY M: Biochemical Characterization of the Inhibitory mediated cytotoxicity by transfectants expressing both cytolysin and Effect of CsA on Cytolytic T Lymphocyte Effector Func- granzyme A strongly suppor& the granule exoczTosis model of cyo tions. J Immunol 1989, 142:37963802. toxicity and weakens one of the objections to this model.

25.

26.

27.

28.

29.

30.

IANEON C, NOwIcKi M, SLJGAWARA S, DENNERT G: Differential Effects of Protein Synthesis on CTL and Targets in Cell- mediated Cytotoxicity. Cellular Immunol 1990, 128:412426.

RUSSELL JH, M&AKOWSKI V, RUCINSKY T, PHILLIPS G: Mecha- nisms of Immune Lysis. III. Characterization of the Nature and Kinetics of the Cytotoxic T Lymphocyte-induced Nu- clear Lesion in the Target. J Immunol 1982, l28:2O87

REDEGEU) FA, CHA~RJEE S, BERGER NA, SITKOVSKV MV: Poly- (ADP-Ribose) Polymerase Partially Contributes to Target CeU Death Triggered by Cytolytic T Lymphocytes. I Im munol 1992, 149:3503-3516.

KRAUT RP, Bosl: R, CR~GOE EJ, GIE:ENBER(; AH The Na+/Caz+ Exchanger Regulates Cytolysin/Perforin-induced Increases in Intracellular CaZ+ and Susceptibility to Cytolysis. J Immunol 1992, 148:248%2496.

SIJGAWARA S, Nowic~l M, XIE S, SONG [I-J. DENN~KI’ G Ef- fects of Stress on Lysability of Tumor Targets by Cyto- toxic T Cells and Tumor Necrosis Factor. J 1mm/~)io1 1990. 145:1991-1998.

410 lymphocyte activation and effector functions

42. ROLMER E, LUCIANI M-F, G~ISTEIN P: Fas Involvement in . . Ca*+-independent T Cell-mediated Cytotoxicity. J Eap Med

1993, 177:195-200. This provides direct evidence of the involvement of the Fas antigen in cell-mediated cytotoxicity and provides the first demonstration of the existance of specific mechanisms and of special surface molecules on the target ceil that mediate triggering for programed cell death. These observations resolve one of the hottly debated issues about the existance of the extracellular Ca2+-independent and granule ex- oqtosis independent mechanism of CTL-mediated cytotoxicity.

43.

44.

45.

46.

47.

48.

49.

50.

51

52

53

54

55

56

57

PODACK ER, KLJPFER A: T-cell Effector Functions: Mechanisms for Delivery of Cytotoxicity and Help. Annu Rev Ceil Biol 1991, 7:47!+504.

HENKAITT PA, MIUARD PJ, REYMOLDS CW, HENKART MP: Cy- tolytic Activity of Purified Cytoplasmic Granules Erom Cy- totoxic Rat LGL Tumors. J Exp Med 1984, 160:75-93.

MUNGER WE, BERRIBI GA, HENKART PA: Possible Involvement of CTL Granule Proteases in Target CeU DNA Breakdown. Immunol Rev 1988, 103:9!+109.

PE’IER~ PJ, BOR~T J, OORXHOT V, F~JKUUDA M, KRAHENRUHL 0, TSCHOPP J, SLOT JW, GEUZE HJ: Cytotoxic T Lympho- cyte Granules Are Secretory Lysosomes, Containing Both Perforin and Granzymes. J Exp Med 1991, 173:109~1109.

DUPUIS M, SCHAEFXR E, KRALJSE K-H, TXZHOPP J: The Calcium- binding Protein CalreticuUn is a Major Constituent of Lytic Granules in Cytolytic T Cells. / Exp Med 1993, 177:1-7.

Lu P, GARCIA-SANZ JA, LICHTENHELD MG, PODACK ER: Perforin Expression in Human Peripheral Blood Mononuclear Cells. J Immunol 1992, 148:335&3360.

KAWA%KI A, SHINKAI Y, YAGITA ,H, OKUMLJRA K: Expression of Perforin in Murine Natural Killer Cells and Cytotoxic T Lymphocytes in Vivo. Eur J Immunol 1992, 22:1215-1219.

JONES J, FRITH S, PIDDLESDEN S, MORGAN BP, COMPS~ON DAS, CAMPBELL AK, HAUETT MB: Imaging Ca*+ Changes in In- dividual Oligodendrocytes Attacked by T-cell Perforin. Im- munology 1991, 74:572%577.

G~UFFITHS GM, ALPERT S, INBERT E, MCGUIRE J, WEISSMAN IL: Perforin and Granzyme A Expression Identifying Cytolytic Lymphocytes in Rheumatoid Arthritis. Proc Nat1 Acad Sci USA 1992, 89:54+553.

GRIFFITHS GM, NAMIKAWA R, MUELLER C, LIU C~C, YOUNG JD-E, BILLINGHAM M, WEISSMAN I: Granzyme A and Perforin as Mark- ers for Rejection in Cardiac Transplantation. EuvJ Immunol 1992, 21687-692.

PODACK ER, HENGERTNER H, LICHTENHELD MG: A Central Role of Perforin in Cytolysis. Annu Rev Immunoll991, 9:12’+157.

HUDIG D, ALLISON NJ, PICKE~T TM, WINKLER U, KAM CM, POWERS JC: The Function of Lymphocyte Proteases. In- hibition and Restoration of Granule-mediated Lysis with Isocoumarin Serine Protease Inhibitors. J Immunol 1991, 147:136&1368.

McGuw MJ, LIPSKY PE, THIELE DL: Generation of Active Myeloid and Lymphoid Granule Serine Proteases Requires Processing by the Granule Thiol Protease Dipeptidyl Pep- tidase I. J Biol Chem 1993, in press.

PASTERNACK MS, BUER JK, MCINERNEY TN: Granzyme A Binding to Target CeU Proteins. Granzyme A Binds To and Cleaves NucleoUn in Vitro. J Biol Chem 1991, Z&14705-14708.

EWOLDT GR, WINKLER U, POWERS JC, HUDIG D: Sulfonyl Flu- oride SerIne Protease Inhibitors Inactivate RNK-16 Lym- phocyte Granule Proteases and Reduce Lysis by Granule Extracts and Perforin. Mol Immunol 1992, 29:713721.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67

68.

69.

70.

71.

72.

TALENTO A, NGLNEN M, LAW S, Wu JK, POE M, BLAKE JT, PATEL M, Wu T-J, MANYAK CL, SILBERKLANG M, ETAL: Transfec- don of Mouse Cytotoxic T Lymphocyte with an Andsense Granzyme A Vector Reduces Lytic Activity. J Immunoll992, 149:40094015.

SAVERS TJ, WILTROUT TA, SOLDER R, MUNGER WL, SMYIH MJ, HENDERSON LE: Purification of a Factor From Granules of Rat Natural Killer CeU Line (RNK) that Reduces Tumor CeU Growth and Changes Tumor Morphology: Molecular Iden- tity with a Granule Serine Protease (RNKP-1). J lmmunol 1992, 148:292-300.

KRAHENBUHL 0, GATIESCO S, TSCHOPP J: Murine Thy-l + Den- dritic Epidermal ‘I’ Cell Lines Express Granule-associated Perforin and a Family of Granzyme Molecules. Immunobi- ology 1992, 184:392401.

THIELE DL, GEISSLER GH, WILLL%MS FH, LIPSKY PE: The Role of Leucyl-leucine Methyl Ester-sensitive Cytotoxic Cells in Skin AlIograft Rejection. Tranplantation 1992, 53:1334-1340.

TRENN G, TAKAYAMA H, SITKOVSKV MV: Exocytosis of Cytolytic Granules May Not be Required for Target CeU Lysis by Cytotoxic T-lymphocytes. Nature 1987, 330:72-74.

TAKAYAMA H, SHINOHARA N, KAWASAKI A, S~MEYA Y, HANAOKA S, KOJIMA H, YAGITA H, OKUMURA K, SHINKAI Y: Antigen-specific Directional Target CeU Lysis by Perforin-negative T Lymphocyte Clones. Int Immunol 1991, 3:114’+1156.

ROMENO C, AMIOT M, SEED B: Sequence Requirements for Induction of Cytolysis by the T CeU Antigen/Fe Receptor 6 Chain. Cell 1992, W389-897.

PRENDERGA~T JA, HELGA~ON CD, BIEACKLEY RC: Quantitative Polymerase Chain Reaction Analysis of Cytotoxic CeU Pro- teinase Gene Transcripts in T Cells. J Biol Chem 1992, 267:509C-5095.

REDEGELD F, FIL~PPINI A, SITKOVSKY M: Comparative Studies of the Cytotoxic T Lymphocyte-mediated Cytotoxicity and of Extracellular ATP-induced CeU Lysis. J Immunol 1991, 147:363%3645.

FIUPPINI A, TAFFS RE, AGUI T, SITKOVSKY MV: Ecto-ATPase Activity in Cytolytic T-lymphocytes. J Biol Cbem 1990, 265:334-340.

FILIPPINI A, TAFFS RF, SITKOVSKY MV: Extracellular ATP in T- lymphocyte Activation: Possible Role in Effector Functions. Proc Natl Acad Sci USA 1990, 87:8267-8271.

MURGIA M, Ptzzo P, SANDONA D, ZANOVEUO P, RIzuTTo R, DIVIRGIUO F: Mitochondrial DNA is not Fragmented During Apoptosis. J Biol Chem, 1992 267:1093‘%10941.

ZANOWLO P, BRONTE V, ROSATO A, PIZZO P, VIRGIIJO F: Re- sponses of Mouse Lymphocytes to Extracellular ATP. II. Extracellular ATP Causes CeU Type-dependent Lysis and DNA Fragmentation. ,/ Immunol 1990, 145:1545-1550.

ZHENG LM, ZYCHLINSKY 4 L1r1 C-C, OJENS DM, YOUNG JD-E: Extracellular ATP as a Trigger for Apoptosis or Programmed CeU Death. J Cell Biol 1991, 112:273-288.

~WENSTEIN CJ, SNYDER SH: Nitric Oxide, a Novel Biologic Messenger. Cell 1992, 70:705-707.

S Apasov, F Redgeld and M Sitkovsky, laboratory of Immunology, Na- tional Institute of Allergy and Infectious Diseases, National Institutes of Health, Room 1 lN-311, Building 10, Bethesda, Matyland 20892, USA.

cell-mediated cytotoxicity: contact and secreted factors

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