CD8+ Effector Cells

advances in immunology, vol. 83

CD81 Effector Cells PIERRE A. HENKART AND MARTA CATALFAMO National Institutes of Health, Bethesda, Maryland 20892-1360

I. Effector Cells Defined

A survey of the literature shows that the term ‘‘effector cells’’ means different things to different immunologists. The word ‘‘effector’’ clearly implies cells with functional activity, but there is no agreement as to what type of activity. For many years, cytotoxicity was the only lymphocyte functional activity that was measurable in short term in vitro assays, and ‘‘effector cells’’ were thus understood to mean cells with such activity. Other in vitro lymphocyte functional activities were clearly more complex, potentially involving multiple cellular activities, and the term ‘‘effector’’ did not seem meaningful if it had to be associated with a particular assay. When it later became possible to quantitatively measure secretion of defined cytokines in short-term in vitro cultures, the term ‘‘effector’’ was logically adopted to describe activity in such assays, and effector cells lost their close association with cytotoxicity. The term effector thus applies to several different types of functions and, for the sake of clarity, the effector function needs to be specified. Confusion can arise from the multiple types of effector function that now can be measured, not only cytotoxic effector function vs cytokine secretion, but also the two different cytotoxicity pathways and the multiple cytokines T cells make, which are not all regulated similarly during differentiation. Thus, we will refer to cytotoxic effector function and cytokine secretory effector function. The term ‘‘effector’’ has often been used to describe lymphocyte differentiation, particularly in the context of distinguishing memory cells from effector cells. Development of TcR transgenic animals and MHC tetramer staining has allowed a functional definition of memory cells as those antigen-reactive cells that persist after an immune response and give rise to the enhanced secondary response characteristic of immunological memory. Memory function is thus defined in an in vivo context over a time span of weeks, months, or years, as opposed to effector function that operates within hours. There is no logical reason why memory function should exclude effector function, and indeed most memory cells seem to have cytokine secretory effector functions and, in some cases, also seem to have cytotoxic effector function (see following text). Even more confusion can occur when the term effector is used by some groups to describe cells that gave rise to longer-term biological functions in vivo, such as tumor therapy after adoptive transfer. While this use of the term effector can be defended as logical, it is very different from more 233 Copyright 2004, Elsevier Inc. All rights reserved. 0065-2776/04 $35.00

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common usage to describe differentiated cells showing immediate and defined functional activity, because many cellular and molecular interactions occur after injecting cells into an animal and observing their effects some days or weeks later. We would argue that since cytotoxicity and cytokine secretion are the only responses that can presently be measured in short-term assays reflecting a defined differentiated T cell phenotype, the term effector should apply only to these functional responses, and we use the term effector accordingly in this chapter. The term effector has also been applied to CD8þ T cell subpopulations defined by phenotypic surface markers, based on correlations of effector function with that subpopulation. This terminology is a result of extensive efforts to identify such correlations, with considerable success for human CD8þ T cells. There is a consensus that naive and cytotoxic effector cells express the CD45RA isoform while memory cells switch to express the smaller CD45RO isoform lacking the CD45RA, B, and C domains. The combination of the CD45 isoform marker with another marker, such as CD27, CD28, CCR7, or CD62L, has been used to delineate four subpopulations, with cytotoxic effector function correlating with low expression of the second marker as well as expression of CD45RA. The same second markers have been proposed to correlate with two functionally distinct subsets of memory cells. The central memory cells bear surface receptors mediating homing to lymphoid organs (such as CD62L) and the CCR7 receptor for chemokines such as CCL19, allowing them to circulate in lymphoid organs. An ‘‘effector memory’’ subpopulation of cells lacking these surface markers is proposed to circulate in nonlymphoid tissue (Sallusto et al., 1999). However, there is presently no consensus as to the precise criteria for defining these memory subpopulations nor, as will be discussed, is there a consensus on whether they also have different effector functions. In any case, to avoid confusion, it should be either specified or clear from the context when the term effector is used to describe a subpopulation based on surface phenotype vs direct measurements of function. While expression of cytoplasmic perforin or granzymes indicates a potential for cytotoxic effector activity, these granule mediators are necessary but not sufficient for cytotoxicity. An interesting transgenic mouse model to identify effector CD8þ T cells has been constructed, using GFP expressed under the control of the CD4 promoter (T-GFP mice) (Manjunath et al., 1999). As CD8þ T cells differentiate into cytotoxic effector cells, the CD4 promoter becomes inactive. Seven days after T-GFP mice were challenged with vaccinia, all the cytotoxic effector activity was found in the GFP CD8þ T cells, while only the GFPþ cells proliferated in response to TcR ligation. Our previously stated functional definition of effector cells raises the important caveat that any strictly defined in vitro assay may not be meaningfully

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extrapolated to the in vivo biological phenomenon one is trying to understand. This limitation must be accepted as part of the process of simplifying systems to allow studies under defined conditions. In vitro functional assays should be viewed as a tool to dissect in vivo immune responses in view of the difficulties that confront manipulating lymphocytes in vivo. There is no argument against the need for better tools for this, and it is hoped that new technologies will provide more sophisticated means to analyze lymphocyte functions in vivo so that correlations can be developed with the in vitro measurements used to define effector function. II. Secretion as the Mechanism of Effector Function

The two major molecular pathways responsible for cytotoxic effector function are now understood, and they both rely on the basic cellular process of secretion (Barry and Bleackley, 2002; Russell and Ley, 2002; Trapani and Smyth, 2002). The perforin-dependent granule exocytosis pathway utilizes a regulated secretory mechanism, which is well known in other cell types but in lymphocytes has been exclusively associated with cytotoxicity. This type of secretion involves previously synthesized proteins that are stored intracellularly in secretory granules. In response to receptor crosslinking, the granule membranes undergo fusion with the plasma membrane, which is exocytosis. Lymphocyte secretory granules are similar to those of other hemapoietic cells in that they are modified lysosomes, containing the usual lysosomal enzymes as well as the secretory proteins and proteoglycans (Stinchcombe and Griffiths, 1999). The FasL/Fas pathway involves membrane receptor expression of FasL, which appears to utilize both the regulated secretory pathway in some circumstances and the constitutive secretory pathway in others (Kojima et al., 2002). In the latter case, newly synthesized protein is deposited in the ER, processed in the Golgi, and shuttled to the plasma membrane in small vesicles, where immediate exocytosis results in surface expression. Soluble FasL can sometimes be released later after cleavage of the membrane form by a metalloprotease. In terms of the stated definition of effector cells, thinking about cytotoxicity in terms of its functional secretory pathways is attractive as a logical connection with functional measurements of immediate cytokine secretion. It should be noted that the major T cell cytokines studied to date all utilize the constitutive secretory pathway. Although their overall secretion is obviously triggered by the TcR, regulation occurs at the level of transcription and protein synthesis, and not the secretory event itself. The classical T cell cytokines are not synthesized previously and stored, as is the case with cytotoxic mediators. Detection of intracellular perforin and granzymes in lymphocytes suggests that they have the potential to be cytotoxic effectors, but it is not clear that

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such cells have the ability to exocytose these mediators after TcR engagement. On the other hand, detection of intracellular cytokine indicates that secretion is occurring. In order for the critical effector secretion events to occur, precursor lymphocytes having the ability to recognize antigen generally must have differentiated into cells that also have the ability to secrete rapidly, allowing their detection in short-term assays. There is unfortunately no universal definition of ‘‘shortterm,’’ although typically 3- to 6-hour incubations are used. Assays of longer duration, e.g., 12 to 24 hours, may be an appropiate readout of effector function in some cases (see following text), but such longer times open the door to measuring antigen-triggered differentiation of precursors into effectors as well as effector function. This issue becomes relevant in studies of memory CD8þ T cells, some of which have no effector function in short-term (4- to 5hour) cytotoxic assays, but acquire it rapidly enough to kill target cells in overnight assays of 16 to 24 hours (Wherry et al., 2003). III. Cytotoxicity In Vivo

Ideally, one would like to be able to measure cytotoxic activity in vivo by measuring target cell death and characterizing the effector cell. One very real problem is that cytotoxic lymphocytes induce an apoptotic response in their target cells, and apoptotic membrane changes trigger phagocytosis by local tissue phagocytes. Thus, any in vivo cytotoxicity assay based on identifying dying target cells faces the difficulty that apoptotic cells may rapidly disappear. In favorable cases, CTL-mediated target cell degeneration can be observed in vivo by histological studies or by in situ assays of DNA fragmentation (Ando et al., 1994). However, such approaches may miss many cases of cytotoxicity in vivo, and the nature of the effector cells may be difficult to elucidate. Instead of looking for dead cells, the disappearance of cells of interest can be assumed to be due to their death, and indeed the rejection of grafts or tumors has classically been used as an in vivo measure of cytotoxicity. The importance of CTL was first recognized when the disappearance of allogeneic tumors injected into the peritoneal cavity correlated with the appearance of CTL displaying cytotoxic function in vitro (Berke, 1980). A newer approach to measure cytotoxicity in vivo is to inject CFSE-labeled target cells into animals and follow their survival by subsequently sacrificing mice and analyzing the labeled cells using flow cytometry. If different levels of CFSE labeling are used to distinguish between antigen-bearing cells and control cells, this approach can assay for antigen-specific survival in vivo (Oehen and Brduscha-Riem, 1998). A significant issue with measurements of loss of cells from a particular in vivo site is that one may be seeing a redistribution to other organs instead of cytotoxicity, requiring a search of likely compartments to check this possibility.

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In all the above in vivo assays, the nature of the effector cells and the molecular pathways responsible for antigen loss in vivo are difficult to assess and must be approached indirectly, e.g., by selectively manipulating the target cells or animals to block the cytotoxic effect. A major problem with such approaches is that while they can implicate a particular cell or molecule as required for the effect, they may not reflect the nature of the direct effector responsible for the depletion of cells in vivo as opposed to one required component in a pathway required for this effect. Effector cells are defined as those directly responsible for a biological phenomenon rather than cells whose activity is required for an upstream step. In some cases, it has been assumed that because effector cells exhibit good cytotoxic activity in vitro, these cells will use the same cytotoxicity pathway to eliminate antigen in vivo. For controlling some viral infections in vivo, the noncytopathic CTL-mediated secretion of IFN-g and TNF-a may be more important than either perforin- or Fas-dependent cytotoxicity (Guidotti and Chisari, 2001). Cell loss in vivo can be due to different mechanisms, and identification of potential effector cells and molecular mechanisms can best be done in vitro. However, it is critically important to go back to in vivo systems to test the effector mechanisms proposed from in vitro studies. In some cases, where particular cytotoxicity pathways predominate in vitro, they were found to be not required in vivo (Barchet et al., 2000; Guidotti et al., 1996). Knockout mice lacking perforin and the gld mutant mice defective in FasL have been used to study a variety of in vivo processes for their dependence on their respective cytotoxicity pathways (Kagi et al., 1996; Russell and Ley, 2002). However, this approach requires caution because these mice have abnormal immune systems, compatible with the idea that these cytotoxicity pathways play an important regulatory role in immune responses. Gld mice, in particular, develop lymphoid hypertrophy and, as they age, they accumulate large numbers of nonfunctional lymphocytes (Cohen and Eisenberg, 1991). Perforin knockout mice do not show a normal homeostatic downregulation of expanded T cell numbers after antigenic challenge (Harty and Badovinac, 2002). Perforin knockout mice have provided clear evidence for the operation of this cytotoxicity pathway in vivo, in particular, in resistance to noncytopathic viruses (Kagi et al., 1996). More recently, a role for perforin-dependent cytotoxicity in tumor surveillance has become clear as older perforin knockout mice develop a high rate of spontaneous tumors (Smyth et al., 2000), although this may be due to NK or NKT cells rather than CD8þ CTL (Hayakawa et al., 2002). Perhaps the most dramatic evidence of perforin’s in vivo role came from the identification of functional perforin mutations as one cause of the human disease familial hematophagocytic syndrome (FHS) (de Saint Basile and Fischer, 2001; Stepp et al., 1999). After viral infections, FHS children with perforin mutations suffer from massive and potentially fatal tissue infiltration

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by activated lymphocytes and macrophages, a phenomenon not described in perforin knockout mice. IV. Cytotoxicity In Vitro

Because of the difficulties previously outlined, cytotoxic effectors have come to be operationally defined as cells whose cytotoxic function can be detected in short-term in vitro assays. These assays of 3 to 6 hours utilize titrated numbers of effector cells incubated with fixed numbers of target cells, with target lysis classically measured by the 51Cr release assay. Other assays measuring cytolysis are also used, including some measuring release of heavy metal chelates or hydrophilic fluorophores. Target cell apoptosis can also be measured using nuclear DNA fragmentation, or caspase activation using fluorogenic substrates and flow cytometry. All these assays quantitate target cell death, but do not enumerate or directly identify the effector cells responsible. Thus, attribution of effector function to a particular lymphocyte population relies on purification of the input effector population in cytotoxicity assays. In the past, cytotoxic effector function was difficult to demonstrate from lymphocytes directly ex vivo, except in patients/animals with acute virus infections or in lymphocytes removed from the peritoneal cavity where allogeneic tumors are undergoing rejection. Cytotoxic function was generally measured from ex vivo lymphocytes (e.g., from human blood) only after culture with antigen and APC for 1 to 2 weeks, followed by a standard short-term assay for cytotoxic function. This approach actually measures an in vitro memory function rather than cytotoxic effector function, as has been defined. However, recent use of TcR transgenic animals has allowed detection of direct ex vivo cytotoxic function since antigen-reactive cells can be identified and target lysis measured after pulsing with appropriate peptides (see following text). V. Perforin-dependent Granule Exocytosis Pathway

The perforin-dependent granule exocytosis pathway often dominates cytotoxicity measured by in vitro assays. This pathway is triggered by TcR recognition of target antigen, leading to a rapid cytoplasmic polarization of the T cell so that many of its cytoplasmic organelles face the bound target cell, and the granules become positioned for exocytosis. Recent studies of microtubules and MTOC in CTL-target interactions provide insights into this process (Kuhn and Poenie, 2002), but many molecular steps between TcR signaling and MTOC reorganization remain unknown. After polarization, secretion of previously synthesized perforin and granzymes stored in lysosomal secretory granules occurs by exocytosis, which is the fusion of the granule membranes with the plasma membrane.

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Rab 27a has been recently identified as a molecule required between the CTL polarization and granule fusion steps. This small GTPase family member is defective in humans with one form of Griscelli’s syndrome, a rare genetic disease associated with pigmentation defects (Menasche et al., 2000). Rab27a mutations give rise to a form of Griscelli’s syndrome that is associated with a hemophagocytic syndrome similar to that seen in perforin-defective humans, and CTL from these patients show defective cytotoxic effector activity. The naturally occurring mouse ashen mutation is similarly associated with a pigmentation defect and a defect in Rab27a (Wilson et al., 2000). CTL and NK cells from ashen mice show minimal cytotoxic activity on Fas target cells in vitro, although cytotoxic activity via the Fas pathway is normal (Haddad et al., 2001; Stinchcombe et al., 2001a). TcR-triggered granule exocytosis is defective in ashen CTL, although TcR-induced granule polarization occurs normally. Thus, Rab27a is required for a late step in the regulated secretory pathway in cytotoxic lymphocytes, perhaps in aligning the granule membrane with the plasma membrane to promote the fusion step. TcR-triggered granule exocytosis occurs within the central region of the immunological synapse in CTL (Stinchcombe et al., 2001b). For this secretion to result in cytotoxicity to the target cell, effector cell granules must contain either perforin and granzymes or FasL on the inner surface of the granule membrane. When triggered by immobilized antibodies against the TcR, CTL degranulation rarely releases more than 50% of the total granule contents. Since CTL can kill multiple target cells within a few hours, antigen on target cells also appears to trigger exocytosis of only a fraction of the granules. It is not clear how many of these granule mediators are required for target cell death, although their local concentration in the small volume between effector and bound target cell can be very high, even after an inefficient degranulation. Perforin and granzymes are required for cytotoxicity via the perforindependent granule exocytosis pathway. Perforin is a water-soluble 65kD protein that undergoes a calcium-dependent aggregation leading to membrane insertion and pore formation. Large porelike structures can be seen by microscopy after CTL or NK killing (Dourmashkin et al., 1980; Podack and Dennert, 1983), although it is not certain that these structures are required for target death. The sieving behavior of macromolecular markers resealed inside red cell ghosts attacked by NK cells indicated that proteins up to 500kD but not larger could escape, compatible with the observed structural pores (Simone and Henkart, 1980), and electrical measurements on planar lipid bilayers demonstrate perforin’s ability to form functional membrane pores (Young and Cohn, 1986). However, no satisfactory molecular mechanism has been elucidated to explain perforin’s oligomerization, membrane insertion, and pore formation. The recognition that a lipid-binding C2 domain is activated by a

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granule proteolytic processing step is an important step in assigning functional domains to this molecule (Uellner et al., 1997). Because perforin knockout CTL show a complete lack of cytotoxic effector function on Fas targets, perforin is clearly necessary for this cytotoxicity pathway. However, pore formation by perforin in target membranes is not the lethal injury resulting in the death of nucleated target cells. When mast cells were transfected to express perforin in their granules, they became highly cytotoxic to red blood cell targets but had minimal ability to kill nucleated target cells (Shiver and Henkart, 1991). Only when granzymes A and B were expressed in addition to perforin did mast cells attain cytotoxic activity comparable to CTL (Nakajima et al., 1995). Thus, perforin’s required role in this cytotoxicity pathway appears to be the facilitation of granzyme entry into target cells. The mechanism of this permeabilization is still not clear. Perforin pores inserted into target membranes may be sealed by normal repair processes to avoid lysis, but while they are open, they may allow granzyme entry that actually leads to target death. However, granzymes are secreted as a complex with large proteoglycans; it is not clear that perforin pores are large enough to allow their passage through the target membrane (Metkar et al., 2002; Raja et al., 2002). An alternative model for perforin’s function is that it permeabilizes endosomal vesicles in target cells after membrane-bound granzymes and perforin are taken up from the surface by endocytosis (Froelich et al., 1996), but whether this occurs in lethally injured CTL target cells is still unclear. Granzymes are a subfamily of serine proteases that are uniquely expressed in CTL and NK cells (Kam et al., 2000), with granzymes A and B the predominant enzymes in CTL in vivo. These proteases have very different substrate specificity, with the former cleaving after lysine and the latter after aspartic acid. Because granzyme B can directly process and activate procaspases that are widely expressed in the cytoplasm of all cells, introduction of granzyme B into the target cytoplasm provides a clear route to target cell apoptosis and death (Darmon et al., 1995). However, it appears that granzyme B-induced apoptosis is more complex, requiring mitochondrial damage to suppress the naturally occurring caspase inhibitors (Goping et al., 2003; Sutton et al., 2003). When caspase activation is blocked by caspase inhibitors, target cell lysis by CTL still occurs normally, although apoptotic nuclear changes are blocked along with some apoptotic cytoplasmic changes (Sarin et al., 1997, 1998). Thus, caspase-independent death pathways can also lead to rapid target cell death. Granzyme B itself cleaves a number of target cell proteins, and it has been suggested that these cleavages might be important in the generation of some forms of autoimmunity (Andrade et al., 1998; Casciola-Rosen et al., 1999). Unlike perforin knockout mice, CTL from granzyme knockout mice retain cytotoxic effector activity in this pathway, although granzyme B knockout CTL

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cause substantially less target apoptotic activity and somewhat less lysis than control CTL (Heusel et al., 1994; Shresta et al., 1995). The lack of a strong cytotoxicity defect in granzyme-deficient CTL can be explained by the potential redundancy of granzyme proteases, which include several that are not well characterized. Granzyme A is the other well-expressed granzyme in primary CTL, and it does not directly process procaspases like granzyme B. When introduced into the cytoplasm of target cells, granzyme A triggers a novel form of apoptosis, associated with DNA single-strand breaks (Beresford et al., 1999, 2001). This target damage pathway is initiated by granzyme A cleavage of SET, part of an ER complex containing an inactive DNAse which becomes active and translocates to the nucleus (Fan et al., 2003a, 2002). Another substrate of granzyme is the oxidative repair protein Ape-1, whose degradation may contribute to the initiation of apoptosis in target cells (Fan et al., 2003b). CTL from granzyme A knockout mice have no detectable defect in cytotoxic effector function (Ebnet et al., 1995), although they are defective in their ability to resist some virus infections (Mullbacher et al., 1996; Pereira et al., 2000; Riera et al., 2000). A model system for studying target apoptosis induced by secreted perforin and granzymes has been extensively investigated by several groups. This involves addition of purified granzymes and sublytic amounts of perforin or other membrane permeabilizing agents to cells in medium. This model allows the use of defined cytotoxic mediators, but their concentration at the target membrane is many orders of magnitude less than occurs after CTL exocytosis. As reviewed elsewhere, granzyme B-induced apoptosis has been extensively studied with this system (Barry and Bleackley, 2002; Smyth et al., 2001). Studies in this model system have led to the proposal that granzyme B binds to target cell surfaces by a mannose-6-phosphate receptor (Motyka et al., 2000), but critical findings supporting this model are controversial (Trapani et al., 2003). The issue of how cytotoxic effector cells themselves are not killed after degranulation has long been a subject of discussion. A recent model to explain this proposes that the lysosomal protease cathepsin B is exposed on the effector surface after exocytosis, allowing proteolytic degradation of cytotoxic mediators coming back to the effector cell (Balaji et al., 2002). In support of this model, it was shown that active surface cathepsin B appears on the CTL surface after degranulation, and its inhibition sensitizes CTL to rapid perforin-dependent suicide upon degranulation. From a practical standpoint, comparing in vitro cytotoxic function of CTL from perforin knockout mice relative to normal mice has often been used to implicate the involvement of this cytotoxicity pathway. Another approach to test the operation of this pathway in vitro is via the use of concanamycin A, a

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drug that blocks the granule proton pump and leads to proteolytic degradation of perforin within the granules (Kataoka et al., 1997). VI. FasL/Fas Death Pathway

The second pathway for cytotoxic effector function is the FasL/Fas pathway. In this case, TcR ligation leads to surface FasL expression, either by de novo transcription and translation followed by FasL surface expression, or by exocytosis of preformed FasL resident on the inside of the lysosomal secretory granule membranes containing perforin and granzymes (Bossi and Griffiths, 1999; Haddad et al., 2001; Kojima et al., 2002). After its biosynthesis, FasL is localized to granules by virtue of a proline-rich motif in its carboxy terminus (Blott et al., 2001). Exocytosis results in surface expression of this FasL as a result of the fusion of the granule membrane with the plasma membrane. Cytotoxicity by the FasL/Fas pathway requires that the target cell express a functional Fas death pathway, which includes not only surface Fas expression measurable by flow cytometry, but also a functionally intact internal death pathway that is not blocked by internal death pathway inhibitors, such as FLIPs. Blocking the surface FasL –Fas interactions with noncrosslinking IgG anti-Fas antibodies or soluble Fas–IgG constructs provides an alternative approach to implicating the Fas pathway in functional cytotoxicity assays. Whether effector FasL expression results from exocytosis of preformed FasL in granules or by de novo synthesis can be addressed by use of inhibitors of protein/RNA synthesis. The relative use of the two cytotoxic effector pathways (perforin-dependent vs Fas-dependent) in vivo will depend on properties of both effector and target cells (Kafrouni et al., 2001). In vivo cytotoxicity by CD8þ T cells may utilize other pathways that do not seem to dominate in vitro. In particular, TNF-a was originally identified as a mediator of tumor cytotoxicity in vitro, but although CTL secrete this cytokine, it does not seem to contribute to short term CTL-mediated cytotoxicity in vitro (Ratner and Clark, 1993). However, when the two major cytotoxicity pathways are inoperative, longer-term (16- to 24-hour) assays may detect cytotoxicity due to membrane-bound or secreted forms of TNF-a expressed by CTL. Although such cytotoxicity could be due to antigen-induced differentiation followed by mediator secretion, some target cells die slowly in the presence of high concentrations of purified TNF-a, so it is possible that longer assays are required to measure this cytotoxicity pathway. In vivo CD8þ T cell-mediated tumor cell killing by this route could be important, particularly for immunotherapy. With target cells that are susceptible to TNF-a-induced apoptosis, the potential for cytotoxicity by secreted TNF-a would appear to be best assayed by measuring TNF-a secretion as will be described.

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VII. Cytokine Secretory Effector Cells

Detection of cytokine mRNA by in situ hybridization or RT-PCR can be used to show activation of cytokine genes in tissue sections, principally to aid in clinical diagnosis. However, since cytokine secretion may not be tightly linked to transcription or mRNA levels, this would not appear to be a satisfactory readout of effector function. Immunohistochemical detection of cytokine protein in tissues has also been reported, but this appears technically demanding and is not quantitative. More satisfying results have come from analysis of cytokine secretion in short cultures of the effector cells in the presence of antigenic stimuli, with measurement of the resulting cytokine secretion by several approaches. Various protein or peptide antigenic stimuli have been used, along with various APC anti costimulating antibodies. Other stimuli used include a combination of phorbol myristyl acetate (PMA) and ionomycin, anti-CD3 with or without costimuling antibodies such as antiCD28, or superantigens. These assays are parallel to cytotoxicity assays except that secretion is measured, using several different approaches as will be described. 1. A straightforward assay of cytokine secreted into the medium by ELISA or proliferation assays, which is homologous to cytotoxicity assays in that a quantitative measure of secretion by the input cell population is obtained. Supernatant-induced proliferation of selected cell lines was formerly widely used to measure cytokines, but the identity of the cytokine responsible may be in doubt because some responding cell lines have multiple cytokine receptors, each of which triggers proliferation. Neutralization by specific antibodies can help satisfy such concerns, but proliferation-based cytokine detection assays have largely been replaced by molecularly based detection systems like ELISA. 2. The first assay developed which read out the number of cells secreting a specific cytokine of interest was the ELISPOT assay, in which cytokinesecreting cells are cultured with antigen or other stimulus (typically, for 24 hours) on a surface containing immobilized anticytokine antibody so that the secreted cytokine is captured and detected with a second anticytokine antibody (Czerkinsky et al., 1991). The readout is a visual count of positive cells based on spots of captured cytokine. 3. Detection of cytokines secreted by individual living activated T cells using a capture technique employing heteroconjugated antibodies between the CD45 surface antigen and an anticytokine antibody. The captured cytokine is then detected with a second, fluorochrome-conjugated anticytokine antibody (Brosterhus et al., 1999). This method can permit simultaneous characterization of surface phenotype, but depends on the availability of the appropriate heteroconjugated antibodies.

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4. A very powerful approach for assaying cytokine-secreting effectors is based on flow cytometric detection of cytoplasmic cytokine after treatment with drugs that block release of the newly synthesized mediators from cells (Jung et al., 1993). Typical protocols call for stimulation of effector cells with antigen, anti-CD3, or PMA and ionomycin for 6 hours, with Brefeldin A added for the last 4 hours to block release from the Golgi. (Some protocols use monensin in place of Brefeldin A.) The cells are then fixed and permeabilized, stained with fluorophore-labeled anticytokine antibody, and analyzed by flow cytometry. This approach allows the enumeration of effector cells and analysis of relative levels of secretion on a per-cell basis. Like the previously cited approach, it permits simultaneous characterization of the effector cell surface phenotype by staining first for surface antigens detectable in different channels. Kits are available (e.g., from BD Biosciences) for such assays of interferon-g, TNF-a, IL-2, IL-4, IL-5, IL-10, and others, along with antibodies for codetection of surface markers. The possible secretion of cytokines such as TNF-a and IL-10 by APC can be eliminated from consideration if cytokine-positive cells are identified by appropriate T cell surface markers. One definitional issue for cytokine secretory effector cells is which cytokines are being measured, and in particular whether IL-2 should be considered as an ‘‘effector cytokine.’’ IL-2 is secreted by naive and memory human CD4þ and CD8þ T cells after TcR ligation, but not by differentiated CD8þ cells that kill and secrete IFN-g (Hamann et al., 1997; Sallusto et al., 1999). This different pattern has led to the exclusion of IL-2 as an effector cytokine, and we will not consider it further. Parallel to the Th1 vs Th2 cytokine secretion patterns of CD4þ T cells, CD8þ T cells follow a tendency to become differentiated along similar patterns (Mosmann et al., 1997). After in vitro activation in the appropriate cytokines, both Tc1 and Tc2 cells have potent in vitro cytotoxic activity via the perforin-dependent granule exocytosis pathway and the FasL/Fas pathway. However, TcR engagement triggers Tc1 cells to secrete predominantly IFN-g and IL-2, whereas Tc2 cells secrete predominantly IL-4, IL-5, and IL-10. Although no phenotypic surface markers have been described that completely correlate with the Tc1 vs Tc2 cytokine secretion patterns, a 2003 report indicates that within the CD8þ central memory subset of human blood lymphocytes (see following text), CCR4 is expressed on cells that secrete IL-4 within 24 hours after activation, while the CCR4 cells make low amounts of IL-4 (but do make IFN-g) under the same conditions (Geginat et al., 2003). When tested in various animal models, Tc1 and Tc2 cells have been shown to have different activities, presumably due to the differences in their cytokine

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secretion. For example, in a mouse tumor therapy model, Tc1 cells were considerably more potent than Tc2 cells, an effect that depended on their ability to secrete IFN-g (Helmich and Dutton, 2001). For immunotherapy of leukemias, Tc2 cells were found to be advantageous over Tc1 cells because of their greater tumor protective graft-vs-leukemia effect vs a deleterious graftvs-host effect (Fowler and Gress, 2000). Tc1 vs Tc2 differentiation has not been shown to play a crucial role in in vivo immune responses, but this is an area of considerable clinical interest.

VIII. CD81 T Cell Differentiation

The role of cytotoxic effector cells in the antigen-triggered differentiation pathway from naive to memory CD8þ T cells has long been a subject of debate. The possibility that functional memory cells may also have cytotoxic or cytokine secretory effector function makes for possible difficulties with interpreting simplified differentiation schemes such as those depicted in Fig. 1. The simplest differentiation model (Fig. 1A) is that there is a linear antigen-induced differentiation from naive to cytotoxic effector cells, followed by survival of some of the latter as memory cells. Opposed to this is the branched model (Fig. 1B) which proposes that antigen induces the formation of separate lineages of effector cells and pre-memory cells, and none of the memory cells derive from cytotoxic effector cells with time. There is evidence in favor of both the linear and branched models (Kaech et al., 2002), which we will not attempt to summarize here, except to point out that there is no strong evidence against the simple linear model. The dissection of memory cells into two subpopulations based on homing and chemokine receptor expression (central and effector memory cells) demands for more complex models, depicted in Fig. 1C–F. Some experiments strongly support a progression from effector memory to central memory cells in the mouse LCMV system (Wherry et al., 2003), compatible with models C or D. A. Effector Functions of CD8þ T Cell Phenotypic Subsets 1. Naive Phenotype Dozens of published studies are in general agreement that when antigen is first encountered, naive CD8þ T cells from humans and mice respond by secreting IL-2 but not other cytokines. One exception to this is a report that naive mouse CD8þ T cells do make TNF-a, although not IFN-g (Walzer et al., 2000). There is a consensus of reports that naive CD8þ T cells do not kill antigen-bearing target cells in short-term assays, nor do they express detectable cytoplasmic perforin or granzymes.

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Fig 1 Possible CD8þ T cell differentiation pathways. N, naive; E, effector; EM, effector memory; CM, central memory.

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2. Central Memory Phenotype As defined by high expression of CCR7 but low expression of CD45 RA, human central memory CD8þ T cells from blood were reported to express undetectable levels of cytoplasmic perforin and to secrete negligible amounts of IFN-g in response to anti-CD3 and PMA (Sallusto et al., 1999). When EBVspecific CD8þ T cells from chronically EBV-infected human donors were examined for their ability to secrete IFN-g, the CCR7þ and CD62Lþ subsets did not respond while the CCR7 and CD62L subsets did, confirming the previously stated depiction of central memory cells as lacking cytokine secretory effector function (Hislop et al., 2001; Tussey et al., 2000). A similar but perhaps not identical subpopulation of human CD8þ cells as defined by high expression of CD27 but low expression of CD45RA was found to express no cytoplasmic perforin and low levels of cytoplasmic granzyme B, and to lack cytotoxic effector activity (Hamann et al., 1997). In contrast to the report using CCR7 expression, the CD27lo subpopulation was reported to be capable of rapidly secreting IFN-g, TNF-a, IL-4 after stimulation by PMA and ionomycin. Further evidence that human CD8þ central memory cells have cytokine secretory effector function was provided by a report that both CCR7þ and CCR7 memory cells against EBV and flu make IFN-g in response to peptide antigens (Ravkov et al., 2003). In the mouse, earlier reports examined memory cells from spleen or a combination of spleen and lymph nodes, without defining their surface phenotype. They found that such memory cells expressed perforin and were cytotoxic to antigen-pulsed target cells in short-term assays (Cho et al., 1999; Opferman et al., 1999), and gave rapid IFN-g secretion after activation (Cho et al., 1999). Similar cytotoxic effector function was reported in human memory cells (Hislop et al., 2001). Another study showed that mouse memory CD8þ T cells from spleen were not cytotoxic in short-term assays, as opposed to CD8þ memory cells from liver, lung, and lamina propria (Masopust et al., 2001). However, these memory cells from spleen as well as other organs were capable of rapid IFN-g secretion after activation. As defined by CCR7 expression, murine central memory CD8þ T cells were reported to be cytotoxic effectors and capable of secreting IFN-g and TNF-a (Unsoeld et al., 2002). It appeared that the cytotoxic activity of these cells may have been declining over a period of weeks, however. Another study defined murine central memory cells based on high CD62L expression, and found that such cells expressed low levels of cytoplasmic granzyme B and were cytotoxic in 18-hour but not 5-hour assays (Wherry et al., 2003). When isolated from either spleen or lymph node, these cells secreted IFN-g and TNF-a rapidly after activation.

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3. Effector Memory Phenotype Among the different laboratories studying CD8þ memory cells, there is a consensus that effector memory cells have cytokine secretory effector activity and, indeed, this ability gave rise to the name ‘‘effector memory.’’ The consensus includes both the mouse (Masopust et al., 2001; Unsoeld et al., 2002; Wherry et al., 2003) and human systems (Hamann et al., 1997; Sallusto et al., 1999), defined by the different approaches already discussed, and applies to IFN-g, TNF-a, IL-4, and IL-5, although not all laboratories examined all these cytokines. With respect to the cytotoxic effector activity of effector memory cells, the limited published data does not show a consensus. In patients infected with hepatitis B and hepatitis C, tetramer staining of blood cells showed a predominance of CD45RA, CCR7 antigen-reactive cells for both viruses during and after the acute viral infection (Urbani et al., 2002). However, the cytoplasmic perforin content was clearly lower in the CD45RA, CCR7 subpopulation after hepatitis C infection than after hepatitis B infection, suggesting that the surface phenotype may not correlate with function. In mice, CD8þ CCR7 memory cells were reported to have cytotoxic effector activity in short-term assays (Unsoeld et al., 2002), and CD8þ memory cells from nonlymphoid organs such as the liver and lung were also described as having such activity (Masopust et al., 2001). On the other hand, CD62Llow memory cells from mice infected with LCMV more than a month previously were found to be active in 18-hour but not 5-hour cytotoxic assays (Wherry et al., 2003). No satisfying generalizations can be made at present regarding either cytotoxic or cytokine secretory effector activities of memory phenotype cells. Part of the problem is a lack of consensus on the definition of memory cells and subsets in both mouse and human, but it also seems likely that different biological systems give different patterns of differentiation. While many reports indicate that memory cells have cytokine secretory effector activity, their cytotoxic effector activity seems to depend on the system being studied. 4. Effector Phenotype Cytotoxic effector function in a subset of CD8þ T cells from normal human blood was first demonstrated in CD11b cells using aCD3-redirected cytotoxicity (Azuma et al., 1993). Subsequently, the CD8þ effector phenotype population, defined by expression of CD45RA but low expression of CD27, also showed potent cytotoxic effector function by redirected cytotoxicity, as well as the ability to rapidly secrete IFN-g and INF-a, but not IL-2 (Hamann et al., 1997). When stained with an anti-granzyme A antibody, this effector subpopulation shows a bright granular stain similar to that of NK cells (Baars et al., 2000). The same study showed that these effectors secrete granzyme

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A and B after treatment with PMA and ionomycin, and that they express intracellular FasL. Interestingly, the frequency of CD45RAþCD27 CD8þ T cells in the blood of healthy children was recently found to correlate with prior infection with CMV but not EBV, varicella-zoster, or with MMR vaccination (Kuijpers et al., 2003). The frequency of these effector phenotype cells rose to 20% of the CD8þ T cells during the acute CMV infection and remained stable over a period of 3 years. In parallel to the these results, a CD45RAþ CCR7 human blood CD8þ effector phenotype subpopulation was reported to express high levels of intracellular perforin and to rapidly make IFN-g but not IL-2 (Sallusto et al., 1999). In the mouse, no useful CD45RA antibodies are available, and no equivalent surface markers have been developed to define this population of CD8þ T cells. However, as has been discussed, cytotoxic effector function is measurable from CD8þ T cells isolated directly from mice under a variety of circumstances. IX. Conclusions

Although the term ‘‘effector cell’’ has been used by immunologists in different ways, the term best describes differentiated T cells with immediate TcR-triggered cytotoxicity or cytokine secretion, as detected in short-term assays, which are currently only feasible in vitro. All investigators agree that antigen recognition by naive CD8þ T cells does not result in either type of effector function (the IL-2 produced by naive T cells is not considered an effector cytokine). After an initial encounter with antigen, a differentiation process occurs enabling effector function in subsequent antigen encounters. However, different effector functions have different differentiation pathways, and some subsets of post-naive CD8þ T cells have potent TcR-triggered secretory function for some cytokines but not for cytotoxic effector function. Major cellular and molecular questions about these differentiation pathways remain unanswered. Cytotoxic effector cells function by two different molecular pathways, both of which utilize TcR-triggered exocytosis of secretory granules. Cytotoxic effector cells are differentiated to express the preformed mediators perforin, granzymes, and Fas Ligand, intracellularly in granules. Differentiation further provides for a functional TcR-induced cytoplasmic polarization and granule exocytosis, with Rab27 playing a role in the latter. In the case of cytokine secretory effector cells, differentiation alters transcriptional regulatory elements to allow rapid cytokine gene expression, protein synthesis, and secretion by the constitutive pathway in response to TcR signaling. Even in fully differentiated cytotoxic effector cells, the different secretory processes for cytotoxicity and cytokine secretion are controlled by different branches of

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