The Low Affinity Fc Receptor for IgG Functions as an Effective Cytolytic Receptor for Self-Specific CD8 T Cells

This information is current as Salim Dhanji, Kathy Tse and Hung-Sia Teh of September 28, 2021. J Immunol 2005; 174:1253-1258; ; doi: 10.4049/jimmunol.174.3.1253 http://www.jimmunol.org/content/174/3/1253 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Low Affinity Fc Receptor for IgG Functions as an Effective Cytolytic Receptor for Self-Specific CD8 T Cells1

Salim Dhanji, Kathy Tse, and Hung-Sia Teh2

We have recently described a population of self-Ag-specific murine CD8؉ T cells with a memory phenotype that use receptors of both the adaptive and innate immune systems in the detection of transformed and infected cells. In this study we show that upon activation with IL-2 with or without Ag, between 10 and 20% of the activated self-specific CD8؉ T cells express the low affinity FcR for IgG. By contrast, all IL-2-activated NK cells express high levels of this FcR. The FcR comprises the Fc␥RIII␣ and FcR␥ subunits. However, the FcR␥ subunit also associates with the CD3 complex, and this association probably contributes to the low expression of FcR in activated cells. Although the FcR is expressed at a low level on activated self-specific CD8؉ T cells, it functions very efficiently as a cytolytic receptor in ADCC. FcR-dependent killing occurred in the absence of TCR stimulation, but could be ؉ augmented by concurrent stimulation of the TCR. In addition to mediating ADCC, engagement of the FcR on self-specific CD8 Downloaded from T cells results in the production of both IFN-␥ and TNF-␣. This is the first report of an activating FcR on self-specific murine CD8؉␣␤ TCR؉ T cells and establishes the importance of innate immune system receptors in the function of these self-specific .CD8؉ T cells. The Journal of Immunology, 2005, 174: 1253–1258

atural killer cells represent a highly specialized lym- Fc␥RIII␣ associates mainly with ITAM-containing homo- or het- ␨ ⑀ ␥ ␥

phoid population characterized by potent cytolytic ac- erodimers of CD3 and Fc RI (FcR ) in humans (8) or solely http://www.jimmunol.org/ N tivity against tumor or virally infected cells. Their func- with FcR␥ homodimers in mice (9). The binding of IgG to CD16 tion is finely regulated by a series of inhibitory or activating results in the phosphorylation of ITAMs in the signaling chains, receptors (1). The inhibitory receptors, specific for MHC class I leading to the recruitment of kinases such as ZAP-70 and Syk (10). molecules, allow NK cells to discriminate between normal cells These kinases initiate a signaling cascade resulting in the lysis of and cells that have lost the expression of MHC class I (e.g., tumor Ab-coated target cells. T cells also use ITAM-containing receptors cells). Inhibitory receptors, such as those belonging to the Ly49 and Syk and ZAP-70 for their signal transduction (reviewed in Ref. family in mice and the killer Ig-like receptors in humans, contain 11). Thus, the signals transduced by the engagement of CD16 on ITIM motifs in their cytoplasmic domains (2). Engagement of NK cells are very similar to those transduced by the engagement of these receptors results in the recruitment of inhibitory phospha- TCR on T cells. by guest on September 28, 2021 tases that prevent NK cell activation. The activating receptors re- CD16 expression is not limited to NK cells; other cell types sponsible for NK cell triggering include NKp46, NKp30, NKp44, have been described that also express this receptor. ␥␦ T cells have and NKG2D in humans and NKp46 and NKG2D in mice (3, 4). been shown to express CD16, as have a population of large gran- The activating receptors, some of which belong to the same fam- ϩ Ϫ Ϫ ular lymphocytes (mostly ␣␤TCR CD4 CD8 T cells) in hu- ilies, do not contain activation motifs, but, rather, associate with mans (12, 13). In addition, some memory phenotype adaptor molecules or signaling partners important for signal trans- ␣␤TCRϩCD8ϩ T cells in humans have been shown to express duction (5). CD16 (14). We have previously described a population of One of the first activating receptors described on NK cells is ␣␤ ϩ ␣␤ϩ Fc␥RIII␣ or CD16. CD16 is a low affinity FcR that binds to IgG TCR CD8 T cells in normal B6 mice that exhibit a mem- and is involved in Ab-dependent cell-mediated cytotoxicity ory phenotype characterized by the expression of high levels of ␤ (ADCC),3 in which an Ab-coated target cell is destroyed by NK CD44, IL-2R , and Ly6C (15). These cells can be activated by cells (6). Stimulation of CD16 on NK cells also results in the cytokines such as IL-2 and IL-15, and upon activation they express production of cytokines, such as IFN-␥, TNF-␣, and GM-CSF (7). several functional NK receptors, including 2B4, CD94, and NKG2D as well as the NK adaptor protein DAP12. Using an H-Y TCR transgenic model, we have shown that the development of CD8ϩCD44high T cells is driven by the high affinity interaction of Department of Microbiology and Immunology, University of British Columbia, Van- couver, Canada the ␣␤TCR with cognate self-Ag (16). The H-Y TCR is specific b b Received for publication May 7, 2004. Accepted for publication November 9, 2004. for a male Ag (H-Y) presented by H-2D , and in H-2 H-Y female The costs of publication of this article were defrayed in part by the payment of page mice, the lack of male Ag results in the development of H-Y CD8 charges. This article must therefore be hereby marked advertisement in accordance T cells with a naive phenotype (CD44low). In H-2b H-Y male mice, with 18 U.S.C. Section 1734 solely to indicate this fact. in contrast, the presence of the cognate (H-Y) Ag results in the 1 This work was supported by grants from the Canadian Cancer Society and the development of a population of CD8ϩ T cells that is virtually Canadian Institutes of Health Research (to H.S.T.). S.D. is supported by the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foun- identical in cell surface and functional phenotypes with the mem- dation of Health Research. ory phenotype CD8ϩCD44high cells in normal B6 mice (16). Be- 2 Address correspondence and reprint requests to Dr. Hung-Sia Teh, Department of cause memory phenotype CD8ϩ T cells in normal and H-Y TCR Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: transgenic mice are specific for self-Ags, we will refer to these ϩ [email protected] cells as self-specific CD8 T cells to distinguish them from con- ϩ ϩ 3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity. ventional memory CD8 T cells. Self-specific CD8 T cells from

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1254 SELF-SPECIFIC CD8ϩ T CELLS EXPRESS AN ACTIVATING FcR both H-Y TCR transgenic (16) and nontransgenic (15) mice pref- sciences). CD3 immunoprecipitation was preformed by treating lysates erentially kill syngeneic tumor cells. This killing of syngenic tu- with preconjugated anti-CD3⑀/protein G-Sepharose beads for2hat4°C, mors involves the MHC-restricted ␣␤ TCR as well as the activat- followed by several washes. Complexes were then removed from the beads by resuspension in 2ϫ protein sample buffer, followed by boiling for 5 ing NK receptor, NKG2D, which results in non-MHC-restricted min. The samples were then run in a 4–15% Tris-HCl gel and immuno- lysis of target cells that express the NKG2D ligand, Rae-1 (16). blotted as described above. In this report we have described the expression and function of CD16 in self-specific CD8ϩ cells from B6 and male H-Y TCR Results transgenic mice. We showed that this FcR, comprising the Expression of Fc␥RIII␣/FcR␥ on activated self-specific Fc␥RIII␣/FcR␥ subunits, is similar in composition to the NK FcR. CD8ϩ cells Although this FcR is expressed at a low level in self-Ag-specific ϩ ϩ We have previously shown that self-specific CD8 T cells express CD8 T cells, it is particularly efficient at initiating the destruction an activated/memory phenotype (15, 16). Initially, ex vivo self- of Ab-coated target cells and can induce the production of two key specific CD8ϩ cells from B6 and male H-2b H-Y mice did not ␥ ␣ inflammatory cytokines, IFN- and TNF- . These observations express significant levels of CD16 (Fig. 1a and data not shown). underscore the importance of innate immune system receptors in ϩ However, upon activation with IL-2 alone (B6) or Ag and IL-2 the function of these self-specific CD8 T cells. (H-Y male) for 5–6 days, between 10 and 15% of the activated self-specific CD8ϩ cells from B6 (CD8ϩCD44high) and male H-2b Materials and Methods H-Y mice expressed CD16 (Fig. 1a). In contrast, all IL-2-activated Mice NK cells expressed high levels of CD16 (Fig. 1a). Because naive Downloaded from Breeders for C57BL/6 (B6) mice were obtained from The Jackson Labo- (CD8ϩCD44low) cells from B6 and female H-Y TCR transgenic ratory. The H-Y TCR transgenic mice (17) were bred to the B6 back- mice did not proliferate in response to stimulation with IL-2 alone, ground. Mice, 6–12 wk of age, were used for the experiments described. these cells were activated with anti-CD3 and IL-2 (B6) or Ag plus Abs and flow cytometry IL-2 (H-Y female) to induce activation and proliferation. It is clear that activated CD8ϩCD44low cells from B6 and female H-Y TCR The following Abs were used: anti-CD4 (GK1.5), anti-CD8␣ (53.67), anti- CD8␤ (53.38), anti-NK1.1 (PK136), anti-CD3⑀ (2C11), anti-CD90 (T24, transgenic mice do not express CD16 (Fig. 1a), consistent with the Rat IgG) (18), anti-CD16/32 (2.4G2, Rat IgG2b), anti-CD44 (PGP1), anti- conclusion that conventional CD8 T cells do not express CD16 http://www.jimmunol.org/ FcR␥, anti-CD3␨ (19), and anti-H-Y TCR␣ (T3.70) (17). All Abs were upon activation. ␥ purchased from BD Pharmingen, except anti-FcR (Upstate Biotechnol- We have previously described the bystander expansion and ac- ogy), anti-CD16/32 (American Type Culture Collection), and anti-H-Y TCR␣ (eBioscience). The CellQuest software program (BD Biosciences) tivation of self-specific CD8 T cells in response to infection with was used for data acquisition and analysis. the bacterial pathogen Listeria monocytogenes (15, 16). Therefore. we wanted to determine whether the frequency of CD16ϩ Cell lines The cell lines used were the RMA lymphoma (H-2bϩ, Rae-1␦Ϫ) and Tap- deficient RMAS (H-2bϪ, Rae-1␦Ϫ). The cell lines were cultured in IMEM (Invitrogen Life Technologies) supplemented with 10% (v/v) FBS (Invitro- by guest on September 28, 2021 gen Life Technologies), 5 ϫ 105 ␮M 2-ME, and antibiotics (I-medium). Cell purification and activation CD8ϩCD44high T cells from B6 mice were purified and cultured in IL-2 (200 U/ml) for 5 days as previously described (15). NK cells were enriched by depletion of CD4ϩCD8ϩCD3ϩIgϩ cells using Dynabeads (Dynal Bio- tech) and then cultured in IL-2 for 5 days, resulting in a pure population of activated NK cells (15). Purified naive CD8ϩ (CD44low) cells do not re- spond to IL-2 alone and were activated for 5 days on plate-bound anti- CD3⑀ (10 ␮g/ml) and IL-2 (20 U/ml). Purified H-Y TCRϩCD8ϩ T cells (1 ϫ 106) were activated by culture with 1 ϫ 107 B6 splenocytes, 1 ␮M H-Y peptide, and 20 U/ml IL-2 for 5–6 days. RT-PCR RNA was extracted from activated cells and reverse transcribed as previ- ously described (15). PCR was preformed using previously described prim- ers and reaction conditions (20). FIGURE 1. Activation of CD8ϩCD44high cells results in the expression CTL assays of CD16. a, Purified CD8ϩCD44high cells from B6 and CD8 cells from CTL assays against RMA and RMAS target cells were performed as pre- male H-2b H-Y TCR transgenic mice were used as a source of self-specific viously described (15). For ADCC, the target cells were pretreated with CD8 T cells. Purified CD8ϩCD44low cells from B6 and female H-2b H-Y anti-CD90 mAb (10 ␮g/ml) for 15 min at room temperature before use. For TCR transgenic mice were used as a source of naive CD8 T cells. Self- the FcR-blocking experiment, anti-FcR mAb (2.4G2 used at 15 ␮g/ml) was specific B6 CD8ϩ T cells and NK cells were activated by culture with IL-2 added to the effector cells 15 min before the addition of targets and was for 5 days. Naive CD8 T cells from B6 mice were activated by culture with present throughout the assay. Spontaneous release varied from 8–15% of anti-CD3 and IL-2 for 5 days, and CD8 cells from male and female H-Y the maximum. All assays were performed in triplicate. The percent specific mice were activated with Ag and IL-2 for 6 days. The activated cells were lysis was calculated as 100% ϫ [cpm (experimental well) Ϫ cpm (spon- f taneous release)]/[(cpm (maximum release) Ϫ cpm (spontaneous release)]. stained for the expression of CD16/CD32. , CD16/CD32 profile of the indicated cell type; Ⅺ, unstained controls. b, Naive B6 mice were infected Immunoprecipitation and immunoblot analysis with 10,000 CFU of L. monocytogenes. On day 5 the mice were killed, and the spleens were removed and stained. The histograms depict the expres- Cells were activated as described above and pelleted/lysed in 10 mM Tris sion of CD8 and CD16 on gated CD8 T cells from infected (right)or (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and protease and ϩ ϩ phosphatase inhibitors. The lysates were separated on a 4–15% Tris-HCl uninfected (left) mice, and the percentages of CD16 CD8 T cells are ϩ ϩ polyacrylamide gel and transferred to a polyvinylidene difluoride mem- indicated. Three-color analysis revealed that the CD8 CD16 cells also brane. Blots were developed using an ECL system (Amersham Bio- expressed high levels of CD44 (data not shown). The Journal of Immunology 1255

CD8ϩCD44high increased upon infection with L. monocytogenes. CD8ϩ cells from B6 (CD8ϩCD44high) and H-Y male mice. By In uninfected B6 mice, there was a small percentage of CD8ϩ T contrast, self-specific CD8ϩ cells from B6 and H-Y male mice cells that expressed CD16, but this number increased by ϳ2-fold express both CD3␨ and FcR␥ (Fig. 2b). As expected, activated upon infection with Listeria (Fig. 1b). In addition, all conventional CD8 (CD8ϩCD44low) cells only express CD3␨. CD8ϩCD16ϩ cells in both infected and uninfected mice expressed These findings suggest that the low levels of CD16 surface ex- high levels of CD44. pression in self-specific CD8ϩ cells are probably due to the inhi- The Ab used to detect CD16 expression also bound to CD32, bition of binding of FcR␥ homodimers to CD16 by CD3␨/FcR␥ and it was important to determine which FcR subunits were actu- heterodimers in these cells. ally expressed by the self-specific CD8ϩ cells. RT-PCR with prim- In conventional T cells, the ␣␤ TCR pairs with the CD3 family ers specific for various FcR subunits was used to determine the of signaling chains, including CD3⑀ and CD3␨ (22). Because FcR␥ composition of the expressed FcR. It is clear from this analysis that is expressed in self-specific CD8ϩ cells, we determined whether activated self-specific CD8ϩ cells from B6 and male H-2b H-Y this signaling molecule could associate with the TCR/CD3 com- mice as well as NK cells from B6 mice expressed the mRNA for plex in these cells. To determine whether FcR␥ associates with the only Fc␥RIII␣ (CD16) and FcR␥, not Fc␥RI or Fc␥RIIB (Fig. 2a). CD3 complex, we immunoprecipitated the CD3 complex from IL- A macrophage cell line was used as a positive control for the 2-activated self-specific CD8ϩ cells from B6 and male H-Y mice expression of Fc␥R1 and Fc␥RIIB (Fig. 2a). using an anti-CD3⑀ Ab and immunoblotted with either an anti- In murine NK cells, CD16 can only pair with FcR␥ homodimers CD3␨ or anti-FcR␥ Ab. Fig. 2c clearly shows that anti-CD3⑀ pre- (9). Furthermore, atopic expression of CD3␨ in murine NK cells cipitates both CD3␨ and FcR␥ in self-specific CD8ϩ cells from B6

actually interfered with the surface expression and function of and male H-Y mice. As expected, anti-CD3⑀ precipitates only Downloaded from CD16 through the formation of CD3␨/FcR␥ heterodimers, which CD3␨ in conventional CD8ϩCD44low cells. This association of cannot associate with CD16 (21). This finding suggests that the FcR␥ with the CD3 complex in self-specific CD8ϩ cells probably low cell surface expression of CD16 on self-specific CD8ϩ cells interferes with the association of CD16 with FcR␥, resulting in low may be due to the high expression of CD3␨ in these cells. To expression of CD16 in these cells. ␨ address this possibility, we compared the total amount of CD3 ϩ ϩ IL-2-activated self-specific CD8 cells can mediate ADCC and FcR␥ in activated NK and self-specific CD8 cells by Western http://www.jimmunol.org/ blot. Fig. 2b shows that activated NK cells express an undetectable After observing the expression of CD16 on IL-2-activated self- level of CD3␨ and the most FcR␥ compared with self-specific specific CD8ϩ cells, we determined whether this receptor could mediate ADCC. To this end, we activated self-specific CD8ϩ cells and NK cells from B6 mice with IL-2, then tested their ability to kill Ab-coated RMAS targets cells. Anti-CD3- and IL-2-activated naive CD8 (CD8ϩCD44low) cells were included as a negative con- trol. TAP-deficient RMAS cells were used as target cells to rule out contribution by MHC class I molecules in the killing reaction. The RMAS cells were left untreated or were pretreated with anti- by guest on September 28, 2021 CD90 (clone T24; 10 ␮g/ml) mAb before use as target cells. Fig. 3a clearly shows that IL-2-activated self-specific CD8ϩ and NK cells can efficiently kill Ab-coated RMAS cells, whereas anti- CD3- and IL-2-activated conventional CD8 (CD8ϩCD44low) cells show absolutely no activity. We noted that anti-CD90 was more efficient in promoting the killing of RMAS targets by self-specific CD8ϩ cells compared with NK cells. This finding is remarkable considering that only a small fraction of the self-specific CD8ϩ cells express CD16, and the level of CD16 expressed per cell is significantly lower than that for NK cells (Fig. 1a). Because RMAS cells are killed efficiently by activated NK cells, the lack of killing of untreated RMAS cells by activated self-specific CD8ϩ cells also indicates the lack of contaminating NK cells in the kill- ing assay. After observing that self-specific CD8ϩ cells from B6 mice could efficiently lyse RMAS targets, we determined whether the presence of MHC class I had any effect on lysis by using TAP- sufficient RMA targets. In addition, we determined whether block- ing the CD16 receptor with an anti-CD16 mAb on the activated self-specific CD8ϩ cells could block killing of Ab-coated target FIGURE 2. Activated self-specific CD8 T cells express a low affinity cells. Fig. 3b demonstrates that self-specific CD8ϩ cells efficiently FcR similar to NK cells. Cells were activated as described in Fig. 1. a, killed Ab-coated RMA cells. Furthermore, the killing of Ab-coated RT-PCR analysis of Fc␥RIII␣, FcR␥,Fc␥RI, and Fc␥RIIB transcripts target cells was greatly reduced by blocking the CD16 receptor on present in activated cells. RNA from the J774 macrophage cell line was self-specific CD8ϩ cells before culture with Ab-coated RMA tar- used as a positive control for Fc␥RI and Fc␥RIIB. b, Whole cell lysates of gets. For NK cells, the lysis of Ab-coated RMA cells was only the activated cells were subjected to immunoblot analysis for the detection of FcR␥ and CD3␨ protein. Blots were stripped and reprobed with anti- partially inhibited by blocking CD16 on NK cells. This was prob- ERK2 as a loading control. c, Activated cells were lysed, and the lysates ably due to the high expression of CD16 on NK cells, which could were immunoprecipitated (IP) with anti-CD3⑀ Ab. The immunoprecipitates not be blocked completely by the anti-CD16 Ab treatment. An were then immunoblotted (IB) with anti-CD3␨ or anti-FcR␥ mAbs. N.D., alternative explanation for the inefficient blocking of killing of not determined. Ab-coated target cells by NK cells is that the anti-CD16 mAb 1256 SELF-SPECIFIC CD8ϩ T CELLS EXPRESS AN ACTIVATING FcR

FIGURE 4. CD16-mediated killing of target cells by self-specific CD8 Downloaded from T cells is TCR independent. Self-specific CD8 T cells from H-2b H-Y male mice and naive CD8 T cells from female H-2b H-Y mice were activated as described in Fig. 1 and used in a standard chromium release assay against RMA targets either pretreated with anti-CD90 mAb (10 ␮g/ml) or left untreated. The assay was performed at a constant 10:1 E:T cell ratio with addition of the indicated concentrations of H-Y peptide. Error bars repre-

sent the SD of triplicate cultures. http://www.jimmunol.org/

self-specific CD8ϩ cells from male H-Y mice killed Ab-coated RMA targets very efficiently even in the absence of the H-Y pep- tide. The killing of anti-CD90-coated target cells was slightly en- FIGURE 3. CD8ϩCD44high cells efficiently kill Ab-coated targets via hanced by the addition of H-Y peptide (Fig. 4). The activated ϩ an Fc-dependent mechanism. a, NK, self-specific CD8 (CD8ϩCD44high), self-specific CD8 cells from male H-Y mice required almost 10 and naive CD8 (CD8ϩCD44low) cells from B6 mice were activated as nM exogenous H-Y peptide to attain the same level of killing as described in Fig. 1 and used as effectors in a standard chromium release that seen with anti-CD90-coated target cells in the absence of H-Y by guest on September 28, 2021 assay against TAP-deficient RMAS target cells that had been treated with peptide. This is remarkable because the entire population of H-Y ␮ anti-CD90 mAb (10 g/ml) or left untreated. b, Activated NK and male cells expressed the H-Y TCR, whereas at most 20% of the ϩ high CD8 CD44 cells were used as effectors in a chromium release assay cells expressed CD16. By contrast, activated conventional CD8ϩ against MHC-sufficient RMA target cells that had been treated with anti- T cells from female H-Y mice could only kill peptide-loaded target CD90 mAb (10 ␮g/ml) or left untreated. The effector cells were also pre- treated with anti-CD16 mAb (10 ␮g/ml) or left untreated to block the FcR. cells, and the presence of Ab on the targets had no effect on this Error bars represent the SD of triplicate cultures. killing. These results suggest that CD16 functions independently of the TCR as an effective cytolytic receptor on self-specific CD8ϩ cells. Furthermore, the FcR and the TCR can act in an additive functions as an agonistic mAb. However, this is unlikely, because manner in the killing reaction.

the treatment of NK cells with anti-CD16 mAb did not have any ϩ effect on the lysis of untreated RMA target cells (data not shown). Engagement of CD16 on self-specific CD8 cells induces These results clearly demonstrate that self-specific CD8ϩ cells ex- cytokine production press a functional FcR, which can mediate ADCC. Furthermore, CD16 engagement on NK cells has been shown to induce the ex- the expression of MHC class I molecules on the target cells does pression of several cytokines in addition to being able to induce not affect FcR-mediated killing. ADCC (7). To test whether CD16 engagement on self-specific

ϩ CD8 T cells could also mediate the production of cytokines, we FcR on self-specific CD8 cells from H-Y male mice functions cultured Ag-activated H-Y male CD8 cells with RMA targets that independently of the TCR had been pretreated with anti-CD90 or left untreated. We found Because self-specific CD8ϩ cells from male H-Y mice express that these cells showed a significant increase in IFN-␥ production only the male-specific H-Y TCR, we used these cells to determine in response to anti-CD90-coated RMA cells (7.2% IFN-␥ϩ) over whether the FcR can function independently of the TCR. Self- untreated RMA cells (1.3%). Furthermore, this increase in IFN-␥ specific CD8ϩ cells from male H-Y mice and conventional CD8ϩ production was reduced to near basal levels by the inclusion of cells from female H-Y mice were activated with Ag and IL-2 for soluble anti-CD16 mAb in this assay (Fig. 5a). This result suggests 6 days. The activated cells were then assessed for cytolytic activity that the Fc portion of the bound anti-CD90 mAb on RMA cells against untreated or anti-CD90-treated RMA (H-2b) target cells. induces the production of IFN-␥ by H-Y male CD8 cells. The killing of anti-CD90-coated target cells in the absence of ex- To obtain more direct evidence for FcR-mediated cytokine pro- ogenous H-Y peptide was used as a measure for the contribution of duction by H-Y male CD8 cells, we used Ab cross-linking to stim- the FcR in the killing reaction. Inclusion of the H-Y peptide in the ulate CD16 directly. We cultured day 6 Ag-activated H-Y male assay allows an estimation of the contribution of H-Y TCR in the CD8 T cells in wells coated with no Ab (Fig. 5b, upper row), killing reaction. It is clear from the data presented in Fig. 4 that anti-CD16 (middle row), or anti-CD3⑀ (bottom row). After a 5-h The Journal of Immunology 1257

CD8ϩ T cells to detect infected or transformed target cells, which might not be detected by NK cells. It is interesting to note that even though the H-Y male cells represent a clonal population in which all cells should have been activated equivalently, only a fraction of the activated cells express CD16. The low cell surface expression of CD16 on self-specific CD8ϩ cells is probably due to the high expression of CD3␨ in these cells. In murine NK cells, CD16 can only pair with FcR␥ homodimers (9). Furthermore, the atopic expression of CD3␨ in murine NK cells interferes with the surface expression and func- tion of CD16 through the formation of CD3␨/FcR␥ heterodimers, which cannot associate with CD16 (21). We found that the FcR␥- chain in self-specific CD8ϩ T cells is coprecipitated with CD3␨ by the anti-CD3⑀ mAb. It is likely that the resulting CD3␨/FcR␥ het- erodimers interfere with the expression of the FcR on IL-2-acti- vated self-specific CD8ϩ T cells. The association of the FcR␥ chain with CD3␨ is not unique to self-specific CD8ϩ T cells, be- cause in large granular lymphocytes and in T cells from tumor-

bearing mice, the CD3 complex has been shown to associate with Downloaded from FcR␥ (23, 24). The association of CD3␨ with the FcR␥-chain also FIGURE 5. Cytokine production by self-specific CD8 T cells in re- provides a potential explanation for the high efficiency of lytic sponse to CD16 engagement. a, Day 6 Ag- and IL-2-activated H-Y male activity of the FcR in these cells. We speculate that engagement of ␮ CD8 cells were cultured with RMA cells treated with anti-CD90 (10 g/ the FcR causes conformational changes in CD3␨, resulting in ac- ␮ ml) or left untreated. Soluble anti-CD16 (10 g/ml) was added to some tivation of signaling pathways associated with the ␣␤ TCR. A cultures to block Fc binding. IFN-␥ production was assessed by intracel-

second, nonmutually exclusive explanation is that the differences http://www.jimmunol.org/ lular staining after a 5-h incubation period. b, Day 6 Ag- and IL-2-activated H-Y male CD8 cells were cultured with plate-bound anti-CD16 (10 ␮g/ml) in the lytic activity of CD16 in these two cell types may be cell or anti-CD3⑀ (10 ␮g/ml) or without any Ab for 5 h. The cells were then intrinsic, which reflects differences in their physiology and/or de- stained for intracellular IFN-␥, TNF-␣, and GM-CSF. The numbers in the velopmental pathway. ϩ dot plots represent the percentage of cytokine-positive CD8ϩ T cells. We have previously shown that self-specific CD8 cells un- dergo bystander expansion in vivo in response to Listeria infec- tion, probably as a consequence of the high expression of IL-2R␤ by these cells, which enabled these cells to proliferate in response incubation period, we fixed and stained the cells for CD8 and to IL-2 or IL-15 (16). Furthermore, self-specific CD8ϩ T cells that IFN-␥ (left column), TNF-␣ (middle column) or GM-CSF (right proliferate in response to bacterial infection exhibit a heightened by guest on September 28, 2021 column). CD16 engagement resulted in a large increase in both ability to produce IFN-␥ (16). These properties of self-specific IFN-␥ and TNF-␣ production, with the percentage of cytokine- CD8ϩ T cells would enable them to detect infected cells and pro- ϩ positive cells being similar to the percentage of CD16 cells in the vide an early source of IFN-␥. The expression of a self-specific sample; CD16 engagement did not induce the production of GM- TCR and NKG2D on these cells would allow them to focus on host CSF (Fig. 5b). We also found that soluble anti-CD16 is inefficient cells that expressed ligands induced by infection or transformation. in inducing IFN-␥ production by H-Y male CD8 cells (data not In this study we have provided evidence for the expression and shown). It is likely that soluble anti-CD16 mAb is less efficient functional significance of another activating NK receptor that adds than plate-bound anti-CD16 mAb in aggregating FcRs, and that to the arsenal of self-specific CD8ϩ cells. We have shown that receptor aggregation is required for efficient activation. By contrast CD16 is expressed on a significant fraction of CD8ϩCD44high T to FcR stimulation, stimulation of the CD3 complex resulted in the cells upon Listeria infection in vivo or upon activation with IL-2 majority of the cells expressing IFN-␥ and TNF-␣. In addition, or Ag and IL-2 in vitro. Furthermore, engagement of CD16 on anti-CD3-stimulated cells produced significant amounts of GM- self-specific CD8ϩ T cells results in efficient lysis of Ab-coated CSF (Fig. 5b). These results indicate that FcR and ␣␤ TCR func- targets as well as in the production of inflammatory cytokines. The tion as directly activating receptors, which transduce qualitatively possession of activating receptors of the innate as well as the adap- and quantitatively distinct signals upon activation. tive immune system distinguished these cells from NK cells and suggests that this interesting cell type may be particularly adept in Discussion providing an early response to infected and transformed cells. In this study we showed that upon activation with IL-2 or Ag plus These cells will also provide an early source of cytokines, such as IL-2, self-specific CD8ϩ cells express an FcR similar in compo- IFN-␥ and TNF-␣, which would prime the adaptive immune sys- sition to the low affinity FcR for IgG that is found on NK cells. tem for the elimination of infected and transformed cells. Even though these cells express only a relatively low level of this receptor compared with NK cells, it functions very efficiently in Acknowledgments the lysis of Ab-coated target cells. FcR-mediated killing is inde- We thank Soo-Jeet Teh for excellent technical assistance, and pendent of Ag expression on the target cells. Furthermore, the Dr. Hao Shen (University of Pennsylvania) for the kind gift of wild-type expression of MHC class I molecules on target cells did not affect Listeria monocytogenes strain 10403s. the efficiency of FcR-mediated killing. The FcR can also act in an additive manner with the TCR in the lysis of susceptible target cells. This FcR not only mediates efficient lysis of susceptible tar- References ␥ ␣ 1. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359. gets, but it also induces the production of both IFN- and TNF- . 2. Long, E. O. 1998. Regulation of immune responses by inhibitory receptors. Adv. The combination of these properties would enable self-specific Exp. Med. Biol. 452:19. 1258 SELF-SPECIFIC CD8ϩ T CELLS EXPRESS AN ACTIVATING FcR

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