Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Lineage‐independent activation of immune system effector function by myeloid Fc receptors.

Lineage‐independent activation of immune system effector function by myeloid Fc receptors. The EMBO Journal vol.1 1 no.13 pp.4861 -4868, 1992 Lineage-independent activation of immune system effector function by myeloid Fc receptors Waldemar Kolanus, association of Charles Romeo and the receptor with surface IgM by co-capping (Amigorena et B Brian Seed al., 1992). Both forms of FcRII are capable of inhibiting B-cell activation mediated by the IgM antigen Department of Molecular Biology, Massachusetts General Hospital, receptor (Amigorena et al., 1992). Boston, MA 02114, USA A low affinity receptor of the third class, Fc'yRIHA, Communicated by R.Kamen mediates immune cell activation through its association with one or more members of a small family of 'trigger An molecules' (Hibbs et al., 1989; Kurosaki and Ravetch, 1989; emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain Lanier et al., 1989; Ra et al., 1989; Anderson et al., 1990; complexes in which the ligand recognition elements Bonnerot et al., 1992). associate with one or more 'trigger molecules' whose These trigger molecules, T cell receptor (TCR) r chain aggregation initiates a signal transduction cascade. The (Samelson et al., 1985; Weissman et al., 1988) TCR v chain sequence motifs et al., 1990) and Fc receptor 'y chain (Blank et al., 1989; constituting the active sites of these (Jin trigger molecules are Ra et al., 1989; Bonnerot et al., 1992) interact with ligand found in the T cell and B cell antigen recognition domains of different immune system receptors receptors, and some Fc receptors, and appear to be and can central to effector function activation. For example, of autonomously initiate cellular effector programs, the many molecules that mimic or potentiate the action including tyrosine kinase activation, cytokine secretion and of the T cell antigen receptor (TCR), none have yet been cytolysis, following their aggregation (Irving and Weiss, found to initiate effector programs autonomously in cells 1991; Letourneur and Klausner, 1991; Romeo and Seed, lacking TCR. We have devised two strategies to study 1991; Bonnerot et al., 1992). Within the ¢, v and FcR -y activation intracellular domains are sequence motifs also represented mediated by myeloid Fc receptors, which in CD3 -y, 6 and e chains, in the B cell antigen receptor mbl appear not to associate with trigger molecules: the use and B29 proteins, and in the FccRI 13 of primary human cytolytic T cells as surrogate effector chain (Kinet, 1989; Reth, 1989; Wegener et al., 1992). Recent studies have cells for genetically delivered receptors, and the use of vaccinia virus vectors to shown that CD3e introduce genetically modified (Letourneur and Klausner, 1992) and a receptors into primary human monocytes. complex comprising CD3-y, 6 and e Using these (Wegener et al., 1992) we have the to activate T approaches, have found that the cytoplasmic domains capacity cells, suggesting that the of two Fc function of the receptors show comparable function to sequence motif common to these chains may domains of be to initiate a common or related activation pathway. equivalent the trigger molecule family, but in are not Early events the activation of receptors associating with homologous to members of that family. words: trigger molecules include tyrosine phosphorylation, Key cytolysis/Fc-yRII/Fc receptor/macrophages formation of inositol phosphates and accumulation of free intracellular Ca2+ Sefton and (e.g. Gardner, 1989; In Campbell, 1991; Keegan and Paul, 1992). many cases Introduction the later stages of activation are accompanied by a change in in Receptors for the constant portion of immunoglobulin G transcriptional potential which results cytokine synthesis (Fc'y form a class of cell surface or a developmentally activated cell state. The causative chain receptors) complex proteins of immune of events which translates cell surface mediating phagocytosis complexes, transcytosis receptor aggregation understood even in and various forms of cellular into cellular activation remains poorly antibody dependent cytotoxicity Unkeless et et the best studied examples. (Meilman, 1988; al., 1988; Fanger al., 1989; The of cell effector function can Human and murine triggering type-specific Ravetch and Kinet, 1991). Fc-y receptors be distinguished from the diverse which either to a signals have been divided into three classes, corresponding high influence to central or a of low responsiveness activating signals affinity type (Fc-yRI), family affinity receptors (the third low mediate activation which do not effector Fc'yRHl receptors) and a affinity type (Fc-yRIH) pathways engage in or chemotaxis. As in mice. Some as found adhesion which has two forms in humans, and one function, e.g. yet, have well delineated. no molecules from members of the of function are apart trigger family aspects receptor fairly activate T the been shown to most has been shown to mediate autonomously cells, recently phagocytosis Fc7yRIIA of immune effector et Odin et characterized cells et Indik extensively system (Engelhardt al., 1991; al., 1991; al., and of of (Yokoyama Shevach, 1989). Aggregation while mediates internalization 1991), Fc-yRIIB2 Ig-coated et Yeh and phospholipid-linked proteins (Kroczek al., 1986; into clathrin coated targets pits, (Hunziker Mellman, and or treatment of et al., Balk CD2 related 1987; Terhorst, 1989) et The 1989; Miettinen al., 1989). closely Fc-yRIIB1 antibodies et with of monoclonal of the of but is from pairs (Meuer al., 1984) bears all sequences Fc'yRIIB2, prevented function in T but this can activate effector activation the a 47 amino cells, association with endocytotic apparatus by of one or more members of on the the in the domain et depends presence acid insertion al., cytoplasmic (Amigorena et et in B favors trigger family (Schmitt-Verhulst al., 1987; Moingeon Miettinen cells, 1992; al., 1992) which, Press Oxford University W.Kolanus, C.Romeo and B.Seed :: :::.: .. ... *, ^ r -- ; ar .: ::-:.:.:. ~~. ..... ::-.::..... .... ...., ~~~~~~~~~~~~~.. _. .. ...... * ........... t: - - :~~....... ..:--..:..:-:-:.: .~~~~~~~~~~~~. ......... , :1 , L-.. ~~~~~~~~~~~.. ~~~~~~~~~~~. ~~~~~~~~~~~~~.. ....... X .... .,.... ..............................-x. B-: ([| r;L 5|i ..... . . . .. .. ... ... . . ... -,:- '::: ..:. .-, t * 9 ~~~~~~~~~....... .. -- -__ ~~~~~. ._. .. ... . .... S._.. __ .9 1.. ~~~~~~~~~~~~~. ~~~~~~~~~~~~. I_r ..1.. Fig. 1. Creation and analysis of FcyRII chimeras. Schematic of CD16: and chimeras. The intracellular domains (a) diagram FcyRII CD4:FcyRll of and C differ by a single residue (268 from the amino-terminus of the while the intracellular domains of and B2 Fc'yRIIA precursor), Fc-yRIIB1 differ by the presence or absence of an additional exon of chimeras. An of (light stippling). (b) Immunoprecipitation CD16:Fc-yRII a autoradiogram reducing gel of Bi and B2 chimeras is shown with molecular mass standards on the left. At we believe immunoprecipitated Fc-yRIIA, C, present that the lower molecular mass in the and C tracks species Fc-yRIIA represent partially glycosylated precursors. In less characterized fusing DNA segments encoding the different extracellular et al., 1992). extensively cell there types the of is relatively little information as to domains to fragments encoding Fc receptor indispensability sequences In the trigger molecules. no small part this of obtained from previously described clones (Stengelin et scarcity al., absence of suitable information can be attributed to the cell 1988) or from polymerase chain reactions based on other lines which both possess effector function and are amenable reported sequences (Stuart et al., 1989; Qiu et al., 1990; to biochemical Figure 1). The gene fusions were constructed in genetic and analyses. vaccinia initiation of Fc in virus expression vectors and subsequently inserted into wild The cytolysis by receptors macrophages type vaccinia by recombination at the thymidine kinase (or monocytes, their blood-borne is an locus, precursors) example of has been for using selection for cointegration of Escherichia an activation process whose study coli gpt stymied want of a suitable in vitro system. several (Falkner and Moss, 1988; Boyle and Coupar, Although myeloid 1988) to cell lines have been none the facilitate identification of the desired developed, display cytolytic recombinants. activity of freshly isolated or Immunoprecipitation of Jurkat (T cell monocytes macrophages. leukemia) cells infected Conversely, monocytes in culture exhibit little with the recombinant viruses revealed chimeric primary molecules of proliferative potential and are to the expected molecular masses (Figure 1 and data not sufficiently refractory transfection to make transient shown). expression approaches infeasible. In this work we have pursued two alternative Calcium in T strategies to explore Fc receptor mediated activation: the use mobilization cells is independent of T of primary human cytolytic T cells as surrogate effector cells cell receptor for genetically delivered Fc receptors, and the use of Introduction of the chimeras vaccinia into a TCR-negative mutant of virus vectors to introduce Fc receptors into primary human the Jurkat line (Weiss and Stobo, 1984) showed that the monocytes. Using these approaches we have found that intracellular domains of and Fc-yRflC were capable FcyRIHA protein of an chimeras based on Fc-yRIIA as well as similar mediating increase in cytoplasmic free calcium ion after chimeras based on the closely related Fc-yRIIC trigger the extracellular domains were crosslinked by antibody, T cell effector whereas cytolytic programs in a manner whose efficacy the intracellular domains of FcoyRIIB 1 and B2 were and lineage are inactive independence reminiscent of the action of under comparable conditions (Figure 2). The CD4 the TCR/Fc receptor-associated v and -y chains. and CD16 of hybrids Fc-yRIIA shared essentially equal to capacity promote the calcium response (Figure 2 and data not shown). These data are consistent with studies Results calcium documenting mobilization by intact in a Fc7yRIIA Construction of Fc receptor chimeras murine macrophage cell line (Odin et al., 1991), although To evaluate the actions of the different receptor subtypes, in the latter case the possible association with zeta-family we created chimeric molecules in which the extracellular trigger molecules has not been excluded. domain of the human CD4 or CD16 antigens were joined to the transmembrane and intracellular domains of the Cytolytic activation in primary human T cells Fc,yRHA, B1, B2 and C subtypes (nomenclature of Ravetch The ability to promote accumulation of free Ca2+ following and Kinet, 1991). The chimeras were created by genetically crosslinking suggested that the active receptors might be 4862 Initiation of cytolysis by myeloid Fc receptors 3.0 * CD16:IIA * CD4:IIA 2.5 a oil .< o 1.9 .. 8t-0... - co am ON .. .H4 4 5 CD16:IIC %% ; 1.0 -4 CD4:IIB1 .am 1.5 . A CD 16:IIB 1 U1. 1.1 so o 1.0 CD4:IIB2 0.8 CD16:IIB2 0.5 0.5 -100.0 0.0 100.0 200.0 -100.0 0.0 100.0 200.0 300.0 time in seconds time in seconds Fig. 2. Calcium mobilization following crosslinking of CD4:Fc-yRll and CD16:Fc-yRII chimeras. (a) The ratio of violet to blue fluorescence emitted by TCR- mutant Jurkat cells loaded with the calcium sensitive fluorophore Indo-l is shown as a function of time following crosslinking of the CD16 extracellular domain with antibodies. (b) Similar analysis of the increase in ratio of violet to blue fluorescence of cells bearing CD4:Fc-yRII chimeras, following crosslinking with antibodies. -o a) a) -r 0n a) a) 0) * CD16:11A * CD4:IIA CD16:11C CD4:IIB2 CD16:11Bl *CD4:11Bl E CD16:IIB2 -20- -0 -10 .2 I. -. i. .01 1 1 1. e/t ratio e/t ratio Fig. 3. Cytolysis assay of chimeras (a) and 5ICr released from anti-CD16 CD16:Fc-yRII CD4:FcyRII chimeras (b). (a) The percent of hybridoma cells (target) when the cells were exposed to increasing numbers of cytotoxic T lymphocytes expressing CD16:Fc-yRII chimeras (effector cells) is plotted as a function of the ratio of the number of effector cells to target cells. (b) Cytolysis experiments were conducted as in (a) except that the effector cells expressed CD4 chimeras, and the targets were HeLa cells expressing HIV gpl20/41. The expression of fusion protein by the vaccinia infected cells was measured by indirect immunofluorescence and flow cytometry. Mean fluorescence intensities for the experiment in panel (a) were: CD16:Fc-yRIIA, 752; CD16:Fc-yRIIC, 777; CD16:Fc-yRIIBI, 1194; and CD16:7:Fc-yRHB2, 1204. Mean fluorescence intensities for the experiment in panel (b) were: CD4:Fc-yRIIA, 467; CD4:Fc-yRIIBI, 120; and CD4:Fc-yRIIB2, 299. capable of initiating a cytolytic response. Because there were expressing HIV envelope were susceptible to lysis by T cells no satisfactory systems for evaluating the cytolytic potential expressing CD4:Fc-yRIIA chimera, but not Fc-yRIIB1 or B2 of gene constructs introduced into myeloid cells, we initially (Figure 3). opted to evaluate their in potential a human cytolytic T cell line previously shown to respond to Analysis of Fc in the Fc-yRIIHA-associated receptor chimeras monocytes ¢ and -y chains (Romeo - and Seed, 1991). Human cytotolytic Primary monocytes represent 15 % of the mononuclear lymphocytes (CTL) were infected with vaccinia fraction of blood leucocytes, and exhibit little or no recombinants expressing IB 1, IIB2 and IIC in proliferative capacity culture. However, they can be CD16:Fc7yRHA, chimeras, and the infected cells were cocultured with 51Cr- on relatively easily purified, based their tendency to adhere loaded hybridoma cells (3G8 clone 10-2; Fleit et al., 1982; to solid surfaces and each other (Connor et al., 1990). Using Shen et of al., 1989) which expressed cell surface to purified preparations we were able to antibody monocytes, CD 16. In this assay (Graziano and Romeo document expression of Fc chimeras delivered Fanger, 1987a,b; receptor by et al., 1992) CTL bearing the CD16 chimera kill vaccinia virus expression vectors. Figure shows that hybridoma if the CD16 for target cells release of free primary monocytes represent good vaccinia virus (allowing 51Cr) targets extracellular domain of of can be the chimera has been joined to an infection, and that high levels expressed protein intracellular ion segment capable of activating the achieved in the infected cells. Free calcium accumulates lymphocyte effector programs. Figure 3 shows that CTL armed with in the monocyte cytoplasm of the Fc following crosslinking that in CD16:FcRyIIA and C but not or B2 are receptor chimeras in a fashion similar to observed Fc-yRIJBl capable of the Jurkat T cell line lysing target cells cell surface anti-CD16 (Figure 5). expressing Purified in culture also show antibody. monocytes cytolytic activity, To that the was albeit at lower levels than The eliminate the possibility cytolytic lymphocytes. specific cytolysis in interaction with the are some way attributable to CD16 effects of confounding nonspecific cytotoxicity higher, in that adhere moiety, we also conducted which which we attribute to the fact non- cytolysis experiments monocytes attached to CD4 cells more than T cells do. the FcRII intracellular domains were to specifically target cytotoxic In the cells were HeLa these it is to extracellular domain. this case target However, despite limitations, possible of the cells HIV demonstrate mobilization of expressing envelope gpl20/41 proteins (Romeo cytotoxic potential Fc chimeras CD4 and As in the CD16 cells primary monocytes Seed, 1991). by receptor bearing system, target 4863 W.Kolanus, C.Romeo and B.Seed (a) CD4: 4 control CD4 CD4:IIA E 40 CD4:11B2 .A CD4:1lBl CD4 CD4:; a) s U0 a) CD4:1 IA cJ -20- . 1 .01 .1~~~~~~~~~~.1 I1r0 .0 1 1 ,00 e/t ratio CD4:11B2 in human expressing CD4 intensity Fig. 6. Cytolysis primary monocytes log fluorescence human were isolated from whole blood chimeras. Primary monocytes with recombinant vaccinia viruses. The infected and infected 4. Primary human monocytes are good targets for vaccinia virus Fig. then cultured with 51Cr-loaded CVI cells which had monocytes were vectors. Human monocytes were freshly isolated from expression infected with recombinant vaccinia viruses expressing previously been infected with vaccinia viruses bearing the indicated whole blood and The figure displays the % of chromium HIV envelope glycoproteins. fusion proteins under the control of the p7.5 promoter. The infected function of the effector to target ratio. Mean fluorescence release as a cells were stained with phycoerythrin-conjugated antibody to CD4 and treated with anti-CD4 intensities for cells phycoerythrin-conjugated by flow cytometry. The panels depict the increase in specific analyzed were: for CD4:¢, 706; for CD4:Fc-yRIIA, antibody in this experiment fluorescence over that shown by uninfected monocytes, which express for CD4:FcyRIIB2, 912; and for 1000; for CD4:Fc-yRIIBl, 75.6; some CD4. CD4 alone, 28.6. 5.0 In the in which the amino- experiments and cytolysis assays. * CD4:IIA of the intracellular domain was removed, terminal portion 4.1 domain of Fc'yRH was replaced with the the transmembrane 0 * u3| domain of the unrelated CD7 antigen, to transmembrane .) CD4:t 3.2 the contribution of interactions mediated eliminate possible domain (Figure 7a). by the membrane-spanning es ag Sd U> 14 carboxyl-terminal residues, including Removal of the 2.3 CD4:IIB2 in a complete loss of cytolytic capacity and Tyr298, resulted reduction in to mobilize a substantial but incomplete ability Mae 1.4 T cell 7b). calcium in mutant cells lacking receptor (Figure CD4:IIB1 cells are often hypersensitive to calcium-eliciting Such 0.5 and a nearly complete loss of activity was stimuli, however, 200.0 300.0 400.0 -100.0 0.0 100.0 recorded in cells retaining T cell antigen receptor 7c). The latter data give results comparable to those (Figure time in seconds with intact in which the removal of 17 obtained receptor, was found to eliminate calcium mobilization in residues mobilization in primary human monocytes expressing Fig. 5. Calcium et al., 1991). Further truncation from human were isolated from whole cells (Odin CD4 chimeras. Primary monocytes P388D, with recombinant vaccinia viruses expressing blood and infected to just before Tyr282 gave an identical the carboxyl-terminus Gated calcium flux analyses were various CD4 fusion proteins. phenotype. after the infected monocytes with conducted by flow cytometry loading from the amino-terminus of the intracellular Deletion of violet to blue fluorescence (a surrogate the dye Indo-1. The ratio 268 had no substantial effect on either domain to residue for the positive cell population (gated for both marker for calcium ion) and using CD14 expression to establish scatter CD4 expression scatter, the calcium profile or the cytolytic potency, whereas deletion is shown as a function of time. windows) to residue 275 markedly impaired free calcium release but had little effect on cytolysis. At present we suspect that the extracellular domains joined to Fc receptor transmembrane inability of the Fc-yRIIA(276-31 1) fragment to mobilize and intracellular domains (Figure 6). As is the case for T calcium in the flow cytometric assay does not reflect its are essentially as effective as cells, Fc receptor chimeras ability to support calcium ion accumulation over the longer chimeras in the redirected cytolysis assay TCR r chain time span of the cytolysis assay, inasmuch as the cytotoxicity (Figure 6). in this assay is inhibited by the inclusion of EGTA in the medium (data not shown). However, it is possible that the Analysis of functional subdomains of FcyRll effects of EGTA are limited to inhibition of exocytosis, rather intracellular sequences than triggering. An EGTA-insensitive cytolytic activity has The intracellular portions of Fc-yRIIA and C share no been reported for some (but not all) cytolytic T cell lines sequence homology with other proteins, appreciable (Ostergaard et al., 1987; Trenn et al., 1987, 1989; Young including the members of the extended TCR r family. To et al., 1987). Further deletion from the amino-terminus, to define the sequence elements responsible for induction of residue 282, produced Fc-yRII tails that lacked the ability we prepared 5' and 3' deletions of the intracellular cytolysis, either to mobilize calcium or to trigger cytolysis. domain coding sequences, and evaluated the efficacy of the The 'active element' defined in this manner is relatively resulting deleted fusion proteins in the calcium mobilization large (36 amino acids) and contains two tyrosines separated 4864 Initiation of cytolysis by myeloid Fc receptors . CD16:11A 20o UCD16:7:11A I , 2.0..* (269-31: LCD16:11AY298- ....... RQLEETNNDY RKKRISANST DPVKAAQFEP PGRQMIAIRK s<-. ::: ; CD 116:LY282- (276-311) (283-311) (299-311) CCD16:1IB1 . * " Fr HVNSNN 276 ETADGGYMTL NPRAPTDDDK NIYLTLPPND 1000 200 0 3000 -100,0 0.0 in seconds Y298, time Y282* 4.0 TO 3.5 e .cno:oo * CD16:7I11A X0 so d a) 0 3 CD16:7:11A 2.5 C 2.5 CD16:7:IIA(269-311) (O -* CDt6 {IA CD16.7 1A 2.0 5 o .+CD16:7:A(276-311) 1.00 CD16.11A298- --- CD16:11A282- g 1.5. c .,.v'"v'-'t-"--'---+.x CD C)2 16:7:UA(283-31 1) -105 0 0.0 2 W CD16:11AY285. ,0- co m in ecnd 0 CCD16:7:IIA(299-311) 10~ ~ t5 i 0.0 100.0 200.0 300.0 -IQ0.0 / rati time in seconds time in seconds eAt ratio * CD16:71A 2.5 oo CDIS:7:HA(269-31 1) a) 2.0 *'* E) * 5. .. CD16:11A I CD18:7:HIA(276-311) CD16:7:11A .E_ CO 1.5 Ooo .!OOCo * CD1 6:7:11A(269-31 1) a; oop 1) CD16:7:1A(276-31 x 1) I CD16:7:1A(283-31 fi C016:7:11A(283-311) CD1 6:7:IIA(299-31 1) 50 : ?~00sS.. R2.20.5 0 CD16:7:IIA(299-31 1) -100.0 0.0 100.0 2c3n .0 .0 ratio time in seconds elt Fig. 7. Deletion mapping of residues in the Fc-yRIIA tail which are important for cytolysis. (a) Schematic diagram of the deletion constructs. Deletions of the amino-terminal portion of the extracellular domain (arrows above sequence) were constructed by addition of the Fc-yRII fragment to the transmembrane domain of CD7, which in turn was joined to the extracellular domain of CD16. (b, c and d) Calcium mobilization in TCR- (b) and TCR+ (c) variants of the Jurkat cell line and cytolysis (d) by carboxyl-terminal deletion variants of CD16:Fc-yRIIA. (e, f and g) Calcium mobilization in TCR- (e) and TCR+ (f) variants of the Jurkat cell line and cytolysis (g) by tripartite chimeras bearing progressively less of the amino terminus of the intracellular tail of CD16:Fc-yRIIA. The mean fluorescence intensities for CD16 expression by the cells in (d) and (g) were: CD16:Fc-yRIIA, 850; CD16:Fc-yRIIA Y298*, 695; CD16:Fc-yRIA Y282*, 319; CD16:7:Fc-yRIIA, 419; CD16:7:Fc-yRIIA(269-311), 908; CD16:7:Fc-yRIIA(276-311), 799; CD16:7:Fc-yRIIA(283- 311), 490; and CD16:7:FcyRIIA(299-311), 333. earlier (Romeo et al., 1992), the presence of a studied by 15 residues. To evaluate the potential role of the tyrosine leucine residue immediately preceding the amino-terminal residues, point mutations converting each tyrosine codon to was found to be important for signalling, but a a serine codon were introduced into the cytoplasmic domain tyrosine of full length tripartite chimeras bearing the CD4 similar residue is not found in the same position for Fc-yRHA However, one similarity between the Fc-yRII family domain, CD7 transmembrane domain and or C. extracellular and the r family is the appearance of a leucine (or tail. In each case, mutation of the Fc-yRIIA cytoplasmic occasionally another aliphatic residue, among r family abolished the ability of the resulting chimeras to tyrosine members) three residues carboxyl-terminal, resulting in a T cells and monocytes, and also mobilize calcium in TCR- consensus of the form Y-X-X-L. There is not a great deal their capacity to mediate HIV envelope-directed blocked of inherent in this pattern, however, and similar either CTL or monocytes (Figure 8). specificity killing by Y-X-X-M or Y-X-X-Aliphatic sequences are found among the phosphorylation sites recognized by src tyrosine Discussion and SH3) domains of tyrosine homology 2 and 3 (SH2 kinases and other proteins. we establish that the and HC subtypes In this report Fc-yRHA there is no evidence pointing to an evolutionary Although activation potential previously thought to a cellular possess between the r family proteins and the Fc-yRII of the family of trigger molecules. relationship be limited to members ¢ recent data support the idea that both classes of motifs characteristic of the r family subtypes, Although the sequence function or engage, tyrosine molecule through, in the domains of Fc-yRIIA and activation are not found cytoplasmic of activation. In (Huang et al., kinases in the platelets process there are structural similarities between the two classes C, cell line U937 (Liao et al., and the monoblastoid 1992) of activation domain, notably the requirement for two of FcyRfl leads to de novo accumulation crosslinking for both calcium mobilization and cytolysis 1992), residues tyrosine the case cannot be made Although of tyrosine phosphate. et al., 1992). The spacing between the tyrosines is (Romeo the results make it likely that it is present unambiguously, for and C than for members Fc-yRIIA substantially greater which is for the increased subtype responsible as to 10 residues), and the the Fc-yRHA opposed of the r family (15 in both Odin et al. kinase examples. Similarly, activity acidic residues flanking the distal tyrosines in characteristic that in a cell line, crosslinking have shown myeloid (1991) are not found in Fc'yRIIA or C. In the r motif the family 4865 W.Kolanus, and C.Romeo B.Seed 5.0 16:IIA owes -%.. ana %% 3.9 0 [ 4) ft-%o% 4) cc ft...] CD1 6:1 IA o 16:IIAY282S cc aD4 CD16:11AY282S CD16:11AY298S .E * L0 1.6 16:IIAY298S -00 goal . e &*a 66" .- 0.5 -100.0 0.0 100.0 200.0 300.0 400.0 time in seconds e/t ratio 2.5 * CD4:¢ 2.1 * CD4:IIA (U co 1.7 CD4:11A _ s . _ .^~~a' CD4: 4 A CD4:7:IIB2 CD4:7:11B2 CD4:7:11AY282S ..-.* 'a~~~~~~~a a) CD4:7:11AY298S .°, *'**_ o CD4:7:IIAY282S N.I. U0 0.9 i6 .* A CD4:7:11AY298S 0.5 -100.0 0.0 000.0 200.0 300.0 400.0 e/t ratio time in seconds of the of residues within the domain. Calcium mobilization in the TCR- Fig. 8. Analysis importance tyrosine cell Fc-yRIIA cytoplasmic (a) analysis line JRT3T3.5. The of cells chimera substituted with and chimera response bearing wild-type chimera, bearing Tyr282 Ser, bearing Tyr298 substituted with Ser is shown. in CTL directed and the mutants and Calcium mobilization (b) Cytolysis by FcyRIIA, in Tyr282Ser Tyr298Ser. (c) monocytes CD4 chimeras CD4: or chimeras CD7 transmembrane and either intact by , CD4:FcyRIIA, tripartite bearing Fc-yRIB2 sequences intracellular mutant or mutant Calcium mobilization was on cells domain, Fc-yRIIA Tyr282Ser Fc'yRIIA Tyr298Ser. for analysis performed gated CD4 and scatter as described in the to 5. in the chimeric molecules described in legend Figure (d) Cytolysis N.I., not monocytes expressing (c). infected. Mean fluorescence intensities for CD16 the cells in were: expression by (b) 246; Y298S, 314; and CD16:Fc-yRIIA, CD16:Fc-yRIIA 304. Mean fluorescence intensities for CD4 the cells in were: CD16:Fc-yRIIA Y282S, 109; 162; expression by (d) CD4:Fc'yRIIA, CD4:D, and not CD4:7:Fc-yRIIA Y298S, 75; Y282S, 120; 26. CD4:7:FcyRIIB2, 84; infected, CD4:7:Fc7RIIA ion the of the Fc-yRIIA can mediate free calcium Whatever explanation, the absence of cell subtype appropriate work an idea and extended our lines is a substantial technological obstacle. In this work we accumulation, supported by and to with chimeras. Thus both the r family trigger molecules have introduced two related strategies study cytolytic the or C calcium transients and pathways initiated by myeloid Fc One is the Fc'yRIIA subtypes produce receptors. use to are known increase of primary human T cells as effector and appear (or to) tyrosine phosphorylation surrogate cells, a stimulus. The that the other is the use of vaccinia virus vectors to introduce following crosslinking finding is receptors into primary human human phospholipase specifically phosphorylated following monocytes. Primary C'y1 Fc-yRll clustering in U937 cells (Liao et al., cytolytic T a renewable 1992) provides lymphocytes represent population a reasonable candidate for the of effector cells with explanation similarities, high target specificity and high cytolytic suggesting that tyrosine and results potential. not to phosphorylation precedes Although especially susceptible transfection, in inositol phosphate production and calcium mobilization are infected with they easily recombinant vaccinia viruses, A of and their does through activation of similar cytolytic capacity not appear to be impaired phosphorylation PLC-'yl. has been observed of the infection. following crosslinking by Moreover, as this study shows, they have the PLC-'yj high affinity IgE receptor (Park et al., 1991). In U937 to to cells, ability respond exogenous receptors whose natural tyrosine of can be distribution phosphorylation PLC-'yl induced by does not fall in their lineage. or crosslinking either Fc-yR1 Fc-yRHl et the More (Liao al., 1992); problematic is the analysis of cytolytic effector mechanism may not be similar, however, since the function in cell types other than CTL. For example, primary domain of no cytoplasmic Fc-yRI shows relatedness to that natural killer cells, although potently cytotoxic, are also of Fc-yRHl, and in no particular contains tyrosine residues. substantially less specific than CTL; and primary monocytes Although activation of cytolysis is a central feature of offer the worst of both worlds, being both less cytolytic and cellular immunity, historically it has been more difficult to less specific than CTL. However, it is possible by suitable study than activation of cellular helper function. In large part choice of target cell and monocyte isolation conditions to this stems from the relative scarcity of cell lines which are make the of study monocyte cytolysis amenable to genetic amenable to transfection and which are also capable of methods of analysis. Like CTL, primary monocytes are mediating a functional cytolytic response. It may be that this easily infected with vaccinia viruses, and give good is inevitable, and that loss of function is an unavoidable expression of cell surface proteins under the control of the consequence of attempting to propagate cells with a high vaccinia p7.5 mixed early/late promoter. Although the cytotoxic potential. tendency of vaccinia to shut off host protein and RNA 4866 Initiation of Fc cytolysis by myeloid receptors must be synthesis counted a liability, there the PE positive cell which is reason to be (infected) population, typically represented 40-80% of the cells. The violet/blue ratio to the emission prior addition that the and of optimistic scope variety subjects which are of was used to antibody establish the normalized initial ratio, set equal to to in susceptible study monocytes will continue to expand. unity. Lymphocyte cytolysis assay Materials and methods A CD8+ CD4- HLA B44 restricted cytolytic line (WH3), kindly provided J.Kurnick and of by F.Harris, was maintained in IMDM, 10% human serum chimeras Preparation with 100 U/ml of IL-2 and was periodically To prepare the cDNA stimulated with irradiated chimeras, sequences corresponding to the (3000 rad) mononuclear transmembrane and domains of cells having the HLA B44 haplotype. Cells were cytoplasmic Fc-yRI and the IIB 1 grown Fc-yRIIA, for at least 10 and IIB2 isoforms were days following stimulation before use in cytotoxicity amplified using synthetic oligonucleotides primers assays. The cells either from clones et were infected with recombinant vaccinia at an m.o.i. of at least existing (Stengelin al., 1988; Allen and Seed, 1989) 10 for h in serum-free or from a human tonsil cDNA 1 medium, followed by incubation in complete medium library. The cDNA fragment corresponding to the FcRIIC which for 3 h. cytoplasmic domain, differs from the IIA isoform at one amino acid residue for P at Cells were harvested by centrifugation and at a (L residue 268; Stuart et al., was resuspened density of 1989) 1 x 107/ml. 100 were added to each well of a U-bottom site directed ytl microtiter generated PCR. The forward plate by mutagenesis by overlap and containing 100 1I/well of medium. Cells were diluted reverse contained complete in 2-fold primers sites for the BamHI cleavage enzymes and NotI indented serial steps. Two wells for each did not respectively, six residues from the 5' end. sample contain lymphocytes, to The Notl site was allow spontaneous followed a chromium release and total chromium uptake to be immediately by stop anticodon, either CTA or TTA. All primers measured. An of contained 18 or more residues aliquot 106 target cells, either 3G8 10-2 (Shen et complementary to the 5' and 3' ends of the al., desired 1989) or HeLa cells infected with vaccinia recombinant vPE16 (Earl et fragments. al., 1990) was centrifuged and resuspended The PCR were inserted into in 50 of sterile fragments expression vectors which contained [5lCr]sodium A1 chromate (1 CD16 or CD4 extracellular domains mCi/mi, DuPont) for 1 h at 37°C with intermittent in a BamHI mixing, terminating site just then washed three times with PBS. 100 1I of to the membrane domain labeled cells resuspended in proximal spanning (Romeo and Seed, 1991; Romeo medium at 105 cells/ml were added et The of to each well. The microtiter plate was al., 1992). identity all isoforms was confirmed by dideoxy spun at 750 g for 1 min and incubated for 4 h at 37°C. At the end of the sequencing. incubation period the cells in each well were resuspended by gentle pipetting, a sample was removed to determine the total counts incorporated and the Immunoprecipitation JRT3.T3.5 microtiter plate was spun at 750 for 1 min. 100 107 and cells were td aliquots of supematant Approximately (Weiss Stobo, 1984) infected g for 1 h in serum-free IMDM were removed and counted in a gamma ray scintillation counter. The medium with recombinant effector vaccinia at a of infection to target ratio was corrected for the of effector of at least 10. Twelve percent cells infected (usually multiplicity (m.o.i.) hours after infection, the cells were 1251 >70%). harvested and surface labeled with 0.5 107 mCi cells per the oxidase method. The labeled using lactoperoxidase/glucose cells were collected Monocyte cytolysis and in 1% assay by centrifugation lysed NP-40, 0.1% SDS, 0.15 M Cytolysis assays using human peripheral blood as effector 0.05 M Tris 5 mM 5 mM 0.2 M iodoacetamide monocytes cells NaCI, pH 8, MgCl2, KCI, were out in fashion and 1 mM PMSF. Nuclei carried the same as for the CTL assays, with the were removed and following by centrifugation CD16 fusion were modifications: monocytes, cultured overnight in IMDM, 20% fetal with bovine proteins immunoprecipitated antibody 3G8 and anti-mouse IgG were serum, were detached by and infected with recombinant 10% scraping vaccinia agarose. Samples electrophoresed through polyacrylamide-SDS under at a of infection of at least 10 for conditions. multiplicity 1 h in serum free medium, gels reducing incubation followed by for 4 h in Teflon beakers in 20% fetal IMDM, bovine serum. CVI cells infected with vaccinia recombinant vPE16 (Earl et al., Isolation of blood peripheral monocytes 1990) were used as targets. The microtiter plate containing effector/target were isolated either from coats or from fresh Monocytes blood as buffy mixtures at 10 ratios from 50: 1 to 0.09: 1 control plus wells for determination described Connor et al. Cells were over Ficoll- by (1990). centrifuged of and maximal 5ICr release was incubated at 37°C spontaneous for 16h. and the interface was collected. After three washes in Hypaque layer IMDM, the cells were in IMDM fetal 20% bovine serum at suspended containing In vitro mutagenesis cells ml in 15 tubes and were 107 ml rotated at 20 per polypropylene r.p.m. FcRUIA deletion mutants were constructed Carboxyl-terminal by PCR in Tektator about an axis to the axis of the (Lab-Quake, Inc.) parallel long the same fashion as for the full length constructs, converting the sequences tubes for 15-25 at to induce min The 4°C monocyte aggregation. aggregated at 282 and encoding tyrosine positions 298 into stop codons (TAA). The cells were sedimented on ice at for 15 in 2 I ml of g min, resuspended amino-terminal deletions were generated by amplifying fragments encoding medium and onto an volume of ice-cold fetal bovine serum. layered equal less of the intracellular domain successively by PCR, using oligonucleotides After sedimentation the fetal bovine serum for 20 min at the through 4°C, which allowed the to be inserted between MluI resulting fragments and NotI lower contained between 80 and 97% the remainder phase monocytes, being restriction sites into a previously constructed expression plasmid encoding cells the interface were lymphocytes. Alternatively, from Ficoll-Hypaque the CD16 extracellular domain fused to the CD7 transmembrane domain, x cells into 100 washed three times in IMDM and at 5 106 ml plated per the latter terminating in a MluI site at the junction between the transmembrane mm tissue culture dishes. were allowed to adhere to the Monocytes plastic and the intracellular domain. for at least 2 h and were then detached for further by scraping analysis. The of was > 90 as determined with purity monocytes usually by staining Leu-M3 antibody (Becton-Dickinson). Acknowledgements We thank Michael and Fanger Jay Unkeless for the 3G8 10-2 cells, Franco Calcium flux analysis Harris and Jim for the Kurnick WH3 line, and members of the laboratory of Cells the Jurkat mutant subline JRT3.T3.5 and or Stobo, (Weiss 1984) for discussion and criticism. C.R. was in supported part by grant 1309-9-RG blood as described above were infected with peripheral monocytes prepared from the American Foundation for AIDS Research. This was study supported recombinant vaccinia viruses for 1 h in serum-free IMDM at an m.o.i. of NIH A127849 and an award to the Massachusetts by grant by General 10% 10 and incubated for 3-9 h in FBS. Cells were collected IMDM, Hospital by Hoechst AG. and at 3 in medium 106/ml by centrifugation resuspended complete 1 mM Indo-l et al., containing acetomethoxyester (Grynkiewicz 1985) and incubated at for 45 The loaded min. (Molecular Probes) 37°C Indo-l References x in serum-free IMDM cells were and at and 1 106/ml pelleted resuspended at room in dark. Cells were for free Ca2 et stored the + Amigorena,A. al. 1808-1812. (1992) Science, 256, temperature analyzed of the violet and blue fluorescence emission simultaneous measurement Anderson,P., Caligiuri,M., O'Brien,C., Manley,T., Ritz,J. and by et To initiate calcium Proc. Natl. Acad. Sci. flow either Schlossman,S.F. (1990) USA, 87, 2274-2278. al., flux, by cytometry (Rabinovitch 1986). and Immunol. monoclonal was Balk,S. Terhorst,C. 411-416. added (1989) Ser., 45, 3G8 (at 1 unconjugated (anti-CD16) antibody ug/ml) 10 of and Kinet,J.-P. to the cell followed Blank,U., Ra,C., Miller,L., White,K., Metzger,H. (1989) suspension by 1g/ml phycoerytdrin (PE)-conjugated Fab'2 goat anti-mouse IgG at time or a anti-CD4 Nature, 337, 187-189. 0, PE-conjugated antibody (Leu-3a, Becton Dickinson) was added, followed by unconjugated secondary Pavlovich,R., Choukroun,V. Bonnerot,C., Amigorena,S., Choquet,D., and collected EMBO antibody. Histograms of the violet/blue emission ratio were from J., 11, 2747-2757. Fridman,W.H. (1992) 4867 W.Kolanus, C.Romeo and B.Seed Boyle,D.B. and Coupar,B.E.H. (1988) Gene, 65, 123-128. Wegener,A.-M.K., Letourneur,F., Hoeveler,A., Brocker,T., Luton,F. and Connor,R.I., Shen,L. and Fanger,M.W. (1990) J. 83-95. Immunol., 145, Malissen,B. (1992) Cell, 68, 1483-1489. and Stobo,J.D. J. Weiss,A. (1984) Exp. Med., 160, 1284-1299. Engelhardt,W., Gorczytza,H., Butterweck,A., Monkemann,H. and Frey,J. Weissman,A.M., Baniyash,M., Hou,D., Samelson,L.E., Burgess,W.H. (1991) Eur. J. Immunol., 21, 2227-2238. and 1018-1020. Klausner,R.D. (1988) Science, 239, Falkner,F.G. and Moss,B. (1988) J. Virol., 62, 1849-1854. and Yeh,E.T., J. Reiser,H., Daley,J. Rock,K.L. (1987) Immunol., 138, Fanger,M.W., Shen,L., Graziano,R.F. and Guyre,P.M. (1989) Immunol. 91-97. Today, 10, 92-99. and Year Yokoyama,W.M. Shevach,E.M. (1989) Immunol., 4, 110-146. Fleit,H.B., Wright,S.D. and Unkeless,J.C. (1982) Proc. Natl. Acad. Sci. and J. Young,J.D., Clark,W.R., Liu,C.C. Cohn,Z.A. (1987) Exp. Med., USA, 79, 3275-3279. 1894-1899. 166, Gardner,P. (1989) Cell, 59, 15-20. Graziano,R.F. Received on and Fanger,M.W. (1987a) J. Immunol., revised on 1992 138, 945-950. July 24, 1992; September 11, Graziano,R.F. and Fanger,M.W. (1987b) J. Immunol., 139, 3536-3541. Grynkiewicz,G., Poenie,M. and Tsien,R.Y. J. (1985) Biol. Chem., 260, 3440-3450. Hibbs,M.L., Selvaraj,P., Carpen,O., Springer,T.A., Kuster,H., Jouvin,M.- H.E. and Kinet,J.-P. (1989) Science, 246, 1608-1611. Huang,M.M., Indik,Z., Brass,L.F., Hoxie,J.A., Schreiber,A.D. and Brugge,J.S. (1992) J. Biol. Chem., 267, 5467-5473. Hunziker,W. and Mellman,I. (1989) J. Cell Biol., 109, 3291-3302. Indik,Z., Kelly,C., Chien,P., Levinson,A.I. and Schreiber,A.D. (1991) J. Clin. Invest., 88, 1766-1771. Irving,B.A. and Weiss,A. (1991) Cell, 64, 891-901. Jin,Y.J., Clayton,L.K., Howard,F.D., Koyasu,S., Sieh,M., Steinbrich,R., Tarr,G.E. and Reinherz,E.L. (1990) Proc. Natl. Acad. Sci. USA, 87, 3319-3323. Keegan,A.D. and Paul,W.E. (1992) Im,munol. Today, 13, 63-68. Kinet,J.-P. (1989) Cell, 57, 351-354. Kroczek,R.A., Gunter,K.C., Germain,R.N. and Shevach,E.M. (1986) Nature, 322, 181-184. Kurosaki,T. and Ravetch,J.V. (1989) Nature, 342, 805-807. Lanier,L.L., Yu,G. and Phillips,J.H. (1989) Nature, 342, 803-805. Letourneur,F. and Klausner,R.D. (1991) Proc. Natl. Acad. Sci. USA, 88, 8905-8909. Letourneur,F. and Klausner,R.D. (1992) Science, 255, 79-82. Liao,F., Shin,H.S. and Rhee,S.G. (1992) Proc. Natl. Acad. Sci. USA, 89, 3659-3663. Mellman,I. (1988) Curr. Opin. Immunol., 1, 16-25. Meuer,S.C., Hussey,R.E., Fabbi,M., Fox,D., Acuto,O., Fitzgerald,K.A., Hodgdon,J.C., Protentis,J.P., Schlossman,S.F. and Reinherz,E.L. (1984) Cell, 36, 897-906. Miettinen,H.M., Rose,J.K. and Mellman,I. (1989) Cell, 58, 317-327. Miettinen,H.M., Matter,K., Hunziker,W., Rose,J.K. and Mellman,I. (1992) J. Cell Biol., 116, 875-888. Moingeon,P., Lucich,J.L., McConkey,D.J., Letourneur,F., Malissen,B., Kochan,J., Chang,H.C., Rodewald,H.R. and Reinherz,E.L. (1992) Proc. Natl. Acad. Sci. USA, 89, 1492-1496. Odin,J.A., Edberg,J.C., Painter,C.J., Kimberly,R.P. and Unkeless,J.C. (1991) Science, 254, 1785-1788. Ostergaard,H.L., Kane,K.P., Mescher,M.F. and Clark,W.R. (1987) Nature, 330, 71-72. Park,D.J., Min,H.K. and Rhee,S.G. (1991) J. Biol. Chem., 266, 24237-24240. Qiu,W.Q., de-Bruin,D., Brownstein,B.H., Pearse,R. and Ravetch,J.V. (1990) Science, 248, 732-735. Ra,C., Jouvin,M.-H.E. and Kinet,J.-P. (1989) J. Biol. Chem., 264, 15323-15327. Rabinovitch,P.S., June,C.H., Grossmann,A. and Ledbetter,J.A. (1986) J. Immunol., 137, 952-961. Ravetch,J.V. and Kinet,J.P. (1991) Annu. Rev. Immunol., 9, 457-492. Reth,M. (1989) Nature, 338, 383-384. Romeo,C. and Seed,B. (1991) Cell, 64, 1037-1046. Romeo,C., Amiot,M. and Seed,B. (1992) Cell, 68, 889-897. Samelson,L.E., Harford,J.B. and Klausner,R.D. (1985) Cell, 43, 223-231. Schmitt-Verhulst,A.M., Guimezanes,A., Boyer,C., Poenie,M., Tsien,R., Buferne,M., Hua,C. and Leserman,L. (1987) Nature, 325, 628-631. Sefton,B.M. and Campbell,M.A. (1991) Annu. Rev. Cell Biol., 7, 257-274. Shen,L., Graziano,R.F. and Fanger,M.W. (1989) Mol. Immunol., 26, 959-969. Stengelin,S., Stamenkovic,I. and Seed,B. (1988) EMBO J., 7, 1053-1059. Stuart,S.G., Simister,N.E., Clarkson,S.B., Kacinski,B.M., Shapiro,M. and Mellman,I. (1989) EMBO J., 8, 3657-3666. Trenn,G., Takayama,H. and Sitkovsky,M.V. (1987) Nature, 330, 72-74. Trenn,G., Taffs,R., Hohman,R., Kincaid,R., Shevach,E.M. and Sitkovsky,M. (1989) J. Immunol., 142, 3796-3802. and Unkeless,J.C., Annu. Rev. Scigliano,E. Freedman,V.H. (1988) 251-281. Immunol., 6, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Lineage‐independent activation of immune system effector function by myeloid Fc receptors.

Kolanus, W.; Romeo, C.; Seed, B.
The EMBO Journal , Volume 11 (13) – Dec 1, 1992
Download PDF
8 pages

Loading next page...
 
/lp/springer-journals/lineage-independent-activation-of-immune-system-effector-function-by-uGAB0gW4m4

References (0)

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 1992
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1002/j.1460-2075.1992.tb05592.x
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal vol.1 1 no.13 pp.4861 -4868, 1992 Lineage-independent activation of immune system effector function by myeloid Fc receptors Waldemar Kolanus, association of Charles Romeo and the receptor with surface IgM by co-capping (Amigorena et B Brian Seed al., 1992). Both forms of FcRII are capable of inhibiting B-cell activation mediated by the IgM antigen Department of Molecular Biology, Massachusetts General Hospital, receptor (Amigorena et al., 1992). Boston, MA 02114, USA A low affinity receptor of the third class, Fc'yRIHA, Communicated by R.Kamen mediates immune cell activation through its association with one or more members of a small family of 'trigger An molecules' (Hibbs et al., 1989; Kurosaki and Ravetch, 1989; emerging theme in immunology finds receptors which initiate cellular effector programs forming multichain Lanier et al., 1989; Ra et al., 1989; Anderson et al., 1990; complexes in which the ligand recognition elements Bonnerot et al., 1992). associate with one or more 'trigger molecules' whose These trigger molecules, T cell receptor (TCR) r chain aggregation initiates a signal transduction cascade. The (Samelson et al., 1985; Weissman et al., 1988) TCR v chain sequence motifs et al., 1990) and Fc receptor 'y chain (Blank et al., 1989; constituting the active sites of these (Jin trigger molecules are Ra et al., 1989; Bonnerot et al., 1992) interact with ligand found in the T cell and B cell antigen recognition domains of different immune system receptors receptors, and some Fc receptors, and appear to be and can central to effector function activation. For example, of autonomously initiate cellular effector programs, the many molecules that mimic or potentiate the action including tyrosine kinase activation, cytokine secretion and of the T cell antigen receptor (TCR), none have yet been cytolysis, following their aggregation (Irving and Weiss, found to initiate effector programs autonomously in cells 1991; Letourneur and Klausner, 1991; Romeo and Seed, lacking TCR. We have devised two strategies to study 1991; Bonnerot et al., 1992). Within the ¢, v and FcR -y activation intracellular domains are sequence motifs also represented mediated by myeloid Fc receptors, which in CD3 -y, 6 and e chains, in the B cell antigen receptor mbl appear not to associate with trigger molecules: the use and B29 proteins, and in the FccRI 13 of primary human cytolytic T cells as surrogate effector chain (Kinet, 1989; Reth, 1989; Wegener et al., 1992). Recent studies have cells for genetically delivered receptors, and the use of vaccinia virus vectors to shown that CD3e introduce genetically modified (Letourneur and Klausner, 1992) and a receptors into primary human monocytes. complex comprising CD3-y, 6 and e Using these (Wegener et al., 1992) we have the to activate T approaches, have found that the cytoplasmic domains capacity cells, suggesting that the of two Fc function of the receptors show comparable function to sequence motif common to these chains may domains of be to initiate a common or related activation pathway. equivalent the trigger molecule family, but in are not Early events the activation of receptors associating with homologous to members of that family. words: trigger molecules include tyrosine phosphorylation, Key cytolysis/Fc-yRII/Fc receptor/macrophages formation of inositol phosphates and accumulation of free intracellular Ca2+ Sefton and (e.g. Gardner, 1989; In Campbell, 1991; Keegan and Paul, 1992). many cases Introduction the later stages of activation are accompanied by a change in in Receptors for the constant portion of immunoglobulin G transcriptional potential which results cytokine synthesis (Fc'y form a class of cell surface or a developmentally activated cell state. The causative chain receptors) complex proteins of immune of events which translates cell surface mediating phagocytosis complexes, transcytosis receptor aggregation understood even in and various forms of cellular into cellular activation remains poorly antibody dependent cytotoxicity Unkeless et et the best studied examples. (Meilman, 1988; al., 1988; Fanger al., 1989; The of cell effector function can Human and murine triggering type-specific Ravetch and Kinet, 1991). Fc-y receptors be distinguished from the diverse which either to a signals have been divided into three classes, corresponding high influence to central or a of low responsiveness activating signals affinity type (Fc-yRI), family affinity receptors (the third low mediate activation which do not effector Fc'yRHl receptors) and a affinity type (Fc-yRIH) pathways engage in or chemotaxis. As in mice. Some as found adhesion which has two forms in humans, and one function, e.g. yet, have well delineated. no molecules from members of the of function are apart trigger family aspects receptor fairly activate T the been shown to most has been shown to mediate autonomously cells, recently phagocytosis Fc7yRIIA of immune effector et Odin et characterized cells et Indik extensively system (Engelhardt al., 1991; al., 1991; al., and of of (Yokoyama Shevach, 1989). Aggregation while mediates internalization 1991), Fc-yRIIB2 Ig-coated et Yeh and phospholipid-linked proteins (Kroczek al., 1986; into clathrin coated targets pits, (Hunziker Mellman, and or treatment of et al., Balk CD2 related 1987; Terhorst, 1989) et The 1989; Miettinen al., 1989). closely Fc-yRIIB1 antibodies et with of monoclonal of the of but is from pairs (Meuer al., 1984) bears all sequences Fc'yRIIB2, prevented function in T but this can activate effector activation the a 47 amino cells, association with endocytotic apparatus by of one or more members of on the the in the domain et depends presence acid insertion al., cytoplasmic (Amigorena et et in B favors trigger family (Schmitt-Verhulst al., 1987; Moingeon Miettinen cells, 1992; al., 1992) which, Press Oxford University W.Kolanus, C.Romeo and B.Seed :: :::.: .. ... *, ^ r -- ; ar .: ::-:.:.:. ~~. ..... ::-.::..... .... ...., ~~~~~~~~~~~~~.. _. .. ...... * ........... t: - - :~~....... ..:--..:..:-:-:.: .~~~~~~~~~~~~. ......... , :1 , L-.. ~~~~~~~~~~~.. ~~~~~~~~~~~. ~~~~~~~~~~~~~.. ....... X .... .,.... ..............................-x. B-: ([| r;L 5|i ..... . . . .. .. ... ... . . ... -,:- '::: ..:. .-, t * 9 ~~~~~~~~~....... .. -- -__ ~~~~~. ._. .. ... . .... S._.. __ .9 1.. ~~~~~~~~~~~~~. ~~~~~~~~~~~~. I_r ..1.. Fig. 1. Creation and analysis of FcyRII chimeras. Schematic of CD16: and chimeras. The intracellular domains (a) diagram FcyRII CD4:FcyRll of and C differ by a single residue (268 from the amino-terminus of the while the intracellular domains of and B2 Fc'yRIIA precursor), Fc-yRIIB1 differ by the presence or absence of an additional exon of chimeras. An of (light stippling). (b) Immunoprecipitation CD16:Fc-yRII a autoradiogram reducing gel of Bi and B2 chimeras is shown with molecular mass standards on the left. At we believe immunoprecipitated Fc-yRIIA, C, present that the lower molecular mass in the and C tracks species Fc-yRIIA represent partially glycosylated precursors. In less characterized fusing DNA segments encoding the different extracellular et al., 1992). extensively cell there types the of is relatively little information as to domains to fragments encoding Fc receptor indispensability sequences In the trigger molecules. no small part this of obtained from previously described clones (Stengelin et scarcity al., absence of suitable information can be attributed to the cell 1988) or from polymerase chain reactions based on other lines which both possess effector function and are amenable reported sequences (Stuart et al., 1989; Qiu et al., 1990; to biochemical Figure 1). The gene fusions were constructed in genetic and analyses. vaccinia initiation of Fc in virus expression vectors and subsequently inserted into wild The cytolysis by receptors macrophages type vaccinia by recombination at the thymidine kinase (or monocytes, their blood-borne is an locus, precursors) example of has been for using selection for cointegration of Escherichia an activation process whose study coli gpt stymied want of a suitable in vitro system. several (Falkner and Moss, 1988; Boyle and Coupar, Although myeloid 1988) to cell lines have been none the facilitate identification of the desired developed, display cytolytic recombinants. activity of freshly isolated or Immunoprecipitation of Jurkat (T cell monocytes macrophages. leukemia) cells infected Conversely, monocytes in culture exhibit little with the recombinant viruses revealed chimeric primary molecules of proliferative potential and are to the expected molecular masses (Figure 1 and data not sufficiently refractory transfection to make transient shown). expression approaches infeasible. In this work we have pursued two alternative Calcium in T strategies to explore Fc receptor mediated activation: the use mobilization cells is independent of T of primary human cytolytic T cells as surrogate effector cells cell receptor for genetically delivered Fc receptors, and the use of Introduction of the chimeras vaccinia into a TCR-negative mutant of virus vectors to introduce Fc receptors into primary human the Jurkat line (Weiss and Stobo, 1984) showed that the monocytes. Using these approaches we have found that intracellular domains of and Fc-yRflC were capable FcyRIHA protein of an chimeras based on Fc-yRIIA as well as similar mediating increase in cytoplasmic free calcium ion after chimeras based on the closely related Fc-yRIIC trigger the extracellular domains were crosslinked by antibody, T cell effector whereas cytolytic programs in a manner whose efficacy the intracellular domains of FcoyRIIB 1 and B2 were and lineage are inactive independence reminiscent of the action of under comparable conditions (Figure 2). The CD4 the TCR/Fc receptor-associated v and -y chains. and CD16 of hybrids Fc-yRIIA shared essentially equal to capacity promote the calcium response (Figure 2 and data not shown). These data are consistent with studies Results calcium documenting mobilization by intact in a Fc7yRIIA Construction of Fc receptor chimeras murine macrophage cell line (Odin et al., 1991), although To evaluate the actions of the different receptor subtypes, in the latter case the possible association with zeta-family we created chimeric molecules in which the extracellular trigger molecules has not been excluded. domain of the human CD4 or CD16 antigens were joined to the transmembrane and intracellular domains of the Cytolytic activation in primary human T cells Fc,yRHA, B1, B2 and C subtypes (nomenclature of Ravetch The ability to promote accumulation of free Ca2+ following and Kinet, 1991). The chimeras were created by genetically crosslinking suggested that the active receptors might be 4862 Initiation of cytolysis by myeloid Fc receptors 3.0 * CD16:IIA * CD4:IIA 2.5 a oil .< o 1.9 .. 8t-0... - co am ON .. .H4 4 5 CD16:IIC %% ; 1.0 -4 CD4:IIB1 .am 1.5 . A CD 16:IIB 1 U1. 1.1 so o 1.0 CD4:IIB2 0.8 CD16:IIB2 0.5 0.5 -100.0 0.0 100.0 200.0 -100.0 0.0 100.0 200.0 300.0 time in seconds time in seconds Fig. 2. Calcium mobilization following crosslinking of CD4:Fc-yRll and CD16:Fc-yRII chimeras. (a) The ratio of violet to blue fluorescence emitted by TCR- mutant Jurkat cells loaded with the calcium sensitive fluorophore Indo-l is shown as a function of time following crosslinking of the CD16 extracellular domain with antibodies. (b) Similar analysis of the increase in ratio of violet to blue fluorescence of cells bearing CD4:Fc-yRII chimeras, following crosslinking with antibodies. -o a) a) -r 0n a) a) 0) * CD16:11A * CD4:IIA CD16:11C CD4:IIB2 CD16:11Bl *CD4:11Bl E CD16:IIB2 -20- -0 -10 .2 I. -. i. .01 1 1 1. e/t ratio e/t ratio Fig. 3. Cytolysis assay of chimeras (a) and 5ICr released from anti-CD16 CD16:Fc-yRII CD4:FcyRII chimeras (b). (a) The percent of hybridoma cells (target) when the cells were exposed to increasing numbers of cytotoxic T lymphocytes expressing CD16:Fc-yRII chimeras (effector cells) is plotted as a function of the ratio of the number of effector cells to target cells. (b) Cytolysis experiments were conducted as in (a) except that the effector cells expressed CD4 chimeras, and the targets were HeLa cells expressing HIV gpl20/41. The expression of fusion protein by the vaccinia infected cells was measured by indirect immunofluorescence and flow cytometry. Mean fluorescence intensities for the experiment in panel (a) were: CD16:Fc-yRIIA, 752; CD16:Fc-yRIIC, 777; CD16:Fc-yRIIBI, 1194; and CD16:7:Fc-yRHB2, 1204. Mean fluorescence intensities for the experiment in panel (b) were: CD4:Fc-yRIIA, 467; CD4:Fc-yRIIBI, 120; and CD4:Fc-yRIIB2, 299. capable of initiating a cytolytic response. Because there were expressing HIV envelope were susceptible to lysis by T cells no satisfactory systems for evaluating the cytolytic potential expressing CD4:Fc-yRIIA chimera, but not Fc-yRIIB1 or B2 of gene constructs introduced into myeloid cells, we initially (Figure 3). opted to evaluate their in potential a human cytolytic T cell line previously shown to respond to Analysis of Fc in the Fc-yRIIHA-associated receptor chimeras monocytes ¢ and -y chains (Romeo - and Seed, 1991). Human cytotolytic Primary monocytes represent 15 % of the mononuclear lymphocytes (CTL) were infected with vaccinia fraction of blood leucocytes, and exhibit little or no recombinants expressing IB 1, IIB2 and IIC in proliferative capacity culture. However, they can be CD16:Fc7yRHA, chimeras, and the infected cells were cocultured with 51Cr- on relatively easily purified, based their tendency to adhere loaded hybridoma cells (3G8 clone 10-2; Fleit et al., 1982; to solid surfaces and each other (Connor et al., 1990). Using Shen et of al., 1989) which expressed cell surface to purified preparations we were able to antibody monocytes, CD 16. In this assay (Graziano and Romeo document expression of Fc chimeras delivered Fanger, 1987a,b; receptor by et al., 1992) CTL bearing the CD16 chimera kill vaccinia virus expression vectors. Figure shows that hybridoma if the CD16 for target cells release of free primary monocytes represent good vaccinia virus (allowing 51Cr) targets extracellular domain of of can be the chimera has been joined to an infection, and that high levels expressed protein intracellular ion segment capable of activating the achieved in the infected cells. Free calcium accumulates lymphocyte effector programs. Figure 3 shows that CTL armed with in the monocyte cytoplasm of the Fc following crosslinking that in CD16:FcRyIIA and C but not or B2 are receptor chimeras in a fashion similar to observed Fc-yRIJBl capable of the Jurkat T cell line lysing target cells cell surface anti-CD16 (Figure 5). expressing Purified in culture also show antibody. monocytes cytolytic activity, To that the was albeit at lower levels than The eliminate the possibility cytolytic lymphocytes. specific cytolysis in interaction with the are some way attributable to CD16 effects of confounding nonspecific cytotoxicity higher, in that adhere moiety, we also conducted which which we attribute to the fact non- cytolysis experiments monocytes attached to CD4 cells more than T cells do. the FcRII intracellular domains were to specifically target cytotoxic In the cells were HeLa these it is to extracellular domain. this case target However, despite limitations, possible of the cells HIV demonstrate mobilization of expressing envelope gpl20/41 proteins (Romeo cytotoxic potential Fc chimeras CD4 and As in the CD16 cells primary monocytes Seed, 1991). by receptor bearing system, target 4863 W.Kolanus, C.Romeo and B.Seed (a) CD4: 4 control CD4 CD4:IIA E 40 CD4:11B2 .A CD4:1lBl CD4 CD4:; a) s U0 a) CD4:1 IA cJ -20- . 1 .01 .1~~~~~~~~~~.1 I1r0 .0 1 1 ,00 e/t ratio CD4:11B2 in human expressing CD4 intensity Fig. 6. Cytolysis primary monocytes log fluorescence human were isolated from whole blood chimeras. Primary monocytes with recombinant vaccinia viruses. The infected and infected 4. Primary human monocytes are good targets for vaccinia virus Fig. then cultured with 51Cr-loaded CVI cells which had monocytes were vectors. Human monocytes were freshly isolated from expression infected with recombinant vaccinia viruses expressing previously been infected with vaccinia viruses bearing the indicated whole blood and The figure displays the % of chromium HIV envelope glycoproteins. fusion proteins under the control of the p7.5 promoter. The infected function of the effector to target ratio. Mean fluorescence release as a cells were stained with phycoerythrin-conjugated antibody to CD4 and treated with anti-CD4 intensities for cells phycoerythrin-conjugated by flow cytometry. The panels depict the increase in specific analyzed were: for CD4:¢, 706; for CD4:Fc-yRIIA, antibody in this experiment fluorescence over that shown by uninfected monocytes, which express for CD4:FcyRIIB2, 912; and for 1000; for CD4:Fc-yRIIBl, 75.6; some CD4. CD4 alone, 28.6. 5.0 In the in which the amino- experiments and cytolysis assays. * CD4:IIA of the intracellular domain was removed, terminal portion 4.1 domain of Fc'yRH was replaced with the the transmembrane 0 * u3| domain of the unrelated CD7 antigen, to transmembrane .) CD4:t 3.2 the contribution of interactions mediated eliminate possible domain (Figure 7a). by the membrane-spanning es ag Sd U> 14 carboxyl-terminal residues, including Removal of the 2.3 CD4:IIB2 in a complete loss of cytolytic capacity and Tyr298, resulted reduction in to mobilize a substantial but incomplete ability Mae 1.4 T cell 7b). calcium in mutant cells lacking receptor (Figure CD4:IIB1 cells are often hypersensitive to calcium-eliciting Such 0.5 and a nearly complete loss of activity was stimuli, however, 200.0 300.0 400.0 -100.0 0.0 100.0 recorded in cells retaining T cell antigen receptor 7c). The latter data give results comparable to those (Figure time in seconds with intact in which the removal of 17 obtained receptor, was found to eliminate calcium mobilization in residues mobilization in primary human monocytes expressing Fig. 5. Calcium et al., 1991). Further truncation from human were isolated from whole cells (Odin CD4 chimeras. Primary monocytes P388D, with recombinant vaccinia viruses expressing blood and infected to just before Tyr282 gave an identical the carboxyl-terminus Gated calcium flux analyses were various CD4 fusion proteins. phenotype. after the infected monocytes with conducted by flow cytometry loading from the amino-terminus of the intracellular Deletion of violet to blue fluorescence (a surrogate the dye Indo-1. The ratio 268 had no substantial effect on either domain to residue for the positive cell population (gated for both marker for calcium ion) and using CD14 expression to establish scatter CD4 expression scatter, the calcium profile or the cytolytic potency, whereas deletion is shown as a function of time. windows) to residue 275 markedly impaired free calcium release but had little effect on cytolysis. At present we suspect that the extracellular domains joined to Fc receptor transmembrane inability of the Fc-yRIIA(276-31 1) fragment to mobilize and intracellular domains (Figure 6). As is the case for T calcium in the flow cytometric assay does not reflect its are essentially as effective as cells, Fc receptor chimeras ability to support calcium ion accumulation over the longer chimeras in the redirected cytolysis assay TCR r chain time span of the cytolysis assay, inasmuch as the cytotoxicity (Figure 6). in this assay is inhibited by the inclusion of EGTA in the medium (data not shown). However, it is possible that the Analysis of functional subdomains of FcyRll effects of EGTA are limited to inhibition of exocytosis, rather intracellular sequences than triggering. An EGTA-insensitive cytolytic activity has The intracellular portions of Fc-yRIIA and C share no been reported for some (but not all) cytolytic T cell lines sequence homology with other proteins, appreciable (Ostergaard et al., 1987; Trenn et al., 1987, 1989; Young including the members of the extended TCR r family. To et al., 1987). Further deletion from the amino-terminus, to define the sequence elements responsible for induction of residue 282, produced Fc-yRII tails that lacked the ability we prepared 5' and 3' deletions of the intracellular cytolysis, either to mobilize calcium or to trigger cytolysis. domain coding sequences, and evaluated the efficacy of the The 'active element' defined in this manner is relatively resulting deleted fusion proteins in the calcium mobilization large (36 amino acids) and contains two tyrosines separated 4864 Initiation of cytolysis by myeloid Fc receptors . CD16:11A 20o UCD16:7:11A I , 2.0..* (269-31: LCD16:11AY298- ....... RQLEETNNDY RKKRISANST DPVKAAQFEP PGRQMIAIRK s<-. ::: ; CD 116:LY282- (276-311) (283-311) (299-311) CCD16:1IB1 . * " Fr HVNSNN 276 ETADGGYMTL NPRAPTDDDK NIYLTLPPND 1000 200 0 3000 -100,0 0.0 in seconds Y298, time Y282* 4.0 TO 3.5 e .cno:oo * CD16:7I11A X0 so d a) 0 3 CD16:7:11A 2.5 C 2.5 CD16:7:IIA(269-311) (O -* CDt6 {IA CD16.7 1A 2.0 5 o .+CD16:7:A(276-311) 1.00 CD16.11A298- --- CD16:11A282- g 1.5. c .,.v'"v'-'t-"--'---+.x CD C)2 16:7:UA(283-31 1) -105 0 0.0 2 W CD16:11AY285. ,0- co m in ecnd 0 CCD16:7:IIA(299-311) 10~ ~ t5 i 0.0 100.0 200.0 300.0 -IQ0.0 / rati time in seconds time in seconds eAt ratio * CD16:71A 2.5 oo CDIS:7:HA(269-31 1) a) 2.0 *'* E) * 5. .. CD16:11A I CD18:7:HIA(276-311) CD16:7:11A .E_ CO 1.5 Ooo .!OOCo * CD1 6:7:11A(269-31 1) a; oop 1) CD16:7:1A(276-31 x 1) I CD16:7:1A(283-31 fi C016:7:11A(283-311) CD1 6:7:IIA(299-31 1) 50 : ?~00sS.. R2.20.5 0 CD16:7:IIA(299-31 1) -100.0 0.0 100.0 2c3n .0 .0 ratio time in seconds elt Fig. 7. Deletion mapping of residues in the Fc-yRIIA tail which are important for cytolysis. (a) Schematic diagram of the deletion constructs. Deletions of the amino-terminal portion of the extracellular domain (arrows above sequence) were constructed by addition of the Fc-yRII fragment to the transmembrane domain of CD7, which in turn was joined to the extracellular domain of CD16. (b, c and d) Calcium mobilization in TCR- (b) and TCR+ (c) variants of the Jurkat cell line and cytolysis (d) by carboxyl-terminal deletion variants of CD16:Fc-yRIIA. (e, f and g) Calcium mobilization in TCR- (e) and TCR+ (f) variants of the Jurkat cell line and cytolysis (g) by tripartite chimeras bearing progressively less of the amino terminus of the intracellular tail of CD16:Fc-yRIIA. The mean fluorescence intensities for CD16 expression by the cells in (d) and (g) were: CD16:Fc-yRIIA, 850; CD16:Fc-yRIIA Y298*, 695; CD16:Fc-yRIA Y282*, 319; CD16:7:Fc-yRIIA, 419; CD16:7:Fc-yRIIA(269-311), 908; CD16:7:Fc-yRIIA(276-311), 799; CD16:7:Fc-yRIIA(283- 311), 490; and CD16:7:FcyRIIA(299-311), 333. earlier (Romeo et al., 1992), the presence of a studied by 15 residues. To evaluate the potential role of the tyrosine leucine residue immediately preceding the amino-terminal residues, point mutations converting each tyrosine codon to was found to be important for signalling, but a a serine codon were introduced into the cytoplasmic domain tyrosine of full length tripartite chimeras bearing the CD4 similar residue is not found in the same position for Fc-yRHA However, one similarity between the Fc-yRII family domain, CD7 transmembrane domain and or C. extracellular and the r family is the appearance of a leucine (or tail. In each case, mutation of the Fc-yRIIA cytoplasmic occasionally another aliphatic residue, among r family abolished the ability of the resulting chimeras to tyrosine members) three residues carboxyl-terminal, resulting in a T cells and monocytes, and also mobilize calcium in TCR- consensus of the form Y-X-X-L. There is not a great deal their capacity to mediate HIV envelope-directed blocked of inherent in this pattern, however, and similar either CTL or monocytes (Figure 8). specificity killing by Y-X-X-M or Y-X-X-Aliphatic sequences are found among the phosphorylation sites recognized by src tyrosine Discussion and SH3) domains of tyrosine homology 2 and 3 (SH2 kinases and other proteins. we establish that the and HC subtypes In this report Fc-yRHA there is no evidence pointing to an evolutionary Although activation potential previously thought to a cellular possess between the r family proteins and the Fc-yRII of the family of trigger molecules. relationship be limited to members ¢ recent data support the idea that both classes of motifs characteristic of the r family subtypes, Although the sequence function or engage, tyrosine molecule through, in the domains of Fc-yRIIA and activation are not found cytoplasmic of activation. In (Huang et al., kinases in the platelets process there are structural similarities between the two classes C, cell line U937 (Liao et al., and the monoblastoid 1992) of activation domain, notably the requirement for two of FcyRfl leads to de novo accumulation crosslinking for both calcium mobilization and cytolysis 1992), residues tyrosine the case cannot be made Although of tyrosine phosphate. et al., 1992). The spacing between the tyrosines is (Romeo the results make it likely that it is present unambiguously, for and C than for members Fc-yRIIA substantially greater which is for the increased subtype responsible as to 10 residues), and the the Fc-yRHA opposed of the r family (15 in both Odin et al. kinase examples. Similarly, activity acidic residues flanking the distal tyrosines in characteristic that in a cell line, crosslinking have shown myeloid (1991) are not found in Fc'yRIIA or C. In the r motif the family 4865 W.Kolanus, and C.Romeo B.Seed 5.0 16:IIA owes -%.. ana %% 3.9 0 [ 4) ft-%o% 4) cc ft...] CD1 6:1 IA o 16:IIAY282S cc aD4 CD16:11AY282S CD16:11AY298S .E * L0 1.6 16:IIAY298S -00 goal . e &*a 66" .- 0.5 -100.0 0.0 100.0 200.0 300.0 400.0 time in seconds e/t ratio 2.5 * CD4:¢ 2.1 * CD4:IIA (U co 1.7 CD4:11A _ s . _ .^~~a' CD4: 4 A CD4:7:IIB2 CD4:7:11B2 CD4:7:11AY282S ..-.* 'a~~~~~~~a a) CD4:7:11AY298S .°, *'**_ o CD4:7:IIAY282S N.I. U0 0.9 i6 .* A CD4:7:11AY298S 0.5 -100.0 0.0 000.0 200.0 300.0 400.0 e/t ratio time in seconds of the of residues within the domain. Calcium mobilization in the TCR- Fig. 8. Analysis importance tyrosine cell Fc-yRIIA cytoplasmic (a) analysis line JRT3T3.5. The of cells chimera substituted with and chimera response bearing wild-type chimera, bearing Tyr282 Ser, bearing Tyr298 substituted with Ser is shown. in CTL directed and the mutants and Calcium mobilization (b) Cytolysis by FcyRIIA, in Tyr282Ser Tyr298Ser. (c) monocytes CD4 chimeras CD4: or chimeras CD7 transmembrane and either intact by , CD4:FcyRIIA, tripartite bearing Fc-yRIB2 sequences intracellular mutant or mutant Calcium mobilization was on cells domain, Fc-yRIIA Tyr282Ser Fc'yRIIA Tyr298Ser. for analysis performed gated CD4 and scatter as described in the to 5. in the chimeric molecules described in legend Figure (d) Cytolysis N.I., not monocytes expressing (c). infected. Mean fluorescence intensities for CD16 the cells in were: expression by (b) 246; Y298S, 314; and CD16:Fc-yRIIA, CD16:Fc-yRIIA 304. Mean fluorescence intensities for CD4 the cells in were: CD16:Fc-yRIIA Y282S, 109; 162; expression by (d) CD4:Fc'yRIIA, CD4:D, and not CD4:7:Fc-yRIIA Y298S, 75; Y282S, 120; 26. CD4:7:FcyRIIB2, 84; infected, CD4:7:Fc7RIIA ion the of the Fc-yRIIA can mediate free calcium Whatever explanation, the absence of cell subtype appropriate work an idea and extended our lines is a substantial technological obstacle. In this work we accumulation, supported by and to with chimeras. Thus both the r family trigger molecules have introduced two related strategies study cytolytic the or C calcium transients and pathways initiated by myeloid Fc One is the Fc'yRIIA subtypes produce receptors. use to are known increase of primary human T cells as effector and appear (or to) tyrosine phosphorylation surrogate cells, a stimulus. The that the other is the use of vaccinia virus vectors to introduce following crosslinking finding is receptors into primary human human phospholipase specifically phosphorylated following monocytes. Primary C'y1 Fc-yRll clustering in U937 cells (Liao et al., cytolytic T a renewable 1992) provides lymphocytes represent population a reasonable candidate for the of effector cells with explanation similarities, high target specificity and high cytolytic suggesting that tyrosine and results potential. not to phosphorylation precedes Although especially susceptible transfection, in inositol phosphate production and calcium mobilization are infected with they easily recombinant vaccinia viruses, A of and their does through activation of similar cytolytic capacity not appear to be impaired phosphorylation PLC-'yl. has been observed of the infection. following crosslinking by Moreover, as this study shows, they have the PLC-'yj high affinity IgE receptor (Park et al., 1991). In U937 to to cells, ability respond exogenous receptors whose natural tyrosine of can be distribution phosphorylation PLC-'yl induced by does not fall in their lineage. or crosslinking either Fc-yR1 Fc-yRHl et the More (Liao al., 1992); problematic is the analysis of cytolytic effector mechanism may not be similar, however, since the function in cell types other than CTL. For example, primary domain of no cytoplasmic Fc-yRI shows relatedness to that natural killer cells, although potently cytotoxic, are also of Fc-yRHl, and in no particular contains tyrosine residues. substantially less specific than CTL; and primary monocytes Although activation of cytolysis is a central feature of offer the worst of both worlds, being both less cytolytic and cellular immunity, historically it has been more difficult to less specific than CTL. However, it is possible by suitable study than activation of cellular helper function. In large part choice of target cell and monocyte isolation conditions to this stems from the relative scarcity of cell lines which are make the of study monocyte cytolysis amenable to genetic amenable to transfection and which are also capable of methods of analysis. Like CTL, primary monocytes are mediating a functional cytolytic response. It may be that this easily infected with vaccinia viruses, and give good is inevitable, and that loss of function is an unavoidable expression of cell surface proteins under the control of the consequence of attempting to propagate cells with a high vaccinia p7.5 mixed early/late promoter. Although the cytotoxic potential. tendency of vaccinia to shut off host protein and RNA 4866 Initiation of Fc cytolysis by myeloid receptors must be synthesis counted a liability, there the PE positive cell which is reason to be (infected) population, typically represented 40-80% of the cells. The violet/blue ratio to the emission prior addition that the and of optimistic scope variety subjects which are of was used to antibody establish the normalized initial ratio, set equal to to in susceptible study monocytes will continue to expand. unity. Lymphocyte cytolysis assay Materials and methods A CD8+ CD4- HLA B44 restricted cytolytic line (WH3), kindly provided J.Kurnick and of by F.Harris, was maintained in IMDM, 10% human serum chimeras Preparation with 100 U/ml of IL-2 and was periodically To prepare the cDNA stimulated with irradiated chimeras, sequences corresponding to the (3000 rad) mononuclear transmembrane and domains of cells having the HLA B44 haplotype. Cells were cytoplasmic Fc-yRI and the IIB 1 grown Fc-yRIIA, for at least 10 and IIB2 isoforms were days following stimulation before use in cytotoxicity amplified using synthetic oligonucleotides primers assays. The cells either from clones et were infected with recombinant vaccinia at an m.o.i. of at least existing (Stengelin al., 1988; Allen and Seed, 1989) 10 for h in serum-free or from a human tonsil cDNA 1 medium, followed by incubation in complete medium library. The cDNA fragment corresponding to the FcRIIC which for 3 h. cytoplasmic domain, differs from the IIA isoform at one amino acid residue for P at Cells were harvested by centrifugation and at a (L residue 268; Stuart et al., was resuspened density of 1989) 1 x 107/ml. 100 were added to each well of a U-bottom site directed ytl microtiter generated PCR. The forward plate by mutagenesis by overlap and containing 100 1I/well of medium. Cells were diluted reverse contained complete in 2-fold primers sites for the BamHI cleavage enzymes and NotI indented serial steps. Two wells for each did not respectively, six residues from the 5' end. sample contain lymphocytes, to The Notl site was allow spontaneous followed a chromium release and total chromium uptake to be immediately by stop anticodon, either CTA or TTA. All primers measured. An of contained 18 or more residues aliquot 106 target cells, either 3G8 10-2 (Shen et complementary to the 5' and 3' ends of the al., desired 1989) or HeLa cells infected with vaccinia recombinant vPE16 (Earl et fragments. al., 1990) was centrifuged and resuspended The PCR were inserted into in 50 of sterile fragments expression vectors which contained [5lCr]sodium A1 chromate (1 CD16 or CD4 extracellular domains mCi/mi, DuPont) for 1 h at 37°C with intermittent in a BamHI mixing, terminating site just then washed three times with PBS. 100 1I of to the membrane domain labeled cells resuspended in proximal spanning (Romeo and Seed, 1991; Romeo medium at 105 cells/ml were added et The of to each well. The microtiter plate was al., 1992). identity all isoforms was confirmed by dideoxy spun at 750 g for 1 min and incubated for 4 h at 37°C. At the end of the sequencing. incubation period the cells in each well were resuspended by gentle pipetting, a sample was removed to determine the total counts incorporated and the Immunoprecipitation JRT3.T3.5 microtiter plate was spun at 750 for 1 min. 100 107 and cells were td aliquots of supematant Approximately (Weiss Stobo, 1984) infected g for 1 h in serum-free IMDM were removed and counted in a gamma ray scintillation counter. The medium with recombinant effector vaccinia at a of infection to target ratio was corrected for the of effector of at least 10. Twelve percent cells infected (usually multiplicity (m.o.i.) hours after infection, the cells were 1251 >70%). harvested and surface labeled with 0.5 107 mCi cells per the oxidase method. The labeled using lactoperoxidase/glucose cells were collected Monocyte cytolysis and in 1% assay by centrifugation lysed NP-40, 0.1% SDS, 0.15 M Cytolysis assays using human peripheral blood as effector 0.05 M Tris 5 mM 5 mM 0.2 M iodoacetamide monocytes cells NaCI, pH 8, MgCl2, KCI, were out in fashion and 1 mM PMSF. Nuclei carried the same as for the CTL assays, with the were removed and following by centrifugation CD16 fusion were modifications: monocytes, cultured overnight in IMDM, 20% fetal with bovine proteins immunoprecipitated antibody 3G8 and anti-mouse IgG were serum, were detached by and infected with recombinant 10% scraping vaccinia agarose. Samples electrophoresed through polyacrylamide-SDS under at a of infection of at least 10 for conditions. multiplicity 1 h in serum free medium, gels reducing incubation followed by for 4 h in Teflon beakers in 20% fetal IMDM, bovine serum. CVI cells infected with vaccinia recombinant vPE16 (Earl et al., Isolation of blood peripheral monocytes 1990) were used as targets. The microtiter plate containing effector/target were isolated either from coats or from fresh Monocytes blood as buffy mixtures at 10 ratios from 50: 1 to 0.09: 1 control plus wells for determination described Connor et al. Cells were over Ficoll- by (1990). centrifuged of and maximal 5ICr release was incubated at 37°C spontaneous for 16h. and the interface was collected. After three washes in Hypaque layer IMDM, the cells were in IMDM fetal 20% bovine serum at suspended containing In vitro mutagenesis cells ml in 15 tubes and were 107 ml rotated at 20 per polypropylene r.p.m. FcRUIA deletion mutants were constructed Carboxyl-terminal by PCR in Tektator about an axis to the axis of the (Lab-Quake, Inc.) parallel long the same fashion as for the full length constructs, converting the sequences tubes for 15-25 at to induce min The 4°C monocyte aggregation. aggregated at 282 and encoding tyrosine positions 298 into stop codons (TAA). The cells were sedimented on ice at for 15 in 2 I ml of g min, resuspended amino-terminal deletions were generated by amplifying fragments encoding medium and onto an volume of ice-cold fetal bovine serum. layered equal less of the intracellular domain successively by PCR, using oligonucleotides After sedimentation the fetal bovine serum for 20 min at the through 4°C, which allowed the to be inserted between MluI resulting fragments and NotI lower contained between 80 and 97% the remainder phase monocytes, being restriction sites into a previously constructed expression plasmid encoding cells the interface were lymphocytes. Alternatively, from Ficoll-Hypaque the CD16 extracellular domain fused to the CD7 transmembrane domain, x cells into 100 washed three times in IMDM and at 5 106 ml plated per the latter terminating in a MluI site at the junction between the transmembrane mm tissue culture dishes. were allowed to adhere to the Monocytes plastic and the intracellular domain. for at least 2 h and were then detached for further by scraping analysis. The of was > 90 as determined with purity monocytes usually by staining Leu-M3 antibody (Becton-Dickinson). Acknowledgements We thank Michael and Fanger Jay Unkeless for the 3G8 10-2 cells, Franco Calcium flux analysis Harris and Jim for the Kurnick WH3 line, and members of the laboratory of Cells the Jurkat mutant subline JRT3.T3.5 and or Stobo, (Weiss 1984) for discussion and criticism. C.R. was in supported part by grant 1309-9-RG blood as described above were infected with peripheral monocytes prepared from the American Foundation for AIDS Research. This was study supported recombinant vaccinia viruses for 1 h in serum-free IMDM at an m.o.i. of NIH A127849 and an award to the Massachusetts by grant by General 10% 10 and incubated for 3-9 h in FBS. Cells were collected IMDM, Hospital by Hoechst AG. and at 3 in medium 106/ml by centrifugation resuspended complete 1 mM Indo-l et al., containing acetomethoxyester (Grynkiewicz 1985) and incubated at for 45 The loaded min. (Molecular Probes) 37°C Indo-l References x in serum-free IMDM cells were and at and 1 106/ml pelleted resuspended at room in dark. Cells were for free Ca2 et stored the + Amigorena,A. al. 1808-1812. (1992) Science, 256, temperature analyzed of the violet and blue fluorescence emission simultaneous measurement Anderson,P., Caligiuri,M., O'Brien,C., Manley,T., Ritz,J. and by et To initiate calcium Proc. Natl. Acad. Sci. flow either Schlossman,S.F. (1990) USA, 87, 2274-2278. al., flux, by cytometry (Rabinovitch 1986). and Immunol. monoclonal was Balk,S. Terhorst,C. 411-416. added (1989) Ser., 45, 3G8 (at 1 unconjugated (anti-CD16) antibody ug/ml) 10 of and Kinet,J.-P. to the cell followed Blank,U., Ra,C., Miller,L., White,K., Metzger,H. (1989) suspension by 1g/ml phycoerytdrin (PE)-conjugated Fab'2 goat anti-mouse IgG at time or a anti-CD4 Nature, 337, 187-189. 0, PE-conjugated antibody (Leu-3a, Becton Dickinson) was added, followed by unconjugated secondary Pavlovich,R., Choukroun,V. Bonnerot,C., Amigorena,S., Choquet,D., and collected EMBO antibody. Histograms of the violet/blue emission ratio were from J., 11, 2747-2757. Fridman,W.H. (1992) 4867 W.Kolanus, C.Romeo and B.Seed Boyle,D.B. and Coupar,B.E.H. (1988) Gene, 65, 123-128. Wegener,A.-M.K., Letourneur,F., Hoeveler,A., Brocker,T., Luton,F. and Connor,R.I., Shen,L. and Fanger,M.W. (1990) J. 83-95. Immunol., 145, Malissen,B. (1992) Cell, 68, 1483-1489. and Stobo,J.D. J. Weiss,A. (1984) Exp. Med., 160, 1284-1299. Engelhardt,W., Gorczytza,H., Butterweck,A., Monkemann,H. and Frey,J. Weissman,A.M., Baniyash,M., Hou,D., Samelson,L.E., Burgess,W.H. (1991) Eur. J. Immunol., 21, 2227-2238. and 1018-1020. Klausner,R.D. (1988) Science, 239, Falkner,F.G. and Moss,B. (1988) J. Virol., 62, 1849-1854. and Yeh,E.T., J. Reiser,H., Daley,J. Rock,K.L. (1987) Immunol., 138, Fanger,M.W., Shen,L., Graziano,R.F. and Guyre,P.M. (1989) Immunol. 91-97. Today, 10, 92-99. and Year Yokoyama,W.M. Shevach,E.M. (1989) Immunol., 4, 110-146. Fleit,H.B., Wright,S.D. and Unkeless,J.C. (1982) Proc. Natl. Acad. Sci. and J. Young,J.D., Clark,W.R., Liu,C.C. Cohn,Z.A. (1987) Exp. Med., USA, 79, 3275-3279. 1894-1899. 166, Gardner,P. (1989) Cell, 59, 15-20. Graziano,R.F. Received on and Fanger,M.W. (1987a) J. Immunol., revised on 1992 138, 945-950. July 24, 1992; September 11, Graziano,R.F. and Fanger,M.W. (1987b) J. Immunol., 139, 3536-3541. Grynkiewicz,G., Poenie,M. and Tsien,R.Y. J. (1985) Biol. Chem., 260, 3440-3450. Hibbs,M.L., Selvaraj,P., Carpen,O., Springer,T.A., Kuster,H., Jouvin,M.- H.E. and Kinet,J.-P. (1989) Science, 246, 1608-1611. Huang,M.M., Indik,Z., Brass,L.F., Hoxie,J.A., Schreiber,A.D. and Brugge,J.S. (1992) J. Biol. Chem., 267, 5467-5473. Hunziker,W. and Mellman,I. (1989) J. Cell Biol., 109, 3291-3302. Indik,Z., Kelly,C., Chien,P., Levinson,A.I. and Schreiber,A.D. (1991) J. Clin. Invest., 88, 1766-1771. Irving,B.A. and Weiss,A. (1991) Cell, 64, 891-901. Jin,Y.J., Clayton,L.K., Howard,F.D., Koyasu,S., Sieh,M., Steinbrich,R., Tarr,G.E. and Reinherz,E.L. (1990) Proc. Natl. Acad. Sci. USA, 87, 3319-3323. Keegan,A.D. and Paul,W.E. (1992) Im,munol. Today, 13, 63-68. Kinet,J.-P. (1989) Cell, 57, 351-354. Kroczek,R.A., Gunter,K.C., Germain,R.N. and Shevach,E.M. (1986) Nature, 322, 181-184. Kurosaki,T. and Ravetch,J.V. (1989) Nature, 342, 805-807. Lanier,L.L., Yu,G. and Phillips,J.H. (1989) Nature, 342, 803-805. Letourneur,F. and Klausner,R.D. (1991) Proc. Natl. Acad. Sci. USA, 88, 8905-8909. Letourneur,F. and Klausner,R.D. (1992) Science, 255, 79-82. Liao,F., Shin,H.S. and Rhee,S.G. (1992) Proc. Natl. Acad. Sci. USA, 89, 3659-3663. Mellman,I. (1988) Curr. Opin. Immunol., 1, 16-25. Meuer,S.C., Hussey,R.E., Fabbi,M., Fox,D., Acuto,O., Fitzgerald,K.A., Hodgdon,J.C., Protentis,J.P., Schlossman,S.F. and Reinherz,E.L. (1984) Cell, 36, 897-906. Miettinen,H.M., Rose,J.K. and Mellman,I. (1989) Cell, 58, 317-327. Miettinen,H.M., Matter,K., Hunziker,W., Rose,J.K. and Mellman,I. (1992) J. Cell Biol., 116, 875-888. Moingeon,P., Lucich,J.L., McConkey,D.J., Letourneur,F., Malissen,B., Kochan,J., Chang,H.C., Rodewald,H.R. and Reinherz,E.L. (1992) Proc. Natl. Acad. Sci. USA, 89, 1492-1496. Odin,J.A., Edberg,J.C., Painter,C.J., Kimberly,R.P. and Unkeless,J.C. (1991) Science, 254, 1785-1788. Ostergaard,H.L., Kane,K.P., Mescher,M.F. and Clark,W.R. (1987) Nature, 330, 71-72. Park,D.J., Min,H.K. and Rhee,S.G. (1991) J. Biol. Chem., 266, 24237-24240. Qiu,W.Q., de-Bruin,D., Brownstein,B.H., Pearse,R. and Ravetch,J.V. (1990) Science, 248, 732-735. Ra,C., Jouvin,M.-H.E. and Kinet,J.-P. (1989) J. Biol. Chem., 264, 15323-15327. Rabinovitch,P.S., June,C.H., Grossmann,A. and Ledbetter,J.A. (1986) J. Immunol., 137, 952-961. Ravetch,J.V. and Kinet,J.P. (1991) Annu. Rev. Immunol., 9, 457-492. Reth,M. (1989) Nature, 338, 383-384. Romeo,C. and Seed,B. (1991) Cell, 64, 1037-1046. Romeo,C., Amiot,M. and Seed,B. (1992) Cell, 68, 889-897. Samelson,L.E., Harford,J.B. and Klausner,R.D. (1985) Cell, 43, 223-231. Schmitt-Verhulst,A.M., Guimezanes,A., Boyer,C., Poenie,M., Tsien,R., Buferne,M., Hua,C. and Leserman,L. (1987) Nature, 325, 628-631. Sefton,B.M. and Campbell,M.A. (1991) Annu. Rev. Cell Biol., 7, 257-274. Shen,L., Graziano,R.F. and Fanger,M.W. (1989) Mol. Immunol., 26, 959-969. Stengelin,S., Stamenkovic,I. and Seed,B. (1988) EMBO J., 7, 1053-1059. Stuart,S.G., Simister,N.E., Clarkson,S.B., Kacinski,B.M., Shapiro,M. and Mellman,I. (1989) EMBO J., 8, 3657-3666. Trenn,G., Takayama,H. and Sitkovsky,M.V. (1987) Nature, 330, 72-74. Trenn,G., Taffs,R., Hohman,R., Kincaid,R., Shevach,E.M. and Sitkovsky,M. (1989) J. Immunol., 142, 3796-3802. and Unkeless,J.C., Annu. Rev. Scigliano,E. Freedman,V.H. (1988) 251-281. Immunol., 6,

Journal

The EMBO Journal – Springer Journals

Published: Dec 1, 1992

There are no references for this article.