Signal Transduction by HLA-DR Is Mediated by Tyrosine Kinase(s) and Regulated by CD45 in Activated T Cells Niels 0dum, Paul J. Martin, Gary L Schieven, Nancy A. Norris, Laura S. Grosnmire, John A. Hansen, and Jeffrey A. Ladbetter

ABSTRACT: Recently, it was shown that HLA class II molecules on B cells and activated human T cells can transmit signals involving tyrosine phosphorylation of specific proteins, activation of the inositol phospholipid pathway, and release of cymsolic free Ca~'(Ca:' I,. The regulation of class II induced signals is poorly understood, however, and it remained unknown whether theee pathways were coupled or activated independently. Here we show that a specific inhibitor of protein tyrosine kin~ses (F'gKL herbimycin, abrogated DR-induced elevation of (Caz+)i in activated human T cells. Genistein, belonging m another family of PTK inhibltots, had weaker but significantinhibitory effects on DR-induced

ABBREVIATIONS (Ca2+), intracellular free calcium IL-2 MHC

concentration interleukin-2 major histocomp~tibilitycomplex

(Ca2+)i responses. CD45 crossiinklsg with DR almost completely abrogated DR-induced (Ca:+ Ii res~onses and profoundly changed the FrK profiles. In coatra.st, CD4 cross[inkingwith DR enhanced the (Ca-"')i responses, but the inhibitory effect of CD45 don~namd over the enhancingeffect of CD4. These data indicme that PTK activation is obligatory for DR.induced (CaZ÷)~responses, suggesting a linkage between these pathways in class II s/gnalrransduction. This conclusioni¢ consha'entwith our observation that in activated human T cells,class II signals are up regulated by CD4, whkh is associated with p56kk, and down regulated by CI~5, whkh is a tyrosine phosphatase. Humar~ Imra#ge/~gy32, 85-94 (19911

mAb PKC PTK TCR

moaodonal antibody protein ldnase C protein tyrosine kinase T-cell receptor

INTRODUCTION Major histocompatibilltycomplex ( M H C ) classII molecules function as restriction elements in antigen-spedfic activation of CD4 T cells [reviewed in ref. 1]. In addition, it was recently shown that class II molecules can function as signal transductio~t structures. Thus, in murine B cells, class II molecules can transmit signals involving translocation of protein kinase C (PKC) to the From tt~ Clinical Rcstarch Divisien, Fred HutcblnJon Cancer Re$¢#rfb Center (N.~.; PJ.M,;J.A.H.). the Del~urtmcnt of /ffedicine. Uui. r¢rsity of Washingto~ CPJ.M.: J.A.H.), a ~ OncogtnlBristal-AI)em Squibb (G.L.$.: N.A.N.: L.S.G.rJ.A.L.L Stattle, Wasbingto,t, Add~ss reprint requests to Nids ~dxm. Tissue Typlug Laboratory. Division of Clinical lmtausology. Rigshospitala Tage~srej20. 2200 Copin.gin, Dtnm~rk, Reuivcd D¢¢cm&r31, 1990; ace*MadApril 1 I. 199L

Humanlmmunolc~32, 85-94 (1991) © gmeriamSocieWfez Histucomp.z*ibility~d l~unogen~lics.1991

nucleus, an increase in ~.AMP levels, and initiation of phosphatidyl inlsitol turnover [2-4]. Furthermore, crnsslinking class II molecules on human B ceils induces Wrosine phosphorylation of specific proteins, irfifiates phosphatidyl iaosiml turnover, and g/yes rise to an increase in intraceUul~x free calcium concentration (Ca2+)~ [5, 6L In contrast to resting T ceils, which generally do not express class lI antigens, activated human T cells synthesize and express class II antigens [for review see ref. 7]. Under cetxa/n circumstances, activated "r cells can present (allo)andgeu to (nile)antigen-specific T cells [ 8 Z2], but processing and presentation of soluble antigens is generally deficient mM the functions of class II mole85 0198-8859/gt/s3.50

86

cules expressed by activated T cells remain largely unknown. A possible role of class II antigens in signal transduction in T cells was suggested by the findings that class lI monoclonal antibody (mAb) inhibited proliferation of activated T cells [ 13-15] and by the observation that crossliukiug anti-DR mAb induced interleukin-2 (IL-2) production in T leukemia cells (HUT78) [16]. More recently, it was shown that crosslinkiug DR molecules on activated human T-cell clones and T leukemia cells caused an increase in tyrosine phosphorylation of several substrates and induced elevations in (Ca2+)i [17]. Furthermore, it was suggested that DR molecules may play a regulatory role in CD4 activation. Thus, crosslinking of DR and CD4 generated synergistic (Ca2+)i responses [17]. These findings raised the question of whether DR-induced tyrosine phosphorylation and (Ca2+)~responses were coupled or independent pathways. Tyrosine kinases are involved in signal transduction in many receptor systems and in regulation of growth of normal and tumor cells. In T ceils, tyrosiue kinases are of importance for signaling by growth hormones (IL-2) [18, 19] and, recently, a linkage was demonstrated between protein tyrosine kinase (PTK) activation and initiation of the phosphotidyl inositol pathway after T-cell receptor (TCR) stimulation [19, 20]. The TCR-CD3 complex is physically associated with the protein tyrosine kinase p59 iv" [21], a member of the src family of PTKs. p56 I(k,another member of this family of tyrosine kinases, is well known for its association with CD4 and CD8 accessory molecules [22-24]. Interactions between the corresponding PTKs are likely to account for the regulatory effects of CD4/CD8 on signal transduction by the TCR-CD3 complex [25]. Thus, when brought into close proximity on the cell surface, CD4/ CD8 enhanced the generation of TCR-CD3-induced second messengers [24-26]. CD45, which is a tyrosine phosphatase, was shown to down regulate TCR-CD3 stimulation when CD45 and CD3 were crosslinked together [27-30], suggesting that tyrosine pbosphatases (CD45) and tyrosine kiuases (CD4/8-p56 l~k) had antagonistic effects on TCR stimularion in T cells [31]. Although CD45 inhibited some signals, CD45 enhanced TCR-CD3 responses in resting T cells when CD3, CD4, and CD45 were crosslinked together [27-29]. Moreover, in cell-free reactions CD45 could activate p56 ~ck by dephospborylation of tyrosine 505, a putative negative regulatory site on p56 Iek[32]. and recent data indicate that CD45 plays an essential role in T-cell functions by coupling the TCR to the phosphatidyl inositol pathway [33]. Thus, the regulatory effects of tyrosine phosphorylation and dephosphorylation comprise a complex system in T-cell activa-

N. Odum et al.

tion. In the present study, we obtained evidence that signal transduction by DR molecules induced PTK activation. Second, this PTK activation was essential for induction of (Ca2+)i responses suggesting a linkage between PTK activation and initiation of phosphaxidyl inositol turnover after DR stimulation. Third, CD45 profoundly changed the DR-induced tyrosine phosphorylation events and almost completely blocked (Ca2+)i responses. Since the inhibitory effects of CD45 dominated over the enhancing effects of CD4, it appears ~hat CD4 and CD45 might have different effects on DRmediated versus TCR-CD3-mediated signals. MATERIALS AND METHODS

Cells. The CD4-positive human T-cell clones specific for DQw9 (IE6) and Dwl4 (EM25, EM36) and T-cell lines specific for DPw3 (P572), DPw4 (P30), and DPw6 (P506) have been described previously [ 12, 17].

Monoclonalantibodiesand reagents. CD3 amibodies 38.1 (igM), G19-4 (lgGl), CD4 mAb G17-2 (IgG1), CD45 mAb 9.4 (lgG2a), CD7 mAb G3-7 (IgG1), and HLADR mAbs p4.1 and L243 (both lgG2a) were previously described [5, 26]. These antibodies were purified from ascites fluid by salt precipitation and DEAE chromatography followed by dialysis and filtration through 0.45-m filters prior to use. F(ab'h fragments of p4.1 was prepared as described [26, 27]. Biotin coniugams were prepared by reacting antibodies with biotin-succinimide (Sigma Chemical Co., St. Louis, MO) as described [26, 27]. Avidin (Sigma) was used to crosslink biotin-couiugated mAbs at a 4 : 1 wt/wt ratio. Protein ryrosiue kinase inhibitor, herbimycin A, was a kind gift from the Drug Synthesis and Cancer Branch, Division of Cancer Treatment, National Cancer Institute, and enzyme inhibitor specificity was described by Uehara et el. [34]. Genistein was purchased from ICN (Irvine, CA).

preparation of soluableconjugatesof CD4 (G17-2), CD45 (9.4), and CD7 (G3-7) mAbs. Antibody homocoujugates were prepared by derivitization with maleimidobutyrloxysucciuimide (GMBS, Calbiochem, LaJolla, CA) and imminothiolane HCL (2-1T, Pierce Chemical Co., Rockford, IL) as described [35]. Briefly, one aliquot of mAb was treated with 2-iT at 250 ~g/mg mAb and a second aliquot was treated with GMBS at 7 ~g/ my. Derivatized mAbs were desalted and mixed together to form a stable thioether bond [35]. Conjugates were dialyzed against phosphate-buffered saline before use.

Measurement of cytoplasmic calcium concentrations (Ca2+)i. (Ca2+)iresponsesinindo-1 (Molecular Probes,

Signal Transduction by DR in Activated T Cells

Eugene, OR) loaded cells were measured v,~th a model 50 H H / 2 1 5 0 flow cytometer (Ortho, Westwood, MA) as described [26, 27]. Histograms were analyzed by programs that calculated the mean indo-1 violet/blue fluorescence ratio as a function of time. For each time point, a second program calculated the percentage of cells with an indo-I ratio higher than -two standard deviations above the mean for control cells. There are 100 data points on the X (time) axis on all flow cytometric data, and calibrations were performed as described [36]. Correlations between indo-I ratios and (Ca:+), values are approximately linear at lower indo-I ratios (i.e., below 2) but progressive at higher indo-1 values [36L Indo-I ratios of 1, i.e., resting T cells, correspond to u (CaZ+)i concentration of 131 aM, whereas indo-I ratios o f 2, 3, 4, and ~ correspond to (Ca"+); concentrations of 323, 614, 1005, and 2098 nM, respectively.

87

A 3.0 2.0 1.0

7o

"o tv ,m

B

'T" O

Detection of tyrosine phosphorylation on Western blots. Protein tyrosine phosphorylation was measured by immunoblotdng cell lysares with a purified rabbit antiphosphotyrosine antibody, prepared as previously described [5]. Cells (5 x 106 ml/sample) were stimulated with antibody under the indicated crosslinking conditions and then lysed in 300-/~1 hot sodium dodecyl sulfate (SD$) sample buffer containing 50 tzM orthovanadate. Lysares were boiled for another 5 rain and then stored at -70°C. Samples were electrophoresed on 10% $DS polyacwlamide gels and transferred to immooilon (Millipore Corp., Bedford, MA). Immunoblots were incubated for 3 hr with 0.25 ?~g/ml rabbit anti-phosphotyrosine antibody, followed by washing and development with 1 wCi/ml high-specific-activity 125 1 protein A (ICN Pharmaceuticals) [5]Prestained high-molecular-weight market's (Bethesda Research I2.borarory, Betheada, MD) were included on each gel and migration positions are indicated by arrows, RESULTS

DR-induced (Ca2+)i responsesdo nst require Fc receptorinvoh,ement. Figure 1 shows the increase of intracellular free calcium after crosslinking of HLA-DR molecules on indo-1 loaded CD4-pusirive T cells. In accord with our previous findings in alioacdvated T clones [17], biotinylared D R mAb had no effect without crosslinking with avidin (data not shown). However, when biotinylared D R mAb was added 3 mitt before time 0 and crossfinked by excess of avidin added at time 1 rain, a significant increase in (Ca2+)i was seen (Fig. 1). In contrast, crossfinking CD7 did not induce significant (CaZ+)i responses (data nor shown) [17]. Co-erosslinking CD7 and other surface molecules, such as D R and

i

+EGTA

2.0

t~ ,

1.5

__/-'-.

1.0 i

3.5 Time

7

(rain)

FIGURE 1 Comparison of (Ca2+)i responses of the P572 T-cell line after antibody-mediated crosslinking of DR (p4. l,--) or CD45 and DR (p4.1 + 9.4, ----) molecules in the absence (A) or presence (B) of EGTA (10 raM) to chelate extrarellular Ca2÷. All mAb were biotinylated. Ten micrograms of anti.CD45, 9.4, and/or anti-DR mAb, p4A, was added at time - 3 rain and crosslinked by addition of excess avidin (avidia : mAb ratios were 4 : 1 wt/wtl at time 1 rain. Mean (CaZ+)iresponses are shown. The data shown were from one represeata!.ve experiment of six independent experiments. The second part of this experiment is shown in Fig. 2B. CD4, had no effect on the (Ca2+)i responses when compared to (Ca2+)i responses induced by DR and CD4 crosslinking, respocdveiy [ 17]. Accordingly, CD7 mAb was used as a control in the following experiments. Since some effects o f anti-class II mAbs (e.g., inhibition of mitogen-induced lg production in B cells [37]) have been ascribed to stimulation of Fc receptors, we tested in parallel the effects of a DR-specific mAb and its F(ab')2 fragments. As shown in Table 1 (cop), crosslinking DR-specific mAb, p4.1, and F(ab')2 fragments induced almost identical (Ca2+)i responses in allospecJfic T cells (P572). S/milar findings were obtained with HLA class ll-posidve leukemia cells (HUT78) (data not shown), indicating that D R mAb-ieduced signals did not require involvement of Fc receptors.

88

N. Odum et aL

lease both from internal stores and from the external medium. Thus, with addition o f EGTA to chelate extracellular calcium, CD45 co-crosslinking inhibited D R responses by more than 95% (Fig. 1B) with fewer than 5% showing any measurable response (data not shown).

A 3.0 2.0

Maximal inhibition of (Ca2+)i responsesrequires that DR and CD45 are erosslinkcd together. In order to test

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2.0 1.0

,'

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3.5 Time (rain)

FIGURE 2 The effects of (A) perturbation (without cresslinking) and (B) simultaneous but independent crosslinking of CD45 and CD4 antigens on DR-induced (Ca2+)i respouses in the T clone IE6 (A) and T line P572 (B). A: Unconjugated anti-CD45 (9.4, ---); CD4 (G17-2, - -); or CD7 (G :3-7, --) (as a control) mAbs (50 ~.g) were added together with biotinylated anti-DR (p4.1) mAb (10 my)at - 3 rain followed by addition of avidin at time l rain. B: (Continued from Fig. 1) biotinylated anti-DR mAb (10 izg) was crosslinked alone (p4.1, --) or with simultaneous bur independent crosslinking by CD7 x CD7 homoconjugates (p4.1 + G3-7 x G3-7 homoconjugates, --); CD45 x CD45 homoconjugates (p4.1 + 9.4 x 9A homoconjugates, --); or CD4 x CD4 homoconjugates (p4.1 + G17-2 x G17-2 homoconjugates, ---), respectively. Bintinylated mAb was added at time - 3 rain and crosslinked at time 1 rain (of Fig. 1) and homoconjugates (50 ,u.g)were added at time 1 rain. The data shown were from one representative experiment of three independent experiments.

Inhibition by CD45 of DR-induced (Ca2+)¢ responses. CD45 is a tyrosine phosphatase which modulates activation events in T cells [31]. Crosslinking of CD45 did not induce (CaZ+)i responses (data not shown) [27}. However, as seen in Fig. 1 and Table 2, co-crosslinking of CD45 almost cotoplerely abrogated the DR-induced (Ca2+)i. The CD45 co-crosslinking inhibited calcium re-

whether inhibitory effects o f CD45 on D R responses required cell surface apposition of CD45 with D R toolecules or, alternatively, whether independent simultaneous crosslinking of CD45 and D R had a similar effect, we employed two different crosslinking techniques. Biotinylated toAb and avidin was used to crosslink D R molecules and CD45, CD4, or CD7 molecules were independently crosslinked by homoconjngates of the respective antibodies. CD45 homoconjugates had some inhibitory effects which were significantly weaker than those obtained when CD45 and D R were crosslinked together (Fig. 1). Likewise, a non-crosslinking pertobalion o f CD45 molecules induced by the binding of unconjugated mAb weakly inhibited DR-induced (Ca2+)i responses (Fig. 2A). As seen in Fig. 2B, CD7 hotooconjngates had no effect on (CaZ+)i responses obtained after D R crosslinking. In contrast to the findings with CD45, co-crosslinking of CD4-enhanced DR-induced (Ca2+)i responses (cf Table 2 and ref. 15) but as seen in Fig. 2B, independent crosslinking of CD4 by aufibod:, homoconjngate and pertubation of CD4 by unconjugated mt~b inhibited DR-induced (CaZ+)i responses (Fig. 2A).

Inhibition of DR- and CD44nduc~4 (Cw~+)i responsesby herbimycin. Herbimycin A is a benanquinonoid ansatoycin antibiotic which has been shown to inhibit tyrosine kinase activity but not serine and threonine kinases in human T cells [20]. Preincubation of CD4 T cells with herbimycio A at I/~g/ml for 4 hr partly inhibited DR-induced (Ca2+)i responses (Fig. 3A) and responses were almost completely abrogated after 18 hr preincubation with herbimycin (Fig. 3B). We considered the possibility that the inhibition o f DR-induced (Ca2+), responses by herbimycin could be du~ to toxic effects. Previous data, however, indicated that when used under condkions similar to those for the present study, herbimycin inhibited TCR signaling and left cellular responses to phorbol esters and ionomycin relatively intact [20]. As shown in Fig. 3B, both control cultures and cultures preiw:ubated with herbimycin for 18 hr responded equally well to stitoulation with calcium ionophore. Genistein, belonging to another family of PTK inbibitors which inhibits phytobetoaggludnin-induced phospholipase activation [19], had a weaker yet

Signal Traasduction by D R in Activated T Cells

TABLE 1

89

Effects o f P T K inhibitors o n D R - and C D 4 - i n d u c e d (C,a:+)i responses

Stimulus

Inhibitor

Responding cells (~')

(Cg~'L (aM1

iahib/tio~ (%) of (Ca:*)~

p4.1 IDR) p4.1 (Fab.~) L243(DR) IA43 L243 G17-2 (CD4) G17-2 G17-2

--4 hr control (medium) 4 hr hetbimycin A (FrK) 4 hr genistein (PTK) 4 hr control (mediuml 4 hr herbimycin A (PTK) 4 hr genestein (Xc~"K)

67.7 70.5 79.7 40.2 69.1 50.0 10.2 41.2

515 455 725 240 580 365 145 265

--81.6 24.4 94.0 ~2.7

The effects of cmmfinkitxgDR :,titigeas on (Ca-'*),responses of T cells (allosFcci[i*.CD4' T