Modulation of cytokine patterns of human autoreactive T cell clones by a single amino acid substitution of their peptide ligand

Immunity,

Vol. 2, 373-380,

April, 1995, Copyright

0 1995 by Cell Press

Modulation of Cytokine Patterns of Human Autoreactive T Cell Clones by a Single Amino Acid Substitution of Their Peptide Ligand An/a Windhagen; Christian Scholz,’ Per H(lllsberg,* Hikoakl F&aura,* Alessandro Sette,t and David A. Hager’ *Laboratory of Molecular Immunology Center for Neurologic Disease Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts 02115 tCytel Corporation 3250 John Hopkins Court San Diego, California 92121

Summary We demonstrate that cognate peptide llgands altered at T cell receptor (TCR) contact residues and bound to class II major histocompatablllty complex can change the cytokine pattern of mature T cell clones. Myelln basic protein peptlde 8888-reactive ThO T cell clones were stimulated wlth altered peptlde ligands, which acted both as TCR antagonist and Induced new mRNA synthesis and proteln secretion of TGF-81, while no longer inducing mRNA synthesis or protein secretlon of IL-2, IL-4, IL-lo, and IFNT. The modffied peptldes falled to induce a detectable caklum flux, ~88” actlvation, or thymldine Incorporatlon, yet triggered nearfy equal amounts of IL-4 secretlon in the presence of ionomycin. Antigen-Induced modulation of T cell cytoklne secretion may be important In regulating the Immune response.

lntroductlon Naive T cells differentiate into distinct populations defined by their cytokine secretion pattern and regulatory function. Thl cells secrete predominantly interleukin-2 (IL-2) and interferon y(lFNy) and are involved in classic delayed-type hypersensitivity reactions, while Th2 cells, which secrete predominantly IL4 and 11-10, induce selected immunoglobulin secretion and can down-regulate Thl-mediated immune responses (Mossmann et al., 1986; Romagnani, 1994). Thus, cytokines secreted by T cells after activation qualitatively influence the nature of the immune response. Recent reports have suggested that the dominant factors determining the maturation of naive T cells into either predominantly Thl or Th2 cells are cytokines, particularly IL12 and IFNy for Thl and IL4 for Th2 (Seder and Paul, 1994). Other factors include the dose and type of antigen, the type of antigen-presenting cell (APC), and costimulatory molecules involved in activation. Once T cells have differentiated into Thl or Th2 type T cells, they are thought to be committed to the production of a given set of lymphokines upon restimulation. T cell secretion of certain cytokines is also implicated in

the induction and regulation of autoimmune inflammatory disease, which is mediated by activated autoreactive T cells that recognize tissue-specific antigen in the context of major histocompatibility complex (MHC) class II mola cules on APCs. In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, evidence suggests that T cell secretion of 11-2, IFNT, and tumor necrosis factor (TNF) may mediate inflammation and tissue damage, while the secretion of IL4, IL-lo, and transforming growth factor-61 (TGF-61) by myelin basic protein (MBP)-reactive T cells is associated with potent suppressor activity and down-regulation of central nervous system inflammation (Miller et al., 1992). The induction of TGF-81 secretion appears to be of particular importance in regulating EAE, as anti-TGF-61 monoclonal antibodies inhibit the suppressor effect of regulatory MBP-reactive T cells (Chen et al., 1994). Thus, autoreactive T cells are not necessarily pathologic and can function to down-regulate immune responses associated with tissue inflammation locally. It has recently become clear that T cell activation is not a simple on/off response. Allen and coworkers (Evavold and Allen, 1991; Evavold et al., 1993a) demonstrated that stimulation of a T cell clone wtth modified peptide-antigens that trigger a suboptimal TCR signal can induce cytokine secretion or IL-2 receptor expression and cytolysis without thymidine incorporation. Altered peptides have also been shown to act as TCR antagonists or to induce anergy in Thl and Th2 clones (Alexander et al., 1993; De Magistris et al., 1992; Jameson et al., 1993; Ostrov et al., 1993; Ruppert et al., 1993; Sloan-Lancaster et al., 1993, 1994). The ability of altered peptide ligands to induce different signaling events prompted us to examine their effects on cytokine secretion using human autoreactive T cell clones recognizing the immunodominant myelin basic protein epitope 85-99 (MBPp85-99).

Results Blndlng of Altered Peptlde Llgando to DRBl l 1501 and Peptlde Fine Specificity of T Cell Clone Obl Al 2 Altered peptide ligands derived from MBPp85-99 were examined for binding to DRBl’1501 in an MHC competition assay and for the ability to stimulate MBPp85-99 reactive T cell clone OblAl2 (Table 1). This clone did not proliferate or incorporate thymidine after stimulation with MBPp85-99 substituted at amino acid positions 90 and 91, while an alanine substitution at position 93 did not significantly alter the T cell response, indicating that positions 90 and 91 are the critical TCR contact points for this clone. Whereas substitutions at positions 69 and 92 significantly reduced binding of the altered peptides to DRBl “1501, substitutions at positions 90,91, and 93 did not reduce binding, confirming positions 89 and 92 in MBPp85-99 are the major MHC contact points.

Immunity 374

Table 1. Altered

Peptide

MBPpE5-QQ

Sequence

05

07

86

00

Ligand

Stimulation

of MBP Reactive

T Cell Clone OblA12

and Binding

to Class

II MHC

Binding to DRB’ISOI 09

90

91

92

93

94

95

96

97

98

QQ

[SH]Thymidine uptake

ENPVVHFFKNIVTPR+tt K

+ -

A D K A A

f ++

A

IC50% nM

Ratlo binding to p&5-99

4.1 404 5.2 76 7 2.1 283 3.5

1 0.01 0.8 0.055 0.59 2.0 0.025 1.2

APCs were pulsed with peptide at l-100 us/ml for 2 hr, washed, and irradiated with 5000 rads. T cells (5 x 10’) and APCs (2 x 10’) were then cocultured in triplicate for 3 days in 200 ul medium at 37OC. T cell proliferation was measured as [‘H]thymidine incorporation after an 18 hr pulse. Trfple pluses represent proliferation (stimulation index >3) at 1 uglml; double pluses represents prollferatlon at IO uglml; and a single plus represents proliferation only at 100 ug/ml of peptide.

TCR Antagonist Properties of Altered Peptide Ligands The biologic activity of peptides with substitutions at position 90 were examined in a prepulse TCR-antagonist experiment in which T cells are stimulated by APCs that were pulsed with a limited concentration of native peptide, followed 2 hr later by stimulation with increasing concentrations of peptide antagonist. This assay was developed to avoid competition between antigen and antagonist at the MHC level and thus allows measurement of events at the TCR level (De Magistris et al., 1992). TCR antagonism has been attributed to active signaling events resulting in the inhibition of early biochemical events required for T ceil activation rather than just competition for TCR binding with the native peptide (Ruppert et al., 1993). As shown in Figure 1, MBPp65-99 peptides altered at position 90 could inhibit proliferation induced by the native peptide, suggesting that they could act as a TCR ligand and signal in the context of class II MHC. These altered peptide ligands did not induce clonal unresponsiveness as the T cells showed a full proliferative response to MBPp65-99 7 days after the initial peptide stimulation (data not shown). MBPp65-99 with an alanine substitution at position 91 (MBPp65-99[91A]) inhibited proliferation of OblA12 to a much lesser degree, even at concentrations of 100 ugl ml, suggesting that this peptide-MHC complex had a low TCR affinity as compared with the peptides with substitutions at position 90 (Figure 1). As expected, peptide MBPp65-99(93A) acted as an agonist similar to the native peptide. Signaling PropertIes of Altered Peptide Llgands Antigen-specific activation of T cells results in a cascade of events involving Ca2+ influx and activation of the CD4associated protein tyrosine kinase ~56~ leading to a mobility shift of ~56~ to p6olck on gel electrophoresis (Berridge, 1993; Rudd et al., 1994). To examine further the signaling properties of the modified peptides and to provide definitive evidence of TCR ligation, we investigated the biochemical consequences of ligating the TCR with MBPp65-99 and the substituted peptides. CDeassociated ~56~ was immunoprecipitated and tested in an in

vitro autophosphoryiation assay. Whereas MBPp65-99 and p(93A), both of which induced thymidine incorporation in OblAl2, were able to induce a mobility shift in psSM to p6tY, p(9OA) and p(91A) did not (Figure 2A). Likewise, MBPp65-99 induced a strong Ca2+ influx in contrast with p(9OA) (Figure 28). T cells can bs activated by a combination of ionomycin and phorbol myrtstate acetate (PMA), which induce a Ca2+ flux and direct protein kinase C acttvation (resulting in ~56~ activation), respectively. As p(9OA) signaled the T cell without a measurable Caa flux, it was of interest to examine 13H]thymidine incorporation and cytokine secretion in the presence of either ionomycin or PMA with antigen. Stimulation of OblA12 with p(9OA) in the presence of ionomycin induced significant amounts

QlA QOA QOD QOK 93A m-99 no Ag PdEPULSE

ALTERED

0.1

1:o

PEPTfDE

lb UGAND

IQ0

’

(&ml]

Figure I. MBP 85-99 Peptides Substituted at Position QO Act as TCR Antagonists APCs prepulsed for 2 hr with limiting concentrations of native pepttde (2ugIml)followed byasecondpulseofantigenwtth theafteredpeptlde ligands at 0.1, 1, 10, and 100 pg/ml for 2 hr were added to the T cell clone CblAl2 (5 x l(r APCs and 2 x 10 T cells) and cocultured for 3 days as described in Experimental Procedures A representattve of three experiments is shown. Standard error bars indicate the varlation within the triplicate wellsof each condition. The native MBPp85-QQ and MBPpM%QQ(Q3A), which were agonist peptides (open symbols), were used as positive controls. Closed symbols denote peptides that did not induce primary proliferation. Note that MBPp85-QQ(QlA) is effects.

;;~ed

Peptide

Ligand

Modulated

Cytokine

Secretion

Figure 2. Signaling Events Associated with Altered Peptide Ligand Stimulation (A) MBPp9599 and p(9SA) but not p(9OA) and p(91A) trigger phosphorylattn of pFW in OblA12.OblA12 wasstimulated with peptide pulsed APCs for 1 hr; CD4 immunopracipltated followed by in vitro kinase assay as described in Experimental Procedures. (B) MEPp99-99 but not p(9OA) induces a Ca2+ flux in CblA12. Addition of ionomycin (4 ug/ ml) was used as a positive control and EGTA (5 &ml) as negative control. (C)Addition of ionomycin but not PMA top(9OA) and p(9OK) reconstitutes IL4 secretion of OblA12. T cells(2 x 109 and APCs (109 were cocultured in triplicate wells in medium supplemented with ionomycin at 109 nglml or PMA at 10 rig/ml. A representative of three experiments is shown.

p60 Ick+ ~56 ‘=k-)

I

I

0

mo

400

I

0

I

200

Seconds

I

PMA

of IL4 (Figure 2C) but not IL-10 or IFNy secretion or PH]thymidine incorporation. These findings suggest that the p@OA)/DRBl l 1501 complex provided a partial TCR signal. T Cell Stimulation with Altered Peptlde Llgands Speclfically Induces TGF-fll mRNA Direct measurement of cytokine mRNA levels following stimulation with altered peptide ligands substituted at the TCR contact residues were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Stimula-

lonomycin

tion of OblA12 with MBPp65-990r p(93A) induces mRNA expression of 11-2, IL4, IL-lo, and IFNy but not TGF-31 as shown for IL4 and TGF-61 in Figure 3. The altered peptide ligands that failed to induce thymidine incorporation also failed to induce IL-2,114, IL-lo, and IFNy mRNA synthesis at different timepoints after T cell stimulation (data not shown). However, TGF-91 mRNA was induced after stimulation of OblA12 with MBPp66-99(9OA) (Figure 3A). A second human MBPp66-99reactive T cell clone, Ob3D1, from the same patient with a different peptide fine specificity was examined. This clone also has a ThO

Immunity 376

MBP (85-99) I I

A.

~365 bp

ObiA12.8 ~336 bp

B.

Ob3D 1.7

~336 bp

Figure 3. Modulation of TGF)3-1 and IL-4 mRNA with Altered Peptide Ligand Stimulation Expression of TGF-f.tl and IL-4 mRNA in human MSP 85-W-reactive T cell clones OblA12 (A) and ObSDl (6) following stimulation with MSPpEb-99, p(goA) and p(g3A). Total RNA was extracted after 4 hr, reverse transcribed into cDNA, and assayed by semiquantltative PCR for expression of cytokine mRNA. These findings were reproduced in four independent experiments for OblA12 and twice for Ob3Dl.

cytokine profile and proliferates to MBPp88-99 and (gOA), while substitutions at position 93 result in loss of reactivity, indicating that the lysine at this position is a critical TCR contact residue. Stimulation with MBPp85-99(93A) but not MBPp85-99 or (SOA) induced significant mRNA expression of TGF-81 (Figure 38). Switch in Cytokine Secretion after T Cell Stimulation with Altered Peptide Ugands The secretion of cytokines into the culture supernatant after stimulation of T cell clone OblA12 with altered peptides was measured by enzyme-linked immunosorbent assay (ELISA) (Figure 4). In accordance with the findings of mRNA expression, 11-2, IL+, 11-10, and IFNy were secreted after stimulation with the wild-type peptide. Secretion of these cytokines correlated with T cell proliferation and was abolished when peptides with substitutions at TCR contact points were used for stimulation. Reduced cytokine secretion after stimulation with MBPp88-99(89K) and p(92A) corresponds to the reduced MHC binding of these peptides as shown in Table 1. Only MBPp88-99(99A) and (9OD) induced TGF-81 secretion, corresponding to the induction of mRNA synthesis. Hence, these MBP analog peptides switched cytokine secretion patterns by selectively inducing mRNA synthesis and protein secretion of TGF-81 in the absence of thymidine incorporation or secretion of IL-2,114 IL-lo, or IFNy. TGF-81 secretion could not be detected by ELISA in clone Ob3Dl in spite of specific induction of TGF-81 mRNA synthesis, which might be due to either posttranscriptional regulation or autocrine usage of the secreted TGF-81 by this T cell clone. It was possible that the induction of TGF-81 secretion by the altered peptide ligand was due to an attenuated T cell activation. The consequences of lowering the native peptide concentration for the induction of TGF-81 synthe-

sis was examined. OblA12 was stimulated with either the native MBPp88-99 peptide or (99A) peptide at lower concentrations and the secretion of IL-t, IFNy, and TGF-81 measured. With lowered peptide concentrations, T cell activation was decreased as measured by IL4 or IFNr secretion (Figure 5) or PH]thymidine incorporation (data not shown) without the induction of TGF-81 secretion. This suggests that the induction of TGF-81 secretion by altered peptide ligands was not due to attenuated Tcell activation. In addition, costimulation with ionomycin and PMA with the altered ligands did not induce TGF8 secretion. The production of active TGF-81 is not exclusively transcriptionally regulated, since a latent form of the cytokine is secreted from the cell and postsecretional activation must occur (Wahl, 1991). Thus, in these experiments, we measured the production of the active TGF-81 by ELISA. Since TGF-81 can be released from dying cells, cell viability after 3 days in culture was examined by trypan blue uptake and was found to be similar in all conditions, indicating that the altered peptide ligands did not induce cell death. Although we cannot exclude the possibility that peptide analogs are inducing T cells to signal the APCs that then secondarily secrete TGF-81 this seems unlikely, since comparable results were obtained using DRBl l 1891transfected L cells as APCs (data not shown).

Discussion In this report, we show that the stimulation of human autoreactive T cell clones by altered peptide ligand-MHC complexes induces a switch in the cytokine pattern from a ThO to a TGF-81 secreting phenotype. This demonstrates that the antigen itself plays an important role in the regulation of T cell function even after T cell differentiation. Recent studies have shown that the stimulation of T cells with peptide ligands substituted at so-called primary TCR contact residues fails to elicit any measurable response, while peptides substituted at so-called secondary TCR contact residues can function as partial agonists (Evavold et al., 1993b). Phe-91 appears to represent the primary TCR contact residue for the clones OblA12 and Ob3D1, since peptides with substitutions at this position do not induce any response. Indeed, none of the previously tested DRBl’1501-restricted MBPp88-99-reactive T cell clones tolerate a substitution at this position (Wucherpfennig et al., 1994). Histidine at position 90 for clone OblA12 and lysine at position 93 for clone Ob3Dl appear to serve as secondary TCR contact residues. These results are consistent with the paradigm of partial agonist peptides having alterationsof secondary TCR contact residues, whereas no response or only weak TCR antagonism was observed with peptides modified at the primary TCR contact point. Altered peptide ligand signaling through the TCR may be due to differential allosteric changes in the TCRaf3 chains resulting in selective triggering of secondary messengers (Rojo et al., 1989). The molecular mechanism involved in the switch of cytokine secretion after stimulation of the T cell clones with altered peptide ligands re-

$I&

Peptide

Ligand Modulated

Cytokine

Secretion

IL-4

y-IFN

IL-1 0

‘-F-l

0 .02 .M .06

0 025 0.5 075

c

L,

0 2.5 5.0 7.5

0 0.5 1.0 1.5

pg/ml

secreted

cytokine

,

x 10-J

mains unclear. Weshowed here that MHC-altered peptide complexes do not activate early intracellular signaling events of antigen-dependent T cell activation such as Ca% influx or ~56~ activation. However, these altered peptides efficiently inhibit antigen-dependent T cell proliferation and can induce IL-4 but not TGFP secretion in the presence of ionomycin. Additionally, induction of TGF$l secretion by the altered peptide ligand was not simply due to an attenuated T cell activation. Thus, our findings are compatible with a model in which the altered peptide engages the TCR with the same affinity as native peptide but instead triggers a different set of signals leading to alterations in the final cytokine secretion pattern. A change in cytokine secretion pattern from a ThO type clone to a TGFf3-1 or IL-4 secreting clone may have important physiologic consequences. TGF-Pl has a broad spectrum of cellular targets and affects many functions in nearly all cells. It is a potent regulator of the immune system, generally functioning as an immunosuppressant, inhibiting the proliferation of T and B lymphocytes at femtomolar concentrations (Kehrl et al., 1966a, 1966b). A prominent role for TGF-pl in the normal homeostasis of the immune system is suggested by the excessive inflammatory response and multifocal inflammatory cell infiltration seen in transgenic TGF-Pl knockout mice (Kulkarni et al., 1993; Shull et al., 1992). Moreover, in the animal model EAE, TGF-Bl administration to mice has been shown to protect and ameliorate disease (Racke et al., 1991). Oral administration of low doses of MBP resulting in suppression of EAE is associated with the generation of T cells, which secrete TGF-fil after antigen-specific stimulation (Miller et al., 1992, 1993). Similarly, IL-4 and TGF-bl have both been associated with recovery from

2500

..ntl~

2000 -g 1500 ‘0 = 1000 500 0.

0

1.5

15

150

0

1.5

15

PEPTIDE CONCENTRATION

150 bg/mll

!b

1

0 0.5 1.0 1.5 2.0

Figure 4. Cyiokine Secretion of TGF+l, 11-2, 11-4, 11-10, and IFNy of T Cell Clone OblA12 Following Stimulation with MBPpB54 and Altered Peptide Ligands Tcells(lO”)fromOblA12werestimulatedwith 4 x 1Q pepMe-pulsed APCs. Supematants for the ELlBAs were taken after 40 hr for 11-2, IL-l, IL-lo, and IFNy and for TGF+l after 72 hr from the stalt of experiment. Assays were performed in duplicate; experiments shown are representative of four independent experiments (standard error bars).

EAE (Khoury et al., 1992), and selective secretion of IL-4 by altered peptide ligands is therefore likely to be important in the regulation of immune responses to self-antigen. Natural modifications of MBP amino acids such as the arginine derivative, citrulline, or phosphorylation of arginine and hi&dine, which occur as posttranslational modifications or during the breakdown and metabolism of MBP (Smith et al., 1976; Wold, 1961), might be expected to regulate and limit autoimmune responses to self-proteins by the mechanism shown here. Recent studies implicate a role of altered peptide ligands in T cell selection in the thymus (Ashton-Rickardt et al., 1994; Hogquist et al., 1994) and naturally occurring variants of virus epitopes that act as T cell antagonists in vivo can be isolated from humans (Bertoletti et al., 1994; Klenerman et al., 1994). The obsewations reported here have obvious therapeutic implications for antigen-specific modifications of immune responses. Adoptive transfer of MBP-reactive T cell clones from orally tolerized mice using the same TCR as encephalitogenic T cell clones but secreting TGF+31 has been shown to inhibit experimental autoimmune encephalomyelitis (Chen et al., 1994). Altered peptide ligands can effectively modify the course of EAE, as recently shown using peptides derived from proteolipid protein peptide 139-151 and substituted at the primary TCR contact points (Kuchroo et al., 1994). These modified peptides protect mice from EAE and induce protective T cells that cross-react with the native peptide (L. 8. Nicholson and V. K. Kuchroo, personal communication). Thus, theadministration of MBP analog peptides may similarly allow the in vivo generation of TGF-pl -secreting autoreactive Tcells capable of migrating to the central nervous system and suppressing local inflammatory responses.

Figure 5. Cytokine Secretionof 11-4, IFNy, and TGF-Bl of T Cell Clone OblA12 Following Stimulation with MBPpS5-gg and Altered Pep tide Ligand MBPp&%gg@OA) at Merent Concentrations T cells (lg from Obl Al2 were stimulated with diirent concentrations of peptide-pulsed APCs. Supematants for the ELlSAs weretaken after 40 hr for IL-4 and IFNy and after 72 hr from the start of the experiment for TGF-Bl. Assays were performed in duplicate, and experiments shown are representative of two in-

- .-

--

less than 10% and are not shown).

Immunity 370

Experlmentel

Procedures

T Cell Clones T cell clones OblA12 and Ob3Dl were generated from peripheral blood mononuclear cells of a multiple sclerosis patient by limiting dilution cloning as described previously (Cta et al., 1990). The clones were maintained in fetal calf serum, 10% DMSC in liquid nitrogen. Aliquots of frozen T cell clones were thawed, washed in RPM1 1640, 213% fetal calf serum, and restimulated with 1 pg/ml PHA.P and allogenit PBMCs as feeder cells in complete medium (RPM1 1640 supplemented with 10% pooled human A/B serum, 2 mM Lglutamine, 10 mM HEPES, and 100 U/l 00 pg psr ml psnicillin/streptomycin). After 3 days, IL-2 (human T cellStim, Becton-Dickinson, Mountain View, California) was added to a final concentration of 5%. The medium was changed every 3 to 4 days. Cells were expanded for 8-10 days: 16 hr before the experiments, cells were put into IL-2-free medium. Bfndlng of MBP PePttdes to MHC MBPp85-99 (ENPWHFFKNIVTPR) and altered peptides were synthesized in the Biopolymer Laboratory, Harvard Medical School, by automated solid-phase methods using FMCC-protected amino acid precursors and pu rifted by revere phase high pressure liquid chromatography. Peptides were greater than 98% pure on the basis of high pressure liquid chromatography analysis. Binding of MBP peptides to purtfted DRB1’1501 molecules was determined as previously described (Wucherpfennig et al., 1994). In brief, purified DRBl’l501 molecules (l-5 uM) were incubated for 48 hr with 100 nM ‘PI-radiolabeled peptides and various doses of unlabeled MBP analog peptides in the presence of a protease inhibitor cocktatl. Relative capacity of MBP peptides to inhibit the binding of MHC molecules and radiolab&d ligands, expressed asaconcentration of MBP peptide(micromolar) yielding half-maximat inhibition, is the measure of the affinity of MBP peptides for MHC isotypes. T till Prollkratlon Assays For all T cell stimulation assays, the homoxygous B cell line 9010 (DR2/DD6) was used as APCs. APCs were pulsed wtth psptide at I-100 @ml for 2 hr, washed and irradiated with 5000 rads. T cells (5 x IO’) and APCs (2 x IO’) were then cocultured in triplicate for 3 days in 200 pl complete medium at 37OC. During the last 18 hr of culture, 1 nCi of PHphymidine was added to each well and T cell proliferation measured as PHjthymidine incorporation. Three pluses represent proliferation (stimulation index > 3) at 1 ug/ml. two pluses represent proliferation at 10 pglml and one plus represents proliferation only at 100 rg/ml of peptide. TCR Antagonlst Amy Live APCs (B cell line 9010 expressing, DRBI ‘1501) were prepulsed for 2 hr with a suboptimal dose of MBPp65-99 peptide (2 u@ml) determined to provide a less than maximal T cell stimulation. Pulsed APCs were irradiated and washed, followed by a second pulse of antigen with the altered peptide ligands at 0.1, 1, 10, and 100 pg!ml for 2 hr. After another wash, the APCs were plated in 9&well microtiter plates and cultured with the T cell clone OblA12 (5 x IO4 APCs and 2 x l(rTcellsperwell in tripkcates)andcocultursdfor3days. Proliferation was measured as PH]thymidine incorporation after an 18 hr pulse. We used the native MBPp65-99 and MBPp85-99(93A), which were agonist peptides, as a positive control. Phoophoryletlon of ~58’ lmmunoprecipitation of CD4 and in vitro kinase assay were performed as previously described (Hollsberg et al., 1994; Rudd et al., IWS). In brief, OblA12 was stimulated with peptidepulsed APCs for 1 hr. washed in phosphatebuffered saline, and tysed in a buffer (pH 8.3) containing 1% Triton X-100, 20 mM Tris, 150 mM NaCI, and 1 mM phenylmethylsulfonyt fluoride. Lysateswere p&eared with Staphylococcus aureus cells and incubated with OKT4-coated protein A Sepharose beads for 1 hr. The immunoprecipitate was washed twice in lysis buffer and incubated in a [ySPPpITP kinase buffer for IO min. The reaction was stopped by the addition of 2x sample buffer. Samples were boiled for 5 min and separated on a 10% SDS gel. Autoradiography was done for 30 min on Kodak XAR 5 film.

ce+ Flux T cells (1 07/ml) were loaded with indo-l acetoxymethyl ester (Molecular Probes, Junction City, Oregon) at 2 pg/ml in cuiture medium for 45 min at 37OC and then diluted I:10 in culture medium. APCs were pulsed with peptide at 100 pg’ml for 2 hr, washed in RPMI, and resuspended in culture medium. T cells (lm snd APCs (5 x lm were run on an Epics V flow cytometer to establish a baseline. Then, cells were spun for 1 min at 1200 rpm to establish cell-to-cell contact and rerun on the flow cytometer for up to 600 s. After Ca% binding, the In-1 dye exhibits changes in fluorescence emission wavelength from 480 to 410 nM. The ratio of 41WSft nm in&l fluorescence was recorded versus time and expressed in arbitrary unlts, one arbttrary unit representing approximately 200 nM increase of Ca-. Addition of ionomycin (4~g/ml)wasusedasapositiiwntrolandEGTA(5~g/ml)asnegatlve control. Meeeurement of Cytofdne mRNA B cells (9010) were pulsed with the psptides as described above. T cells(106)were thencocuftursdwlth 4 x l(rAPCs ln2OOnl medium in U-bottomed wells for 4 hr, followed by RNA extraction and cDNA synthesis. Total cellular RNA was extracted using the RNAzol B method (Teltest, Incorporated, Friendswood, Texas). RNA was coprecipttated with 10 ng of transfer RNA in isopropanol overnight. For cDNA synthesis, the pellet was resuspended in 6 ul of sterile double-distilled water, 5 pl of oligodT (Sigma) and random hexamers (Pmmega) were added, and the samples heated to 70°C for 10 min. Then, 4 pl of 5x buffer, 2 ul of 0.1 M DTT, and 1 pl each of 10 mM dNTPs, RNA&, and M-MLV reverse transcriptase (all from Promega) were added. cDNA synthesis was carried out at 37OC for 80 min and water added to a final volume of 200 cl. Sampfes were normalized to the Bgctin cDNA wncentration as determined by wmpsttttve RT-PCR using a nonhomologous DNA fragment of known concentration that has the same primer annealing sites and amplifies with the same efficiency as the internal standard (data not shown). The PCR for TGF-5 and IL-4 was semiquantitative, with 25 amplification cycles of denaturation at 94OC for 1 mitt, of annealing at 60°C for 1 min, and of extension at 72OC for 90 s, which was determined to be within the linear range of the PCRs. Concentrations in the PCRs were as previously reported (Wucherpfennig et al., 1990), except for the addition of 0.6 pCi of PP-labefed dCTP to each reaction. Forward primer for TGF+l was 5%CC CTG GAC ACC MC TAT TGC-3’; reverse primer was 5’GCT GCA ClT GCA GGA GCG CAC-3’; PCR product length is 336 bp. Primers for IL-4 were as follows: forward primer was 5’CTG CTA GCA TGT GCC GGC AAC llT GTC CAC-3’; reverse primer was 5’GAA GlTlTCCAACGTACTCTGGlTGGCTTC-3’; IengthofPCRproduct is 365 bp. Radioactive PCR products were separated on 5% potyacrylamide gel and visualized by autoradiography. Cytoldne EUBA Tcells(l@)from OblAl2wsrestimulated in5ml polypropylenstubes with 4 x 10 peptiwulsed APCs (9010 B cells) pulsed wfth peptkfe for 2 hr. Culturesupernatantsfor 11-2. IL4, IL-IO, and IFNyweretaken after 40 hr. To measure secretion of actlve TGF-51, the cells were then washed carefully three times in RPM1 to remove all serum and resuspended in serum-free medium X-Vivo 20 supplemented with 2 U rlL2/ml and 2 U rlL4/ml (Soehringer Mannheim). Supernatant for TGF-fJl ELISA was harvested after 72 hr from start of exPsrlment. The ELISA was done for active TGF-ftl and was performed as described previously (Miller et al., 1993). For the detection of IL4 and IL-IO. primary and secondary antibodii were purchased from Pharmingen and used following the suggestions of the manufacturer. IL-2 was assayed using the ELISA kit from Endogen. For IFNy, the primary antibody was obtained from Genzyme; the secondary from Bkeource International. Assays were done in duplicate. To study the effect of ionomycin and PMA on IL4 secretion T cells (2 x 106) and APCs (106) were cccuitursd in triplicate welts for 40 hr in 200 ul complete medium supplemented wtth kmomycin at 100 nglml or PMA at 10 rig/ml.

We thank S. Southwood

for excellent

technical

assistance.

This work

$tttted

Peptide

Ligand Modulated

Cytokine

Secretion

was supported in part by the National lnstttutes of Health, the National Multiple Sclerosis Society, the Boehringer Ingetheim Btipendienfonds, Stuttgart, Germany and the Qertrud Reemtsma StiftunglMax Planck Society, Munich, Germany. Received

November

9, 1994; revised

February

21, 1995.

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