Immunology and Cell Biology

(2003)

81

, 8–19

Research Article

IL-4 responsive CD4

+

T cells specific for myelin basic protein: IL-2 confers a prolonged postactivation refractory phase

M A R K D M A N N I E , D A N A J F R A S E R a n d T H O M A S J M C C O N N E L L

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina, USA

Summary

This study compared myelin basic protein-specific T cells from Lewis rats that were derived in thepresence of either rat IL-4 or IL-2. Interleukin-4 was a maintenance factor that enabled derivation of long-termT cell lines. When activated, IL-4 dependent lines were lacking in IL-2 production capacity but maintained highlevels of responsiveness to IL-2 and recognized IL-2 as a dominant growth factor. Activated IL-4 dependent T cellsrapidly reverted to a quiescent phenotype in the presence of IL-4 and rapidly regained myelin basic proteinreactivity. In contrast, activated IL-2 dependent T cells that were propagated in IL-2 had a more persistentblastogenic phenotype and a prolonged refractory phase. Interleukin-4 dependent lines that were propagated in IL-2up-regulated the capacity to produce IL-2 and also acquired prolonged postactivation refractoriness. Thus, IL-2 wasa dominant growth factor that conferred prolonged activation-dependent non-responsiveness. The coupling ofdominant growth factor activity with prolonged postactivation refractoriness may be associated with the requisiterole of IL-2 in homeostatic self-tolerance.

Key words

:

antigen presentation, experimental autoimmune encephalomyelitis, major histocompatibility complex,multiple sclerosis, T cells, T cell receptors.

Introduction

Interleukin-2 and IL-4 are type I cytokines that together withIL-7, IL-9 and IL-15 utilize the common cytokine receptorgamma chain (CD132) to transduce growth-promoting anddifferentiation signals via autocrine and paracrine pathways.

1,2

Interleukin-2 is produced by activated Th0/Th1 T cells topromote activation, proliferation and differentiation of abroad spectrum of activated T cells. Even though IL-2represents the dominant T cell growth factor

in vitro

, micedeficient in IL-2,

3–7

IL-2 receptor chains, IL-2R

α

/IL-2R

β

, orIL-2R signalling proteins Stat5a/5b,

8–11

exhibit a lympho-proliferation syndrome together with lethal CD4

+

T cell-mediated autoimmune disease. The dominant pro-mitogenicactivity of IL-2

in vitro

is apparently compensated

in vivo

byother growth factors that promote T cell expansion but lackthe normal homeostatic checks necessary for self-tolerance.

Interleukin-4 is produced by activated Th2 T cells as wellas CD4

+

NK1.1 cells, mast cells, and basophils to stimulategrowth and differentiation of Th0/Th2 T cells and B cells.

12–15

Interleukin-4 is associated with the differentiation of regula-tory T cell subsets that inhibit Th1-mediated immuneresponses.

16

For example, neuroantigen-specific CD4

+

T cellsthat differentiate under the influence of IL-12 mediateautoimmune pathogenesis,

17–20

whereas Th2 T cells that dif-ferentiate in the presence of IL-4 or IL-10 may exhibit

regulatory activity that antagonizes pathogenesis of disease.

21–23

Furthermore, administration of Th2 cytokines IL-4 or IL-10or generation of Th2 T cells is associated with the inhibitionof experimental autoimmune encephalomyelitis (EAE).

24–27

The balance between pathogenic versus regulatory activitymay be influenced by the environment because activated Th2T cells express pathogenic activity upon adoptive transfer intoimmunodeficient hosts.

28

Mice deficient in IL-4 or IL-4receptor chain IL-4R

α

(i.e. also component of IL-13 receptor)have marked deficiencies in Th2 immunity,

29,30

but do notexhibit a lack of regulatory homeostasis or enhanced suscep-tibility to EAE.

31,32

The pro-regulatory activity of IL-4 isapparently compensated

in vivo

by other growth factors thatenable homeostatic self-tolerance.

Hence, a compelling question pertains to the followingparadox. Interleukin-2 is a dominant T cell growth factor thatis known to generate effector T cells that when deficient, isparadoxically associated with lymphoproliferation of CD4

+

effector T cells, a lack of immune homeostasis, and auto-immune disease. In contrast, IL-4 is a key differentiationfactor for regulatory Th2 T cells that when deficient, isnonetheless associated with the maintenance of homeostaticself-tolerance.

Relevant to this paradox is the observation that exposureof activated T cells to IL-2 results in unresponsiveness toantigenic re-stimulation

33,34

and an accumulation of low affin-ity IL-2 receptors.

35

Indeed, activation of T cells results in atransient period of unresponsiveness that gradually attenuatesas T cells revert to a quiescent resting phase.

36–42

Post-activation refractoriness is evident upon re-exposure of acti-vated T cells to immunogenic antigen and is manifest as adesensitized IL-2 production pathway, a consequent lack of

Correspondence: Dr Mark D Mannie, Department of Microbiologyand Immunology, Brody Medical Sciences Building, East CarolinaUniversity School of Medicine, Greenville, NC 27858-4354; Email: manniem@mail.ecu.edu

Received 26 August 2002; accepted 14 October 2002.

IL-2 prolongs postactivational refractoriness

9

proliferative activity, and a phase of heightened susceptibilityto tolerance induction.

37,43,44

This study provides evidence that myelin basic protein(MBP)-specific T cells derived in the presence of IL-2 had asubstantially prolonged postactivation refractory phasecompared to MBP-specific T cells derived in the presence ofIL-4. Hence, T cell growth factors differentially regulate theduration of the refractory phase by a mechanism that corre-lates with cytokine-specific homeostatic activity. That is,IL-2 is associated with a prolonged refractory phase that maybe required for homeostatic self-tolerance whereas IL-4 isassociated with an abbreviated refractory phase that may beassociated with sustained effector activity.

Materials and Methods

Animals and reagents

Lewis rats were bred and maintained at East Carolina UniversitySchool of Medicine. Myelin basic protein was purified from rat orguinea pig spinal cords (Rockland Immunochemicals, GilbertsvillePA). The anti-IL-4 OX81 IgG1 mAb

45

was concentrated by ultrafil-tration of B cell hybridoma supernatants through Amicon spiralwound membranes (100 kDa exclusion). Phorbol myristate acetatewas purchased from Sigma (St. Louis, MO).

Cloning and baculovirus expression of rat IL-2 and IL-4 genes

Oligonucleotides specific for the 3

′

region of the IL-2 and IL-4 genes(5

′

-GAGAAGCTTTTACTGAGTCATTGTTGAGAT-3

′

and 5

′

-GAG-AAGCTTTTAGGACATGGAAGTGCAGGACTGC-3

′

, respectively)

46–48

were used to prime the synthesis of cDNA from 1

µ

g of RNA thatwas isolated from activated GP2E5.30 T cells. First strand synthesisof cDNA was performed with SuperScript II according to manufac-turer’s recommended conditions (Life Technologies, Gaithersburg,MD). Interleukin-2 and IL-4 upstream primers (5

′

-GAGGGATC-CATGTACAGCATGCAGCTCGCA-3

′

and 5

′

-GAGGGATCCAAAT-GGGTCTCAGCCCCCACCTT-3

′

) were added to the correspondingreaction tubes and amplified with

Taq

DNA polymerase (Promega,Madison, WI) for 30 cycles (94

°

C for 1 min, 62

°

C for 2 min, 72

°

Cfor 2 min) for IL-2. An annealing temperature of 56

°

C was used forIL-4. The PCR products were cloned into pGEM-T (Promega) andsequenced. The inserts were isolated by digestion with

Bam

H I and

Hin

dIII and cloned into pBlueBac III vector (Invitrogen, Carlsbad,CA) predigested with the same restriction endonucleases. Sf9 insectcells were transfected with AcMNPV linear DNA and either IL-2 orIL-4 recombinant pBlueBac III plasmid by use of Cationic Liposomesolution according to manufacturer’s protocol (Invitrogen). Viralstock was harvested after 48 h. Recombinant IL-2 and IL-4 baculo-virus (bv) clones were isolated from plaques identified with the Bluo-gal colour selection method (Invitrogen) and placed into tissueculture plates seeded with Sf9 cells for generation of recombinantviral stock. After another round of plaque purification, PCR analysisconfirmed the presence of either IL-2 or IL-4 DNA in the baculovirusstocks.

Cell lines and culture conditions

The GP3 line was derived from guinea pig (GP) MBP-sensitizedLewis rats and selected

in vitro

with GPMBP. After an initial 9 daysin the presence of IL-4 (see Fig. 4 legend), GP3-IL2 and GP3-IL4lines were derived by subsequent propagation in IL-2 or IL-4,

respectively. RsL.IL4 and RsL.IL2 lines were derived from rat (R)MBP-sensitized Lewis rats that were activated for 3 days with RMBPand then maintained in the presence of IL-4 (RsL.IL4) or IL-2(RsL.IL2). The RsL.11 clone was derived from RsL.IL2 by limitingdilution at 0.5 cells/well. Other T cell lines were derived as previ-ously described.

49–52

The R72sL line, the R2 line and the R2.2F4clone were derived from Lewis rats sensitized with the R72-86peptide (P Q K S Q R T Q D E N P V) in CFA. The GP2 clonesGP2.E5/R1, GP2.4E5, GP2.3H3.16, and GP2.5F3 were derived fromLewis rats sensitized with GPMBP in CFA. Guinea pig MBP-specificBN-GP, BNxLe, PVGxLe, PVG-GP lines were derived from BN(Brown Norway),

F

1(BN

×

Lewis),

F

1(PVG

×

Lewis), and PVG rats,respectively. The conalbumin-specific BN-conal and 8D9 T cell lineswere derived from BN and Lewis rats, respectively. The

PVG-GP-trans

,

PVGxLe-trans

,

5F3-trans

,

R1-trans

8D9-trans

and

2F4-trans

clones were IL-2-dependent, transformed variants of the PVG-GP,PVGxLe, GP2.5F3, GP2.E5/R1, 8D9 and R2.2F4 T cells, respectively.

Assays were performed in complete Roswell Park MemorialInstitute (RPMI) medium [10% heat-inactivated fetal bovine serum(Summit, Boulder, CO), 2 mmol/L glutamine, 100

µ

g/mL streptomy-cin, 100 U/mL penicillin (Whittaker Bioproducts, Walkersville, MD),50

µ

mol/L 2-ME (Sigma)]. Interleukin-2 and IL-4 dependent T cellswere propagated in complete RPMI supplemented with recombinantrat IL-2 (0.4% v/v Sf9 supernatant) or rat IL-4 (0.5–1.0% v/v). T cells(1.25

×

10

5

/mL in complete RPMI) were activated by culture withirradiated (3000 rads) syngeneic splenocytes (SPL) (2.5

×

10

6

mL) and1

µ

mol/L GPMBP or RMBP for a duration of 3 days unless desig-nated otherwise.

In vitro proliferation and IL-2 production

Responder T cells (2.5

×

10

4

/well) were cultured with irradiated SPL(5

×

10

5

/well) with or without antigen in complete RPMI and werepulsed with 1

µ

Ci of [

3

H]thymidine [6.7 Ci/mmol, Perkin-Elmer(Boston, MA)] after 24–48 h of culture in a 3 or 4 day assay asdesignated. Supernatants (100

µ

L/well) were transferred to replicateplates to measure IL-2 bioactivity, and T cells were harvested ontofilters to measure [

3

H]thymidine incorporation by scintillation count-ing. Cells from the CTLL cells (10

4

/50

µ

L cRPMI/well) were cul-tured with 100

µ

L of supernatant for 3 days, and 10

µ

L of a MTS/PMS solution [2.9 mg/mL MTS (Promega) and 0.1 mg/mL PMS(Sigma)] were added to each well during the last 24 h of culture.Optical density was recorded at 492 nm. Guinea pig MBP-stimulatedIL-2 production was calculated as the mean OD values from experi-mental cultures minus the mean OD values from control non-stimulated cultures. Error bars portray standard deviations in figuresof proliferation or IL-2 production data.

Flow cytometric analysis

Dead cells were excluded from analysis by forward versus sidescatter profiles and by incorporation of propidium iodide. Data wereacquired with a Becton Dickinson FACScan flow cytometer and wereanalysed with CELLQuest software programs.

Results

Expression of rat IL-2 and rat IL-4

To derive IL-2 dependent and IL-4 dependent T cell lineswithout influence from other rat cytokines, rat IL-2 and IL-4genes were expressed in bv expression systems. Sf9 insectcells infected with the rat IL-2 bv secreted high levels of rat

10

MD Mannie

et al.

IL-2 into culture supernatants as detected by SDS-PAGE andby a CTLL viability assay (not shown). Supernatant dilutionsin the range of 1/10

4

–1/10

5

typically yielded half-maximalstimulation of mouse CTLL T cells. These supernatants alsopotently stimulated activated rat T cells. Insect cells frommock infected cultures or from Sf9 cells infected with ratIL-4 or wildtype bv did not exhibit stimulatory activity in theCTLL assay (not shown).

Sf9 cells infected with the rat IL-4 bv secreted rat IL-4 intothe supernatant. Interleukin-4 activity was detected in culturesof Lewis rat thymocytes that were costimulated with PMA(Fig. 1) as previously described.

24,25

At high cell densities(3

×

10

6

thymocytes/well), IL-4 bv supernatants directly stim-ulated mitogenesis by a mechanism that was promoted byPMA (Fig. 1a). At lower cell densities (for example, 1

×

10

6

/well), PMA was required to detect mitogenic activity of bvIL-4 (Fig. 1b). Indeed, PMA exhibited increasing potency asa costimulatory agent at progressively higher cell densities.Mitogenic activity of IL-4 bv supernatants was attributed torat IL-4 and not another undefined activity because mitogen-esis was neutralized by the antirat IL-4 OX81 mAb (Fig. 1a).Overall, these data document the successful expression ofbioactive rat IL-2 and IL-4 gene products.

T cell reactivity to IL-2 and IL-4

We then tested normal and transformed T cells for reactivityto IL-2 and IL-4. As expected, IL-2 stimulated a number oftransformed T cell clones that constitutively expressed activa-tion markers such as I-A and the IL-2 receptor alpha chain(Fig. 2a top). Interleukin-2 also stimulated normal activated Tcells (Fig. 2b top) but did not stimulate T cells that were in aquiescent resting phase (Fig. 2a top). These data indicatedthat, as expected, IL-2 was a selective growth factor foractivated T cells. We used a commercial source of rat IL-4 toassess IL-4 responsiveness of each line and found that theseT cells were largely unresponsive to IL-4 (Fig. 2a bottom). Ofthe 12 lines tested, only the transformed variant of theGP2.E5/R1 clone (i.e. the

R1-trans

clone) exhibited apprecia-ble reactivity to IL-4. Like

R1-trans

T cells, activatedGP2.E5/R1 T cells uniquely reacted to IL-4 (Fig. 2b, bottom).However, rat IL-4 did not stimulate rested GP2.E5/R1 T cells(Fig. 2a bottom). Hence, IL-4 responsiveness was not atypical attribute of T cell lines that were derived by propaga-tion in IL-2.

Rat IL-4 was less effective than IL-2 as a growth factor inthat the magnitude of responses stimulated by IL-4 wastypically less than 10% of those stimulated by IL-2. This isshown by the comparison of the x-axes in the top versus thebottom graphs in Fig. 2a,b. Also, IL-4 stimulated a shortproliferative burst but did not sustain proliferative responses(not shown). Hence, short 2-day proliferative assays wereused to detect the mitogenic activity of IL-4. A concentrationof 10 ng/mL IL-4 was optimal whereas higher concentrationsdid not augment proliferative activity (Fig. 2c top). Like thethymocyte assay, IL-4 activity was optimal in the presence ofanother stimulus (for example, antigen). For example, theresponse attributable to IL-4 was approximately 2.5-foldhigher in the presence of 1

µ

mol/L GPMBP (delta cpm = 19 278)than in the absence of antigen (delta cpm = 7268) (Fig. 2cbottom). These findings indicate that IL-4 behaved as a

costimulus for certain clones. Although

R1-trans

T cellsexhibited responsiveness to IL-4, these T cells did not dependupon IL-4 production for antigen-stimulated autocrine growth(Fig. 3). That is, proliferative responses of

R1-trans

T cells toeither GPMBP (Fig. 3a) or IL-2 (Fig. 3b) were not affected bythe neutralizing anti-IL-4 mAb OX81 whereas responses toIL-4 were abrogated by this mAb. The observation that OX81did not affect GPMBP-stimulated proliferation indicated thatIL-4 did not contribute to this response. These findings reveala clone that retains responsiveness to IL-4 but appears largelydependent upon IL-2 for autocrine growth.

Figure 1

Bioactivity of rat IL-4 expressed in a recombinantbaculovirus (bv) system. (a) Lewis rat thymocytes (3

×

10

6

/well)were cultured with designated dilutions of the IL-4 bv supernatantin the presence or absence of 500 nmol/L PMA with or withoutthe antirat IL-4 mAb OX81.

�

, PMA;

�

, none;

�

, PMA + OX81;

�

, OX81. (b) Designated densities (per well) of Lewis rat thymo-cytes were cultured with designated concentrations of PMA in thepresence or absence of 1% dilution of the IL-4 bv supernatant.Cultures were pulsed with [

3

H]thymidine during the last 2 days ofa 4-day assay to measure proliferation. These data are representa-tive of three experiments. IL-4:

�

, 2

×

10

6

;

�

, 1

×

10

6

; ,0.5

×

10

6

. None:

�

, 2

×

10

6

;

�

, 1

×

10

6

;

�

, 0.5

×

10

6

.

IL-2 prolongs postactivational refractoriness

11

Derivation of IL-4-dependent T cells specific for MBP

To derive MBP-reactive T cells with optimal reactivity to IL-4,we obtained lymph node cells from GPMBP-sensitized Lewisrats and activated these T cells in the presence of bv IL-4 andGPMBP. These T cells were then cultured for 7 days in IL-4and re-activated with GPMBP in the absence of an exogenousgrowth factor. These T cells were then split into two groupsand were propagated in either bv IL-2 or IL-4 to derive theGP3-IL2 and GP3-IL4 lines, respectively.

Both GP3-IL2 and GP3-IL4 T cells proliferated exten-sively in the presence of irradiated SPL and GPMBP(Fig. 4a). After passage from the activation culture, activatedGP3-IL2 T cells continued to proliferate extensively inresponse to IL-2, whereas activated GP3-IL4 T cells rapidlyreverted to a quiescent resting phase when propagated in IL-4(Fig. 4a). Hence, activated GP3-IL4 T cells did not exhibit aphase of IL-4 dependent expansion. Nonetheless, IL-4 wassufficient to maintain the viability of GP3-IL4 T cells duringlong-term culture and thereby enabled derivation of an IL-4responsive line. When re-stimulated with GPMBP in thepresence of irradiated SPL, GP3-IL2 T cells that had beenrested for 40 days produced high levels of IL-2 whereassimilarly rested GP3-IL4 T cells did not produce detectablelevels of IL-2 (Fig. 4b). Both T cell lines exhibited vigorousMBP-specific proliferative responses (Fig. 4c). As describedfor other IL-2 dependent T cell lines, GP3-IL2 T cellsexhibited a postactivation refractory phase that was apparenteven 13 days after activation (Fig. 4b). At this time-point,

refractoriness was marked by decreased antigenic responsive-ness in IL-2 production assays but not in proliferative assays(Fig. 4b,c).

In accordance with previous studies

53,54

these data indi-cated that IL-4 had a maintenance activity

in vitro

withoutexhibiting an overt or independent growth factor activity. Totest responsiveness to IL-4, GP3-IL4 T cells and control R2T cells were each cultured for 6 days in IL-4, IL-2 or nogrowth factor. The GP3-IL4 T cells exhibited cell enlarge-ment in response to IL-4 whereas R2 T cells exhibitedmarginal or no enlargement in response to IL-4 (Fig. 5a,b).Both T cell lines exhibited cell enlargement in response to IL-2.Indeed, GP3-IL4 T cells were more responsive to IL-2 thanR2 T cells. Thus, T cells derived in the presence of IL-4retained high levels of reactivity to IL-2 even when assayed11 days after the last activation.

Similar findings were noted in an independent set of MBP-specific T cell lines that were derived from RMBP-sensitizedrats and were propagated in the presence of either IL-2 (RsL-IL-2 line) or IL-4 (RsL-IL-4 line). These T cells exhibitedhighly specific, equipotent reactivity to RMBP after a single

in vitro

selection with antigen (Fig. 5c). RsL-IL-4 T cellsexhibited rapid expansion in the presence of MBP and irradi-ated SPL but like GP3-IL4 T cells rapidly reverted to aquiescent phase when cultured in IL-4 (not shown). Fullyactivated RsL-IL-4 T cells showed high levels of proliferativeresponsiveness to IL-2 but did not exhibit detectableresponses to IL-4 in a 3-day proliferative assay (Fig. 5d).Thus, derivation of two separate MBP-specific lines

Figure 2

The guinea pig (GP) 2.E5/R1 clone and the derivative R1-trans clone uniquely exhibit responsiveness to rat IL-4. Designatedclones (2.5

×

10

4

/well) were cultured with 0.4% v/v rat IL-2 baculovirus (bv) supernatant or 10 ng/mL recombinant rat IL-4 (Peprotech) or nogrowth factor. (a) The ‘trans’ designation refers to clones that constitutively had a blastogenic I-A

+

phenotype whereas the remaining cloneswere normal rested T cells.

�

, IL-2; , IL-4; �, none. (b) Likewise, R1-trans T cells or antigen-activated T cells were cultured withIL-2, IL-4 (Peprotech), or no growth factor. �, IL-2; , IL-4; �, none. (c) R1-trans T cells were cultured with or without designatedconcentrations of rat IL-4 (top) or were cultured with or without IL-4 and no antigen, 1 µmol/L guinea pig myelin basic protein (GPMBP), or5 µmol/L GPMBP. T cells were pulsed with [3H]thymidine during the last day of a 2-day proliferative assay. These data are representative ofthree experiments. CPM, counts per minute. (c) top: concentration of IL-4. , 100 ng/mL; , 32 ng/mL; , 10 ng/mL. (c) bottom: , IL-4;�, none.

12 MD Mannie et al.

(GP3-IL4 and RsL-IL-4) by propagation in rat IL-4 yieldedT cells that maintained high levels of IL-2 reactivity and thatrecognized IL-4 largely as a maintenance factor in vitro.

Figure 3 The anti-IL-4 mAb OX81 does not block antigen-stimulated proliferation of R1-trans T cells. (a) R1-trans T cellswere cultured with designated concentrations of guinea pigmyelin basic protein (GPMBP) (x-axis) in the presence or absenceof the OX81 anti-IL-4 mAb. Designated groups were also cul-tured with rat IL-4 (1% titration of baculovirus [bv] supernatant).�, IL-4; �, IL-4 + OX81; , none; �, OX81. (b) R1-transT cells were cultured with rat IL-4 (1% v/v), rat IL-2 (0.4%titration of bv supernatant), or no cytokine in the presence orabsence of the OX81 anti-IL-4 mAb. Cultures were pulsed with[3H]thymidine during the last day of a 2-day proliferative assay.CPM, counts per minute. , none; �, OX81. These data arerepresentative of three experiments.

Figure 4 Rat IL-4 maintains myelin basic protein (MBP)-specific T cells that lack capacity for antigen-induced IL-2 pro-duction. Draining lymph node cells from guinea pig myelin basicprotein (GPMBP)-sensitized Lewis rats were cultured withGPMBP and a 0.5% rat baculovirus (bv) IL-4 supernatant for2 days. Activated blast T cells were then propagated for 7 days inrat IL-4. T cells were re-activated for 3 days with irradiatedsplenocytes (SPL) and GPMBP (without exogenous growthfactor) and were then propagated in either IL-2 (GP3-IL2 line, �)or IL-4 (GP3-IL4, ) line for another 9 days. (a) T cells werethen re-activated for 2 days with GPMBP and irradiated SPL. Thecell yield is shown for GP3-IL2 and GP3-IL4 lines during re-activation and during the next three 3 passages (2, 4 and 5 daypassages) in IL-2 or IL-4, respectively. Cell yields were calculatedas the number of viable (trypan blue exclusion test) T cellsrecovered from a culture divided by the input number of T cellsfor that culture. (b) The GP3-IL2 and GP3-IL4 lines were rested inIL-2 or IL-4 for designated durations. These T cells were re-activated with irradiated SPL and designated concentrations ofGPMBP to measure IL-2 production (b) and proliferation (c).GP3-IL-2 T cells: �, IL-2 (day 40); �, IL-2 (day 13); GP3-IL-4T cells: �, IL-4 (day 40); �, IL-4 (day 13). CPM, counts perminute. These data are representative of three experiments.

IL-2 prolongs postactivational refractoriness 13

IL-2 dependent T cells exhibit a prolonged refractory phase

RsL-IL-2 T cells, like the remainder of our IL-2 dependentlines exhibited a postactivation refractory phase (Fig. 6).36–39

Activated RsL-IL-2 T cells that were ‘rested’ for 2 days inIL-2 lacked antigenic responsiveness in subsequent prolifera-tive and IL-2 production assays compared to T cells that hadbeen ‘rested’ for 18 days in IL-2. Unlike the anergic pheno-type of activated RsL-IL-2 T cells, activated RsL-IL-4 T cellsthat had been rested for 2 days in IL-4 exhibited high levels ofproliferative reactivity to RMBP. Direct comparisons of acti-vated T cell lines propagated in IL-2 versus IL-4 revealedsubstantial differences in the extent of postactivation refracto-riness (Fig. 7). Activated R2.2F4 T cells that were propagatedfor 4 days in IL-2 were ∼ 100-fold less reactive to MBP thanfully rested R2.2F4 T cells when re-stimulated in proliferativeassays. Activated RsL-IL-4 T cells also exhibited postactiva-tion refractoriness although the magnitude of refractorinesswas substantially less than that observed for R2.2F4 T cells.

In accordance, previous studies showed that postactivationrefractoriness primarily affected MBP-stimulated productionof IL-2 mRNA but did not adversely affect accumulation ofIL-4 mRNA.37 The extent of postactivation refractoriness mayreflect dependency on IL-2 as an autocrine growth factor. For

example, R2.2F4 and RsL-IL-2 T cells expressed IL-2 as adominant autocrine growth factor, and both lines exhibitedprofound postactivation refractoriness. In contrast, GP3-IL4and RsL-IL-4 T cells expressed only marginal or undetectablelevels of IL-2, (for example, see the bottom of Fig. 7). Hence,the lack of postactivation refractoriness in IL-4 dependent Tcells may reflect a relatively low dependence upon IL-2 forautocrine growth.

IL-4 dependent T cells lack defining characteristics of the Th2 subset

Even though IL-4 did not directly stimulate proliferation ofactivated GP3-IL4 and RsL-IL-4 T cells, IL-4 may augmentproliferation in the presence of other stimulatory agents suchas PMA (Fig. 1), antigen (Fig. 2), or other costimulatorymolecules such as IL-1.13 Thus, IL-4 may not be sufficient asan independent growth factor but may nonetheless augmentantigen-specific clonal expansion. However, like R1-trans Tcells (Fig. 3), the OX81 anti-IL-4 mAb did not affect MBP-stimulated proliferation of RsL-IL-4 T cells (not shown).Thus, RsL-IL-4 T cells that were derived by propagation inIL-4 did not appear to depend upon IL-4 for antigen-specific

Figure 5 Interleukin-4 causes cell enlargement but is not a sufficient stimulus for proliferation. Rested GP3-IL4 and R2 T cell lines(2.5 × 105/mL) were propagated for 6 days in the presence of no growth factor, 0.5% baculovirus (bv) rat IL-2, or 0.5% bv rat IL-4. (a,b)Histograms show forward scatter profiles of R2 and GP3-IL4 T cells after propagation with designated growth factors. (c) Draining lymphnode cells from RMBP-sensitized Lewis rats were cultured with RMBP for 4 days. Activated T cells were then cultured for 8 days in eitherIL-2 (RsL-IL-2 line) or IL-4 (RsL-IL-4 line). These T cells were then assayed for proliferative reactivity to GPMBP and RMBP in thepresence of irradiated splenocytes (SPL). RsL-IL-4: �, GPMBP; �, RMBP. RsL-IL-2: �, GPMBP; �, RMBP. (d) RsL-IL-4 T cells werecultured for 3 days with RMBP and irradiated SPL. These activated T cells were washed and were cultured with 0.5% bv IL-2 or IL-4 inthe presence or absence of the anti-IL-4 mAb OX81. Cultures were pulsed with [ 3H]thymidine during the last 2 days of a 3-day assay.CPM, counts per minute. These data are representative of three experiments.

14 MD Mannie et al.

clonal expansion and thereby lacked a defining characteristicof the Th2 subset.

To assess the ability of activated RsL.IL4 T cells toproduce IL-4, T cells were stimulated with mitogen in thepresence of brefeldin A, and intracellular cytokine stainingwas measured with FITC-conjugated antirat IFN-γ or PE-conjugated antirat IL-4 mAb (Fig. 8). Concentrations of PMAand ionomycin used in these experiments were optimal foreliciting proliferation of RsL.IL4 T cells (not shown). Acti-vated RsL.IL4 T cells accumulated intracellular IFN-γ but didnot exhibit intracellular IL-4 (Fig. 8). R72sL and RsL.11T cell lines exhibited a bimodal pattern of IFN-γ accumula-tion and did not express detectable intracellular IL-4. RestingT cells did not exhibit these intracellular cytokines. ARsL.IL4 subline that had been cultured in IL-2 for over amonth exhibited essentially the same profile as RsL.IL4T cells maintained in IL-4. These findings suggest that in vivosensitization with MBP in CFA may have driven differentia-tion RsL.IL4 T cells as non-Th2 memory T cells beforeexposure to exogenous IL-4 in vitro. Overall, RsL.IL4 T cellswere distinguished from IL-2 dependent lines by severalcriteria; including minimal exposure to IL-2 during in vitroderivation, maintenance of IL-4 responsiveness, deficient IL-2production, and reduced susceptibility to postactivationrefractoriness.

RsL.IL4 T cells had functional activities typical of MBP-specific T cell lines. When activated, these T cells possessedmild encephalitogenic activity in adoptive transfer assays.After convalescence from EAE, recipient rats exhibited partialresistance to a subsequent bout of EAE (data not shown).

IL-2 regulates the duration of postactivation refractoriness

Previous studies have shown that IL-2 primes IL-2 productionpathways in resting T cells but prolongs the postactivationrefractory phase by suppressing IL-2 pathways in activatedT cells.39 Likewise, exposure of RsL.IL4 T cells to highconcentrations of IL-2 enhanced levels of IL-2 producedduring a subsequent activation (Fig. 9). In these experiments,RsL.IL4 T cells were propagated for more than 25 days ineither low (0.01% titration of bv IL-2 supernatant) or high(1%) concentrations of IL-2 and then were stimulated withMBP in the presence of irradiated SPL. Supernatants werecollected after 1, 2, 3 and 4 days of culture to measureIL-2 bioactivity, shown in the left three panels of Fig. 9.Control R72sL T cells produced high levels of IL-2 that were

Figure 6 Activated RsL-IL-2 T cells show a profound post-activation refractory phase. RsL-IL-2 or RsL-IL-4 T cells werepropagated for 18 days or 2 days of ‘rest’ in the presence of therespective growth factor. These T cells and irradiated splenocyteswere cultured with designated concentrations of guinea pigmyelin basic protein and were pulsed with [3H]thymidine duringthe last day of a 3-day assay. Supernatants were assayed for IL-2activity via the CTLL bioassay. These data are representative ofthree experiments. GPMBP, guinea pig myelin basic protein; �,RsL-IL-2 day 18; �, RsL-IL-2 day 2; �, RsL-IL-4 day 2.

Figure 7 The postactivation refractory phase of RsL-IL-2 Tcells is more profound than that of RsL-IL-4 T cells. RsL-IL-2 orRsL-IL-4 T cells were propagated in the respective growth factorfor more than 1 month (rested) or for 4 days since the lastantigenic activation. These T cells and irradiated splenocytes werecultured with designated concentrations of guinea pig myelinbasic protein and were pulsed with [3H]thymidine during the lastday of a 3-day assay. Supernatants were assayed for IL-2 activityvia the CTLL bioassay. �, R2.2F4 (rested); �, R2.2F4 (day 4);�, RsL-IL-4 (rested); �, RsL-IL-4 (day 4). These data are repre-sentative of three experiments.

IL-2 prolongs postactivational refractoriness 15

detectable through 3 days of culture. RsL.IL4 T cells previ-ously cultured in high concentrations of IL-2 showedintermediate levels of IL-2 production that were detectable

through 2 days of culture. RsL.IL4 previously cultured in lowconcentrations of IL-2 produced marginal levels of IL-2 thatwere detectable only after the first day of culture. Despite

Figure 8 Activated RsL.IL4 T cellsexpress intracellular IFN-γ but donot express detectable intracellularIL-4. RsL.IL4 T cells, a subline ofRsL.IL4 T cells that were propa-gated in IL-2 for over 1 month,R72sL T cells, or RsL.11 T cellswere cultured for 5 h in the pres-ence of brefeldin A in the presenceor absence of 1 µmol/L PMA and2 µmol/L ionomycin. Cells werewashed in cold HBSS, were fixedin 1% paraformaldehyde, andwere permeabilized in 0.1% sapo-nin. Cells were stained with FITC-conjugated mouse antirat IFN-γ,DB-1 (BD Biosciences, San Diego,CA) mAb or PE-conjugatedmouse antirat OX81 mAb inpermeabilization buffer and thenwere thoroughly washed in perme-abilization buffer. Histogramsshow intracellular staining of IFN-γ(left-most two columns) or IL-4(right column). These data are rep-resentative of five experiments.

Figure 9 Propagation of RsL.IL4T cells in IL-2 partially enables theIL-2 production pathway. RsL.IL4T cells were propagated for> 25 days in either 0.01% IL-2(low IL-2) or 1% IL-2 (high IL-2),and control R72sL T cells weremaintained in 1% IL-2. T cells(25 000/well) and irradiated Lewissplenocytes (SPL) (500 000/well)were cultured with designated con-centrations of guinea pig myelinbasic protein (GPMBP). Cultureswere pulsed with [3H]thymidine at12 h and were harvested at 24 h(�, day 1) or were pulsed 24 hbefore harvesting the respectivecultures at 48, 72 or 96 h (�, day2; �, day 3; �, day 4, respec-tively). Interleukin-2 productionwas measured by the CTLL bio-assay (left three panels), and pro-liferation was measured by[3H]thymidine incorporation (rightthree panels). These data are rep-resentative of three experiments.

16 MD Mannie et al.

profound differences in IL-2 production, the three lines exhib-ited similar antigen-specific proliferation responses. This isshown in the right three panels of Fig. 9. A positive correla-tion was noted between levels of IL-2 production during days1–3 and the magnitude of proliferation on day 4. Overall,these data provide evidence that the lack of IL-2 productioncapacity by RsL.IL4 T cells was related, at least in part, to alack of IL-2 exposure during derivation and maintenance ofthe line.

Because activated RsL.IL4 T cells produced low or mar-ginal levels of IL-2, the accelerated recovery of antigenicreactivity during the postactivation phase may be due in partto reduced IL-2 exposure and a more rapid reversion to aresting quiescent phase. In accordance, activated RsL.IL4T cells that were exposed to high concentrations of IL-2exhibited a substantially prolonged phase of postactivationrefractoriness compared to control RsL.IL4 T cells cultured inlow concentrations of IL-2 (Fig. 10). In these experiments,RsL.IL4 T cells or control R72sL T cells were activated withMBP and irradiated SPL for 3 days and then were propagatedfor 3, 7 or 25 days in either low or high levels of IL-2.RsL.IL4 T cells cultured for 7 days in low concentrations ofIL-2 had equivalent antigenic reactivity as T cells rested for25 days whereas RsL.IL4 T cells cultured for 7 days in highconcentrations of IL-2 had markedly reduced reactivity(Fig. 10a). Like GP3-IL2 T cells (Fig. 4), RsL-IL-2 T cells(Fig. 6), and other IL-2 dependent T cell lines,36–39 the R72sLline exhibited profound postactivation refractoriness (Fig. 10b),and IL-2 substantially depressed recovery of antigenic reac-tivity (Fig. 10c). Overall, these findings indicate that the levelof IL-2 signalling in activated T cells quantitatively regulatesthe extent and duration of postactivation refractoriness.

Discussion

This study revealed that a subset of MBP-specific T cellsretained reactivity to IL-4 and could be maintained in vitro asa continuous antigen-specific line (e.g. RsL.IL4 T cells) in thepresence of IL-4 even though these T cells did not exhibitovert attributes of Th2 T cells. These IL-4 dependent T cellsexhibited an abbreviated postactivation refractory phasecompared to IL-2 dependent T cells. Interleukin-4 dependentT cells were highly reactive to IL-2, but these T cellsproduced only marginal levels of IL-2 during activation.These T cells were maintained by exogenous IL-4 and there-fore had minimal in vitro exposure to IL-2; however, whencultured for several weeks in exogenous IL-2, RsL.IL4 T cellsup-regulated the capacity for IL-2 production and showedsubstantially elevated IL-2 production during subsequentactivations. Interleukin-2 had the opposite effect on activatedT cells. Exposure of activated RsL.IL4 T cells to highexogenous concentrations of IL-2 promoted profound post-activation refractoriness. These findings support a modelwhereby many memory/effector T cells may primarily dependupon growth factors such as IL-4, IL-7 or IL-15 and maymediate sustained effector activity in the presence of persist-ent antigen. Exposure to IL-2 during the course of an anti-genic response may cause differentiation/up-regulation of theIL-2 pathway and may impose a temporal refractory phasedesigned to limit ongoing effector activity against persistentantigens. In as much as IL-2 represents a dominant growth

factor that causes a profound refractory phase on a localizedT cell response, this model is consistent with the observationthat genetic deficiency in IL-2 results in uncontrolled CD4+

lymphoproliferation and a lethal autoimmunity.Interleukin-4 may enable more rapid recovery of anti-

genic responsiveness and may therefore promote a more

Figure 10 Interleukin-2 prolongs the refractory phase of IL-4dependent T cells. RsL.IL4 or R72sL T cells (1.25 × 105/mL)were activated for 3 days with irradiated splenocytes (SPL)(1.25 × 106/mL) in the presence of 1 µmol/L guinea pig myelinbasic protein (GPMBP). Activated T cells were then propagatedfor 3 (c), 7 or 25 days (a,b) in the presence of no growth factor,0.01% IL-2 (low IL-2), or 0.4% IL-2 (high IL-2). T cells werethen stimulated for 3 days with designated concentrations ofGPMBP (x-axis) and irradiated SPL. Cultures were pulsed with[3H]thymidine during the last day of a 3-day culture. RSL.IL-4T cells (a) �, day 25, low IL-2; �, day 7, low IL-2; �, day 7, highIL-2. R72sL T cells (b) �, day 25, high IL-2; �, day 7, high IL-2.R72sL T cells (c) �, day 3, none; �, day 3, low IL-2; �, day 3,high IL-2. These data are representative of four experiments.

IL-2 prolongs postactivational refractoriness 17

sustained immune response in the presence of persistentantigen. Accordingly, Th2 T cells appear better suited asmediators of immunity against persistent foreign antigensand appear less susceptible to tolerance induction.55 Con-versely, Th1 T cells depend on IL-2 as the primary autocrinegrowth factor and may be thereby limited by a postactiva-tion refractory phase that represents a phase of heightenedsusceptibility to tolerance induction.37 By prolongingpostactivation refractoriness, IL-2 may promote apoptosis,anergy, and long-term desensitization of chronically acti-vated T cells. Hence, IL-2 may promote adaptive cell-mediated immune responses followed by an aftermath suitedto the re-establishment of self-tolerance.

A unique aspect of this study was that IL-4 dependentT cells were derived in IL-4 without reliance on the use ofexogenous IL-2 for T cell growth. Most continuous Th2 celllines have been derived via an initial differentiation in Th2-biased conditions (IL-4 and anti-IL-12) followed by propaga-tion in IL-2. Hence, IL-2 may predispose many differentsubsets toward prolonged postactivation refractoriness andsusceptibility to tolerance. For example, Th2 and Th1 T cellsthat were derived and propagated in IL-2 showed essentiallyequivalent postactivation refractoriness.42 Exposure of T cellsto IL-4 induced the IL-4Rα chain and increased T cellresponsiveness to IL-4.56,57 Likewise, exposure of T cells toIL-2 induced IL-2Rα and augmented responsiveness to IL-2by a mechanism that involved an IL-2Rα-mediated auto-induction loop.58–60 Even though RsL.IL4 T cells producedmarginal levels of IL-2, these T cells expressed high levels ofIL-2Rα chain (not shown) and showed high levels of IL-2responsiveness. Because activated T cells of the Th0, Th1 andTh2 subsets have high levels of IL-2 reactivity, IL-2 may be adominant paracrine growth factor that imposes a prolongedrefractory phase and susceptibility to tolerance in a broadarray of T cell subsets.

The observation that IL-4 acts as a maintenance factorand does not independently cause overt T cell blastogenesisand proliferation may be related to the association of IL-4with an abbreviated refractory phase. Activated T cellscultured without any exogenous growth factor also exhibitedan abbreviated refractory phase due to a rapid deactivationand reversion to a small quiescent phenotype. Interleukin-4,however, maintains T cell viability while enabling T celldeactivation. In contrast, IL-2 maintains the blastogenicphase of activated T cells such that the strong stimulatoryactivity of IL-2 is coupled with relatively inefficient deacti-vation. Activated T cells maintained in IL-2 require 7–14days to revert to a resting state and require essentially thesame duration to fully recover antigenic reactivity. Imposi-tion of an IL-2 dependent refractory phase may represent acritical checkpoint limiting progression of cell-mediatedimmune responses in vivo.

Acknowledgements

This study was supported by a research grant from theNational Multiple Sclerosis Society. The authors acknowl-edge the expert technical assistance of Gregory A. White,John P. Nardella, Michelle M. LaHair and Jason R. Harris.

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