JOURNAL OF VIROLOGY, June 1994, p. 3650-3655 Vol. 68, No. 60022-538X/94/$04.00+0Copyright ©D 1994, American Society for Microbiology

Vesicular Stomatitis Virus Indiana Glycoprotein asa T-Cell-Dependent and -Independent Antigen

GIULIA FREER,* CHRISTOPH BURKHART, ILJA CIERNIK, MARTIN F. BACHMANN,HANS HENGARTNER, AND ROLF M. ZINKERNAGEL

Department of Pathology, Institute for Experimental Immunology, University of Zurich, CH-8091 Zurich, Switzerland

Received 10 January 1994/Accepted 18 February 1994

The neutralizing immunoglobulin M (IgM) response to vesicular stomatitis virus (VSV) has been shown tobe largely T-cell independent in several T-cell-deficient models of mice. By using different antigen forms ofVSV,VSV antigen doses could be graded in vivo (infectious > UV inactivated > formalin inactivated). The presentstudy reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenousinjections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice aretransfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, whichleads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. Theseresults indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nudemice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. AVSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, andlonger lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (thelatter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude miceproceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominantrole of helper T cells may be to expand and maintain the individual steps of differentiating B cells.

Vesicular stomatitis virus (VSV) is very efficient at inducingan antibody response in mice (7, 19). When injected intrave-nously (i.v.), VSV induces a short-lived neutralizing immuno-globulin M (IgM) response, known to be largely helper-T-cellindependent (3), and a virtually lifelong, strictly helper-T-cell-dependent neutralizing IgG response starting from days 6 to 8after infection (9). All neutralizing antibodies are exclusivelydirected toward the glycoprotein (G) of VSV (7), whereascytotoxic T cells can also recognize internal components suchas nucleoprotein (17). In contrast to the well-studied cytotoxiclymphocyte specificity, virus-specific helper T cells are less wellknown. Helper T cells have been particularly well defined inthe influenza virus system in vitro (6, 10, 24), but there havebeen few additional studies on their in vivo helper activity (20,21).Immune responses can be divided into T-cell dependent, the

most numerous, or T-cell independent (4). T-independentantigens usually exhibit a repetitive structure or are polyclonalB-cell activators in the absence of T-cell help (14). VSV is anexcellent antibody stimulus since it can induce such a T-independent IgM and a lifelong IgG response without being apolyclonal activator of B cells (1). The spacing of the Gmolecules on the surface of VSV has been recently proposedto play a role in inducing T-independent IgM: VSV-G is tightlypacked on the viral envelope and behaves as a polymericantigen (1). These properties are not unique to VSV but havealso been described for other viral antigens such as thenucleocapsid antigen of hepatitis B virus (13). The aim of thepresent study was to evaluate the role of helper T cells duringthe immune response against VSV infection in mice: athymicnu/nu BALB/c mice, which lack T cells, were infected withVSV serotype Indiana (VSV-IND) and then transfused with a

* Corresponding author. Mailing address: Department of Pathology,Institute for Experimental Immunology, University of Zurich, Stern-wartstrasse 2, CH-8091 Zurich, Switzerland. Phone: 41 1 255 29 89.Fax: 41 1 255 44 20.

VSV-IND-specific CD4+ T-cell line. The results show that,although the IgM response to large doses of VSV-G is mostlyT-cell independent, IgM and IgG responses are enhanced byhelper T cells with respect to titer and duration if limitingamounts of antigen are used.

MATERIALS AND METHODS

Mice. Inbred BALB/c (H-2d), C57BL/6 (H-2"), and B10.BR(H-2k) mice were obtained from the breeding colony of theInstitut fur Zuchthygiene, Tierspital Zurich, Zurich, Switzer-land. nu/nu BALB/c mice were from Bomholdgard, Copenha-gen, Denmark, and were used between 8 and 12 weeks of age.

Immunization. Animals were infected i.v. with either infec-tious, UV-inactivated, or formalin-inactivated VSV-IND at thedose specified for each experiment or with 2 x 106 PFU ofrecombinant vaccinia virus expressing the glycoprotein ofVSV-IND (vacc-IND-G).

Viruses. VSV-IND (Mudd-Summers isolate) and VSV NewJersey (VSV-NJ) (Pringle isolate) seeds were originally ob-tained from D. Kolakofsky, University of Geneva, Geneva,Switzerland; they were grown on BHK 21 cells infected with alow multiplicity of infection, plaques were allowed to developon Vero cells, and the viruses were purified as describedpreviously (12). The purified preparation contained approxi-mately 1.5 mg of protein per ml and 10i"' PFU/ml. Thegeneration of vacc-IND-G has been described elsewhere (11).vacc-IND-G was a gift of B. Moss, Laboratory of ViralDiseases, National Institutes of Health, Bethesda, Md. Re-combinant viruses were grown on BSC 40 cells at a lowmultiplicity of infection, and plaques were allowed to developon the same cells. The recombinant baculovirus expressing theglycoprotein of VSV-IND was a generous gift of D. H. L.Bishop, Natural Environment Research Council Institute ofVirology, Oxford, United Kingdom. The recombinant baculo-viruses expressing VSV nucleoprotein (VSV-N) and VSVmatrix protein (VSV-M) were a generous gift of Yan Li, John

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Robarts Research Institute, London, Ontario, Canada. Theywere derived from nuclear polyhedrosis virus and grown at280C in Spodoptera frugiperda (Sf9) cells in spinner cultures inTC-100 medium.

Inactivation of VSV. For both inactivation procedures, thevirus concentration was 5 x 108 PFU/ml in minimal essentialmedium containing 2% fetal calf serum. UV inactivation wasperformed under a 15-W UV lamp (type 7 UV; Philips) at 10cm from the source for 2 min. For formalin inactivation, 16 RIof 4% formalin was added to 1 ml of VSV (final concentration,0.0625%) and the mixture was incubated at 40C for 16 h.Before injection, inactivated virus was diluted so that 200 ,ucould be injected in every experiment (1).

Antigens. A crude preparation of recombinant protein frombaculovirus-infected Sf9 cells was obtained as follows. Cells ata density of 2 x 106 cells per ml in spinner flasks were infectedat a multiplicity of infection of 10 with recombinant baculovi-rus expressing the VSV-IND G, N, or M protein. Infected cellswere harvested after 2 days at 28°C, disrupted by sonication,and stored at -20°C (2). The presence of recombinant proteinwas confirmed by Western immunoblot, and the concentrationwas estimated by sodium dodecyl sulfate-gel analysis.Serum neutralization test. The sera obtained on days 4, 8,

and 12 after infection were each prediluted 40-fold in supple-mented minimal essential medium and then heat inactivatedfor 30 min at 56°C. Serial twofold dilutions were mixed withequal volumes of virus diluted to contain 500 PFU/ml. Themixture was incubated for 90 min at 37°C in an atmospherewith 5% CO2. Then 100 ,u of the serum-virus mixture wastransferred onto Vero cell monolayers in 96-well plates andincubated for 1 h at 37°C. The monolayers were then overlaidwith 100 [lI of Dulbecco's modified Eagle's medium-5% fetalcalf serum containing 1% methylcellulose. After incubation for24 h at 37°C, the overlay was flicked off and the monolayer wasfixed and stained with 0.5% crystal violet. The highest dilutionof serum that reduced the number of plaques by 50% wastaken as the titer. Because of the addition of an equal volumeof virus, the titer of serum was considered to be one stephigher. To determine IgG titers, we pretreated undilutedserum with an equal volume of 0.1 M 2-mercaptoethanol insaline. This treatment has been shown to eliminate only IgMbut not IgG from serum (22). Unreduced samples were takenas IgM titers only, if the corresponding reduced samples had atleast a fourfold-lower titer, i.e., when the IgG present in theunreduced sample could be neglected.ELISA. The VSV-specific enzyme-linked immunosorbent

assay (ELISA) was determined as described previously (3). Inbrief, 96-well plates (Petra Plastic) containing purified VSV-IND (1 ,ug/ml) in 0.1 M NaHCO3 (pH 9.6) were incubated at4°C overnight. The plates were blocked with 1% bovine serumalbumin (BSA) in phosphate-buffered saline for 2 h, washed,and incubated for 1 h with serial dilutions of serum samples in1% BSA. Plates were washed and incubated with horseradishperoxidase-labeled goat anti-mouse IgG (Sigma) or goat anti-mouse IgGl, IgG2a, IgG2b, or IgG3. After 1 h, the plates werewashed and developed with 5 mg of 2,2'-azino-bis(3-ethylben-zthiazoline-6-sulfonic acid) (ABTS)-20 RI of H202 in 50 ml ofNaH2PO4 (pH 4.0). Optical density was determined at 405 nm.For gamma interferon (IFN--y)-specific ELISA, the protocolrecommended by Pharmingen was followed. In this method,plates were coated with the anti-IFN--y monoclonal antibodyR4-6A2 (Pharmingen, San Diego, Calif.). Supernatants fromday 1 cultures of the SPI T-cell line, performed as forproliferation assays, were left in the washed plates overnight.In the second stage, the biotinylated anti-IFN--y monoclonalantibody XMG1.2 (Pharmingen) was added. Horseradish per-

oxidase-labeled streptavidin was added after washing, and theplates were developed as above.

Proliferation assays. To evaluate T-cell line specificity, wegrew quiescent T cells (3 x 104 cells per well) in RPMI-5%fetal calf serum-1% glutamine-5 x 10-5 M 2-mercaptoetha-nol in the presence of irradiated syngeneic splenocytes (8 x105 cells per well) and different dilutions of antigens, as shownin the figures. [3H]thymidine (1 ,uCi per well) was added after48 h, and incorporation of radioactivity was measured after anadditional- 12 h (10).

T-cell restriction assays. To evaluate the restriction of theT-cell line used, we grew 4 x 104 T cells in the presence of 5x 104 fibroblasts transfected either with I-Ad (RT2.3.3H) orwith I-Ed (RT10.3H2) (8) and 1 pRg of purified UV-inactivatedVSV-IND per ml in RPMI supplemented with antibiotics, 5%fetal calf serum, 1% glutamine, and 5 x 10-5 M 2-mercapto-ethenol. Both lines were kindly provided by Peter Erb, Institutfur Hygiene, University of Basel, Basel, Switzerland. Interleu-kin-2 (IL-2) release by the T-cell line was assessed as follows:150 ,ul of the supernatant from each well was transferred toanother plate, and 104 IL-2-dependent CILL cells were added,grown for another 24 h, and pulsed as described for theproliferation assay.

Establishment of T-cell lines. The VSV-IND-specific SPIT-cell line was established as described by Morrison et al. withminor modifications (15). In brief, BALB/c mice were immu-nized with 5 x 106 PFU of VSV-IND i.v. 8 days before beingkilled. One day before being killed, they were depleted ofCD8+ T cells by intraperitoneal injection of the anti-CD8monoclonal antibody YTS 169.4.2 (9). Their spleens were thenremoved and cultured in the presence of 0.75 ,ug of purifiedUV-inactivated VSV-IND per ml. Cells were restimulatedevery 12 days with irradiated syngeneic splenocytes and UV-inactivated VSV-IND. The medium was replaced after 6 dayswith fresh medium containing 7 to 10% rat concanavalin Asupernatant.

Adoptive transfer of T cells. To remove antigen from theT-cell cultures, we discarded the VSV-containing mediumafter 6 days of restimulation. At day 10 after restimulation, Tcells were centrifuged on a Ficoll cushion and the viable cellsat the interface were washed twice in balanced salt solution.Between 2 x 106 and 3 x 106 cells were resuspended in 200

RI of Iscove's modified Dulbecco's medium and injected intoeach mouse i.v. 1 day after immunization or infection with 106PFU of VSV.

RESULTS

A VSV-M-specific and H-2Ed-restricted CD4+ T-cell line. Toobtain a T-cell line that would exert a helper activity in vivo, wetried the following approaches. Mice differing at major histo-compatibility complex (MHC) and non-MHC genes (BALB/cand C57BV/6) were immunized with different forms of viralantigens, such as wild-type VSV-IND, recombinant or purifiedVSV-IND-G, or vacc-IND-G. These attempts were not suc-cessful except for one line obtained from a BALB/c mouseimmunized i.v. with 5 x 106 PFU of VSV-IND. On day 8 afterinfection, the spleen was removed and grown in the presenceof purified UV-inactivated VSV-IND. Culturing immunesplenocytes in the presence of noninfectious antigen has beenproven to select for MHC class II-restricted T cells, which aremostly CD4+ (14). However, to obtain a CD4+ helper line,mice infected with VSV were also depleted of CD8+ T cellsbefore immunization. The line obtained (SPI line) was CD4+CD8-, as shown by fluorescence-activated cell sorter analysis

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0

x

E0.0

60i

x

m 0.1 x

ER 0.01 x

FI] 0.001 x

40

20

0J

medium uninf N G U VSV-IND

FIG. 1. SPI is a T-cell line specific for VSV-IND-M. We cultivated3 x 104 cells of the SPI line in the presence of 8 x 105 syngeneicsplenocytes alone or with 1/10 dilutions of crude recombinant G, N(approximate 1x concentration, 50 ,ug/ml), and M (approximate 1xconcentration, 30 ,ug/ml) proteins and 1 ,ug of purified UV-inactivatedVSV-IND per ml in 96-well plates. Data are expressed as the mean ofduplicate determinations that did not differ by more than 10%.

(data not shown). Also, its proliferation was blocked by GK1.5,an anti-CD4 monoclonal antibody (data not shown).

In proliferation tests, SPI proved to be specific for theVSV-M protein (Fig. 1). SPI was VSV-IND specific since it didnot proliferate in the presence of VSV-NJ-pulsed splenocytes.This fits the finding that BALB/c helper T cells against VSVfail to cross-react between the IND and NJ serotypes (1, 18).MHC restriction of SPI was assessed by growing the line in

the presence of syngeneic and allogeneic (H-2b) splenocytesand different dilutions of VSV-IND: SPI did not recognizeVSV-IND-pulsed H-2b splenocytes (Fig. 2a). The SPI linesecreted IL-2 in the presence of I-Ed-transfected fibroblasts(but not in the presence of I-Ad-transfected fibroblasts) plusUV-inactivated VSV-IND. Thus, SPI was I-Ed restricted.

SPI helper T activity in vivo. To evaluate the helper activityin vivo, SPI cells were transfused into nu/nu BALB/c mice thathad been infected on day -1 with the equivalent of 5 x 105PFU of purified VSV, either UV inactivated or infectious. Onday 0, 2 x 106 SPI cells were transfused i.v. All mice were bledon days 4, 8, 12, and 21 to evaluate their neutralizing IgM andIgG titers against VSV. Infectious VSV elicited a high neu-tralizing response even in the absence of transfused T cells onday 4 (Fig. 3a). This response is totally accounted for by IgM,which has been proven to be predominantly T-cell independent(3). In the presence of T cells, the IgM titer induced byinfectious VSV could be only marginally enhanced on day 4but remained until day 8. Surprisingly, specific IgM was stillpresent on day 21, whereas in normal mice it has usuallydisappeared by this time (results not shown).To evaluate the response induced with limiting antigen

doses, VSV was UV inactivated; whereas infectious VSVreplicates abortively (but nevertheless produces great amountsof viral antigen in vivo), UV inactivation leads to a reduction inthe generation of antigen produced in vivo (1) and permits amore precise determination of the antigen titers. After injec-tion of a low dose of UV-inactivated VSV, the VSV-neutral-izing IgM titer was very low in nude mice, unless, as shownhere, T cells were present; transfer of 2 x 106 SPI cellsrestored the IgM response, which lasted as long as in miceinfected with live VSV (Fig. 3b). As can be seen best in Fig. 3a,SPI also induced specific neutralizing IgG in nude recipients.

a

medium unintected

1 ug/mi0 0.1

=1O 0.01

iSV-lND VSV-NJ INDH-2d H-2 d H-2b

60T

0

xEQ.a

40+

bU I.E

Eli-A¶

20+

medium uninfected VSV-IND

FIG. 2. The SPI T-cell line is VSV-IND specific and H-2Ed re-stricted. (a) We grew 3 x 104 SPI T cells in the presence of 8 x 105irradiated syngeneic splenocytes and different dilutions of purifiedVSV-IND or VSV-NJ or in the presence of splenocytes of the H-2"haplotype and 0.1 ,ug of VSV-IND per ml. (b) We grew 4 x 104 SPI Tcells in the presence of 5 x 104 fibroblasts (7) transfected with eitherthe I-Ad or the I-Ed molecule and 1 ,ug of VSV-IND per ml. One daylater, 150 ,ul of supernatant was transferred to a new plate and 104IL-2-dependent CTLL cells were added to assess IL-2 content bymeasuring [3H]thymidine uptake. Data are expressed as the mean ofduplicate determinations that did not differ by more than 10%.

To evaluate the specificity of T-cell help to B cells by SPI, weinfected nude mice i.v. with vacc-IND-G (Fig. 3c). There wasno difference between the VSV-IND-neutralizing antibodytiters in mice transfused with SPI and in those given no cells.This result indicated that SPI was not VSV-IND-G specific.Thus, SPI was specific for IND-M, and help was cognate.As an additional control, nude mice were transfused with

SPI and infected with VSV-NJ. The level of their neutralizingIgM or IgG to VSV-NJ was not enhanced (data not shown),and no neutralizing-antibody titer was detected against VSV-IND, showing that there was no detectable carryover ofantigen with T cells.

Infection with high doses of VSV induces IgG in athymicmice. In preliminary experiments, nude mice had been immu-nized with higher doses of infectious VSV-IND prior totransfer of T cells (107 to 108 PFU). In such experiments, evenmice that had not been transfused with T cells had a lowVSV-IND-neutralizing IgG titer [-log2 (titer x 40) between 3and 4] (Fig. 4), which could also be measured by ELISA onVSV-IND-coated plates (results not shown). This VSV-IND-neutralizing IgG was short-lived in the absence of T cells,

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08 4 8 12 21x b * ~uv-vsv

b

64-

2-

0

0 4 8 12 21

Time after immunization (days)

FIG. 3. Neutralizing-antibody responses in nude mice transfused

with SPI T cells, flu/flu BALBIc mice were infected i.v. on day -1 with

x i05 PFUJ of VSV-IND (a) or UV-inactivated VSV-IND (b) or 2 x

106 PFU of vacc-IND-G (C) and then transfused on day 0 with 2 x 106

T cells of the SPI line (+T; solid symnbols) or not transfused (open

symbols). The serum titer of unreduced Ig (total Ig [A, *]) or reduced

Ig (IgG [0, 0]) inhibiting VSV plaque formation by 50% was

determined on Vero cells on the days indicated. Data shown are

representative of two experiments, and each point is the mean result

from experiments with two animals. Variation between values was + 1

dilution step (not shown).

disappearing at the latest by day 30 (results not shown). The

low T-cell-independent IgG titer was also detectable in otherstrains of nude mice such as C57BL/6 and, to a much lower

extent,Pfu/nfu ICR (not shown). To assess the virus dose

dependence of IgG and IgM in nude mice, we immunized

flu/flu BALB/c mice with different doses of VSV-IND, either

UV or formalin inactivated or infectious. The doses ranged

from i04 to 108 PFU per mouse (Fig. 4). The day 4 IgM titer

is stringently dose dependent when inactivated virus is used.

Since infectious VSV replicates abortively in the mouse,

different doses of infectious VSV did not correlate with theIgM titer. In this dose-response experiment, the IgG titer wasdetermined on days 6 to 8, even though it usually peaks on day

12, because 107 to 108 PFU of infectious VSV killed all nude

mice by days 6 to 8. Low neutralizing IgG titers were detectedonly when high doses of inactivated or infectious virus wereused (Fig. 4a).

Interestingly, a VSV-neutralizing IgG could also be detectedin BALB/c nude mice after immunization with 2 x 106

vacc-IND-G (Fig. 3c). No IgG specific for vaccinia virus couldbe measured by ELISA in the same sera (results not shown),

suggesting that IgG induction in nude mice was a property of

VSV-G.

x

0

ci*-

104 105 10 6 10 7 108pfu/mouse

FIG. 4. A T-cell-independent anti-VSV-IND IgG response in nudemice injected with high doses of VSV. nulnu BALB/c mice were

injected with different doses of infectious (IND; El) UV-inactivated(UV-IND; 0), or formalin-inactivated (F-IND; *) VSV-IND. Day 8neutralizing IgG (a) and day 4 neutralizing IgM (b) titers were

determined after dilution of the sera 1:40. Data are the mean of threemice per group. Variation between values was ± 1 dilution step and isnot shown.

Thus, infection of nude mice with high doses of VSV-IND orvacc-IND-G induced low titers of VSV-IND-specific IgG in theabsence of T cells.A partially T-cell-dependent IgM response to low doses of

VSV-IND-G in normal mice. Because the results found withnude mice may not be representative of those with euthymicmice, we tested whether normal mice also had a helperT-cell-dependent component in their neutralizing IgM re-

sponse.It has been shown that helper T cells from BIO.BR and

C57BL/6 mice cross-react between VSV-IND and VSV-NJ,whereas their neutralizing antibodies are strictly serotype-specific (18). We therefore primed euthymic B1O.BR andC57BL/6 mice i.v. with 2 x 106 PFU of VSV-NJ. Four dayslater, the same mice were given the equivalent of 2 x 106 PFUof formalin-inactivated VSV-IND i.v. Formalin treatment ofVSV abolishes replication of VSV, thereby defining andlimiting the amount of antigen accumulating in vivo withoutdestroying the virus particles (1). Recent experiments hadrevealed that injection of 2 x 106 PFU of formalin-inactivatedVSV-IND i.v. fails to prime naive T cells, but leaves theneutralizing IgM mostly unaffected in C57BL/6 and B1O.BRmice (1). Mice that had been previously infected with VSV-NJtherefore exhibited cross-reacting specific T-cell help; if chal-lenged with formalin-inactivated VSV, these mice showed an

enhanced IgM titer on days 4 and 8 compared with naive mice

(Fig. 5). Results for B1O.BR were comparable (not shown).Thus, cross-reactive primed T cells enhanced the neutraliz-

ing IgM response against VSV-IND in euthymic mice under

5-a IgG4 X3

2

12I0 - b0

12.

8

6-

4 M/ IgM2n

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Time after infection (days)

FIG. 5. Normal mice express a T-cell-dependent IgM titer toVSV-IND under conditions of limiting antigen. C57BL/6 mice were

immunized i.v. with 2 x 106 PFU of VSV-NJ (a) or with balanced saltsolution (O) and challenged 4 days later with 2 x 106 PFU offormalin-inactivated VSV-IND. Their serum-neutralizing Ig titer wasmeasured on days 4 and 8 of challenge. Titers for individual mice fromone of two similar experiments are shown.

conditions where antigen was limiting and T-cell help was inexcess.

SPI T cells mediate help mainly for IgG2a. The isotypes ofthe IgG were characterized in VSV-IND-infected nude micethat were transfused with the SPI T-cell line and comparedwith normal mice by a VSV-IND-specific ELISA. Figure 6shows that the levels of IgG2a specific for VSV-IND in theSPI-transfused nu/nu animals almost reached the levels ineuthymic mice, whereas IgGl was completely missing. Theother two isotypes tested, IgG2b and IgG3, were present intransfused nude mice, but their levels were relatively de-creased.The fact that most of the IgG present in transfused animals

is IgG2a suggests that IFN--y was released upon activation ofthe SPI T cells (5). IFN--y was found in the supernatant of SPIT cells after activation in the presence of splenocytes and

id

6

1 2 3 4 5 6 7 1 2 3 4 5 6 7

-log (titer x 40)2

FIG. 6. The main IgG isotype in SPI-transfused nude mice isIgG2a. The isotype of the IgG present in the sera from nude micetransfused with the SPI cell line on day 12 after infection was

determined by standard ELISA as described in Materials and Meth-ods. The data represent the mean from three mice per group.

Variation between values was ±5% and is not shown. Symbols: *,nu/nu mice plus SPI; 0, normal mice; O, nu/nu mice. O.D. 405, opticaldensity at 405 nm.

1

LOl

CD

uninf IND NJFIG. 7. IFN--y release by SPI T cells. We grew 4 x 104 SPI T cells

in the presence of 5 x 105 irradiated splenocytes (uninf) and 1 pLg ofVSV-IND or VSV-NJ per ml. One day later, 100 ,ul of supernatant wasanalyzed for IFN--y by ELISA as described in Materials and Methods.The line represents the standard curve obtained with twofold dilutionsof recombinant IFN-y starting from 800 U/ml. Data are expressed as

the mean of duplicate determinations. O.D. 405, optical density at 405nm.

VSV-IND (Fig. 7). The release was maximal after 24 h and was

specific, since it was not found after restimulation withVSV-NJ or if no antigen was added.

DISCUSSIONThe present study with a VSV-IND-M-specific T-cell line

confirmed that the IgM response to VSV in nude mice ismostly T-cell independent but revealed some T-cell depen-dency as far as the titer and duration of the response are

concerned. The transfused T-cell line also helped to build upan IgG titer, mainly of the IgG2a isotype, in nude mice,provided that the mice were given sufficient antigen, i.e.,infectious VSV.The help provided by the SPI T-cell line is of the intermo-

lecular type, since the line is specific for the M protein and thetiter measured was against the neutralizing epitopes of theVSV, which are on the G protein (7). In this study, VSVbehaved as a classical hapten-carrier complex, as has beenfound for influenza virus (20) and postulated for VSV in a

study with VSV-G transgenic mice, which are tolerant forVSV-G at the T-cell level but can still mount a T-cell-dependent anti-VSV-G IgG titer after infection with VSV(27). The isotype of the IgG produced in mice immunized withVSV and transfused with the M-specific SPI T-cell line was

IgG2a, suggesting that IFN--y is involved. Indeed, SPI specifi-cally released high levels of IFN-,y on activation, as seen byIFN-y-specific ELISA.A neutralizing IgG titer was seen when nude mice transfused

with the M-specific line were injected with infectious VSV andnot with UV-inactivated VSV; the latter also does not inducean anti-VSV IgG titer in normal mice at this dose (1). It istempting to speculate from these results that the response toVSV, as a model antigen, occurs in gradually increasing levelsthat are regulated by the amount of antigen and not in anall-or-none fashion as the term "switch" may suggest: in a firstphase, at a very low antigen dose (5 x 105 PFU of UV-inactivated VSV), there would be a T-cell-dependent IgMsecretion. This can be seen only in nude mice (i) injected witha low level of inactivated virus and (ii) transfused with immuneT cells (Fig. 3b). In naive euthymic mice, as a result of the lowfrequency of specific helper T cells, this cannot be seenbecause more antigen is needed to prime helper T cells than is

IgG2a IgG2b

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T-CELL HELP IN VSV INFECTION 3655

provided by 5x 105 PFU of inactivated virus. In normal naivemice, such an amount is enough to prime only B cells but notT cells (1). When the antigen level reaches a critical point,IgMis induced in the absence of T-cell help but still can beincreased and prolonged if specific T cells are added bytransfusion (Fig. 3a). This is not readily seen in normal miceimmunized with the same dose, because most B cells haveswitched to IgG production by day 8 andIgM cannot bedetected by ELISA any more (data not shown).A further increase in antigen dose causes the appearance of

IgG in the serum in the presence of T cells; if the antigen doseis even greater (2x 106 to 4x 106 PFU of live VSV [Fig. 4]),IgG is produced even in the absence of helper T cells in nudemice. The exact role of T cells in isotype switching is notknown, but T cells have been assumed to be responsible for itin most antigen systems (25). Interestingly, for the VSV-Gprotein, switching to IgG occurs inefficiently but significantlyalso in athymic mice; this suggests that, at least for VSV, theswitch itself could either be partially T-cell independent (e.g.,Fig. 3c) or depend on poorly defined nude T cells (see below).The reason why vacc-IND-G induced a relatively higher T-cell-independent IgG titer in nude mice than did VSV could bethat vacc-IND-G replicates in mice, in contrast to VSV, whichusually replicates productively only in neuronal tissue of miceinfected i.v., but undergoes abortive replication in extraneuro-nal tissue (16, 26). Therefore, the data obtained here suggestthat one role of T cells may be to amplify the IgG response inan as yet undefined way. In this respect, VSV-G seems to be anunusual antigen in that it gives rise to a low but significantneutralizing IgG titer that can be seen even in the absence ofT-cell help. Because nude mice have been reported to haveatypical T cells, analysis of their possible role in switchingduring VSV infection was attempted, even though the func-tionality of such T cells has not been shown (23). Depletionof T cells in infected nude mice with anti-T-cell antibodiesor with cyclophosphamide was performed, but such experi-ments proved unsuccessful because nude mice did not survivefor long enough after the treatment and immunization withvsv.

In conclusion, this study reports on T-cell help in the VSVsystem, showing that secretion of both IgG and T-cell-inde-pendent IgM is enhanced by T cells. This may suggest that theresponse to VSV, as a model antigen, is gradual and occursstepwise in an antigen-dose-dependent fashion; in this process,T cells facilitate expansion and prolongation of the B-cellresponse.

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