Vesicular Stomatitis Virus Indiana Glycoprotein as a T-Cell-Dependent and -Independent Antigen. GIULIA FREER,* CHRISTOPH BURKHART, ILJA CIERNIK, ...
Vol. 68, No. 6
JOURNAL OF VIROLOGY, June 1994, p. 3650-3655
0022-538X/94/$04.00+0 Copyright ©D 1994, American Society for Microbiology
Vesicular Stomatitis Virus Indiana Glycoprotein as a 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 to be largely T-cell independent in several T-cell-deficient models of mice. By using different antigen forms of VSV, VSV antigen doses could be graded in vivo (infectious > UV inactivated > formalin inactivated). The present study reveals a T-cell-dependent component of the neutralizing IgM response in nude mice given intravenous injections of low doses of noninfectious UV-inactivated VSV serotype Indiana (VSV-IND) only if the mice are transfused with VSV-IND-specific helper T cells. Instead, nude mice immunized with infectious VSV, which leads to greater antigen doses in vivo, were able to mount an IgM response in the absence of T cells. These results indicate that the IgM response to low doses of VSV-IND glycoprotein (G) is T-cell dependent. Nude mice immunized with infectious VSV also made a variable but low VSV-IND-neutralizing IgG response. A VSV-IND matrix (M)-specific helper T-cell line rendered this response more consistent, much higher, and longer lasting. Thus (i) VSV-G induces a mostly T-cell-independent but partially T-cell-dependent IgM (the latter can be visualized best at low doses of antigen) and (ii) the antibody response to VSV in nude mice proceeds through steps, i.e., IgM and IgG, that are dose dependent. The results suggest that the predominant role of helper T cells may be to expand and maintain the individual steps of differentiating B cells.
VSV-IND-specific CD4+ T-cell line. The results show that, although the IgM response to large doses of VSV-G is mostly T-cell independent, IgM and IgG responses are enhanced by helper T cells with respect to titer and duration if limiting amounts of antigen are used.
Vesicular stomatitis virus (VSV) is very efficient at inducing an antibody response in mice (7, 19). When injected intravenously (i.v.), VSV induces a short-lived neutralizing immunoglobulin M (IgM) response, known to be largely helper-T-cell independent (3), and a virtually lifelong, strictly helper-T-celldependent neutralizing IgG response starting from days 6 to 8 after infection (9). All neutralizing antibodies are exclusively directed toward the glycoprotein (G) of VSV (7), whereas cytotoxic T cells can also recognize internal components such as nucleoprotein (17). In contrast to the well-studied cytotoxic lymphocyte specificity, virus-specific helper T cells are less well known. Helper T cells have been particularly well defined in the influenza virus system in vitro (6, 10, 24), but there have been few additional studies on their in vivo helper activity (20,
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 the Institut fur Zuchthygiene, Tierspital Zurich, Zurich, Switzerland. nu/nu BALB/c mice were from Bomholdgard, Copenhagen, Denmark, and were used between 8 and 12 weeks of age.
Immunization. Animals were infected i.v. with either infectious, UV-inactivated, or formalin-inactivated VSV-IND at the dose specified for each experiment or with 2 x 106 PFU of recombinant vaccinia virus expressing the glycoprotein of VSV-IND (vacc-IND-G). Viruses. VSV-IND (Mudd-Summers isolate) and VSV New Jersey (VSV-NJ) (Pringle isolate) seeds were originally obtained from D. Kolakofsky, University of Geneva, Geneva, Switzerland; they were grown on BHK 21 cells infected with a low multiplicity of infection, plaques were allowed to develop on Vero cells, and the viruses were purified as described previously (12). The purified preparation contained approximately 1.5 mg of protein per ml and 10i"' PFU/ml. The generation of vacc-IND-G has been described elsewhere (11). vacc-IND-G was a gift of B. Moss, Laboratory of Viral Diseases, National Institutes of Health, Bethesda, Md. Recombinant viruses were grown on BSC 40 cells at a low multiplicity of infection, and plaques were allowed to develop on the same cells. The recombinant baculovirus expressing the glycoprotein of VSV-IND was a generous gift of D. H. L. Bishop, Natural Environment Research Council Institute of Virology, Oxford, United Kingdom. The recombinant baculoviruses expressing VSV nucleoprotein (VSV-N) and VSV matrix protein (VSV-M) were a generous gift of Yan Li, John
21). Immune responses can be divided into T-cell dependent, the most numerous, or T-cell independent (4). T-independent antigens usually exhibit a repetitive structure or are polyclonal B-cell activators in the absence of T-cell help (14). VSV is an excellent antibody stimulus since it can induce such a Tindependent IgM and a lifelong IgG response without being a polyclonal activator of B cells (1). The spacing of the G molecules on the surface of VSV has been recently proposed to play a role in inducing T-independent IgM: VSV-G is tightly packed on the viral envelope and behaves as a polymeric antigen (1). These properties are not unique to VSV but have also been described for other viral antigens such as the nucleocapsid antigen of hepatitis B virus (13). The aim of the present study was to evaluate the role of helper T cells during the immune response against VSV infection in mice: athymic nu/nu BALB/c mice, which lack T cells, were infected with VSV serotype Indiana (VSV-IND) and then transfused with a * Corresponding author. Mailing address: Department of Pathology, Institute for Experimental Immunology, University of Zurich, Sternwartstrasse 2, CH-8091 Zurich, Switzerland. Phone: 41 1 255 29 89. Fax: 41 1 255 44 20.
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Robarts Research Institute, London, Ontario, Canada. They were derived from nuclear polyhedrosis virus and grown at 280C in Spodoptera frugiperda (Sf9) cells in spinner cultures in TC-100 medium. Inactivation of VSV. For both inactivation procedures, the virus concentration was 5 x 108 PFU/ml in minimal essential medium containing 2% fetal calf serum. UV inactivation was performed under a 15-W UV lamp (type 7 UV; Philips) at 10 cm from the source for 2 min. For formalin inactivation, 16 RI of 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 ,u could be injected in every experiment (1). Antigens. A crude preparation of recombinant protein from baculovirus-infected Sf9 cells was obtained as follows. Cells at a density of 2 x 106 cells per ml in spinner flasks were infected at a multiplicity of infection of 10 with recombinant baculovirus expressing the VSV-IND G, N, or M protein. Infected cells were harvested after 2 days at 28°C, disrupted by sonication, and stored at -20°C (2). The presence of recombinant protein was confirmed by Western immunoblot, and the concentration was 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 supplemented minimal essential medium and then heat inactivated for 30 min at 56°C. Serial twofold dilutions were mixed with equal volumes of virus diluted to contain 500 PFU/ml. The mixture was incubated for 90 min at 37°C in an atmosphere with 5% CO2. Then 100 ,u of the serum-virus mixture was transferred onto Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. The monolayers were then overlaid with 100 [lI of Dulbecco's modified Eagle's medium-5% fetal calf serum containing 1% methylcellulose. After incubation for 24 h at 37°C, the overlay was flicked off and the monolayer was fixed and stained with 0.5% crystal violet. The highest dilution of serum that reduced the number of plaques by 50% was taken as the titer. Because of the addition of an equal volume of virus, the titer of serum was considered to be one step higher. To determine IgG titers, we pretreated undiluted serum with an equal volume of 0.1 M 2-mercaptoethanol in saline. This treatment has been shown to eliminate only IgM but not IgG from serum (22). Unreduced samples were taken as IgM titers only, if the corresponding reduced samples had at least a fourfold-lower titer, i.e., when the IgG present in the unreduced sample could be neglected. ELISA. The VSV-specific enzyme-linked immunosorbent assay (ELISA) was determined as described previously (3). In brief, 96-well plates (Petra Plastic) containing purified VSVIND (1 ,ug/ml) in 0.1 M NaHCO3 (pH 9.6) were incubated at 4°C overnight. The plates were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline for 2 h, washed, and incubated for 1 h with serial dilutions of serum samples in 1% BSA. Plates were washed and incubated with horseradish peroxidase-labeled goat anti-mouse IgG (Sigma) or goat antimouse IgGl, IgG2a, IgG2b, or IgG3. After 1 h, the plates were washed and developed with 5 mg of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)-20 RI of H202 in 50 ml of NaH2PO4 (pH 4.0). Optical density was determined at 405 nm. For gamma interferon (IFN--y)-specific ELISA, the protocol recommended by Pharmingen was followed. In this method, plates were coated with the anti-IFN--y monoclonal antibody R4-6A2 (Pharmingen, San Diego, Calif.). Supernatants from day 1 cultures of the SPI T-cell line, performed as for proliferation assays, were left in the washed plates overnight. In the second stage, the biotinylated anti-IFN--y monoclonal antibody XMG1.2 (Pharmingen) was added. Horseradish per-
3651
oxidase-labeled streptavidin was added after washing, and the plates were developed as above. Proliferation assays. To evaluate T-cell line specificity, we grew quiescent T cells (3 x 104 cells per well) in RPMI-5% fetal calf serum-1% glutamine-5 x 10-5 M 2-mercaptoethanol in the presence of irradiated syngeneic splenocytes (8 x 105 cells per well) and different dilutions of antigens, as shown in the figures. [3H]thymidine (1 ,uCi per well) was added after 48 h, and incorporation of radioactivity was measured after an additional- 12 h (10). T-cell restriction assays. To evaluate the restriction of the T-cell line used, we grew 4 x 104 T cells in the presence of 5 x 104 fibroblasts transfected either with I-Ad (RT2.3.3H) or with I-Ed (RT10.3H2) (8) and 1 pRg of purified UV-inactivated VSV-IND per ml in RPMI supplemented with antibiotics, 5% fetal calf serum, 1% glutamine, and 5 x 10-5 M 2-mercaptoethenol. Both lines were kindly provided by Peter Erb, Institut fur Hygiene, University of Basel, Basel, Switzerland. Interleukin-2 (IL-2) release by the T-cell line was assessed as follows: 150 ,ul of the supernatant from each well was transferred to another plate, and 104 IL-2-dependent CILL cells were added, grown for another 24 h, and pulsed as described for the proliferation assay. Establishment of T-cell lines. The VSV-IND-specific SPI T-cell line was established as described by Morrison et al. with minor modifications (15). In brief, BALB/c mice were immunized with 5 x 106 PFU of VSV-IND i.v. 8 days before being killed. One day before being killed, they were depleted of CD8+ T cells by intraperitoneal injection of the anti-CD8 monoclonal antibody YTS 169.4.2 (9). Their spleens were then removed and cultured in the presence of 0.75 ,ug of purified UV-inactivated VSV-IND per ml. Cells were restimulated every 12 days with irradiated syngeneic splenocytes and UVinactivated VSV-IND. The medium was replaced after 6 days with fresh medium containing 7 to 10% rat concanavalin A supernatant. Adoptive transfer of T cells. To remove antigen from the T-cell cultures, we discarded the VSV-containing medium after 6 days of restimulation. At day 10 after restimulation, T cells were centrifuged on a Ficoll cushion and the viable cells at 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 into each mouse i.v. 1 day after immunization or infection with 106 PFU of VSV.
RESULTS A VSV-M-specific and H-2Ed-restricted CD4+ T-cell line. To obtain a T-cell line that would exert a helper activity in vivo, we tried the following approaches. Mice differing at major histocompatibility complex (MHC) and non-MHC genes (BALB/c and C57BV/6) were immunized with different forms of viral antigens, such as wild-type VSV-IND, recombinant or purified VSV-IND-G, or vacc-IND-G. These attempts were not successful except for one line obtained from a BALB/c mouse immunized i.v. with 5 x 106 PFU of VSV-IND. On day 8 after infection, the spleen was removed and grown in the presence of purified UV-inactivated VSV-IND. Culturing immune splenocytes in the presence of noninfectious antigen has been proven to select for MHC class II-restricted T cells, which are mostly CD4+ (14). However, to obtain a CD4+ helper line, mice infected with VSV were also depleted of CD8+ T cells before immunization. The line obtained (SPI line) was CD4+ CD8-, as shown by fluorescence-activated cell sorter analysis
3652
J. VIROL.
FREER ET AL.
60i 1
0
a
=1O
ug/mi
0.1 0.01
40 x
E
m 0.1 x ER 0.01 x
0
FI]
0.001
0 x
E0.
0
x
20
0J
medium
uninf
N
G
U
medium
unintected
H-2d
VSV-IND
FIG. 1. SPI is a T-cell line specific for VSV-IND-M. We cultivated 3 x 104 cells of the SPI line in the presence of 8 x 105 syngeneic splenocytes alone or with 1/10 dilutions of crude recombinant G, N (approximate 1x concentration, 50 ,ug/ml), and M (approximate 1x concentration, 30 ,ug/ml) proteins and 1 ,ug of purified UV-inactivated VSV-IND per ml in 96-well plates. Data are expressed as the mean of duplicate determinations that did not differ by more than 10%.
VSV-NJ H-2 d
IND H-2b
60T
b 0
40+
U I.E
Eli-A¶
x
(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 the VSV-M protein (Fig. 1). SPI was VSV-IND specific since it did not proliferate in the presence of VSV-NJ-pulsed splenocytes. This fits the finding that BALB/c helper T cells against VSV fail 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) splenocytes and different dilutions of VSV-IND: SPI did not recognize VSV-IND-pulsed H-2b splenocytes (Fig. 2a). The SPI line secreted IL-2 in the presence of I-Ed-transfected fibroblasts (but not in the presence of I-Ad-transfected fibroblasts) plus UV-inactivated VSV-IND. Thus, SPI was I-Ed restricted. SPI helper T activity in vivo. To evaluate the helper activity in vivo, SPI cells were transfused into nu/nu BALB/c mice that had been infected on day -1 with the equivalent of 5 x 105 PFU of purified VSV, either UV inactivated or infectious. On day 0, 2 x 106 SPI cells were transfused i.v. All mice were bled on days 4, 8, 12, and 21 to evaluate their neutralizing IgM and IgG titers against VSV. Infectious VSV elicited a high neutralizing response even in the absence of transfused T cells on day 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 by infectious VSV could be only marginally enhanced on day 4 but remained until day 8. Surprisingly, specific IgM was still present on day 21, whereas in normal mice it has usually disappeared by this time (results not shown). To evaluate the response induced with limiting antigen doses, VSV was UV inactivated; whereas infectious VSV replicates abortively (but nevertheless produces great amounts of viral antigen in vivo), UV inactivation leads to a reduction in the generation of antigen produced in vivo (1) and permits a more precise determination of the antigen titers. After injection of a low dose of UV-inactivated VSV, the VSV-neutralizing IgM titer was very low in nude mice, unless, as shown here, T cells were present; transfer of 2 x 106 SPI cells restored the IgM response, which lasted as long as in mice infected with live VSV (Fig. 3b). As can be seen best in Fig. 3a, SPI also induced specific neutralizing IgG in nude recipients.
iSV-lND
E Q.
a
20+
medium
uninfected
VSV-IND
FIG. 2. The SPI T-cell line is VSV-IND specific and H-2Ed restricted. (a) We grew 3 x 104 SPI T cells in the presence of 8 x 105 irradiated syngeneic splenocytes and different dilutions of purified VSV-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 T cells in the presence of 5 x 104 fibroblasts (7) transfected with either the I-Ad or the I-Ed molecule and 1 ,ug of VSV-IND per ml. One day later, 150 ,ul of supernatant was transferred to a new plate and 104 IL-2-dependent CTLL cells were added to assess IL-2 content by measuring [3H]thymidine uptake. Data are expressed as the mean of duplicate determinations that did not differ by more than 10%.
To evaluate the specificity of T-cell help to B cells by SPI, we infected nude mice i.v. with vacc-IND-G (Fig. 3c). There was no difference between the VSV-IND-neutralizing antibody titers 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 neutralizing IgM or IgG to VSV-NJ was not enhanced (data not shown), and no neutralizing-antibody titer was detected against VSVIND, showing that there was no detectable carryover of antigen with T cells. Infection with high doses of VSV induces IgG in athymic mice. In preliminary experiments, nude mice had been immunized with higher doses of infectious VSV-IND prior to transfer of T cells (107 to 108 PFU). In such experiments, even mice that had not been transfused with T cells had a low VSV-IND-neutralizing IgG titer [-log2 (titer x 40) between 3 and 4] (Fig. 4), which could also be measured by ELISA on VSV-IND-coated plates (results not shown). This VSV-INDneutralizing IgG was short-lived in the absence of T cells,
VOL. 68, 1994
T-CELL HELP IN VSV INFECIION
x 0
4
8
x08bb *
12
21
5-a 4 3 2
3653
IgG X
120
~uv-vsv
I0 - b
*-
ci
12.
8
6
6-
42-
4 2
0
n
M/ 104
0
4
8
12
21
Time after immunization (days) FIG.
Neutralizing-antibody responses in nude mice transfused
3.
with SPI T cells, x
flu/flu BALBIc mice were infected i.v. on day -1 with
i05 PFUJ of VSV-IND
106 PFU
(a) or
UV-inactivated VSV-IND (b) or 2 x
105 10 6 10 7 108 pfu/mouse
FIG. 4. A T-cell-independent anti-VSV-IND IgG response in nude mice 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 8 neutralizing IgG (a) and day 4 neutralizing IgM (b) titers were determined after dilution of the sera 1:40. Data are the mean of three mice per group. Variation between values was ± 1 dilution step and is not shown.
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, Ig
IgM
(IgG
[0,
0])
inhibiting
VSV
plaque
formation
by
or reduced 50%
was
disappearing at the latest by day 30 (results not shown). The low T-cell-independent IgG titer was also detectable in other
Thus, infection of nude mice with high doses of VSV-IND or vacc-IND-G induced low titers of VSV-IND-specific IgG in the absence of T cells. A partially T-cell-dependent IgM response to low doses of VSV-IND-G in normal mice. Because the results found with nude mice may not be representative of those with euthymic mice, we tested whether normal mice also had a helper T-cell-dependent component in their neutralizing IgM re-
strains
sponse.
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).
of nude mice such as C57BL/6 and, to a much lower
Pfu/nfu
extent,
(not
ICR
dependence of IgG flu/flu BALB/c mice
and
shown).
IgM in nude
i04
to
assess
the
virus
dose
mice, we immunized
with different doses of VSV-IND, either
UV or formalin inactivated or
from
To
infectious. The doses ranged The day 4 IgM titer
108 PFU per mouse (Fig. 4).
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 the IgM titer. In this dose-response experiment, the IgG titer was determined 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 detected only when high doses of inactivated or infectious virus were used (Fig. 4a). Interestingly, a VSV-neutralizing IgG could also be detected in BALB/c nude mice after immunization with 2 x 106 vacc-IND-G (Fig. 3c). No IgG specific for vaccinia virus could be measured by ELISA in the same sera (results not shown), suggesting that IgG induction in nude mice was a property of VSV-G.
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 serotypespecific (18). We therefore primed euthymic B1O.BR and C57BL/6 mice i.v. with 2 x 106 PFU of VSV-NJ. Four days later, the same mice were given the equivalent of 2 x 106 PFU of formalin-inactivated VSV-IND i.v. Formalin treatment of VSV abolishes replication of VSV, thereby defining and limiting the amount of antigen accumulating in vivo without destroying the virus particles (1). Recent experiments had revealed that injection of 2 x 106 PFU of formalin-inactivated VSV-IND i.v. fails to prime naive T cells, but leaves the neutralizing IgM mostly unaffected in C57BL/6 and B1O.BR mice (1). Mice that had been previously infected with VSV-NJ therefore exhibited cross-reacting specific T-cell help; if challenged 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 neutralizing IgM response against VSV-IND in euthymic mice under
3654
J. VIROL.
FREER ET AL. 12.
1
x
9-
LOl R
S'2 6-
CD
~3-
4
8
Time after infection (days) FIG. 5. Normal mice express a T-cell-dependent IgM titer to VSV-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 salt solution (O) and challenged 4 days later with 2 x 106 PFU of formalin-inactivated VSV-IND. Their serum-neutralizing Ig titer was measured on days 4 and 8 of challenge. Titers for individual mice from one of two similar experiments are shown.
uninf
IND
NJ
FIG. 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 of VSV-IND or VSV-NJ per ml. One day later, 100 ,ul of supernatant was analyzed for IFN--y by ELISA as described in Materials and Methods. The line represents the standard curve obtained with twofold dilutions of recombinant IFN-y starting from 800 U/ml. Data are expressed as the mean of duplicate determinations. O.D. 405, optical density at 405 nm.
conditions where antigen
was
limiting and T-cell help
was
in
excess.
SPI T cells mediate help mainly for IgG2a. The isotypes of the IgG were characterized in VSV-IND-infected nude mice that were transfused with the SPI T-cell line and compared with normal mice by a VSV-IND-specific ELISA. Figure 6 shows that the levels of IgG2a specific for VSV-IND in the SPI-transfused nu/nu animals almost reached the levels in euthymic mice, whereas IgGl was completely missing. The other two isotypes tested, IgG2b and IgG3, were present in transfused nude mice, but their levels were relatively decreased. The fact that most of the IgG present in transfused animals is IgG2a suggests that IFN--y was released upon activation of the SPI T cells (5). IFN--y was found in the supernatant of SPI T cells after activation in the presence of splenocytes and
IgG2a
IgG2b
I gG1
IgG-3
.4
.68 id
6
l1.2-
I4 _2
o -
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 is IgG2a. The isotype of the IgG present in the sera from nude mice transfused with the SPI cell line on day 12 after infection was determined by standard ELISA as described in Materials and Methods. 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, optical density at 405 nm.
VSV-IND (Fig. 7). The release was maximal after 24 h and was specific, since it was not found after restimulation with VSV-NJ or if no antigen was added.
DISCUSSION The present study with a VSV-IND-M-specific T-cell line confirmed that the IgM response to VSV in nude mice is mostly T-cell independent but revealed some T-cell dependency as far as the titer and duration of the response are concerned. The transfused T-cell line also helped to build up an 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 intermolecular type, since the line is specific for the M protein and the titer measured was against the neutralizing epitopes of the VSV, which are on the G protein (7). In this study, VSV behaved as a classical hapten-carrier complex, as has been found for influenza virus (20) and postulated for VSV in a study with VSV-G transgenic mice, which are tolerant for VSV-G at the T-cell level but can still mount a T-celldependent anti-VSV-G IgG titer after infection with VSV (27). The isotype of the IgG produced in mice immunized with VSV and transfused with the M-specific SPI T-cell line was IgG2a, suggesting that IFN--y is involved. Indeed, SPI specifically released high levels of IFN-,y on activation, as seen by IFN-y-specific ELISA. A neutralizing IgG titer was seen when nude mice transfused with the M-specific line were injected with infectious VSV and not with UV-inactivated VSV; the latter also does not induce an anti-VSV IgG titer in normal mice at this dose (1). It is tempting to speculate from these results that the response to VSV, as a model antigen, occurs in gradually increasing levels that are regulated by the amount of antigen and not in an all-or-none fashion as the term "switch" may suggest: in a first phase, at a very low antigen dose (5 x 105 PFU of UVinactivated VSV), there would be a T-cell-dependent IgM secretion. This can be seen only in nude mice (i) injected with a low level of inactivated virus and (ii) transfused with immune T cells (Fig. 3b). In naive euthymic mice, as a result of the low frequency of specific helper T cells, this cannot be seen because more antigen is needed to prime helper T cells than is
T-CELL HELP IN VSV INFECTION
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provided by 5x 105 PFU of inactivated virus. In normal naive mice, such an amount is enough to prime only B cells but not
T cells (1). When the antigen level reaches a critical point,IgM
is induced in the absence of T-cell help but still can be increased and prolonged if specific T cells are added by transfusion (Fig. 3a). This is not readily seen in normal mice immunized with the same dose, because most B cells have switched to IgG production by day 8 and IgM cannot be detected 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 dose is 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 nude mice. The exact role of T cells in isotype switching is not known, but T cells have been assumed to be responsible for it in most antigen systems (25). Interestingly, for the VSV-G protein, switching to IgG occurs inefficiently but significantly also in athymic mice; this suggests that, at least for VSV, the switch 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-cellindependent IgG titer in nude mice than did VSV could be that vacc-IND-G replicates in mice, in contrast to VSV, which usually replicates productively only in neuronal tissue of mice infected i.v., but undergoes abortive replication in extraneuronal tissue (16, 26). Therefore, the data obtained here suggest that one role of T cells may be to amplify the IgG response in an as yet undefined way. In this respect, VSV-G seems to be an unusual antigen in that it gives rise to a low but significant neutralizing IgG titer that can be seen even in the absence of T-cell help. Because nude mice have been reported to have atypical T cells, analysis of their possible role in switching during VSV infection was attempted, even though the functionality of such T cells has not been shown (23). Depletion of T cells in infected nude mice with anti-T-cell antibodies or with cyclophosphamide was performed, but such experiments proved unsuccessful because nude mice did not survive for long enough after the treatment and immunization with vsv. In conclusion, this study reports on T-cell help in the VSV system, showing that secretion of both IgG and T-cell-independent IgM is enhanced by T cells. This may suggest that the response to VSV, as a model antigen, is gradual and occurs stepwise in an antigen-dose-dependent fashion; in this process, T cells facilitate expansion and prolongation of the B-cell response. REFERENCES 1. Bachmann, M. F., T. M. Kundig, C. Kalberer, H. Hengartner, and R. M. Zinkernagel. 1993. Formalin inactivation of vesicular stomatitis virus impairs T-cell- but not T-help-independent B-cell responses. J. Virol. 67:3917-3922. 2. Bailey, M. J., D. A. McLeod, C. Y. Kang, and D. H. L. Bishop. 1989. Glycosylation is not required for the fusion activity of the G protein of vesicular stomatitis virus in insect cells. Virology
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