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The Journal of Immunology, 1999, 162: 2867-2874.
Copyright © 1999 by The American Association of Immunologists

Qualitative and Quantitative Requirements for CD4+ T Cell-Mediated Antiviral Protection1

Kevin J. Maloy2,3,*, Christoph Burkhart2,*, Giulia Freer*, Thomas Rülicke*, Hanspeter Pircher*, Dwight H. Kono{dagger}, Argyrios N. Theofilopoulos{dagger}, Burkhard Ludewig*, Urs Hoffmann-Rohrer*, Rolf M. Zinkernagel* and Hans Hengartner*

* Department of Pathology, Institute of Experimental Immunology, Zurich, Switzerland; and {dagger} The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ Th cells deliver the cognate and cytokine signals that promote the production of protective virus-neutralizing IgG by specific B cells and are also able to mediate direct antiviral effector functions. To quantitatively and qualitatively analyze the antiviral functions of CD4+ Th cells, we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of vesicular stomatitis virus (VSV). The elevated precursor frequency of naive VSV-specific Th cells in tg7 mice led to a markedly accelerated and enhanced class switching to virus-neutralizing IgG after immunization with inactivated VSV. Furthermore, in contrast to nontransgenic controls, tg7 mice rapidly cleared a recombinant vaccinia virus expressing the VSV-G (Vacc-IND-G) from peripheral organs. By adoptive transfer of naive tg7 CD4+ T cells into T cell-deficient recipients, we found that 105 transferred CD4+ T cells were sufficient to induce isotype switching after challenge with a suboptimal dose of inactivated VSV. In contrast, naive transgenic CD4+ T cells were unable to adoptively confer protection against peripheral infection with Vacc-IND-G. However, tg7 CD4+ T cells that had been primed in vitro with VSV-G peptide were able to adoptively transfer protection against Vacc-IND-G. These results demonstrate that the antiviral properties of CD4+ T cells are governed by the differentiation status of the CD4+ T cell and by the type of effector response required for virus elimination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The variety of strategies that viruses use to infect and replicate in mammalian hosts is paralleled by the diversity of immune mechanisms that these hosts have evolved to protect against infection. The particular effector mechanisms required depend primarily on the nature of the virus and the route of infection 1 . In general, for noncytopathic viruses such as lymphocytic choriomeningitis virus (LCMV),4 CD8+ CTL are the major effectors in the eradication of primary infection, while neutralizing Abs may assist in protecting against reinfection 2, 3 . In the case of cytopathic viruses, T cell-dependent cytokines together with neutralizing Abs are usually essential for viral eradication and protection against reinfection 1, 4 . CD4+ T cells play a crucial role in many of these antiviral responses. As well as their direct antiviral effects via the production of cytokines such as IFN-{gamma} and TNF-{alpha} 5, 6, 7, 8, 9, 10 , they provide the cognate signals that induce neutralizing IgG responses 11 , and they also enhance the magnitude and longevity of antiviral CTL responses 12, 13, 14, 15, 16 .

A number of experimental models of viral infection have emphasized the important role of CD4+ T cells. For example, mice deficient in CD8+ CTL can clear influenza A virus, and adoptively transferred CD4+ T cell clones have been shown to be able to promote recovery from lethal infection 17, 18, 19 . In addition, CD8+ CTL-deficient mice can also effectively control vaccinia virus infection 20, 21 . Furthermore, poliovirus-specific CD4+ T cell clones are capable of adoptively transferring protection against lethal infection by stimulating neutralizing Ab production 22 . Thus CD4+ T cell responses may play a key role in the eradication of viruses by both humoral and cell-mediated mechanisms. Currently, however, little is known about the quantitative and qualitative characteristics of these anti-viral CD4+ T cell responses. Such information would have important implications for adoptive immunotherapy of viral diseases in immunocompromised hosts, where it has already been demonstrated that anti-viral CD8+ CTL responses were more efficiently reconstituted when virus-specific CD4+ Th cells were cotransferred 23, 24, 25 and adoptively transferred CD4+ T cells also contributed to the clearance of persistent LCMV infection from carrier mice 26 . It may also have implications for the maintenance of the protective T cell repertoire to viral infections, since it has recently been shown that the memory T cell pool specific for a given virus is altered by subsequent viral infections 27 .

To address these issues we generated transgenic mice (tg7) expressing an MHC class II (I-Ab)-restricted TCR specific for a peptide derived from the glycoprotein (G) of VSV serotype Indiana (VSV-IND) 28 . VSV infection of immunocompetent mice induces a rapid neutralizing IgM response that occurs independently of T cell help, followed by production of neutralizing IgG Abs that are strictly dependent on CD4+ T cell help 11 . The neutralizing IgG response seems to be crucial for recovery from primary infections and for protection against reinfection 29, 30, 31, 32 . Furthermore, previous work in this laboratory has demonstrated that H-2b mice that have been primed with VSV rapidly eliminate a recombinant vaccinia virus expressing the VSV-IND-G from ovaries or brain in a CD4+ T cell-dependent manner 7, 8, 33 . Thus, using the same population of VSV-G-specific Th cells, we have analyzed qualitative and quantitative characteristics of different classes of CD4+ T cell-mediated antiviral responses, the induction of VSV-neutralizing IgG Abs, and the cell-mediated clearance of the recombinant vaccinia virus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (H-2b) and TCR transgenic (SMARTA) mice recognizing a peptide from the LCMV-G 34 , were obtained from the breeding colony of the Institut für Zuchthygiene, Tierspital (Zurich, Switzerland). Mice rendered T cell deficient by targeted disruption of the TCR{alpha} gene (TCR{alpha}-/-) 35 were obtained from Dr. M. J. Owen, Imperial Cancer Research Fund (London, U.K.) and were backcrossed five times to C57BL/6 mice to ensure a H-2b background. Mice were between 8 and 16 wk of age when first used.

Generation of {alpha}ß TCR transgenic mice (tg7)

For cloning of the TCR {alpha}-chain, cDNA obtained from the VSV-G-specific T cell hybridoma 31.2.10.4 28 was used as a template for PCR amplification with a downstream primer (C{alpha}-912; 5'-ATCCGGCTACTTTCAGCAGCAG-3') harboring in the C{alpha} region and an upstream primer (5'V{alpha}4-1; 5'-TTTTGAATTCGCTAAGAATCATGAACACTGTCGA-3') hybridizing with the 5' end of the leader sequence, producing a 0.65-kb fragment containing the leader sequence (L), the rearranged V{alpha}4, J{alpha}TA72, and part (224 bp) of the C{alpha} gene segment. This fragment was then cloned into the pDPL13-derived plasmid p14{alpha}2AR containing a full C{alpha} gene (H. Pircher, unpublished observations). The resulting 1.4-kb construct (L-V{alpha}4-J{alpha}TA72-C{alpha}) was then cloned into the BamHI/SalI site of the expression vector pHSE3' 36 (Fig. 1GoA). The TCR ß-chain gene construct was prepared as previously described 37 (Fig. 1GoB). For microinjection, the linearized expression vectors depicted in Fig. 1Go were isolated with silica-based beads (Prep-A-Gene, Bio-Rad, Hercules, CA). The DNA was precipitated with ethanol and diluted in 10 mM Tris-HCl (pH 7.5) containing 0.1 mM EDTA to a final concentration of 100 ng/µl. Approximately 1 ng (500 molecules) of each expression vector was injected into the pronucleus of fertilized eggs derived from C57BL/6 mice 38, 39 . Surviving microinjected eggs were transferred into the oviducts of pseudopregnant C57BL/6 foster females. Offspring were analyzed for integration of the transgenic TCR {alpha}- and ß-chain constructs by PCR using the following primers; for V{alpha}4, 5'-TTTTGAATTCGCTAAGAATCATGAACACTGTCGA-3' and 5'-AGAGGGTGCTGTCCTGAGAC-3'; for Vß2, 5'-AGAACCTTGTACTGCACCTGC-3' and 5'-CTGTGTGACAGTTTGGGTGA-3'. Double transgenic founder mice (tg7) were selected that had cointegrated the transgenic TCR constructs and transmitted them in a Mendelian manner.



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  		FIGURE 1. Schematic depiction of the linearized TCR{alpha} (A) and TCRß (B) transgene constructs. Important restriction sites are given. Arrows indicate DNA stretches amplified by PCR with specific primer pairs for the identification of the TCR transgenes.

 
Cytofluorometric analysis

Surface expression of the TCR Vß2 transgene was determined by cytofluorometric analysis. Spleen or blood cells (5 x 105) from tg7 mice were incubated for 30 min at 4°C in FACS buffer (PBS containing 2% FCS and 20 mM EDTA) containing FITC-labeled anti-Vß2 Ab (B20.6) and PE-conjugated anti-CD4 (RM4-5, both from PharMingen, Hamburg, Germany). For three-color analysis of surface activation markers, spleen cells were first stained with biotinylated Abs against CD25 (7D4), CD62-L (MEL-14), or CD69 (H1.2F3; all from PharMingen) followed by washing with FACS buffer and detection with Tri-Color-conjugated streptavidin (Caltag Laboratories, Burlingame, CA). After washing in FACS buffer, samples were analyzed using a FACScan flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA). Forward and side scatter characteristics were used to distinguish the lymphocyte population. Before analysis of peripheral blood samples, RBC were lysed with FACSlyse solution (Becton Dickinson).

CD69 Up-regulation by CD4+ T cells

Lymph node cells from C57BL/6 or transgenic mice were isolated, and CD8+ T cells and B cells were removed using magnetic Ab cell sorting (MACS) with anti-CD8 and anti-B220 microbeads according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Remaining CD4+ lymph node cells (>90% pure) were resuspended in RPMI/10% FCS at 2.5 x 106/ml. C57BL/6 dendritic cells were prepared as previously described 40 and were cultured at 5 x 106 cells/ml in RPMI/10% FCS with 100 µg/ml p8 for 2 h at 37°C and then washed in RPMI/10% FCS. Aliquots of 2.5 x 105 CD4+ T cells were added to an equal number of C57BL/6 dendritic cells in 96-well U-bottom plates, pelleted for 2 min by centrifugation, and incubated at 37°C for 6 h. Samples were then transferred into tubes containing 2 ml of ice-cold FACS buffer, vortexed for 30 s, and put on ice for 10 min to allow dissociation of dendritic cell-T cell clusters, which was verified by light microscopy. Samples were washed in FACS buffer, stained with PE-anti-CD4 plus FITC-anti-CD69 for 30 min on ice, and analyzed as described above.

Viruses and inactivation of VSV

VSV-IND (Mudd-Summers isolate) and VSV-NJ (Pringle Isolate) were originally obtained from D. Kolakovsky, University of Geneva (Geneva, Switzerland). They were grown on BHK 21 cells infected at low multiplicity of infection, and plaqued on Vero cells 41, 42 . UV light inactivation of VSV was performed under a 15-W UV lamp (type 7 UV, Phillips, Mahway, NJ) at 10 cm from the source for 4 min 43 . Inactivation of VSV was verified by plaquing on Vero cells. Recombinant vaccinia virus expressing VSV-IND-G (Vacc-IND-G) were a gift from B. Moss, Laboratory of Viral Diseases, National Institutes of Health (Bethesda, MD), and were grown on BSC 40 cells at low multiplicity of infection and plaqued on the same cells 44 .

Immunizations

Mice were immunized i.v. with 2 x 106 pfu of live or UV-inactivated VSV-IND or VSV-NJ. Sera were collected by bleeding from the retro-orbital plexus at different time points after injection for determination of VSV-specific neutralizing Ab titers.

Serum neutralization test

VSV-neutralizing IgM and IgG Ab titers were assayed as previously described 45 . Briefly, sera were prediluted 40-fold in MEM containing 5% FCS, then heat-inactivated for 30 min at 56°C. Serial 2-fold dilutions were mixed with equal volumes of VSV diluted to contain 500 pfu/ml. The mixture was incubated for 90 min at 37°C in an atmosphere with 5% CO2. One hundred microliters of the serum-virus mixture was then transferred onto Vero cell monolayers in 96-well plates and incubated for 1 h at 37°C. The monolayers were overlaid with 100 µl of DMEM 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. To determine IgG titers, undiluted serum was pretreated with an equal volume of 0.1 M 2-ME in saline 46 .

Vaccinia protection assay

Female transgenic tg7 or control C57BL/6 mice were primed with either VSV-IND wild-type virus i.v. or VSV-G peptide 415–433 (50 µg in IFA i.p.) or were left unprimed. Eight to ten days later mice were challenged with 5 x 106 pfu Vacc-IND-G i.p. Both ovaries were harvested 5 days later, and the titer of Vacc-IND-G was determined on BSC 40 monolayers as described previously 7 .

T cell proliferative assays

Single cell suspensions of spleen cells from normal or transgenic mice were prepared in RPMI 1640 medium (Life Technologies, Paisley, U.K.) containing 10% FCS, penicillin, streptomycin, L-glutamine, and 5 x 10-5 M 2-ME. Spleen cells(4 x 105/well) were cultured in 96-well round-bottomed plates (Falcon, Becton Dickinson) at 37°C in 5% CO2 in the presence of serial threefold dilutions of Ag. After 72 h, cells were pulsed with [3H]thymidine (1 µCi/well) for 16 h and harvested, and the incorporated radioactivity was measured using a beta counter (Wallac, Turku, Finland). Results are expressed as a stimulation index that was calculated as (cpmsample/cpmspontaneous), with spontaneous counts per minute obtained using cells cultured in the absence of Ag.

In vitro priming of transgenic CD4+ T cells

Naive transgenic tg7 CD4+ spleen cells were obtained at a purity of 98% by MACS purification with anti-CD4 microbeads (Miltenyi Biotech). Aliquots of 106 CD4+ T cells were cultured in six-well tissue culture plates (TPP, Wohlen, Switzerland) in 5 ml of RPMI 1640 medium containing 10% FCS, penicillin, streptomycin, L-glutamine, 5 x 10-5 M 2-ME, and 50 U/ml recombinant murine IL-2 (PharMingen) together with 107 irradiated C57BL/6 spleen cells and antigenic peptide p8 (amino acids 415–433; 1 µg/ml). After 4 days of culture at 37°C in 5% CO2, cells were washed, split 1:2 in fresh medium containing 50 U/ml recombinant murine IL-2, and cultured for an additional 3–4 days. Primed cells were then harvested and washed twice in balanced salt solution before adoptive transfer.

Cytokine analysis

Aliquots of 106 naive or primed transgenic tg7 CD4+ T cells were cultured in 24-well tissue culture plates (TPP) in 1 ml of RPMI 1640 medium containing 10% FCS, penicillin, streptomycin, L-glutamine, and 5 x 10-5 M 2-ME together with 5 x 106 irradiated C57BL/6 spleen cells and peptide p8 (10 µg/ml). Supernatants were harvested after 24 h (for IL-2) or 72 h (for IFN-{gamma} and IL-4) of culture at 37°C in 5% CO2. IL-2 production was determined by assaying growth of the IL-2-dependent cell line CTLL-2, with quantification of viable cells using the AlamarBlue color reaction (BioSource International, Camarillo, CA) measured by fluorescence emission at 590 nm using a CytoFluor 2350 fluorometer (Millipore, Bedford, MA). IFN-{gamma} and IL-4 were measured by ELISA, using Abs and protocols provided by PharMingen. In all cases standard curves were prepared using recombinant cytokines (PharMingen) assayed in parallel. Results are expressed as the mean cytokine concentration (units per milliliter) ± SEM of samples assayed in triplicate.

Adoptive transfer of antiviral immunity

TCR{alpha}-/- mice or C57BL/6 mice were adoptively transferred i.v. with the indicated numbers of naive or primed transgenic CD4+ T cells and challenged 24 h later with 2 x 106 pfu UV-VSV i.v. or with 5 x 106 pfu Vacc-IND-G i.p. Neutralizing Ab responses and vaccinia titers in ovaries were measured as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotype and Ag specificity of tg7 transgenic CD4+ T cells

To assess quantitative and qualitative aspects of antiviral CD4+ T cell function, we generated transgenic mice (tg7) expressing a I-Ab-restricted TCR that recognized a peptide (p8; amino acids 415–433) derived from the VSV-G 28 . FACS analysis of tg7 mice demonstrated that there were no gross abnormalities in T cell subsets present in either thymus or spleen (data not shown) and that the transgenic Vß2 TCR chain was expressed on 60–70% of mature CD4+ T cells (Fig. 2GoA). The transgenic Vß2+CD4+ T cells exhibited a phenotype characteristic of normal naive T cells, i.e., CD25low CD69low CD62Lhigh (Fig. 2GoB). Since a mAb against the V{alpha}4 transgene product was not available, and as allelic exclusion at the TCR{alpha} locus is much weaker than that at the TCRß locus 47 , it was not certain that all the Vß2+CD4+ cells also expressed the transgenic {alpha}-chain. To quantify the true proportion of p8-reactive T cells in tg7 mice, we performed CD69 up-regulation studies on CD4+ T cells from tg7 mice. After culture for 6 h with p8-bearing dendritic cells, about 50% of tg7 CD4+ T cells expressed high levels of CD69 compared with only 5% of those cultured with dendritic cells in the absence of p8 (Fig. 3GoA). CD4+ T cells from nontransgenic C57BL/6 mice showed no increase in CD69 expression after culture with p8-bearing dendritic cells (Fig. 3GoB). Thus, around 45% of mature CD4+ T cells in tg7 mice were VSV-G specific. The accuracy of this method was verified using another CD4+ T cell transgenic line, SMARTA, recognizing a peptide derived from the LCMV-G. Approximately 90% of SMARTA CD4+ T cells up-regulated CD69 when cultured with dendritic cells bearing the LCMV-G peptide (Fig. 3GoC), which is equivalent to the proportion of cells bearing the transgenic V{alpha}2Vß8.3 TCR 34 .



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  		FIGURE 2. Phenotypic analysis of transgenic tg7 CD4+ T cells. A, Spleen cells from tg7 transgenic (solid line) or control C57BL/6 (dashed line) mice were stained with FITC-anti-Vß2 and PE-anti-CD4 mAbs. The graph shows the expression of the Vß2 TCR on gated CD4+ T cells. B, Expression of CD25, CD69, and CD62-L on naive (dashed lines) or p8-primed (solid lines) Vß2+CD4+ cells from tg7 mice. Numbers shown are percentages of Vß2+CD4+ T cells contained within the respective markers. Plots are representative of four similar experiments.

 


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  		FIGURE 3. CD69 up-regulation by transgenic tg7 CD4+ T cells. Purified CD4+ T cells from tg7 transgenic (A), control C57BL/6 (B), or SMARTA transgenic (C) mice were stimulated for 6 h with dendritic cells in the absence (dashed lines) or the presence (solid lines) of 100 µg/ml VSV-G p8 (A and B) or LCMV-G p13 (C) and then stained with PE-anti-CD4 plus FITC-anti-CD69 mAbs. Plots show the expression of CD69 on gated CD4+ T cells and are representative of three separate experiments.

 
The Ag specificity of the transgenic CD4+ T cells was confirmed using in vitro proliferative assays. In contrast to nontransgenic C57BL/6 cells, splenocytes from tg7 mice exhibited strong proliferative responses when stimulated with UV-VSV, purified VSV-G, or peptide p8 (Fig. 4Go). The tg7 T cells did not mount in vitro proliferative responses when cultured with another VSV-G Th epitope (p41) which is also presented in H-2b mice (Fig. 4Go) 28 . These findings confirmed that tg7 mice contained a high frequency of specific T cells that recognized the VSV-G-derived p8 peptide.



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  		FIGURE 4. Ag-specific proliferative responses of transgenic tg7 splenocytes. Spleen cells from tg7 transgenic were cultured for 72 h in the presence of UV-VSV ({circ}), VSV-G ({square}), p8 (VSV-G415–433; {triangleup}), or p41 (VSV-G52–71; •), and proliferation was monitored by overnight incorporation of [3H]thymidine. Results shown are means from duplicate cultures and are representative of two similar experiments.

 
Tg7 mice exhibit enhanced neutralizing IgG responses after immunization with inactivated VSV-IND

We next analyzed whether the increased precursor frequency of VSV-G-specific CD4+ T cells present in tg7 mice correlated with an enhanced responsiveness to VSV. After immunization with live VSV, both C57BL/6 mice and tg7 mice mounted rapid, T-independent, VSV-neutralizing IgM responses followed by class switching to neutralizing IgG Abs, which are known to be CD4+ T cell dependent (Fig. 5GoA) 11 . However, when immunized with UV-inactivated VSV, which efficiently triggers B cells while poorly priming CD4+ Th cells 43 , the C57BL/6 mice produced very low levels of neutralizing IgG, whereas the tg7 mice again rapidly produced high titers of neutralizing IgG Abs (Fig. 5GoA). This enhanced sensitivity to suboptimal immunization with inactivated VSV-IND was dependent on the p8-specific T cells in tg7 mice, since there was no augmentation in the neutralizing IgG response to immunization with inactivated VSV-NJ, which does not contain the p8 epitope (Fig. 5GoB) 48 .



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  		FIGURE 5. The tg7 mice exhibit enhanced VSV-IND-G-specific Th cell reactivity in vivo. Groups of naive tg7 transgenic (squares) or C57BL/6 control (circles) mice were immunized i.v. with 2 x 106 pfu of live (open symbols) or UV-inactivated VSV-IND (filled symbols; A) or with live or UV-inactivated VSV-NJ (B). On the indicated days, blood was taken, and serum was analyzed for the presence of VSV-IND neutralizing IgM and IgG. Mean titers from groups of two or three mice are shown, and intragroup variations were two titer steps or less. One of two similar experiments is shown.

 
Adoptively transferred tg7 CD4+ T cells mediate isotype switching in vivo

We next attempted to quantify the number of VSV-G-specific CD4+ T cells required to mediate isotype switching in vivo. Thus, naive tg7 CD4+ T cells were purified using MACS and adoptively transferred into syngeneic T cell-deficient recipients (TCR{alpha}-/- mice), which were then challenged with UV-VSV-IND. As shown in Fig. 6Go, TCR{alpha}-/- mice adoptively transferred with C57BL/6 CD4+ T cells made high levels of neutralizing IgM, but failed to produce neutralizing IgG Abs after immunization with UV-VSV-IND. In contrast, adoptive transfer of as few as 105 tg7 CD4+ T cells enabled the TCR{alpha}-/- mice to produce VSV-neutralizing IgG responses (Fig. 6Go), demonstrating that the virus-specific CD4+ T cells could efficiently transfer help for antiviral humoral immunity.



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  		FIGURE 6. Adoptively transferred naive tg7 CD4+ T cells mediate Ig class switching in vivo. Graded numbers of purified C57BL/6 or tg7 CD4+ T cells were adoptively transferred i.v. into syngeneic TCR{alpha}-/- mice. One day later recipients were immunized with 2 x 106 pfu UV-VSV-IND i.v. On the indicated days, blood was taken, and serum was analyzed for the presence of VSV-IND neutralizing IgM and IgG. Mean titers from groups of two or three mice are shown, and intragroup variations were two titer steps or less. One of two similar experiments is shown.

 
In vitro primed, but not naive, tg7 CD4+ T cells can adoptively transfer cell-mediated immunity against recombinant Vacc-IND-G

We next examined whether tg7 CD4+ T cells could mediate cell-mediated immunity against a recombinant vaccinia virus expressing the VSV-IND glycoprotein (Vacc-IND-G). Previous work in this laboratory has shown that C57BL/6 mice primed with VSV-IND were resistant against challenge with Vacc-IND-G and that this protection was mediated by CD4+ T cells 33 . In agreement with those results, we found that naive tg7 mice were as resistant to Vacc-IND-G challenge as VSV-immune or p8-primed C57BL/6 mice (Fig. 7GoA), showing that the p8-specific tg7 CD4+ T cells could indeed mediate cell-mediated immune protection. This protection was Ag specific, since the tg7 mice were not protected against challenge with a recombinant vaccinia virus expressing an irrelevant Ag Vacc-LCMV nucleoprotein (Fig. 7GoA).



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  		FIGURE 7. Primed, but not naive, tg7 CD4+ T cells can adoptively transfer protection against peripheral vaccinia virus infection. A, Naive female C57BL/6 (open circles) or tg7 (filled circles) mice were left untreated or were primed with 2 x 106 pfu of VSV-IND i.v. or with 50 µg of p8 in IFA i.p. Eight to ten days later mice were challenged with 5 x 106 pfu of Vacc-IND-G or Vacc-LCMV nucleoprotein (Vacc-LCMV-NP) i.p. B, C57BL/6 female mice were left untreated or were primed with 2 x 106 pfu of VSV-IND i.v. or were adoptively transferred with 107 purified naive tg7 CD4+ T cells i.v. One day later recipients were challenged with 5 x 106 pfu of Vacc-IND-G i.p. C, Graded numbers of in vitro primed tg7 CD4+ T cells were adoptively transferred i.v. into C57BL/6 female mice. One day later recipients were challenged with 5 x 106 pfu Vacc-IND-G i.p. In all cases, 5 days after challenge ovaries were removed, and vaccinia titers were determined. Symbols represent individual mice, and bars represent mean titers. One representative experiment of three is shown. D and E, Cytokine secretion by naive and primed tg7 CD4+ T cells. Supernatants collected after in vitro culture of 106 CD4+ T cells with 5 x 106 syngeneic irradiated spleen cell APC in the absence (filled bars) or the presence (hatched bars) of p8 (10 µg/ml) were assayed for IL-2 (D) and IFN-{gamma} (E). Results are expressed as the mean ± SEM of samples assayed in triplicate and are representative of two similar experiments.

 
To quantify the number of VSV-G-specific CD4+ T cells required for this cell-mediated protection, we adoptively transferred naive tg7 CD4+ T cells into C57BL/6 recipients and challenged them with Vacc-IND-G. However, adoptive transfer of as many as 107 naive tg7 CD4+ T cells failed to offer any significant protection against Vacc-IND-G (Fig. 7GoB). As there is evidence that the trafficking of lymphocytes through peripheral solid organs may depend on the differentiation status of the cell 49, 50 , we primed tg7 CD4+ T cells with p8 peptide in vitro before adoptive transfer. The primed tg7 CD4+ T cells exhibited the phenotypic characteristics of effector/memory T cells, i.e., CD25high, CD69high, CD62-Llow (Fig. 2BGo). Adoptive transfer of >= 106 primed tg7 CD4+ T cells mediated protection against Vacc-IND-G (Fig. 7GoC).

To examine whether the ability to confer protection against vaccinia correlated with different patterns of cytokine secretion, we measured the cytokines produced by naive and primed tg7 CD4+ T cells following stimulation with p8. Naive tg7 CD4+ T cells produced high levels of IL-2 and only low amounts of IFN-{gamma}, while primed tg7 CD4+ T cells produced similar levels of IL-2 but around 80- to 100-fold higher levels of IFN-{gamma} (Fig. 7Go, D and E). Neither naive nor primed tg7 CD4+ T cells produced detectable amounts of IL-4 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we generated TCR transgenic mice (tg7) that allowed us to examine the roles of CD4+ T cells of identical specificity in humoral or cell-mediated antiviral protection. Our results confirmed that CD4+ T cells can efficiently mediate protection against viruses that are controlled by T help-dependent neutralizing IgG Abs (VSV) or cytokines (Vacc-IND-G). However, the number and activation state of CD4+ T cells required for protection depended on the virus and the protection assay used.

The tg7 mice exhibited enhanced responsiveness both in vitro and in vivo to VSV-IND, but not to VSV-NJ, which does not share the p8 peptide sequence of the IND-G 48 . The augmentation of the IgG responses against suboptimal Ag challenge with UV-VSV confirmed that many of the characteristics of T cell memory can be mimicked by enhanced precursor frequency in transgenic mice 37 . Although cytofluorometric analysis revealed that in tg7 mice 60–70% of the peripheral CD4+ T cells expressed the transgenic TCR ß-chain, analysis of CD69 up-regulation following TCR ligation showed that the actual frequency of p8-specific CD4+ T cells was around 45%. Hence, adoptive transfer of humoral immunity required 105 tg7 CD4+ T cells, representing about 4.5 x 104 Ag-specific CD4+ T cells. Again, the number of these cells actually involved in mediating class switching is probably considerably less, as factors such as homing after transfer will also affect the availability of CD4+ T cells to help B cells 51 . Thus, our results suggest that relatively few Ag-specific naive CD4+ T cells are capable of promoting antiviral IgG responses.

In contrast to the neutralizing IgG responses, naive tg7 CD4+ T cells were unable to adoptively transfer protection against recombinant vaccinia virus infection of peripheral solid organs (ovaries). However, tg7 CD4+ T cells that had been preactivated with p8 in vitro were able to rapidly eradicate Vacc-IND-G from peripheral organs after adoptive transfer. This qualitative difference in the protective capacities of naive and primed CD4+ T cells may be explained by at least two major mechanisms. First, the migratory patterns of naive and primed lymphocytes differ such that naive T cells recirculate through secondary lymphoid organs, while primed T cells may traffic through peripheral tissues 49, 50, 52 . Second, the cytokine production characteristics of the cells may also be qualitatively and quantitatively different 53, 54, 55 . In support of the latter possibility, we observed that primed CD4+ T cells produced 80- to 100-fold higher levels of IFN-{gamma} than naive CD4+ T cells. This difference is probably a major factor in the ability of primed CD4+ T cells to protect against vaccinia, since previous studies have shown that IFN-{gamma} plays an essential role in protection against vaccinia mediated by CD4+ T cells 4, 7, 9 .

Adoptive transfer of about 20-fold more CD4+ T cells was required for eradication of peripheral vaccinia virus than for the induction of isotype switching. Two main factors could explain this quantitative difference; Ag form and localization. Class switching was assayed following i.v. injection of nonreplicating virus (UV-VSV), which is efficiently filtered out in the spleen and presented to naive T cells 56 . Cell-mediated protection was assayed in the ovaries after i.p. challenge with live vaccinia, in which case requirements for antiviral protection are probably much more stringent. During the time that it takes virus Ag to reach lymphoid tissue, naive T cells to be primed, and protective T cells to emigrate to the peripheral organ and mediate effector function, the virus may continuously replicate. Interestingly, this qualitative difference in the protective capacities of naive and primed T cells has also been observed for virus-specific CD8+ T cells. Thus, recently primed CD8+ T cells are also able to mediate protection against peripheral vaccinia infection, but peripheral protection is relatively short-lived in the absence of further antigenic restimulation, which enhances extravasation of CD8+ memory T cells 57, 58, 59 . Thus, it appears that both CD4+ and CD8+ T cell-mediated antiviral protection in peripheral tissues is dependent on the presence of recently Ag-activated effector T cells rather than merely an elevated frequency of Ag-specific T cells.

One paradoxical finding of this study was that naive tg7 mice were resistant against challenge with Vacc-IND-G, while naive tg7 CD4+ T cells isolated from these mice were unable to adoptively transfer protection against Vacc-IND-G. Two factors may contribute to this discrepancy. First, since the adoptively transferred transgenic CD4+ T cells must compete with endogenous lymphocytes for space in lymphoid tissues, the B6 recipient mice will still have many fewer VSV-G-specific CD4+ T cells than the tg7 transgenic mice. A second factor may be the expression of endogenous TCR{alpha}-chains by tg7 CD4+ T cells, which could pair with the transgenic TCR Vß2 chain, giving rise to T cells bearing two different TCRs 47, 60, 61 . Normal mice and humans also contain a small population of dual TCR-bearing T cells, which may be triggered via either TCR 62, 63, 64 . This situation has been reported in other TCR transgenic mice, where the existence of transgenic T cells exhibiting a memory phenotype was observed in the absence of specific Ag priming 61 . Priming may have occurred through environmental Ag stimulation of the second TCR, since T cells with a memory phenotype were not observed when the tg TCR mice were crossed onto a recombination-activating gene-/- background 61 . In the tg7 mice most environmentally primed CD4+ T cells capable of trafficking through tissues will also possess the transgenic TCR and thus be able to mediate peripheral protection against the recombinant vaccinia virus.

Most class II MHC-restricted TCR transgenic mouse strains described to date express TCRs specific for model protein Ags, and while these have proved very useful for studying the development and differentiation of CD4+ T cells, they do not permit analysis of protective immune responses mediated by CD4+ T cells in vivo. Recently, a few class II-restricted TCR transgenic mouse strains specific for Ags derived from pathogens have been described 34, 65, 66, 67 . Mice expressing a transgenic TCR recognizing a class II MHC-restricted epitope of the influenza hemagglutinin exhibited only slightly higher resistance to lethal influenza infection than nontransgenic littermates 65 . Interestingly, these mice did not make higher titers of hemagglutinin-specific Abs, and the protective effect seemed to be due to enhanced class I MHC-restricted and class II MHC-restricted cytotoxicity 65 . Similarly, a recombinant influenza virus expressing a class II MHC-restricted peptide from hen egg lysozyme was cleared with similar kinetics by normal and HEL-specific TCR{alpha}ß transgenic mice 66 . Furthermore, previous studies in our laboratory have shown that mice transgenic for a class II MHC-restricted TCR recognizing an epitope in the LCMV-G fail to efficiently control LCMV infection 34 , confirming that clearance of LCMV is crucially dependent on CD8+ CTL 2, 21 . Conversely, it has been shown that mice expressing a single transgenic TCR-{alpha}ß recognizing a Leishmania Ag class II MHC-restricted epitope could significantly control Leishmania major infection, and in this case protection correlated with the development of a Th1 response in both CD4+ and CD4-8- transgenic T cells 67 . These studies, together with the results described in this paper, show that CD4+ T cells may mediate a wide range of immune responses to pathogens and that their protective capabilities may differ depending on the type of response required for pathogen elimination. This transgenic model should permit further analyses of antiviral properties of CD4+ T cells during different viral infections, particularly in terms of phenotype and effector function of the various subpopulations of CD4+ T cells.


   Footnotes
 
1 This work was supported by grants from the Swiss National Science Foundation (31-32179.91) and the Kanton Zurich. Back

2 These authors contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Kevin J. Maloy, Department of Pathology, Institute of Experimental Immunology, Schmelzbergstrasse 12, CH-8091 Zurich, Switzerland. E-mail address: Back

4 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; G, glycoprotein; VSV, vesicular stomatitis virus; Vacc-IND-G, recombinant vaccinia virus expressing the vesicular stomatitis virus serotype Indiana glycoprotein; PE, R-phycoerythrin; MACS, magnetic antibody cell sorting; VSV-IND, vesicular stomatitis virus serotype Indiana; VSV-NJ, vesicular stomatitis virus serotype New Jersey; pfu, plaque-forming units; UV-VSV, ultraviolet-inactivated vesicular stomatitis virus; Vacc-LCMV-NP, recombinant vaccinia virus expressing the lymphocytic choriomeningitis virus nucleoprotein. Back

Received for publication September 11, 1998. Accepted for publication November 13, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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