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The Journal of Immunology, 1999, 162: 5178-5182.
Copyright © 1999 by The American Association of Immunologists

A Novel Approach to Visualize Polyclonal Virus-Specific CD8 T Cells In Vivo1

Christine Zimmermann and Hanspeter Pircher2

Department of Immunology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent technical breakthroughs in generating soluble MHC class I-peptide tetramers now allow the direct visualization of virus-specific CD8 T cells after infection in vivo. However, this technique requires the knowledge of the immunodominant viral epitopes recognized by T cells. Here, we describe an alternative approach to visualize polyclonal virus-specific CD8 T cells in vivo using a simple adoptive transfer system. In our approach, C57BL/6 (Thy1.2) mice were infected with lymphocytic choriomeningitis virus, vesicular stomatitis virus, or vaccinia virus to induce virus-specific memory T cells. Tracer T cells (2 x 106) from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. After infection of the F1-recipient mice with the appropriate virus, the transferred cells expanded vigorously, and on day 8 postinfection 60–80% of total CD8 T cells were of donor T cell origin. Under the same conditions memory CD4 T cells gave rise to at least 10 times less cell numbers than memory CD8 T cells. The transfer system described here not only allows to visualize effector and memory CD8 T cells in vivo but also to isolate them for further in vitro characterization without knowing the epitopes recognized by these Ag-specific CD8 T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Viral infections can induce extensive T cell proliferation that is either due to clonal expansion of virus–Ag-specific T cells or to polyclonal stimulation mediated by cytokines (1, 2, 3, 4). Using adoptive transfer protocols, we found that TCR transgenic (tg)3 T cells specific for the lymphocytic choriomeningitis virus (LCMV) expanded about 103- to 104-fold after LCMV infection in recipient mice (5). In this system 70–80% of CD8 T cells in the acute phase of the response expressed the LCMV-specific tg TCR. Recent studies using the same virus have provided definite evidence that the majority of the CD8 T cell expansion seen during acute infection represents Ag-driven proliferation of specific T cells (6, 7). The infection of mice with LCMV seems not to be an exception in this respect since it has been shown that frequencies of virus-specific CD8 T cells have been greatly underestimated by limiting dilution analysis in the acute and memory phase after different viral infections (8, 9, 10, 11, 12).

Adoptive transfer experiments using tracer populations of TCR tg T cells in recipient mice represent one approach to follow Ag-specific T cells directly during an immune response (13, 14, 15). However, the generation of TCR tg mice is tedious and analysis of a single, monoclonal T cell response may not always reflect the physiological, polyclonal situation. Soluble peptide MHC class I tetramers can be used to directly visualize Ag-specific CD8 T cells by flow cytometry (16). However, this powerful technique requires knowledge of the immunodominant T cell epitopes and involves laborious construction of the appropriate reagents. Cytoplasmic staining of cytokines or enzyme-linked immunospot (ELISPOT) represent two other methods to quantify virus-specific T cells. However, these methods cannot be used to isolate viable Ag-specific T cells for further biological assays.

In the present study we describe a simple adoptive transfer system to visualize and isolate polyclonal antiviral CD8 T cells and demonstrate with three viruses—LCMV, vesicular stomatitis virus (VSV), and vaccinia virus (VV)—that the massive proliferation of T cells after viral infection is mainly due to clonal expansion of Ag-specific CD8 T cells.


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

C57BL/6 (Thy1.2) mice (B6) were obtained from Harlan Winkelmann (Borchen, Germany), B6.PL-Thy-1a (B6.Thy1.1) mice were a generous gift of Dr. H. Mossmann (Max Planck Institute of Immunobiology, Freiburg, Germany). LCMV TCR tg mice (line 318) have been described previously (17, 18). (C57BL/6 x B6.PL-Thy-1a)F1 mice were bred locally in a conventional mouse house facility.

Viruses

The LCMV-WE isolate was originally provided by F. Lehmann-Grube (Hamburg, Germany) and was grown on L929 cells with a low multiplicity of infection. Vesicular stomatitis virus Indiana (VSVIND) (Mudd-Summers isolate) was originally obtained from D. Kolakofsky (University of Geneva) and was grown with a low multiplicity of infection on BHK cells. Stocks of VV strain WR were produced by infecting BSC 40 cells with a low multiplicity of infection.

Adoptive transfers

Virus immune B6 mice were generated by i.v. injection of LCMV-WE (200 pfu), VSVIND (2 x 106 pfu), or VV (2 x 106 pfu). Sex-matched spleen cells from these virus-immune mice (4–6 wk after infection) containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells were injected i.v., in a volume of 0.5 ml medium without FCS, into normal, nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. One day after cell transfer the F1 recipient mice were challenged with the corresponding virus: LCMV-WE (200 pfu), VSVIND (2 x 106 pfu), or VV (2 x 106 pfu). LCMV TCR tg memory T cells were generated by transferring spleen cells of naive LCMV TCR tg mice containing 1 x 105 tg CD8 T cells i.v. into (C57BL/6 x B6.PL-Thy-1a)F1 mice and infecting the recipient mice 1 day later with LCMV-WE (200 pfu). Mice were designated as LCMV TCR tg memory mice when the period following infection exceeded 4 wk.

Flow cytometry

For detection of C57BL/6- or B6.PL-Thy-1a-derived donor CD8 T cells in (C57BL/6 x B6.PL-Thy-1a)F1 recipient mice PBL were incubated on ice with biotinylated anti-CD8 (Life Technologies, Gaithersburg, MD), PE-labeled anti-CD90/Thy1.1 (PharMingen, San Diego, CA), and FITC-labeled anti-CD90/Thy1.2 (Caltag, South San Francisco, CA) followed by Tricolor-streptavidin (Caltag). For detection of CD4 T cells, PBL were incubated with FITC-labeled anti-CD4, PE-labeled anti-CD90/Thy1.1, and biotinylated anti-CD90/Thy1.2 (all from PharMingen) followed by Tricolor-streptavidin. Staining of PBL was performed in PBS containing 2% FCS, 0.1% NaN3, and 10 U/ml heparin (Liquemin, Hoffmann-La Roche, Basel, Switzerland). PBL were analyzed after lysis of red blood cells using FACS Lysing Solution (Becton Dickinson, Mountain View, CA) according to the instructions of the manufacturer. Cells were analyzed using a FACSort (Becton Dickinson) and CellQuest software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clonal expansion of LCMV TCR tg naive and memory CD8 T cells after LCMV infection

We have previously shown that adoptively transferred naive LCMV TCR tg CD8 T cells expanded in B6 recipient mice by a factor of 103–104 after LCMV infection (5). To examine whether LCMV TCR tg memory T cells proliferate to the same extent, spleen cells containing 105 naive or memory LCMV TCR tg cells (Thy1.2) were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy1a)F1 recipient mice that were infected 1 day later with LCMV. LCMV TCR tg memory T cells were generated in vivo using the same adoptive transfer protocol and were isolated 6–12 wk after infection.

Clonal expansion of the TCR tg T cells in the recipient mice was monitored by flow cytometry using the allelic Thy1 marker to distinguish donor (Thy1.2+) and host (Thy1.1+, Thy1.2+) T cells. As shown in Fig. 1Go, naive and memory LCMV TCR tg T cells proliferated to the same extent in the recipient mice, and 8 days after infection >40% of PBL (Fig. 1Go, a and c) and >70% of CD8 T cells (Fig. 1Go, b and d) were of donor T cell origin (Thy 1.2). As a control, CD8 T cells were also stained with Abs specific for the transgenic LCMV TCR (V{alpha}2/Vß8) which confirmed that >95% of Thy1.2+ cells found in these mice expressed the tg TCR (data not shown). Even at the earliest time point after infection (day 6), when sizable numbers of the donor T cells were first detected in the recipient mice, no significant difference in clonal expansion of naive or memory T cells was observed. After the acute phase of the infection the number of TCR tg T cells derived from either naive or memory cells declined with similar kinetics. Thus, in vivo-generated bona fide CD8 memory T cells exhibit the same characteristics in clonal expansion and decline after adoptive transfer and Ag challenge as naive T cells.



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  		FIGURE 1. Kinetics of adoptively transferred naive and memory LCMV TCR tg T cells in LCMV-infected hosts. (C57BL/6 x B6.PL-Thy-1a)F1 mice that had received spleen cells containing 1 x 105 naive (a and b) or memory (c and d) Thy1.2+ TCR tg CD8 T cells were infected with LCMV on day 0. a and c, Percentages of Thy1.2+ T cells of PBL are shown. b and d, Percentages of Thy1.2+ of CD8 T cells are shown. PBL of single mice were analyzed at the time points indicated by flow cytometry using Thy1.2-, Thy1.1-, and CD8-specific mAbs.

 
Experimental design to visualize polyclonal virus-specific T cells in vivo

Recent studies using MHC/peptide tetramers, cytoplasmic cytokine stainings, or ELISPOT have demonstrated that precursor frequencies of memory CD8 T cells after viral infections are about 10-fold higher than determined previously by limiting dilution analysis. In the LCMV system, Murali-Krisha et al. (6) found that 5–10% of memory CD8 T cells in LCMV immune B6 mice were virus-specific. As shown above in the LCMV TCR tg model, both naive and memory LCMV-specific CD8 T cells were able to undergo the same vigorous clonal expansion after Ag (re)challenge in vivo. Therefore, it should be possible to also visualize polyclonal LCMV-specific memory CD8 T cells from nontransgenic but LCMV immune mice after adoptive transfer and LCMV infection using appropriate allelic markers (Thy1) to distinguish donor from host T cells. To test this hypothesis, B6 mice (Thy1.2) were infected with LCMV, VSV, or VV to induce virus-specific memory T cells (Fig. 2Go). Four to 6 wk after infection tracer spleen cells from these virus-immune mice were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice. One day after transfer the F1-recipient mice were challenged with the corresponding virus, and the kinetics of the adoptively transferred tracer T cells (Thy1.2) was followed in the F1-recipient mice (Thy1.1, Thy1.2) by flow cytometry of PBL.



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  		FIGURE 2. Experimental design to visualize polyclonal memory CD8 T cells. B6 (Thy1.2+) mice were infected with virus (LCMV, VSV, or VV). Four to 6 wk later spleen cells of these mice containing CD4 and CD8 (1–2 x 106) T cells were adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 (Thy1.2+, Thy1.1+) mice that were infected 1 day later with the corresponding virus.

 
Visualization of polyclonal LCMV-specific T cells during infection

In the first set of experiments we determined the in vivo proliferation of T cells from B6 mice immune to LCMV (=B6/LCMV). Tracer spleen cells from B6/LCMV mice containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells were adoptively transferred into F1-recipient mice. A small population of Thy1.2+ Thy1.1- cells (3–5%) could be detected in normal (C57BL/6 x B6.PL-Thy-1a)F1 mice, and this percentage did not significantly increase after cell transfer (Fig. 3Goa). After LCMV infection, donor Thy1.2+ T cells from B6/LCMV mice proliferated strongly in the F1-recipient mice, and on day 8 postinfection 30–50% of PBL (Figs. 3Gob and 4a) and 70–80% of total CD8 T cells (Fig. 4Gob) were of donor T cell origin. The extent and the kinetics of the clonal expansion of donor T cells from B6/LCMV mice (Fig. 4Go, a and b) was strikingly similar to the data obtained with naive or memory donor T cells from LCMV TCR tg mice (Fig. 1Go).



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  		FIGURE 3. Flow cytometric analysis of PBL from (C57BL/6 x B6.PL-Thy-1a)F1-recipient mice on day 0 (a and d), day 8 (b and e), and on day 50 (c and f) after LCMV infection. One day before LCMV infection, the F1 mice had received spleen cells containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells from LCMV immune B6 mice. The dot plots (a–c) show anti-Thy1.1/Thy1.2 double staining of PBL from the F1-recipient mice, and the histograms (d–f) display CD8 expression of the gated Thy1.2+ Thy1.1- cells.

 


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  		FIGURE 4. Kinetics of adoptively transferred T cells in PBL of LCMV-infected hosts. a–c, (C57BL/6 x B6.PL-Thy-1a)F1 mice that had received spleen cells containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells from LCMV immune B6 mice were infected with LCMV. Flow cytometry with Abs specific for Thy1.1, Thy1.2, CD8, and CD4 was performed at the time points indicated after infection. The percentage of donor Thy1.2+ T cells of PBL (a), CD8 (b), and CD4 (c) T cells from individual mice is shown. In d and e, F1 mice that had received spleen cells containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells from LCMV immune (d) or from control B6 (e) mice were infected with VV (d) or LCMV (e) and analyzed as described above.

 
Flow cytometric analysis also revealed that the LCMV-induced clonal expansion of T cells from B6/LCMV mice was almost exclusively restricted to the CD8 subset, despite the fact that the same number of CD4 and CD8 T cells were transferred (Figs. 3Goe and 4c). Donor T cells from B6/LCMV mice did not expand in the F1 recipient mice after VV infection (Fig. 4God) and donor T cells from normal B6 mice did not proliferate noticeably in F1 recipient mice after LCMV infection (Fig. 4Goe). These control experiments indicated that the virus-induced in vivo proliferation of donor T cells in the F1 recipient mice was Ag-specific.

Visualization of polyclonal VSV- and VV-specific T cells during infection

Once the adoptive transfer protocol with LCMV had been successfully established, the same basic approach was used to visualize VSV- and VV-specific T cells. Tracer spleen cells from VV or VSV immune B6 mice were adoptively transferred into F1-recipient mice, which were then challenged with the corresponding virus 1 day later. Flow cytometric analysis revealed that donor T cells from B6/VSV or B6/VV mice expanded massively in the F1-recipient mice (Fig. 5Go, a, b, e, and f). Importantly, Thy1.2+ donor T cells from VSV or VV immune mice did not expand after infection with the unrelated LCMV (Fig. 5Go, d and h). The slight proportional decrease of these VSV or VV immune T cells in the recipient mice observed after LCMV infection was probably due to the massive expansion of host LCMV-specific T cells.



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  		FIGURE 5. Kinetics of adoptively transferred T cells in PBL of VSV- or VV- infected hosts. (C57BL/6 x B6.PL-Thy-1a)F1 mice that had received spleen cells containing CD8 (2 x 106) and CD4 (2–4 x 106) T cells from B6 mice immune to VSV (a–d) or to VV (e–h) were infected with VSV (a–c) or VV (e–g). As control for unspecific expansion mice were infected with LCMV after transfer of VSV- (d) or VV- (h) specific memory CD8 T cells. Flow cytometry with Abs specific for Thy1.1, Thy1.2, CD8, and CD4 was performed at the time points indicated after infection. The percentage of donor Thy1.2+ Thy1.1- T cells of PBL (a and e), CD8 (b, f, d, and h), and CD4 (c and g) T cells from individual mice is shown.

 
The extent of the in vivo proliferation of donor T cells during acute VV or VSV infection, the kinetics of the response and the virtually exclusive expansion of the CD8 subset from the donor cell inoculum (Fig. 5Go, c and g), was remarkably similar to the results obtained in the LCMV system. As observed for PBL, donor T cells from virus immune mice were also found in significant quantities in the spleens of F1 recipient mice during acute infection and also in the memory phase (data not shown). Thus, despite obvious differences between LCMV, VV and VSV with respect to viral replication (1, 3, 4, 19, 20) and CD28-dependence (21) the clonal expansion of virus-specific T cells was strikingly similar with these three viruses.

Competitive expansion of LCMV- and VSV-specific memory T cells

CD8 memory T cells but not naive cells were shown to be driven to activation and proliferation by IFNs induced during viral infections (22). Since memory CD8 T cells secrete higher levels of IFN-{gamma} than naive cells after stimulation (10, 23), it was important to test bystander expansion of VV-specific memory CD8 T cells in the presence of vigorously proliferating LCMV-specific memory CD8 T cells and vice versa. (C57BL/6 x B6.PL-Thy-1a)F1 mice were given a 1:1 mixture of spleen cells containing 1 x 106 CD8 and 1–2 x 106 CD4 T cells from LCMV immune B6.PL-Thy-1a (Thy 1.1) and from VV immune B6 (Thy 1.2) mice (Fig. 6GoA). Afterward, the recipient mice were infected with either LCMV or VV and PBL were analyzed 8 days after infection. As shown in Fig. 6GoB, only donor T cells from LCMV but not from VV immune mice expanded after LCMV infection and vice versa. Importantly, immune CD8 T cells being specific for the unrelated virus did not increase above background levels.



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  		FIGURE 6. A, Experimental design to visualize specific expansion of memory CD8 and CD4 T cells. B6.PL-Thy-1a (Thy1.1) and B6 (Thy1.2) mice were infected with LCMV or VV, respectively. Four to 6 wk later spleen cells containing CD8 (1 x 106) and CD4 (1–2 x 106) T cells were mixed at a 1:1 ratio and adoptively transferred into nonirradiated (C57BL/6 x B6.PL-Thy-1a)F1 mice that were infected 1 day later with LCMV or VV. B, Flow cytometric analysis of PBL from F1 mice that had received a 1:1 mix of spleen cells from LCMV immune B6.PL-Thy-1a mice and VV immune B6 mice. Analysis was performed 8 days after LCMV (a and c) or VV (b and d) infection. The dot plots show anti-Thy1.1/Thy1.2 double staining of PBL from the F1 recipient mice gated on CD8 (a and b) and CD4 (c and d) T cells.

 
Interestingly, only CD8 T cells from the cell inoculum proliferated extensively in the recipient mice after infection, although similar numbers of CD4 and CD8 T cells from virus primed mice were transferred. In the "mixed" experiment shown in Fig. 6Go we observed an increase of donor Thy1.1+ CD4 T cells from LCMV immune mice from 0.8% (percent Thy1.1+ CD4 T cells after infection with the unrelated VV, Fig. 6GoBd) to 5.1% after transfer and LCMV infection (Fig. 6GoBc). Similarly, Thy1.2+ CD4 T cells from VV immune mice increased from 2% (percent Thy1.2+ CD4 T cells after infection with the unrelated LCMV, Fig. 6GoBc) to 6.3% after VV infection (Fig. 6GoBd).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data, which imply that most (70–80%) CD8 T cells found during acute LCMV, VSV, or VV infection were Ag-specific, agree well with two recent studies (6, 7) performed in the LCMV system that used MHC/peptide-tetramer-stainings and assays measuring IFN-{gamma} production at the single-cell level. In our experimental approach, whole virus was used as antigenic challenge whereas the former studies used the three known antigenic peptides of LCMV in B6 mice to visualize virus-specific CD8 T cells. The finding that both approaches yielded similar numbers of reactive T cells during acute infection further indicates that all the major peptide epitopes of LCMV in B6 mice are now known. Following the acute phase of the infection the frequency of LCMV-specific T cells per CD8 cell decreased roughly by a factor of 2 in our system (Fig. 4Gob), whereas Murali-Krisha et al. (6) found a drop by a factor of 4–5. This relatively small disparity could be explained by the different methodology, the different LCMV strain (WE vs Armstrong) used, and/or the different virus dose (200 pfu vs 105 pfu) injected.

The small degree of virus-specific CD4 T cell expansion found here might be due to 1) a lower frequency of virus-specific T helper cells when compared with CD8 T cells, 2) differences in viral Ag presentation, and/or 3) a lower potential of CD4 memory T cells to proliferate in vivo. Recently, the frequency of CD4 memory T cells has been determined in the LCMV system by two groups using limiting dilution analysis, ELISPOT, and intracellular IFN-{gamma} expression (24, 25, 26). These studies revealed frequencies of LCMV-specific CD4 T cells ranging from 1/10 to 1/600 CD4 T cells during the acute infection and from 1/100 to 1/1200 CD4 T cells in long-term memory. Thus, in comparison to CD8 T cells, 10- to 100-fold lower numbers of LCMV-specific CD4 T cells are induced in the course of a LCMV infection. Therefore, the selective expansion of CD8 T cells in the transfer system described here using LCMV is readily explained by the higher frequency of LCMV-specific T cells in the CD8 subset when compared with CD4 T cells.

Frequencies of VSV or VV-specific CD4 or CD8 T cells have not yet been determined precisely. Our data revealed vigorous CD8 T cell expansion but only minimal Ag-specific proliferation of CD4 T cells after VSV or VV infection. This finding may also indicate that in these viral infections the numbers of the induced VSV- or VV-specific CD8 T cells are significantly higher than of the corresponding CD4 T cells.

The result that adoptively transferred T cells from virus-immune mice did not expand after infection with the unrelated viruses indicated that bystander proliferation induced by IFNs was not sufficient to induce detectable T cell expansion in this system. This finding appears to be in contrast with a previous study by Tough et al. (22), who reported induction of bystander proliferation of CD8 memory T cells by viral infections. However, it is important to note that bystander proliferation of memory CD8 T cells was examined by Tough et al. using the 5-bromo-2'-deoxyuridine (BrdU) incorporation technique. Doubling of cells is easily detected by this method, whereas in our system doubling of cells would not result in significant clonal expansion in the context of the vigorous proliferation of the Ag-specific CD8 T cells.

In initial experiments we directly transferred LCMV immune spleen cells from B6 (Thy1.2) into B6.PL-Thy-1a (Thy1.1) mice. Clonal expansion of the transferred T cells after infection was similar to that described above; however, donor T cells disappeared rapidly in some recipient mice, suggesting rejection of donor T cells by the host immune system due to allelic Thy1- and/or other unknown differences of the two "congenic" mouse strains used.

In conclusion, we describe a simple transfer system which allows us to visualize a polyclonal virus-specific T cell response in vivo. This experimental approach may also be useful to directly follow T cell responses against other infectious agents or autoantigens where the epitopes recognized by T cells are not yet well characterized. By using different Thy1 alleles of donor and recipient mice, Ag-specific memory T cells can easily be isolated for further characterization.


   Acknowledgments
 
We thank S. Batsford for comments on the manuscript, M. Rawiel for excellent technical assistance, and S. Denkler and T. Imhof for animal husbandry.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Pi-295/2–1). Back

2 Address correspondence and reprint requests to Dr. Hanspeter Pircher, Department of Immunology, Institute for Medical Microbiology and Hygiene, Hermann-Herder-Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address: Back

3 Abbreviations used in this paper: tg, transgenic; LCMV, lymphocytic choriomeningitis virus; ELISPOT, enzyme-linked immunospot; B6, C57BL/6 mice; pfu, plaque- forming units; VSV, vesicular stomatitis virus; VV, vaccinia virus. Back

Received for publication December 17, 1998. Accepted for publication February 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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