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The Journal of Immunology, 2001, 167: 2437-2440.
Copyright © 2001 by The American Association of Immunologists

Cutting Edge

Cutting Edge: Culture with High Doses of Viral Peptide Induces Previously Unprimed CD8+ T Cells to Produce Cytokine1

Janice M. Riberdy2, Amy Zirkel, Sherri Surman, Julia L. Hurwitz and Peter C. Doherty

Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, 38105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Culturing naive T cells with 50 µM selected HIV-1 envelope peptides for 6 days in the presence of IL-2 drives the emergence of a substantial CD8+ population that secretes IFN-{gamma} following short-term stimulation with 1 µM peptide. This response is H-2Kb restricted, epitope specific, and requires the continuing presence of peptide. The same effect was found for known H-2Db-restricted peptides from two influenza virus proteins. The great majority of these influenza-specific CD8+IFN-{gamma}+ T cells neither stained with the cognate tetramer nor expressed the TCR V{beta} bias that is characteristic of the CD8+ set expanded in vivo during an infection. Thus, multipoint binding of low affinity TCRs on naive CD8+ T cells can drive peptide-specific cytokine production. However, at least for two influenza-derived epitopes, the avidity of the TCR-MHC peptide interaction appears to be insufficient to stabilize a tetrameric complex of MHC class I glycoprotein plus peptide on the lymphocyte surface.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The analysis of cell-mediated immunity has been revolutionized by the development of tetrameric complexes of MHC class I glycoprotein plus peptide (tetramers) for the direct staining of Ag-specific CD8+ T cells (1, 2). However, before this approach can be used, it is obviously necessary to know the identity of both the non-self peptide and the self MHC class I allele. Another method of detecting Ag-specific T cells involves short-term (5-h) stimulation of activated/memory CD8+ T cells in vitro with cognate peptide in the presence of brefeldin A. This treatment induces the production of cytokines that can be detected in fixed and permeabilized cells. When used to quantify virus-specific CD8+ T cell responses by measuring IFN-{gamma} production, the flow cytometric peptide/IFN-{gamma} (Pep{gamma})3 assay generally gives results that are comparable to those found by tetramer staining (2, 3).

Intracellular cytokine staining has been successfully used by our laboratory to identify five epitopes in H-2b mice infected with the murine gammaherpesvirus 68 and two novel epitopes derived from influenza A viruses (DbPA224–233 and KbPB1703–711; Refs. 4, 5, 6). The present experiments started with efforts to define the CD8+ T cell response in H-2b mice primed and boosted with HIV-1 envelope glycoproteins in the form of a DNA vaccine, followed by infection with a recombinant vaccinia virus (Vacc) (7, 8). To identify candidate epitopes, we used a 6-day in vitro culture system that was intended to expand any epitope-specific CD8+ T cells before screening our panel of HIV-1 envelope peptides with the Pep{gamma} assay. Two peptides from different regions of the HIV-1 envelope molecule elicited substantial IFN-{gamma} production. The surprising finding was that these HIV-1 envelope peptides were equally effective at stimulating CD8+ T cells from mice that had not previously been exposed to the HIV envelope glycoprotein. The effect was analyzed further with known influenza virus peptides. The experiments provide insight into the nature of the naive CD8+ T cell repertoire and provide a cautionary note for using the Pep{gamma} assay with cultured cells to identify novel immunogenic epitopes.


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

Female C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions. Experiments were done with mice that were first "rested" under specific pathogen-free conditions for at least 1 mo before being used as "naive" lymphocyte donors at 10–12 wk of age. Some mice were primed with DNA encoding sequences of the HIV-1 envelope gp140 molecule by immunizing i.m. with 100 µg DNA. Primed mice were boosted i.p. 30–60 days later with 107 PFU of a Vacc construct (Vacc-gp140) encoding the same HIV gp140 (7, 8). Influenza memory mice had been infected intranasally (i.n.) 1–6 mo previously with 106.8 egg infectious doses of the A/HKx31 influenza A virus (referred to hereafter as HKx31) (3).

Cultures

The responder lymphocyte populations were derived from single-cell suspensions of spleen that were treated with Gey’s solution to lyse RBCs and were then incubated for 1 h at 37°C on flasks that had been precoated with goat anti-mouse Ig to remove B cells and adhere macrophages. The APCs were prepared by incubating naive, RBC-depleted splenocytes with a mAb to Thy 1.2 (AT83) for 30 min on ice, followed by washing and a 1-h incubation at 37°C in a mix of rabbit and guinea pig complement (Cedarlane Laboratories, Hornby, Ontario, Canada) diluted in HBSS (Life Technologies, Grand Island, NY). A total of 1 ml rabbit and 5 ml guinea pig complement was added to a final volume of 40 ml in HBSS, and <0.1% CD3+ T cells remained after this treatment. The APCs were irradiated with 2500 rad, then 0.5–1 x 106 responders and 1 x 106 APCs were cultured for 6 days (unless otherwise noted) in 2 ml complete S-MEM (Life Technologies) incorporating 10 U/ml recombinant human IL-2 (R&D Systems, Minneapolis, MN) and graded doses (50–0.0005 µM) of peptide. All peptides were synthesized at the Hartwell Center for Biotechnology (St. Jude Children’s Research Hospital, Memphis, TN). Although the initial screening was done with unpurified mimotope peptide preparations, all experiments shown here used peptides purified by HPLC (70% purity). The HIV-1 envelope peptide sequences are LPCRIKQIINMWQEV for the 208 epitope and REKRAIGLGALFLGF for the 388 epitope. The full-length envelope sequences have been previously described (8). Control experiments were done using the influenza A virus nucleoprotein366–374 peptide (ASNENMETM; NP366) (9) and the influenza acid polymerase PA224–233 peptide (SSLENFRAYV; PA224) (5).

Flow cytometry

Cultured cells were harvested from the plates, washed, and incubated for another 5 h in the presence of brefeldin A plus peptide (2). The lymphocytes were then washed, blocked for 10 min on ice with purified anti-mouse CD16/CD32 (Fc{gamma}RIII/II; BD PharMingen, San Diego, CA), and stained for 20 min on ice with anti-CD8-tricolor (Caltag Laboratories, Burlingame, CA). Some were also stained with a FITC-conjugated mAb to the TCR {beta}-chain (H57-597; BD PharMingen). The T cells that were analyzed by the Pep{gamma} assay were then fixed in 1% paraformaldehyde, permeabilized in saponin, and stained with PE-conjugated anti-IFN-{gamma} (XMG1.2; BD PharMingen). Tetramer staining was performed by treating cultured cells with anti-CD16/CD32 for 10 min on ice, staining with anti-CD8-tricolor for 20 min on ice, washing, and incubating for 1 h at room temperature with either the PE-conjugated tetrameric complex of H-2Db and NP366 or the PE-conjugated tetrameric complex of H-2Db and PA224 (3, 5). All tetramers were made at the St. Jude Children’s Research Hospital Tetramer Facility. To test for TCR V{beta} gene usage, viable IFN-{gamma}+ cells were isolated from the cultures (see following section for purification) and were stained for 20 min on ice with CD8-tricolor and either V{beta}8.3-FITC or V{beta}7.1-FITC (BD PharMingen).

Separation of IFN-{gamma}-producing cells

IFN-{gamma}-producing CD8+ T cells were recovered from 6-day cultures using a mouse IFN-{gamma} cell enrichment and detection kit (Miltenyi Biotec, Auburn, CA). Briefly, live cells were purified on a gradient and then labeled with a chimeric capture Ab that binds to a cell surface protein and captures IFN-{gamma} secreted during a 45-min incubation at 37°C. The cells were then washed and coated with a PE-conjugated mAb to IFN-{gamma}. Finally, cells were incubated with anti-PE microbeads and enriched magnetically.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Characterization of CD8+ T cell responses to HIV-1 envelope peptides

More than 400 overlapping 15-mers (10) spanning four different HIV-1 envelope sequences were tested by culturing lymphocytes from DNA/Vacc-gp140-primed mice with pools of 10 peptides at 50 µM. The final concentration for each 15-mer was 5 µM, a level that (under these conditions) effectively stimulates immune T cells specific for known peptides. This led to the identification of nine candidates (data not shown) that stimulated optimally at 5–50 µM as individual peptides. The surprise came when, as a control, lymphocytes from naive mice were also cultured with two peptides that elicited the strongest responses, 208 (LPCRIKQIINMWQEV) and 388 (REKRAIGLGALFLGF). The levels of IFN-{gamma} staining for naive T cells cultured with the 208 and 388 peptides were equivalent to those found for the populations from vaccinated mice (Fig. 1Go).



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  		FIGURE 1. The CD8+IFN-{gamma}+ staining profiles of lymphocytes cultured in the presence (208) or absence (Nil) of 50 µM of the 208 peptide. Lymphocytes were stimulated with 1 µM peptide for analysis by the Pep{gamma} assay at the end of the 6-day culture. The B6 mice were either naive (upper panels) or had been immunized by a DNA/Vacc-gp140 prime and boost regime (lower panels).

 
Further analyses were done to address whether the IFN-{gamma} production by CD8+ T cells in the naive T cell cultures was peptide specific. Thus, lymphocytes were cultured for 6 days with 50 µM of 208 or 388 and then stimulated for 5 h with 1 µM of either peptide. The percentage of CD8+IFN-{gamma}+ T cells detected in the gated CD8+ set was 7.4% (for 208 cultures) and 2.6% (for 388 cultures) following exposure to the homologous peptide and 0.9% in each case after the heterologous stimulation. Repeat experiments with 208, 388, and other candidate peptides showed comparable specificity for a variety of peptide combinations. The responding IFN-{gamma}+ cells were also shown to be reproducibly TCR{alpha}{beta}+ (data not shown).

When single residue truncations were made from either the amino or carboxyl termini of the original 15-mers, maximum IFN-{gamma} production by naive CD8+ T cells was found for the IKQIINMWQEV and RAIGLGALFLGF peptides (208D and 388C, respectively; data not shown). Neither 208 nor 388 perfectly match the MHC class I binding motifs (11) for H-2Kb, XXXXF(Y)XXL(M)(I)(V), or H-2Db, XXXXNXXX(M)(I)(V). To map the MHC restriction element, truncated versions of 208 (IKQIINMWQEV) and 388 (RAIGLGALFLGF) were used to stimulate naive T cells from B10 (KbDb)-, B10.A(5R) (KbDd)-, and B10.A(4R) (KkDb)-congenic mice. Cultures with 388C clearly mapped to Kb (data not shown). Lymphocytes cultured with the truncated 208D peptide showed a Kb restriction in that the CD8+IFN-{gamma}+ response with 5R CD8+ T cells (4.6%) was greater than that for the 4R population (1.6%, data not shown). However, 1.6% CD8+IFN-{gamma}+ cells is slightly higher than our average background (generally <1%) and may suggest a small portion of Db reactivity.

Comparing naive and memory responses

An obvious question was whether these in vitro CD8+ T cell responses from naive mice (Fig. 1Go and data not shown) reflect some special property of MHC class I-peptide complexes when selected HIV-1 envelope peptides bind to H-2Kb. Thus, the analysis was repeated with the H-2Db-restricted NP366 and PA224 influenza peptides (5, 9) using lymphocytes from uninfected and virus-primed mice. Again, it was demonstrated that naive CD8+ T cells could be specifically stimulated to produce substantial amounts of IFN-{gamma} (Fig. 2Go). As expected, memory CD8+ T cells needed much less Ag. The concentration required for optimal stimulation of the naive set was 50 µM compared with 0.05 µM for the immune population (Fig. 2Go). A further difference between naive and immune T cells was the requirement for soluble peptide throughout the incubation period. When peptide was present continuously during the 6-day culture period, both naive and immune cultures exhibited a vigorous IFN-{gamma} response to PA224 and 208. In contrast, peptide-pulsed stimulators failed to induce an IFN-{gamma} response, except for the influenza-derived epitope PA224, in cultures using T cells from an influenza-immune mouse (Fig. 3Go). Thus, a much greater and sustained Ag dose is clearly required to drive these naive responses.



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  		FIGURE 2. Differential dose requirements and response profiles for CD8+IFN-{gamma}+ T cells from naive (upper panels) and immune (lower panels) mice following in vitro culture with the influenza A virus NP366 peptide. The peptide concentrations present at the beginning of the 6-day culture period are shown above the FACS plots, and the percentage of CD8+ T cells producing IFN-{gamma} is shown in the upper right quadrants. The immune mice had been infected i.n. with the HKx31 virus 6 wk previously.

 


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  		FIGURE 3. The continued presence of peptide is required for the stimulation of naive but not immune CD8+IFN-{gamma}+ T cells. Upper panels, The response following continuous culture with the 208 and PA224 peptides, as described in Materials and Methods. Lower panels, The data for lymphocytes exposed to APCs that were incubated with peptide for 2 h at 37°C, washed three times, and placed in culture. All cultures had a final concentration of 50 µM peptide or had APCs pulsed with 50 µM peptide except the immune PA224 cultures, in which 0.05 µM peptide was used. The immune mice had been primed i.n. with the HKx31 virus 6 wk previously.

 
Do the IFN-{gamma}-producing cells in the naive cultures reflect the properties of the virus-specific CD8+ set that would normally be induced by in vivo priming with respect to TCR affinity/avidity and V{beta} gene usage? This was found not to be the case. Although the percentage of CD8+IFN-{gamma}+ T cells was generally comparable to the percentage of CD8+tetramer+ cells in the immune cultures, the naive cultures did not show any staining with cognate tetramer above background, even though a substantial IFN-{gamma} response was clearly detected (Expt. 1, Table IGo). NP366- and PA244-specific cells generated in vivo during an influenza infection show a bias toward the use of V{beta}8.3+ and V{beta}7.1+ TCRs, respectively, with bias toward V{beta}7.1 in the PA224-specific population being much stronger than that seen for V{beta}8.3 in the NP366 response (5, 12). Whereas the CD8+IFN-{gamma}+ set from the immune cultures showed the appropriate V{beta} bias, this skewing of TCR usage was not found in the naive cultures (Expt. 2, Table IGo). Thus, only a minority of the CD8+ T cells in the naive cultures were selected to express the TCR profile that emerges in a virus-specific immune response. Furthermore, staining with the entire panel of available TCR V{beta} mAbs (purchased from BD PharMingen) did not indicate any obvious bias in the naive in vitro response (data not shown).


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  		Table I. Comparison of T cell phenotypes for lymphocytes cultured from naive or HKx31-immune B6 mice1

 
In conclusion, the combination of high peptide dose with 6 days of in vitro culture in the presence of IL-2 stimulates a diverse spectrum of low affinity/avidity CD8+ T cells. The effect obeys the rules for MHC restriction, but only a minority of these lymphocytes show the TCR V{beta} phenotypes or bind the tetramers that define the Ag-specific CD8+ set in adaptive immunity (3, 5, 9, 12). Clearly, other defined CTL epitopes need to be examined to ascertain whether or not any peptide can elicit such broad low affinity responses from naive CD8+ T cells. How this in vitro response to high doses of peptide relates to the in vivo situation is remains open to question. One intriguing possibility is that although these low affinity populations can produce ample amounts of IFN-{gamma} to specific stimuli, their ability to mediate other functions such as cytotoxicity may be influenced by a variety of parameters such as antigenic dose during the initial encounter with Ag, costimulation by the APC, and an ability to interact effectively with a target cell. Preliminary experiments (data not shown) have shown potent CTL activity when cells from influenza-immune mice were cultured with influenza-specific peptides and assayed on peptide-pulsed EL4 (H-2b) targets. In contrast, the comparable cultures from naive mice caused little, if any, 51Cr release in a 6-h assay. However, both naive and immune cell cultures showed potent cytoxicity when tested by the redirected CTL assay (13) using FcR+ P815 (H-2d) targets coated with an Ab to CD3{epsilon} (data not shown). Lastly, there is evidence that memory CD8+ T cells may show unpredicted cross-reactivity patterns (14). However, it is unlikely that the IFN-{gamma} production seen in the naive cell cultures is due to cross-reactive memory T cells, because CD8+ T cells sorted for the CD44low naive phenotype show the same pattern of reactivity under those culture conditions (data not shown).

Immune responses develop in defined microenvironments in which it is difficult to measure local Ag concentrations. Depending on the dose in a particular anatomical niche, a much broader spectrum of naive CD8+ T cells may be involved than those that ultimately emerge as the virus-specific effector/memory populations (3, 15). The phenotype of CD8+ T cells that respond during the initial phases of infection has generally been impossible to study because the frequency of such cells is too low to detect with current methodology. The in vitro culture system described here should allow us to further analyze both the plasticity of the naive T cell repertoire and the possible consequences of inducing such low affinity/avidity peptide-specific effector T cells.


   Acknowledgments
 
We thank Vicki Henderson for help in preparing this manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI45142, CA21765, and AI29579, and by the American Syrian Lebanese-Associated Charities. Back

2 Address correspondence and reprint request to Dr. Janice M. Riberdy, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis TN 38105. E-mail address: janice.riberdy{at}stjude.org Back

3 Abbreviations used in this paper: Pep{gamma}, peptide stimulation/IFN-{gamma}; Vacc, vaccinia virus; NP366, influenza nucleoprotein366–374 peptide; PA224, influenza acid polymerase PA224–233 peptide; i.n., intranasally. Back

Received for publication April 17, 2001. Accepted for publication July 10, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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