T Cells + and CD4 + Percentage of Virus-Specific CD8 NK Markers ...

NK Markers Are Expressed on a High Percentage of Virus-Specific CD8ⴙ and CD4ⴙ T Cells1 Mark K. Slifka,2 Robb R. Pagarigan, and J. Lindsay Whitton NK cells have been phenotypically defined by the expression of specific markers such as NK1.1, DX5, and asialo-GM1 (ASGM1). In addition to NK cells, a small population of CD3ⴙ T cells has been shown to express these markers, and a unique subpopulation of NK1.1ⴙCD3ⴙ T cells that expresses an invariant TCR has been named “NKT cells.” Here, we describe NK marker expression on a broad spectrum of MHC class I- and MHC class II-restricted T cells that are induced after acute viral infection. From 5 to >500 days post lymphocytic choriomeningitis virus (LCMV) infection, more than 90% of virus-specific CD8ⴙ and CD4ⴙ T cells coexpress one or more of these three prototypical NK markers. Furthermore, in vivo depletion of NK cells with anti-ASGM1 Ab resulted in the removal of 90% of virus-specific CD8ⴙ T cells and 50 – 80% of virus-specific CD4ⴙ T cells. This indicates that studies using in vivo depletion to determine the role of NK cells in immune defense could potentially be misinterpreted because of the unintended depletion of Ag-specific T cells. These results demonstrate that NK Ags are widely expressed on the majority of virus-specific T cells and indicate that the NK and T cell lineages may not be as distinct as previously believed. Moreover, the current nomenclature defining NKT cells will require comprehensive modification to include Ag-specific CD8ⴙ and CD4ⴙ T cells that express prototypical NK Ags. The Journal of Immunology, 2000, 164: 2009 –2015. atural killer cells play an important role in the innate immune response against a variety of microbial pathogens (1– 4), and NK cell deficiencies in humans often result in an increased susceptibility to both bacterial and viral infections (5, 6). Surface Ags such as NK1.1, DX5, and asialo-GM1 (ASGM1)3, thought to be specific for NK cells, have been used to characterize the effector functions of this lymphocyte population. However, these markers are not exclusively expressed by NK cells. For example, ASGM1 expression has also been found on naive CD8⫹ T cells (7) and antiviral CD8⫹ T cells (8, 9), but expression appears to vary depending on the type of stimulation that they receive (10). In addition, conflicting studies have reported that alloreactive CD8⫹ T cells are either ASGM1⫹ (11) or ASGM1⫺ (10, 12). NK1.1 is expressed by a unique subpopulation of CD3⫹ T cells, termed NKT cells, that represent ⬍2% of T lymphocytes (13). These cells have a limited TCR repertoire (V␣14 J␣281; Refs 14 –16) and therefore cannot be the source of the polyclonal T cell response typically seen during viral infection (17–19). DX5 is a marker that is used extensively to identify NK cells in mouse strains that do not express NK1.1 (e.g., BALB/c), and the expression of this NK marker has been observed on only a small number of splenic T cells (20). These published studies

have identified only small populations of NK-marker⫹ T cells and have not directly quantitated the extent of NK marker expression. Moreover, they have focused only on CD8⫹ T cells, and the expression of NK markers on CD4⫹ T cells has not been explored. Here, we used flow cytometry to quantitate the expression of all three prototypical NK markers on virus-specific and nonspecific CD8⫹ T cells and CD4⫹ T cells at each stage of the antiviral immune response. Our study demonstrates that these “NK markers” are found on a remarkably high number of virus-specific CD8⫹ and CD4⫹ T cells. In addition, these T cells are depleted in vivo by administration of anti-ASGM1 Ab, a procedure commonly believed to specifically remove NK cells. Based on these results, the present definition of NKT cells will require considerable restructuring to include Ag-specific T cells that express the NK markers DX5, NK1.1, and ASGM1.

Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037

Peptides and in vitro stimulation

N

Received for publication October 15, 1999. Accepted for publication December 1, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health (NIH) Grant AI-27028 to J.L.W. M.K.S. was supported by NIH Training Grant T32 MH-19185-09. This is manuscript number 12625-NP from the Scripps Research Institute. 2 Address correspondence and reprint requests to Dr. Mark K. Slifka, Department of Neuropharmacology, CVN-9, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] 3 Abbreviations used in this paper: ASGM1, asialo-GM1; LCMV, lymphocytic choriomeningitis virus.

Copyright © 2000 by The American Association of Immunologists

Materials and Methods Virus and mice C57BL/6 mice (5–15 wk of age) were injected i.p. with 2 ⫻ 105 PFU of lymphocytic choriomeningitis virus (LCMV)-Armstrong (Arm-53b) and were used at the indicated time points. C57BL/6 mice were either purchased from The Jackson Laboratory (Bar Harbor, ME) or obtained from The Scripps Research Institute breeding facility.

HPLC-purified (⬎95% pure) MHC class I epitope peptides, GP33– 41 (KAVYNFATM), NP396 – 404 (FQPQNGQFI), GP276 –286 (SGVENPG GYCL), and NP205–212 (YTVKYPNL), and MHC class II epitope peptides NP309 –328 (SGEGWPYIACRTSIVGRAWE), and GP61– 80 (GLKGP DIYKGVYQFKSVEFD) were purchased from Peptidogenic (Livermore, CA), or synthesized at The Scripps Research Institute Core Facility and stored at ⫺80°C until use. Peptides were pooled and used at the following concentrations: 1 ⫻ 10⫺7 M (of each peptide) to stimulate CD8⫹ T cells or 1 ⫻ 10⫺5 M (of each peptide) to stimulate CD4⫹ T cells. Spleen cells (2 ⫻ 106/well) from LCMV-infected or naive mice were cultured at 37°C, 6% CO2 for 6 h in the presence or absence of pooled peptide in RPMI 1640 containing 10% FBS, 20 mM HEPES, L-glutamine, and antibiotics. Brefeldin A (Sigma, St. Louis, MO) was added at a final concentration of 2 ␮g/ml. No IFN-␥ production was observed after stimulation of 0022-1767/00/$02.00

2010

NK MARKERS ON VIRUS-SPECIFIC T CELLS

FIGURE 1. Expression of NK markers on virus-specific CD8⫹ T cells at 8 days postinfection. Adult C57BL/6 mice were infected i.p. with 2 ⫻ 105 PFU of LCMV-Armstrong. At 8 days postinfection, mice were euthanized, and spleen cells were harvested and cultured in the presence or absence of CD8 epitope peptides (GP33, NP396, GP276, and NP205) for 6 h directly ex vivo. The cells were surface-stained for CD8, and the NK markers DX5, NK1.1, ASGM1, or an irrelevant PE-conjugated Ab before they were fixed, permeabilized, and stained intracellularly for IFN-␥. Cytokine production by virusspecific CD8⫹ T cells was observed only in the presence of peptide, demonstrating that IFN-␥ synthesis was strictly peptide/MHC class I specific. Naive CD8⫹ T cells from uninfected mice did not produce IFN-␥ in the presence or absence of peptide stimulation, demonstrating that only virus-specific T cells are detected by this assay. Each dot plot is gated on CD8⫹ T cells, and the numbers represent the percentage of T cells in each quadrant. The data are representative of six independent experiments.

virus-specific T cells with an irrelevant peptide restricted by MHC class I (SMIKNLEYM; Ref. 21) or by MHC class II (VSV415– 433, SSKAQVFE HPHIQDAASQL; Ref. 22).

as the number of splenocytes required to exhibit 30% lysis of peptidecoated targets.

Results

Intracellular cytokine staining and flow cytometry After in vitro stimulation, cells were immediately placed on ice, washed, and stained overnight at 4°C with Ab combinations of CD8-cychrome, CD4-cychrome, NK1.1-PE, and/or DX5-biotin (followed by streptavidinPE) (PharMingen, San Diego, CA). Some samples were also stained with rabbit anti-ASGM1 (Cedarlane, Ontario, Canada) followed by polyclonal anti-rabbit-PE (PharMingen). The cells were washed and permeabilized using a Cytofix/Cytoperm kit (PharMingen) according to the manufacturer’s directions and stained with FITC-conjugated anti-IFN-␥ (PharMingen). Samples were resuspended in PBS containing 2% formaldehyde and acquired on a FACScan flow cytometer (100,000 –500,000 events acquired per sample) and analyzed using Cellquest software (Becton Dickinson, San Jose, CA). An irrelevant PE-conjugated Ab (Rat IgG1 PE isotype control, PharMingen,) or streptavidin PE alone (the secondary reagent for DX5 staining) was used to determine the quadrant line settings, and nonspecific staining with these negative controls was subtracted from the numbers described in the figures.

In vivo depletion of ASGM1⫹ cells C57BL/6 mice were infected with LCMV at 8 days or 105 days before the assay and were injected i.v. with 30 ␮L of rabbit polyclonal anti-ASGM1 (Cedarlane) or PBS in a total volume of 300 ␮l at 36 h and again at 24 h before harvest. 51

Cr release assays

At 8 days postinfection, direct ex vivo cytolytic activity against peptidecoated (1 ⫻ 10⫺7 M NP396 – 404 and 1 ⫻ 10⫺7 M GP33– 41) MC57 target cells was performed as previously described (23). Lytic units were defined

Expression of NK markers on activated and memory CD8⫹ T cells LCMV infection of adult mice is typically resolved within 1–2 wk postinfection, and it is well established that viral clearance is mediated by CD8⫹ T cells (24 –30). The peak of the cellular immune response against LCMV occurs at about 8 days postinfection, and, consistent with previous results (31–33), we found that ⬃60% of CD8⫹ T cells produced IFN-␥ after direct ex vivo stimulation with the four major CD8 epitope peptides (GP33, GP276, NP205, and NP396) (Fig. 1). IFN-␥ production was not observed in the absence of peptide, indicating that cytokine synthesis is not constitutive in virus-specific T cells and requires direct contact with specific Ag to be induced and maintained (33). No IFN-␥ production was observed after stimulation of virus-specific T cells with irrelevant MHC class I or class II peptides (data not shown). In addition, IFN-␥ production was not observed after peptide stimulation of naive T cells, indicating that only virus-specific T cells are detected by this assay (Fig. 1 and Refs. 32, 34, and 35). Expression of the three most commonly used NK markers (DX5, NK1.1, and ASGM1) was determined using flow cytometry. Approximately 6 – 8% of naive CD8⫹ T cells expressed DX5, 1–3% expressed NK1.1, and nearly 30% expressed ASGM1. In contrast, 30 – 40% of virus-specific (IFN-␥⫹) CD8⫹ T cells were positive for DX5 or NK1.1 and ⱖ 90% expressed ASGM1 at 8 days postinfection. The proportion of nonspecific (IFN-␥⫺) CD8⫹ T cells expressing NK markers also increased, although not to the degree observed with Ag-specific T cells (described in detail below). The addition of

The Journal of Immunology

2011

FIGURE 2. Expression of NK markers on virus-specific memory CD8⫹ T cells at 50 days postinfection. At 50 days post LCMV infection, spleen cells were cultured directly ex vivo in the presence or absence of CD8 epitope peptides for 6 h. The cells were then surface stained for CD8 and the NK markers before intracellular staining for IFN-␥. Virus-specific T cells were identified by IFN-␥ production in the presence of peptide stimulation. Each dot plot is gated on CD8⫹ T cells, and the numbers represent the percentage of T cells in each quadrant. The data are representative of five independent experiments.

brefeldin A during the in vitro stimulation period not only blocked cytokine secretion but also inhibited the transport of new molecules to the surface of the cells (data not shown). Consistent with this, the total percentage of CD8⫹ T cells expressing each NK marker remained essentially the same, both in the presence or absence of peptide stimulation. Thus, the high percentage of virusspecific T cells expressing NK markers is not the result of in vitro peptide stimulation, but is instead a reflection of the in vivo NKmarker⫹ status of these cells. CD8⫹ T cell expression of DX5, NK1.1, and ASGM1 was analyzed at 50 days postinfection to determine whether there were any differences in NK marker expression between activated and memory CD8⫹ T cells (Fig. 2). About 20% of memory CD8⫹ T cells expressed DX5, whereas NK1.1 was expressed on ⱖ50%,

and ASGM1 was expressed on 98% of the virus-specific memory T cells. This shows that, compared with activated T cells at 8 days postinfection, the percentage of virus-specific memory T cells expressing DX5 declined, whereas NK1.1 expression increased and ASGM-1 expression remained nearly unchanged. Expression of NK markers on activated and memory CD4⫹ T cells Similar to naive CD8⫹ T cells, 6 – 8% of naive CD4⫹ T cells expressed DX5, 1–3% expressed NK1.1, and ⬃20% expressed ASGM1 (Fig. 3). By 8 days postinfection with LCMV, between 15–20% of the CD4⫹ T cells in the spleen respond to LCMVspecific MHC class II peptides (Fig. 3; Refs. 34 and 35). At this time point, DX5 and NK1.1 were expressed on 40 – 60% of the

FIGURE 3. Expression of NK markers on virus-specific CD4⫹ T cells at 8 days postinfection. At 8 days postinfection, spleen cells were cultured in the presence or absence of the two known CD4 epitope peptides (GP61 and NP309) for 6 h directly ex vivo. The cells were surface-stained for CD4 and the NK markers DX5, NK1.1, and ASGM1, or an irrelevant PE-conjugated Ab before they were fixed, permeabilized, and stained intracellularly for IFN-␥. Cytokine production by virus-specific CD4⫹ T cells was observed only in the presence of peptide, demonstrating that IFN-␥ synthesis was peptide/MHC class II restricted. In vitro stimulation of naive CD4⫹ T cells from uninfected mice did not result in detectable levels of IFN-␥ production. Each dot plot is gated on CD4⫹ T cells, and the numbers represent the percentage of T cells in each quadrant. The data are representative of five independent experiments.

2012

NK MARKERS ON VIRUS-SPECIFIC T CELLS

FIGURE 4. Expression of NK markers on virus-specific memory CD4⫹ T cells. At 50 days post LCMV infection, spleen cells were cultured directly ex vivo in the presence or absence of CD4 epitope peptides for 6 h. The cells were then surface stained for CD4 and the NK markers before intracellular staining for IFN-␥. Virus-specific T cells were identified by IFN-␥ production in the presence of peptide stimulation. Each dot plot is gated on CD4⫹ T cells, and the numbers represent the percentage of T cells in each quadrant. The data are representative of five independent experiments.

peptide-specific CD4⫹ T cells, and nearly 90% of the Ag-specific CD4⫹ T cells expressed ASGM1. By 50 days postinfection, DX5 was still expressed on 35–50% of memory CD4⫹ T cells whereas NK1.1 was expressed on only 15–25% of the memory cells (Fig. 4). Almost 90% of the Ag-specific CD4⫹ T cells continued to express ASGM1. Together, these results indicate that virus-specific CD4⫹ T cells express prototypical NK markers at frequencies similar to those observed on virus-specific CD8⫹ T cells. Kinetics of NK marker expression on virus-specific and nonspecific T cells To further characterize the kinetics of NK marker expression on T cells during the course of acute viral infection, we determined the percentage of virus-specific T cells and nonspecific T cells expressing NK Ags at time points ranging from 5 days to 580 days postinfection (Fig. 5). Following acute LCMV infection, there was a rapid expansion of virus-specific CD8⫹ and CD4⫹ T cells that peaked at 8 days postinfection. In accord with previous studies (32, 35), virus-specific CD8⫹ T cells numbers declined 10- to 20-fold by 15 days postinfection, and virus-specific CD4⫹ T cell numbers dropped by 4- to 5-fold (Fig. 5, A and E). By 30 – 40 days postinfection, each virus-specific T cell population had declined to a plateau that was maintained essentially for the life of the immune animal. Fig. 5, B–D, and Fig. 5, F–H, show the percentage of virus-specific and nonspecific CD8⫹ and CD4⫹ T cells that express NK markers during each stage of the immune response. Compared with naive T cells, the percentage of virus-specific CD8⫹ T cells expressing DX5 increased 14-fold and peaked by 5 days postinfection before declining to a stable, but smaller, population of DX5⫹ cells by day 15. Nonspecific CD8⫹ T cells exhibited only a 4-fold increase in DX5 expression by day 5 before returning to near normal levels by about 15 days postinfection. In contrast to DX5, NK1.1 expression on Ag-specific CD8⫹ T cells increased by 40-fold at 15 days postinfection and then declined slowly over the following month. NK1.1 expression increased about 7-fold on nonspecific CD8⫹ T cells by 15 days postinfection and remained elevated in comparison with naive CD8⫹ T cells. Similar to previous observations (7), we found that ⬃30% of naive CD8⫹ T cells expressed ASGM1 (Fig. 1). In contrast, by 5 days postinfection and at all later time points examined, nearly 100% of virus-specific CD8⫹ T cells expressed this marker, whereas only 40 – 60% of the nonspecific CD8⫹ T cells in LCMV-infected mice expressed the ASGM1 Ag.

Acquisition of NK markers on virus-specific CD4⫹ T cells (Fig. 5, F–H) followed kinetics similar to those observed on virus-specific CD8⫹ T cells. Compared with naive CD4⫹ T cells, the percentage of virus-specific CD4⫹ T cells expressing DX5 increased 7-fold by 5 days postinfection, and the DX5 Ag was maintained on over half of the antiviral CD4⫹ T cells for ⬎500 days. The percentage of virus-specific CD4⫹ T cells expressing NK1.1 increased by 18-fold at 8 days postinfection before declining slowly over the next several months. ASGM1 expression on virus-specific CD4⫹ T cells was maximal by day 5 and was then maintained on 85–90% of antiviral CD4⫹ T cells at all other time points examined. In contrast to Ag-specific CD4⫹ T cells, NK marker expression on nonspecific CD4⫹ T cells was not greatly altered. On average, there was less than a 2- to 3-fold increase in NK marker expression on nonspecific CD4⫹ T cells. In vivo depletion of NK cells also depletes virus-specific T cells Since a surprisingly high percentage of virus-specific T cells expressed ASGM1, we determined whether these lymphocytes could be depleted in vivo with the same anti-ASGM1 Ab that is commonly used to deplete NK cells. Using the criterion of in vitro cytotoxicity, others have addressed this issue, but with conflicting results. One study found reduced CTL activity following antiASGM1 depletion of normal mice (10), whereas a second investigation suggested that such depletion is of concern only in previously immunosuppressed animals (9). Furthermore, these studies focused solely on CD8⫹ T cells. We chose to determine the effects of anti-ASGM1 depletion on both virus-specific CD4⫹ and CD8⫹ T cells; and we used flow cytometry to directly quantitate the frequency of NK marker expression in both groups, as well as evaluating the cytolytic activity of the latter population. LCMV-specific T cell responses were analyzed at either 8 days postinfection or 105 days postinfection to compare the susceptibility of activated and memory T cells to in vivo depletion. Based on flow cytometry, anti-ASGM1 treatment was reasonably effective at depleting NK cells; on average, about 75% of the NK1.1⫹CD3⫺ NK cells were removed by this procedure (data from representative mice are shown in Fig. 6A). However, NK1.1⫹CD3⫹ T cells also were severely depleted by this procedure, indicating that NK1.1⫹ T cells in LCMV-infected mice coexpress the ASGM1 Ag. We next evaluated the effects of anti-ASGM1 treatment on virus-specific T cells (Fig. 6, B–D). In mice infected 8 days previously, CD8⫹ T cellmediated CTL activity against peptide-coated targets was greatly

The Journal of Immunology

2013

FIGURE 5. Summary of CD8⫹ and CD4⫹ T cell responses, and NK marker expression, during and after virus infection. Adult C57BL/6 mice were infected with LCMV, and splenic T cells were assayed directly ex vivo for peptide-specific IFN-␥ production. The total number of virus-specific CD8⫹ T cells (A) and CD4⫹ T cells (E) per spleen were determined by IFN-␥ production after 6 h of in vitro stimulation. At the indicated time points, splenic CD8⫹ (B–D) or CD4⫹ (F–H) T cells were assayed for peptide-specific IFN-␥ production, and the percentages of virus-specific and nonspecific T cells expressing prototypical NK markers were determined. Day 0 indicates the percentage of naive CD8⫹ or CD4⫹ T cells that express the indicated NK marker. The data show the average and SD of 2–7 mice per time point after subtracting the background staining observed with the irrelevant Ab controls.

diminished after in vivo administration of anti-ASGM1 (Fig. 6B), and lytic units per 106 spleen cells were reduced by approximately 8-fold. In addition, we used intracellular cytokine staining to directly quantitate the efficiency of anti-ASGM1 depletion of Agspecific T cells, and found that about 90% of virus-specific CD8⫹ T cells and 50 – 80% of virus-specific CD4⫹ T cells were depleted by this treatment (Fig. 6, C and D). Both activated and memory T cells were enumerated, and both populations were greatly depleted in vivo using Abs to this NK marker.

Discussion In this study, we determined the frequency with which CD8⫹ and CD4⫹ T cells express the prototypical NK markers DX5, NK1.1, and ASGM1 following acute viral infection. In striking contrast to the small number of naive T cells that express NK Ags (15, 16), we identified all three NK markers on a remarkably high number of virus-specific T cells at time points as early as 5 days postinfection and as late as 580 days postinfection with LCMV. Following acute viral infection, the percentage of virus-specific T cells expressing NK markers was preferentially increased, but nonspecific T cells also demonstrated an increase in NK marker expression. In vivo depletion with anti-ASGM1 Ab removed ⬃75% of NK1.1⫹CD3⫺

NK cells and also resulted in a similar loss of virus-specific CD8⫹ and CD4⫹ T cells. Together, these results demonstrate that the majority of virus-specific T cells express one or more of the three most commonly used NK markers. In light of our observations and others (9, 10, 36), the results of in vivo NK cell depletion studies should be viewed conservatively unless the appropriate T cell controls have been included. Our results show that a high proportion of LCMV-specific and nonspecific T cells in C57BL/6 mice express NK markers following LCMV infection. The high-frequency expression of NK markers on Ag-specific T cells was not limited to the specific mouse strain or the virus used in this particular study. We examined DX5 and ASGM1 expression on virus-specific T cells in BALB/c mice following acute LCMV infection (the NK1.1 marker is not expressed in this mouse strain). DX5 was expressed on ⬎80% of LCMV-specific CD8⫹ T cells by 5 days postinfection in BALB/c mice, and on ⬎20% of the virus-specific memory T cells analyzed at ⬎100 days postinfection (data not shown). ASGM1 was expressed on 80 –100% of virus-specific CD8⫹ T cells in BALB/c mice at all time points examined. Furthermore, NK marker expression was not restricted to LCMV-specific T cells; recombinant vaccinia virus (rVV-NP) infection of BALB/c mice also resulted in

2014

FIGURE 6. In vivo depletion of virus-specific T cells expressing ASGM1. Polyclonal rabbit anti-ASGM1 Ab or sterile PBS was injected i.v. into LCMV-infected mice at 36 and 24 h before harvest. A, Flow cytometry was used to measure the depletion of traditional NK1.1⫹CD3⫺ NK cells after anti-ASGM1 injection. The dot plots show spleen cells from representative mice at 105 days postinfection that received either PBS or polyclonal anti-ASGM1 Ab. Approximately 75% of the total splenic NK cells were removed by this procedure. Note that the NK1.1⫹CD3⫹ T cell subset was also heavily depleted after anti-ASGM1 injection, indicating that these cells coexpress both NK1.1 and ASGM1. B, Direct ex vivo lysis of peptidecoated MC57 cells was determined in a standard 5-h 51Cr release assay using spleen cells from mice at 8 days postinfection. Depletion of ASGM1⫹ cells (squares) resulted in about 8-fold lower cytolytic activity compared with PBS-treated controls (circles). Filled symbols represent Agspecific lysis of peptide-coated targets, and open symbols represent nonspecific lysis of uncoated targets. C and D, Peptide-specific IFN-␥ production was determined after 6 h of in vitro stimulation with CD8 epitope peptides (C) or CD4 epitope peptides (D) using spleen cells from mice at either 8 days or 105 days postinfection. The data show the average and SD of 3 mice per group at 8 days postinfection and 2 mice per group at 105 days postinfection.

high-level DX5 and ASGM1 expression on virus-specific CD8⫹ T cells (M. K. Slifka and J. L. Whitton, unpublished results). This suggests that the high expression of NK markers on virus-specific T cells, especially during the early stages of infection, may be much more common than previously believed. Following acute LCMV infection, NK markers were preferentially expressed on virus-specific T cells (Fig. 5), although a substantial number of nonspecific CD8⫹ and CD4⫹ T cells also showed an increase in NK marker expression. Up-regulation of NK markers was more pronounced on nonspecific CD8⫹ T cells than on nonspecific CD4⫹ T cells. One explanation for this result may be the differences in the overall expansion of these two T cell subsets. CD8⫹ T cells undergo a massive expansion during the course of LCMV infection in which ⱖ50% of the CD8⫹ T cells are specific for viral peptide Ags by 8 days postinfection (Fig. 1 and Refs. 31–33). In contrast, CD4⫹ T cells undergo a more modest

NK MARKERS ON VIRUS-SPECIFIC T CELLS expansion, resulting in 15–20% of the CD4⫹ T cell population becoming virus-specific by 8 days postinfection (Fig. 3 and Refs. 34 and 35). At the peak of the antiviral immune response, the total number of virus-specific CD4⫹ T cells per spleen is roughly 10fold lower than the number of CD8⫹ T cells (Fig. 5). We speculate that the more extensive expansion of a particular T cell subset may correlate with an increase in NK marker expression. This may be due, in part, to bystander activation (37). Studies are underway to determine whether the number of cell divisions and/or the cytokine milieu play a role in the induction of NK marker expression on CD8⫹ and CD4⫹ T cells. NKT cells have been considered a novel lymphoid lineage that is distinct from T cells and NK cells. In mice, NKT cells have been characterized as NK1.1⫹ cells that express an invariant Ag receptor encoded by V␣14 and J␣281 (15, 16) in association with a variety of V␤ genes, but mainly V␤8.2 (38, 39). These cells can be stimulated with anti-CD3 or the CD1-restricted Ag, ␣-galactosylceramide (40). In contrast, the virus-specific “NKT cells” described in this study have polymorphic TCR, express either the CD8 or CD4 coreceptors, and have specificity for several distinct viral peptide Ags that are either MHC class I or MHC class II restricted. For these reasons, it is unlikely that the “NKT cells” generated after LCMV infection are similar to V␣14 NKT cells. It will be important to determine the functional and phenotypic characteristics of each of these subsets of NKT cells and clarify their role in the immune response to a variety of Ags. Moreover, the “NKT cell” nomenclature will require extensive revision to distinguish between these different lymphocyte populations. This study demonstrates that the great majority of virus-specific CD8⫹ and CD4⫹ T cells express one or more prototypical NK markers at every stage of the antiviral immune response. Although ␣␤ T cells and NK cells are quite distantly related in terms of lineage commitment, our results suggest that, at least phenotypically, these two cell types have a great deal in common. Other NK receptors such as KIRs (killer inhibitory receptors) have also been identified on T cells (41), and it will be interesting to learn what role these receptors play in T cell-mediated immunity. In addition to expanding our phenotypic analysis, we are currently investigating whether virus-specific T cells that express NK markers also respond to signals known to regulate NK cell activity. Together, these studies will help clarify the important functional roles of T cells, NK cells, and NKT cells in providing protective antimicrobial immunity.

Acknowledgments We thank Annette Lord for excellent secretarial support and Dr. Rolf Kiessling for critical review of the manuscript.

References 1. Welsh, R. M., and R. M. Zinkernagel. 1977. Heterospecific cytotoxic cell activity induced during the first three days of acute lymphocytic choriomeningitis virus infection in mice. Nature 268:646. 2. Kiessling, R., E. Eriksson, L. A. Hallenbeck, and R. M. Welsh. 1980. A comparative analysis of the cell surface properties of activated vs. endogenous mouse natural killer cells. J. Immunol. 125:1551. 3. Biron, C. A. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9:24. 4. Lanier, L. L. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359. 5. Biron, C. A., and R. T. Gazzinelli. 1995. Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome. Curr. Opin. Immunol. 7:485. 6. Biron, C. A., K. S. Byron, and J. L. Sullivan. 1989. Severe herpesvirus infections in an adolescent without natural killer cells. N. Engl. J. Med. 320:1731. 7. Lee, U., K. Santa, S. Habu, and T. Nishimura. 1996. Murine asialo GM1⫹CD8⫹ T cells as novel interleukin-12-responsive killer T cell precursors. Jpn. J. Cancer Res. 87:429. 8. Parker, S. E., Y. H. Sun, and D. W. Sears. 1988. Differential expression of the ASGM1 antigen on anti-reovirus and alloreactive cytotoxic T lymphocytes (CTL). J. Immunogenet. 15:215.

The Journal of Immunology 9. Doherty, P. C., and J. E. Allan. 1987. Anti-asialo GM-1 eliminates both inflammatory process and cytotoxic T-cell function in the lymphocytic choriomeningitis adoptive transfer model. Cell. Immunol. 107:1. 10. Stitz, L., J. Baenziger, H. Pircher, H. Hengartner, and R. M. Zinkernagel. 1986. Effect of rabbit anti-asialo GM1 treatment in vivo or with anti-asialo GM1 plus complement in vitro on cytotoxic T cell activities. J. Immunol. 136:4674. 11. Charley, M. R., A. Mikhael, J. Hackett, V. Kumar, and M. Bennett. 1988. Mechanism of anti-asialo GM1 prevention of graft-vs-host disease: identification of allo-antigen activated T cells. J. Invest. Dermatol. 91:202. 12. Kasai, M., M. Iwamori, Y. Nagai, K. Okumura, and T. Tada. 1980. A glycolipid on the surface of mouse natural killer cells. Eur. J. Immunol. 10:175. 13. Bendelac, A. 1995. Mouse NK1⫹ T cells. Curr. Opin. Immunol. 7:367. 14. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for V␣14 NKT cells in IL-12mediated rejection of tumors. Science 278:1623. 15. Makino, Y., R. Kanno, T. Ito, K. Higashino, and M. Taniguchi. 1995. Predominant expression of invariant V␣14⫹ TCR ␣ chain in NK1.1⫹ T cell populations. Int. Immunol. 7:1157. 16. Lantz, O., and A. Bendelac. 1994. An invariant T cell receptor ␣ chain is used by a unique subset of major histocompatibility complex class I-specific CD4⫹ and CD4⫺8⫺ T cells in mice and humans. J. Exp. Med. 180:1097. 17. Yanagi, Y., A. Tishon, H. Lewicki, B. A. Cubitt, and M. B. A. Oldstone. 1992. Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule. J. Virol. 66:2527. 18. Lin, M. Y., and R. M. Welsh. 1998. Stability and diversity of T cell receptor repertoire usage during lymphocytic choriomeningitis virus infection of mice. J. Exp. Med. 188:1993. 19. Sourdive, D. J., K. Murali-Krishna, J. D. Altman, A. J. Zajac, J. K. Whitmire, C. Pannetier, P. Kourilsky, B. Evavold, A. Sette, and R. Ahmed. 1998. Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection. J. Exp. Med. 188:71. 20. Ortaldo, J. R., R. Winkler-Pickett, A. T. Mason, and L. H. Mason. 1998. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3⫹ cells. J. Immunol. 160:1158. 21. von Herrath, M. G., B. Coon, H. Lewicki, H. Mazarguil, J. E. Gairin, and M. B. A. Oldstone. 1998. In vivo treatment with a MHC class I-restricted blocking peptide can prevent virus-induced autoimmune diabetes. J. Immunol. 161: 5087. 22. Burkhart, C., G. Freer, R. Castro, L. Adorini, K. H. Wiesmuller, R. M. Zinkernagel, and H. Hengartner. 1994. Characterization of T-helper epitopes of the glycoprotein of vesicular stomatitis virus. J. Virol. 68:1573. 23. Whitton, J. L., and A. Tishon. 1990. Use of CTL clones in vitro to map CTL epitopes. In Animal Virus Pathogenesis: A Practical Approach. M. B. A. Oldstone, ed. Oxford University Press, Oxford, p. 104. 24. Byrne, J. A., and M. B. A. Oldstone. 1984. Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus in vivo. J. Virol. 51:682. 25. Moskophidis, D., S. P. Cobbold, H. Waldmann, and F. Lehmann-Grube. 1987. Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2⫹ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response. J. Virol. 61:1867.

2015 26. Whitton, J. L. 1990. Lymphocytic choriomeningitis virus CTL. Sem. Virol. 1:257. 27. Fung-Leung, W. P., T. M. Kundig, R. M. Zinkernagel, and T. W. Mak. 1991. Immune response against lymphocytic choriomeningitis virus infection in mice without CD8 expression. J. Exp. Med. 174:1425. 28. Walsh, C. M., M. Matloubian, C. C. Liu, R. Ueda, C. G. Kurahara, J. L. Christensen, M. T. Huang, J. D. Young, R. Ahmed, and W. R. Clark. 1994. Immune function in mice lacking the perforin gene. Proc. Natl. Acad. Sci. USA 91:10854. 29. Kagi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, and H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31. 30. Shen, H., M. K. Slifka, M. Matloubian, E. R. Jensen, R. Ahmed, and J. F. Miller. 1995. Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity. Proc. Natl. Acad. Sci. USA 92:3987. 31. Butz, E. A., and M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⫹ T cells during an acute virus infection. Immunity 8:167. 32. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177. 33. Slifka, M. K., F. Rodriguez, and J. L. Whitton. 1999. Rapid on/off cycling of cytokine production by virus-specific CD8⫹ T cells. Nature 401:76. 34. Varga, S. M., and R. M. Welsh. 1998. Detection of a high frequency of virusspecific CD4⫹ T cells during acute infection with lymphocytic choriomeningitis virus. J. Immunol. 161:3215. 35. Whitmire, J. K., M. S. Asano, K. Murali-Krishna, M. Suresh, and R. Ahmed. 1998. Long-term CD4 Th1 and Th2 memory following acute lymphocytic choriomeningitis virus infection. J. Virol. 72:8281. 36. Ehl, S., R. Nuesch, T. Tanaka, M. Myasaka, H. Hengartner, and R. M. Zinkernagel. 1996. A comparison of efficacy and specificity of three NK depleting antibodies. J. Immunol. Methods 199:149. 37. Sprent, J., X. Zhang, S. Sun, and D. Tough. 1999. T-cell turnover in vivo and the role of cytokines. Immunol. Lett. 65:21. 38. Budd, R. C., G. C. Miescher, R. C. Howe, R. K. Lees, C. Bron, and H. R. Macdonald. 1987. Developmentally regulated expression of T cell receptor ␤ chain variable domains in immature thymocytes. J. Exp. Med. 166:577. 39. Fowlkes, B. J., A. M. Kruisbeek, H. Ton-That, M. A. Weston, J. E. Coligan, R. H. Schwartz, and D. M. Pardoll. 1987. A novel population of T-cell receptor ␣␤-bearing thymocytes which predominantly expresses a single V␤ gene family. Nature 329:251. 40. Cui, J., N. Watanabe, T. Kawano, M. Yamashita, T. Kamata, C. Shimizu, M. Kimura, E. Shimizu, J. Koike, H. Koseki, Y. Tanaka, M. Taniguchi, and T. Nakayama. 1999. Inhibition of T helper cell type 2 cell differentiation and immunoglobulin E response by ligand-activated V␣14 natural killer T cells. J. Exp. Med. 190:783. 41. Mingari, M. C., A. Moretta, and L. Moretta. 1998. Regulation of KIR expression in human T cells: a safety mechanism that may impair protective T-cell responses. Immunol. Today 19:153.