Lymphocytic choriomeningitis virus (LCMV), like many viruses, induces a profound activation and expansion of CD8+ T cells. In contrast, CD4+ T cells do not increase in total number during the acute infection. We show here that mice infected with LCMV have a low but detectable frequency (<1/300) of CD4+ T cells, as detected by IL-2 production in limiting dilution assays, to each of two class II peptides during the peak of the acute LCMV response and into long-term memory. However, during the peak of the acute CD4+ T cell response, >20% of the CD4+ T cells secreted IFN-γ after stimulation with PMA and ionomycin, and >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound Ag-specific CD4+ T cell response during viral infections.

Virus infections are potent stimulators of the immune system (1, 2, 3). During an acute lymphocytic choriomeningitis virus (LCMV)3 infection, the CD8+ T cells expand as much as 5- to 10-fold, resulting in a conversion of the CD4 to CD8 ratio from 2:1 to 1:2–3 (4). Coincident with this expansion is an increase in the frequency of virus-specific CTL precursors as detected by limiting dilution analysis (LDA) (5, 6). However, either due to a low efficiency or because a low percentage of Ag-specific cells are cytotoxic, LDA only accounts for a small fraction (5–10%) of the activated CD8+ T cells during the acute LCMV infection (6, 7). Nevertheless, because there is little expansion in the number of T cells not specific to the virus during infection (8), we have concluded that the great majority of activated cells during LCMV infection is virus specific. A similar conclusion has been reached in recent studies using MHC class I tetramers loaded with immunodominant viral peptides and intracellular staining for IFN-γ following virus-peptide stimulation (9, 10, 11). These techniques can now account for nearly 70% of the CD8+ T cells that respond during an acute LCMV infection (9).

Although usually not as vigorously stimulated as CD8+ T cells, CD4+ T cells also become activated and respond during acute viral infections (1). In the LCMV system, about 25% of the CD4+ T cells are blast-sized at day 7 postinfection (p.i.). In addition, 20 to 30% of the CD4+ T cells express increased cell surface levels of activation markers and adhesion molecules (12, 13, 14). Thus, a significant percentage of the CD4+ T cells are activated during the acute LCMV infection, but <1% of these cells scored in LDA as being virus specific (13). Here we show, by using a sensitive assay to measure IFN-γ production at the single cell level, that >10% of the CD4+ T cells are virus specific during an acute LCMV infection.

Male C57BL/6 (H-2b) mice from The Jackson Laboratory (Bar Harbor, ME) were inoculated i.p. with 7 × 104 plaque-forming units of LCMV, Armstrong strain, diluted 1:100 in PBS in 0.1 ml vol/mouse. Splenic leukocytes were obtained by preparing single-cell suspensions from spleens and treating them with 0.84% NH4Cl to lyse E (15). For CD4+ T cell cycle analysis, splenic leukocytes were stained with FITC-conjugated anti-CD4 (H129.19) from PharMingen (San Diego, CA). After washing in staining buffer (2% FCS and 0.02% NaN3 in PBS), the cells were subsequently fixed in ice-cold 95% ethanol, washed in PBS, and stained with propidium iodide (Sigma, St. Louis, MO). Approximately 1 × 106 cells were stained, and >20,000 events were acquired from each preparation. The data were analyzed using Cell Quest software (Becton Dickinson, San Jose, CA).

The LDA for determining LCMV-specific CD4+ Thp frequency has been described previously in detail (13). To determine the frequency of peptide-specific Thp, peritoneal exudate cells (PEC) were pulsed with 5 μg/ml of one of the two known LCMV class II-restricted peptides, GP61-80 and NP309-328 (16). Frequencies were calculated using χ2 analysis according to the method of Taswell (17) on a computer program kindly provided by Dr. Richard Miller (University of Michigan, Ann Arbor, MI).

Intracellular IFN-γ staining was performed based on modifications of recently published protocols (9, 18). To assess intracellular cytokine expression in cells activated nonspecifically, 2 × 106 spleen cells were cultured for 4 h in 50 ng/ml of PMA (Sigma) and 500 ng/ml of ionomycin (Sigma), with 10 μg/ml of Brefeldin A (Sigma) added for the final 2 h to enable intracellular proteins to accumulate. For Ag-specific expression of cytokines, splenocytes were cultured for 5 h at a concentration of 2 × 106 cells/tube in a volume of 1 ml of complete medium supplemented with 10 U/ml of recombinant mouse IL-2 (PharMingen) and 10 μg/ml of Brefeldin A in the presence (5 μg/ml) or absence of one of the two LCMV peptides. After 5-h culture, the cells were harvested, washed once in staining buffer, blocked with purified anti-FcγRII/III mAb (2.4G2; PharMingen) for 10 min on ice, and surface stained with either FITC-conjugated anti-CD4 (H129.19; PharMingen) or tricolor-conjugated anti-CD4 (CT-CD4; Caltag, Burlingame, CA) for 30 min on ice. In some experiments, the cells were also stained with FITC-conjugated anti-CD44 (IM7; PharMingen). The cells were then washed in staining buffer and fixed with 2% formaldehyde in PBS for 20 min at room temperature. Following two washes with permeabilization buffer (staining buffer containing 0.5% saponin, Sigma), the cells were resuspended in 100 μl of permeabilization buffer and incubated for 10 min at room temperature, followed by an additional 30-min incubation after the addition of phycoerythrin conjugated anti-mouse IFN-γ (XMG1.2; PharMingen) or an isotype control (IgG1; PharMingen). Cells treated with PMA and ionomycin were also stained with FITC-conjugated anti-mouse IL-4 (BVD4-1D11; PharMingen) or the appropriate isotype control (IgG2b; PharMingen). Approximately 2 × 106 cells were stained, and between 30,000 and 60,000 events were acquired from each sample. The data were analyzed using Cell Quest software (Becton Dickinson).

Table I shows the Thp frequencies for the two known LCMV class II-restricted peptides (16) during the peak of the acute CD4+ T cell response and into long-term memory. The stability of the virus-specific Thp frequencies up to 18 mo p.i. following stimulation with LCMV-infected PEC shown here extends on our previous work regarding the stability of CD4+ Thp frequencies into long-term memory (13). The Thp frequency to each of the two LCMV class II-restricted peptides dropped only 2- to 7-fold from the peak of the CD4+ T cell response into memory (Table I). Quantitation of virus-specific Thp frequencies using this established LDA method for IL-2 in several virus systems has yielded Thp frequencies that range from 1/300 to 1/2000 from the peak of the acute response into long-term memory (13, 19, 20, 21). Thus, <1% of the CD4+ T cells can be identified by LDA as Ag specific during these infections.

Table I.

LCMV-specific Thp frequencies to the LCMV class II-restricted peptides

Day p.i.Reciprocal of Thp Frequency\E
Uninfected APCLCMV-infected APCGP-pulsed APCNP-pulsed APC
10 >120,000 1,536 (1,191–2,160)a 931 (740–1,256) 5,460 (3,945–7, 242)\E 
450 47,551 (345,919–74,502) 1,505 (1,172–2,100) 2,055 (1,666–2,680) 10,762 (3,945–7,242)\E 
10 >240,000 5,402 (4,199–7,572) 1,423 (1,112–1,977) 3,785 (3,148–4,747)\E 
450 60,986 (33,657–324,385) 6,125 (4,665–8,912) 5,118 (4,137–6,707) 12,264 (9,265–18,134)\E 
10 22,243 (16,222–35,370) 1,073 (854–1,444) 301 (230–434) 2,028 (1,590–2,799)\E 
540 >240,000 1,288 (1,046–1,674) 2,108 (1,641–2,941) 6,859 (5,444–9,263) 
Day p.i.Reciprocal of Thp Frequency\E
Uninfected APCLCMV-infected APCGP-pulsed APCNP-pulsed APC
10 >120,000 1,536 (1,191–2,160)a 931 (740–1,256) 5,460 (3,945–7, 242)\E 
450 47,551 (345,919–74,502) 1,505 (1,172–2,100) 2,055 (1,666–2,680) 10,762 (3,945–7,242)\E 
10 >240,000 5,402 (4,199–7,572) 1,423 (1,112–1,977) 3,785 (3,148–4,747)\E 
450 60,986 (33,657–324,385) 6,125 (4,665–8,912) 5,118 (4,137–6,707) 12,264 (9,265–18,134)\E 
10 22,243 (16,222–35,370) 1,073 (854–1,444) 301 (230–434) 2,028 (1,590–2,799)\E 
540 >240,000 1,288 (1,046–1,674) 2,108 (1,641–2,941) 6,859 (5,444–9,263) 
a

95% confidence limits.

Although the total CD4+ T cell number remains relatively stable during the acute LCMV infection, there is an increase in the percentage of blast-sized CD4+ T cells and of CD4+ T cells expressing various activation and adhesion molecules (13). Using enzyme-linked immunospot (ELISPOT) assays (in the absence of added IL-2) to assess IFN-γ production at the single-cell level, we previously found that the frequency of sorted CD4+ T cells that produced IFN-γ when stimulated with LCMV-infected PEC was similar to the Thp frequency in the IL-2-based LDA (13). In contrast, Figure 1 shows that a much higher frequency (>20%) of CD4+ T cells from mice acutely infected with LCMV make IFN-γ when nonspecifically stimulated with PMA and ionomycin. We find no increase in the percentage of IL-4-producing CD4+ T cells during the acute LCMV infection, confirming that LCMV induces primarily a Th1 response (13, 22, 23). Analysis of DNA content by propidium iodide staining revealed an increase in the percentage of CD4+ T cells in the cell cycle. The mean percentages of CD4+ T cells in G2 + M or S phase from six individual mice per group were as follows: day 0, 2 ± 1%; day 7, 7 ± 2%, day 9, 6 ± 2%; day 11, 5 ± 1%; 7 mo, 2 ± 1%; 11 mo, 2 ± 1%; 19 mo, 2 ± 1%. Thus, there is a much larger percentage of CD4+ T cells that are expressing an activated cell phenotype, that are blast-sized and have entered the cell cycle, and that produce IFN-γ on stimulation, than can be accounted for by using LDA (Table I and 13 .

FIGURE 1.

Intracellular IFN-γ and IL-4 expression of virus-induced CD4+ T cells following stimulation with PMA and ionomycin. Splenocytes from the indicated days postinfection were stimulated with PMA and ionomycin for 2 h before the addition of Brefeldin A for an additional 2 h as described in Materials and Methods. The cells were gated on CD4+ cells, and the values shown represent the percentage of CD4+ cells that expressed a given cytokine. Means ± SDs for time points from two separate experiments with two individual mice per experiment are shown (n = 4/group).

FIGURE 1.

Intracellular IFN-γ and IL-4 expression of virus-induced CD4+ T cells following stimulation with PMA and ionomycin. Splenocytes from the indicated days postinfection were stimulated with PMA and ionomycin for 2 h before the addition of Brefeldin A for an additional 2 h as described in Materials and Methods. The cells were gated on CD4+ cells, and the values shown represent the percentage of CD4+ cells that expressed a given cytokine. Means ± SDs for time points from two separate experiments with two individual mice per experiment are shown (n = 4/group).

Close modal

The above experiments show that at least 20 to 25% of the CD4+ T cells produce IFN-γ when nonspecifically stimulated with PMA and ionomycin during the acute LCMV infection. The peptide-specific intracellular IFN-γ assay, displayed in Figure 2, shows that >10% of the CD4+ T cells at day 9 p.i. were specific for either of two class II-restricted peptides. CD4+ T cells produced no IFN-γ in the absence of peptide, and peptide-stimulated CD4+ T cells did not demonstrate any intracellular staining above background with the isotype control Ab. Figure 3 shows that virtually all of the GP61-80-specific CD4+ T cells that stained positive for IFN-γ were also CD44high, consistent with an activated cell phenotype (24). Figure 4 shows a time course in which the IFN-γ-producing cells peak at day 9 p.i. but are still detectable in long-term memory.

FIGURE 2.

Intracellular IFN-γ expression of LCMV peptide-specific CD4+ T cells. Splenocytes from LCMV-infected (day 9) C57BL/6 mice were stimulated with or without one of the two LCMV class II-restricted peptides in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. The numbers shown indicate the percentage of cells that fall into each gate. Data are representative of four experiments.

FIGURE 2.

Intracellular IFN-γ expression of LCMV peptide-specific CD4+ T cells. Splenocytes from LCMV-infected (day 9) C57BL/6 mice were stimulated with or without one of the two LCMV class II-restricted peptides in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. The numbers shown indicate the percentage of cells that fall into each gate. Data are representative of four experiments.

Close modal
FIGURE 3.

CD44 expression on peptide-specific CD4+ T cells staining positive for intracellular IFN-γ. Splenocytes from LCMV-infected (day 9) mice were cultured in vitro with GP61-80 in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. Top panel, numbers shown in the R1 gate indicate the percentage of splenocytes that are positive for the A isotype control mAb, B intracellular IFN-γ stain, or C control IFN-γ staining of cells from day 9-infected mice prestained with unlabeled anti-IFN-γ mAb. The histograms in the bottom panel (D and E) represent the cell surface expression of CD44 on the two gated populations from B. Data are representative of four separate experiments.

FIGURE 3.

CD44 expression on peptide-specific CD4+ T cells staining positive for intracellular IFN-γ. Splenocytes from LCMV-infected (day 9) mice were cultured in vitro with GP61-80 in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. Top panel, numbers shown in the R1 gate indicate the percentage of splenocytes that are positive for the A isotype control mAb, B intracellular IFN-γ stain, or C control IFN-γ staining of cells from day 9-infected mice prestained with unlabeled anti-IFN-γ mAb. The histograms in the bottom panel (D and E) represent the cell surface expression of CD44 on the two gated populations from B. Data are representative of four separate experiments.

Close modal
FIGURE 4.

Intracellular IFN-γ expression of LCMV peptide-specific CD4+ T cells. Splenocytes from the indicated days postinfection were stimulated with one of the two LCMV class II-restricted peptides in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. Background staining with the appropriate isotype-matched control mAb was subtracted from each individual. Means ± SDs for time points from four separate experiments with two individual mice per experiment are shown (n = 8/group).

FIGURE 4.

Intracellular IFN-γ expression of LCMV peptide-specific CD4+ T cells. Splenocytes from the indicated days postinfection were stimulated with one of the two LCMV class II-restricted peptides in the presence of IL-2 and Brefeldin A for 5 h as described in Materials and Methods. Background staining with the appropriate isotype-matched control mAb was subtracted from each individual. Means ± SDs for time points from four separate experiments with two individual mice per experiment are shown (n = 8/group).

Close modal

Quantitation of virus-specific Thp frequencies using LDA in several virus systems, including LCMV, has yielded Thp frequencies that range from 1/300 to 1/2000 from the peak of the acute response into long-term memory (13, 19, 20, 21). However, here we report that >10% of the CD4+ T cells are virus specific during an acute LCMV infection (Figs. 2 and 4). This result suggests that there is a more significant CD4+ T cell response during acute virus infections than may have previously been realized. There is a similar disparity in the frequency of virus-specific CD8+ T cells obtained using LDA vs using MHC class I tetramers loaded with immunodominant viral peptides or intracellular staining for IFN-γ following virus-peptide stimulation (9). These discrepancies may either be due to a low efficiency in LDA or because a low percentage of Ag-specific cells are CTL precursors or IL-2 producing Thp. Although earlier studies have detected significant frequencies (>40%) of CD4+ T cells expressing IL-2 mRNA by in situ hybridization (25), we have been unable to detect IL-2-secreting CD4+ T cells by FACS following peptide stimulation. In addition, a recent report (23) failed to detect IL-2-secreting CD4+ T cells by FACS following anti-CD3 treatment of splenocytes during LCMV infection. This result suggests that the frequency of virus-specific CD4+ T cells that secrete IL-2 may be lower than the frequency capable of producing IFN-γ.

In the present study with sensitive assays to measure IFN-γ production at the single-cell level, >20% of the CD4+ T cells secreted IFN-γ after stimulation with PMA and ionomycin, and >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV peptides. The discrepancy between these two numbers could be accounted for either by PMA and ionomycin stimulation detecting lower affinity responses or responses specific to undefined virus-encoded class II peptides. This discrepancy could also be due to a nonspecific bystander activation of CD4+ T cells, although this would seem unlikely based on other studies directly examining bystander activation (8).

We thank C. Kamperschroer for his helpful suggestions, Keith Daniels for his technical assistance, and Tammy Krumpoch and Barbara Fournier for help with the FACS analysis.

1

This work was supported by U.S. Public Health Service Training Grant AI07439 to S.M.V. and by Research Grants AI17672 and AR35506 to R.M.W.

3

Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; LDA, limiting dilution analysis; Thp, Th precursor; PEC, peritoneal exudate cell; p.i., postinfection.

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