Progressive Differentiation and Commitment of CD8⁺ T Cells to a Poorly Cytolytic CD8^low Phenotype in the Presence of IL-4

T cell preparation and analysis

All animal studies were approved by the Queensland Institute of Medical Research Animal Ethics Committee. Specific pathogen-free female C57BL/6 mice from the Animal Resources Centre (Western Australia) were used at 6–9 wk. Cell suspensions from brachial, axillary, inguinal, and lumbar lymph nodes were passed through stainless steel mesh, followed by Ficoll-Paque (Amersham Biosciences) separation. Cells were incubated with PE- or FITC-conjugated anti-CD8α Ab (53.6; BD Pharmingen) and biotinylated anti-CD44 Ab (IM7.81; BD Pharmingen), followed by FITC- or PE-conjugated streptavidin (Caltag Laboratories), respectively, then resuspended in balanced salt solution with 5% heat-inactivated FCS (CSL) and 1 μg/ml propidium iodide (Calbiochem). Viable naive cells were purified using a FACSVantage with Lysis II software (BD Biosciences) based on CD8⁺ and CD44^low (lowest 30%) expression; cells were >97% CD8⁺ on reanalysis. For cloning, individual cells were sorted twice, and on the second sort were deposited in 96-well round-bottom plates (Falcon; BD Biosciences) using an automated cell deposition unit attached to the FACSVantage. For purification of activated CD8^high and CD8^low cells, viable T cells were sorted for high (>80) and low or nil (<10) fluorescence intensity of CD8 expression. For analysis without sorting, a FACSCalibur was used with CellQuest V3.1f software (BD Biosciences).

Activation of CD8⁺ T cells with anti-CD3/8/11a Ab

Single or 1–5 × 10³ CD8⁺CD44^low T cells were cultured either in 200 μl in 96-well plates or in 2 ml in 24-well plates previously coated with protein G-purified hamster anti-mouse CD3ε (10 μg/ml; 145-2C11), rat anti-mouse CD8α (10 μg/ml; 53.6), and rat anti-mouse CD11a (5 μg/ml; I21/7.7) Ab (26, 31, 32). Cultures were established in growth medium (modified DMEM supplemented with 50 μM 2-ME, 216 mg/L l-glutamine, and 10% heat-inactivated FCS) containing human rIL-2 (20 IU/ml; Hoffmann-LaRoche) (neutral conditions) or rIL-2, mouse rIL-4 (3.3 U/ml, supernatant of Sf9 insect cells infected with murine IL-4 cDNA-encoding baculovirus (33)), and anti-IFN-γ Ab (1 μg/ml, XMG1.2) (type 2-polarizing conditions). For cultures initiated with single cells, clone size was assessed microscopically after 8 days. For restimulation of previously activated cells from bulk cultures, live cells were sorted for high or low CD8 expression, and bulk or individual cells were activated with immobilized anti-CD3/8/11a Ab as above, except that anti-CD3 Ab was reduced to 1 μg/ml.

Activation of CD8⁺ T cells in MLR

CD8⁺CD44^low T cells (1.5–10 × 10³) from C57BL/6 mice were incubated with 1–2 × 10⁷ allogeneic spleen cells (gamma-irradiated with 3000 rad) from DBA/2 mice in 2 ml of growth medium in neutral or type 2 culture conditions in 24-well plates for 8–9 days. For long-term MLR, T cells were restimulated with irradiated DBA/2 spleen cells weekly.

Cytokine induction assays

For clonal assays, cells were washed three times in situ and incubated overnight in the same wells containing immobilized anti-CD3/8/11a Ab with 200 μl of growth medium and 20 IU/ml IL-2. After 18–22 h, supernatants were assayed for cytokines at a single concentration (100%). For bulk assays, T cell populations (5–10 × 10⁴) were incubated for 22–29 h in 200 μl with IL-2 and 10 μg/ml immobilized anti-CD3 Ab, 5 × 10⁴ allogeneic target cells, or medium only. Duplicate or triplicate serial dilutions of supernatant were assayed for IL-4 and IFN-γ by ELISA using the anti-IL-4 Ab BVD4 and biotinylated BVD6 (34) or the anti-IFN-γ Ab R4-6A2 and biotinylated XMG1.2, respectively. In Fig. 1⇓, IFN-γ was measured by growth inhibition of the IFN-γ-sensitive cell line WEHI-279 in a colorimetric assay (35). IL-4 and IFN-γ activities were standardized by reference to titrations of baculovirus-derived murine rIL-4 or purified murine rIFN-γ (Sigma-Aldrich). For IL-4 neutralization, anti-IL-4 (clone 11B11 or BVD4) or control Ab were added at 10 μg/ml at the start of incubation; these concentrations completely neutralized the secreted IL-4.

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FIGURE 1.

Effect of delayed IL-4 addition on the development of cytokine-producing and cytolytic T cell clones. Single CD8⁺CD44^low T cells were cultured with IL-2 and immobilized Ab to CD3, CD8, and CD11a. IL-4 and neutralizing anti-IFN-γ Ab (type 2 conditions) were added to some cultures on day 0, 3, or 6, as indicated. After 8 days, cells were washed in situ and incubated in the same wells in medium with IL-2 for 1 day before functional analysis. Cloning efficiencies at day 8 were 89.6% (neutral), 86.5% (type 2 stimuli from day 0), 88.5% (type 2 stimuli from day 3), and 92.7% (type 2 stimuli from day 6). A, Culture supernatants were assayed for IL-4 by ELISA and for IFN-γ in a colorimetric assay measuring inhibition of WEHI-279 cell growth. The scale of the IFN-γ assay is inverted because low OD reflects high activity. Each dot represents a single clone. The broken line indicates the detection threshold of each assay, defined as the mean activity + 3 SD of supernatants from control wells in which no clone had developed. The upper and lower figures in each panel indicate the percentage of clones positive for IFN-γ and IL-4, respectively. B, Clones from the upper panels estimated to contain at least 10³ cells were pooled and titrated in a redirected ⁵¹Cr release assay using P815 target cells in the absence (○) or presence (•) of bridging anti-CD3 Ab.

⁵¹Cr release assay for cytolytic T cells

Cells of the FcR⁺ mastocytoma line P815 were labeled with Na⁵¹CrO₄ (Amersham Biosciences) for 60 min at 37°C and washed twice in growth medium. Labeled target cells (2–5 × 10³) were incubated for 4–5 h at 37°C with duplicate serial dilutions of T cells in 200 μl in round-bottom 96-well plates. In the case of anti-CD3/8/11a Ab-stimulated T cells, 1 μg/ml anti-CD3 Ab was added to bridge T cells with target cells (redirected assay). Harvested supernatants were dried onto 96-well solid Lumaplates (Packard Instrument), and radioactivity was counted in a Topcount Microplate Scintillation Counter (Packard Instrument).

RT-PCR and quantitative competitive PCR (QC-PCR)³ analysis

Primer sequences and protocols for mRNA preparation, cDNA conversion, and amplification were published recently (26, 36, 37). Briefly, RNA was extracted by Nonidet P-40 hypotonic lysis of 1–5 × 10³ cells, and cDNA was prepared using avian myeloblastosis virus reverse transcriptase (Promega), oligo(dT) primers, and RNase inhibitors. QC-PCR of single or duplicate cDNA samples was performed with Red Hot polymerase (Advanced Biotechnologies), dNTPs, competitor plasmid, and one of the primer pairs specific for cDNA of perforin, granzyme A, granzyme B, granzyme C, IL-4, and IFN-γ, with the reverse primers being biotinylated at their 5′ ends. Each competitor plasmid encoded the corresponding PCR product sequence with a small deletion and was tested in 5-fold dilutions against a fixed amount of cDNA. PCR products were captured on streptavidin-coated plates, hybridized with FITC-labeled probes specific for either the cDNA or competitor product, and quantitated with an alkaline phosphatase-conjugated anti-FITC Ab and 4-nitrophenylphosphate (Roche Diagnostics Australia). PCR amplification (25 μl assay in 96-well plates) was performed using an Omnigene thermal cycler (Thermo Hybaid), as follows: 1 cycle of 96°C for 4 min, then 60°C for 1 min and 72°C for 1.5 min, followed by 39 cycles of 94°C for 0.5 min, then 60°C for 1 min and 72°C for 1.5 min. For expression controls, CD3ε or β₂-microglobulin cDNA was amplified by QC-PCR or by PCR using serial dilutions of cDNA, respectively.

Statistical analyses

The Prism 4.0 software package (GraphPad) was used for statistical analyses.

Requirement for early and sustained exposure to IL-4 for the development of poorly cytolytic IL-4-producing CD8^low cells

We have previously reported that activation of naive CD8⁺ T cells in the presence of IL-4 led to development of a subpopulation of IL-4-producing effector cells with poor cytolytic activity and low CD8 expression (26). To determine whether this response required exposure to IL-4 early in primary activation, we studied the effects of delayed IL-4 addition on development of cytolytic and cytokine-producing clones from individual CD8⁺ T cells. As in our earlier studies (26), a single cell, accessory cell-free culture system was used to allow tracking of the progeny of individual cells and to limit the influence of endogenous cytokines on cell differentiation. Single CD8⁺ cells of naive (CD44^low) phenotype were purified from lymph nodes of C57BL/6 mice and cultured with IL-2 and immobilized Ab to CD3, CD8, and CD11a, either in the absence of other cytokines (neutral conditions) or in the presence of IL-4 and neutralizing anti-IFN-γ Ab (type 2-polarizing conditions) added at various times after culture initiation. Cultures were washed after 8 days, then assayed on day 9 for cytokine production and cytolytic activity in a redirected ⁵¹Cr release assay. For the latter assay, FcR-bearing P815 tumor cells were used as target cells with bridging anti-CD3 Ab to bypass the requirement for peptide/MHC recognition.

Analysis of individual clones grown in neutral conditions showed that most secreted IFN-γ and very few secreted IL-4 (Fig. 1⇑A). Addition of IL-4 and anti-IFN-γ Ab at day 0 did not significantly affect cloning efficiency, but caused a modest reduction in the frequency of IFN-γ-secreting clones and markedly increased the frequency that secreted IL-4. When addition of type 2 stimuli was delayed by 3 or 6 days, the frequency of IL-4-secreting clones was reduced by 21 and 84%, respectively. Fig. 1⇑B shows the cytolytic activity of pools of clones from these cultures. Whereas those grown in neutral conditions were highly cytolytic in the presence of bridging anti-CD3 Ab, pooled clones grown in type 2 conditions from day 0 displayed little lytic activity. CTL activity was higher when addition of type 2 stimuli was delayed to day 3 and was equivalent to that observed in neutral conditions when delayed to day 6. Other experiments showed that the effect of type 2 conditions on both IL-4 production and cytolytic activity was mainly due to exogenous IL-4, not the anti-IFN-γ Ab (26) (data not shown).

Similar results were obtained in two other experiments. Analysis of a total of 1181 clones derived from CD8⁺CD44^low cells in the three experiments showed that exposure to type 2 stimuli from day 0 significantly reduced the frequency of CTL-positive clones and increased the frequency of IL-4 producers compared with neutral conditions (p < 0.001; z test). Compared with addition of type 2 stimuli at day 0, delayed addition by 1 or more days progressively increased the frequency of cytolytic clones and/or reduced the frequency of IL-4 producers; when addition was delayed to day 6, frequencies of all parameters were equivalent to those observed in neutral conditions.

These data support the idea that early exposure to type 2 conditions is required for maximal induction of IL-4-producing noncytolytic T cells in this system. However, an alternative interpretation is that late addition of type 2 stimuli reduced the time available for cells to acquire the noncytolytic IL-4-producing phenotype. To test the latter possibility and to analyze CD8 expression in parallel with effector function, CD8⁺CD44^low cells were activated in bulk cultures in neutral conditions or with addition of IL-4 and anti-IFN-γ Ab at various times between days 0 and 6; after 7 days, cells were harvested and cultured for 1 day in neutral conditions to allow IL-4 secretion, or for 7 days in type 2 conditions before culture for 1 day in neutral conditions. At day 8, most cells cultured in neutral conditions retained high levels of surface CD8α, whereas most cells exposed to type 2 stimuli from day 0 expressed low or undetectable CD8α levels (Fig. 2⇓A). As in our previous study (26), CTL activity was markedly lower in the CD8^low population compared with the CD8^high population when tested in a redirected cytotoxicity assay at day 7. Other experiments indicated that these CD8^low cells were first detectable at day 3–4 and reached maximal frequencies at day 7–8 (data not shown). Delayed addition of the type 2 stimuli by 1–4 days progressively increased the proportion of CD8^high cells and decreased IL-4 titers in the culture supernatants at day 8 (▪, Fig. 2⇓, A and B). Addition of type 2 stimuli at day 5 or 6, however, had no detectable effect on CD8α expression or IL-4 titers. Similar results were obtained in three other experiments; although the proportion of CD8^low cells generated in type 2-polarized cultures varied between experiments, viable cell recoveries were similar under all culture conditions within each experiment, and no loss of CD3ε or TCRαβ and no induction of CD4 expression were detected (data not shown). Progressive diminution of the effects on CD8 expression and IL-4 titers with longer delay in the addition of type 2 stimuli was also seen when the duration of exposure was extended by 7 days (□, Fig. 2⇓, A and B). The data show that the effects of delayed addition reflect a requirement for early, rather than prolonged, exposure to type 2 stimuli to achieve maximal down-regulation of CD8 expression and induction of IL-4 synthesis. Marked down-regulation of surface CD8 and induction of IL-4 expression were also seen in the presence of type 2 conditions when the anti-receptor Ab stimulation regimen was altered, either by omitting the immobilized anti-CD8 Ab or by substituting anti-CD11a with anti-CD28 Ab (data not shown).

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FIGURE 2.

Effect of delayed addition or early removal of IL-4 on CD8 expression and IL-4 synthesis during primary CD8⁺ T cell activation. Left panels, CD8⁺CD44^low T cells were cultured with IL-2 and immobilized anti-CD3/8/11a Ab. IL-4 and anti-IFN-γ Ab (type 2 stimuli) were added to some cultures on day 0 or 1, 2, 3, 4, 5, or 6 days after culture initiation. On day 7, cells were washed and equal numbers were stimulated with either: 1) immobilized anti-CD3 Ab and IL-2 for 1 day, or 2) anti-CD3/8/11a Ab, IL-2, IL-4, and anti-IFN-γ Ab until day 14, then immobilized anti-CD3 Ab and IL-2 for 1 day. Cell surface CD8α expression (A) and IL-4 production (B) were assayed on day 8 (▪) or day 14/15 (□) for cells cultured in neutral conditions (nil) or type 2 conditions from the indicated day of culture. The means and SD of triplicate IL-4 determinations are shown; the broken line indicates the detection threshold of the IL-4 assay. Right panels, CD8⁺CD44^low T cells were cultured with immobilized anti-CD3/8/11a Ab and IL-2 alone (neutral conditions) or in addition with anti-IL-4 Ab, or IL-4 and anti-IFN-γ Ab (type 2 conditions). After 2, 3, 4, or 5 days, cells in type 2 conditions were washed in situ and recultured in the same well in neutral conditions or with anti-IL-4 Ab or type 2 stimuli, as above. On day 8, cells were washed and analyzed for CD8α cell surface expression by FACS (C); equal numbers of cells were recultured with immobilized anti-CD3 Ab and IL-2 for 1 day before IL-4 assay (D).

We next examined the duration of IL-4 exposure needed for induction of IL-4 synthesis and the CD8^low phenotype. CD8⁺CD44^low cells were activated in bulk cultures in neutral or type 2 conditions at day 0; some of the former cultures also received neutralizing anti-IL-4 Ab to block endogenous IL-4, or anti-CD4 Ab as a control. After 2, 3, 4, or 5 days, cells were removed from type 2 stimuli, washed, and recultured in neutral conditions, with anti-IL-4 or anti-CD4 Abs, or with type 2 stimuli. At day 8, all cultures were analyzed for CD8 expression and recultured for 1 day in neutral conditions to allow IL-4 secretion (Fig. 2⇑, C and D). As in previous experiments, continuous culture with type 2 stimuli for 8 days caused significant down-regulation of surface CD8 levels and induction of IL-4 synthesis. Cell transfer from type 2 to neutral conditions showed that these effects were maximal after exposure to exogenous type 2 stimuli for 4 and 3 days, respectively. However, when endogenous IL-4 was neutralized with anti-IL-4 Ab in the second culture, the requirement for exposure to exogenous type 2 stimuli increased to 5 days for both parameters. The control anti-CD4 Ab had no significant effect (data not shown). We conclude that the early and continuous presence of IL-4 is required for maximal induction of CD8^low cells and IL-4 secretion during primary CD8 T cell effector differentiation; endogenous IL-4 can replace the requirement for exogenous IL-4 after 3–4 days.

Loss of CD8 expression by type 2-polarized T cells is a progressive process of differentiation

The experiments described above showed that CD8⁺ T cells exposed to type 2 stimuli for 1–2 wk were heterogeneous in their CD8 expression levels and functional activities. To determine whether the loss of CD8 increased with time, we cultured single CD8⁺CD44^low T cells in neutral or type 2 conditions from day 0. After 7 days, clones were transferred to neutral conditions and sampled sequentially over the next 10 days for analysis of their CD8 expression levels (Fig. 3⇓). Of 24 clones initiated in neutral conditions, all comprised >99.3% CD8^high cells and continued to express CD8 at high frequency (>91.7%) for the remainder of the culture period. In contrast, 23 clones cultured with type 2 stimuli contained variable numbers of CD8^high cells ranging from 0 to 97.9% at day 7. None of these clones regained CD8^high cells with further culture in the absence of type 2 stimuli. Instead, the majority progressively lost CD8 expression such that 16 of the 23 clones contained <15% CD8^high cells and only 4 clones contained >50% CD8^high cells at the last time point analyzed. None expressed surface CD4. Other clonal experiments indicated that progressive loss of CD8 continued even when type 2 stimuli were removed and replaced with neutral conditions as early as day 4 (data not shown).

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FIGURE 3.

Progressive loss of CD8 expression by type 2-polarized T cell clones. Individual CD8⁺CD44^low T cells were cultured with anti-CD3/8/11a Ab and IL-2 in the absence (top panel) or presence of IL-4 and anti-IFN-γ Ab (bottom panel). After 7 days, clones estimated to contain at least 10³ cells were washed in situ to remove type 2 stimuli and reincubated in the same wells in medium containing IL-2. Clones were sampled to determine the percentage of CD8^high cells on days 7, 10, 14, and 17. Each data point represents a single clone. Data were pooled from two separate experiments.

The progressive acquisition of the CD8^low phenotype with continued culture might reflect preferential outgrowth of CD8^low cells. To assess division rates of CD8^high and CD8^low cells, CD8⁺CD44^low T cells were activated in neutral or type 2 conditions, sorted for high or low CD8 expression at day 4, then labeled with the intracellular dye CFSE and restimulated for a further 3 days with anti-CD3/8/11a Ab in either neutral or type 2 conditions. In two independent experiments, analysis of CFSE levels by FACS showed that, on average, CD8^low cells proliferated at a similar or slightly lower rate (half a division over 3 days) than CD8^high cells from either neutral or type 2 cultures, regardless of whether the CFSE-labeled cells were cultivated in neutral or type 2 conditions (data not shown). We conclude that the progressive accumulation of CD8^low cells in type 2 cultures was not due to a growth advantage over CD8^high cells.

The finding that the continuing loss of CD8α expression by clones removed from type 2-polarizing conditions was not due to faster growth of CD8^low cells suggested prior commitment of the cells to a continuing process of differentiation initiated by IL-4. To examine the reversibility of this process, CD8^low and CD8^high cells were purified from polyclonal type 2-polarized cultures at day 4 and restimulated with anti-CD3/8/11a Ab in the presence or absence of IL-4, neutralizing anti-IL-4 Ab (11B11), or a control anti-CD4 Ab. Fig. 4⇓ shows that some cells in the CD8^low population regained CD8 expression and, conversely, some cells in the CD8^high population lost CD8 expression after 2 days in neutral conditions. Exogenous IL-4 decreased the frequency of CD8^high cells in the CD8^low population, while anti-IL-4 Ab markedly increased CD8 expression compared with neutral conditions. CD8 expression by the CD8^high population was not strongly affected by any of the culture conditions, although the modest effect of IL-4 in decreasing CD8 levels may indicate the persistence of some residual IL-4-responsive cells. Similar results were obtained in two experiments using a different anti-IL-4 Ab (BVD4); both 11B11 and BVD4 increased CD8 expression in the CD8^low population, while control anti-IFN-γ or anti-CD4 Ab had no significant effect (data not shown). The data suggested that some CD8^low cells had the potential to regain CD8 expression when endogenous IL-4 was neutralized at days 4–6 after primary activation. However, because these experiments were performed in bulk cultures, it remained a formal possibility that rare contaminating CD8^high cells accumulated because of their slight growth advantage over CD8^low cells and/or preferential apoptosis of the CD8^low cells; neither possibility was likely given the high purity of the day 4 FACS-purified CD8^low cells (>99%) and the culture time of 2 days.

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FIGURE 4.

Reversibility of CD8^low phenotype early in effector cell differentiation. CD8⁺CD44^low T cells were cultured with anti-CD3/8/11a Ab, IL-2, IL-4, and anti-IFN-γ Ab (type 2 conditions). A, On day 4, cells were separated into CD8^low and CD8^high cells. CD8 expression is shown before (thick line in upper panel; thin line is negative control binding of anti-rat IgG) and immediately after purification of the lowest 10% and highest 18% of cells (lower panels). B, CD8 expression is shown after reculture of the purified populations with anti-CD3/8/11a Ab and IL-2 (neutral) or, in addition, IL-4 or anti-IL-4 Ab (clone 11B11) for 2 days.

To determine definitively whether individual CD8^low or CD8^high cells or their progeny can change their CD8 phenotype during early type 2 polarization, we undertook a clonal analysis of cells that had been FACS purified twice to minimize contamination with cells of the opposite phenotype. CD8 T cells of naive phenotype were activated in neutral or type 2 conditions for 4 days; CD8^low and CD8^high cells were then sorted twice, yielding purities of 99.8 and 98.8%, respectively, before single cell cloning in the presence or absence of IL-4 or neutralizing anti-IL-4 Ab (Fig. 5⇓). As detailed in the legend of Fig. 5⇓, secondary culture conditions did not significantly affect cloning efficiencies. Clones generated in neutral conditions retained high CD8 levels regardless of secondary stimulation conditions. Clones derived from type 2-polarized cells that were CD8^low or CD8^high on day 4, then cultured in neutral conditions contained highly variable percentages of CD8^high cells, although the median and mean values were biased toward the phenotype of the founder cell. Fewer cells of either phenotype gave rise to CD8^high clones when cultured with IL-4, while neutralization of endogenous IL-4 with anti-IL-4 Ab favored re-expression of CD8 by CD8^low cells; for both starting phenotypes, the median percentages of CD8^high cells were significantly different between clones grown in neutral conditions and with IL-4, and between clones grown with IL-4 and with anti-IL-4 Ab (p < 0.02, two-tailed Mann-Whitney U test). Among clones derived from CD8^low cells, 65% of those grown in neutral conditions, 41% grown with IL-4, and 72% grown with anti-IL-4 Ab contained >10% CD8^high cells. Most of the informative clones were tested for surface expression of CD4 and/or CD3; all cells were negative for CD4 and positive for CD3 expression (data not shown). We conclude that, at day 4, many CD8^low cells retained the potential to give rise to CD8^high cells, while some CD8^high cells could lose CD8. Continuous exposure to IL-4 amplified the development of CD8^low cells, while removal of IL-4 promoted reversion to a CD8^high phenotype.

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FIGURE 5.

Clonal analysis of the reversibility of the CD8^low phenotype early in effector cell differentiation. A, CD8⁺CD44^low T cells were cultured with anti-CD3/8/11a Ab and IL-2 alone (neutral) or in addition IL-4 and anti-IFN-γ Ab (type 2). On day 4, CD8^high cells from neutral culturesand CD8^low and CD8^high cells from type 2 cultures were sorted twice and cloned by single cell deposition into wells with anti-CD3/8/11a Ab and IL-2 (neutral) or, in addition, IL-4 or anti-IL-4 Ab (clone 11B11). B, The percentage of CD8^high cells in each clone was determined 8–9 days after cloning. Each dot represents a clone estimated to contain at least 100 cells. The broken line indicates the median percentage of CD8^high cells in each set of clones. Data were pooled from six independent experiments with the following average cloning efficiencies: CD8^high cells from neutral cultures, neutral 5.8%, IL-4 3.2%, anti-IL-4 Ab 5.3%; CD8^high cells from type 2 cultures, neutral 11.5%, IL-4 9.8%, anti-IL-4 Ab 14.1%; CD8^low from type 2 cultures, neutral 11.1%, IL-4 11.6%, anti-IL-4 Ab 16.9%.

We have previously reported that CD8^low cells isolated from type 2-polarized cultures at day 8 displayed markedly lower cytolytic activity and expressed lower levels of perforin, granzyme B, and granzyme C mRNAs than CD8^high cells from either neutral or type 2 cultures; granzyme A expression was lower in both CD8^low type 2-polarized cells and CD8^high cells from neutral cultures than in CD8^high type 2-polarized cells (26). To determine whether these functional parameters were also correlated with CD8 expression in individual clones, gene expression was examined in random selections of large clones from the experiment in Fig. 5⇑. Analysis of 32 clones for expression of cytokine, perforin, and granzyme mRNAs revealed that CD8 levels were positively correlated with the expression of perforin and granzymes A, B, and C, and negatively correlated with IL-4 (Fig. 6⇓). Most clones expressed IFN-γ without any obvious relationship to CD8 levels.

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FIGURE 6.

Relationship between CD8 expression and cytokine, perforin, and granzyme expression profiles among CD8 T cell clones. Clones estimated to contain at least 10³ cells were randomly selected from those shown in Fig. 5⇑B and analyzed by QC-PCR for the presence of the indicated mRNA species. Each dot represents a single clone derived from cultures initiated in neutral (open symbol) or type 2 (filled symbol) conditions. The broken lines indicate the detection threshold of the QC-PCR assay. Exponential regression lines (solid) and Spearman correlation coefficients (r) are shown with two-tailed p values (∗∗∗, p < 0.001; ∗∗, p = 0.001–0.01; ns, not significant).

The CD8^low phenotype is stable in long-term culture

The strong stimulation provided by immobilized anti-CD3/8/CD11a Ab ultimately leads to apoptosis, limiting the use of this system for long-term studies. The long-term stability of the CD8^low phenotype following removal of type 2 stimuli was therefore analyzed in MLR. CD8⁺CD44^low T cells from lymph nodes of untreated C57BL/6 (H-2^b) mice were cultured with irradiated DBA/2 (H-2^d) spleen cells in neutral or type 2 conditions for 9 days (Fig. 7⇓A). Fig. 7⇓B shows that ∼30% of CD8 T cells had lost CD8 expression in type 2, but not neutral, conditions at this stage. CD8^low and CD8^high cells were then purified and propagated with DBA/2 spleen cells in neutral conditions. The CD8 expression profile of the propagated cells was monitored for another 4 wk (Fig. 7⇓C). CD8^high cells maintained their phenotype regardless of their origin. The CD8^low phenotype was also largely maintained over time despite the early emergence of a subpopulation of CD8^high cells. All cells were CD3⁺CD4⁻, and loss of surface CD8 protein correlated with very low CD8α mRNA expression levels in CD8^low cells when compared with the corresponding CD8^high siblings (data not shown). The CD8^low and CD8^high subpopulations were isolated at day 36 and recultured in neutral conditions (Fig. 7⇓A, cultures f and g). Subsequent analysis of all populations revealed significant functional differences. The long-term CD8^low cells (f) were poor CTL in contrast to the CD8^high populations, all of which lysed H-2^d target cells in a ⁵¹Cr release assay (Fig. 7⇓D). Both CD8^low and CD8^high cells from type 2 cultures secreted IL-4 in response to P815 cells, whereas CD8^high cells from neutral cultures did not; in contrast, the type 2-polarized populations produced lower levels of IFN-γ than CD8^high cells from neutral cultures (Fig. 7⇓E). Population g displayed the classical Tc2 phenotype (CD8⁺ IL-4⁺ CTL⁺) previously described by others and shown to exert a variety of effects in mouse models (16, 17, 18). From this and two other experiments analyzing long-term cultures between days 20 and 73, it was concluded that the CD8^low phenotype showed a high degree of stability through many rounds of cell division in MLR and was associated with high IL-4 synthesis, limited IFN-γ synthesis, and negligible CTL function. Additional experiments showed that CD8 expression in long-term clonal and polyclonal cultures of CD8^low or CD8^high cells in MLR was not influenced by exogenous IL-4 or anti-IL-4 Ab (data not shown), supporting the conclusion that these are stable differentiated phenotypes.

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FIGURE 7.

Long-term stability of the CD8^low phenotype. A, CD8⁺CD44^low T cells from C57BL/6 mice were cultured in MLR with irradiated DBA/2 spleen cells and IL-2 (neutral conditions) or in addition with IL-4 and anti-IFN-γ Ab (type 2 conditions). On day 9, CD8^high and CD8^low cells were purified and recultured weekly with irradiated DBA/2 spleen cells and IL-2 for up to 50 days; where indicated, cells (d) were resorted for CD8^high and CD8^low cells. ○, Represent CD8^high cells; •, represent CD8^low cells. a–g, Indicate populations analyzed in the following panels. B, FACS histograms are shown for surface CD8α expression in populations a and b at day 9. The percentage of CD8^high cells is indicated above the M1 bar. C, The percentage of CD8^high cells is shown in populations derived from CD8^high cells of neutral culture origin (c) and from CD8^low cells (d) and CD8^high cells (e) of type 2 culture origin on the indicated days of culture. D, CD8^high cells of neutral (c) or type 2-polarized (e) culture origin, and CD8^high cells (g) or CD8^low cells (f) of type 2-polarized origin repurified on day 36, were assayed on day 42 for cytolytic function against ⁵¹Cr-labeled allogeneic P815 target cells. E, In an independent experiment, the indicated populations were incubated on day 50 with allogeneic P815 target cells for 20 h before assay of supernatants for IL-4 and IFN-γ by ELISA. The broken line depicts the detection limit of the assay.