We have shown previously that T cells activated by optimal TCR and CD28 ligation exhibit marked proliferative heterogeneity, and ∼40% of these activated cells fail entirely to participate in clonal expansion. To address how prior cell division influences the subsequent function of primary T cells at the single cell level, primary CD4+ T cells were subjected to polyclonal stimulation, sorted based on the number of cell divisions they had undergone, and restimulated by ligation of TCR/CD28. We find that individual CD4+ T cells exhibit distinct secondary response patterns that depend upon their prior division history, such that cells that undergo more rounds of division show incrementally greater IL-2 production and proliferation in response to restimulation. CD4+ T cells that fail to divide after activation exist in a profoundly hyporesponsive state that is refractory to both TCR/CD28-mediated and IL-2R-mediated proliferative signals. We find that this anergic state is associated with defects in both TCR-coupled activation of the p42/44 mitogen-activated protein kinase (extracellular signal-related kinase 1/2) and IL-2-mediated down-regulation of the cell cycle inhibitor p27kip1. However, these defects are selective, as TCR-mediated intracellular calcium flux and IL-2R-coupled STAT5 activation remain intact in these cells. Therefore, the process of cell division or cell cycle progression plays an integral role in anergy avoidance in primary T cells, and may represent a driving force in the formation of the effector/memory T cell pool.

The events that drive the development of naive T cells into effector and/or memory cells are unclear. While individual primary T cells exhibit a relatively uniform pattern of early signal transduction and activation in response to optimal TCR and costimulatory receptor ligation (1, 2), a large degree of heterogeneity can be observed with respect to functional behavior such as cytokine production (3, 4, 5, 6, 7). A similar degree of diversity characterizes the proliferative behavior of individual activated T cells. Several days following in vitro stimulation, one can detect individual T cells that have divided between one and eight times, while as many as 40% of these activated cells do not participate at all in clonal expansion (8). This limitation in the frequency of activated T cells that respond by proliferating is observed both in vitro and in vivo in response to either mitogens or peptide Ag, and occurs even when signals from TCR, CD28, and IL-2R are optimal (8, 9). Interestingly, this limitation in responder frequency is not observed in TCR-transgenic T cell populations that have matured on a recombination-deficient background, suggesting that maximal T cell proliferative potential requires allelic exclusion at the TCR-α locus (9).

This diversity observed in primary T cell responses has suggested that an individual T cell’s proliferative behavior during primary culture might influence its eventual effector function (10, 11, 12). To further address this question, we have labeled T cells with the fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE),3 stimulated these cells in vitro, and sorted them by flow cytometry based upon their proliferative history. Our data demonstrate that the responsiveness of individual T cells to secondary TCR engagement is quantitatively tuned to the number of mitotic events achieved during primary stimulation. We also define a T cell fate that is refractory to both TCR- and IL-2R-mediated signals, and is associated with the failure to proliferate following primary activation in the presence of CD28 costimulation. These data support a model in which T cells must divide after Ag encounter to avoid anergy.

Pooled spleen and lymph node cells from female BALB/c mice, aged 8–12 wk, were used for all experiments. mAbs against CD3 (145-2C11) and CD28 (37.51) were purified from hybridomas obtained from J. Bluestone (University of Chicago, Chicago, IL) and J. Allison (University of California, Berkeley, CA), respectively. Purified, fluorochrome-conjugated mAb against CD16/CD32 (Fc-block), Thy-1.2, CD4, CD25, CD122, common γ-chain (γc), IL-2, and IFN-γ, and biotinylated anti-CD3 and anti-CD28 were purchased from PharMingen (San Diego, CA). Abs reactive with both the phosphorylated and unphosphorylated forms of extracellular signal-related kinase (ERK)1, ERK2, and STAT5 were purchased from Zymed Laboratories (San Francisco, CA). mAb specific for p27kip1 was purchased from Transduction Laboratories (Lexington, KY). Abs specifically reactive with only the phosphorylated forms of ERK1 and ERK2 were purchased from New England Biolabs (Beverly, MA), and Ab reactive with the phosphorylated form of STAT5 was purchased from Zymed Laboratories (San Francisco, CA). Rabbit antiserum against actin was purchased from Sigma (St. Louis, MO). CFSE, Indo-1 AM, FuraRed AM, and TOPRO-3 were purchased from Molecular Probes (Eugene, OR). PMA and ionomycin were purchased from Sigma and were used at 5 ng/ml and 0.25 μM, respectively. Murine rIL-2 was obtained from Genzyme (Cambridge, MA), and was used at 15–30 U/ml.

Cell isolation and fluorescent labeling of cells with CFSE were performed as previously described (8). Briefly, pooled spleen and lymph node cells were incubated with CFSE in PBS at a final concentration of 2 μM for 3 min. CFSE-labeled cells were stimulated with soluble anti-CD3 mAb (1 μg/ml, unless otherwise stated) at 2–5 × 106/ml in either 24-well plates or 75-cm2 flasks. Where indicated, cultures contained anti-CD28 mAb (1 μg/ml) and/or IL-2 (15 U/ml). For primary cultures, the cells were stimulated for 4 days, washed, and replated in fresh medium for 48 h, then sorted based on CFSE fluorescence. Sorted T cells were restimulated in 96-well round-bottom plates with soluble Abs, as indicated, for 4 days in the presence of a 4-fold excess of irradiated syngeneic splenocytes, and secondary proliferation (CFSE fluorescence) was analyzed by flow cytometry.

Cell surface marker staining was performed as described (8), and flow cytometric analysis was performed on a FACScalibur flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA). Methods using CFSE labeling to calculate the responder frequency (defined as the proportion of input T cells that undergo one or more cell divisions during the culture period) and the absolute number of mitotic events occurring in the culture have been described (8). The vital dye TOPRO-3 was used to discriminate live and dead cells (8). Live, Thy-1.2+, or CD4+ T cells were sorted based on CFSE fluorescence using a FACSvantage flow cytometer/sorter (Becton Dickinson).

Cytokine expression was assessed at the single cell level, as described previously (5), with some modifications. Cells that had been activated in primary culture for 4 days and rested for 48 h were cultured for 5 h with plate-bound anti-CD3 plus anti-CD28 mAbs (5 μg/ml each) in the presence of 2 μM monensin (Sigma). As primed T cells do not divide during this 5-h restimulation period (data not shown), cytokine production can be assessed as a function of primary proliferative history without the use of cell sorting.

Primed T cells were loaded with either Indo-1 AM (2 μg/ml) or FuraRed AM (10 μg/ml) (Molecular Probes) for 30 min at 30°C in RPMI 1640 with 1% serum (13). Agonistic, biotinylated anti-CD3 and anti-CD28 Abs were also added at this time. Cells were washed, resuspended in RPMI 1640 lacking serum and sodium bicarbonate, and warmed to 37°C for 5 min, and Indo-1 or FuraRed fluorescence before and after receptor cross-linking was assessed on a FACStarPlus or FACScalibur flow cytometer, respectively (13). Receptor cross-linking was achieved by the addition of streptavidin (0.2 μg/ml, final concentration). Maximal [Ca2+]i flux was achieved by the addition of ionomycin (1 μM). Kinetic analysis of [Ca2+]i was achieved using FloJo flow cytometry software (Tree Star Software, San Carlos, CA).

CD4+ T cells sorted based on division cycle were rested at 37°C for 2–4 h in medium, and a portion of each pool was set aside for analysis of total ERK content. The remaining cells were restimulated for 10 min with polystyrene beads coated with anti-CD3 mAb ± anti-CD28 mAbs (14), or with PMA. Whole cell lysates (6–8 × 105 cell equivalents per lane) were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-ERK or anti-phospho-ERK antisera (1/1000 dilution). Bands were quantified by densitometric analysis using NIH Image software, and the phospho-ERK signals were normalized to the corresponding total ERK signals to determine relative ERK activation. For the assessment of p27kip1 degradation, cells primed and rested as above were cultured for 48 h in the presence of 50 U/ml IL-2. The cells were then sorted into divided and undivided fractions, and the whole cell lysates were subjected to immunoblot analysis as above using a mAb against p27kip1. For the assessment of STAT5 activation, splenic T cells were primed for 4 days with anti-CD3 (1 μg/ml), rested for 24 h, and restimulated by the addition of IL-2 (100 U/ml) for 10 min. The cells were fixed for 5 min in cold 1% formaldehyde, permeabilized for 10 min in cold methanol, and washed with PBS containing 3% nonfat dry milk. The fixed/permeabilized cells were then stained for either total STAT5 or phosphorylated STAT5 using 1 μg of specific primary Ab for 1 h at room temperature. Cells were washed four times with PBS containing 3% milk, and Cy5-conjugated donkey anti-mouse IgG F(ab′)2 (0.3 μg) was added for 30 min at room temperature. Cells were washed twice in PBS and subjected to flow cytometric analysis.

Previously, we have shown that while optimal stimulation of freshly isolated T cells by anti-CD3 ± anti-CD28 mAb induces the activation of 95–98% of the T cell population, as assessed by CD25 and CD69 expression, a large proportion of the activated T cells (up to 50%) fails to undergo even a single round of cell division (8). This raised the question as to whether there might be an association between the heterogeneity in proliferative behavior observed within the CD4+ T cell pool and heterogeneity in subsequent effector function (i.e., cytokine production and proliferation). To test this hypothesis, murine spleen and lymph node cells were labeled with CFSE and cultured in vitro with an optimal concentration of T cell mitogenic anti-CD3 Ab for 4 days. The cultures were then washed and rested for 48 h, and T cells that had divided once, twice, or had remained undivided following primary stimulation were purified by FACS and restimulated (Fig. 1 A).

FIGURE 1.

Quantitative assessment of secondary T cell proliferative responses as a function of prior cell division. CFSE-labeled BALB/c T cells were stimulated with soluble anti-CD3 Ab (2 μg/ml) for 4 days, washed, and rested in fresh medium for 48 h. A, On day 6, Thy-1.2+ cells were sorted based on CFSE fluorescence into discrete populations corresponding to 0, 1, or 2 rounds of cell division. A total of 2 × 104 sorted T cells was cultured with 4 × 104 irradiated syngeneic splenocytes in medium alone (B) or in the presence of anti-CD3 Ab (1 μg/ml) (C), and cell division by the CD4+ T cell subset was assessed 4 days later by flow cytometry. The division profiles depicted in B and C were used to calculate the frequency of CD4+ T cells that proliferated (D), as well as the absolute number of mitotic events accumulated (E) in response to medium alone (□) or anti-CD3 (•). Values are shown graphed as a function of primary division cycle. The results shown are representative of six independent experiments.

FIGURE 1.

Quantitative assessment of secondary T cell proliferative responses as a function of prior cell division. CFSE-labeled BALB/c T cells were stimulated with soluble anti-CD3 Ab (2 μg/ml) for 4 days, washed, and rested in fresh medium for 48 h. A, On day 6, Thy-1.2+ cells were sorted based on CFSE fluorescence into discrete populations corresponding to 0, 1, or 2 rounds of cell division. A total of 2 × 104 sorted T cells was cultured with 4 × 104 irradiated syngeneic splenocytes in medium alone (B) or in the presence of anti-CD3 Ab (1 μg/ml) (C), and cell division by the CD4+ T cell subset was assessed 4 days later by flow cytometry. The division profiles depicted in B and C were used to calculate the frequency of CD4+ T cells that proliferated (D), as well as the absolute number of mitotic events accumulated (E) in response to medium alone (□) or anti-CD3 (•). Values are shown graphed as a function of primary division cycle. The results shown are representative of six independent experiments.

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The experiment shown in Fig. 1 demonstrates this proliferative heterogeneity. After the peak of the clonal expansion phase (day 4), 25.7% of the input T cells responded to primary stimulation by dividing once, 18.7% divided twice, 11% divided three times, and less than 4% divided four or more times, while 41% remained undivided. Following cell sorting, T cells were cultured with fresh syngeneic accessory cells in medium alone, or in the presence of anti-CD3 mAb. As the cells remained labeled with CFSE, we were able to monitor their proliferative response to restimulation. Sorted CD4+ T cells cultured with feeders in medium alone did not divide during the 4-day restimulation period (Fig. 1,B), confirming that all of the T cells had exited the cell cycle following the initial phase of activation and rest. When subjected to TCR reengagement by the addition of anti-CD3 to the restimulation cultures, those CD4+ T cells that had undergone at least a single round of cell division during primary activation initiated a second burst of proliferation (Fig. 1,C, middle and lower panels). In contrast, those cells that failed to divide following primary activation were unable to proliferate in response to TCR reengagement (Fig. 1 C, upper panel).

Quantitative, single cell analysis of the dynamics of secondary clonal expansion shows that the proportion of input CD4+ T cells that responded to restimulation by proliferating (i.e., the responder frequency) was less than 5% in the undivided pool, whereas 65% of the input CD4+ T cells that divided once during primary activation, and 75% of the CD4+ T cells that divided twice were able to reenter the cell cycle when subjected to TCR ligation again (Fig. 1,D). Measuring the accumulation of cell divisions within the CD4+ subset during the restimulation period shows that 10,000 undivided CD4+ T cells cultured in the presence of anti-CD3 gave rise to <1,500 total mitotic events. In contrast, the same number of cells from the pool of CD4+ T cells that divided once gave rise to 50,000 mitotic events, while the CD4+ T cells that divided twice during primary activation gave rise to >130,000 mitotic events (Fig. 1 E). These absolute mitotic event values correspond to the generation of an average of between three and four daughter cells per responder from the pool of precursors with a prior division history of one, while the average CD4+ T cell that divided twice during the primary response generated 10 daughters during secondary clonal expansion.

Therefore, we find that both the probability that an individual CD4+ T cell will participate in the secondary phase of clonal expansion, and the number of rounds of cell division it subsequently achieves are linked to its proliferative behavior during the primary response.

To explore the potential basis for the observed relationship between primary and secondary proliferative behavior, we analyzed the production of IL-2 by individual CD4+ T cells as a function of their primary division status. CFSE-labeled splenocytes were primed as described above, and then briefly (5 h) restimulated by coligation of TCR and CD28, after which cytokine production was assessed by flow cytometry. As was the case for proliferation, the frequency of CD4+ T cells that produced IL-2 upon secondary TCR ligation increased with each successive division cycle (Fig. 2, A and C), such that while only 3% of undivided CD4+ T cells could secrete IL-2, 12% of those cells that had divided three times were able to produce IL-2. We have observed a similar relationship between IL-2 production and cell division in an MHC class II-restricted, TCR-transgenic T cell model, in which the frequency of IL-2 producers is <5% within the undivided Ag-specific CD4+ T cell pool, but increases with each cell division such that up to 80% of the cells that have divided five or six times are able to produce IL-2 (9). This trend in cell division-associated IL-2 production does not generalize to all cytokines in this in vitro model, as all primed CD4+ T cells exhibited a similar capacity to secrete IFN-γ, regardless of their previous proliferative behavior (Fig. 2, B and D). These results suggest that T cells that fail to divide after activation are subsequently unable to produce IL-2. This defect is a hallmark of T cell clonal anergy (15) and could possibly explain the inability of the undivided cells to proliferate in response to restimulation.

FIGURE 2.

Assessment of IL-2 and IFN-γ production by CD4+ T cells as a function of prior division history. Primary, CFSE-labeled T cells were stimulated and rested as described in Fig. 1, and intracellular cytokine expression was detected by flow cytometry with PE-conjugated anti-IL-2 (A) or anti-IFN-γ (B) Abs. Cytokine expression is shown as a function of CFSE fluorescence. CFSE fluorescence is shown also as a frequency histogram above each plot. The dotted lines in each plot represent the maximal fluorescence of CD4+ T cells stimulated, fixed, and permeabilized as above, but stained with PE-conjugated isotype control Ab. The data in A and B are also shown plotted as the specific proportion of CD4+ T cells in each division secreting IL-2 (C) or IFN- γ (D) following restimulation with medium alone (○) or plate-bound Abs (•). Background positive events, as defined by isotype control staining (<1.5%), were subtracted from the anti-cytokine Ab-stained sample values to obtain the specific proportion of cytokine-positive CD4+ T cells. The data shown are representative of two independent experiments.

FIGURE 2.

Assessment of IL-2 and IFN-γ production by CD4+ T cells as a function of prior division history. Primary, CFSE-labeled T cells were stimulated and rested as described in Fig. 1, and intracellular cytokine expression was detected by flow cytometry with PE-conjugated anti-IL-2 (A) or anti-IFN-γ (B) Abs. Cytokine expression is shown as a function of CFSE fluorescence. CFSE fluorescence is shown also as a frequency histogram above each plot. The dotted lines in each plot represent the maximal fluorescence of CD4+ T cells stimulated, fixed, and permeabilized as above, but stained with PE-conjugated isotype control Ab. The data in A and B are also shown plotted as the specific proportion of CD4+ T cells in each division secreting IL-2 (C) or IFN- γ (D) following restimulation with medium alone (○) or plate-bound Abs (•). Background positive events, as defined by isotype control staining (<1.5%), were subtracted from the anti-cytokine Ab-stained sample values to obtain the specific proportion of cytokine-positive CD4+ T cells. The data shown are representative of two independent experiments.

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The failure of the undivided CD4+ T cells to produce IL-2 and proliferate in response to mitogenic anti-CD3 Abs, despite normal expression of the αβ TCR (data not shown), suggested a biochemical defect in TCR-coupled signal transduction. As a first step to localize this defect, we examined TCR-proximal signals involved in the elevation of [Ca2+]i following engagement of TCR/CD3. All T cells exhibited an increase in [Ca2+]i in response to CD3 ligation (Fig. 3,A), and the relative increase in [Ca2+]i was comparable in T cells with varied proliferative history (Fig. 3,B). The ability of the nonproliferative cells to flux calcium and secrete IFN-γ (Fig. 2) in response to TCR ligation emphasizes that these cells are not globally unresponsive.

FIGURE 3.

Kinetic analysis of TCR-coupled [Ca2+]i flux as a function of cell division cycle. Primary, CFSE-labeled T cells were stimulated and rested, as described in Fig. 1. A, The primed cells were then stained with biotinylated anti-CD3 (1 μg/ml) and loaded with Fura Red, and resting [Ca2+]i was assessed by flow cytometry. During each kinetic experiment, TCR cross-linking was achieved by the addition of streptavidin (0.2 μg/ml; solid arrow), and maximal [Ca2+]i flux was then induced by the addition of ionomycin (1 μM; open arrow). The plot shown is gated on the entire, live CD4+ T cell population. B, [Ca2+]i flux by CD4+ T cells was analyzed as a function of time and division cycle by gating on discrete CD4+ CFSE populations, corresponding to cells that divided 0, 1, 2, or 3 times during the primary stimulation, and comparing the median traces of each population over time in response to TCR cross-linking (solid arrow), followed by ionomycin (open arrow). Similar results were obtained when CD3 and CD28 were cocross-linked (data not shown), and experiments in which [Ca2+]i flux was measured ratiometrically using the calcium probe Indo-1 AM also generated similar results (data not shown). The data shown are representative of six independent experiments.

FIGURE 3.

Kinetic analysis of TCR-coupled [Ca2+]i flux as a function of cell division cycle. Primary, CFSE-labeled T cells were stimulated and rested, as described in Fig. 1. A, The primed cells were then stained with biotinylated anti-CD3 (1 μg/ml) and loaded with Fura Red, and resting [Ca2+]i was assessed by flow cytometry. During each kinetic experiment, TCR cross-linking was achieved by the addition of streptavidin (0.2 μg/ml; solid arrow), and maximal [Ca2+]i flux was then induced by the addition of ionomycin (1 μM; open arrow). The plot shown is gated on the entire, live CD4+ T cell population. B, [Ca2+]i flux by CD4+ T cells was analyzed as a function of time and division cycle by gating on discrete CD4+ CFSE populations, corresponding to cells that divided 0, 1, 2, or 3 times during the primary stimulation, and comparing the median traces of each population over time in response to TCR cross-linking (solid arrow), followed by ionomycin (open arrow). Similar results were obtained when CD3 and CD28 were cocross-linked (data not shown), and experiments in which [Ca2+]i flux was measured ratiometrically using the calcium probe Indo-1 AM also generated similar results (data not shown). The data shown are representative of six independent experiments.

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Next, we examined the Ras-coupled, Raf-dependent mitogen-activated protein kinase (Ras-Raf-MAPK) pathway, a particularly important TCR-coupled signaling cascade involved in several aspects of T cell activation, including IL-2 production (16, 17, 18, 19, 20). CFSE-labeled splenocytes were primed as described above, and the CD4+ T cells were sorted into a fraction that had divided one or more times, and into another fraction that had remained undivided throughout primary activation. The cells were then restimulated with polystyrene beads coated with anti-CD3 Ab, and phosphorylation of the p42/44 extracellular signal-regulated kinase (ERK1/2) was analyzed as a reliable and quantitative indicator of the activation of the Ras-Raf-MAPK pathway (21). The total levels of both ERK isoforms (ERK 1 and 2) were reduced ∼4-fold (ERK1) and 2-fold (ERK2) in undivided CD4+ T cells compared with the levels in divided CD4+ T cells (Fig. 4, A and B, upper panels). In addition, we observed a ligand density-dependent defect in the capacity to activate these ERK species in the undivided compared with the divided CD4+ T cell pool. In response to restimulation with a relatively high density of TCR ligand (1 μg/ml anti-CD3), the undivided CD4+ T cells exhibited a 3.5-fold reduction in the relative activation of ERK1 compared with the activation of this isoform in divided cells, but ERK2 was activated to a comparable degree (Fig. 4,A, middle panel). At half this ligand density (0.5 μg/ml anti-CD3), the defect was more profound: activation of ERK1 was not detected in the undivided CD4+ T cells, and ERK2 activation was reduced ∼4-fold compared with the activation of this isoform in the divided cells (Fig. 4 A, lower panel). Therefore, we find that CD4+ T cells that fail to proliferate in response to primary stimulation suffer from a quantitative defect in TCR-coupled activation of the Ras-Raf-MAPK pathway. This signaling defect has been described previously in anergic T cell clones (22, 23, 24, 25), and could explain the inability of the undivided cells to produce IL-2.

FIGURE 4.

Assessment of MAPK phosphorylation by divided vs undivided CD4+ T cells following secondary receptor cross-linking. Primary, CFSE-labeled T cells were stimulated and rested, as described in Fig. 1, and the CD4+ T cell population was sorted into a divided pool and an undivided pool. The cells were restimulated for 10 min with polystyrene beads coated with anti-CD3 (A) or anti-CD3 and anti-CD28 (B), and lysates were analyzed by immunoblotting for total ERK and phospho-ERK, as described in Materials and Methods. The densitometric ratios of the phosphorylated ERK to total ERK species for A are as follows: ERK1 undivided, 1 μg; 0.24, ERK1 divided, 1 μg; 0.82, ERK2 undivided, 1 μg; 3.05, ERK divided, 1 μg; 2.9, ERK1 undivided, 0.5 μg; undetectable, ERK1 divided, 0.5 μg; 0.73, ERK2 undivided, 0.5 μg; 0.44, ERK2 divided, 0.5 μg; 1.7. Extracts from unstimulated T cells contained no phosphorylated ERK (data not shown). C, Assessment of secondary proliferation in response to TCR and CD28 coligation. Sorted T cells that had divided two times (▦) or had remained undivided throughout primary stimulation (□) were cultured with irradiated syngeneic splenocytes in medium, with anti-CD3 (1 μg/ml), or anti-CD3 and anti-CD28 (1 μg/ml each). Four days later, the absolute number of mitotic events accumulated by each sorted CD4+ T cell population was quantified by flow cytometry. The data shown are representative of three separate experiments.

FIGURE 4.

Assessment of MAPK phosphorylation by divided vs undivided CD4+ T cells following secondary receptor cross-linking. Primary, CFSE-labeled T cells were stimulated and rested, as described in Fig. 1, and the CD4+ T cell population was sorted into a divided pool and an undivided pool. The cells were restimulated for 10 min with polystyrene beads coated with anti-CD3 (A) or anti-CD3 and anti-CD28 (B), and lysates were analyzed by immunoblotting for total ERK and phospho-ERK, as described in Materials and Methods. The densitometric ratios of the phosphorylated ERK to total ERK species for A are as follows: ERK1 undivided, 1 μg; 0.24, ERK1 divided, 1 μg; 0.82, ERK2 undivided, 1 μg; 3.05, ERK divided, 1 μg; 2.9, ERK1 undivided, 0.5 μg; undetectable, ERK1 divided, 0.5 μg; 0.73, ERK2 undivided, 0.5 μg; 0.44, ERK2 divided, 0.5 μg; 1.7. Extracts from unstimulated T cells contained no phosphorylated ERK (data not shown). C, Assessment of secondary proliferation in response to TCR and CD28 coligation. Sorted T cells that had divided two times (▦) or had remained undivided throughout primary stimulation (□) were cultured with irradiated syngeneic splenocytes in medium, with anti-CD3 (1 μg/ml), or anti-CD3 and anti-CD28 (1 μg/ml each). Four days later, the absolute number of mitotic events accumulated by each sorted CD4+ T cell population was quantified by flow cytometry. The data shown are representative of three separate experiments.

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Signals emanating from the CD28 costimulatory receptor normally act in part to amplify TCR-coupled signals, including the Ras-Raf-MAPK pathway (26, 27). Therefore, we examined whether provision of maximal Ag and costimulatory receptor cross-linking (10 μg/ml anti-CD3 and anti-CD28) could induce maximal ERK activation in nonproliferative CD4+ T cells. Interestingly, coligation of TCR and CD28 induced comparable ERK phosphorylation in both divided and undivided T cells (Fig. 4,B, lower panel). This result suggests that the diminished activation of ERK in the undivided cells is not due to an intrinsic defect in the Ras-Raf-MAPK cascade, but rather that this pathway is inefficiently coupled to the TCR. Because maximal signals through TCR and CD28 cooperated to overcome the defect in ERK activation, and because CD28 costimulation is also important for the activation of other signal transduction pathways required for optimal IL-2 production and proliferation (28, 29), we tested whether coligation of TCR and CD28 by agonistic Abs could overcome the proliferative defect exhibited by previously undivided CD4+ T cells. While the combination of agonistic anti-CD3 and anti-CD28 Abs induced a nearly 4-fold expansion in the pool of previously divided cells, these signals failed to restore efficient clonal expansion by the undivided CD4+ T cells (in this experiment, 20,000 T cells gave rise to 76,100 mitoses within the previously divided pool vs 10,660 within the undivided pool; a 7-fold difference) (Fig. 4,C). This is consistent with the fact that coligation of CD28 and TCR did not result in IL-2 production by the undivided cells (Fig. 2). These results suggest that an additional defect(s) exists either downstream of ERK, or more likely, within a signal transduction pathway distinct from the Ras-Raf-MAPK pathway, and that this pathway is required for T cell proliferation.

The inability of the previously undivided CD4+ T cells to activate the Ras-Raf-MAPK cascade, produce IL-2, and proliferate in response to restimulation constitutes a phenotype that is remarkably similar to that of anergic T cell clones. However, another hallmark of T cell clonal anergy is that it can be readily reversed by the addition of IL-2 (30). Therefore, we examined whether the hypoproliferative phenotype of the undivided CD4+ T cells could be reversed by the addition of exogenous IL-2. Previously divided or undivided CD4+ T cells were restimulated by TCR ligation in the presence or absence of exogenous IL-2 (Fig. 5,A). In response to TCR ligation alone, the previously divided and undivided CD4+ T cell pools exhibited differences in responder frequency and proliferative capacity similar to those shown in Fig. 1, which together translated to a greater than 20-fold difference in the number of total mitotic events. The previously undivided CD4+ T cells were likewise refractory to IL-2 alone. Surprisingly, even the combination of TCR or TCR/CD28 ligation and exogenous IL-2, which induced a 30-fold expansion of the previously divided T cell pool, failed to induce comparable expansion of the previously undivided population (Fig. 5,A). Therefore, the apparent link between primary T cell proliferative behavior at the single cell level and subsequent proliferative responsiveness upon reactivation could be explained by the fact that the capacity of an individual CD4+ T cell to not only produce IL-2 (Fig. 2), but also to respond to IL-2 (Fig. 5), is quantitatively associated with cell division. The inability of the undivided cells to respond to IL-2 represents a distinction between this division-associated hyporesponsive state and anergy in T cell clones.

FIGURE 5.

Assessment of high-affinity IL-2R expression and IL-2 utilization by CD4+ T cells as a function of prior division history. A, Primary CFSE-labeled T cells, stimulated and rested as in Fig. 1, were sorted into fractions that had divided twice (▦) or had remained undivided during primary activation (□). The cells were then cultured with irradiated, syngeneic splenocytes in the presence of either anti-CD3 Ab (1 μg/ml), IL-2 (15 U/ml), or anti-CD3 and IL-2. Four days later, the absolute number of mitotic events accumulated by the CD4+ T cell subset was quantified by flow cytometry. The data shown are representative of three independent experiments. B, Primary CFSE-labeled T cells were primed, rested, and then restimulated with anti-CD3 and irradiated splenic accessory cells. Cultures were harvested after 24 h, and CD25 expression on the CD4+ T cell subset was assessed by flow cytometry as a function of CFSE fluorescence. No further division of CD4+ T cells was observed during this 24-h period. The data shown are representative of two independent experiments. C, In a separate experiment, primary CFSE-labeled T cells were primed, rested, and sorted into fractions that had divided twice (right panel) or had remained undivided (left panel). The sorted cells were then cultured in medium with (solid lines) or without (dashed lines) exogenous IL-2 (20 U/ml). After 24 h, CD25 expression was assessed by flow cytometry. The data shown are representative of two independent experiments.

FIGURE 5.

Assessment of high-affinity IL-2R expression and IL-2 utilization by CD4+ T cells as a function of prior division history. A, Primary CFSE-labeled T cells, stimulated and rested as in Fig. 1, were sorted into fractions that had divided twice (▦) or had remained undivided during primary activation (□). The cells were then cultured with irradiated, syngeneic splenocytes in the presence of either anti-CD3 Ab (1 μg/ml), IL-2 (15 U/ml), or anti-CD3 and IL-2. Four days later, the absolute number of mitotic events accumulated by the CD4+ T cell subset was quantified by flow cytometry. The data shown are representative of three independent experiments. B, Primary CFSE-labeled T cells were primed, rested, and then restimulated with anti-CD3 and irradiated splenic accessory cells. Cultures were harvested after 24 h, and CD25 expression on the CD4+ T cell subset was assessed by flow cytometry as a function of CFSE fluorescence. No further division of CD4+ T cells was observed during this 24-h period. The data shown are representative of two independent experiments. C, In a separate experiment, primary CFSE-labeled T cells were primed, rested, and sorted into fractions that had divided twice (right panel) or had remained undivided (left panel). The sorted cells were then cultured in medium with (solid lines) or without (dashed lines) exogenous IL-2 (20 U/ml). After 24 h, CD25 expression was assessed by flow cytometry. The data shown are representative of two independent experiments.

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To understand the basis of the association between cell division and IL-2 responsiveness, we first assessed the expression of IL-2R chains on CD4+ T cells of varying proliferative histories. The majority of primed CD4+ T cells, regardless of prior division history, were induced to express IL-2R α-chain (CD25) upon TCR reengagement (Fig. 5,B, •), although the relative level of CD25 per cell was successively higher on cells that had undergone more rounds of cell division (Fig. 5,B, ○). Flow cytometric analysis of IL-2R β-chain (CD122) and γc-chain expression on primed CD4+ T cells showed no significant differences as a function of division cycle (data not shown). Furthermore, both divided and undivided CD4+ T cells were able to up-regulate CD25 in response to IL-2 (Fig. 5 C). Therefore, the differential responsiveness of the divided vs the undivided CD4+ T cells to IL-2 cannot be explained simply by the presence vs absence of high-affinity IL-2R, suggesting that the undivided CD4+ T cell pool suffers from a biochemical defect(s) in signal transduction downstream of IL-2R.

Two major IL-2R-coupled signaling pathways have been reported to be absolutely necessary for the transduction of IL-2-mediated proliferative signals in T cells. One pathway involves the Janus kinase (JAK)3-mediated activation of the transcription factor STAT5 (31), while the other pathway involves the phosphatidylinositol-3 kinase (PI3K)-mediated activation of protein kinase B (PKB), also known as Akt (32). Interestingly, up-regulation of CD25 in response to IL-2, which occurs normally in both the divided and undivided T cell populations (Fig. 5, B and C), requires the functional activity of STAT5 (33). This suggests that the JAK3/STAT5 pathway is not compromised in the undivided CD4+ T cell pool. To confirm this biochemically, we assessed the activation of STAT5 in IL-2-stimulated CD4+ T cells by measuring STAT5 phosphorylation. A relatively large amount of total, intracellular STAT5 could be detected by flow cytometry in all CD4+ T cells (Fig. 6,A). Specific phosphorylation of STAT5 could likewise be detected, in an IL-2-dependent manner, in all CD4+ T cells (Fig. 6,A). Quantitative analysis of phospho-STAT5 content as a function of cell division (Fig. 6, B and C) showed that all CD4+ T cells could activate STAT5 to a similar degree in response to IL-2, regardless of their proliferative behavior during the primary stimulus. These data suggest that the inability of the previously undivided CD4+ T cells to proliferate in response to IL-2 results from a biochemical defect in a pathway distinct from the JAK3/STAT5 pathway. To test the integrity of the PI3K/Akt pathway as a function of cell division, we compared the ability of divided vs nondivided CD4+ T cells to down-regulate the cyclin-dependent kinase inhibitor p27kip1 in response to IL-2. IL-2-mediated down-regulation of p27kip1 is crucial for the progression of T cells from the G1 phase to the S phase of the cell cycle (34), and activation of the PI3K/Akt pathway is both necessary and sufficient to achieve p27kip1 down-regulation in T cell lines (35). Interestingly, we found that while those CD4+ T cells that participated in clonal expansion during the primary stimulus contained very little p27kip1 after restimulation with IL-2 (Fig. 6,D, right lane), CD4+ T cells that failed to divide were unable to down-regulate this cell cycle inhibitor (Fig. 6 D, left lane). These results suggest that the inability of the undivided CD4+ T cells in this model may be due to a defect in IL-2R-coupled signal transduction involving the PI3K, PKB/Akt pathway.

FIGURE 6.

IL-2R-coupled STAT5 activation and p27kip1 down-regulation in CD4+ T cells as a function of division cycle. A, Primary CFSE-labeled T cells were primed, rested, and then cultured for 10 min in the presence of 100 U/ml IL-2. The cells were fixed, permeabilized, and stained for either total STAT5 (solid black line) or phosphorylated STAT5 (solid gray line). The dashed gray line depicts the phospho-STAT5 content of CD4+ T cells cultured in medium alone for 10 min, and the dashed black line depicts the isotype control staining of the IL-2-stimulated CD4+ T cells. B, Density plot of phospho-STAT5 vs CFSE content in the IL-2-stimulated CD4+ T cells. C, Quantitation of phospho-STAT5 relative to total STAT5 content in unstimulated (○) and IL-2-stimulated (•) CD4+ T cells. The pSTAT5/STAT5 ratio was calculated by dividing the phospho-STAT5 MFI/isotype control MFI ratio by the total STAT5 MFI/isotype control MFI ratio. The data shown are representative of two independent experiments. D, Primary, CFSE-labeled T cells were primed with anti-CD3, rested, and restimulated with 50 U/ml IL-2 for 48 h. The live, CD4+ cells were then sorted into fractions that had divided two or more times (right lane), or had remained undivided during the culture period (left lane). The cells were lysed, equal cell equivalents were subjected to SDS-PAGE, and p27kip1 content was assessed by immunoblot analysis (top panel). The blot was then stripped and reprobed for actin as a loading control (lower panel). Although the divided and undivided subsets contained similar levels of p27kip1 before restimulation (see Fig. 7), the relative level of p27kip1 (normalized to actin) in the undivided CD4+ T cell fraction following IL-2 stimulation is nearly 15-fold higher than the level of p27kip1 in the divided CD4+ T cell fraction. The data shown are representative of two independent experiments.

FIGURE 6.

IL-2R-coupled STAT5 activation and p27kip1 down-regulation in CD4+ T cells as a function of division cycle. A, Primary CFSE-labeled T cells were primed, rested, and then cultured for 10 min in the presence of 100 U/ml IL-2. The cells were fixed, permeabilized, and stained for either total STAT5 (solid black line) or phosphorylated STAT5 (solid gray line). The dashed gray line depicts the phospho-STAT5 content of CD4+ T cells cultured in medium alone for 10 min, and the dashed black line depicts the isotype control staining of the IL-2-stimulated CD4+ T cells. B, Density plot of phospho-STAT5 vs CFSE content in the IL-2-stimulated CD4+ T cells. C, Quantitation of phospho-STAT5 relative to total STAT5 content in unstimulated (○) and IL-2-stimulated (•) CD4+ T cells. The pSTAT5/STAT5 ratio was calculated by dividing the phospho-STAT5 MFI/isotype control MFI ratio by the total STAT5 MFI/isotype control MFI ratio. The data shown are representative of two independent experiments. D, Primary, CFSE-labeled T cells were primed with anti-CD3, rested, and restimulated with 50 U/ml IL-2 for 48 h. The live, CD4+ cells were then sorted into fractions that had divided two or more times (right lane), or had remained undivided during the culture period (left lane). The cells were lysed, equal cell equivalents were subjected to SDS-PAGE, and p27kip1 content was assessed by immunoblot analysis (top panel). The blot was then stripped and reprobed for actin as a loading control (lower panel). Although the divided and undivided subsets contained similar levels of p27kip1 before restimulation (see Fig. 7), the relative level of p27kip1 (normalized to actin) in the undivided CD4+ T cell fraction following IL-2 stimulation is nearly 15-fold higher than the level of p27kip1 in the divided CD4+ T cell fraction. The data shown are representative of two independent experiments.

Close modal

The data above suggest that the integrity of both TCR-coupled and IL-2R-coupled signal transduction pathways is linked to cell division, and that activated T cells that fail to divide suffer from defects in both these pathways that render them unable to proliferate upon further mitogenic stimulus. We next tested whether these functional defects could be overcome by bypassing T cell surface receptors. The combination of the phorbol ester PMA and the calcium ionophore ionomycin can uncouple the Ras-Raf-MAPK cascade from the Ag receptor by directly activating PKC (36, 37, 38). Receptor-independent activation of PKC using PMA resulted in comparable ERK1/2 phosphorylation in both divided and undivided CD4+ T cells (Fig. 7,A). PKC also lies downstream of PI3K in the IL-2R signal transduction pathway (39, 40), and is normally activated in response to IL-2 (41). To test whether receptor-independent activation of PKC could overcome the defect in IL-2-mediated p27kip1 down-regulation exhibited by the undivided CD4+ T cell subset, we restimulated primed cells with PMA and ionomycin instead of IL-2. This combination of phorbol ester and calcium ionophore was able to induce comparable p27kip1 down-regulation in both the divided and the undivided CD4+ T cells (Fig. 7,B). We next compared the ability of the divided vs the undivided CD4+ T cells to proliferate upon restimulation with anti-CD3 vs PMA/ionomycin. As seen before, the previously undivided CD4+ T cells exhibited little proliferation in response to TCR engagement compared with the divided population (Fig. 7,C). However, the combination of PMA and ionomycin was able to stimulate a large and equal degree of proliferation by both the divided and the undivided subsets (Fig. 7 C). These results suggest that receptor-independent activation of PKC is able to bypass the defect in PI3K-mediated signal transduction normally exhibited by the undivided CD4+ subset.

FIGURE 7.

PMA and ionomycin induce normal MAPK activation, p27kip1 down-regulation, and proliferation in the previously undivided CD4+ T cell subset. A, Primary CFSE-labeled T cells, stimulated and rested as above, were sorted into fractions that had divided twice (right lane), or had remained undivided during primary activation (left lane). The cells were then restimulated by the addition of PMA and ionomycin for 10 min, and the phospho-ERK content of the cell lysates was assessed as in Fig. 4. B, In a separate experiment, primed and rested cells were sorted by division cycle and either assessed immediately for p27kip1 content (upper panel; resting), or were restimulated with PMA and ionomycin for 48 h before assessment of p27kip1 content (middle panel; PMA/IONO). The blot was stripped and reprobed for actin as a loading control (lower panel). C, In a separate experiment, cells were primed, rested, and sorted as above, and the undivided (□) and divided (▦) fractions were restimulated for 4 days with either anti-CD3 Ab (1 μg/ml) or PMA/ionomycin (3 ng/ml and 250 μM, respectively). The data shown are representative of two independent experiments.

FIGURE 7.

PMA and ionomycin induce normal MAPK activation, p27kip1 down-regulation, and proliferation in the previously undivided CD4+ T cell subset. A, Primary CFSE-labeled T cells, stimulated and rested as above, were sorted into fractions that had divided twice (right lane), or had remained undivided during primary activation (left lane). The cells were then restimulated by the addition of PMA and ionomycin for 10 min, and the phospho-ERK content of the cell lysates was assessed as in Fig. 4. B, In a separate experiment, primed and rested cells were sorted by division cycle and either assessed immediately for p27kip1 content (upper panel; resting), or were restimulated with PMA and ionomycin for 48 h before assessment of p27kip1 content (middle panel; PMA/IONO). The blot was stripped and reprobed for actin as a loading control (lower panel). C, In a separate experiment, cells were primed, rested, and sorted as above, and the undivided (□) and divided (▦) fractions were restimulated for 4 days with either anti-CD3 Ab (1 μg/ml) or PMA/ionomycin (3 ng/ml and 250 μM, respectively). The data shown are representative of two independent experiments.

Close modal

Previously, we have found that even with optimal TCR ligation and CD28 costimulation, a large proportion of the activated (i.e., CD25+CD69+) T cells fails to divide both in vitro (8) and in vivo (9). We demonstrate in this work that individual, primed CD4+ T cells exhibit distinct secondary response patterns that depend upon their prior division history. Specifically, CD4+ T cells that undergo more rounds of cell division during primary stimulation are more likely to produce IL-2, and respond with a greater degree of proliferation, upon restimulation. Furthermore, we show that the integrity of both TCR- and IL-2R-coupled signal transduction pathways is quantitatively tuned to cell division, such that the activated cells that fail to divide are unable to respond to receptor-coupled mitogenic signals. The failure of this subset of CD4+ T cells to participate in the primary clonal expansion phase is not due to limitations in CD28 costimulation or IL-2, as a similar proportion of the activated CD4+ T cells fails to divide whether or not agonistic anti-CD28 or exogenous IL-2 is included in the primary cultures (8). Furthermore, CD4+ T cells that remain undivided following primary stimulation in the presence of agonistic anti-CD28 Ab are also unable to proliferate in response to restimulation (our unpublished results). Finally, primary stimulation of T cells with PMA and ionomycin does not induce all of the activated T cells to proliferate, suggesting that at least a significant component of this pool fails to divide despite sufficient receptor-proximal signal transduction (8).

An association between cell division and cytokine production has been observed previously during the primary phase of clonal expansion in CD4+ T cells in vitro (10, 11) and in vivo (9), leading to the hypothesis that multiple rounds of cell division may be required to render chromosomal loci associated with T cell effector function accessible to specific transcription factors induced during earlier activation (10, 12, 42). Our data show that this division-associated trend in T cell effector differentiation (i.e., cytokine production and proliferation) is stably maintained within the pool of relatively long-lived cells present after a period of primary stimulation and rest in vitro, and is again operative when these cells reencounter Ag.

We show that those CD4+ T cells that fail entirely to divide in response to primary activation exist in a hyporesponsive state that is refractory to TCR ligation, and is associated with a quantitative defect in TCR-coupled Ras-Raf-MAPK signal transduction. A rest period of at least 2 days following primary stimulation is required for the development of this MAPK defect (our unpublished observations), suggesting either that this defect arises only after cessation of primary signal transduction, or possibly that cell death due to growth factor withdrawal may select for cells that exhibit this phenotype. However, this defect is selective, as although TCR-coupled MAPK activation is attenuated in these cells, the TCR-coupled calcium response appears intact. In normal T cells, the most TCR-proximal events following Ag recognition are initiated by the nonreceptor tyrosine kinases Lck and ZAP-70 (43). The Ras-Raf-MAPK cascade is coupled to the TCR via adaptor molecules such as linker of activated T cells (LAT), SH2 domain-containing leukocyte protein of 76 kDa (SLP-T6), and Grb2/Sos (44), and involves initial events mediated by the SH3 domain of Lck (45). TCR-induced increase in [Ca2+]i, which is mediated by phosphatidylinositol, is dependent on the activation of PLC-γ, and is coupled to the TCR through ZAP-70 and Rho/Vav (45, 46). TCR-coupled PLC-γ activation, generation of phosphatidylinositol, and [Ca2+]i elevation are not dependent on the SH3 domain of Lck (45). The selective defect in MAPK activation, but not PLC-γ activation, in the undivided CD4+ T cells suggests a defect in the coupling of the SH3 domain of Lck to the Ras-Raf-MAPK pathway. Also, we observe in this study that signal transduction through the costimulatory receptor CD28, which has been shown to augment TCR-coupled ERK activation in normal T cells (27), and phorbol ester, which bypasses the most proximal TCR-coupled events through the direct activation of PKC, both restored maximal activation of the MAPK cascade in the undivided T cells. This also suggests that the defect is not in the MAPK pathway itself, but rather is in a pathway that couples Ras-Raf-MAPK to the TCR. PI3K, Raf, and mitogen-activated protein/extracellular signal-related kinase kinase are capable of binding to the SH3 domain of Lck (47, 48, 49), suggesting that uncoupling of these factors from Lck in undivided cells could result in this selective defect in MAPK activation. The ability of PMA/ionomycin to induce proliferation in these cells (see below) may also suggest that PKC-mediated activation of the NF-κB pathway, which is required for the production of IL-2, could be defective in these cells. Alternatively, such negative regulatory factors as Ras-GAP and c-Cbl also bind to SH3 domain of Lck (50, 51), suggesting that selective coupling of these factors to the TCR in undivided cells, but not divided cells, could likewise explain this selective MAPK defect. Differential coupling of negative regulatory molecules to the TCR has been demonstrated previously in anergic human T cell clones, in which the TCR is coupled not to Ras, but to Rap1 (25), a Ras homologue that binds Raf, but is unable to activate the MAPK cascade. Interestingly, the Ras-Raf-MAPK defect exhibited by the undivided cells in this model appears to preferentially affect the ERK1 isoform, as TCR-coupled activation of ERK2, but not ERK1, remains relatively intact. The presence of a significant amount of activated ERK2 in the undivided pool following TCR ligation might suggest that the Ras-Raf-MAPK pathway is not significantly compromised in these cells; however, recent studies in ERK1−/− mice have defined an obligatory role for ERK1 in T cell activation that cannot be fulfilled by ERK2 (52). Together, these results support a model in which a selective defect in ERK1 might result in a highly attenuated response to TCR ligation.

The inability of maximal TCR-coupled calcium and Ras-Raf-MAPK signaling to overcome the proliferative defect in the undivided CD4+ T cells suggests the presence of further defect(s) in growth factor-mediated signal transduction. Unlike the defect in TCR-coupled MAPK activation, a rest period following primary stimulation is not required for the development of this IL-2 refractory phenotype (our unpublished observations). CD4+ T cells from both the divided and undivided pools were able to express all components of the high-affinity IL-2R in response to both TCR engagement and IL-2 itself, demonstrating that the inability of the undivided subset to proliferate is not due to a lack of growth factor receptor expression. This suggests that the integrity of signal transduction from the IL-2R must be coupled to cell division. Two major IL-2R-coupled signaling pathways have been reported to be absolutely necessary for the transduction of IL-2-mediated proliferative signals in T cells. One pathway, which is specifically coupled to the γc-chain of the IL-2R, involves the JAK3-mediated activation of the transcription factor STAT5 (31), while the other pathway is coupled to the IL-2R β-chain and involves the PI3K-mediated activation of PKB/Akt (32). All T cells were able to phosphorylate STAT5 in response to IL-2 stimulation, regardless of their prior division history, suggesting that the defect in IL-2R-coupled signal transduction does not reside in this JAK/STAT pathway. PI3K is an integral component of many growth factor receptor signal transduction pathways (53), and is absolutely required for normal T cell responses (54). D3 phosphoinositides generated by PI3K recruit PKB/Akt and phosphoinositide-dependent kinase-1 (PDK1) to the cell membrane, in which PDK1 phosphorylates and activates PKB/Akt (55). The PKB/Akt kinase is also a crucial signal transduction component for many growth factor receptors (56, 57), and has been shown to be both necessary and sufficient for the down-regulation of p27kip1, hyperphosphorylation of the retinoblastoma tumor suppressor protein, and release of active E2F in T cell lines (35). However, the mechanism by which PKB/Akt activity leads to these downstream effects is unclear. Our studies show that CD4+ T cells that fail to divide following activation are neither able to down-regulate p27kip1 nor able to proliferate in response to restimulation with IL-2, suggesting that PI3K and/or PKB/Akt are not coupled properly to the IL-2R, or that another biochemical step(s) leading to p27kip1 is not coupled properly to PKB/Akt in these cells. In this sense, the defect in IL-2 responsiveness exhibited by the nonproliferative CD4+ T cell subset resembles the phenotype of T cells treated with the pharmacologic agent rapamycin (34), which blocks IL-2R signal transduction by binding to mammalian target of rapamycin (mTOR)/FK506-binding protein-rapamycin-associated protein (FRAP) (58), a downstream target of PKB/Akt.

Interestingly, receptor-independent activation of PKC in the undivided CD4+ T cells using phorbol ester and calcium ionophore resulted in efficient down-regulation of p27kip1 and normal proliferative capacity. These results suggest that the IL-2 refractory phenotype of the previously undivided CD4+ T cells is due to a biochemical defect that lies somewhere upstream of the action of PKC within the PI3K-PKB/Akt pathway. PKC is normally coupled to the IL-2R through PI3K, as PKC is activated by PDK1-dependent phosphorylation (40), and a role for PKC in IL-2-mediated signal transduction has been defined using the IL-2-dependent cell line CTLL (59, 60). Interestingly, full activation of the PKC-δ isoform requires the activity of mTOR/FRAP (61), which is in turn dependent on PKB/Akt (62). These two distinct PI3K-dependent pathways leading to PKC activation, which can be bypassed using phorbol esters, represent a set of biochemical events that could potentially be defective in the undivided pool. Additionally, PKC can phosphorylate other downstream targets of PKB/Akt, including GSK-3 and cAMP response element binding protein (56). Therefore, the defect in IL-2-mediated signal transduction exhibited by the undivided CD4+ T cells in this model might result specifically from the failure to activate downstream targets of PKB/Akt or PKC.

The phenotype of the CD4+ T cells that fail to divide following primary activation, i.e., an inability to secrete IL-2 and proliferate upon TCR reengagement, closely resembles that of clonal anergy, a phenomenon normally associated with TCR occupancy in the absence of CD28 costimulation (15, 63, 64). The cell division-associated hyporesponsive state that we observe also shares similarities with clonal anergy at the molecular level. Similar to the undivided CD4+ T cells described in this work, both mouse and human T cell clones rendered anergic by TCR engagement in the absence of CD28 costimulation exhibit a normal increase in intracellular calcium following TCR engagement (15), but suffer from quantitative defects in Ras-coupled MAPK activation (22, 23, 24, 25). Also, the cyclin-dependent kinase inhibitor p27kip1, which we show is highly elevated in the CD4+ T cells that fail to divide after activation, has recently been shown to function as a molecular anergy factor in human T cell clones stimulated in the absence of CD28 costimulation (65). In addition, the authors discovered that p27kip1 could actively inhibit IL-2 gene transcription by sequestering the AP-1 coactivator JAB1, an activity not previously attributed to this cyclin-dependent kinase inhibitor. Therefore, the inability of the undivided CD4+ T cells in our model to down-regulate p27kip1 in response to IL-2 could explain not only why these cells are unable to progress from the G1 phase to the S phase of the cell cycle, but when paired with the coincident defect in TCR-coupled ERK1 activation, could also explain why these cells are unable to produce IL-2 as well. Finally, the combination of phorbol ester and calcium ionophore, which was able to overcome the molecular and functional defects exhibited by the undivided T cells in this study, has been shown to reverse anergy in T cell clones (15).

While these two refractory states share many functional and biochemical similarities, they differ in at least two respects. As mentioned above, classical T cell clonal anergy results from TCR occupancy in the absence of CD28 costimulation, whereas the division-associated unresponsiveness described in this work occurs despite the presence of sufficient CD28 costimulatory signals. Second, the failure of anergic T cell clones to proliferate in the models described above is due to a defect in the production, but not in the utilization, of IL-2. The provision of IL-2 during restimulation in both models restored clonal responsiveness (30, 65). This is in contrast to the phenotype of the undivided CD4+ T cells described in this work, which not only fail to produce IL-2 during restimulation, but are also refractory to IL-2 provided exogenously. This difference may point to an important distinction between the division-associated, hypoproliferative state and clonal anergy induced by TCR occupancy in the absence of costimulation. Alternatively, these two phenotypes could arise from analogous biochemical circumstances, and the differential IL-2 responsiveness may reflect differences in the capacity of IL-2R-coupled signal transduction pathways (e.g., PI3K or PKB/Akt) to remain poised in primary T cells vs long-term clones. Notably, IL-2 unresponsiveness has also been observed in T cells from mice chronically treated with staphylococcal superantigen (66). However, in this case, IL-2 unresponsiveness apparently resulted from a defect in γc-coupled activation of the JAK3-STAT5 pathway.

These data show that cell division or a process associated with cell cycle progression controls the ability of a T cell to respond to both Ag and growth factor by modulating the integrity of the signals transduced through both the Ras-Raf-MAPK cascade and the PI3K, PKB/Akt pathway. This further suggests that activated T cells must proliferate to avoid the induction of anergy. The hypothesis that cell cycle progression may be required for anergy avoidance by T cells was originally proposed by Schwartz and Jenkins (15, 67). These investigators have suggested that CD28 costimulation promotes anergy avoidance indirectly by mediating efficient IL-2 production. In this scenario, it is the subsequent IL-2-driven cell division that allows T cells to escape anergy. Several lines of evidence, including our results in this study, support this model. Partial agonist ligands, which signal through the TCR, but do not cause proliferation, induce anergy in T cells despite the presence of CD28 costimulatory signals (68). Furthermore, whether a given peptide ligand induces productive activation or anergy correlates with its capacity to induce IL-2 (and proliferation), not earlier signaling patterns such as TCR-ζ phosphorylation (69). Second, overt blockade of IL-2-mediated signal transduction, using either anti-IL-2/IL-2R Abs (67) or the pharmacologic agents butyrate (70) or rapamycin (71), induces anergy. Interestingly, primary T cells arrested with butyrate during in vitro stimulation with agonistic anti-CD3 and anti-CD28 Abs are also rendered anergic, and like the T cells that naturally fail to divide during primary stimulation, these butyrate-treated cells are also refractory to IL-2 (our unpublished results). These results are consistent with previous studies using T cells clones; however, not all pharmacological inhibitors of cell cycle induce anergy in T cells (70, 71) (our unpublished observations). More specifically, anergy avoidance may be associated with the G1 to S phase transition, as opposed to mitosis per se, as drugs that block mitosis, but allow G1 to S phase transition (e.g., hydroxyurea), do not induce anergy (71).

The use of primary T cells isolated from normal mice has allowed us to assess the size of the nonresponsive pool within a normal T cell repertoire. Surprisingly, this pool represents between 30 and 40% of the mature, peripheral T cells in the mouse. Although we believe that this population is generated during in vitro stimulation (see below), because our analysis involves a heterogeneous starting T cell population, we cannot eliminate the possibility that the nonproliferative cells in our study represent a subset of the peripheral CD4+ T cell repertoire with previous antigenic experience, or that has otherwise been rendered anergic in vivo before our analysis. However, several lines of evidence argue against this deterministic model. First, the use of mitogenic Abs against the monotypic CD3 component of the TCR effectively bypasses the Ag specificity, and therefore the affinity, of the individual T cells. We have also detected this nonproliferative subset in populations of TCR-transgenic T cells stimulated in vitro and in vivo with specific peptide (9). These data suggest that the nonproliferative population is not merely a subset of the peripheral repertoire with reduced TCR affinity. We have addressed the possibility that the nonproliferative pool represents a subset of T cells with previous antigenic exposure by fractionating the peripheral T cell repertoire of normal mice and D011.10 TCR transgenic mice into naive and memory subsets based on surface phenotype. In these experiments, both pools exhibited a similar degree of proliferative heterogeneity and gave rise to both divided and undivided cells after stimulation through TCR/CD28 (our unpublished observations). This suggests to us that the nonproliferative pool is not comprised preferentially of cells with any given antigenic history. Our previous studies using Ag-specific T cells from TCR-transgenic Rag2−/− mice also argue against this possibility. These cells represent a population of purely clonal, naive T cells, and despite this, these cells still exhibit marked heterogeneity in proliferative behavior at the single cell level, and although they exhibit higher responder frequencies than T cells from recombination-competent mice, the few Rag2−/− D011 T cells that fail to divide in response to antigenic peptide also fail to produce IL-2 upon restimulation (9). Finally, the studies described above using cell cycle inhibitors (butyrate and rapamycin) show that T cells that would normally proliferate in response to TCR/CD28 stimulation can be rendered unresponsive by overtly blocking their cell cycle progression. For these reasons, we favor a stochastic model in which any given naive T cell has the potential to respond to an antigenic stimulus by dividing, but that the probability that any given cell will achieve this goal is <1; from our data, we estimate this number to be between 0.5 and 0.7. The failure to divide in this scenario fixes the cell in a state that is refractory to further stimulation. Support for this stochastic model can be observed in our data in this study, which show that even among previously divided cells, a sizeable proportion (∼25%) fails to divide upon restimulation; i.e., a pure population of responders can give rise to both responders and nonresponders (see Fig. 1). This indicates that the responsive phenotype can be transient, and leaves open the possibility that random events that control gene expression in complex systems may regulate proliferation (72). However, whether the origin of this nonresponsive population is stochastic or deterministic, its presence is remarkable, as it represents approximately one-third of the peripheral CD4+ T cell pool in a normal mouse.

The results reported in this work, together with several recent studies (9, 10, 11), suggest that secondary T cell responses are influenced by a mitotic clock (73, 74). Specifically, T cells that fail to divide following activation are refractory to secondary stimulation, while T cells that do proliferate remain responsive. Additionally, those cells that progress through many division cycles during primary stimulation tend to exhibit better secondary responses than those T cells that divide fewer times. Thus, the role of T cell clonal expansion in generating effective Ag-specific immunity may be 2-fold: The progression of naive T cells through a phase of exponential growth not only increases the frequency of Ag-specific T cells, but may also ensure that the resultant T cell pool consists preferentially of those T cells that have undergone multiple rounds of cell division and therefore carry the greatest potential to respond to future antigenic challenge.

We thank J. Moore and C. Pletcher at the University of Pennsylvania Cancer Center Flow Cytometry and Cell Sorting Resource for excellent technical advice and support. We also thank B. Freedman for technical advice concerning calcium experiments, M. Carroll for technical advice concerning intracellular STAT5/pSTAT5 staining, and X.-C. Li, T. Laufer, T. Judge, and S. Reiner for critical review of this manuscript.

1

This work was supported by National Institutes of Health Grants AI-37691, AI-41521, and P30-CA16520-24. L.A.T. is an Established Investigator of the American Heart Association. A.D.W. was supported by National Institutes of Health Training Grants CA 09140 and K01DK02771-01.

3

Abbreviations used in this paper: CFSE, 5- and 6-carboxyfluorescein diacetate succinimidyl ester; [Ca2+]i, intracellular Ca2+ concentration; ERK, extracellular signal-related kinase; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; MFI, mean fluorescence intensity; PDK, phosphoinositide-dependent kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C; γc, common γ-chain; LAT, linker of activated T cells; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; mTOR, mammalian target of rapamycin; FRAP, FK506-binding protein-repamycin-associated protein.

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