Continued Antigen Stimulation Is Not Required During CD4⁺ T Cell Clonal Expansion

Animals

The BALB/c ByJ and DO11.10 (12) mice used in these experiments were bred and maintained at the Wadsworth Center Animal Core Facility (Albany, NY) under specific pathogen-free conditions. The majority of T cells in the DO11.10 mice are CD4⁺ cells which bear a TCR that recognizes a chicken OVA-derived peptide, OVA_323–339 (hereafter referred to as OVA), presented by I-A^d (12). This TCR is encoded by transgenes encoding Vβ8.2/Vα13.1 chains and can be identified by the anticlonotypic mAb, KJ1-26 (13). The DO11.10 mice were originally obtained from Dr. D. Loh (Kamakura, Japan). Unless otherwise indicated, the experiments were performed using 6- to 8-wk-old mice. Both male and female mice were used in different experiments with no discernible differences in the results. All mice used in these studies were bred and maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources (National Research Council, Washington, DC).

Reagents and Abs

Chicken OVA peptide (OVA_323–339) was synthesized and supplied by the Wadsworth Center Peptide Synthesis Core Facility. Polyclonal rabbit anti-mouse Ig was prepared by affinity purification on mouse Ig-Sepharose columns. mAbs GK1.5 (14) and 2B6 (15) (anti-CD4), 3.155 (anti-Lyt-2 (CD8)) (16), M1/70.15 (anti-MAC-1) (17), J11d.8 (anti-J11d) (18), HO13.4 (anti-Thy1.2) (19), Mar 18.5 and MARK-1 (anti-rat κ-chain) (20), and KJ1-26 (anti-DO11.10 clonotype) (13) were prepared from the supernatants of hybridoma cell lines, as previously described (21). Biotinylated anti-CD25 (mAb 3C7) and anti-CD69 (mAb H1.2F3) were purchased from BD PharMingen (San Diego, CA). CFSE was purchased from Molecular Probes (Eugene, OR). Mitomycin C was purchased from Sigma-Aldrich (St. Louis, MO).

Preparation of cells

In all experiments, enriched populations of CD4⁺ T cells were prepared as previously described (22). Briefly, RBCs were removed by hypotonic lysis. B cells were depleted using rabbit anti-mouse Ig followed by goat anti-rabbit Ig-coated magnetic beads (Advanced Magnetics, Cambridge, MA) and adherence to a magnet. Residual B cells, macrophages, and CD8⁺ T cells were removed by Ab and complement depletion using anti-J11d, anti-MAC-1 plus MAR18.5, and anti-CD8, respectively. Baby rabbit serum (Wadsworth Center Animal Core Facility) was used as a source of complement. When these procedures were used, cells were 90–95% CD4⁺ and <3% surface Ig⁺ as determined by flow cytometric analyses. APCs were prepared by T cell depletion of splenocytes using anti-Thy1-1.2 and complement followed by anti-CD4 (mAb 2B6) and anti-CD8 plus complement. Unless otherwise indicated, APCs were treated with mitomycin C (25 μg/ml) for 20 min at 37°C.

Cell labeling and culture

CD4⁺ T cells were labeled with CFSE using previously described procedures (9, 11). After enrichment for CD4 cells, the cells (5 × 10⁷ cells/ml) were incubated with CFSE (1 μM) in PBS for 10 min at 37°C. After washing three times with tissue culture medium containing 10% FBS (Life Technologies, Grand Island, NY), the cells were placed into tissue culture. Unless otherwise indicated, the unfractionated CD4 cells were cultured either in 96-well plates (1 × 10⁵/well; Falcon Labware, Oxnard, CA) with 2 × 10⁵/well APCs in 0.2 ml of RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-ME, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine or were cultured in bulk at a concentration of 1 × 10⁶/ml with 2 × 10⁶/ml APCs. Where indicated, OVA_323–339 was added into the cultures. In all experiments, unless otherwise indicated, the data depicted are representative results from a minimum of three separate experiments.

Immunofluorescence staining, analysis, and sorting

Where indicated, mAbs were directly labeled with Cy5 (Biological Detection Systems, Pittsburgh, PA). Alternatively, biotinylated mAbs were detected using Cy5-streptavidin (Biological Detection Systems). Fluorescence staining was performed at 4°C in 100 μl with a predetermined optimal amount of primary Ab in balanced salt solution containing 2% FBS, 20 mM HEPES, and 0.1% NaN₃. Staining with the secondary reagent was performed in a similar manner after washing the cells. Flow cytometry analyses and sorting of stained cells were performed using either a FACSCalibur or a FACSVantage (BD Biosciences, Mountain View, CA). With the latter cytometer, the Cy5 was excited using a helium-neon laser (632.6 nm emission). For sorting experiments, power of the 488-nm argon laser was reduced to 75 mW. Flow cytometry data was analyzed using CellQuest software (BD Biosciences). Percentages of cells at specific rounds of division were calculated using Modfit LT software (Verity Software House, Topsham, ME).

Adoptive transfer of DO11.10 T cells

The procedure is done as described by Kearney et al. (23) with some modifications. CFSE-labeled, DO11.10 CD4⁺ T cells (2.5 × 10⁶) were suspended in sterile PBS and injected i.v. into 6- to 8-wk-old BALB/c mice. For sorted cells, lymph node cells were analyzed using flow cytometry to determine CFSE fluorescence on KJ1-26⁺ cells 48 h after adoptive transfer. For sorted cells, 1 day after adoptive transfer, some of the host mice were immunized s.c. with OVA_323–339 (150 μg) in CFA while others were not immunized. For both groups, 48 h after the immunization period the axillary, brachial, and cervical lymph nodes (draining nodes for the immunized mice) were removed, pooled, and analyzed by flow cytometry.

Ag dose influences the percentage of CD4⁺ T cells activated but not the rate of cell proliferation

To measure proliferation in response to culture with specific peptide Ag, CD4 T cells obtained from DO11.10 mice (12) were labeled with CFSE before culture with unlabeled APCs and OVA_323–339, and at different times the cells were collected and examined by flow cytometry. As previously reported, 50–80% of the CD4⁺ T cells in DO11.10 mice express both the transgenic TCRα and TCRβ chains and are stimulated by OVA/I-A^d (24). Hence, mAb KJ1-26 (13) was used to identify these clonotype-bearing T cells. These cells proliferated when exposed to OVA and cumulative cell division was indicated by the appearance of cells with decreased fluorescence intensity. Cell division was limited to the KJ1-26⁺ cells (Fig. 1⇓, a and b), and in the absence of peptide the cells remained undivided, exhibiting a single peak of high fluorescence intensity (Fig. 1⇓, c and d). Culture with OVA induced early activation of the KJ1-26⁺ cells, as evidenced by expression of CD69 (Fig. 1⇓c) and CD25 (Fig. 1⇓d). Expression of both markers was largely limited to the KJ1-26⁺ cells after stimulation (data not shown). Indeed, at higher peptide doses (e.g., 200 ng/ml) the percentages of KJ-126⁺ cells and the percentages of CD25⁺ and CD69⁺ cells after stimulation were similar. These data indicated that with these higher doses virtually all of the KJ1-26⁺ cells had responded to peptide within 24 h. However, even at the highest tested peptide dose (2000 ng/ml), little proliferation was observed before 36 h. After this time rapid cell division occurred such that by 60 h most cells had divided, with the majority of cells having undergone three to four cell divisions. Cell division was asynchronous; cells were distributed among multiple divisions, including small numbers of KJ1-26⁺ cells that remained undivided through 60–66 h in culture. At lower peptide doses, activation markers were expressed on smaller numbers of KJ1-26⁺ cells and fewer cells ultimately proceeded to divide. In addition, at lower doses, the beginning of cell division was slightly delayed. Comparison of the cell distribution among rounds of division indicates that cells stimulated with 20 ng/ml OVA were approximately one cycle behind cells stimulated with 10-fold higher amounts of peptide (Fig. 1⇓b). However, once cell division had begun, the rate of division appeared similar, regardless of peptide dose (see Fig. 1⇓b; 20 ng/ml at 66 h vs 200 ng/ml at 60 h). Peptide dose appeared to more profoundly affect the percentage of cells that ultimately divided (Fig. 1⇓, a and b). These data indicate that Ag dose regulates the percentage of cells that will proliferate but has a more modest effect on the rate of clonal expansion. Furthermore, cell division is preceded by activation marker (CD69 and CD25) expression.

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FIGURE 1.

Activation and proliferation of DO11.10 clonotype-bearing CD4⁺ T cells. CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁵/well) were cultured with unlabeled APCs (2 × 10⁵/well) and the indicated concentrations of OVA. At the indicated times the cells were collected and stained with mAbs specific for DO11.10 clonotype (mAb KJ1-26) (a), CD69 (c), or CD25 (d). The cells were immediately analyzed using the FACSCalibur cytometer. Data are gated to show CFSE and surface marker expression on live lymphocytes based upon forward and side light scatter profiles. ∗, Surface marker expression at 24 h in the absence of OVA, which were similar to the levels observed at 24 h in the presence of 5 ng/ml OVA. Without stimulation, similar profiles were observed at all time points. In selected panels, cells of interest are enclosed in boxes and the percentages of CFSE⁺ cells are indicated. Data in b are calculated by ModFit LT analysis of KJ1-26⁺ cell division at the indicated times for cells stimulated with 20 ng/ml OVA (open symbols, solid line) and cells stimulated with 200 ng/ml OVA (closed symbols, dashed line). Numbers in parentheses are percentages of KJ1-26⁺ cells that remained undivided.

Ag is not required to maintain clonal expansion

The use of CFSE to measure cell growth enabled us to identify proliferating cells based upon the numbers of cell divisions that had occurred after initial stimulation. Using this technique, we wished to determine whether the requirements to activate resting cells and progress to the first cell division are the same as those required to progress into each subsequent round of cell division. In the current study we have focused on the role of Ag in both inducing and sustaining clonal expansion. While it is clear that Ag is essential for initial CD4 cell activation, the requirement for Ag in ongoing proliferation is less clear. To determine whether TCR signaling is required for each round of cell division, we established a model in which Ag could be removed after initial cell activation. DO11.10 CD4 T cells were labeled with CFSE before culture with unlabeled APCs and OVA. After 60 h, the dividing cells were collected and were fluorescence cell-sorted based on CFSE fluorescence. Before sorting, the T cell population contained a mixture of cells that had undergone up to five rounds of cell division (Fig. 2⇓a). Furthermore, a portion of the cells remained undivided, although the induction of activation markers indicated that they had been exposed to OVA during the culture period (Fig. 1⇑ and data not shown). The undivided cells were isolated (Fig. 2⇓b) and returned to culture with fresh, unlabeled APCs but in the absence of OVA. At different times, the cells were analyzed by flow cytometry. As indicated in Fig. 2⇓c, the cells continued to divide after the removal of Ag. These data suggest that cell division is triggered by interaction of the resting cell with Ag and that a proliferation program begins such that clonal expansion can proceed in the absence of further exposure to Ag.

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FIGURE 2.

Proliferative response of stimulated DO11.10 CD4⁺ in the absence of Ag. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and OVA (2 μg/ml) for 60 h. a, Analysis of viable cells before FACS sorting based upon CFSE fluorescence profiles. Undivided cells (boxed) were collected. b, Undivided cells after sorting. The cells were cultured with fresh, unlabeled APCs at the original concentrations. c, At the indicated times, the cells were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data in c are gated to show CFSE staining on viable KJ1-26⁺ cells. Omitting APCs in the secondary culture gave similar results.

To ensure that the Ag-independent proliferation was not due to unusual or excessive TCR signaling, the amounts of OVA needed to stimulate proliferation of DO11.10 CD4 T cells were established (Fig. 1⇑). To determine whether similar concentrations of Ag would program cells to proliferate after Ag was withdrawn, CFSE-labeled DO11.10 T cells were cultured for 36 h with varying amounts of OVA. The cells were then sorted to collect only the undivided T cells, which were then returned to culture with fresh APCs. In parallel, freshly prepared CFSE-labeled DO11.10 CD4 T cells were similarly cultured with varying amounts of OVA. Cell proliferation was assessed by flow cytometry after a 60-h culture period. As indicated in Fig. 3⇓, proliferation occurred when the cells were exposed to as little as 20 ng/ml OVA, regardless of whether OVA was removed before the first cell division. As in Fig. 1⇑, the Ag dose appeared to exert a greater effect on the proportion of cells that participated in clonal expansion and only a modest effect on the rate of proliferation. At low Ag doses most cells did not divide even if Ag was maintained in the cultures. At high Ag doses greater numbers of cells were stimulated to proliferate and were programmed to proliferate in the absence of Ag. Hence, Ag-independent proliferation does not require any more extensive TCR clustering than proliferation that occurs in the continuous presence of Ag.

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FIGURE 3.

Dose response leading to Ag-independent proliferation of DO11.10 CD4⁺ cells. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and either 20 ng/ml (a) or 200 ng/ml (b) OVA for 36 h before FACS sorting to obtain undivided (based upon CFSE fluorescence) cells. The sorted T cells were returned to culture in the absence of additional peptide. At that time, freshly prepared DO11.10 CD4⁺ T cells (1 × 10⁵/well) were also cultured with unlabeled APCs (2 × 10⁵/well) and 0 (c), 20 (d), or 200 ng/ml (e) OVA. After 60 h the cells were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26⁺ cells.

We also wished to ensure that Ag-induced proliferation was not a consequence of artificial, in vitro culture. Thus, it was important to show that proliferation in the absence of prolonged signaling by Ag could occur in vivo. To demonstrate in vivo proliferation, we used a previously described adoptive transfer model (23, 25) whereby DO11.10 cells that were transferred into congenic BALB/c hosts could be identified using the anticlonotypic mAb, KJ1-26 (13). In vivo proliferation of these cells can be visualized by adoptive transfer of CFSE-labeled DO11.10 cells. In the absence of immunization, the KJ1-26 cells remain as a single fluorescent peak, similar to that observed in analyses of unstimulated CD4 cells in vitro (Fig. 4⇓a). Immunization with OVA, as with culture with peptide, promoted specific cell division in the KJ1-26⁺ population isolated from the lymph nodes of the host mice (Fig. 4⇓b). To determine whether the host mice would support Ag-independent proliferation, CFSE-labeled DO11.10 CD4 T cells were cultured with unlabeled APCs and OVA before cell sorting and isolation of peptide-exposed, undivided T cells. These cells were injected into BALB/c mice. Lymph nodes were isolated from the host mice 48 h after transfer and the KJ1-26⁺ cells were identified by flow cytometry. As indicated in Fig. 4⇓, c and d, if the cells were exposed to peptide before transfer, proliferation occurred in vivo.

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FIGURE 4.

Proliferative response of DO11.10 CD4⁺ cells after adoptive transfer. Purified CFSE-labeled DO11.10 CD4⁺ T cells were injected into BALB/c mice (a and b) or were cultured with unlabeled APCs and OVA (2 μg/ml) (c and d) for 48 h before FACS sorting, using CFSE fluorescence profiles to purify undivided cells. The sorted cells were immediately transferred into BALB/c mice. After 48 h LN cells were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. b, For nonsorted cells, the mice were analyzed 60 h after immunization with OVA. c, Data are gated to show CFSE and marker expression on live lymphocytes based upon forward and side light scatter profiles. d, Data within the box in c are gated to show CFSE staining on viable KJ1-26⁺ cells.

It was possible that Ag-independent cell proliferation was due to carryover of small numbers of Ag-bearing APCs that were present in the primary culture and were isolated with the CFSE-labeled T cells during the sorting procedure. We considered this unlikely for several reasons. First, the sorting gates were focused on viable cells (determined by forward and 90° light scatter) and CFSE⁺ cells. Because potential APCs would largely be nonviable 60 h after the start of the culture (6), and because only CD4 T cells would be labeled with CFSE and proliferate in response to OVA, it was likely that only CD4 T cells were collected during the sorting procedure. Second, CD4⁺ T cell proliferation was similar both in vitro and in vivo. Because the CD4 cells were transferred into recipient mice via systemic administration, both these cells and potential contaminating APCs would need to traffic to the same lymph node to maintain contact and Ag presentation. However, to directly ensure that no contaminating OVA-bearing APCs were carried into the secondary culture, CFSE-labeled DO11.10 CD4 T cells were cultured for 60 h before sorting as above. The cells were stained for MHC class II (I-A^d) to identify potential APCs, and they were sorted to collect only undivided T cells and to eliminate APCs. The cells were returned to culture with freshly isolated APCs and freshly isolated, unlabeled DO11.10 CD4⁺ T cells. After 24 h the cultures were examined using flow cytometry for the expression of the activation markers CD69 and CD25. As shown in Fig. 5⇓, a and b, in the absence of added OVA, the unlabeled CD4 cells did not express either CD69 or CD25 (Fig. 5⇓, a and b, boxed). However, even in the absence of exogenous OVA the sorted cells proliferated in the secondary culture (Fig. 5⇓, a and b, circled). If OVA was introduced into the cultures, CD69 and CD25 were readily induced on the unlabeled indicator DO11.10 cells (Fig. 5⇓, c and d). Indeed, activation marker expression was observed at concentrations as low as 5 ng/ml OVA, an amount less than that needed to induce extensive cell proliferation (Fig. 1⇑ and data not shown). Taken together, these data indicate that continued cell division can proceed in an Ag-independent fashion after initial stimulation.

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FIGURE 5.

Activation marker expression on unlabeled DO11.10 CD4⁺ T cells. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and OVA (2 μg/ml) for 60 h before staining with anti-I-A^d (to identify APCs) and FACS sorting to isolate undivided (based upon CFSE fluorescence) T cells from MHC class II⁺ cells. The labeled T cells (1 × 10⁵/well) were cultured with freshly prepared, unlabeled DO11.10 CD4⁺ T cells (1 × 10⁵/well) and unlabeled APCs (2 × 10⁵/well) in the absence (a and b) or presence (c and d) of OVA (0.02 μg/ml). After 24 h the cells were collected, stained with mAbs reactive with I-A^d and either CD69 (a and c) or CD25 (b and d), and analyzed by flow cytometry. MHC class II⁺ and CFSE-labeled T cells are on the right. The cells on the left of the panels are largely (>80%) CD4⁺. The indicator cells that express activation markers are boxed, and the proliferating sorted T cells are circled. Data are gated to show CFSE and marker expression on live lymphocytes based upon forward and side light scatter profiles.

Ag-independent proliferation proceeds after only a brief exposure to peptide

A program for Ag-independent CD4 cell proliferation was induced rapidly upon culture initiation. CFSE-labeled cells were cultured with unlabeled APCs and peptide for various times (0–16 h). No cell division was observed during these culture times. At the end of the primary cultures, all of the cells populations, including noncultured cells (Fig. 6⇓a), were labeled with Abs directed against MHC class II molecules (I-A^d) to identify the APCs before sorting the undivided T cells. The T cells were returned to culture for 3 days in the absence of peptide before analysis by flow cytometry to determine the extent of cell proliferation in the secondary cultures. As indicated in Fig. 6⇓b, as little as 2 h of exposure to peptide was sufficient to induce proliferation in the secondary culture. There was little difference between the cell division profiles of cells cultured with OVA for 2 h as compared with cells cultured for 16 h (data not shown), which suggested that signaling through the TCR occurred on all cells shortly after the primary cultures were begun.

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FIGURE 6.

Exposure times leading to Ag-independent proliferation of DO11.10 CD4⁺ cells. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and OVA (2 μg/ml) for 0 (a), 2 (b), or 4 h (c). The cells, including the unstimulated cells, were stained with mAb MKD6 (anti-I-A^d) before FACS sorting to purify undivided (based upon CFSE fluorescence profiles) MHC class II⁻ cells. The cells were then cultured with fresh, unlabeled APCs for 60 h before they were collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26⁺ cells. Omitting APCs in the secondary culture gave similar results.

Clonal expansion is promoted by the continued presence of Ag

The experiments described thus far showed that signaling through the TCR on resting CD4 T cells was sufficient to stimulate multiple rounds of cell division. However, it was unclear whether Ag was needed solely to begin the begin the expansion process. We next wished to examine the consequences of TCR ligation and the effects of continuous Ag stimulation during clonal expansion. As shown in Fig. 1⇑, TCR expression remained elevated during peptide-induced proliferation. Such expression might indicate that dividing T cells might still be capable of responding to Ag. To address whether or not T cells become refractory to Ag during clonal expansion, we stimulated DO11.10 CD4 T cells with OVA and isolated daughter cells from each specific round of cell division. The cells were then returned to culture in the presence or absence of OVA. After 48 h the cells were collected and analyzed by flow cytometry to determine the extent of proliferation in secondary culture. As expected, cells from each round of cell division could continue to divide in the absence of Ag (Fig. 7⇓, upper panels). Interestingly, cell division was not uniform, as cells early in the expansion process (originally undivided cells) proliferated more in the secondary culture than did cells derived from the latter portion of the initial proliferation. Indeed, after division three cells generally did not divide more than twice within the 48-h secondary culture period (W. T. Lee, unpublished observations). This result suggests that as clonal expansion proceeds, each successive division possesses a diminishing capacity for further division. However, growth could be renewed by again signaling through the TCR. At all cell divisions the addition of Ag resulted in an increased cell division (Fig. 7⇓, lower panels). Hence, although Ag is not required for proliferation, the TCR can still be stimulated to promote growth throughout clonal expansion.

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FIGURE 7.

Effect of continued exposure to peptide on CD4⁺ T cell clonal expansion. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and OVA (2 μg/ml) for 60 h before FACS sorting based upon CFSE fluorescence profiles. Undivided cells or daughter cells from the indicated cell divisions were isolated and cultured with fresh, unlabeled APCs in the absence (upper panels) or presence (lower panels) of OVA (2 μg/ml) for 48 h. The cells were then collected, stained with mAb KJ1-26, and analyzed by flow cytometry. Data are gated to show CFSE staining on viable KJ1-26⁺ cells.

When collecting cells for analysis after the secondary culture, we consistently observed that more cells were present if Ag was maintained throughout the culture. Although part of the increased cell recoveries could be attributed to the additional rounds of cell division promoted by Ag, comparison of cultures without Ag addition at later time points to shorter cultures with Ag added indicated that more cells were in the latter cultures (W. T. Lee, unpublished observations). Furthermore, the cultures containing Ag appeared more robust as assessed by changes in culture media color. To more directly measure the effect of Ag stimulation on T cell numbers, DO11.10 T cells were stimulated with OVA for 48 h before FACS sorting. Cells before (undivided cells) or undergoing (first cell division) clonal expansion were isolated and equal numbers were recultured in the presence or absence of OVA for various times before enumeration of viable cells. As indicated in Fig. 8⇓, for both undivided and dividing cells a greater number was recovered at any time if Ag was present in the culture. Analysis at later time points is complicated by the additional cell division induced by Ag; however, within the first 25–36 h of reculture the division profiles, as indicated by CFSE fluorescence, were similar (data not shown). Thus, these data suggest that a contribution toward increased cell numbers is made by positive effects of Ag on cell viability. A further example of the effects of Ag on cell vigor is indicated by increased cytokine secretion. The culture supernatants were collected after 48 h of the secondary culture and the amount of IL-2 secreted by the cells was determined using ELISA. As shown in Fig. 8⇓, higher levels of IL-2 were found when OVA was present in the cultures. Hence, we conclude that although cell division per se is Ag independent, cytokine secretion and cell accumulation is maximal when Ag is continuously present.

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FIGURE 8.

Effect of Ag on cell survival and lymphokine secretion during CD4⁺ T cells clonal expansion. Purified CFSE-labeled DO11.10 CD4⁺ T cells (1 × 10⁶/ml) were cultured with unlabeled APCs (2 × 10⁶/ml) and OVA (2 μg/ml) for 48 h before FACS sorting, using CFSE fluorescence profiles to purify undivided cells and daughter cells from the first cell division. The cells were then cultured with fresh, mitomycin C-treated APCs in the presence or absence of OVA. a, At the indicated times the number of cells in two pooled wells were determined where the starting T cell populations were originally undivided (○ and •) or from the first cell division (□ and ▪). Open symbols represent cultures without OVA; closed symbols represent cultures with OVA (0.2 μg/ml). b, After 48 h culture supernatants from wells containing the indicated cell types were collected and assayed for IL-2. Cells were cultured in the presence (▦) or absence (▪) of OVA (0.2 μg/ml). This experiment is representative of four independent experiments, all of which gave similar results.