IL-16, in a CD4-dependent manner, induces high affinity IL-2R (CD25) selectively on CD4+ T cells. Based on this observation, we determined the relative effects of IL-16 on IL-2Rα, β, and γ expression on CD4+ T cells and of IL-16/IL-2 cotreatment of resting human PBMC obtained from normal individuals on CD4+ T cell proliferation and cytokine production, in vitro. IL-16 increased CD4+ T cell IL-2Rα and β expression, but had no effect on expression of IL-2Rγ. There was marked synergy of thymidine uptake and expansion of CD4+ T cell numbers in the presence of IL-16 and IL-2 or IL-16 and IL-15 compared with the responses to any of the cytokines alone. By 4 wk, IL-16/IL-2-cotreated PBMC cultures were predominantly CD4+, CD25+ CD45RO T cells. Of the cytokines measured, IL-16 treatment alone was sufficient to induce synthesis of granulocyte-macrophage CSF by 2 wk. IL-16/IL-2 cotreatment did not appear to induce selective proliferation of any Th subset, as cytokines of both Th1 (e.g., IFN-γ) and Th2 (e.g., IL-5) types were synthesized by the expanded cell populations at 2 and 4 wk. These results suggest that IL-16 can prime CD4+ T cells for IL-2 responsiveness, and therefore may be a useful adjunct to IL-2 therapy for immune reconstitution in disease or therapeutic conditions resulting in CD4+ T cell depletion.

Depletion of CD4+ T cells is a major predictive factor for the development of certain types of opportunistic infections and malignancies. This is true in individuals infected with HIV-1 (1) and in those undergoing chemotherapy or chronic immunosuppressive therapy (2, 3). In regard to chemotherapy, there has been successful reconstitution of neutrophils with the administration of GM-CSF,3 shortening the time period of susceptibility to bacterial and fungal infections (4, 5, 6). Following that logic, IL-2 therapy has been explored as a means to increase CD4+ T cell numbers in HIV-1-infected individuals (7, 8). This expansion presumably results from proliferation of mature peripheral T cells. While increases in CD4+ T cells have been noted, the changes were limited to an average of a twofold increase in circulating T cells (7, 8). The magnitude of the increase was most likely restricted by the dose of IL-2 that can be administered without toxicity, as well as the relatively low percentage of circulating IL-2R+ T cells present at the beginning of therapy. Furthermore, concerns have been raised that the expanded CD4+ T cell pool may represent a limited number of TCR specificities depending upon the repertoire of CD4+ T cells that spontaneously express IL-2R at the beginning and during therapy (9).

One of the functional consequences of IL-16 interaction with CD4 on T cells is the induction of IL-2Rα (CD25) (10, 11). Because there is an absolute requirement for cell surface expression of CD4 for signaling and functional responses to IL-16 (10, 12, 13, 14, 15), we hypothesized that IL-16 might provide a selective synergistic signal for IL-2-induced proliferation of CD4+ T cells over all other T cell phenotypes. In the current studies, we determined the effects of IL-16 on IL-2Rβ and γ, and then determined the effects of sequential IL-16/IL-2 treatment of human blood T cells obtained from normal individuals on CD4+ T cell proliferation, in vitro. IL-16 increased IL-2Rα and β expression, but had no effect on IL-2Rγ. We observed selective enhancement of the proliferative response of blood CD4+ T cells in the presence of both IL-16 and IL-2 compared with the responses to either cytokine alone. A similar effect was noted in the presence of IL-16 and IL-15, while there was no synergy with IL-4. Following 4-wk exposure to IL-16/IL-2, the expanded cell population was predominantly CD4+CD25+CD45RO CD95+. By 2 wk, IL-16-treated cells synthesized GM-CSF, while IL-16/IL-2-cotreated cells synthesized significant levels of IFN-γ, GM-CSF, and IL-5; low levels of IL-10; but undetectable levels of IL-4. By 4 wk, the synthesis of IL-10 increased following IL-16/IL-2 stimulation. The presence of both Th-type cytokines suggests that IL-16/IL-2 treatment does not result in any bias toward a single Th subtype over time in culture. These results suggest that IL-16 may be a useful adjunct to IL-2 therapy for immune reconstitution in states associated with CD4+ T cell depletion.

rIL-16 containing an NH4-terminal polyhistidine tag linked via a factor Xa-susceptible cleavage site was produced in Escherichia coli, purified by metal-chelation chromatography, and isolated from the polyhistidine tag by enzymatic cleavage, as previously described (11). Thoroughness of removal of endotoxin by polymyxin binding was determined by assay with a BioWhittaker QCL 1000 LAL testing kit (Walkersville, MD). rIL-16 was stored in 7.5% glycerol with 0.1 mM HCl at −80°C and thawed immediately before use. The bioactivity of rIL-16 preparations was confirmed by chemotaxis of human T cells, as previously described (11). rIL-2 was purchased from Genzyme Corp. (Cambridge, MA); rIL-4 and rIL-15 were purchased from Biosource (Camarillo, CA).

As per institutional review board protocol, blood was obtained from volunteers after a description of the intent of the experiments was given and the informed consent forms were signed. Cells were isolated according to previously reported protocols (10, 12). Briefly, blood from normal volunteers was collected in heparinized syringes, and the PBMC were isolated using Ficoll-Hypaque density centrifugation. PBMC were washed and resuspended in RPMI 1640 supplemented with 25 mM HEPES buffer, 100 U/ml penicillin and streptomycin (HPS), and 10% FBS.

To determine the effects of IL-16 on [3H]thymidine incorporation into T cells, rIL-16 (10−10 M) or molar equivalent of a recombinant protein, β-galactosidase, produced in an identical fashion in E. coli (11), was added to PBMC 24 h before the addition of rIL-2 (10 U/ml). Cells were cultured in presence of rIL-2 for 5 days and then pulsed with [3H]thymidine (1 uCi/well) for 24 h, harvested, and assessed by scintillation counting. In experiments in which we determined the proliferation of T cells in culture, human PBMC were plated at a concentration of 1 × 106 cells/ml in 3 ml of RPMI HPS/10% FBS. The cells were treated with either media alone (every Monday, Wednesday, and Friday); media (every Monday) plus rIL-2 (10 U/ml every Wednesday and Friday); media (every Wednesday and Friday) plus rIL-16 (10−10 M every Monday), or a combination of weekly rIL-16 (every Monday) and twice weekly rIL-2 (every Wednesday and Friday). Volumes of added media and cytokines were adjusted so that a total of 200 μl by volume was added each week. Equal volumes were maintained throughout the culture period as 200 μl was aspirated weekly for cell counts. Cell density was maintained at 1 × 106 cells/ml. The cells were enumerated by hemocytometer and FACS analyses. The data are expressed as the average cell counts from each culture dish (±SD) and then meaned with counts from cultures from other experiments under similar conditions and time intervals.

PBMC were incubated with a combination of phycoerythrin (PE)-conjugated or fluorescein-conjugated Abs directed at membrane-expressed Ags. In general for each Ab, 200 μl of cell suspension (1 × 106 cells/ml) was washed, resuspended in PBS with 0.1% azide, and incubated with 5 μl of Ab for 30 min at 4°C. Cells were washed three times, fixed with 10% Formalin, and protected from light at 4°C until analysis with a Becton Dickinson FACS cytometer. Conjugated Abs, CD3 FITC, CD4FITC, CD8PE, CD16 PE, CD14 FITC, CD45RO FITC, and CD45RA FITC were obtained from Biosource; CD3 PE and CD25 PE from Becton Dickinson (Bedford, MA); and CD95 PE, CD57 PE, and CD40L PE from PharMingen (San Diego, CA). Anti-IL-2Rβ was a gift from Lee Anne Beausang (Endogen, Cambridge, MA). Anti-IL-2Rγ was purchased from PharMingen (San Diego, CA). Calculation of the total numbers of CD4 cells in culture was based on the percentage of CD3+CD4+ cells by FACS analyses.

One-hundred-microliter aliquots of supernatants of cultures were used to assess cytokine concentrations, as determined by ELISA. The IL-4, IL-5, IL-10, IFN-γ, and GM-CSF ELISA assays were obtained from Biosource. In general, the ELISA kits had a lower limit of detection of 20 pg/ml. Assays were conducted as specified by the manufacturer.

We have shown previously that IL-16 induces cell surface expression of CD25 protein on T cells within 24 to 48 h (10, 11). To demonstrate that this response was selective for CD4+ T cells, as predicted by IL-16’s requirement for CD4 to initiate its functions (10, 11, 12, 13, 14, 15), we exposed PBMC to 10−10 M rIL-16 for 24 and 48 h and labeled the cells for expression of CD4 and CD25. The induction of CD25 at both the 24- and 48-h time points occurred exclusively in the CD4+ T cell population (Fig. 1, upper panel). Under these conditions, IL-16 stimulated the expression of CD25 on 18% of the CD4+ cells at 24 h, which increased to 30% of the CD4+ cells at 48 h. As the IL-2R high affinity complex is comprised of two other chains, we also determined whether IL-16 stimulation had an effect on IL-2Rβ and IL-2Rγ expression. The level of IL-2Rγ expression was high on all of the T cells cultured under control, untreated conditions (Fig. 1, lower panel). The addition of IL-16 did not induce a significant change in IL-2Rγ expression. Conversely, IL-2Rβ was expressed at relatively low levels in untreated T cells. The addition of IL-16 resulted in an increase in IL-2Rβ expression from an average of 2% positive cells for untreated cells to 11% positive cells exposed to IL-16 for 24 h (Fig. 1, lower panel). In general, the magnitude for the up-regulation of IL-2Rβ was not as great nor as consistent as was seen for IL-2Rα. The average increase for IL-2Rβ was 11%; however, the range was from 1.5 to 23% for the 24-h period. By 48 h, the percentage of positive cells was 16% (data not shown). There was no significant change in IL-2Rγ expression at any time point examined.

FIGURE 1.

IL-16 induces IL-2Rα and β in CD4+ cells. Upper panel, PBMC were treated with media alone, or 10−10 M IL-16 for 24 and 48 h. Cells from each condition were incubated with anti-CD4 and anti-CD25 fluorescently conjugated Abs for 30 min, washed, and fixed. To eliminate the debris, events were collected after gating on viable lymphocytes, as determined by forward and side scatter dot blots. The lower panel depicts the double labeling of PBMC with fluorescently conjugated anti-CD4 and either anti-IL-2Rβ or anti-IL-2Rγ Abs following a 24-h incubation period either in the presence or absence of IL-16 stimulation. Each graph is representative of three separate experiments.

FIGURE 1.

IL-16 induces IL-2Rα and β in CD4+ cells. Upper panel, PBMC were treated with media alone, or 10−10 M IL-16 for 24 and 48 h. Cells from each condition were incubated with anti-CD4 and anti-CD25 fluorescently conjugated Abs for 30 min, washed, and fixed. To eliminate the debris, events were collected after gating on viable lymphocytes, as determined by forward and side scatter dot blots. The lower panel depicts the double labeling of PBMC with fluorescently conjugated anti-CD4 and either anti-IL-2Rβ or anti-IL-2Rγ Abs following a 24-h incubation period either in the presence or absence of IL-16 stimulation. Each graph is representative of three separate experiments.

Close modal

We next determined whether IL-16 priming of CD4+ T cells resulted in the expression of functional high affinity IL-2R. For these experiments, we cultured PBMC from normal individuals in the presence of either media alone, rIL-2, rIL-16, or rIL-16, followed 24 h later by rIL-2. Thymidine incorporation was assessed at 7 days in each culture. As shown in Table I, rIL-16 had no effect on thymidine uptake, while rIL-2 resulted in a minimal increase, probably representing a response of the low percentage of IL-2R+ cells in the starting cell population (1–7% in these experiments). Sequential treatment with rIL-16 and rIL-2 resulted in a sixfold increase in [3H]thymidine incorporation, indicating that the IL-2R induced by IL-16 was functional. In addition, we determined whether the proliferative responses to IL-4 and IL-15 were augmented by preincubation of PBMC with IL-16 under identical conditions. There was no augmentation of IL-4-induced proliferation. The meaned data for an IL-4 concentration of 100 ng/ml are shown in Table I, but there was no response over a wide concentration range (0.5–100 ng/ml). However, IL-16 did prime PBMC to incorporate [3H]thymidine in the presence of IL-15 (50 ng/ml) (Table I). IL-15 (100 ng/ml) resulted in a twofold increase in [3H]thymidine uptake that was further augmented to 10-fold by prior exposure to IL-16 (data not shown). Despite the large range in the magnitude of IL-2Rβ expression, all cultures receiving IL-16 demonstrated enhanced proliferation to IL-15. In the following experiments, we limited our observations to the study of the synergism between IL-16 and IL-2.

Table I.

Synergy of rIL-16 and rIL-2, rIL-4, and rIL-15 on 3[H]thymidine incorporation of normal resting human PBMCa

StimulusbExperiment 1cExperiment 2Experiment 3
Buffer control 983 ± 145 1,074 ± 326 954 ± 197 
rβ-gal 1,032 ± 214 1,042 ± 431 1,012 ± 368 
rIl-16 (10−8 M) 1,203 ± 284 1,054 ± 212 982 ± 301 
rIL-2 (1 U/ml) 2,381 ± 185 2,594 ± 464 2,508 ± 471 
rβ-gal + rIL-2 2,573 ± 572 2,692 ± 485 2,582 ± 278 
rIL-16 + rIL-2 12,664 ± 2,802 15,037 ± 1,088 13,753 ± 2,068 
rIL-4 (100 ng/ml) 2,643 ± 306 2,561 ± 276 2,209 ± 406 
rIL-16 + rIL-4 2,683 ± 402 2,536 ± 381 2,338 ± 260 
rIL-15 (50 ng/ml) 2,408 ± 372 2,721 ± 252 2,338 ± 260 
rIL-16 + rIL-15 18,647 ± 2,346 17,433 ± 1,649 21,174 ± 4,693 
StimulusbExperiment 1cExperiment 2Experiment 3
Buffer control 983 ± 145 1,074 ± 326 954 ± 197 
rβ-gal 1,032 ± 214 1,042 ± 431 1,012 ± 368 
rIl-16 (10−8 M) 1,203 ± 284 1,054 ± 212 982 ± 301 
rIL-2 (1 U/ml) 2,381 ± 185 2,594 ± 464 2,508 ± 471 
rβ-gal + rIL-2 2,573 ± 572 2,692 ± 485 2,582 ± 278 
rIL-16 + rIL-2 12,664 ± 2,802 15,037 ± 1,088 13,753 ± 2,068 
rIL-4 (100 ng/ml) 2,643 ± 306 2,561 ± 276 2,209 ± 406 
rIL-16 + rIL-4 2,683 ± 402 2,536 ± 381 2,338 ± 260 
rIL-15 (50 ng/ml) 2,408 ± 372 2,721 ± 252 2,338 ± 260 
rIL-16 + rIL-15 18,647 ± 2,346 17,433 ± 1,649 21,174 ± 4,693 
a

Human PBMC were incubated with the cytokines for 5 days prior to pulsing with 3[H]thymidine (1 uCi/well) overnight.

b

rIL-16 or β-gal were added 24 h prior to the addition of rIL-2, rIL-4, or rIL-15.

c

The data are expressed as incorporated cpm ± SD and represent five separate wells for each condition.

To determine whether these short-term effects could be maintained over time and if they would result in increased numbers of CD4+ T cells, we quantified cell numbers in extended PBMC cultures in the presence of rIL-2, rIL-16, or rIL-16 and rIL-2 in combination. In these experiments, rIL-16 was added on Mondays, followed by rIL-2 on Wednesdays and Fridays of each week. Figure 2 depicts the total cell numbers from PBMC cultures. There was a steady decline in the untreated and rIL-16-treated cells, although cell numbers declined more slowly in the presence of rIL-16 than in media alone. While twice weekly rIL-2 treatment led to rises in total cell numbers for the first 3 wk, these increases were accentuated significantly by weekly cotreatment with rIL-16 (Fig. 2). In cultures cotreated with rIL-16/rIL-2, the cell numbers continued to rise for the duration of the culture period. Using paired t test analysis, there was a significant difference (p < 0.01) between rIL-16/rIL-2 treatment and rIL-16 alone and media alone at every time point after week 3. By week 4, there were statistically significant increases in total cells in rIL-16/rIL-2-treated cultures compared with IL-2 treatment alone. IL-16/IL-2 costimulation also imparted increased viability to CD4+ T cells, as more than 90% of the cells in the cotreated cultures excluded trypan blue, while up to 50% of cells treated with either cytokine alone did not exclude trypan blue at 2 wk, and 90% of the cells were not viable by 4 wk.

FIGURE 2.

Effect of rIL-16 plus rIL-2 on total cell growth. PBMC were incubated with rIL-16 (10−10 M) (circles), rIL-2 (10 U/ml) (diamonds), or a combination of rIL-16 and rIL-2 (triangles), or left untreated (squares) for 9 wk. A quantity amounting to 3 × 106 cells for each condition was cultured initially, and changes in total cell numbers were monitored each week. The data represent the average numbers for eight different experiments +/− the SD.

FIGURE 2.

Effect of rIL-16 plus rIL-2 on total cell growth. PBMC were incubated with rIL-16 (10−10 M) (circles), rIL-2 (10 U/ml) (diamonds), or a combination of rIL-16 and rIL-2 (triangles), or left untreated (squares) for 9 wk. A quantity amounting to 3 × 106 cells for each condition was cultured initially, and changes in total cell numbers were monitored each week. The data represent the average numbers for eight different experiments +/− the SD.

Close modal

To determine the selective effect on the magnitude of the increase in CD4+ T cells, we quantitated the percentage of CD4+ cells at 2 and 4 wk by FACS analysis. The total numbers of CD4+ T cells are depicted in Figure 3. In addition to the rise in total CD4+ T cell numbers, the percentage of CD4+ T cells rose continuously throughout the culture period in IL-16/IL-2-treated cells (from 40–50% positive at time zero to more than 95% CD4+ by 4 wk) (Table II). There were no significant increases in the total numbers of CD14+ (monocytes) or CD16+ (NK cells) with combination rIL-16/rIL-2 treatment at either the 2- or 4-wk time point (Table II). Therefore, IL-16/IL-2 cotreatment resulted in selective expansion of CD4+ T cells, resulting in a homogeneous population of CD4+ cells, with no apparent effect on either monocytes or NK cells.

FIGURE 3.

Effect of rIL-16 plus rIL-2 on CD4+ T lymphocyte growth. The effect of rIL-16 (circles), rIL-2 (diamonds), a combination of rIL-16 and rIL-2, or no treatment (squares) on CD4+ T cell numbers was determined. Using the cultures described in Figure 2, weekly aliquots from each culture condition were labeled with anti-CD4FITC Abs and total CD4+ T cell numbers were quantitated by FACS analysis. The data represent the average weekly CD4 cell counts from eight different experiments +/− the SD.

FIGURE 3.

Effect of rIL-16 plus rIL-2 on CD4+ T lymphocyte growth. The effect of rIL-16 (circles), rIL-2 (diamonds), a combination of rIL-16 and rIL-2, or no treatment (squares) on CD4+ T cell numbers was determined. Using the cultures described in Figure 2, weekly aliquots from each culture condition were labeled with anti-CD4FITC Abs and total CD4+ T cell numbers were quantitated by FACS analysis. The data represent the average weekly CD4 cell counts from eight different experiments +/− the SD.

Close modal
Table II.

Phenotype of cells in 2- and 4-wk cultures following stimulation with rIL-16, rIL-2, or a combination of the two cytokinesa

ReceptorWeek 0Week 2Week 4
ControlbControlrIL-16rIL-2rIL-16 + rIL-2rIL-2rIL-16 + rIL-2
CD3 76 ± 8.5 88 ± 3.8 86 ± 3 90 ± 6.3 94 ± 3.5 90 ± 9 95 ± 5 
 n = 9 n = 4 n = 4 n = 4 n = 6 n = 6 n = 6 
 (62–92) (82–91) (85–88) (84–98) (92–98) (81–98) (86–99) 
CD4 50 ± 10 62 ± 10 67 ± 14 68 ± 13 70 ± 10 52 ± 18 92 ± 5 
 n = 9 n = 5 n = 3 n = 4 n = 5 n =4 n = 6 
 (35–73) (50–73) (51–78) (52–79) (60–80) (43–76) (84–99) 
CD16 10.7 ± 6 6.3 ± 2 6.7 ± 5.5 5.3 ± 3 4.3 ± 2 3.9 ± 1.9 4.1 ± 1.6 
 n = 7 n = 4 n = 5 n = 4 n = 6 n = 2 n = 4 
 (2–19.4) (5–9.5) (2–16) (8–9.8) (2–7.2) (2.5–5.2) (1.8–5.3) 
CD14 7 ± 4.3 7 ± 2.2 5.5 ± 3 6.6 ± 3 4.5 ± 1.8 2.9 ± 3.1 6 ± 2.7 
 n = 6 n = 4 n = 3 n = 3 n = 4 n = 2 n = 5 
 (2.7–15) (4.6–10) (3.3–9) (2.9–6.1) (2–6.1) (0.6–5.1) (3–10.3) 
ReceptorWeek 0Week 2Week 4
ControlbControlrIL-16rIL-2rIL-16 + rIL-2rIL-2rIL-16 + rIL-2
CD3 76 ± 8.5 88 ± 3.8 86 ± 3 90 ± 6.3 94 ± 3.5 90 ± 9 95 ± 5 
 n = 9 n = 4 n = 4 n = 4 n = 6 n = 6 n = 6 
 (62–92) (82–91) (85–88) (84–98) (92–98) (81–98) (86–99) 
CD4 50 ± 10 62 ± 10 67 ± 14 68 ± 13 70 ± 10 52 ± 18 92 ± 5 
 n = 9 n = 5 n = 3 n = 4 n = 5 n =4 n = 6 
 (35–73) (50–73) (51–78) (52–79) (60–80) (43–76) (84–99) 
CD16 10.7 ± 6 6.3 ± 2 6.7 ± 5.5 5.3 ± 3 4.3 ± 2 3.9 ± 1.9 4.1 ± 1.6 
 n = 7 n = 4 n = 5 n = 4 n = 6 n = 2 n = 4 
 (2–19.4) (5–9.5) (2–16) (8–9.8) (2–7.2) (2.5–5.2) (1.8–5.3) 
CD14 7 ± 4.3 7 ± 2.2 5.5 ± 3 6.6 ± 3 4.5 ± 1.8 2.9 ± 3.1 6 ± 2.7 
 n = 6 n = 4 n = 3 n = 3 n = 4 n = 2 n = 5 
 (2.7–15) (4.6–10) (3.3–9) (2.9–6.1) (2–6.1) (0.6–5.1) (3–10.3) 
a

Human PBMC were cultured for 0, 2, or 4 wk in the presence of rIL-16 (10−10 M), rIL-2 (10 U/ml), a combination of the two cytokines, or left untreated. In the cultures receiving both cytokines, rIL-16 was added 24 h prior to the addition of rIL-2.

b

Cells were labeled with fluorescently conjugated Abs, and the data are expressed as percent positive cells ± SD, with the range for all the experiments shown in parentheses.

We next identified the IL-16-responsive cell population by surface phenotyping. During the first 4 wk of culture, it was observed that unstimulated cells, while decreasing in viability and cell number, demonstrated an increase in IL-2R and CD45RO expression. The rise in CD40L expression was not statistically significant at 4 wk (Table III). Our starting cell populations had high CD45RA expression that decreased over time. The addition of rIL-16, rIL-2, or the two in combination had the effect of further increasing the levels of IL-2R and CD95 expression at all time points. The combination of rIL-16/rIL-2 also increased the expression of CD45RO and CD40L (Table III). By the 6-wk time point, only the cultures receiving both rIL-16/rIL-2 were viable. FACS analysis of the population of the cells at 6 wk revealed that they were predominantly CD4+, CD25+, CD45RO, and CD95+ (Fig. 4). In addition, at this time point the cells were analyzed for their expression of CD57, a marker of lymphokine-activated killer cells. Less than 10% of all of the cells in these cultures were CD57+ (Fig. 4). By 6 wk, the percentage of CD40L-expressing cells rose to 25% in a pattern that suggested a continuum of expression of CD40L. While the data shown in Figure 4 are a representative experiment, the percentage of cells expressing these patterns differed by less than 10% among all individual experiments. We observed a high percentage of CD45RA cells following isolation. CD45RO expression increased over time in response to rIL-16/rIL-2 treatment, which was associated with the presence of significant numbers of CD45RO CD45RA double-positive cells by 2 and 4 wk, which were not observed at 6 wk. Beyond week 4, the expression of CD45RO routinely increased to approximately 90%, while expression of CD45RA decreased to less than 20% of the cells. We did not address the mechanism of this phenotypic change in these experiments.

Table III.

Surface expression of activation-dependent molecules following stimulation by rIL-16, rIL-2, or a combination of the two cytokinesa

IL-2RbCD95CD45 RACD45 ROCD40L
4.6 ± 2.2 37 ± 18.8 91.8 ± 10 18.6 ± 8.4 1.8 ± 1.6 
 n = 8 n = 8 n = 4 n = 8 n = 3 
 (1.2–7.7) (19.5–56.9) (78–99.7) (7.5–32.7) (0–3.9) 
Week 2, Control 22 ± 6.7 69 ± 12 70 ± 25 52 ± 13 3.4 ± 1.1 
 n = 5 n = 3 n = 3 n = 4 n = 3 
 (10.8–27.3) (61–83) (50–97.5) (34–65) (2.3–4.5) 
Week 2 34 ± 18 86 ± 12 53 ± 15 47 ± 28 2.5 ± 0.7 
rIL-16 n = 5 n = 5 n = 3 n = 3 n = 2 
 (24–67) (66–98) (36.3–65) (22–77) (2–3.1) 
rIL-2 44 ± 11 91 ± 7.5 41 ± 10 52 ± 23 2.6 ± 1.9 
 n = 4 n = 3 n = 3 n = 3 n = 3 
 (28–54) (83–98) (30–50) (30–77) (1.4–4.8) 
rIL-16 + rIL-2 45 ± 13 93 ± 5 45 ± 5 75 ± 13 2.8 ± 1 
 n = 5 n = 3 n = 3 n = 6 n = 2 
 (34.5–59.5) (88–98.8) (40–49) (50–86) (1.8–3.9) 
Week 4 30 ± 10 88 ± 9.6 28 ± 17 59 ± 21 4 ± 4.3 
rIL-2 n = 3 n = 3 n = 2 n = 4 n = 2 
 (23–42.6) (79–98) (16–40) (36–85) (1–7) 
rIL-16 + rIL-2 68.7 ± 18.9 88 ± 17.7 64 ± 26 69 ± 17 17.3 ± 10 
 n = 6 n = 5 n = 3 n = 4 n = 3 
 (48.3–98.6) (57.2–99.5) (34–85) (51–91) (11.4–29) 
IL-2RbCD95CD45 RACD45 ROCD40L
4.6 ± 2.2 37 ± 18.8 91.8 ± 10 18.6 ± 8.4 1.8 ± 1.6 
 n = 8 n = 8 n = 4 n = 8 n = 3 
 (1.2–7.7) (19.5–56.9) (78–99.7) (7.5–32.7) (0–3.9) 
Week 2, Control 22 ± 6.7 69 ± 12 70 ± 25 52 ± 13 3.4 ± 1.1 
 n = 5 n = 3 n = 3 n = 4 n = 3 
 (10.8–27.3) (61–83) (50–97.5) (34–65) (2.3–4.5) 
Week 2 34 ± 18 86 ± 12 53 ± 15 47 ± 28 2.5 ± 0.7 
rIL-16 n = 5 n = 5 n = 3 n = 3 n = 2 
 (24–67) (66–98) (36.3–65) (22–77) (2–3.1) 
rIL-2 44 ± 11 91 ± 7.5 41 ± 10 52 ± 23 2.6 ± 1.9 
 n = 4 n = 3 n = 3 n = 3 n = 3 
 (28–54) (83–98) (30–50) (30–77) (1.4–4.8) 
rIL-16 + rIL-2 45 ± 13 93 ± 5 45 ± 5 75 ± 13 2.8 ± 1 
 n = 5 n = 3 n = 3 n = 6 n = 2 
 (34.5–59.5) (88–98.8) (40–49) (50–86) (1.8–3.9) 
Week 4 30 ± 10 88 ± 9.6 28 ± 17 59 ± 21 4 ± 4.3 
rIL-2 n = 3 n = 3 n = 2 n = 4 n = 2 
 (23–42.6) (79–98) (16–40) (36–85) (1–7) 
rIL-16 + rIL-2 68.7 ± 18.9 88 ± 17.7 64 ± 26 69 ± 17 17.3 ± 10 
 n = 6 n = 5 n = 3 n = 4 n = 3 
 (48.3–98.6) (57.2–99.5) (34–85) (51–91) (11.4–29) 
a

Human PBMC were incubated with rIL-16 (10−10 M), rIL-2 (10 U/ml), or a combination or the two cytokines for 2 or 4 wk.

b

Surface expression was determined using fluorescently conjugated Abs. The data are expressed as percent positive cells ± SD, with the range of expression for the different cultures given in parentheses.

FIGURE 4.

Phenotype of the expanded CD4+ T cell population at 6 wk. PBMC cultured for 6 wk in the presence of rIL-16 and rIL-2 were labeled with fluorescently conjugated Abs to IL-2R, CD95, CD45RO, CD40L, or CD57. The cells were also double labeled with fluorescently conjugated Abs to CD4. The percentage of positive cells is indicated numerically for each quadrant.

FIGURE 4.

Phenotype of the expanded CD4+ T cell population at 6 wk. PBMC cultured for 6 wk in the presence of rIL-16 and rIL-2 were labeled with fluorescently conjugated Abs to IL-2R, CD95, CD45RO, CD40L, or CD57. The cells were also double labeled with fluorescently conjugated Abs to CD4. The percentage of positive cells is indicated numerically for each quadrant.

Close modal

Taken together, these data demonstrate that long-term culture of PBMC with rIL-16 and rIL-2 results in selective growth of CD4+CD25+CD45RO+ T cells with increased levels of CD40L and CD95 expression, suggesting that the expanded cell population are activated memory lymphocytes, capable of proliferating to IL-2 and participating in B cell help.

We next determined whether the expanded cell population represented a bias in Th subtype by measuring cytokines synthesized over time in the presence of rIL-16, rIL-2, or rIL-16/IL-2 under the identical conditions noted above. rIL-16 treatment alone did not result in synthesis of any IL-2 at any time point (data not shown). By 2 wk, rIL-16 treatment resulted in significant GM-CSF synthesis (Table IV). rIL-2 induced synthesis of GM-CSF, IL-5, and IFN-γ. Cultures treated with both rIL-16 and rIL-2 demonstrated similar cytokine production to rIL-2-treated cultures. By 4 wk, we were able to measure cytokines only in the rIL-16/rIL-2-cotreated cultures because there were so few viable cells remaining under the other culture conditions. These cells secreted IL-5, GM-CSF, and IFN-γ and, at this time point, there were higher levels of IL-10 in the cell supernatants than at 2 wk (Table IV). Addition of exogenous IL-2 and IL-16 precluded measurement of endogenous production of these cytokines. These data indicate that the expanded cell population can synthesize both Th1 and Th2 cytokines, and that there is no obvious bias toward expansion of one cell type over the other.

Table IV.

Cytokine production by 20- and 4-wk cultures stimulated with rIL-16, rIL-2, or a combination of the twoa

CytokineWeek 2Week 4
ControlbrIL-16rIL-2rIL-16 + rIL-2rIL-16 + rIL-2
IL-5 NDc ND 97 ± 29 64 ± 27 120 ± 30 
 n = 2 n = 2 n = 3 n = 3 n = 6 
   (66–124) (40–94) (63–143) 
IL-10 ND ND 27 ± 26 25 ± 24 131 ± 29 
 n = 3 n = 3 n =4 n = 4 n = 5 
   (5–64) (5–63) (86–156) 
IFN-γ ND ND 48 ± 4 99 ± 21 327 ± 63 
 n = 3 n = 3 n = 1 n = 3 n = 3 
    (84–114) (269–394) 
GM-CSF ND 146 ± 93 85 ± 26 87 ± 73 103 ± 54 
 n = 2 n = 3 n = 3 n = 3 n = 2 
  (39–202) (58–110) (45–71) (68–145) 
CytokineWeek 2Week 4
ControlbrIL-16rIL-2rIL-16 + rIL-2rIL-16 + rIL-2
IL-5 NDc ND 97 ± 29 64 ± 27 120 ± 30 
 n = 2 n = 2 n = 3 n = 3 n = 6 
   (66–124) (40–94) (63–143) 
IL-10 ND ND 27 ± 26 25 ± 24 131 ± 29 
 n = 3 n = 3 n =4 n = 4 n = 5 
   (5–64) (5–63) (86–156) 
IFN-γ ND ND 48 ± 4 99 ± 21 327 ± 63 
 n = 3 n = 3 n = 1 n = 3 n = 3 
    (84–114) (269–394) 
GM-CSF ND 146 ± 93 85 ± 26 87 ± 73 103 ± 54 
 n = 2 n = 3 n = 3 n = 3 n = 2 
  (39–202) (58–110) (45–71) (68–145) 
a

Human PBMC were cultured in the presence of rIL-16 (10−10 M), rIL-2 (10 U/ml), or a combination of the two cytokines for 2 or 4 wk.

b

Cytokines were quantitated by ELISA, and data are expressed as pg/ml/106 cells ± SD and the range shown in parentheses.

c

ND designates below detectable levels.

The existence of a cytokine ligand for CD4, IL-16, implies that CD4 provides signals for T cell activation that are independent of MHC/TCR stimulation. This appears to be true, in that IL-16 interaction with CD4 results in activation and autophosphorylation of CD4-associated p56lck (13), and induces rises in intracellular inositol trisphosphate and calcium (12) and translocation of protein kinase C from cytosol to membrane (15). Along with these signals, a motile response occurs in CD4+ cells along with up-regulation of IL-2R and MHC class II expression (10). Prestimulation of CD4+ cells with IL-16 results in temporary loss of TCR responsiveness, as evidenced by decreased proliferation to immobilized anti-CD3 Ab and tetanus toxoid (16) and decreased responses to allo-Ags, as demonstrated by a diminished one-way MLR (17). These immunomodulatory functions are associated with inhibition of Ag-induced CD95 expression (16). Taken together, short-term IL-16 stimulation results in a CD4+CD25+MHCII+CD95+ motile T cell that is transiently and reversibly protected from Ag-induced proliferation and cell death.

This study addresses the long-term consequences of IL-16 stimulation, and in the process determines the functionality of the up-regulated CD25 (resulting in high affinity IL-2R) and the Th subset bias of the responding CD4+ T cell population. In regard to the function of the induced IL-2R, IL-16 pretreatment is markedly synergistic with IL-2, as determined by increased [3H]thymidine incorporation and by increased numbers of CD4+ T cells as compared with treatment with IL-2 or IL-16 alone. The synergistic effect is more prominent with the length of time in culture past 2 wk. By 4 wk, most IL-16- or IL-2-treated PBMC cultures have few surviving cells, while the proliferating cells in IL-16/IL-2-costimulated PBMC are populated almost entirely by CD4+CD45RO CD25+ T cells. IL-16/IL-2 costimulation imparts increased viability to CD4+ T cells, as almost all exclude trypan blue, while in the other cultures, up to 50% of the cells by 2 wk and 90% of the cells by 4 wk do not exclude trypan blue. We believe that the mechanism for the increase in viability of IL-16/IL-2-treated cells relates to the levels of Bcl-2 (18, 19), as IL-16/IL-2-treated cells express high levels of Bcl-2 protein, whereas the levels in cells cultured with either IL-2 alone, IL-16 alone, or untreated are greatly diminished (N.A.P., manuscript in preparation).

IL-16/IL-2 cotreatment also results in rises in the percentages of CD45RO cells. It is not clear from our experiments whether these cells proliferated selectively or whether there was a phenotypic change within cells from CD45RA to CD45RO (20, 21, 22). The presence of double-positive cells by 4 wk (Table III) does not answer this question; and we had insufficient cells to address this issue definitively in the current experiments. Interestingly, IL-16/IL-2 treatment does not appear to bias toward a specific Th subtype. IL-16 treatment alone results in significant levels of GM-CSF. We believe that this is a consequence of the regulation of GM-CSF gene transcription and protein synthesis following CD4-dependent signaling rather than selective activity of one Th subset, because cytokines of both Th1 and Th2 types are synthesized and secreted by cells treated with combination rIL-16 and rIL-2 at similar levels at both 2 and 4 wk. Furthermore, all of these data together indicate that the IL-16/CD4-dependent signals that regulate transcription and expression of IL-2R are not Th subset specific. The IL-16/IL-2-expanded CD4+ T cells do not demonstrate the same TCR unresponsiveness observed in cells cultured for several hours with IL-16, as anti-CD3 stimulation of 4-wk cultures results in increased cytokine synthesis (unpublished observations). This suggests that IL-2 stimulation over an extended period of time is sufficient to facilitate escape of CD4+ T cells from IL-16-induced TCR unresponsiveness in vitro.

These studies did not address any potential role for IL-16 in normal CD4+ T cell maturation or proliferation of mature peripheral T cells. Definitive studies must await the development of IL-16 knockout mice. These studies do imply, however, that IL-16 may be a reasonable adjunct to IL-2-based immune reconstitution in states in which CD4+ T cells are diminished selectively in numbers. In that regard, IL-16 might induce IL-2 responsiveness in unactivated T cells across TCR specificities; and thus, expansion would not be limited to those T cell subsets that express IL-2R at the beginning or during the therapeutic course. The synergism in [3H]thymidine incorporation between IL-16 and IL-15 (Table I) implies that this combination of cytokines might also be of value in immune reconstitution; however, it is not yet clear that IL-16/IL-15 costimulation results in expanded numbers of CD4+ T cells over time. The effect of IL-16 stimulation on both the α- and β-chains of the IL-2R also implies that the cells might be responsive to other cytokines that use various components of the IL-2R as part of their receptor complex, such as IL-7, IL-9, and IL-13 (23). Studies investigating the effect of IL-16 stimulation on expression of these receptor complexes as well as functional responses to cytokine stimulation are currently in progress. As IL-16 and IL-2 are predominantly of T cell origin, while IL-15 is predominantly of macrophage origin, these studies imply that there might be synergy between lymphocyte- and monocyte-derived cytokines in the proliferation of CD4+ T cells.

The present studies used a single weekly low concentration of IL-16 to augment IL-2 responsiveness. Further studies are needed to determine the frequency and doses required to induce maximal effects; to determine whether IL-16/IL-2 combination therapy will be more effective in expanding CD4 populations across TCR specificities than IL-2 alone in PBMC from immunodeficient individuals; and whether IL-16/IL-2 therapy can rescue lost antigenic responsiveness.

1

This work was supported by Grants AI 37368, AI 35680, and HL 32802 from National Institutes of Health. N.A.P. is supported by a Robert Wood Johnson Foundation Minority Faculty Development Award. W.W.C. is a recipient of a Career Investigator Award from American Lung Association.

3

Abbreviations used in this paper: GM-CSF, granulocyte-macrophage CSF; CD40L, CD40 ligand; PE, phycoerythrin.

1
Connor, R., H. Mohri, Y. Cao, D. Ho.
1993
. Increased viral burden and cytopathicity correlate temporally with CD4+ T lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals.
J. Virol.
67
:
1772
2
Mackall, C. L., T. Fleischer, M. Brown, M. Andrich, C. Chen, I. Feurstein, M. Horowitz, I. Magrath, A. Shad, S. Steinberg, L. Wexler, R. Gress.
1995
. Age, thymopoeisis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy.
N. Engl. J. Med.
332
:
143
3
Mackall, C. L., T. Fleischer, M. Brown, I. Magrath, A. Shad, M. Horowitz, L. Wexler, M. Adder, L. McLure, R. Gress.
1994
. Lymphocyte depletion during treatment with intensive chemotherapy for cancer.
Blood
84
:
2221
4
Nemunaitis, J., S. Rabinowe, J. Singer, P. Bierman, J. Vose, A. Freedman.
1991
. Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer.
N. Engl. J. Med.
324
:
1773
5
Advani, R., N. J. Chao, S. J. Horning, K. G. Blume, D. K. Ahn, K. R. Lamborn, N. C. Fleming, E. M. Bonnem, P. L. Greenberg.
1992
. Granulocyte-Macrophage colony-stimulating factor (GM-CSF) as an adjuvant to autologous hematopoeitic stem cell transplantation for lymphoma.
Ann. Intern. Med.
116
:
183
6
Powles, R., C. Smith, S. Milan, J. Treleaven, J. Millar, T. McElwain, E. Gordon-Smith, S. Milliken, C. Tiley.
1990
. Human recombinant GM-CSF in allogeneic bone-marrow transplantation for leukemia: double-blind, placebo-controlled trial.
Lancet
336
:
1417
7
Kovacs, J., M. Baseler, R. Dewer.
1995
. Increases in CD4 T lymphocytes with intermittent courses of interleukin-2 in patients with human immunodeficiency virus infection: a preliminary study.
N. Engl. J. Med.
332
:
567
8
Kovacs, J., S. Vogel, J. Albert, J. Falloon, R. Davey, R. Walker, M. Polis, K. Spooner, J. Metcalf, M. Baseler, G. Fyfe, C. Lane.
1996
. Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus.
N. Engl. J. Med.
335
:
1350
9
Gea-Banacloche, J., E. Weiskopf, J. Lopez, J. Faloon, M. Baesler, R. Stevens, M. Connors.
1997
. HIV infection induces progressive depletions within the CD4+ T cell repertoire that are not immediately restored by anti-viral or immune-based therapies.
J. Allergy Clin. Immunol.
99
:
S110
10
Cruikshank, W., J. Berman, A. Theodore, J. Bernardo, D. M. Center.
1987
. Lymphokine activation of T4+ T lymphocytes and monocytes.
J. Immunol.
138
:
3817
11
Cruikshank, W., D. Center, N. Nisar, M. Wu, B. Natke, A. Theodore, H. Kornfeld.
1994
. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression.
Proc. Natl. Acad. Sci. USA
91
:
5109
12
Cruikshank, W., J. Greenstein, A. Theodore, D. Center.
1991
. Lymphocyte chemoattractant factor induces CD4-dependent intracytoplasmic signaling in lymphocytes.
J. Immunol.
146
:
2928
13
Ryan, T. C., W. W. Cruikshank, H. Kornfeld, T. L. Collins, D. M. Center.
1995
. The CD4-associated tyrosine kinase p56lck is required for lymphocyte chemoattractant factor-induced T lymphocyte migration.
J. Biol. Chem.
270
:
17081
14
Rand, T. H., W. W. Cruikshank, D. M. Center, P. F. Weller.
1991
. CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration.
J. Exp. Med.
173
:
1521
15
Parada, N. A., W. W. Cruikshank, H. L. Danis, T. C. Ryan, D. M. Center.
1996
. IL-16 and other CD4 ligand-induced migration is dependent upon protein kinase C.
Cell. Immunol.
168
:
100
16
Cruikshank, W. W., K. Lim, A. C. Theodore, J. Cook, G. Fine, P. F. Weller, D. M. Center.
1996
. IL-16 inhibition of CD3-dependent lymphocyte activation and proliferation.
J. Immunol.
157
:
5240
17
Theodore, A. C., D. M. Center, J. Nicoll, G. Fine, H. Kornfeld, W. W. Cruikshank.
1996
. CD4 ligand IL-16 inhibits the mixed lymphocyte reaction.
J. Immunol.
157
:
1958
18
Ma, A., J. C. Pena, B. Chang, E. Margosian, L. Davidson, F. W. Alt, C. B. Thompson.
1995
. Bclx regulates the survival of double-positive thymocytes.
Proc. Natl. Acad. Sci. USA
92
:
4763
19
Linette, G. P., M. J. Grusby, S. M. Hedrick, T. H. Hansen, L. H. Glimcher, S. J. Korsmeyer.
1994
. Bcl-2 is up-regulated at the CD4+ CD8+ stage during positive selection and promotes thymocyte differentiation at several control points.
Immunity
1
:
197
20
Tough, D. F., J. Sprent.
1994
. Turnover of naive- and memory-phenotype T cells.
J. Exp. Med.
179
:
1127
21
Johannisson, A., R. Festin.
1995
. Phenotype transition of CD4+ T cells from CD45RA to CD45RO is accompanied by cell activation and proliferation.
Cytometry
19
:
343
22
Beverely, P. C. L..
1990
. Is T-cell memory maintained by crossreactive stimulation?.
Immunol. Today
11
:
203
23
Sugamura, K., H. Asao, M. Kondo, N. Tanaka, N. Ishii, K. Ohbo, M. Nakamura, T. Takeshita.
1996
. The interleukin-2 receptor γ chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID. W. E. Paul, and C. G. Fathman, and H. Metzger, eds.
Annual Review of Immunology
179
Annual Reviews Inc., Palo Alto.