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The Journal of Immunology, 2003, 171: 3459-3466.
Copyright © 2003 by The American Association of Immunologists

Diversity of Clonal T Cell Proliferation Is Mediated by Differential Expression of CD152 (CTLA-4) on the Cell Surface of Activated Individual T Lymphocytes1

Frank Maszyna*, Holger Hoff*, Désirée Kunkel*, Andreas Radbruch*,{dagger} and Monika C. Brunner-Weinzierl2,*,{dagger}

* Deutsches Rheuma-Forschungszentrum, Berlin, Germany; and {dagger} Medizinische Klinik der Humboldt-Universität zu Berlin, Charité, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitory effects of CD152 (CTLA-4) engagement during T cell activation have been described. To date, such effects could only be correlated to CD152 expression at the population level because expression of CD152 on the cell surface is too low to be assessed by conventional immunofluorescence on the single cell level. In this study, we use magnetofluorescent liposomes for the immunofluorescent detection of surface CD152-expressing CD4+ T cells and show that, despite the fact that nearly all cells express intracellular CD152, only a fraction of 12% of activated T cells expresses surface CD152 at any given time point. Surface CD152+ T cells appear with similar kinetics after primary or secondary activation in vitro. However, the frequency of surface CD152+ T cells 48 h postactivation is 2-fold higher during secondary activation. Surface expression of CD152 is independent of the proliferative history of an activated T cell. Instruction of T cells for surface expression of CD152 rather depends on the time elapsed since the onset of activation, with a maximum at 48 h, and requires less than 12 h of Ag exposure. CD152- T cells, when isolated by cell sorting and restimulated, continue to proliferate. CD152 blockade has no effect on their proliferation. Isolated surface CD152+ T cells do not proliferate upon restimulation unless CD152 is blocked. CD152 thus acts directly and autonomously on individual activated and proliferating T lymphocytes. Due to its heterogeneous expression on the cell surface of activated Th cells, CD152 might diversify the T cell response.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A broad repertoire of mature effector T cells with specific, diverse functional capabilities can be generated in adaptive immune responses. Critical for the generation of functional diversity are the costimulatory signals provided during Ag-specific stimulation of T cells by APCs (1). CD28 and CD152, two homologous members of the Ig superfamily, are the receptors for such a costimulation. CD28 and CD152 apparently have different functions during T cell activation. Triggering of CD28 strongly up-regulates IL-2 transcription initiation, IL-2 production, and ultimately T cell proliferation (2, 3) and prevents apoptosis (4). In contrast, CD152 (CTLA-4) seems to be an important down-regulator of immune responses. Most studies indicate that triggering of CD152 down-regulates the proliferation and cytokine production of the entire T cell population (1, 5, 6, 7, 8, 9, 10). Indirect inhibitory effects have been described involving the induction of TGF-{beta} or IL-10 expression by CD152 (11, 12, 13, 14). Some groups have even provided evidence that CD152 engagement may play an activating role (12, 15, 16).

CD28 and CD152 bind to the same ligands on APCs, CD80, and CD86; however, CD152 possesses a 20- to 50-fold higher binding affinity (17, 18). Although CD28 is constitutively expressed on naive CD4+ T cells, the expression of CD152 is induced upon T cell activation. In naive T cells, CD152 mRNA is detectable, but not the intracellular or surface protein (19). Already 4 h after onset of T cell activation cross-linking of CD152 by specific Abs shows that it is expressed functionally by at least some cells (5, 20) and prevents complete T cell activation. The main effect of CD152 engagement during T cell activation is probably the inhibition of transcription of the IL-2 gene by preventing NF-AT translocation to the nucleus (5). The expression of activation molecules such as CD69 and CD25 is also prevented by cross-linking of CD152 (7, 21). Debate concerns the inhibition of T cells by CD152 after a successful T cell stimulation at the peak of CD152 expression when T cells already proliferate and produce growth factors such as IL-2. A quantitative increase in the expression of CD152 has been suggested to correlate with the number of cell cycles that a cell has undergone (22). This would imply that the transcription machinery of the CD152 gene would have the ability to count cell cycles and convert this information into expression. However, it is not clear whether CD152 engagement is able to inhibit the proliferation of such T cells.

In activated T lymphocytes, CD152 molecules are stored in intracellular vesicles (23), where they are found in most of the cells at 48 h after onset of stimulation. It has been shown that vesicles containing CD152 molecules are mobilized toward the sites of Ag receptor engagement and CD152 is probably displayed at the immunological synapse (23, 24, 25). The localization of CD152 at the cell surface is regulated by the association of clathrin-coated pit adaptor protein AP-2 to the intracellular tyrosine-based motif of CD152 (26, 27, 28, 29). Because CD152 protein can be detected intracellularly 24–48 h after onset of T cell activation, it has been suggested that the molecule is probably also expressed on the cell surface of these cells and functional (14, 30). But a highly restricted regulation of CD152 localization in a cell suggests that the restricted surface expression of CD152 represents a major control point for the regulation of the inhibitory function of CD152 on T cells. Due to a lack of sensitivity of CD152-specific immunofluorescence for the detection of surface CD152 on activated T cells, the expression of surface CD152 has not been analyzed at the level of single cells to date, nor have CD152-expressing cells been isolated for functional studies at a single cell level. Also, only functional studies at the population level have been performed, dismissing individual responses of activated surface CD152-expressing T cells (5, 6, 7, 8, 9, 10, 20, 21, 30, 31, 32, 33).

In this study, we have used magnetofluorescent liposomes to detect CD152 on the surface of activated T cells with a sensitivity of less than 200 molecules per cell (34). Unlike intracellular CD152 expression, the expression of surface CD152 is restricted to a small fraction of activated T cells. This expression is independent of the proliferative history of the cell, but dependent on the time elapsed since the onset of activation of the T cell. The commitment of a T cell to express surface CD152 upon restimulation requires less than 12 h of T cell stimulation. T cells expressing surface CD152 are inhibited in their proliferation, when isolated and restimulated with APCs ex vivo, demonstrating that CD152 does directly block the proliferation of those individual cells expressing it.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice transgenic (tg)3 for the DO11.10 {alpha}/{beta}-TCR (OVA-specific TCRtg/tg) on BALB/c background (gift from D. Loh, Washington University School of Medicine, St. Louis, MO) and BALB/c mice were bred under specific pathogen-free conditions in the animal facility of the Bundesgesundheitsamt (Marienfelde, Berlin). Mice were used at the age of 6 wk to 4 mo.

Abs and reagents

The following Abs against murine Ags were used: anti-CD4 (GK-1.5/4), anti-CD8 (196), anti-Thy-1.2 (HO-13), anti-CD62L (MEL-14), anti-B220 (RA3.6B2), anti-CD152 (UC10-4F10-11; BD Biosciences, San Jose, CA), anti-trinitrophenol (A19-3; BD Biosciences), anti-CD69 biotin (H1.2F3; BD PharMingen, San Diego, CA), anti-CD25 (BD PharMingen), anti-TCRtg/tg (KJ1–26.1) in their respective form of FITC, PE, and Cy5 conjugates. Anti-CD152 Ab was also purified from the supernatant of F10 hybridoma cells via a protein G affinity column (9). Sulfate polystyrene latex microspheres of 5 +/- 0.1-µm mean diameters were obtained from Interfacial Dynamics (Portland, OR). Anti-CD152 Fab were prepared with the Immunopure Fab preparation kit (Pierce, Rockford, IL) and used at 200 µg/ml. The Fab were analyzed by HPLC and used when at least twice as much proliferation and 3- to 5-fold more IFN-{gamma} were observed in Ag-specific stimulation of primary CD4+CD62L+ T cells when treated with anti-CD152 Fab compared with those treated with control Fabs.

Cell culture

Cell cultures were set up with 3–4 x 106 cells/ml in a complete RPMI 1640, containing 10% FCS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 0.3 mg/ml glutamine, and 10 µM 2-ME. The antigenic peptide OVA323–339 (Neosystem, Strasbourg, France) was used at 1.0 µg/ml. Irradiated T cell-depleted splenocytes (3000 rad) from BALB/c mice were used as APCs for OVA-specific TCRtg/tg T cells at a 3:1 ratio. Proliferation was determined by CFSE labeling of the cells. Due to fading of the label within the first 10 h, a 12-h time point poststimulation was used to get the cytometric measurement of unproliferated cells.

Latex microspheres were coated, as described (5). Briefly, 1 x 107 microspheres/ml were suspended in PBS with the indicated Abs (anti-CD3, 0.1 µg/ml; anti-CD28, 2 µg/ml; and anti-CD152 or a hamster control (anti-MAC1) Ab was added at 2.9 µg/ml) and incubated for 1.5 h at 37°C, followed by washing with PBS and blocking with 10% FCS. T cells (106/ml) were stimulated in a ratio of 1:1 with the Ab-coupled microspheres.

Preparation and activation of T lymphocytes

Magnetic isolation of naive CD62L+CD4+ T cells from OVA-specific TCRtg/tg mice was performed using MACS with FITC-conjugated anti-CD4 mAb (GK1.5) and MultiSort anti-FITC microbeads (Miltenyi Biotec, Auburn, CA) to a purity of 98–99%. Surface CD152+ cells were isolated by cell sorting from 48-h activated T cells (see above) using a FACSVantage (BD Biosciences). To label T cells with CFSE (Molecular Probes, Eugene, OR), cells were washed and resuspended at a concentration of 107 cells/ml in PBS (35). CFSE was added (5 µM) and incubated for 6 min at room temperature. After washing the cells with PBS/BSA, cells were resuspended in RPMI 1640 (Life Technologies, Andover, U.K.) containing 10% FCS (Sigma-Aldrich, St. Louis, MO).

Four-color cytometric analysis of surface expression of CD152

Surface expression of CD152 protein was detected using magnetofluorescent liposomes (34). T cells were incubated with digoxigenin (Dig)-conjugated anti-CD152 Ab at a concentration of 1 µg/ml for 15 min at 4°C. Cells were washed twice and incubated with fluorescein-filled liposomes, attaching anti-Dig Fab, for 30 min at 4°C. The specificity of the Dig-based CD152 staining was controlled by blocking the staining of the digoxigenized anti-CD152 Ab by incubating the cells with an excess of unconjugated anti-CD152 Ab. Alternatively, cells were incubated with uncoupled anti-CD152 Ab at a concentration of 1 µg/ml for 15 min at 4°C. Cells were washed twice and then incubated with fluorescein-filled liposomes attaching an anti-hamster IgG Ab. Isotype control Abs were used to validate the specificity of the CD152 staining. Specificity of the staining for liposomes was routinely assessed by isotype control Abs, and in addition by a separate incubation of cells with liposomes only. Cytometric analysis was performed using a FACSCalibur (BD Biosciences) and CellQuest software. Dead cells were excluded according to forward and sideward scatter and staining with propidium iodide (0.4 µg/ml).

Four-color cytometric analysis of intracellular CD152

Cells were fixed with 2% formaldehyde and permeabilized with 0.5% saponin (Sigma-Aldrich) in PBS/BSA/azide. The cells were then incubated with unconjugated anti-CD152 Ab at a concentration of 1 µg/ml, followed by incubation with anti-hamster Cy5.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of individual surface CD152-expressing cells

CD152 is a cell surface molecule that is not detectable on resting T lymphocytes. Expression is up-regulated upon activation of T lymphocytes and then maintained at low levels. Labeling of surface CD152 with anti-CD152 Dig and anti-Dig Cy5 results in a dim staining of a fraction of Con A-activated spleen cells (Fig. 1A). The frequency of positive cells cannot be determined due to the lack of optical separation. To identify T cells expressing surface CD152 unambiguously, we used enhanced CD152-specific immunofluorecent liposomes, increasing the sensitivity 1000-fold (34). A subpopulation of CD4 T cells expressing surface CD152 can now clearly be identified (Fig. 1B). The specificity of the staining was confirmed by pretreatment of the cells with unconjugated anti-CD152 Abs, resulting in a background of less than 2% of unspecifically stained cells (Fig. 1C). Routinely, specificity controls of the surface CD152-expressing cells in Ag-specific stimulations of T cells show only 1% or less unspecific staining (Fig. 2).



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  		FIGURE 1. Detection of individual surface CD152-expressing T cells using liposome-enhanced staining technology. OVA-specific TCRtg/tg splenocytes were stimulated with OVA323–339 for 48 h. CD4+ T cells were identified using staining with anti-CD4 Cy5 mAb. Subsequently, surface CD152+ cells were stained using digoxigenized anti-CD152 Ab, followed by anti-Dig Fab conjugated to FITC (A) -digoxigenized anti-CD152 Ab, followed by anti-Dig FITC liposomes (B). The specificity of the staining using liposomes was controlled by blocking with an excess amount of unconjugated anti-CD152 Ab (C).

 


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  		FIGURE 2. Surface CD152-expressing and -nonexpressing T cells appear during a primary activation and express effector molecules. A, CFSE-labeled, CD4+CD62L+ OVA-specific TCRtg/tg T cells were stimulated with OVA323–339 and irradiated APCs. Surface CD152-expressing cells (in %) were analyzed, as described in Fig. 1B, in the respective cell generations at the indicated time points. The specificity of the staining was controlled by blocking of the binding of digoxigenized anti-CD152 Ab with an excess of unconjugated anti-CD152. This represents data from three experiments. B, Gated surface CD152+ (in front) and surface CD152- T cells and their respective staining for CD25 or CD71 were overlaid in a histogram at the indicated time points. One of two similar experiments is shown.

 
Expression of surface CD152 on individual activated T cells in relation to proliferation

Naive CD4 T cells were purified to >99% from the spleens of OVA-specific TCRtg/tg BALB/c mice by MACS for CD4 and CD62L. The isolated cells were labeled with CFSE to determine their proliferation status later on (35). CFSE-labeled, naive, OVA-specific CD4+ T cells were stimulated with the antigenic peptide OVA323–339 and congenic APC using BALB/c spleen cells. Expression of surface CD152 was evaluated at various time points after primary activation. The frequency of surface CD152-expressing Th cells was determined for each generation that had undergone a defined number of cell divisions according to CFSE staining (Figs. 2A and 3A). Among the cells that had divided for up to seven times, we generally observed the expression of surface CD152 with similar frequencies in all generations. Within a given generation, surface CD152+ T cells were detectable at various frequencies over time, from 36 to 72 h of stimulation. Cells that had not divided were present until 48 h of stimulation. In 5.9% of these cells, surface CD152 was clearly detectable, showing that expression of this molecule on the cell surface does not require cell division. The highest frequency of surface CD152+ T cells (4–7%) was detectable 48 h after activation in all generations of activated cells. At all time points, CD152 expression was limited to a minority of activated cells. The majority of CD152-expressing cells also expressed CD69 (74% of surface CD152+ CD4+ cells; data not shown).



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  		FIGURE 3. Expression of intracellular and surface CD152+ on activated T cells in relation to proliferation during a primary and a secondary activation is independent of cell generations. A, CFSE-labeled, CD4+CD62L+ OVA-specific TCRtg/tg T cells were stimulated with OVA323–339 and irradiated APCs. At the indicated time points, CD4+OVA-specific TCRtg/tg T cells were examined for surface CD152 expression in the respective cell generations. The specificity of the staining was controlled by blocking the digoxigenized anti-CD152 Ab with an excess of unconjugated anti-CD152 Ab. The background generated by unspecific binding of liposomes to the cells was determined in a separate sample (data not shown). B, CD4+CD62L+ OVA-specific TCRtg/tg T cells were stimulated with OVA323–339 and irradiated APCs. Cells were harvested on day 7 after onset of stimulation. CD4+ T cells were CFSE labeled, and restimulated with OVA323–339 and irradiated APCs. At the indicated time points, cells were examined for surface CD152 expression in their respective cell generations. The specificity of the staining was controlled by blocking of the binding of digoxigenized anti-CD152 Ab with an excess of unconjugated anti-CD152 Ab. This represents data from three similar experiments. C, CD4+CD62L+ OVA-specific TCRtg/tg T cells were labeled with CFSE and stimulated with 1 µg/ml OVA323–339 peptide and irradiated APCs. After fixation and permeabilization, intracellular CD152 expression and proliferation were measured at the indicated time points. Frequencies of intracellular CD152-expressing cells are shown at a given time point and per cell generation. D, One week after stimulation, cells were fixed, permeabilized, and stained for CD4 and CD152 (open curve) or CD4 and an isotype control (gray curve). This represents data from two experiments.

 
In Fig. 3B, we have analyzed surface CD152 expression during a secondary response. The stimulation of Ag-experienced T cells led to a high frequency of ~10–11% of surface CD152+ T cells at 48 h after activation. The kinetics of expression of surface CD152 in secondary responses was similar to that of a primary response, with a maximum frequency of surface CD152+ T cells at 48 h after stimulation in cells of all generations. As in primarily activated Th cells, the expression of detectable surface CD152 is neither correlated to the proliferative history nor is proliferation a mandatory prerequisite for CD152 expression.

Intracellular and surface expression of CD152 does not correlate at the single cell level

Transcription of CD152 mRNA is synergistically induced by TCR, CD28, and IL-2 signaling. Newly synthesized protein of CD152 is stored intracellularly, until it is released to the cell surface upon phosphorylation at position Y201 (26, 28, 29). It has generally been assumed that those cells expressing intracellular CD152, i.e., most activated T cells, also express functional surface CD152 (31, 36). In this study, we show that this is not the case.

By immunofluorescence, expression of intracellular CD152 was evaluated at various time points after primary activation. The frequency of intracellular CD152-expressing Th cells was determined for each generation that had undergone a defined number of cell divisions, as determined by CFSE staining (Fig. 3C). We observed expression of intracellular CD152 generally with similar frequencies in all generations. The expression of intracellular CD152 increased steadily until 72 h after stimulation. Interestingly, almost all T cells (>75%) store CD152 protein intracellularly 1 wk after activation (Fig. 3D).

Although >30% of the T cells express intracellular CD152 from day 1 to day 7 after stimulation, no more than 12% of the T cells ever express CD152 on the cell surface (Figs. 2, A and B, and 3B). After 72 h of activation with APC and Ag, only 2% of the activated T cells express surface CD152, while >80% of them express intracellular CD152 (Figs. 2A and 3C). Thus, the intracellular expression of CD152 does not imply automatically expression of surface CD152.

Priming for intracellular and surface CD152 instruction requires only a short time of T cell stimulation

To date, it has become clear that the expression of surface CD152 does not correlate with the proliferation status of an activated T cell, the expression rather being dependent on the time elapsed since the onset of activation. We analyzed next whether the overall time of stimulation or the onset of stimulation was decisive for T cells to acquire the ability to express intracellular and surface CD152. Splenocytes, which contain resting, OVA-specific TCRtg T cells, were stimulated with Ag, and at different time points after onset of stimulation the Ag was withdrawn from the cultures (Fig. 4). The frequency of surface CD152+ Th cells was determined 48 h after the onset of activation. At that time point, intracellular CD152 was detectable in the majority of the CD4+ T cells that have received as little as 12 h of antigenic stimulation (Fig. 4B). Interestingly, also surface CD152 expression of 9% of the T cells was apparent 48 h after the onset of stimulation in such cultures (Fig. 4C). The frequency of surface CD152+ T cells increased to 17% after 36 h of antigenic stimulation, but it did not increase much thereafter (22%). Thus, instruction of intracellular and surface expression of CD152 requires less than 12 h of Ag-specific stimuli for the T cells; this also means that continuous stimulation of the T cell until CD152 is expressed at the cell surface is not necessary.



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  		FIGURE 4. Priming for intracellular and surface CD152 expression requires only a short time of T cell stimulation. CD4+ OVA-specific TCRtg/tg T cells were isolated and stimulated with 1 µg/ml OVA323–339 peptide and APCs in three different cultures. The stimulation was stopped by isolation of CD4+ T cells after 12 or 36 h, as indicated. A, OVA-specific TCRtg/tg cells were identified according to the staining with a clonotypic mAb KJ1-26.1. Sorted and unsorted cells were analyzed after an overall culture time of 48 h for intracellular (B) or surface (C) CD152 expression, as described. Tg-TCRhigh (B, bold line; C, filled bars) and tg-TCRlow cells (B, filled curve; C, hatched bars) were plotted separately in relation to CD152 expression. One of two similar experiments is shown.

 
Cross-linking of surface CD152+ does not down-regulate expression of CD25

It has been suggested that cross-linking of CD152 during T cell activation inhibits the up-regulation or down-regulates the expression of activation markers such as CD25, CD69, CD71, and TCR (7, 21). As shown in Fig. 2B, almost all T cells expressing surface CD152 coexpressed the transferrin receptor (CD71) and the IL-2R {alpha}-chain (CD25) as well as the activation marker CD69 at levels comparable to surface CD152- cells, indicating that either their CD152 molecules have not yet been cross-linked or that CD152-mediated functions do not include the down-regulation of expression of activation markers (Fig. 2B; CD69 data not shown). To decide between the two options, naive DO11.10 CD4 T cells were stimulated with OVA peptide and sorted into surface CD152-positive and -negative subpopulations at the peak of surface CD152 expression. Isolated surface CD152+ CD4+ T cells and surface CD152- CD4+ T cells were compared for the expression of CD25 upon restimulation by APC plus Ag (Fig. 5C). Both subpopulations show comparable and high expression of CD25 upon restimulation and comparable loss of CD25 expression in the absence of further stimuli.



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  		FIGURE 5. Only activated, surface CD152-expressing T cells are inhibited to proliferate. A, CD4+CD62L+ OVA-specific TCRtg/tg T cells were stimulated for 48 h, as described, and surface CD152+ and CD152- T cells were separated using a FACS sorter. Sorted cells were labeled with CFSE and cultured with APCs and 1 µg/ml OVA323–339 peptide for 48 h; cells were collected and analyzed for proliferation 12 h (gray curves) and 48 h (open curves) after separation. B, Sorted cells were labeled with CFSE and stimulated with PMA/ionomycin for 5 h. Proliferation of cells was analyzed 12 h (gray curves) and 36 h (open curves) after separation. One of two similar experiments is shown. C, Sorted surface CD152+ and CD152- T cells were also analyzed for CD25 expression cocultured with APCs only (open curves) or in conjunction with OVA peptide (gray curves). One of three similar experiments is shown.

 
CD152 expression is correlated with TCR density on activated T cells

Intracellular as well as surface CD152 expression correlated with the expression of CD25, CD71, and CD69, which resembles the phenotype of activated T cells (Fig. 2B). Another major hallmark of T cell stimulation is the down-modulation of the TCR. As CD152 is targeted toward the immunological synapse after TCR stimulation and probably expressed there at the cell surface, its correlation with the density of the TCR was analyzed (23, 25) (Fig. 4A). Correlation of intracellular CD152 expression and tg-TCR density per cell revealed a significant reduction of intracellular CD152 in tg-TCRlow cells (Fig. 4B). As shown in Fig. 2, all CD4+TCRtg T cells were efficiently stimulated, shown by up-regulated activation-induced molecules CD25, CD69, and CD71. To further confirm that tg-TCRlow cells were indeed efficiently activated T cells, we analyzed tg-TCRlow cells that had also down-regulated CD4 molecules after activation in comparison with tg-TCRhigh cells (tg-TCRlowCD4low) and gained similar results. Correlation of surface CD152 expression and TCR density at 48 h after T cell activation showed that 22.5% of the tg-TCRhigh-expressing cells coexpressed surface CD152, but only 15% of the tg-TCRlow-expressing cells (Fig. 4C). As shown, down-modulation of TCR and CD4 is correlated with less surface CD152-expressing T cells, and therefore, CD152 surface expression is tightly connected to the activation status of the T cell.

Individual responses of already activated T cells toward CD152 engagement

According to the results described above, which show a heterogeneous expression of surface CD152 after stimulation, a heterogeneous response of T cells upon activation plus surface CD152 engagement would be expected. Previous results showed that resting CD4+ T cells stimulated with anti-CD3, anti-CD28, and anti-CD152 had very low, but clearly detectable proliferation, as measured by thymidine incorporation (7, 21). We extended the finding, using naive T cells labeled with CFSE, and stimulated them, as described earlier, by cross-linking with anti-CD3 and anti-CD28 or anti-CD3, anti-CD28, and anti-CD152, showing that 85% of the cells were inhibited by CD152 engagement already in the first generation (Fig. 6A) (5, 7). We have shown above that 48 h after the onset of activation, only a subpopulation of T cells expresses surface CD152 at detectable levels, and that these cells are inhibited in their proliferation. To monitor the effect of CD152 on an equally activated entire CD4 T cell population, naive T cells were stimulated for 48 h with anti-CD3 and anti-CD28, and then Ab-coupled microspheres were used, as mentioned above, to cross-link CD152 in concordance with CD3 and CD28 (Fig. 6B). Eighty-eight percent of the cells proliferated up to five to six times if stimulated with anti-CD3 and anti-CD28 coupled to microspheres. The data in Fig. 6B show that even with possible CD152 engagement at any time of the response after 48 h, 70% of the cells do proliferate, which might be due to surface CD152- T cells or T cells that do not express enough surface CD152. The stimulation with CD3, CD28, and CD152 showed cell cycle arrest in 30% of the T cells, 18% more than in CD3/CD28-stimulated T cells. Thus, only a fraction of activated T cells of the entire T cell population is susceptible to CD152 cross-linking and arrested in cell cycling.



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  		FIGURE 6. CD152 cross-linking on a uniformly preactivated T cell population does not arrest all T cells. A, Naive CD4+CD62L+ T cells were labeled with CFSE and stimulated with anti-CD3 and anti-CD28, or anti-CD3, anti-CD28, and anti-CD152 coupled to microspheres for 72 h. B, Naive CD4+CD62L+ T cells were labeled with CFSE and stimulated with plate-bound anti-CD3 and anti-CD28 for 48 h before stimulation with Ab-coated microspheres, as indicated. Cells were analyzed 72 h later. This represents data from three similar experiments.

 
Isolated surface CD152+ T lymphocytes are limited in their proliferation upon reactivation with APC and Ag

To analyze whether surface CD152+ and CD152- T cells are distinct subpopulations, we characterized their proliferative response separately. Naive DO11.10 CD4 T cells were stimulated with OVA peptide and sorted into surface CD152-positive and -negative subpopulations at the peak of surface CD152 expression. CSFE-labeled isolated CD4+ surface CD152+ and CD4+CD152- T cells were compared for proliferation and the expression of CD25 (see above) upon restimulation by APC plus Ag (Fig. 5). Seventy-five percent of CD152- T cells divided once and twice within 48 h after restimulation (Fig. 5A). Even if these unstained cells express low amounts of surface CD152, it is not inhibiting T cell proliferation in 75% of the cells. In contrast, stimulated surface CD152+ T cells were strongly inhibited in their proliferation. Thus, T cells expressing detectable amounts of surface CD152 (>100 molecules per cell) were immediately inhibited in their proliferation. The inhibition of proliferation was mediated by CD152 engagement during restimulation of the CD152+ T cells, as shown by CD152 blockade with anti-CD152 Fab (Fig. 7). No difference in the proliferative response was seen when CD152 was blocked in isolated, restimulated CD152- T cells.



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  		FIGURE 7. Blockade of CD152 on sorted surface CD152+ T cells reverses inhibition of proliferation. CD4+CD62L+ OVA-specific TCRtg/tg T cells were stimulated for 48 h, as described, and surface CD152+ and CD152- T cells were separated using a FACS sorter. Sorted cells were labeled with CFSE and cultured with APCs, 1 µg/ml OVA323–339 peptide, and anti-CD152 Fab (gray curves) or control Fab (open curves). Cells were collected and analyzed for proliferation 24 h after the onset of restimulation and CFSE labeling. One of two similar experiments is shown.

 
To control downstream signaling of TCR and CD28 in the isolated surface CD152+ T cells, we stimulated the cells with PMA and ionomycin for 6 h (Fig. 5B). Triggering of calcineurin by ionomycin-induced Ca+ flux and protein kinase C by PMA resulted in proliferation of both surface CD152+ and CD152- subpopulations. Thus, the signal transduction machinery for the progression of the cell cycle is intact in surface CD152-expressing cells, and the inhibition of signaling for proliferation in surface CD152+ T cells operates upstream of the protein kinase C and calcineurin in the TCR and CD28 pathway.

A total of 12.5% of surface CD152-expressing, isolated, Ag-activated T cells remained surface CD152 positive, when cultured in vitro without restimulation, whereas only 5.8% of isolated CD152- T cells gained expression of surface CD152 after 2 days (mean of 8 independent experiments). After restimulation with Ag, only some CD152- T cells were able to up-regulate surface CD152 (9.5%). Interestingly, TCR stimulation did stabilize surface CD152 expression of the CD152+ T cells for >36 h in 23% of the cells (mean of 10 independent experiments). It will be interesting to learn whether such cells have distinct functional properties in regulating immune responses.

Altogether, the time-dependent differential expression of CD152 on the cell surface of activated T cells and the specific arrest in cell cycling of surface CD152-expressing cells cause diversification of clonal T cell responses with respect to clonal expansion and differentiation steps that are dependent on extended proliferation, e.g., functional polarization of cytokine expression.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD152 is an important regulator of peripheral T cell responses by virtue of its ability to regulate activation and expansion of T cells. The role of CD152 has been studied extensively in genetic and functional assays at the level of T cell populations. Heterogeneity of surface expression of CD152 of individual T cells has not been analyzed to date, because of the low abundance of surface CD152 expression on cells and the resulting technical problems to identify surface CD152-expressing cells unambiguously. In this study, we compare proliferation of T cells capable or incapable of expressing CD152 on the cell surface in primary and secondary Ag-specific responses using liposome-based immunofluorescence. Surprisingly, only a minority of T cells ever expresses surface CD152 on the cell surface in detectable amounts, independent of their proliferation history at a given time point, but dependent on a short time window of stimulation and dependent on the time elapsed since the onset of activation. When activated T cells were separated according to expression of surface CD152, all cells expressing surface CD152 do not continue to proliferate, nor do they show a proliferative response to restimulation. In contrast, surface CD152-negative cells proliferated upon restimulation. In accordance with our concept, CD152 cross-linking on cells of an entire unseparated T cell population 48 h after activation revealed that not all cells were inhibited in their proliferation. The fraction of the cells that were arrested in the cell cycle or had reduced proliferative capacity in the first generation after restimulation corresponds to the fraction expressing surface CD152 after 48 h of stimulation with anti-CD3 plus anti-CD28 (data not shown).

Instruction for expression of intracellular and surface CD152 is restricted to a narrow time window

It has been reported that the instruction of naive T cells to express genes coding for effector cytokines, such as IL-4 and IFN-{gamma}, requires DNA synthesis of the activated T cell or even completion of several cycles of cell division (37, 38). In this study, we show that expression of the gene for CD152 leading to intracellular storage of CD152 is not correlated to entry of the activated cell into cell cycling or proliferation. The number of cells expressing intracellular CD152 and the amount of intracellular CD152 per cell do not correlate to the number of cell divisions that the cell has performed. This is in contrast to a recent report that the proliferative history of a given cell correlates with a quantitative increase in expression of intracellular CD152 per cell (22). One explanation for this discrepancy could be that in those experiments, continuous, strong stimulation with anti-CD3 had stimulated continuous CD152 synthesis over the period of culture, i.e., that the increased intracellular expression was due to prolonged stimulation of the T cells rather than the number of cell cycles the cell had performed. For cell surface expression of CD152, we show in this work that the proliferative history of an activated naive T cell does not correlate with the frequency of surface CD152-positive cells. Rather, the induction of CD152 expression is restricted to a narrow time window after onset of activation, with no enhancement in frequencies of surface CD152-expressing cells in later generations. This result has major implications for the response of activated T cells, because the cells that receive the instruction to express surface CD152 in this time window will eventually express CD152, with all the consequences.

Surface CD152 is expressed on a subpopulation of T lymphocytes

Expression of CD152 on the surface of T cells has been difficult to detect using conventional cytometric and microscopic techniques (5, 7, 25). The sensitive liposome-based staining technique shown in this work allows for the detection of cells expressing as few as 100–200 molecules of a given molecule per cell (34). We show in this study by CD152-specific liposome staining, that only a fraction of primary and secondary activated T cells, no more than 12% at a given time point, expresses surface CD152. The heterogeneity of activated T cells with respect to surface CD152 expression was also evident from functional analysis using an entire activated T cell population: at the time point of maximal CD152 expression after the onset of activation, activated T cells were provided with a continuously available stimulus for CD152 by cross-linking of CD152 with Abs; 18% of the cells did not proliferate or showed reduced proliferation due to CTLA-4 cross-linking (Fig. 6B). Our data show also that 70% of proliferating cells do not get a CD152 signal or that the signal is not strong enough due to too low or no CD152 expression. Furthermore, the clearly positive surface staining of CD152 on a subpopulation of T cells (12%) was verified when surface CD152+ T cells were isolated and responded functionally distinct to restimulation than surface CD152- cells: surface CD152+ T cells were immediately inhibited in their proliferation (Fig. 5A), whereas >75% of surface CD152- T cells proliferated.

Twenty-two percent of surface CD152-expressing, isolated, staphylococcal enterotoxin B-activated human T cells remained surface CD152 positive, when cultured in vitro without restimulation (Hirseland and Brunner-Weinzierl, unpublished data). Only 3% of isolated CD152- T cells gained expression of surface CD152 after 2 days. TCR stimulation did stabilize surface CD152 expression of the CD152+ T cells for >3 days in 50% of the cells. Using mouse cells, 12.5% of surface CD152-expressing, isolated, Ag-activated T cells remained surface CD152 positive, when cultured in vitro without restimulation for 36 h. TCR stimulation did stabilize surface CD152, with 23% of the cells remaining positive. This result shows that expression of CD152 on the cell surface is transient in some T cells, but can be maintained for several days at least in a substantial fraction of cells, as T cell proliferation of almost all isolated, surface CD152-expressing cells is inhibited, but only a proportion of these cells remain surface CD152 positive of the entire period of stimulation. That shows that even a dynamic surface expression of CD152 during a relatively short time window can lock a T cell's proliferative fate, at least for the remaining duration of the response. Our data also imply that anergy induction in T cells can be initiated by CD152 signaling in T cells independently of their cell generation. Monitoring the frequencies of surface CD152-positive cells over longer periods of time will show whether subpopulations of T cells generated in such cultures can stably express surface CD152 and might qualify as regulatory T cells (39).

The molecular basis for the heterogeneous expression of surface CD152 by Ag-activated primary cells is not addressed in this or previous studies and remains undefined. As CD152 is expressed intracellularly in almost all of the cells 2–3 days after activation, an intrinsic program of the cells to express CD152 is unlikely. It has been shown that CD152 expression needs TCR signaling and is synergistically enhanced by CD28 and IL-2 signals, which make undoubtedly stochastic components of the strength of activation of T cells likely to be involved (25, 40). We show in this study that stimulation of naive T cells with anti-CD3 and anti-CD28 for 48 h and subsequent cross-linking of CD152 result in inhibition of proliferation of only 18% of the T cells, corresponding to 19% of cells stainable for surface CD152 with magnetofluorescent liposomes (F. Maszyna and M. C. Brunner-Weinzierl, data not shown,), and suggesting that the surface CD152-positive cells were the ones inhibited in proliferation. This interpretation is supported by those experiments showing that isolated CD152-positive cells are quantitatively inhibited in proliferation. Thus, even with a very similar stimulus for all of the T cells, only some cells respond to CD152 cross-linking, suggesting that a factor to induce surface expression of CD152 in all of them is still missing or suboptimally available. It has been shown that Th2 clones express more surface CD152 than Th1 clones, pointing to a role of the cytokine milieu for the induction of surface expression, directly or indirectly via the APCs (33). Thus, heterogeneity of the clonal T cell population might reflect heterogeneity in the APC population.

Activated surface CD152+ T cells maintain up-regulated expression of effector molecules

The situation of regulation of effector molecules may be different for CD152 engagement at early steps of activation in which the up-regulation of surface molecules such as CD69 and CD25 was prevented by CD152 cross-linking with specific Abs (7, 9). At that time point, detection of the expression of functional surface CD152 was not possible. In this study, we show that in an Ag-specific system with natural ligands for CD152, a majority of the cells expressing surface CD152 do express the activation markers CD69, CD71, and CD25. When isolated and restimulated, primary surface CD152+ T cells, which were inhibited in their proliferation, possibly due to CD152 engagement, remained positive for CD25. As proliferation of isolated CD152+ T cells could be restored by CD152 blockade, CD152 engagement during an ongoing T cell response does not lead to the down-regulation of CD25 expression of the T cell. Our data and the evidence that CD152 engagement inhibits accumulation of transcription factors, such as NF-AT and NF-{kappa}B in the nucleus (5, 20), lead us to speculate that, as has been suggested elsewhere (9), already up-regulated molecules will not be down-regulated by CD152.

CD152 mediates inhibition of proliferation of a subset of activated T cells

Expression of surface CD152 after the activation of T cells by APC and Ag was detectable on proliferating cells with an activated phenotype, implicating that either CD152 had not been engaged to date or that CD152 engagement did not give a strong enough signal to stop T cell activation yet. Therefore, during an optimal T cell response, the CD152-mediated inhibition of early T cell activation is dispensable (5). According to earlier results, CD152 mRNA was detectable in naive CD4+ T cells (19), but now our enhanced surface staining for CD152 performed on naive T cells did not detect CD152 protein. Therefore, we hypothesize that surface CD152 is either very low expressed (<100–200 CD152 molecules per cell) on naive T cells and functional at this expression level during early TCR triggering or that CD152 is quickly and shortly up-regulated after CD4+ T cell activation, at least from some cells, which has been reported for other molecules, such as IL-4 (41, 42, 43, 44).

Our new results deal mainly with the scenario when CD152 takes up its role during ongoing immune responses, when it is expressed on the surface at detectable levels of >100 molecules per cell. We show in this work that in a stimulated primary T cell population at the peak of surface CD152 expression, cross-linking of CD152 in addition to CD3 and CD28 stopped or reduced the cell cycle progression. Also, when highly activated, proliferating T cell populations were separated according to surface CD152 expression and restimulated, the CD152-expressing cells did not divide even once, while all CD152- cells went through at least one more cell cycle. Thus, CD152-mediated inhibition of cell cycling is strictly and exclusively confined to surface CD152-expressing and stained cells. The inhibition of proliferation was mediated by CD152 engagement during restimulation of the CD152+ T cells, as shown by CD152 blockade with Fab (Fig. 7). No difference in the proliferative response was seen when CD152 was blocked in isolated, restimulated CD152- T cells. The fact that activated T cells express CD152 heterogeneously during an optimal immune response raises the possibility that these cells might also have heterogeneous fates. Whether or not surface CD152-expressing cells represent a distinct pool of memory T cells remains to be shown.


   Acknowledgments
 
We thank G.-R. Burmester for his support and A. Scheffold for technical advice. We also thank J. P. Allison for kindly providing our laboratory with reagents.


   Footnotes
 
1 This study was supported by the Deutsche Forschungsgemeinschaft (Br 1860/3-2), the Gemeinnützige Hertiestiftung (GHZ 191/00/15), and a grant of the Bundesministerium für Bildung und Forschung to M.C.B.-W. Back

2 Address correspondence and reprint requests to Dr. Monika C. Brunner-Weinzierl, Molecular Immunology, Deutsches Rheuma-Forschungszentrum Berlin, Schumannstrasse 21/22; 10117 Berlin, Germany. E-mail address: brunner{at}drfz.de Back

3 Abbreviations used in this paper: tg, transgenic; Dig, digoxigenin. Back

Received for publication June 19, 2003. Accepted for publication July 22, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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