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The Journal of Immunology, 1998, 160: 12-15.
Copyright © 1998 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: CTLA-4 Ligation Delivers a Unique Signal to Resting Human CD4 T Cells That Inhibits Interleukin-2 Secretion but Allows Bcl-XL Induction1

Patrick J. Blair*, James L. Riley{dagger}, Bruce L. Levine{ddagger}, Kelvin P. Lee*, Nancy Craighead*, Tara Francomano{ddagger}, Steven J. Perfetto{ddagger}, Gary S. Gray§, Beatriz M. Carreno§ and Carl H. June2,{ddagger}

* Naval Medical Research Institute, Bethesda, MD 20889; {dagger} Walter Reed Army Institute for Research, Rockville, MD 20850; {ddagger} Henry M. Jackson Foundation for the Advancement of Military Medicine, U.S. Military HIV Research Program, Bethesda, MD 20889; and § Genetics Institute, Inc., Cambridge, MA 02140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We have assessed the functional effects of a panel of CTLA-4 mAbs on resting human CD4+ T cells. Our results demonstrate that some CTLA-4 mAbs can inhibit proliferative responses of resting CD4+ cells and cell cycle transition from G0 to G1. The inhibitory effects of CTLA-4 were evident within 4 h, at a time when cell surface CTLA-4 expression remained undetectable. Other CTLA-4 mAbs had no detectable inhibitory effects, indicating that binding of Ab to CTLA-4 alone is not sufficient to mediate down-regulation of T cell responses. Interestingly, while IL-2 production was shut off, inhibitory anti-CTLA-4 mAbs permitted induction and expression of the cell survival gene bcl-XL. Consistent with this observation, cells remained viable and apoptosis was not detected after CTLA-4 ligation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
CTLA-4 is a counter-regulatory relative of CD28 that delivers an opposing signal (1, 2). CTLA-4 and CD28 bind to both B7 ligands with low affinity but with rapid on and off rates (3). CTLA-4 mRNA expression is confined to the CD28+ subset of T cells, and mRNA is detected within 1 h after T cell activation (4, 5). Cell surface expression of CTLA-4 is not detectable on resting T cells and peaks 2 to 3 days after activation (6, 7). Previously, it has been shown that anti-CTLA-4 mAb plus anti-TCR mAb and suboptimal anti-CD28 mAb augmented proliferation in CD4+ T cell blasts (6). However, other evidence suggests that CTLA-4 activation opposes CD28-mediated costimulation and, in fact, may have a dominant negative regulatory role (7).

We have analyzed the functional effects of a panel of CTLA-4 mAbs coimmobilized with anti-CD3 and anti-CD28 mAb on resting primary human CD4+ T cells. Our results demonstrate that while some CTLA-4 mAbs blocked T cell activation, others had no effect. This CTLA-4-mediated inhibition induces a novel phenotype during the first 4 h of activation whereby IL-2 induction is prevented while enhancement of the cell survival gene bcl-XL is preserved.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Antibodies and cells

Antibodies used to stimulate cells included: the anti-CTLA-4 mAbs ER5.3D8 and ER5.3D6 (8); humanized anti-CD3 OKT3 (kindly provided by Dr. Jeffrey Bluestone); and anti-CD28 9.3 (9); anti-monomorphic HLA class I mAb W6/32 and anti-glycophorin A 10FTMC served as controls. Freshly isolated PBL were isolated from healthy donors, and CD4+ T cells were purified by negative selection (10). Mean cell volume (fl) was determined using a Coulter Counter Channelyzer (Coulter, Miami, FL).

Bead preparation

Anti-CD3, anti-CD28, and anti-CTLA-4 mAb were covalently attached to polyurethan-coated tosyl-activated Dynabeads (Dynal, Lake Success, NY) per manufacturer’s instructions (11) (bead-cell ratio, 1:1). Beads were prepared with a constant amount of anti-CD3 Ab that represented 5% of the total protein bound and a titration of anti-CD28 and anti-CTLA-4 or control mAb to make up the remaining 95%. Ab-coated beads were tested at anti-CD28-CTLA-4 ratios of 10:0, 9:1, 5:5, 3:7, 1:9, and 0:10. Ab loading was verified by staining beads with pretitered amounts of Abs against their specific isotype.

Proliferation and cytokine ELISA assays

Purified CD4+ T cells were cultured in 96-well plates at a density of 2 x 105 cells. Cultures were pulsed with 1 µCi of [3H]TdR for 18 h before harvest. IL-2 was assayed in supernatants by ELISA (Endogen, Inc., Cambridge, MA).

Cell cycle and apoptosis analysis

Bivariate analysis of cellular RNA and DNA content was performed by staining cells with the RNA-specific fluorochrome pyronin Y and the DNA-specific fluorochrome Hoechst 33342. Regions were determined using unstimulated T cells (12). Apoptosis was analyzed using a modified TUNEL-based (Tdt-mediated dUTP-biotin Nick end labeling) procedure (13).

Immunoprecipitation and Western blotting

Antibodies for immunoprecipitating Bcl-X (rabbit anti-Bcl-X polyclonal serum (14)) and Western blotting (ascites from the anti-human Bcl-X mAb, 2A1) were a kind gift from Dr. Craig Thompson. Cells were harvested after 72 h of stimulation and 1 x 107 cells lysed, immunoprecipitated, and Western blotted (13).

PCR-based liquid hybridization assay

We developed quantitative assays to measure steady state mRNA levels of IL-2 and bcl-XL. cDNAs were prepared from total RNA. Twofold dilutions of the RT product were amplified with the following primers to ensure that the PCR reactions were performed in a linear range:

bcl-XL: (S)-5'-GCTCCACATCACCCCAGGGACAGCA-3'

(AS)-3'-GTAGAGTGGATGGTCAGTGTCTGGT-3'

IL-2: (S)-5'-CAACTCCTGTCTTGCATTGC-3'

(AS)-5'-TTCTGTGGCCTTCTTGGG-3'

Liquid hybridization of the PCR products was then performed (15). Briefly, 200,000 counts of the following labeled oligonucleotides were added to 30 µl of each PCR reaction:

Bcl-xL: 5'-TACTTTTGTGGAACTCTATGGGAAC-3'

IL-2: 5'-ACAAGAATCCCAAACTCACCAGG-3'

Following hybridization, samples were separated on a 6% acrylamide gel and then exposed to a phosphorimager screen for 2 h.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
With a single exception (16), previous work with mouse T cells has shown that CTLA-4 opposes the costimulatory effects of CD28 (1, 17, 18, 19, 20, 21). In contrast, reports on human CTLA-4 function are more limited, and most indicate that CTLA-4 enhances costimulation (6, 22, 23). One study indicates that anti-human CTLA-4 mAbs can induce Ag-specific clonal deletion (8). To address this paradox, a panel of five anti-CTLA-4 mAbs was tested on human CD4 cells, with 3D6 and 3D8 chosen for detailed analysis.

We measured proliferation of human CD4+ T cells following stimulation using beads coated with constant amounts of anti-CD3 and varying amounts of anti-CD28/CTLA-4 mAbs (Fig. 1Go). Stimulation of CD4+ cells with the control anti-CD3/28/MHC I or anti-CD3/28/glycophorin A beads led to robust responses similar to those of anti-CD3/28 alone. Very little proliferation was evident when cultures were stimulated with coimmobilized anti-CD3 and anti-CTLA-4 mAbs alone. Increasing the ratio of anti-CTLA-4-anti-CD28 mAb led to markedly decreased proliferation in some of the cultures, most notably with the anti-CTLA-4 mAb 3D6. Inhibition was maintained in cultures cultured for up to 7 days (data not shown). Additionally, expression of CD25 IL-2R and CD69 were greatly diminished (data not shown). Inhibition was dependent on the ratio of anti-CD28 mAb to 3D6, implying that increasing levels of CD28 activation can circumvent inhibitory effects generated by CTLA-4 ligation. In contrast, beads containing the anti-CD3/CD28/CTLA-4 Ab 3D8 were not inhibitory. For all subsequent experiments, beads coated with anti-CD28/CTLA-4 at a ratio of 1:9 were used to explore the mechanism of inhibition by CTLA-4.



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  		FIGURE 1. Distinct effects of CTLA-4 on T cell proliferation. Resting human CD4+ T cells (2 x 105) were stimulated with anti-CD3/28/CTLA-4 or anti-CD3/28/MHC I-coated Ab beads at 1 bead/cell. The ratio of CD28 to CTLA-4 or CD28 to MHC is shown at the left. Cells cultured at a 0:10 ratio are denoted by hatched bars, at 1:9 by filled bars, and at 3:7 by unfilled bars. Cultures were pulsed following 54 h of culture and then harvested after an additional 18 h. Right, IL-2 concentration (pg/ml) measured in culture supernatants after 24 h by ELISA. Data represent the mean ± SEM from three separate experiments using cells from different donors.

 
Previous studies in the mouse showed that anti-CTLA-4 prevented IL-2 secretion and did not affect T cell viability (19). To examine the mechanism of CTLA-4-mediated inhibition of T cell activation in the absence of cell death, we assessed whether CTLA-4 might affect cytokine secretion and bcl-XL expression. IL-2 and bcl-XL are regulated in part by CD28 (24, 25). Perturbation of these genes could account for the observed phenotype induced by CTLA-4 ligation. Therefore, we measured IL-2 in supernatants from cultures of CD4+ T cells stimulated with Ab-coated beads (Fig. 1Go). Cultures stimulated with anti-CD3/28 and the control anti-MHC I or 3D8 Ab secreted moderate amounts of IL-2. In contrast, cross-linking CD4+ T cells with 3D6 strongly inhibited IL-2 production.

We assayed IL-2 mRNA levels using a semiquantitative RT-PCR-based assay. A profound inhibition of IL-2 mRNA expression was induced by ligation with the anti-CTLA-4 mAb 3D6 within 4 h of culture (Fig. 2GoA). Samples that were stimulated with anti-CD3/28 and 3D6 had roughly 35-fold less IL-2 mRNA than samples stimulated with the noninhibitory anti-CTLA-4 3D8 or MHC I mAb. Our results extend the inhibitory effects of CTLA-4 to a much earlier phase of T cell activation than was previously identified (19). These results are important since we and others (6) have been unable to demonstrate surface CTLA-4 expression on resting T cells, although we have found induction of CTLA-4 mRNA in T cells within 1 h after stimulation by anti-CD3/28 (4). Thus, despite low levels of surface expression, CTLA-4 ligation results in potent inhibition. The ability of CTLA-4 to inhibit early IL-2 induction is consistent with the proposed role of CTLA-4 for induction of CD4 T cell anergy (26). As has been previously reported for mouse T cells (19), inhibition of proliferation by inhibitory anti-CTLA-4 Ab can be partially reversed by addition of exogenous IL-2 (data not shown).



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  		FIGURE 2. Distinct effects of CTLA-4 ligation on IL-2 and bcl-XL expression during early T cell activation. (A) CD4+ cells were cultured with Ab-coated beads containing constant amounts of anti-CD3 mAb and anti-CD28/CTLA-4 or CD28/MHC I at a 1:9 ratio. IL-2, and Bcl-XL mRNA expression was measured at 4 h. Semiquantitative RT-PCR utilizing liquid hybridization was performed as described in Materials and Methods. Samples that lacked reverse transcriptase (NO) were included for each condition to exclude contamination with genomic DNA and dilutions of RT products in microliters (2.5, 5, 10) are shown above the lanes. Samples were equilibrated on rRNA levels. B, Results at 48 h following stimulation with anti-CD3 and anti-MHC I or CTLA-4 mAbs alone. C, Induction of Bcl-XL in response to costimulation by anti-CD3/28/glycophorin A, CD3/28/CTLA4 3D8, and CD3/28/CTLA4 3D6. The anti-CD28-CTLA-4 ratio of the beads in this experiment was 1:9. CD4 T cells were stimulated for 72 h as indicated, and extracts from 1 x 107 cells per sample were subjected to sequential immunoprecipitation and immunoblotting. The position of Bcl-XL (29 kDa) is indicated. The percent of apoptosis in cultured CD4+ T cells as determined by TUNEL staining is noted below each sample. CD4+ T cells that had been irradiated with 100 Gy {gamma}-irradiation 24 h previously served as positive controls for the assay. Apoptosis values shown represent the mean of three separate experiments.

 
It is possible that the lack of proliferation noted in cultures with 3D6 results from increased cell death. Others have reported that 3D8 and 3D6 mAbs mediate Ag-specific apoptosis of T cell blasts or T cell clones (8). Since this would be inconsistent with the proposed role for CTLA-4 in anergy, we tested the effect of CTLA-4 engagement on bcl-XL induction early after T cell activation (Fig. 2Go, A and B). Resting T cells had low levels of bcl-XL mRNA, which is substantially enhanced following stimulation with anti-CD3/28 alone or anti-CD3/28/MHC I. A similar amount of bcl-XL was present in cultures stimulated with anti-CD3/28 and either inhibitory (3D6) or noninhibitory (3D8) CTLA-4 mAb. No induction of bcl-XL was evident even after 48 h, when samples were stimulated under conditions that did not include anti-CD28 mAbs (Fig. 2GoB). Further, the addition of beads containing anti-CD3/28 and inhibitory anti-CTLA-4 mAb did not inhibit bcl-XL induction or proliferative responses in CD3/28-stimulated CD4+ T cells (data not shown). These data suggest that cross-linking CTLA-4 has no direct effect on bcl-XL induction in the absence of CD28 and support the dominant role that CD28 exhibits within our system. Finally, immunoprecipitation and Western analysis (Fig. 2GoC) of cultures stimulated with anti-CD3/28/glycophorin A, anti-CD3/28/CTLA4 3D8, or anti-CD3/28/CTLA4 3D6 all induced similar amounts of Bcl-XL. No Bcl-XL was induced in lysates from unstimulated cells. Thus, cells stimulated with anti-CD3/28 under conditions that induce IL-2 expression or stimulated with anti-CD3/28/CTLA-4 to block IL-2 expression express similar amounts of bcl-XL at both the mRNA and protein levels. Additionally, the anti-CTLA-4 mAb 3D6 did not induce apoptosis via a bcl-XL-independent pathway inasmuch as in all experimental samples <4% were apoptotic by TUNEL analysis (Fig. 2CGo, bottom). These data suggest that the pathways regulating IL-2 production and bcl-XL expression are differentially influenced by a CTLA-4-induced block.

Previous studies have shown that CTLA-4 mAbs induce a G-S block in cell cycle progression but did not permit discrimination between a G0 and G1 cell cycle arrest (19). To further characterize the mechanism underlying the pronounced decrease in T cell proliferation and IL-2 secretion noted in cultures stimulated with anti-CD3/28 and 3D6, we measured mean cell volume as T cells progressed through the cell cycle (Fig. 3Go). We found that anti-CD3/28-stimulated CD4 T cells had an increased cell volume 4 h after stimulation with a significant increase at 24 h as cells entered S phase (Fig. 3Go). In marked contrast, anti-CD3/28/CTLA-4 3D6-stimulated cells did not increase cell volume for the first 12 h of culture. Abrogation of this marker of T cell activation is consistent with the timing of the block in IL-2 mRNA induction previously observed. In contrast, most cells stimulated with CD3/28/CTLA-4 3D6 mAb remained in G0 as seen in Fig. 4Go. Bivariate plots of the cell cycle data are consistent with the induction of exponential proliferation in the anti-CD3/28 or CD3/28/CTLA-4 3D8-stimulated cultures, with only 10 to 14% of cells remaining in G0 phase of the cell cycle after a 72-h culture. Together, our data indicate that some CTLA-4 mAbs deliver a potent signal that induces a cell cycle arrest in G0 and prevents the induction of IL-2 secretion, while preserving the induction of the cell survival gene bcl-XL.



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  		FIGURE 3. Kinetics of cell sizing after activation with beads coated with anti-CD3/28 or anti-CD3/28/CTLA-4 3D6 mAb. Cell volume was measured using a Coulter Channelyzer and 256 channel histograms displayed. The anti-CD28-CTLA-4 ratio of the beads in this experiment was 1:9. Note that the gaussian shape of the histogram after 24 h of CD3/28 stimulation is consistent with a modest size increase that occurred in all cells. In contrast, anti-CD3/28/3D6 stimulated cells exhibited minimal size increase during the initial 24 h following stimulation. No increase in cell size was noted in cells cultured in medium alone (data not shown).

 


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  		FIGURE 4. CTLA-4 ligation by mAb 3D6 induces a cell cycle arrest at the G0 to G1. transition in CD4+ T cells during the first 4 h of culture. After 72 h of culture in medium alone, or following stimulation with anti-CD3/28 or anti-CD3/28 and the anti-CTLA-4 mAbs 3D6 or 3D8, CD4+ cells were harvested and subjected to simultaneous analysis of cellular RNA and DNA content by staining with pyronin Y and Hoechst 33342 as described in Materials and Methods. The percentage of cells in G0 is indicated for each condition at the bottom of each panel. Data shown are representative of four separate experiments.

 
In this study, we examined whether TCR, CD28, and CTLA-4 interactions have a specialized role in the regulation of cell cycle progression and T cell-proliferative responses of CD4 T cells. The results presented here are the first to demonstrate potent inhibitory effects of CTLA-4 on resting human T cells. CTLA-4-mediated inhibition is uniquely capable of regulating T cell activation and survival in that it can prevent IL-2 induction while not interfering with CD28-mediated bcl-XL induction, suggesting that CTLA-4 inhibits a subset of TCR and CD28-mediated activation signals. Together, our results suggest that the unique role of CTLA-4 signaling is not merely to down-regulate T cell responses late after activation during the time of clonal contraction but also to regulate the threshold of initial T cell activation. In this regard, our data tend to support the notion proposed by Allison and coworkers that a major function of CTLA-4 may be to set the threshold for activation above the level encountered by T cells as a result of the intrinsic affinity of self MHC (27). It is also possible that CTLA-4 has a role in the later down-regulation of ongoing immune responses and this would be consistent with the observed kinetics of CTLA-4 expression that peak 2 to 3 days after activation in vitro. Indeed, these two potential functions of CTLA-4 are not mutually exclusive. Clearly, understanding the CTLA-4 pathway will lead to insights for novel approaches for use in immunoregulation.


   Acknowledgments
 
We thank Dr. Richard Hodes for constructive comments and Gil McCrary, David Ritchey, and Amy Mosquera for excellent technical and administrative assistance. We are grateful for the help of Jason Jussif, Agnes Ciarletta and the Antibody Technology Group at Genetics Institute for preparation and characterization of the anti-CTLA4 Abs. We thank Julio Cotte and the staff at the National Naval Medical Center Blood Bank for their assistance in drawing blood.


   Footnotes
 
1 Supported by the Naval Medical Research and Development Command and by Army Contract DAMD17-93-V-3004. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Army, Department of Defense or the U.S. government. Back

2 Address correspondence and reprint requests to Dr. Carl H. June, Immune Cell Biology Program (061), Naval Medical Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5607. E-mail address: Back

Received for publication September 12, 1997. Accepted for publication October 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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