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Originally published online as doi:10.1189/jlb.0706474 on October 6, 2006

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(Journal of Leukocyte Biology. 2007;81:161-167.)
© 2007 by Society for Leukocyte Biology

Regulatory T cell-mediated suppression: potential role of ICER

Josef Bodor*,1, Zoltan Fehervari{dagger}, Betty Diamond* and Shimon Sakaguchi{ddagger}

* Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA;
{dagger} Department of Pathology, University of Cambridge, Cambridge, UK; and
{ddagger} Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

1Correspondence: Department of Medicine, Columbia University, College of Physicians and Surgeons, 1130 St. Nicholas Ave., New York, NY 10032, USA. E-mail: jb2562{at}columbia.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 CTLA-4: OUTSIDE-IN "REVERSE"...
 ICER-MEDIATED UNCOUPLING OF CBP:...
 MECHANISMS OF SUPPRESSION IN...
 REFERENCES
 
How regulatory T (TR) cells dampen T cell responses remains unclear. Multiple modes of action have been proposed, including cell contact-dependent and/or cytokine-dependent mechanisms. Suppression may involve direct contact between TR cells and responder T cells. Alternatively, TR cells may act on dendritic cells to reduce their ability to prime T cells by modulating costimulation, inducing the secretion of suppressive cytokines or the increase of tryptophan metabolism. Here, we review emerging, novel mechanisms involved in contact-dependent, TR-mediated suppression of IL-2 production in responder CD25– T lymphocytes and the potential involvement of inducible cAMP early repressor (ICER) in this suppression. Finally, cytokines such as TGF-ß and IL-10, produced by TR cells or other cells, may exert local suppression, which can be conveyed by basic mechanism(s) acting in a similar manner as contact-dependent, TR-mediated suppression.

Key Words: transcriptional repressor • inhibitory receptor • immune regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
CTLA-4: OUTSIDE-IN "REVERSE"...
 ICER-MEDIATED UNCOUPLING OF CBP:...
 MECHANISMS OF SUPPRESSION IN...
 REFERENCES
 
It is now firmly established that T cell-mediated regulation of immune responses is essential for maintaining tolerance to self-antigens and foreign antigens, and among other kinds of regulatory T cells, CD25+CD4+ regulatory T (CD25+ TR) cells seem to play a key role [1 , 2 ]. Peripheral tolerance is sustained by several mechanisms such as deletion, anergy, and ignorance [3 ]. In addition to natural CD25+ TR cells, there is compelling evidence for the existence of active mechanisms of tolerance that operate through IL-10-secreting type 1 TR cells [4 ]. It seems clear that without a better understanding of these mechanisms, therapeutic exploitation of CD25+ TR cells will remain elusive. The majority of murine and human in vitro studies has concluded that CD25+ TR cells mediate suppression by a yet unknown cell, contact-dependent mechanism, which is cytokine-independent. Suppression cannot be abrogated by neutralizing TGF-ß or IL-10, and CD25+ TR cells cultured with CD25– responder T cells in a transwell system are unable to suppress the proliferation of the responder cells [5 , 6 ]. It is interesting that human CD25+ TR cells fixed with paraformaldehyde remained suppressive as long as they had been activated prior to fixation [7 , 8 ]. This suggests the involvement of a surface-bound molecule that is up-regulated on CD25+ TR cells upon activation and mediates a suppressive signal to the responder cell. However, no such agent has been identified beyond reasonable doubt, although CTLA-4 has been proposed as a candidate [9 , 10 ]. Another suggested mechanism of cell contact-dependent suppression is by TGF-ß bound to the cell surface of CD25+ TR cells [11 , 12 ]. Aside from IL-10 [13 ], TGF-ß seems to be one of several major mechanisms of peripherally induced suppression, regardless of whether it is in its bound or secreted form [14 ].

Clearly, IL-2 is crucial for the survival of natural CD25+ TR cells [15 ], and CD25 is not merely a marker for their chronically activated state but an essential, functional molecule for TR cells as a component of the high-affinity IL-2 receptor, whether CD25+ TR cells expressing forkhead/winged-helix transcription factor Foxp3 [16 ] are generated in the thymus or in the periphery [17 , 18 ]. Recently, Shevach and colleagues [6 , 19 ] proposed a model in which CD25+ TR cells do not suppress the initial activation of CD25– responder T cells but mediate their suppressive effect reversibly following production of IL-2 by responder T cells, resulting in the expansion of CD25+ TR cells and the induction of their suppressive function. Based on this model, Foxp3+CD25+ TR cells initially require IL-2 to inhibit IL-2 transcription in Foxp3– responder T cells. In contrast with other TR cells that secrete immunoregulatory cytokines such as TGF-ß or IL-10 [4 , 13 ], the in vitro CD25+ TR cell-mediated suppression is not mediated by far-reaching or long-lasting humoral factors but is dependent on cell-to-cell interaction among CD25+ TR cells, CD25– responder T cells, and APC. This review summarizes our current understanding of the mechanisms of TR-mediated suppression of T cell responses in vitro and in preclinical murine models with emphasis on transcriptional attenuation of IL-2 in the responder T cell population.


   CTLA-4: OUTSIDE-IN "REVERSE" SIGNALING THROUGH THE B7 LIGANDS
TOP
 ABSTRACT
 INTRODUCTION
 CTLA-4: OUTSIDE-IN "REVERSE"...
ICER-MEDIATED UNCOUPLING OF CBP:...
 MECHANISMS OF SUPPRESSION IN...
 REFERENCES
 
CTLA-4 is a member of the CD28-B7 Ig superfamily of immune regulatory molecules [20 ]. It shares its two ligands, B7.1 and B7.2 (CD80, CD86), with its costimulatory counterpart CD28. It is interesting that the only cells in naïve animals or human cord blood that express CTLA-4 in the absence of activation are CD25+ TR cells [21 ]. It is important that CTLA-4 has significantly higher affinities for both B7 ligands than does CD28 [22 ], and its interaction with B7.1 is of higher affinity than that of B7.2, whereas CD28 is predicted to bind to B7.2 more effectively than B7.1 [23 ]. In addition, the structure of cocrystals of CTLA-4 and B7.1 suggests that these molecules might form extended, lattice-like networks, as a result of the distal positioning of CTLA-4-binding sites from the B7.1 dimer interface [24 ]. This is in striking contrast with a likely monovalent interaction between CD28 and B7.2 [23 ]. Such features probably have significance for one of the mechanisms by which CTLA-4 inhibits CD28-mediated signaling and might exclude CD28 from the immunological synapse and out-compete it for the shared ligands, indicating the importance of the tight regulation of CTLA-4 expression on the cell surface [25 ]. Although CTLA-4 undoubtedly has a cell-autonomous role in controlling helper and effector T cell function, an additional, cell-nonautonomous role in the activity of TR cell populations, which express high levels of CTLA-4, remains more controversial. Several groups have investigated the involvement of CTLA-4 in the suppressive mechanism. It is interesting that signaling through the second messenger, cyclic AMP (cAMP), induces up-regulation of CTLA-4 in CD4+ T lymphocytes [26 ]. It is important that cAMP signaling conveyed via G protein-coupled receptor 83 in naïve CD25– responder T cells may lead to the induction of Foxp3+ TR cells in vivo [27 , 28 ]. Moreover, cAMP-elevating agonists, such as PGE2, can also induce Foxp3 gene expression during peripheral conversion to the regulatory phenotype [29 , 30 ]. The addition of CTLA-4 Fab fragments to in vitro cocultures of murine CD25+ TR cells and CD25– responder T cells neutralizes the inhibitory effect [9 ], and administration of CTLA-4 antibody abolished the protective ability of CD25+ TR cells in murine inflammatory bowel disease [10 ]. TR-mediated CTLA-4 engagement of B7 expressed on APC has been suggested to result in the induction of indoleamine 2,3-dioxygenase (IDO), which in turn leads to immune suppression as a consequence of tryptophan depletion and production of proapoptotic metabolites [31 ]. However, IDO does not seem to be an exclusive mechanism of suppression, as TR-mediated suppression can be achieved, even in the absence of APC, at least in vitro [32 ]. Additional evidence supports the possibility of a similar, direct effect on T cells, which can up-regulate B7.1 and B7.2 following TCR activation [33 ]. It is believed that these cells, in particular, naturally occurring Foxp3+CD25+ TR cells, use interaction based on CTLA-4 binding to B7 ligands expressed on activated T cells, resulting in outside-in reverse signaling into the ligand-bearing Foxp3– responder T cell [34 , 35 ]. These data are consistent with the putative model of reverse signaling via CTLA-4-induced inhibition of IL-2 as a mechanism of TR suppression [36 ]. Upon TCR activation, CTLA-4 is deployed to the surface of TR cells, and high-affinity CTLA-4 interaction with B7 expressed in Foxp3– responder T cells can induce expression of a potent transcriptional inhibitor of IL-2 transcription, inducible cAMP early repressor (ICER; Fig. 1 ) [37 ]. After 2–4 h of delay necessary for ICER protein synthesis [38 , 39 ], ICER attenuates the initial IL-2 expression in responder T cells [37 , 40 , 41 ]. Next, TCR-activated responder T cells induce CTLA-4 and deploy it to the surface [42 ]. CTLA-4 in turn engages neighboring, activated T cell responders expressing B7 via reverse signaling of CTLA-4 [9 , 35 ]. This interaction may lead to the induction of ICER, also in the neighboring Foxp3– responders, thus amplifying the original TR-suppressive signal. Therefore, the first suppressed responder T cell induces CTLA-4, B7, and ICER simultaneously, mimicking a TR cell in its ability to induce suppression via CTLA-4/B7 engagement in adjacent, activated responder T cells in an infectious manner, leading thus to "processive," ICER-mediated transcriptional attenuation of IL-2 expression. In addition, CTLA-4/B7 engagement can activate autonomous CTLA-4 signaling inhibiting extracellular signal-related kinases (ERK1 and ERK2) [43 ], which may protect induced ICER from ERK-mediated phosphorylation and degradation by subsequent ubiquitination [44 , 45 ]. However, at the same time, lack of IL-2 production in T cell responders will eventually diminish the suppressive activity of TR cells conveyed through the CD25 receptor, allowing escape from suppression, leading thus to another round of adjustment of self versus nonself discrimination [15 , 46 , 47 ]. This contact-dependent, infectious tolerance acting through ICER-mediated inhibition of IL-2 synthesis may explain how a relatively small population of TR cells can effectively suppress a much larger population of Foxp3– responders and how inducible CTLA-4 and B7 expression on the surface of activated responder T cells contributes to this infectious suppression conveyed by ICER in an antigen-nonspecific manner. At the same time, TR cells may act back on immature dendritic cells to block the up-regulation of B7 expression [48 ]. Thereby, synergy between reverse and autonomous CTLA-4 signaling may lead in parallel to induction and protection of ICER in TR cells, along with stabilization of inhibitory ICER function in a suppressed population of responder T cells and thus, be crucial for maintenance of a suppressed phenotype (Fig. 1) .


Figure 1
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  		Figure 1. Putative model of reverse signaling. CTLA-4-induced amplification of ICER-mediated "infectious" inhibition of IL-2 expression in Foxp3-negative CD25– responder T cells as a mechanism of TR suppression. Upon TCR activation, CTLA-4 is deployed to the surface of TR cells, and a high-affinity CTLA-4/B7 interaction leads to the immediate, early induction of ICER in TCR-activated Foxp3– responder T cells. The initial IL-2 expression, induced by TCR activation in the Foxp3–responder T cell, is subsequently attenuated by ICER after 2–4 h of delay, necessary for ICER synthesis. Next, TCR-activated Foxp3– responder T cells induce CTLA-4 and B7, which in turn engage with neighboring, activated T cell responders via CTLA-4/B7 interaction, thus amplifying the original TR-suppressive signal generated by reverse signaling on the next neighboring TCR-activated Foxp3– responders. Thus, the first suppressed Foxp3– responder mimicks TR cells in its ability to induce ICER in the next neighboring, activated Foxp3– responders via CTLA-4/B7 interaction in an infectious manner, leading to processive, ICER-mediated transcriptional attenuation of IL-2 expression. Furthermore, in the presence of ICER (whether a result of direct, intracellular Foxp3 expression in TR cells or in suppressed T cell responders), autonomous CTLA-4 signaling inhibits ERK and thus protects ICER from ERK-mediated phosphorylation and subsequent ubiquitination. In this model, TR cells may modulate activity of autoreactive T cells and/or APC through high-affinity CTLA-4/B7 and Class II–TCR interactions.

 
In a recent study, TR cells were preactivated in the presence of anti-CTLA-4 Fab fragments before they have been added to cultures with CD25– responders [49 ]. This experiment showed that TR cells were fully competent suppressors, despite blocking of CTLA-4. In light of the model presented above, these results could be resolved by the existence of compensatory mechanisms using TGF-ß-mediated suppression [50 ], which is thought to be mediated through TGF-ß-induced Foxp3 expression in responder T cells, circumventing CTLA-4 blockade by subsequent conversion of Foxp3– responders to adaptive Foxp3+CD25+ TR cells with a regulatory phenotype [50 ]. Such notion is supported further by observations that TGF-ß requires autonomous CTLA-4 signaling early after T cell activation to induce Foxp3 and generate adaptive CD4+CD25+ TR cells [51 ]. Moreover, TGF-ß cannot induce CD25– responders from CTLA-4-deficient mice to express normal levels of Foxp3 or to develop suppressor activity [51 ]. TGF-ß also enhanced CD4+ cell expression of B7.1. Finally, these observations suggest that CD25+ TR cells from CTLA-4-deficient mice may suppress responder T cells via a compensatory mechanism using TGF-ß-mediated Foxp3 induction in responders, thus acquiring regulatory function outside of an "original" TR population [50 ]. Therefore, two seemingly exclusive mechanisms of suppression (i.e., CTLA-4 vs. TGF-ß) could in fact be reconciled by taking into consideration a novel, unifying mechanism implicating ICER in uncoupling of CREB-binding protein (CBP/p300) suggested below.


   ICER-MEDIATED UNCOUPLING OF CBP: A CRITICAL INHIBITORY CHECKPOINT LIMITING TGF-ß-INDUCED Foxp3-MEDIATED CONVERSION TO ADAPTIVE TR CELL
TOP
 ABSTRACT
 INTRODUCTION
 CTLA-4: OUTSIDE-IN "REVERSE"...
 ICER-MEDIATED UNCOUPLING OF CBP:...
MECHANISMS OF SUPPRESSION IN...
 REFERENCES
 
ICER is an inducible, dominant-negative regulator of the CREB protein and cAMP-responsive element (CRE) modulator family of transcription factors [52 ]. As ICER does not possess a transactivation domain required for the recruitment of CBP/p300, the binding of ICER to CRE-like DNA motifs leads to competition with constitutively expressed CREB protein as a result of highly homologous DNA binding through basic leucine zipper (bZIP) domains [52 ]. Thereby, robust ICER induction leads to ICER-mediated uncoupling of CBP/p300, which abrogates early stages of transcriptional initiation as a result of the lack of CBP/p300-associated histone acetyltransferase (HAT) activity and a failure to maintain the transcriptionally competent conformation of chromatin [53 ] (Fig. 2 ). Furthermore, in the absence of CBP/p300 interactions, NFAT and NF-{kappa}B are likely to be affected, as their full transcriptional activity is dependent on interaction with CBP/p300 [54 ]. Therefore, ICER can compete with bZIP proteins (e.g., CREB or Jun) [55 ] bound to CRE-like, DNA-binding motifs positioned adjacent to NFAT and NF-{kappa}B binding sites in the context of numerous cytokine and chemokine promoters [56 ], prevent the recruitment of CBP/p300, and thereby, abort cross-talk between Rel- and bZIP-mediated transcription after T cell activation [57 , 58 ]. Furthermore, CBP/p300 serves as an important transcriptional integrator of IL-2 as well as TGF-ß signaling, acting through CBP interaction with STATs and/or Smads [59 , 60 ] (Fig. 2) . Thus, ICER-mediated uncoupling of CBP/p300 may clearly diminish these responses [53 , 61 ], leading to growth arrest while ICER protects cells against cAMP-induced apoptosis [62 ].


Figure 2
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  		Figure 2. Proposed role of ICER in uncoupling of transcriptional integrator CBP, which is a general mediator of signal-dependent transcription in response to various stimuli and associates with a variety of signal-dependent factors including CREB, Jun, STAT, and Smads, enabling TGF-ß signaling. CREB can be phosphorylated on critical Ser 133 by protein kinase A (PKA)-dependent as well as PKA-independent signaling pathways such as one triggered by TCR. Upon phosphorylation of CREB, CBP is recruited to signal-dependent promoters. CBP can accommodate numerous signal transduction pathways mediated by cytokines such as IL-2 (STAT) and TGF-ß (Smads) and is thought to stimulate the expression of target genes through its association with RNA polymerase (Pol II) and through its intrinsic HAT activities (the addition or removal of acetyl groups; Transcription ON). However, if ICER is induced before CBP recruitment, it competes with CREB or Jun for DNA binding. Therefore, ICER may mask CREB and Jun DNA-binding sites and prevent them from recruitment of CBP, as it lacks the transactivation domain, leading thus to extinguishing transcription (Transcription OFF). CDKs, Cyclin-dependent kinases.

 
TGF-ß plays complex roles in carcinogenesis, as it may exert tumor suppressor and pro-oncogenic activities, depending on the stage of the tumor. Smad proteins transduce signals from the TGF-ß receptors to regulate the transcription of specific target genes. Cross-talk with other signaling pathways may contribute to the specificity of TGF-ß effects. Intracellular cAMP-elevating agonists inducing ICER such as forskolin or PGE2 [39 , 57 ] interfere with TGF-ß-mediated, Smad-specific gene transactivation [63 ]. It is important that upon TCR activation, forskolin treatment in TR assays diminishes Foxp3 expression in the CD4+CD25+ population, suggesting that forskolin-induced ICER might be involved in down-regulation of Foxp3 (our unpublished observations). These data suggest that abrogation of TGF-ß/Smad signaling is likely to occur via ICER-mediated uncoupling of Smad-CBP/p300 complexes, yielding thus disengagement from Foxp3 expression (Fig. 3 ). Likewise, {gamma}{delta} T cells constitutively expressing ICER [64 ] are less prone to induce Foxp3 [65 ]. It is important that TGF-ß was identified as a factor involved in induction of Foxp3 expression in CD25– responder T cells (for review, see ref. [66 ]). As cAMP-elevating agonists inducing ICER may eventually prevent TGF-ß-induced, Smad-specific gene activation, we speculate that already suppressed CD25– responder T cells with elevated levels of ICER are unlikely to induce Foxp3 and thus might be resistant to conversion to a TR phenotype (Fig. 3) . These assumptions correspond with our unpublished observations, indicating that upon TCR activation, forkolin treatment decreases Foxp3 expression in the CD25+ T cell population and does not endow the CD25– population with regulatory properties in cocultivation assays, perhaps, by opposing Smad-mediated transcriptional activation of Foxp3. Furthermore, this is consistent with observations indicating that upon TR-mediated suppression, blockade of CTLA-4 prevents ICER induction in T cell responders [37 ]. We propose that ICER, induced upon CTLA-4/B7 engagement, then uncouples CBP, which impairs recruitment of Smads to the Foxp3 promoter (Figs. 1 and 2) . As ICER is expressed in Foxp3– responders with a suppressed phenotype [37 ], ICER-mediated uncoupling of CBP/p300 may also abrogate TGF-ß-mediated transcription of Foxp3 in the suppressed responder population and thus, prevent peripheral conversion to adaptive TR cells (Fig. 3) . Not all suppressed responder T cells express Foxp3, which is well documented by numerous observations, indicating that T cells do not seem to undergo complete conversion to the regulatory phenotype in the periphery, even under highly suppressive conditions (such as could be found in tumor infiltrates) [36 , 67 , 68 ]. It is important that this does not preclude T cells expressing ICER from breaking a "suppressed" phenotype upon CD28-mediated costimulation (Fig. 3) , presumably by ERK-mediated phosphorylation, leading to ubiquitin-mediated degradation of ICER [44 ]. This creates favorable conditions for ICER replacement by ubiquitously present CREB [69 ]. Upon TCR activation, phosphorylation of CREB [70 ] allows for CBP/p300 recruitment and engagement of Smads, and subsequent peripheral induction of Foxp3 enables conversion to the regulatory phenotype in CD3- and CD28-activated human T cells [71 ]. Such a mechanism would be consistent with the effects of CTLA-4-Ig as a potent inhibitor of CD28-mediated T cell costimulation [72 ]. According to the model depicted in Figure 3A , CTLA-4-Ig could uncouple CBP/p300 in response to CD28 costimulation and thus, compromise TGF-ß-mediated peripheral conversion of TR cells by blocking their development and function. These conclusions are supported further by preliminary data, suggesting that CTLA-Ig prevents peripheral conversion of TR cells, even when administered later in life (Anne Davidson, Columbia University, New York, NY, personal communication and ref. [73 ]). Likewise, the CTLA-4 blockade may abrogate CTLA-4-mediated reverse signaling (Fig. 3B) and thus, interfere with TR cell function [36 , 72 , 74 , 75 ].


Figure 3
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  		Figure 3. TR-triggered, CTLA-4 reverse signaling may lead to ICER-mediated uncoupling of CBP, which in an already suppressed responder T cell population, abrogates conversion to TR cells by TGF-ß-responsive Foxp3 expression. (A) CD28 facilitated TGF-ß-mediated conversion of responders to adaptive TR cells. (B) Lack of TGF-ß-mediated conversion in responders suppressed by TR-triggered CTLA-4 signaling inducing ICER may lead to uncoupling of a CBP-mediated TGF-ß transcriptional response, driving Foxp3 expression in T responders.

 

   MECHANISMS OF SUPPRESSION IN VIVO
TOP
 ABSTRACT
 INTRODUCTION
 CTLA-4: OUTSIDE-IN "REVERSE"...
 ICER-MEDIATED UNCOUPLING OF CBP:...
 MECHANISMS OF SUPPRESSION IN...
REFERENCES
 
Investigations in humans have largely confirmed the expression pattern seen in mice, and Foxp3+CD25+ TR cells have been identified in thymus as well as in adult peripheral blood and cord blood [76 77 78 ]. In contrast to the murine studies, human CD25– responder T cells have been reported to express Foxp3 and to acquire suppressive ability after stimulation in vitro with anti-CD3 and anti-CD28 antibodies [71 ]. Of note, TGF-ß has been shown to induce Foxp3 expression in murine and human CD25– responder T cells [79 ]. There are recent reports indicating that suppressive properties of human TR cells are, at least in part, dependent on CTLA-4 expression [80 ]. These data are consistent with observations that antigen exposure during enhanced CTLA-4 expression promotes allograft tolerance in vivo [81 ]. It is important that CD28 disruption exacerbates inflammation in TGF-ß-deficient mice, indicating that in vivo suppression by TR cells is independent of autocrine TGF-ß1 [82 ].

Costimulation and TGF-ß might be a key for development of suppressive function in peripheral TR cells. Therefore, we believe that improved understanding of a mechanism of peripheral conversion of CD25– responder T cells to Foxp3+CD25+ TR cells, linking CD28-mediated costimulation with TGF-ß-induced expression of Foxp3, could be critically important for our ability to enhance therapeutic strategies using TR cells. It is not surprising that tissue-specific factors, such as local cytokine environment, presence of prostaglandins, or hormones, might be important to optimize the suppressive response of TR cells. Suppression by natural TR cells along with peripheral conversion to a regulatory phenotype in human CD25– responder T cells may thus represent a critical mechanism of action.

Overall, these data indicate that contact-dependent and cytokine-mediated suppression may share similar underlying mechanisms, which are likely to exhibit general features of transcription machinery such as ICER-mediated uncoupling of CBP/p300. In addition, these mechanisms could clarify the discrepancy in the contact-dependent versus cytokine-mediated suppression observed in vivo and might also explain how a relatively small population of TR cells could impose suppression on a much larger population of responder T cells in a CTLA-4- and ICER-dependent, infectious manner.


   ACKNOWLEDGEMENTS
 
We thank Drs. Hiroyuki Yoshitomi, Keiji Hirota, Harvey Cantor, and Anne Davidson for helpful comments and suggestions. The authors have no conflicting financial interests.

Received July 26, 2006; accepted September 12, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 CTLA-4: OUTSIDE-IN "REVERSE"...
 ICER-MEDIATED UNCOUPLING OF CBP:...
 MECHANISMS OF SUPPRESSION IN...
 REFERENCES
 

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