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Intermediate Affinity Interleukin-2 Receptor Mediates Survival via a Phosphatidylinositol 3-Kinase-dependent Pathway*

  • Ana González-García
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma Campus de Cantoblanco, Cantoblanco, 28049-Madrid, Spain
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  • Isabel Mérida
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma Campus de Cantoblanco, Cantoblanco, 28049-Madrid, Spain
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  • Carlos Martinez-A
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma Campus de Cantoblanco, Cantoblanco, 28049-Madrid, Spain
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  • Ana C. Carrera
    Affiliations
    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma Campus de Cantoblanco, Cantoblanco, 28049-Madrid, Spain
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  • Author Footnotes
    * This work was supported in part by grants from Fundación José Carreras, AICR, Comisión Interminesterial de Ciencia y Tecnología (CICyT), Unión Europea and Pharmacia & Upjohn. The department of Immunology and Oncology was founded and is supported by the CSIC and Pharmacia & Upjohn. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:April 11, 1997DOI:https://doi.org/10.1074/jbc.272.15.10220
      Peripheral blood T lymphocytes require two signals to enter and progress along the cell cycle from their natural quiescent state. The first activation signal is provided by the stimulation through the T cell receptor, which induces the synthesis of cyclins and the expression of the high affinity interleukin-2 receptor. The second signal, required to enter the S phase, is generated upon binding of interleukin-2 to the high affinity αβγ interleukin-2 receptor. However, resting T cells already express intermediate affinity βγ interleukin-2 receptors. As shown here, T cell stimulation through intermediate affinity receptors is capable of inducing cell rescue from the apoptosis suffered in the absence of stimulation. Characterization of the signaling pathways utilized by βγ interleukin-2 receptors in resting T cells, indicated that pp56lck, but not Jak1 or Jak3, is activated upon receptor triggering. Compelling evidence is presented indicating that phosphatidylinositol 3-kinase associates with the intermediate affinity interleukin-2 receptor and is activated upon interleukin-2 addition. Bcl-xL gene was also found to be induced upon βγ interleukin-2 receptor stimulation. Finally, pharmacological inhibition of phosphatidylinositol 3-kinase blocked both interleukin-2-mediated bcl-xL induction and cell survival. We conclude that βγ interleukin-2 receptor mediates T-cell survival via a phosphatidylinositol 3-kinase-dependent pathway, possibly involving pp56lck and bcl-xL as upstream and downstream effectors, respectively.

      INTRODUCTION

      Interleukin-2 (IL-2)
      The abbreviations used are: IL-2
      interleukin-2
      IL-2R
      interleukin-2 receptor
      TCR
      T cell receptor
      PI3K
      phosphatidylinositol 3-kinase
      Abs
      antibodies
      PBL
      peripheral blood lymphocytes
      PAGE
      polyacrylamide gel electrophoresis.
      plays a major role in T cell biology, as activated T cells depend on this cytokine for their proliferation and effector functions. IL-2 effects are mediated through interaction with a specific transmembrane receptor (IL-2R) composed of three different molecules, α, β, and γ (
      • Taniguchi T.
      • Minami Y.
      ). The β and γ chains are constitutively expressed in T lymphocytes and bind IL-2 with intermediate affinity. The α chain, in contrast, is only expressed upon T cell activation and, together with the β and γ chains, forms the high affinity IL-2 receptor (
      • Taniguchi T.
      • Minami Y.
      ). It has been shown that heterodimerization of the IL-2R β and γ chains is required for IL-2-induced cellular proliferation (
      • Nakamura Y.
      • Russell S.M.
      • Mess S.A.
      • Friedmann M.
      • Erdos M.
      • Francois C.
      • Jacques Y.
      • Adelstein S.
      • Leonard W.J.
      ,
      • Nelson B.H.
      • Lord J.D.
      • Greenberg P.D.
      ). The IL-2R has no intrinsic enzymatic activity; therefore, its ability to transmit intracellular signals relies on its association with signaling molecules. IL-2R ligation triggers the activation of several tyrosine kinases, including src-kinases (
      • Hatakeyama M.
      • Kono T.
      • Kobayashi N.
      • Kawahara A.
      • Levin S.D.
      • Perlmutter R.M.
      • Taniguchi T.
      ,
      • Kobayashi N.
      • Kono T.
      • Hatakeyama M.
      • Minami Y.
      • Miyazaki T.
      • Perlmutter R.M.
      • Taniguchi T.
      ), Jak kinases (
      • Johnston J.A.
      • Kawamura M.
      • Kirken R.A.
      • Chen Y.
      • Blake T.B.
      • Shibuya K.
      • Ortaldo J.R.
      • McVicar D.W.
      • O'Shea J.J.
      ,
      • Witthuhn B.A.
      • Silvennoinen O.
      • Miura O.
      • Lai K.S.
      • Cwik C.
      • Liu E.T.
      • Ihle J.N.
      ,
      • Miyazaki T.
      • Kawahara A.
      • Fujii H.
      • Nawaka Y.
      • Minami Y.
      • Liu Z-J.
      • Oishi I.
      • Silvennoinen O.
      • Witthuhn B.A.
      • Ihle J.N.
      • Taniguchi T.
      ), and pp72syk (
      • Minami Y.
      • Nakagawa Y.
      • Kawahara A.
      • Miyazaki T.
      • Sada K.
      • Yamamura H.
      • Taniguchi T.
      ). While Jak kinases are essential for IL-2-induced proliferation, the involvement of src-kinases on T cell proliferation following IL-2R binding is not so clear (
      • Hatakeyama M.
      • Kono T.
      • Kobayashi N.
      • Kawahara A.
      • Levin S.D.
      • Perlmutter R.M.
      • Taniguchi T.
      ). IL-2 also regulates phosphatidylinositol 3-kinase (PI3K) activation (
      • Augustine J.A.
      • Sutor S.L.
      • Abraham R.T.
      ,
      • Merida I.
      • Diez E.
      • Gaulton G.N.
      ,
      • Remillard B.
      • Petrillo R.
      • Maslinski W.
      • Tsudo M.
      • Strom T.B.
      • Cantley L.
      • Varticovski L.
      ) and induces p21ras (
      • Satoh T.
      • Minami Y.
      • Kono T.
      • Yamada K.
      • Kawahara A.
      • Taniguchi T.
      • Kaziro Y.
      ), c-raf (
      • Zmuidzinas A.
      • Mamom H.J.
      • Roberts T.M.
      • Smith K.A.
      ), and pp70S6K (
      • Calvo V.
      • Crews C.M.
      • Vik T.A.
      • Bierer B.E.
      ). After the initial phase mediated by these set of cytoplasmic molecules, several transcription factors including Stat3, Stat5, c-myc, c-fos, and c-jun (
      • Johnston J.A.
      • Bacon C.M.
      • Finbloom D.S.
      • Rees R.C.
      • Kaplan D.
      • Shibuya K.
      • Ortaldo J.R.
      • Gupta S.
      • Chen Y.G.
      • Giri J.D.
      • O'Shea J.J.
      ,
      • Shibuya H.
      • Yoneyama M.
      • Ninomiya-Tsuji J.
      • Matsumoto K.
      • Taniguchi T.
      ) are also induced that putatively control gene expression leading to cell division. All these signaling cascades have been described to be triggered by high affinity IL-2 receptors and are involved in inducing cell proliferation. However, it has been recently shown that the intermediate affinity IL-2 receptor mediates protection against radiation-induced cell death (
      • Boise L.H.
      • Minn A.J.
      • June C.H.
      • Lindsten T.
      • Thompson C.B.
      ,
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ). We confirm here that although unable to promote cell division, the intermediate affinity IL-2R induced cell survival. In fact, IL-2 treatment rescued resting T cells from the spontaneous cell death observed when these cells are cultured without stimulus. We subsequently analyzed the signaling pathways triggered by intermediate affinity IL-2R in resting T cells and found that pp56lck is induced upon βγ IL-2R stimulation. In addition, data is presented indicating that the activation of PI3K is required for IL-2-mediated cell survival and that bcl-xL behaves as a downstream effector of this enzyme.

      DISCUSSION

      The results presented in this report indicate that IL-2 binding to the intermediate affinity IL-2R promotes rescue from apoptosis of resting T cells cultured in vitro in the absence of TCR stimulation. Analysis of the signaling molecules implicated in βγ IL-2R-mediated survival, addressed here for the first time, indicates that pp56lck, but not Jak kinases, is induced upon βγ receptor ligation. We have previously described that pp56lck is required for activation of TCR-associated PI3K (
      • Carrera A.C.
      • Rodriguez-Borlado L.R
      • Martinez-A C.
      • Merida I.
      ) and regulates αβγ IL-2R-induced PI3K activation (
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ). Thus, we have also analyzed whether PI3K was stimulated in this system. We found that PI3K is constitutively associated to βγ IL-2R and became activated in response to IL-2 binding. The fact that pharmacological inhibition of PI3K blocked IL-2-mediated survival strongly suggests that PI3K activation is required for the ability of IL-2 to abrogate cell death. Finally, we also show here that bcl-xL is induced upon βγ IL-2R stimulation (Fig. 8) and that this induction is blocked upon PI3K inhibition (Fig. 8). Together these results allow the proposal of the model depicted in Fig. 9: ligation of intermediate affinity βγ IL-2R induces both pp56lck and PI3K activation. PI3K activity is then required for the induction of bcl-xL, an important mediator of cell survival.
      Figure thumbnail gr9
      Fig. 9Schematic representation of a putative model for IL-2-mediated cell survival in resting T cells. The stimulation of intermediate affinity IL-2R induces pp56lck activation, PI3K induction, and bcl-xL expression. Several observations (see Refs.
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ,
      • Carrera A.C.
      • Rodriguez-Borlado L.R
      • Martinez-A C.
      • Merida I.
      ) suggest that the activation of pp56lck could be involved in inducing PI3K activation. PI3K activation seems to be in turn required for bcl-xL increased expression and for cell survival.
      To the best of our knowledge, no previous publication has characterized the effect of βγ IL-2R ligation on the death of normal T cells in the absence of TCR stimulation. However, at least three previous reports have analyzed the IL-2 involvement in T cell rescue from γ radiation-induced cell death (
      • Boise L.H.
      • Minn A.J.
      • June C.H.
      • Lindsten T.
      • Thompson C.B.
      ,
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ,
      • Mor F.
      • Cohen I.R.
      ). Boise et al. (
      • Boise L.H.
      • Minn A.J.
      • June C.H.
      • Lindsten T.
      • Thompson C.B.
      ) describe that preincubation of normal human peripheral T cells with IL-2, IL-4, or IL-7, but not with IL-1, IL-3, or IL-6, decreases the apoptosis observed in response to γ radiation. Mor and Cohen (
      • Mor F.
      • Cohen I.R.
      ), have reported that IL-2 mediates protection to γ radiation in antigen-specific T cells; however, they do not detect protection in resting T cells. Finally, Seki et al. (
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ) show that CD8+ and NK cells are the most radiosensitive of resting T cells but could be rescued by IL-2 treatment. These observations, together with the description here that IL-2 protects from the cell death suffered by normal T cells when cultured in the absence of TCR stimulation (Fig. 1), support the conclusion that intermediate affinity IL-2R is capable of inducing T cell survival. In addition to this mechanism, others are likely to mediate survival of resting T cells, given that a significant proportion of the resting cells survive in the absence of IL-2 (Fig. 1A).
      We have performed a systematic study of βγ IL-2R-induced signaling pathways. As proposed in the model in Fig. 9, these data suggest that pp56lck is one of the signals involved in IL-2R-mediated survival in resting T cells. This is in agreement with the observation that constitutive active pp56lck mutants decrease the apoptosis induced by growth factor removal (
      • Miyazaki T.
      • Liu Z.J.
      • Kawahara A.
      • Minami Y.
      • Yamada K.
      • Tsujimoto Y.
      • Barsoumian E.L.
      • Perlmutter R.M.
      • Taniguchi T.
      ). In contrast, the involvement of pp56lck on IL-2-induced cell division is controversial, since a mutant of the β chain lacking the acidic region (which does not associate pp56lck) is, however, capable of mediating a proliferative signal (
      • Hatakeyama M.
      • Kono T.
      • Kobayashi N.
      • Kawahara A.
      • Levin S.D.
      • Perlmutter R.M.
      • Taniguchi T.
      ). The other tyrosine kinases involved in high affinity IL-2R signaling are the Jak kinases (
      • Johnston J.A.
      • Kawamura M.
      • Kirken R.A.
      • Chen Y.
      • Blake T.B.
      • Shibuya K.
      • Ortaldo J.R.
      • McVicar D.W.
      • O'Shea J.J.
      ,
      • Witthuhn B.A.
      • Silvennoinen O.
      • Miura O.
      • Lai K.S.
      • Cwik C.
      • Liu E.T.
      • Ihle J.N.
      ,
      • Miyazaki T.
      • Kawahara A.
      • Fujii H.
      • Nawaka Y.
      • Minami Y.
      • Liu Z-J.
      • Oishi I.
      • Silvennoinen O.
      • Witthuhn B.A.
      • Ihle J.N.
      • Taniguchi T.
      ), which have been shown to be required for IL-2-mediated proliferation (
      • Nelson B.H.
      • Lord J.D.
      • Greenberg P.D.
      ,
      • Kawahara A.
      • Minami Y.
      • Miyazaki T.
      • Ihle J.N.
      • Taniguchi T.
      ). The mechanism by which these enzymes are activated entails their phosphorylation on tyrosine residues. The current model proposes that Jak1 and Jak3 transphosphorylate each other (
      • Nelson B.H.
      • Lord J.D.
      • Greenberg P.D.
      ). The low levels of Jak3 expression, and the concomitant lack of Jak1 and Jak3 activation in our system suggest that these proteins do not participate in IL-2-mediated survival. In addition to IL-2-mediated survival, bcl-2 induction by IL-2 in BAF/3 also seems to be independent of Jak3 as bcl-2 up-regulation is not altered by overexpression of a dominant negative Jak3 in these cells (
      • Kawahara A.
      • Minami Y.
      • Miyazaki T.
      • Ihle J.N.
      • Taniguchi T.
      ).
      Our conclusion on PI3K activation involvement in T cell rescue from apoptosis (Fig. 6C) concurs with the implication of PI3K in survival discussed by Ward et al. (
      • Ward S.G.
      • June C.H.
      • Olive D.
      ) for CD28-mediated signals. Moreover, studies in rodent fibroblast (
      • Yao R.
      • Cooper G.M.
      ), in neural growth factor receptor signaling in PC12 cells (
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ), and insulin growth factor receptor signaling in myeloid progenitors cells (
      • Minshall C.
      • Arkins S.
      • Freund G.G.
      • Kelley K.W.
      ), in which activation of PI3K prevents apoptosis, also support this view. The mechanism through which PI3K is activated is not evident. In this regard, several cytokine receptors, such as IL-4R and IL-7R that share the γ chain with the IL-2R (
      • Russell S.M.
      • Keegan A.D.
      • Harada N.
      • Nakamura Y.
      • Noguchi M.
      • Leland P.
      • Friedmann M.C.
      • Miyajima A.
      • Puri R.K.
      • Paul W.E.
      • Leonard W.J.
      ,
      • Noguchi M.
      • Nakamura Y.
      • Russell S.M.
      • Ziegler S.F.
      • Tsang M.
      • Cao X.
      • Leonard W.J.
      ) have been shown to enhance the resistance of resting T cells to radiation-induced cell death as effectively as IL-2 (
      • Boise L.H.
      • Minn A.J.
      • June C.H.
      • Lindsten T.
      • Thompson C.B.
      ,
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ). Studies with the IL-7 receptor show that PI3K activation is mediated by the γ chain and its associated Jak3 tyrosine kinase (
      • Sharfe N.
      • Dadi H.K.
      • Roifman C.M.
      ). However, the low levels of Jak3 expression in our system and the fact that this protein is not up-regulated upon IL-2 treatment makes unlikely the implication of this mechanism in PI3K activation. Another possible mechanism for PI3K activation involves the IL-2R β chain and src-kinases (
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ,
      • Karnitz L.M.
      • Sutor S.L.
      • Abraham R.T.
      ). We previously described that pp56lck activation regulates PI3K activation upon IL-2 triggering of high affinity IL-2R (
      • Taichman R.
      • Merida I.
      • Torigoe T.
      • Gaulton G.N.
      • Reed J.C.
      ). In addition, the serine-rich region required for pp56lck activation is also required for activation of PI3K (
      • Merida I.
      • Williamson P.
      • Kuziel W.A.
      • Greene W.C.
      • Gaulton G.N.
      ). Finally, the fact that pp56lck tyrosine kinase and correlatively PI3K are activated upon IL-2 binding to βγ IL-2R (Fig. 3A) suggests that pp56lck may contribute to induce PI3K activation.
      Bcl-2 and bcl-xL have been shown to mediate survival (
      • Boise L.H.
      • Gottschalk A.R.
      • Quintans J.
      • Thompson C.B.
      ) and to be induced upon high affinity IL-2R ligation (
      • Deng G.
      • Podack E.R.
      ,
      • Miyazaki T.
      • Liu Z.J.
      • Kawahara A.
      • Minami Y.
      • Yamada K.
      • Tsujimoto Y.
      • Barsoumian E.L.
      • Perlmutter R.M.
      • Taniguchi T.
      ,
      • Broome H.E.
      • Dargan C.M.
      • Krajewski S.
      • Reed J.C.
      ,
      • Mor F.
      • Cohen I.R.
      ). We therefore tested the putative involvement of bcl-2 and bcl-xL in IL-2-mediated survival. Induction of bcl-xL/bcl-2 in irradiated cells upon βγ IL-2R ligation has not been previously detected (
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ). We found a slight increase in bcl-2 expression that could suggest a partial role of this protein for IL-2-mediated survival. More clearly, the induction of bcl-xL upon βγ IL-2R stimulation and the fact that blocking IL-2-induced survival with LY294002 also results in abrogation of bcl-xL up-regulation suggest that PI3K activation precedes bcl-xL expression and that bcl-xL is probably involved in βγ IL-2R-induced survival.
      IL-2 mediates three different cellular responses. First, it has been involved in mediating the proliferation of cells preactivated via T cell receptor and expressing αβγ IL-2R (
      • Rebollo A.
      • Gomez J.
      • Martinez-A C.
      ). Second, it has been suggested to mediate survival (Fig. 1) (
      • Boise L.H.
      • Minn A.J.
      • June C.H.
      • Lindsten T.
      • Thompson C.B.
      ,
      • Seki H.
      • Iwai K.
      • Kanegane H.
      • Konno A.
      • Ohta K.
      • Ohta K.
      • Yachie A.
      • Taniguchi N.
      • Miyawaki T.
      ), and finally, it has been recently proposed to program T lymphocytes for apoptosis (
      • Lenardo M.J.
      ). Regarding the dual role of IL-2 at promoting either survival or cell proliferation, we describe here that only a set of signals (pp56lck, PI3K, and bcl-xL) of those triggered for IL-2-induced cell division are involved in mediating cell survival. The reason for high affinity IL-2R to trigger different early signals than that derived from intermediate affinity IL-2R in resting T cells could be the presence in the high affinity IL-2R of the α chain. However, chimeric β and γ IL-2R chains transfected in cell lines lacking the α chain behave as high affinity IL-2R, inducing proliferation (
      • Nakamura Y.
      • Russell S.M.
      • Mess S.A.
      • Friedmann M.
      • Erdos M.
      • Francois C.
      • Jacques Y.
      • Adelstein S.
      • Leonard W.J.
      ,
      • Nelson B.H.
      • Lord J.D.
      • Greenberg P.D.
      ). Therefore, it seems that the lack of IL-2R α chain expression is not responsible for the partial signaling of βγ IL-2R in resting T cells. Instead, other proteins not expressed in resting T cells but expressed in cell lines or induced upon TCR cross-linking in normal T cells could be responsible for the different biological responses following TCR ligation. Candidates for these molecules could be c-myc, Jak3, or c-raf, relatively abundant in activated T cell and cell lines but not detectable in resting T cells (our data not shown) (
      • Zmuidzinas A.
      • Mamom H.J.
      • Roberts T.M.
      • Smith K.A.
      ,
      • Kawamura M.
      • McVicar D.W.
      • Johnston J.A.
      • Blake T.B.
      • Chen Y-Q.
      • Lal B.K.
      • Lloyd A.R
      • Kelvin D.J.
      • Staples J.E.
      • Ortaldo J.R.
      • O'Shea J.J.
      ).
      Regarding the ability of IL-2 to program T lymphocytes for apoptosis, it has been described that incubation of T cells in IL-2 or IL-4 is required for ulterior TCR-mediated cell death (
      • Lenardo M.J.
      ). Moreover, IL-2−/− mice developed splenomegaly and lymphoadenophaty (
      • Sadlack B.
      • Merz H.
      • Schorle H.
      • Schimpl A.
      • Feller
      • A C.
      • Horak I.
      ), similar to IL-2Rβ−/− mice (
      • Suzuki H.
      • Kunding T.M.
      • Furlonger C.
      • Wakeham A.
      • Timms E.
      • Matsuyama T.
      • Schmits R.
      • Simard J.J.L.
      • Ohashi P.S.
      • Griesser H.
      • Taniguchi T.
      • Paige C.J.
      • Mak T.W.
      ). We sought to investigate the signaling pathways responsible for the ability of IL-2 to induce susceptibility to TCR-triggered apoptosis. However, in our hands, the conditions that induce susceptibility to apoptosis also trigger cell division. Thus, we favor the hypothesis that the same intracellular signaling program induces both cellular responses.
      The fact that intermediate affinity IL-2 receptors transmit intracellular signals in resting cells, opens the possibility of a biological role for the βγ IL-2R. The circulating levels of IL-2 are normally low. However, encounter with antigen primes T cells for IL-2 production, locally increasing the concentration of this cytokine. Cells that have not been stimulated by the antigen, and therefore do not express the IL-2R α chain, could use IL-2 to increase their survival potential.

      Acknowledgments

      We thank Drs. M. A. Rodriguez and P. Torres from the Centro de Transfusiones de la Comunidad de Madrid for kindly providing the buffy coats, Drs. A. Bernard and V. Calvo for critical reading of the manuscript, and C. Mark for editorial assistance.

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