doi:10.1182/blood-2008-07-1676192008 112: 4776-4777   

 Rita Casetti, Angelo Martino, Alessandra Sacchi, Chiara Agrati, Delia Goletti and Federico Martini 

protein antigens?M tuberculosis T cells respond to δγDo human

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Correspondence

To the editor:

Do human �� T cells respond to M tuberculosis protein antigens?

Recently Li et al1 reported that circulating �� T cells fromtuberculin skin test (TST)-positive patients respond directly in vitroto early secreted antigenic target 6 (ESAT-6) protein from Mycobac-terium tuberculosis by proliferating and by producing interferon �(IFN-�). We think that these results do not agree with the currentmodel of �� T-cell antigen (Ag) recognition and activation.

Human V�9V�2 �� T cells represent 1% to 5% of circulatinglymphocytes in adults and recognize nonpeptidic phosphoantigens,metabolites of isoprenoid pathway.2,3 Although most potent phos-phoantigens, such as hydroxymethylbutyl-pyrophosphate (HMBP),are produced by infectious agents and recognized as nonself,others, such as isopentenyl pyrophosphate (IPP), are normalmetabolites of mammalian mevalonate pathway, and their accumu-lation represents a danger signal from infected or transformedcells.4 A similar mechanism explains V�9V�2 response to ami-nobisphosphonates5 and alkyl amines.6 Due to small size, phospho-antigens are physically unable to cross-link T-cell receptors (TCRs),and a complex of apolipoprotein A (ApoA) and mitochondrialadenosine triphosphatase (ATPase) was proposed as a presentingmolecule.7 However, the recognition mechanism of human V�9V�2TCR remains incompletely defined. Other major histocompatibilitycomplex (MHC) class I–like molecules may stimulate human ��T cells: CD1c can present foreign lipids and glycolipids to V�1� ��T cells, a subset commonly found in humans in periphery.8

On the other hand, ESAT-6 protein is able to induce IFN-�production by CD4 T lymphocytes from patients with activetuberculosis (TB) and latent TB infection (LTBI).9

To evaluate �� T cells’ response to ESAT-6 protein, we tested4 patients with active TB and 4 subjects with LTBI (positive toQuantiFERON TB Gold and TST). Figure 1 shows representativeresults from an active TB patient (Figure 1A) and a TST-positiveLTBI subject (Figure 1B-D). As expected, CD4 T cells produceIFN-� to ESAT-6 in active TB patients (Figure 1A) as well as in

LTBI subjects (Figure 1B). Differently from Li et al,1 in the sameconditions ESAT-6 failed to induce IFN-� production from V�2 orV�1 �� T cells (Figure 1C,D). Accordingly, IFN-� response wasnot associated with CD4� T cells (Figure 1A,B).9

Since no direct specific response of �� T cells to proteinantigen, as described by Li et al,1 is known, the proposed �� T-cellresponse to ESAT-6 should be confirmed by analyzing its depen-dence from a conserved protein antigen 3-dimensional conforma-tion. Alternatively, other triggering agent(s) inducing activation of�� T cells could be proposed. Since, according to Li et al,1 �� T-cellresponse was linked to ESAT-6 in a dose-response manner, acontaminating phosphoantigen could be postulated in that particu-lar ESAT-6 preparation; �� T-cell response should disappear ifmore purified (or dialyzed) ESAT-6 protein is used. Therefore,human �� T-cell direct response to soluble protein Ag is anunexpected result representing a new, direct nonprocessed surveil-lance route regarding extracellular proteins. This mechanismwould be totally different from �� T cells’ response to protein Ag,which requires intracellular or extracellular processing and,respectively, MHC class I or II molecule presentation. Ifconfirmed, �� T cells could, once again, display a unique responsecapability. However, in our view, this possibility deserves a moreaccurate evaluation.

Rita Casetti, Angelo Martino, Alessandra Sacchi, Chiara Agrati, DeliaGoletti, and Federico Martini

This study was supported by grants from Italian Health Ministry.

Conflick-of-interest disclosure: The authors declare no competing financialinterests.

Correspondence: Rita Casetti, Laboratory of Cellular Immunology, NationalInstitute for Infectious Diseases Lazzaro Spallanzani, IRCCS, Rome, Italy;e-mail: casetti@inmi.it.

Figure 1. ESAT-6 protein induces �� but not �� T cells’ response in active TB patients and TST-positive LTBI subjects. Peripheral blood mononuclear cells (PBMCs)from a representative active TB patient (A) and a representative TST-positive LTBI subject (B-D) were stimulated with ESAT-6 protein (5 �g/mL; Lionex, Braunschweig,Germany) in presence of anti-CD28 monoclonal antibody (1 �g/mL; BD Biosciences, San Jose, CA). To detect intracellular expression of IFN-�, brefeldin-A (Sigma-Aldrich, StLouis, MO) at 10 �g/mL was used. After overnight stimulation, cells were stained with CD4-peridinin chlorophyll protein (PerCP), V�2-phycoerythrin (PE), V�1-fluoresceinisothiocyanate (FITC), IFN-� allophycocyanin (APC) anti–human conjugated monoclonal antibodies (BD Biosciences). For all staining procedures, an isotype-matchednegative control was used. Data acquisition and analysis were performed using a FASCalibur flow cytometer and CellQuest software (both BD Biosciences).

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References1. Li L, Wu CY. CD4� CD25� Treg cells inhibit human memory gammadelta

T cells to produce IFN-gamma in response to M tuberculosis antigen ESAT-6.Blood. 2008;111:5629-5636.

2. Hayday AC. [gamma][delta] cells: a right time and a right place for a conservedthird way of protection. Annu Rev Immunol. 2000;18:975-1026.

3. Chien YH, Konigshofer Y. Antigen recognition by gammadelta T cells. ImmunolRev. 2007;215:46-58.

4. Morita CT, Jin C, Sarikonda G, Wang H. Nonpeptide antigens, presentationmechanisms, and immunologic memory of human Vgamma2Vdelta2 T cells:discriminating friend from foe through the recognition of prenyl pyrophosphateantigens. Immunol Rev. 2007;215:59-76.

5. Gober HJ, Kistowska M, Angman L, et al. Human T cell receptor gammadelta

cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med.2003;197:163-168.

6. Kabelitz D. Small molecules for the activation of human gammadelta T cell re-sponses against infection. Recent Patents Anti-Infect Drug Disc. 2008;3:1-9.

7. Scotet E, Martinez LO, Grant E, et al. Tumor recognition followingVgamma9Vdelta2 T cell receptor interactions with a surface F1-ATPase-relatedstructure and apolipoprotein A-I. Immunity. 2005;22:71-80.

8. Russano AM, Bassotti G, Agea E, et al. CD1-restricted recognition of exog-enous and self-lipid antigens by duodenal gammadelta� T lymphocytes. J Im-munol. 2007;178:3620-3626.

9. Goletti D, Butera O, Bizzoni F, et al. Region of difference 1 antigen-specificCD4� memory T cells correlate with a favorable outcome of tuberculosis. J In-fect Dis. 2006;194:984-992.

Response

Human memory but not naïve �� T cells from TST-positive individuals respond toM tuberculosis antigen

We thank Casetti et al for their interest in our recent work,“CD4�CD25� Treg cells inhibit human memory �� T cells toproduce IFN-� in response to M tuberculosis antigen ESAT-6.”1 Inour article, we showed that stimulation of peripheral bloodmononuclear cells (PBMCs) from tuberculin skin test (TST)–positive individuals with ESAT-6 resulted in not only the produc-tion of cytokines but also the activation and division of memory ��T cells. These responding �� T cells displayed the phenotype ofmemory but not naive �� T cells. Most interestingly, CD4�CD25�

Treg cells could inhibit IFN-� production by �� T cells.Casetti et al observed that CD4� but not �� T cells from

4 patients with active tuberculosis (TB) disease and 4 subjects withlatent TB infection (LTBI) responded to ESAT-6 to express IFN-�.In accordance with their and others’ observations,2 in our unpub-lished data from a few active TB patients, we also found that CD4�

T cells, in addition to �� T cells, produced IFN-� in response toESAT-6. Of note, the cells from different individuals with TBinfection had distinct quality of response. The discrepanciesbetween their and our results on the response of �� T cells toESAT-6 might be influenced by many factors. The concern mightbe that the source of ESAT-6 we purchased from suppliers wasdifferent from that Casetti et al used. The various preparations ofrecombinant antigens, including cloning, sequences, expression,and purification process from different companies, might havedifferent biologic activities. Moreover, differences in the classicaland nonclassical major histocompatibility class (MHC) molecules,the affinity to antigenic epitopes, and the distinct biologic featuresbetween eastern and western peoples might lead to distinctreactivity to the same antigen. Clearly, it has been reported that ��T cells from bovines could react to ESAT-6 by IFN-� productionand proliferation.3 In addition, several antigenic epitopes/proteinsrecognized by human �� T cells have been identified via CDR3�peptide–based immunobiochemical strategy.4 These peptides notonly bind to �� T cells but also activate �� T cells. Moreover, inhuman chronic human herpesvirus 8 (HHV-8) infection, purified

viral proteins resulted in �� V�1 T cell activation.5 Taken together,we agree with Casetti et al that human �� T cells recognizenonpeptidic phosphoantigens, metabolites of the isoprenoid path-way.6,7 However, the mechanism by which human �� T cellsrecognized protein antigens remains unclear currently and needsfurther investigation.

Li Li and Chang-You Wu

Approval was obtained from the Zhongshan School of Medicine, Sun Yat-SenUniversity institutional review board for these studies. Informed consent wasobtained in accordance with the Declaration of Helsinki.

Conflict-of-interest disclosure: The authors declare no competing financialinterests.

Correspondence: Changyou Wu, MD, PhD, Department of Immunology,Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2ndRoad, Guangzhou 510080, PR China; e-mail: changyou_wu@yahoo.com.

References1. Li L, Wu CY. CD4�CD25� Treg cells inhibit human memory gammadelta T cells

to produce IFN-gamma in response to M tuberculosis antigen ESAT-6. Blood.2008;111:5629-5636.

2. Goletti D, Butera O, Bizzoni F, Casetti R, Girardi E, Poccia F. Region of differ-ence 1 antigen-specific CD4� memory T cells correlate with a favorable out-come of tuberculosis. J Infect Dis. 2006;194:984-992.

3. Welsh MD, Kennedy HE, Smyth AJ, Girvin RM, Andersen P, Pollock JM. Re-sponses of bovine WC1(�) gammadelta T cells to protein and nonprotein anti-gens of Mycobacterium bovis. Infect Immun. 2002;70:6114-6120.

4. Chen H, He X, Wang Z, et al. Identification of human T cell receptor gammadelta-recognized epitopes/proteins via CDR3delta peptide-based immunobiochemicalstrategy. J Biol Chem. 2008;283:12528-12537.

5. Barcy S, De Rosa SC, Vieira J, et al. gammadelta� T cells involvement in viralimmune control of chronic human herpesvirus 8 infection. J Immunol. 2008;180:3417-3425.

6. Chien YH, Konigshofer Y. Antigen recognition by gammadelta T cells. ImmunolRev. 2007;215:46-58.

7. Hayday AC. [gamma][delta] cells: a right time and a right place for a conservedthird way of protection. Annu Rev Immunol. 2000;18:975-1026.

To the editor:

Is exclusive Skp2 targeting always beneficial in cancer therapy?

We read with great interest the work published in Blood by Chen etal concerning the therapeutic restoration of p27KIP1 protein levels in

cancer after applying the specific Skp2 inhibitor CpdA.1 Skp2 is anE3-ubiquitin ligase that mediates degradation of several cell-cycle

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regulators.2 Among its targets are negative cell-cycle regulators, includ-ing the kinase inhibitor protein (KIP) family member p27KIP1, andpositive ones, such as cyclin E.2 In addition, p27KIP1 also inhibits cyclinE.2 Thus, Skp2, p27KIP1, and cyclin E form a tight network controllingS-phase entry.2

In various tumors Skp2 is frequently overexpressed and repre-sents a major cause of p27KIP1 protein down-regulation.2 However,p27KIP1 gene alterations and transcriptional silencing, due tomicroRNA-dependent repression, promoter methylation, and tran-scriptional suppressors, is not a rare event, as the authors mention,1

but a significant source of p27KIP1 inactivation in several malignan-cies (Table S1, available on the Blood website; see the SupplementalMaterials link at the top of the article). Consequently, we cannot excludethe possibility that Skp2 overexpression coexists with transcriptionallysilenced and/or mutant p27KIP1. We have observed such a condition withthe other KIP member and Skp2 target, p57KIP2.3

Based on the above, Skp2 inhibition, in cases with transcription-ally silenced and/or mutant p27KIP1, could result in increasedexpression of cyclin E with potential deleterious effects. Cyclin E

provokes genomic instability, when overexpressed, by producingeither DNA damage and/or centrosome amplification.4,5 Up-regulation of cyclin E is frequently observed in cancer, and isassociated in various malignancies with poor survival.6

To test this hypothesis we mimicked the above scenario bysilencing Skp2 alone or in combination with p27KIP1 in A549cancer cells, which express high Skp2 levels.3 The experimentshowed that sole Skp2 silencing resulted in elevation of p27KIP1,reduction of cyclin E expression (Figure 1A,B), and a decreasein growth,3 while Skp2/p27KIP1 double knockout, recapitulatingthe proposed scenario, led to increased levels of cyclin E (Figure1A,B), centrosome amplification (Figure 1B), abnormal mito-ses, and pronounced nuclear atypia characterized by micronu-clei, lobulated nuclei, and nucleoplasmic bridges, features thatare indicative of chromosomal instability (Figure 1C).7 Inaddition, marked p53 Ser-15 phosphorylation (Figure 1A),indicating a prominent DNA damage response, provides amechanistic explanation for the observed genomic instability.8,9

Furthermore, the elevated levels of cyclin E comprised not only

Figure 1. Effect of Skp2 silencing alone or in combination with p27KIP1 in A549 carcinoma cell line. (A) Western blot analysis of Skp2, p27KIP1, cyclin E andlow-molecular-weight isoforms, Ser15 phosphorylated p53 levels in A549 mock, siSkp2-, and siSkp2/p27KIP1-treated cells. (B) Impact of deregulated cyclin E expression oncentrosome status after Skp2 and Skp2/p27KIP1 silencing in A549 cells. Immunofluorescence analysis (Texas Red � cyclin E, Oregon Green � �-tubulin) and counterstainingwith DAPI. A549 mock cells demonstrate moderate nuclear staining of cyclin E accompanied by centrosome amplification (top panel, magnification 600). A549 siSkp2 cellsdisplay normal centrosomal profile (arrow) and suppression of cyclin E expression (middle panel). A549 siSkp2/p27KIP1 cells showed increased, cyclin E levels (accumulation ofboth nuclear and cytoplasmic isoforms) and centrosome aggregates (arrows; bottom panel). (C) Abnormal mitoses, micronuclei, nuclear lobulation, and nucleoplasmic bridgesin A549 siSkp2/p27KIP1 cells. Cells were counterstained with DAPI. Histograms depict percentages of abnormal mitoses (P � .001, ANOVA) and micronuclei (P .001,ANOVA) in A549 mock and A549 siSkp2/p27KIP1-treated cells. Images in panels B and C were viewed through a Zeiss Axiolab microscope with 63 times 0.80, Zeiss Achroplanlens (both Carl Zeiss, AntiSel). Cell spreads were mounted in Fluoromount G. Texas Red was used to detect cyclin E, Oregon Green to detect �-tubulin, and DAPI as acounterstain (Invitrogen, AntiSel). Images were photographed with a SenSys camera (Photometrics, Tucson, AZ) and processed with SmartCapture VP software version 1.4(Digital Scientific, Cambridge, United Kingdom).

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the full-length but also the low-molecular-weight isoforms.5,6

These isoforms are present in both cytoplasm and nucleus, and haveincreased affinity for cdk2.5,6 They have been associated with genomicinstability, resistance to CIP/KIP inhibition, and poor outcome ofpatients with various malignancies.6

Similarly, is exclusive Skp2 targeting efficient in patients withp27KIP1 haploinsufficiency?10 In such cases, SKP2 blocking iseffective when the levels of the remaining p27KIP1 allele areup-regulated above a threshold, able to exert its negative effect oncell-cycle progression. Below this threshold the CIP/KIP moleculeswill be sequestered by the cyclin D/cdk complexes, furtherpromoting cyclin E/cdk2 activity.11

In conclusion, although the findings of Chen et al are significantand important, our results present an additional point of view,which stresses the impact of defining the transcriptional and/ormutational status of p27KIP1 before applying a therapeutic approachbased exclusively on Skp2 inhibition.

Marilena Koutsami, Georgia Velimezi, Athanassios Kotsinas,Konstantinos Evangelou, Athanassios G Papavassiliou, Christos Kittas,and Vassilis G. Gorgoulis

The online version of this letter contains a data supplement.

This work was financially supported by the European Commission SeventhFramework Health Programme Genomic instability in cancer and precancer(FP7-GENICA) grant.

Conflict-of-interest disclosure: The authors declare no competing financialinterests.

Correspondence: Vassilis G. Gorgoulis, 53 Antaiou Str, Lamprini, Ano PatisiaAthens, GR-11146, Greece; e-mail: histoclub@ath.forthnet.gr, vgorg@med.uoa.gr.

References1. Chen Q, Xie W, Kuhn DJ, et al. Targeting the p27 E3 ligase SCFSkp2 results in

p27- and SKP2-mediated cell-cycle arrest and activation of autophagy. Blood.2008;111:4690-4699.

2. Hershko DD. Oncogenic properties and prognostic implications of the ubiquitinligase Skp2 in cancer. Cancer. 2007;112:1415-1424.

3. Pateras IS, Apostolopoulou K, Koutsami M, et al. Downregulation of the KIP familymembers p27KIP1 and p57KIP2 by SKP2 and the role of methylation in p57KIP2inactivation in non small cell lung cancer. Int J Cancer. 2006;119:2546-2556.

4. Spruck CH, Won KA, Reed SI. Deregulated cyclin E induces chromosome in-stability. Nature. 1999;401:297-300.

5. Koutsami MK, Tsantoulis PK, Kouloukoussa M, et al. Centrosome abnor-malities are frequently observed in non-small-cell lung cancer and are asso-ciated with aneuploidy and cyclin E overexpression. J Pathol. 2006;209:512-521.

6. Akli S, Keyomarsi K. Cyclin E and its low molecular weight forms in human can-cer and as targets for cancer therapy. Cancer Biol Ther. 2003;2:S38-S47.

7. Liontos M, Koutsami M, Sideridou M, et al. Deregulated overexpression of hCdt1and hCdc6 promotes malignant behavior. Cancer Res. 2007;67:10899-10909.

8. Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of the DNA damagecheckpoint and genomic instability in human precancerous lesions. Nature.2005;434:907-913.

9. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damagemodel for cancer development. Science. 2008;319:1352-1355.

10. Komuro H, Valentine MB, Rubnitz JE, et al. p27KIP1 deletions in childhoodacute lymphoblastic leukemia. Neoplasia. 1999;1:253-261.

11. Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res.2000;60:3689-3695.

To the editor:

Platelet components associated with acute transfusion reactions: the role of platelet-derivedsoluble CD40 ligand

Several independent studies indicate that soluble CD40 ligand(sCD40L) derived and cleaved from platelets is responsible foracute transfusion reactions (ATR).1-3 Ratliff et al4,5 show in thisjournal that platelets modulate innate and adaptive immunity inmice away from the site of activation and impact antibody-mediated immune responses. Having shown that platelet-derivedsCD40L alters human B-cell responses in vitro,6 we examinedwhether sCD40L in platelet concentrates (PCs) associated withclinical ATR could mediate B-cell responses as an indication ofpathophysiologic function. Apheresed PCs were collected andprocessed with leukocyte reduction ( 106 per unit); suspended in35% donor plasma and 65% InterSol platelet additive solution(Fenwal, La Chatre, France); prepared with the amotosalen HClplus UVA light pathogen inactivation procedure (Intercept; Cerus,Concord, CA); and stored at 22°C with shaking for 5 or 7 daysbefore transfusion.7 An active hemovigilance program evaluatedthe response to platelet transfusion.7 Reported ATR episodes wereinvestigated using residual platelet components associated withATR. In the 4 investigated cases of ATR (PCs were older than3 days; Figure 1),8 2 aliquots from each PC (and, for each aliquot,10 controls not associated with ATR) were prepared. One aliquotwas used to assay supernatant fractions and the other to assayplatelet lysates using specific, sensitive ELISAs (R&D SystemsEurope, Lille, France) targeting a panel of cytokines and chemo-kines. IL8, CD62p, and platelet-derived growth factor–AB (PDGF-AB) levels were similar between ATR-associated PCs and PCswithout ATR. In ATR-associated PCs, supernatant fractions con-

tained higher levels of sCD40L than the control component,consistent with release; in an inverse correlation, the correspondingplatelet lysates contained lower levels of sCD40L, consistent withrelease during storage (P .05). To determine whether the re-leased sCD40L (possibly among other costimulators) was biologi-cally active, we incubated purified B cells, isolated from the bloodof healthy donors, with PC supernatants and platelet lysates fromPCs either associated or not with ATR. We then measured B-cellproduction of IL-6, on day 2 of the culture, to identify a productionplateau (F.C., unpublished data, April 6, 2006).

Baseline IL-6 concentrations were consistently less than 5 to10 pg/mL in each control. The addition of 20 �L 1/20 diluted“ATR” supernatant samples to 2 104 purified B cells in 200 �Lculture medium9 resulted in increased IL-6 production comparedwith samples from control PCs (P .05), the correspondingplatelet lysates from ATR-associated PCs failed to elicit IL-6production; recombinant purified sCD40L stimulated IL-6 produc-tion (P .05), a cytokine strongly reactive to B cell stimulation.Preincubation of B cells with 5 �g/mL CD40-blocking antibodies(R&D Systems Europe and ATCC, Manassas, VA) substantiallyabrogated IL-6 secretion, unlike isotype-matched control. Thepartial blocking of CD40 binding on CD40� B cells stronglysuggests a potentially synergistic role in B cells for cytokines otherthan sCD40L (under investigation) and indicates a sustained rolefor PC-derived sCD40L.10

These data prompted us to institute a multicenter collaborativestudy of a larger series of ATR-associated PCs to determine specific

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factors that can be targeted generally or in certain donors or patientsto further reduce proinflammatory sCD40L production duringcollection and storage of platelet components.

Fabrice Cognasse, Jean Marc Payrat, Larry Corash,Jean Claude Osselaer, and Olivier Garraud

Conflict-of-interest disclosure: The authors declare no competing financialinterests.

Correspondence: Olivier Garraud, EFS Auvergne-Loire and GIMAP-EA 3064,Universite de Saint-Etienne, Faculte de Medecine, 15 rue Ambroise Pare,42023 Saint-Etienne cedex 2, France; e-mail: olivier.garraud@efs.sante.fr.

References1. Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derived CD40L:

the switch-hitting player of cardiovascular disease. Circulation. 2002;106:896-899.

2. Kaufman J, Spinelli SL, Schultz E, Blumberg N, Phipps RP. Release of biologi-cally active CD154 during collection and storage of platelet concentrates pre-pared for transfusion. J Thromb Haemost. 2007;5:788-796.

3. Khan SY, Kelher MR, Heal JM, et al. Soluble CD40 ligand accumulates instored blood components, primes neutrophils through CD40, and is a potentialcofactor in the development of transfusion-related acute lung injury. Blood.2006;108:2455-2462.

4. Elzey BD, Schmidt NW, Crist SA, et al. Platelet-derived CD154 enables T-cellpriming and protection against Listeria monocytogenes challenge. Blood. 2008;111:3684-3691.

5. Sprague DL, Elzey BD, Crist SA, Waldschmidt TJ, Jensen RJ, Ratliff TL. Plate-let-mediated modulation of adaptive immunity: unique delivery of CD154 signalby platelet-derived membrane vesicles. Blood. 2008;111:5028-5036.

6. Cognasse F, Hamzeh-Cognasse H, Lafarge S, et al. Human platelets can acti-vate peripheral blood B cells and increase production of immunoglobulins. ExpHematol. 2007;35:1376-1387.

7. Osselaer JC, Messe N, Hervig T, et al. A prospective observational cohortsafety study of 5106 platelet transfusions with components prepared with pho-tochemical pathogen inactivation treatment. Transfusion. 2008;48:1061-1071.

8. Cognasse F, Boussoulade F, Chavarin P, et al. Release of potential immuno-modulatory factors during platelet storage. Transfusion. 2006;46:1184-1189.

9. Cognasse F, Chavarin P, Acquart S, et al. Differential downstream effects ofCD40 ligation mediated by membrane or soluble CD40L and agonistic Ab: astudy on purifed human B cells. Int J Immunopathol Pharmacol. 2005;18:65-74.

10. Heddle NM, Klama L, Singer J, et al. The role of the plasma from platelet con-centrates in transfusion reactions. N Engl J Med. 1994;331:625-628.

Response

Are PDMVs the biologically active source of CD154 in ATR?

In vivo animal data reporting biologic platelet CD154 function,1-4

coupled with reports of direct biologic activity on isolated primaryhuman neutrophils and B cells,5-7 indicate platelet CD154 couldhave clinically significant function. Indeed, recent reports indicatethat acute transfusion reactions (ATRs) may result from solubleCD154 (sCD154) released from platelets during storage.5,8-10 Bydemonstrating that only platelet concentrates (PCs) that resulted in

clinical ATR can stimulate CD154-specific B cell IL-6 production,Cognasse and colleagues make a compelling case for investigation intoimproved platelet storage that minimizes platelet release of CD154.

Upon activation, platelets can release microparticles and exo-somes11 (collectively referred to as platelet-derived membranevesicles [PDMVs]) that can deliver platelet-derived signals invitro 12-14 or in vivo.3 This becomes an important consideration

Figure 1. Concentration and physiologic effects ofsoluble CD40 ligand (sCD40L) in supernatants andlysates of platelet concentrates (PCs) associatedwith ATR(451,715,561,536) and control PCs. (A) Deter-mination of sCD40L levels in PC supernatants andlysates in platelet components associated with ATRversus control components; data obtained from theclinical ATR occurrence with a day-5 (d5) PC sample anda d7 PC sample. (B) PC supernatants and lysates ofindividual components associated with reactions weretested with purified blood B cells from healthy blooddonors stimulated to secrete IL-6; data obtained from theclinical episodes with one d5 and 3 d7 PC samples,respectively. (C) The consistent decrease of IL-6 secre-tion in response to stimulation with a high concentrationof sCD40L in platelet supernatants after preincubation ofB cells with 5 �g/mL CD40-blocking antibody. We ob-served similar results by preincubation of B cells with5 �g/mL of another CD40-blocking antibody and withboth antibodies together (data not shown). All values(pg/mL) were corrected for background levels. Data areexpressed as means plus or minus SD in n � 5 experi-ments. *P .05 in differences between tested valuesand respective controls (Wilcoxon paired test).

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when examining what is thought to be “soluble” CD154. Somestudies use centrifugation at g forces too low to separate PDMVfrom sCD154, while others are unclear about centrifugation speeds.Transwell experiments have also been used, but the small size ofPDMVs (0.04-1.0 �m) would have permitted their free diffusionalong with sCD154. Separating PDMV CD154 from sCD154 is ofsignificance because we have reported that in the absence of wholemurine platelets, the ability to deliver a CD154 signal in vitro or invivo resides almost exclusively with PDMVs and only minimallywith truly soluble CD154.3 This is in spite of the fact that mostplatelet CD154 is in the soluble fraction and very little remains onPDMVs. We have also made the same observation with humanplatelets in vitro (manuscript in preparation). It will be important toidentify if PDMVs are the biologically active source of CD154 inATR when considering methods of collection and storage of PCsdesigned to inhibit release of CD154.

Bennett D. Elzey, Scott A. Crist, Daniel L. Sprague, and Timothy L. Ratliff

Conflict-of-interest disclosure: The authors declare no competing financialinterests.

Correspondence: Timothy L. Ratliff, Director, Purdue Cancer Center, HansenLife Sciences Research Building, Room 145, 201 South University Street,West Lafayette, IN 47907-2064; e-mail: tlratliff@purdue.edu.

References1. Elzey BD, Tian J, Jensen RJ, et al. Platelet-mediated modulation of adaptive

immunity. A communication link between innate and adaptive immune compart-ments. Immunity. 2003;19:9-19.

2. Elzey BD, Grant JF, Sinn HW, Nieswandt B, Waldschmidt TJ, Ratliff TL. Coop-

eration between platelet-derived CD154 and CD4� T cells for enhanced germi-nal center formation. J Leukoc Biol. 2005;78:80-84.

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CORRESPONDENCE 4781BLOOD, 1 DECEMBER 2008 � VOLUME 112, NUMBER 12

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