Nonregulatory) T Cells − CD25 + (Regulatory

Platelet Factor 4 Differentially Modulates CD4 +CD25+ (Regulatory) versus CD4+CD25 − (Nonregulatory) T Cells

This information is current as Chao Yan Liu, Manuela Battaglia, Seon Ho Lee, Qi-Hong of September 23, 2021. Sun, Richard H. Aster and Gian Paolo Visentin J Immunol 2005; 174:2680-2686; ; doi: 10.4049/jimmunol.174.5.2680 http://www.jimmunol.org/content/174/5/2680 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Platelet Factor 4 Differentially Modulates CD4؉CD25؉ Regulatory) versus CD4؉CD25؊ (Nonregulatory) T Cells1)

Chao Yan Liu,*† Manuela Battaglia,*‡ Seon Ho Lee,2* Qi-Hong Sun,*† Richard H. Aster,* and Gian Paolo Visentin3*†

Active suppression mediated by CD4؉CD25؉ T regulatory (Tr) cells plays an important role in the down-regulation of T cell responses to both foreign and self-Ags. Platelet factor 4 (PF4), a platelet-derived CXC chemokine, has been shown to strongly inhibit T cell proliferation as well as IFN-␥ and IL-2 release by isolated T cells. In this report we show that human PF4 stimulates proliferation of the naturally anergic human CD4؉CD25؉ Tr cells while inhibiting proliferation of CD4؉CD25؊ T cells. In coculture experiments we found that CD4؉CD25؉ Tr cells exposed to PF4 lose the ability to inhibit the proliferative response of CD4؉CD25؊ T cells. Our ﬁndings suggest that human PF4, by inducing Tr cell proliferation while impairing Tr cell function, may play a previously unrecognized role in the regulation of human immune responses. Because platelets are the sole source of PF4 Downloaded from in the circulation, these ﬁndings may be relevant to the pathogenesis of certain immune-mediated disorders associated with platelet activation, such as heparin-induced thrombocytopenia and autoimmune thrombocytopenic purpura. The Journal of Immunol- ogy, 2005, 174: 2680–2686.

utoreactive T and B cells can be detected in healthy in- longs to the CXC family of chemokines, but does not share certain

dividuals, but are normally kept in check by regulatory proinflammatory properties of other CXC family members because http://www.jimmunol.org/ mechanisms. Among those is an active suppression of it is missing a critical N-terminal Glu-Leu-Arg sequence, the ELR A 4 naive T cells by T regulatory (Tr) cells (1–3). Several types of Tr motif, that precedes the first cysteine residue (14). Several reports cells exist, including CD4ϩ T cells that express the IL-2R ␣-chain have identified PF4 as an inhibitor of hemopoietic progenitor and (CD25) constitutively, do not secrete IL-10, and suppress immune endothelial cell proliferation and angiogenesis (14, 15). Further- responses via direct cell-to-cell interactions; type 1 T regulatory more, PF4 has been shown to strongly inhibit T cell proliferation (Tr1) cells, which function via secretion of IL-10; and TGF-␤1- as well as IFN-␥ and IL-2 release by isolated T cells (16). producing Th3 cells (4–6). CD4ϩCD25ϩ T regulatory cells rep- In this report we confirm that human PF4 is a potent inhibitor of resent 5–10% of the endogenous CD4ϩ T cell subset (7). Like their CD4ϩCD25Ϫ T cell proliferation and show for the first time that mouse counterparts, human CD4ϩCD25ϩ Tr cells are anergic this chemokine acts quite differently on CD4ϩCD25ϩ Tr cells, in by guest on September 23, 2021 when stimulated in vitro with anti-CD3 mAbs (8) and are able to that it induces proliferation and subsequent loss of regulatory func- suppress CD4ϩ and CD8ϩ T cell responses in vitro and in vivo tion in this T cell subset. We suggest that additional studies of the upon TCR ligation (9). latter activity may provide an explanation for the high incidence of Tr cells may be required for maintenance of homeostasis in the Abs specific for PF4:heparin complexes in patients treated with immune system (10). In addition, Tr cells can prevent pathology in unfractionated heparin and insights into a previously unexpected autoimmune diseases (1), intestinal inflammatory diseases (11), role for PF4 in regulation of human immune responses. and allograft rejection (12). Human PF4, a heparin-binding protein contained in platelet Materials and Methods ␣-granules, is secreted upon activation of platelets (13). PF4 be- Chemicals and reagents These were obtained from the following sources: heparin derived from porcine intestinal mucosa (10,000 IU/ml; sp. act., 176 IU/mg) and prota- *Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, mine sulfate (10 mg/ml) from Wyeth/Lederle/ESI; [methyl-3H]thymidine WI 53226; and †Department of Pediatrics, University at Buffalo-State University of (37 MBq/ml) from PerkinElmer; BSA, phytohemagglutinin (PHA), and ‡ New York, Buffalo, NY 14214; San Raffaele Telethon Institute for Gene Therapy, propidium iodide from Sigma-Aldrich; recombinant human IL-2 and IL-8 Milan, Italy from R&D Systems; mouse mAb anti-CD3 (OKT3) from Ortho Biotech; Received for publication August 26, 2004. Accepted for publication December mAb anti-CD28 (clone CD 28.2) from BD Pharmingen; and rabbit anti- 13, 2004. mouse IgG from Jackson ImmunoResearch Laboratories. All other chem- The costs of publication of this article were defrayed in part by the payment of page icals were reagent grade. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Isolation of T cell populations 1 This work was supported by Grant HL64704 from the National Heart, Lung, and Human peripheral blood, anticoagulated with citrate, was obtained from six Blood Institute. healthy donors and three patients experiencing heparin-induced thrombo- 2 Current address: University of Ulsan College of Medicine, Department of Labora- cytopenia (HIT). Results obtained using cells derived from normal donors tory Medicine, Ulsan University Hospital, 290-3 Jeonha-dong, Dong-gu, Ulsan 682- or HIT patients were fully comparable. The human studies were approved 714, South Korea. by The Blood Center of SE Wisconsin and University at Buffalo institu- 3 Address correspondence and reprint requests to Dr. Gian Paolo Visentin, Depart- tional review board. PBMC were prepared by centrifugation over Ficoll- ment of Pediatrics, University at Buffalo-State University of New York, Biomedical Hypaque gradient (Amersham Biosciences). All T cell subpopulations Research Building, Room 422, Buffalo, NY 14214. E-mail address: visentin@ were obtained by positive and/or negative selection of PBMCs on magnetic buffalo.edu beads (Miltenyi Biotec) according to the manufacturer’s instructions and 4 Abbreviations used in this paper: Tr, T regulatory; HIT, heparin-induced thrombo- previously described methods (17) (Fig. 2A) or FACS sorting on a FACStar ϩ cytopenia; PF4, platelet factor 4; PHA, phytohemagglutinin. (BD Biosciences; Fig. 2B). Briefly, CD3 T cells were purified by positive

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2681 selection with CD3 microbeads (20 ␮l/107 cells). CD4ϩ T cells were iso- tial halving of CFSE fluorescence, generating equally spaced peaks on a lated by positive selection with the CD4 MultiSort kit (20 ␮l/107 cells). logarithmic scale. Six individual peaks and corresponding regions were Eluted/released CD4ϩ T cells were washed once in cold PBS containing identified (0–5, delimited by dashed lines, as shown in Fig. 5A). The pro- 0.5% BSA and 2 mM EDTA and were separated with CD25 microbeads portion of cells responding to the different stimuli was calculated according (10 ␮l/107 cells) into CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cell fractions. to published methods (21, 22). Briefly, the percentage of events (corre- Alternatively, CD4ϩ T cells isolated by positive selection with the CD4 sponding to cells) in a given cycle (n) is divided by 2 raised to the power MultiSort kit were labeled with PE-conjugated anti-CD4 and FITC-conju- n to calculate the percentage of original, undivided cells from which they gated anti-CD25 and sorted on a FACStar to obtain a subset of arose (precursor number). The sums of these give the total precursor cells CD4ϩCD25ϩ Tr cells expressing CD25 at high levels (CD4ϩCD25high) (7, for each culture (Table II). As shown in Fig. 5A, the numbers appearing 18). All purified T cell subpopulations were used immediately after above each peak (0–5) are indicated with the undivided T cells (0) residing isolation. in the rightmost peak, and T cells that have divided five times (5) residing in the leftmost peak, ending at the first minor tick mark between the 101– Culture conditions and proliferation/suppression assays 102 scale. Therefore, the sum of cohorts from divisions 1–5 represents the Isolated T cells, in triplicate wells (1 ϫ 105/well, in a 200-␮l final volume), number of precursor T cells that have been proliferating within the 96-h culture. The leftmost peak represents unlabeled CD4ϩCD25ϩ (Fig. 5, C were cultured in RPMI 1640 medium supplemented with 100 U/ml peni- ϩ Ϫ cillin/streptomycin, 2 mM L-glutamine, 5 mM HEPES (Mediatech), and and D)orCD4 CD25 T cells (Fig. 5, G and H). 10% heat-inactivated human AB serum (Cambrex; complete RPMI 1640) in 96-well, U-bottom plates (Corning Life Sciences). Purified T cells were Cytokine measurement incubated in the absence or the presence of various concentrations of PF4, T cells were stimulated with immobilized anti-CD3 (OKT3; 0.5 ␮g/ml) and heparin, PF4:heparin complexes, protamine, and IL-8. Cells were stimu- soluble anti-CD28 (1 ␮g/ml) mAbs in complete RPMI 1640. Supernatants lated with PHA (2.5 ␮g/ml) or with immobilized (4°C, overnight) mAb from triplicate culture wells were collected after 72 h and frozen at Ϫ80°C anti-CD3 (OKT3) at 0.5 ␮g/ml, either alone or in combination with IL-2

until use. Cytokine concentrations in supernatants were assessed using a Downloaded from (100 U/ml), soluble (nonimmobilized) anti-CD28 mAb (1 ␮g/ml), soluble commercially available ELISA kit for human TGF-␤1 (R&D Systems) and anti-CD28 mAb (1 ␮g/ml) and IL-2 (100 U/ml), or soluble anti-CD28 mAb a cytometric bead array (Th1/Th2 Cytokine CBA 1; BD Pharmingen) for (1 ␮g/ml) together with secondary rabbit anti-mouse IgG (10 ␮g/ml) as a IL-2, IL-4, IL-5, IL-10, IFN-␥, and TNF-␣, following the manufacturer’s cross-linker. Human PF4, either isolated from human platelets or expressed protocols. The limits of sensitivity were as follows: 3 pg/ml for IL-2, IL-4, as a recombinant protein, was purified in our laboratory as previously de- IL-5, IL-10, and TNF-␣; and 7 pg/ml for IFN-␥ and TGF-␤1. scribed (19, 20). In preliminary experiments we determined that recombinant human PF4 is indistinguishable from human PF4 isolated from plate- Statistical analysis lets with respect to its effects on stimulated T cell subpopulations. All http://www.jimmunol.org/ subsequent experiments were performed using human recombinant PF4 at All analyses for statistically significant differences were performed with a concentration of 5 ␮g/ml, unless otherwise indicated. PF4 preparations Student’s paired t test with a two-tailed distribution. A value of p Ͻ 0.05 were shown to be free of endotoxin using a Limulus amebocyte lysate gel was considered significant. All cultures were performed in triplicate, and clot assay (Cambrex). error bars represent 1 SD. Reported results were obtained from multiple Cell proliferation/DNA synthesis was assessed by the level of TdR in- experiments with similar results. Only a representative experiment is re- corporation. After 72 h of culture, [3H]TdR (1 ␮Ci/well) was added for an ported for each figure. additional 16 h. The medium was then discarded, and the cells were harvested using a FilterMate 196 harvester (Packard). Radioactivity was mea- Results sured using a Matrix 9600 (Packard) gas scintillation beta counter. Assays PF4 inhibits proliferation of purified CD3ϩ T cells were performed in triplicate. To analyze the effect of PF4 on the suppres-

ϩ by guest on September 23, 2021 sive activity of CD4ϩCD25ϩ Tr cells or CD4ϩCD25Ϫ T cells, a constant CD3 T cells were isolated from buffy coats of HIT patients or number of CD4ϩCD25Ϫ T cells (5 ϫ 104) was cocultured with different ϩ ϩ normal donors using magnetic bead separation. The resulting pu- ratios of CD4 CD25 Tr cells (from 1/1, e.g., 5 ϫ 104 cells, to 1/32, e.g., ϩ Ͼ 3 rity of CD3 T cells was 95% as determined by flow cytometry 1.5 ϫ 10 cells) as indicated in the figures. Cocultures were stimulated with ϩ allogeneic APCs (PBMCs that had been depleted of CD3ϩ T cells by (data not shown). The freshly purified CD3 T cells were stimu- negative selection on CD3 microbeads; Miltenyi Biotec) and exposed to lated with immobilized anti-CD3 mAb (0.5 ␮g/ml) in the absence 3000 rad of gamma irradiation. Alternatively, immobilized anti-CD3, in the or the presence of increasing concentrations of PF4, PF4:heparin absence or the presence of PF4, was used as the stimulus. complexes, heparin, or protamine. Consistent with a previous re- Flow cytometric analysis port (16), PF4 strongly inhibited T cell proliferation in a dose- dependent manner (Fig. 1). Lesser, but statistically significant, T cells (1 ϫ 105) in 0.02 M PBS, pH 7.4, containing 0.1% sodium azide and 1% BSA were stained for 30 min at 4°C in a 50-␮l final volume with optimal dilution of FITC-conjugated anti-CD3 (UCHT1) or anti-CD4 (RPA-T4), PE-conjugated anti-CD25 (M-A251), and mouse subclass-specific isotype controls (BD Pharmingen). Cells were then washed and acquired by flow cytometry (FACScan, FACSCalibur, and CellQuest software (BD Biosciences)). Data were analyzed with WinList software (Verity Software House). CFSE labeling and flow cytometric analysis CFSE (Molecular Probes) labeling and analysis were performed according to the manufacturer’s recommendations and previously described methods (21, 22). Briefly, freshly purified CD4ϩCD25ϩ and CD4ϩCD25Ϫ T cells (1 ϫ 107/ml) were labeled with 5 ␮M CFSE for 15 min at 37°C in PBS containing 0.1% BSA. Cells were quenched with ice-cold PBS containing 10% autologous plasma for 5 min, then washed extensively three times. CFSE-labeled T cells (5 ϫ 104) were cultured in 96-well, U-bottom plates FIGURE 1. PF4 inhibits the proliferation of purified CD3ϩ T cells stim- coated with mAb anti-CD3 (OKT3; 0.5 ␮g/ml) in the absence or the pres- ulated by anti-CD3 mAb. Total CD3ϩ T cells were freshly isolated from ence of 5 ␮g/ml PF4. In coculture experiments, CFSE-labeled HIT patients or normal donors’ PBMCs (purity, Ͼ95%). CD3ϩ T cells ϩ Ϫ ϫ 4 CD4 CD25 T cells (5 10 ) were mixed with the same number (1:1 (1 ϫ 105 cells/well) were stimulated with immobilized anti-CD3 mAb (0.5 ratio) of unlabeled CD4ϩ ϩ CD25 (and vice versa). Only viable cells, as ␮g/ml) in the absence or the presence of the indicated concentrations of determined by propidium iodide staining exclusion, were analyzed. Briefly, PF4, PF4:heparin complexes, heparin, or protamine. After 72-h culture, after 96-h culture, cells were harvested and washed three times in ice-cold 3 PBS containing 1% BSA. After the last washing step, cells were resus- [ H]thymidine was added for an additional 16 h. Individual data points p Ͻ 0.05 vs ,ء .pended in 50 ␮l of PBS containing 1% BSA and 10 ␮g of propidium represent the average of triplicate determinations Ϯ 1 SD iodide, and acquired by flow cytometry. Data were analyzed with WinList medium (by t test). Results are representative of three independent software (Verity Software House). Division was characterized by sequen- experiments. 2682 MODULATORY EFFECT OF PF4 ON T CELLS inhibition was also seen with PF4:heparin complexes. Conversely, we found that heparin alone and protamine, a positively charged heparin-binding protein used as a control for any putative interaction mediated only by positive charges, were without effect. In three independent experiments, an average of 82.4 Ϯ 4.1% ( p Յ 0.0006) inhibition was observed in the presence of 5 ␮g/ml PF4, 4.8 Ϯ 6.5% ( p Յ 0.64) in the presence of 5 ␮g/ml protamine, 3.9 Ϯ 3.0% ( p Յ 0.40) in the presence of 5 U/ml heparin, and 53.4 Ϯ 2.4% ( p Յ 0.002) in the presence of 5 ␮g/ml PF4:0.25 U/ml heparin complexes. These findings indicate that PF4 and PF4:heparin complexes inhibit the proliferation of human T cells in response to TCR cross-linking. The absence of APCs, such as B cells and monocytes, in these cultures shows that APCs are not required for this effect.

PF4 inhibits proliferation of CD4ϩCD25Ϫ T cells, but stimulates proliferation of CD4ϩCD25ϩ Tr cells It has been convincingly demonstrated that CD4ϩCD25ϩ Tr cells are present in the peripheral blood of humans and exert inhibitory Downloaded from regulatory function on naive T cells in a cell-cell contact-dependent manner (8, 17). To investigate whether PF4 acts on a specific T cell subset, we isolated CD4ϩCD25Ϫ nonregulatory T cells and CD4ϩCD25ϩ regulatory T cells from HIT patients and normal healthy donors using a two-step magnetic separation. Purity Ͼ94% was achieved for both T cell subpopulations, as determined by http://www.jimmunol.org/ flow cytometry (Fig. 2). Freshly isolated CD4ϩCD25Ϫ T cells and CD4ϩCD25ϩ Tr cells were cultured and activated with different stimuli in the absence or the presence of 5 ␮g/ml PF4. As shown in Fig. 3A, PF4 strongly inhibited the proliferation of CD4ϩCD25Ϫ T cells in response to anti-CD3 mAb alone or com- FIGURE 3. PF4 inhibits the proliferation of CD4ϩCD25Ϫ T cells, but ϩ ϩ ϩ Ϫ bined with anti-CD28 mAb, but was without effect on the response stimulates the proliferation of CD4 CD25 Tr cells. CD4 CD25 (A) and ϩ ϩ to anti-CD3/anti-CD28 cross-linked (with rabbit anti-mouse IgG), CD4 CD25 (B) T cell populations, in the absence (Ϫ) or the presence ϩ ␮ ϫ 5 anti-CD3 plus IL-2, and anti-CD3/CD28 plus IL-2. In five inde- ( ) of PF4 (5 g/ml), were tested (1 10 cells/well) for their ability to

␮ by guest on September 23, 2021 pendent experiments (three with T cells from normal donors and proliferate in response to different stimuli, including PHA (2.5 g/ml), immobilized mAb anti-CD3 at 0.5 ␮g/ml, either alone (␣CD3) or in com- two from HIT patients), in the presence of PF4 an average of ␮ ␣ Ϯ Յ bination with soluble anti-CD28 mAb at 1 g/ml ( CD3/28), soluble anti- 14.5 2.6% ( p 0.02) inhibition was observed when these T CD28 mAb at 1 ␮g/ml together with secondary rabbit anti-mouse IgG (10 Ϯ Յ cells were activated by PHA, 52.3 5.1% ( p 0.0008) when ␮g/ml) used as a cross-linker (␣CD3/28/CL), IL-2 at 100 U/ml activated by anti-CD3 alone (anti-CD3), and 39.7 Ϯ 1.5% ( p Յ (␣CD3ϩIL-2), IL-2 at 100 U/ml, and soluble anti-CD28 mAb at 1 ␮g/ml (␣CD3/28ϩIL-2). CD4ϩCD25Ϫ (C)orCD4ϩCD25high (D) T cell populations were stimulated with immobilized mAb anti-CD3 at 0.5 ␮g/ml either alone (␣CD3) or in combination with soluble anti-CD28 mAb at 1 ␮g/ml (␣CD3/28) in the absence (medium only (M)) or the presence of PF4 (5 g/ml), PF4:heparin complexes (PF4:H; 5 ␮g/ml:0.25 U/ml) or IL-8 (5 ␮g/ml). After 72-h culture, [3H]thymidine was added for an additional 16 h. Individual data points represent the average of triplicate determina- p Ͻ 0.05 vs medium (by t test). Results are representative ,ء .tions Ϯ 1 SD of ﬁve independent experiments for A and B, and three for C and D.

0.002) when activated by anti-CD3 together with anti-CD28 (␣CD3/28). Addition of anti-CD3, anti-CD28, and rabbit anti- mouse IgG as a cross-linker (␣CD3/28/CL), or anti-CD3 together with IL-2 (␣CD3ϩIL-2), resulted in a slight, but not statistically significant, inhibition mediated by PF4 (12.9 Ϯ 5.2% ( p Յ 0.16) and 11.5 Ϯ 3.9% ( p Յ 0.11), respectively). When this T cell subpopulation was activated by anti-CD3 together with anti-CD28 ϩ Ϫ ϩ ϩ FIGURE 2. Isolation and purity of human CD4 CD25 , CD4 CD25 , and IL-2 (␣CD3/28ϩIL-2), a slight, but not statistically signifi- ϩ high ϩ and CD4 CD25 T cells. Total CD4 T cells were freshly isolated from cant, increase in the proliferation in the presence of PF4 was ob- HIT patients or normal donors’ PBMCs (positive selection) and separated Ϯ Յ ϩ Ϫ served (11.7 4.6%; p 0.20). These data indicate that PF4 has into CD25 (positive selection) and CD25 (negative selection) fractions ϩ Ϫ inhibitory properties on CD4 CD25 T cell proliferation, as ob- by magnetic beads (A) or by anti-CD4 magnetic bead enrichment followed by FACS sorting to recover CD4ϩ expressing high levels of CD25 served for unfractionated T cells (Fig. 1). Moreover, IL-2 and (CD4ϩCD25high; B). The purity of the T cell subpopulations was deter- cross-linker interfere with PF4 inhibitory effects on T cell mined by flow cytometry (A: CD4ϩ, Ͼ97%; CD25Ϫ, Ͼ95%; CD25ϩ, proliferation. ϩ ϩ Ͼ94%; B: CD4ϩ, Ͼ95%; CD25high, Ͼ98%). Results are representative of CD4 CD25 Tr cells do not ordinarily respond to PHA or anti- nine independent experiments for A and three for B. CD3 mAb (8, 23). As shown in Fig. 3B, in contrast to what was The Journal of Immunology 2683 observed for CD4ϩCD25Ϫ T cells, PF4 (5 ␮g/ml) caused values (picograms per milliliter) of pooled data from three inde- CD4ϩCD25ϩ Tr cells to become responsive to anti-CD3 and pro- pendent experiments; SDs were Ͻ20%). Mirroring the prolifera- moted their increased response to the other agents used for stim- tion results (Fig. 3, A and B), PF4 strongly inhibited the release of ulation, except for PHA. IL-2, IL-4, IL-5, IL-10, IFN-␥, and TNF-␣ by CD4ϩCD25Ϫ T In five independent experiments (three using T cells isolated cells without affecting TGF-␤1 production. In contrast, PF4 sig- from normal donors and two using cells from HIT patients), in the nificantly increased the release of IL-2, IFN-␥, and TNF-␣, but not presence of PF4 an average of 86.8 Ϯ 13.1% ( p Յ 0.009) up- IL-10 or TGF-␤1, by CD4ϩCD25ϩ Tr cells. Taken together, these regulation (increased proliferation vs medium) was observed when findings demonstrate a previously unrecognized role for PF4 in these T cells were activated by anti-CD3 alone (␣CD3; 47.2 Ϯ modulating the response of human T cell subpopulations to TCR 7.0%; p Յ 0.002), when stimulated by anti-CD3 together with cross-linking. anti-CD28 (␣CD3/28; 24.7 Ϯ 6.6%; p Յ 0.03), with the addition PF4 induces proliferation, rather than suppression, of of anti-CD3 together with anti-CD28 and rabbit anti-mouse IgG as ϩ Ϫ ϩ ϩ a cross-linker (␣CD3/28/CL; 42.7 Ϯ 1.9%; p Յ 0.003) when anti- cocultures of CD4 CD25 and CD4 CD25 T cells CD3 and IL-2 (␣CD3ϩIL-2) were combined together, and 30.2 Ϯ It is known that CD4ϩCD25ϩ Tr cells suppress the proliferation 3.1% ( p Յ 0.01) when IL-2 was added to anti-CD3 and anti-CD28 and cytokine production of CD4ϩCD25Ϫ T cells (8, 17). To fur- (␣CD3/28ϩIL-2). These data provide the first evidence that PF4 ther characterize the suppressive effect of PF4 on mixed T cell differentially modulates human T cell subsets. populations, we studied the effect of PF4 on the response to allo- It has been reported that CD4ϩCD25ϩ Tr cells can be distin- genic APCs (Fig. 4A; representative of four independent experi- guished on the basis of the CD25 levels of expression (high and ments) or immobilized anti-CD3 (Fig. 4B; representative of three Downloaded from low) being the CD25high subset a homogeneous population con- independent experiments) of T cell mixtures containing known sisting of Tr cells (7, 18). We therefore repeated the experiment ratios of CD4ϩCD25ϩ and CD4ϩCD25Ϫ T cells. As expected, in shown above with the CD4ϩCD25high Tr cell subset obtained by the absence of PF4, CD4ϩCD25ϩ Tr cells, at a 1:1 ratio, sup- FACS sorting. The antithetic effects of PF4 on CD4ϩCD25Ϫ T pressed the proliferative response of CD4ϩCD25Ϫ T cells to al- cells and CD4ϩCD25high Tr cells in response to anti-CD3 and loantigens (40.2 Ϯ 6.3%; p Յ 0.028; Fig. 4A) and to anti-CD3 anti-CD3/28 mAbs were comparable to those obtained with (52.9 Ϯ 3.5%; p Յ 0.029; Fig. 4B). http://www.jimmunol.org/ CD4ϩCD25ϩ Tr cells isolated by a two-step magnetic separation Unexpectedly, however, PF4 accentuated the proliferative re- and appear to be specific because these effects were not mimicked sponse of a 1:1 coculture of CD4ϩCD25ϩ and CD4ϩCD25Ϫ T by equivalent quantities of IL-8, a CXC chemokine that is signif- cells to alloantigens (74.9 Ϯ 6.3%; p Յ 0.004) and to anti-CD3 icantly homologous with PF4 in amino acid composition (Fig. 3, C (34.3 Ϯ 5.4%; p Յ 0.006). Because in a coculture system, TdR and D). In three independent experiments (two using T cells iso- incorporation cannot discriminate between CD4ϩCD25Ϫ and lated from normal donors and one from HIT patients) confirming CD4ϩCD25ϩ T cell proliferation, the increased proliferation ob- our observations, PF4 strongly inhibited proliferation of served in the presence of PF4 can be ascribed to either an increased CD4ϩCD25Ϫ T cells (Fig. 3C) induced by anti-CD3 (48.4 Ϯ response of CD4ϩCD25ϩ Tr cells to PF4 (as our previous findings 6.7%; p Յ 0.01) or induced by anti-CD3 together with anti-CD28 could suggest) or a loss of the suppressive activity of CD4 CD25ϩ by guest on September 23, 2021 (36.5 Ϯ 6.5%; p Յ 0.02), whereas heparin partially neutralized the Tr cells on CD4ϩCD25Ϫ T cells, which, in turn, proliferate. Ϯ Յ ␣ inhibitory effects of PF4 (41.1 6.2% ( p 0.04) for -CD3; ϩ Ϫ Ϯ Յ ␣ Suppression of the proliferative response of CD4 CD25 T 25.6 6.2% ( p 0.04) for -CD3/28). Similarly to what is ϩ ϩ shown in Fig. 3B, PF4 strongly up-regulated the proliferation of cells to anti-CD3, mediated by CD4 CD25 Tr cells, is CD4ϩCD25high Tr cells (Fig. 3D) induced by anti-CD3 (81.5 Ϯ impaired in the presence of PF4 15.7%; p Յ 0.007) or induced by anti-CD3 together with anti- To dissect which subset of T cells proliferated in cocultures of CD28 (48.3 Ϯ 8.0%; p Յ 0.01), whereas in the presence of heparin CD4ϩCD25ϩ and CD4ϩCD25Ϫ T cells exposed to PF4, we re- the up-regulatory effects of PF4 were reduced, but were still sig- peated the study shown in Fig. 4B using CFSE labeling to track the nificant (50.7 Ϯ 16.1% ( p Յ 0.05) for ␣-CD3; 31.7 Ϯ 6.3% ( p Յ proliferation of the two T cell subpopulations. In these studies, 0.04) for ␣-CD3/28). IL-8 failed to affect the proliferation of either CFSE-labeled CD4ϩCD25Ϫ (5 ϫ 104) T cells were cultured alone T cell population. (Fig. 5, A and B) or with an equal number of unlabeled ϩ ϩ ϩ Ϫ CD4 CD25 Tr cells (Fig. 5, C and D). In the converse experi- PF4 inhibits cytokine release of CD4 CD25 T cells, but ϩ ϩ ϫ 4 ϩ ϩ ment, CFSE-labeled CD4 CD25 (5 10 ) Tr cells were cul- up-regulates cytokine release of CD4 CD25 Tr cells tured alone (Fig. 5, E and F) or with an equal number of unlabeled Cytokine profiles of CD4ϩCD25Ϫ T cells and CD4ϩCD25ϩ Tr CD4ϩCD25Ϫ T cells (Fig. 5, G and H). Each of the cell mixtures cells stimulated with immobilized anti-CD3 and soluble anti-CD28 was stimulated with immobilized anti-CD3 mAb in the absence or mAbs are summarized in Table I (numbers represent the average the presence of PF4. After a 96-h culture, cells were harvested and

Table I. Cytokine production (picograms per milliliter) by CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cells in the absence or the presence of PF4a

Cells PF4 IL-2 IL-4 IL-5 IL-10 IFN-␥ TNF-␣ TGF-␤

CD25Ϫ Ϫ 2,234 113 219 1,184 218,867 6,621 221 ϩ 643 27 89 51 39,536 2,732 199 CD25ϩ Ϫ 296 23 142 29 1,368 246 187 ϩ 893 61 291 37 2,527 641 183

a Freshly isolated CD4ϩCD25Ϫ and CD4ϩCD25ϩ (1 ϫ 105/well) were stimulated with immobilized mAb anti-CD3 at 0.5 ␮g/ml together with soluble anti-CD28 mAb at 1 ␮g/ml (anti-CD3/28), in the absence (Ϫ) or the presence (ϩ) of PF4. Supernatants were collected after 72 h and then analyzed by cytometric bead array for IL-2, IL-4, IL-5, IL-10, TNF-␣, and IFN-␥ or ELISA for TGF-␤1. Numbers represent the average values (picograms per milliliter) of pooled data from three independent experiments. SDs were Ͻ20%. 2684 MODULATORY EFFECT OF PF4 ON T CELLS Downloaded from http://www.jimmunol.org/ FIGURE 4. CD4ϩCD25ϩ regulatory T cells suppress the proliferation of CD4ϩCD25Ϫ T cells in a dose-dependent manner. In the presence of ϩ Ϫ ϩ ϩ PF4, the proliferation of mixed CD4 CD25 and CD4 CD25 cell cul- ϩ ϩ ϩ ϩ FIGURE 5. PF4 induces the proliferation of CD4 CD25 Tr cells and ture increases. Freshly puriﬁed CD4 CD25 Tr cells, at the indicated ra- ϩ Ϫ ϩ Ϫ impairs their ability to suppress the response of CD4 CD25 T cells to tio, were added to autologous CD4 CD25 T cells (5 ϫ 104) stimulated anti-CD3. Freshly isolated CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cells were with allogeneic APCs (A;1ϫ 105) or immobilized mAb anti-CD3 (B; 0.5 ␮ ␮ labeled with CFSE and stimulated with immobilized mAb anti-CD3. g/ml) in the absence or the presence of PF4 (5 g/ml). After 72-h culture, ϩ Ϫ ϫ 4 3 CFSE-labeled CD4 CD25 T cells (5 10 ) were cultured in the absence [ H]thymidine was added for an additional 16 h. Individual data points ϩ Ϫ ϫ Ͻ (A) or the presence of PF4 (B). CFSE-labeled CD4 CD25 T cells (5 ء Ϯ represent the average of triplicate determinations 1 SD. , p 0.05 (by 4 ϩ ϩ

10 ) mixed with the same number (1:1 ratio) of unlabeled CD4 CD25 Tr by guest on September 23, 2021 t test). Results are representative of four independent experiments for A and cells were cultured in the absence (C) or the presence (D) of PF4. CFSE- three for B. labeled CD4ϩCD25ϩ T cells (5 ϫ 104) were cultured in the absence (E)or the presence of PF4 (F). CFSE-labeled CD4ϩCD25ϩ Tr cells (5 ϫ 104) mixed with the same number (1:1 ratio) of unlabeled CD4ϩCD25Ϫ T cells analyzed by flow cytometry. Allogeneic APCs alone were not used were cultured in the absence (G) or the presence (H) of PF4. After 96-h in this assay, because the levels of autofluorescence they generated culture, cells were harvested and analyzed by flow cytometry. Only viable and the relatively weak stimulus provided greatly limited the num- cells, as determined by propidium iodide staining exclusion, are shown. ber of cell cycles that could be resolved by flow cytometry (data Dashed lines (shown only in A) indicate the boundaries of each division not shown). cycle. The numbers appearing above each peak (0–5) denote each division In four independent experiments, as expected, CD4ϩCD25Ϫ T population, with the undivided T cells (0) residing in the rightmost peak, and the T cells that have divided five times (5) residing in the leftmost peak cells proliferated in response to anti-CD3 (Fig. 5A and Table II), and ending at the first minor tick mark between the 101–102 scale. The leftmost this response was inhibited by PF4 (Fig. 5B and Table II). Unexpect- ϩ ϩ ϩ Ϫ ϩ ϩ peak represents unlabeled CD4 CD25 (C and D) and CD4 CD25 T edly, however, in the presence of unlabeled CD4 CD25 Tr cells, cells (G and H). Results are representative of four independent ϩ Ϫ the proliferation of CD4 CD25 T cells was moderately enhanced experiments. by PF4, rather than suppressed (Fig. 5D and Table II). CD4ϩCD25ϩ Tr cells were unresponsive to anti-CD3 (Fig. 5E and Table II); however, they proliferated in response to this stimulus when PF4 was present (Fig. 5F and Table II). This response was unaffected by the the presence of PF4, CD4ϩCD25ϩ Tr cells lose their potent sup- ϩ Ϫ presence of unlabeled CD4 CD25 T cells (Fig. 5H and Table II). pressive capacity on conventional CD4ϩCD25Ϫ T cells in re- ϩ ϩ Together, these data show that PF4 acts on CD4 CD25 Tr cells to sponse to alloantigen or TCR engagement (anti-CD3; Figs. 4 and stimulate their proliferation (Fig. 5H) and that this response, paradox- 5). Therefore, in this study we report that human PF4 reverts the ϩ ϩ ically, impairs the ability CD4 CD25 Tr cells to inhibit the prolif- anergic Tr cell phenotype and impairs their suppressive activity, ϩ Ϫ eration of CD4 CD25 T cells (Fig. 5D) in response to anti-CD3. suggesting a previously unrecognized role of PF4 in the regulation of human immune responses. Discussion Although it is known that CD4ϩCD25ϩ Tr cells exert their sup- CD4ϩCD25ϩ Tr cells are essential for the maintenance of self- pressive effect by inhibiting IL-2 production and promoting cell tolerance in mouse models (1, 11, 12). A large body of evidence cycle arrest (7, 24), the specific mechanism governing the suppres- suggests that human CD4ϩCD25ϩ T cells share many of the char- sive activity of CD4ϩCD25ϩ Tr cells is still undefined. Signaling acteristics of murine CD4ϩCD25ϩ Tr cells (18). In our experi- through a few receptors appears to be critical for the regulatory mental conditions, PF4 stimulates the proliferation of activity of CD4ϩCD25ϩ Tr cells. A glucocorticoid-induced TNF CD4ϩCD25ϩ regulatory T cells (Fig. 3, B and D). Moreover, in receptor (TNFRSF18) is highly expressed on mouse CD4ϩCD25ϩ The Journal of Immunology 2685

Table II. Responder frequency of CD4ϩCD25Ϫ and CD4ϩCD25ϩ T cells stimulated with anti-CD3 mAb, in the absence or the presence of PF4a

Culture Condition

CD4ϩCD25Ϫ CD4ϩCD25ϩ

ABCDEFGH Division (n) Events PN Events PN Events PN Events PN Events PN Events PN Events PN Events PN

0 704 704 1372 1372 864 864 684 684 2753 2753 1697 1697 2370 2370 1576 1576 1 421 210.50 433 216.50 546 128.00 368 184.00 66 33.00 144 71.75 69 34.50 178 89.00 2 487 121.75 552 138.00 341 85.25 476 119.00 68 16.94 176 44.00 44 11.00 168 42.00 3 745 93.13 699 87.38 486 60.75 680 85.00 87 10.84 254 31.75 50 6.25 220 27.50 4 1375 85.94 1004 62.75 711 44.44 849 53.06 119 7.45 392 24.50 62 3.88 437 27.31 5 936 29.25 666 20.81 535 16.72 347 10.84 80 2.50 225 7.03 18 0.56 241 7.53 P 1–5 540.56 525.44 335.16 451.91 70.73 179.03 56.19 193.34 TP 0–5 1244.5 1897.4 1199.2 1135.9 2823.5 1876.4 2426.2 1769.3 % Div. 43.43 Ϯ 0.6 27.69 Ϯ 0.8 27.95 Ϯ 0.8 39.78 Ϯ 0.7 2.51 Ϯ 1.1 9.54 Ϯ 0.9 2.32 Ϯ 1.2 10.93 Ϯ 0.9

a Data from Fig. 5, A–H, were used to generate the table. The proportion of cells responding to anti-CD3 mAb in the absence (A, C, E, G) or the presence (B, D, F, H) of PF4 was calculated as described in Materials and Methods. Brieﬂy, the number of cells (events) in a given cycle (division (n)) was divided by 2 raised to the power n, to calculate the percentage of original, undivided cells from which they arose (precursor number (PN)). The sum of these gave the total precursor cells for each culture (TP 0–5). The sum of precursors from divisions 1 to 5 (P

1–5) represents the number of precursor T cells which have been proliferating within the 96-h culture. The percent divided (% Div.) represents the proportion [(P 1–5)/(TP 0–5) ϫ 100] of Downloaded from the original CFSE-labeled T cell population induced into cell division. Numbers represent the average values of pooled data from four independent experiments. SDs, relative to the % Div., are reported (Ϯvalue).

Tr cells (25). In a mouse model, glucocorticoid-induced TNF re- lial cell proteoglycans (36). Our results are in agreement with those ceptor stimulation abrogated CD4ϩCD25ϩ Tr cell-mediated sup- of Fleischer et al. (16), who reported half-maximal inhibition of T pression in vitro and in vivo (25, 26). 4-1BB, another TNF receptor cell proliferation at ϳ0.7 ␮M PF4 (e.g., ϳ5 ␮g/ml). The finding by http://www.jimmunol.org/ superfamily member, has been shown to modulate the suppressor Taub et al. (37) that PF4 lacks any capacity to modulate purified ϩ ϩ function of activated, but not resting, CD4 CD25 Tr (27). Li- human T cells does not appear to contradict our present observa- gation of CD28 or blockade of CTLA-4 expressed on tions, because the authors used PF4 at physiological concentrations ϩ ϩ CD4 CD25 Tr cells can also abrogate suppression (28, 29). (Ͻ1 ␮g/ml). Interestingly, PF4-mediated modulation of T cell pro- ϩ ϩ How PF4 modulates CD25 CD4 Tr cell-mediated suppression liferation occurs at the same concentration range (2–10 ␮g/ml) as remains to be determined. As a possible mechanism of action, that required for inhibition of endothelial cells (38) and activation direct signaling of PF4 via CXCR3B, a recently identified spliced of human neutrophils (39) and monocytes (40) by this chemokine. variant of CXCR3 to which PF4 binds with high affinity (30), or Such plasma concentrations of PF4 could occur during heparin another yet to be identified receptor may interfere with the capacity by guest on September 23, 2021 ϩ ϩ therapy, because heparin releases PF4 from endothelial cell gly- of CD25 CD4 Tr cells to deliver to other T cells a negative cosaminoglycans and directly from circulating platelets (41). signal for activation and proliferation. Alternatively, PF4 could Moreover, platelet activation that occurs during and after specific physically interfere (in an antagonistic or synergistic manner) with invasive procedures, such as cardiopulmonary bypass (convention- the interaction of costimulatory molecules on T cells and APCs by ally associated with heparin administration) (42), also leads to in- competing for glycosaminoglycan binding. In our experiments, creased PF4 release into the circulation. Therefore, a transient im- heparin partially affected the proliferative response induced by PF4 pairment of Tr suppressor activity might be postulated in otherwise on the two T cell subsets. It is known that PF4 has a high affinity healthy individuals experiencing acute and severe platelet destruc- for cell surface heparan sulfate proteoglycans, on which it recog- tion, hence possibly explaining why different patient groups ex- nizes a specific structural motif (31). Although proteoglycans gen- posed to heparin become sensitized at such different rates (43, 44). erally serve as coreceptors (32), an intrinsic signaling capacity has Of interest, the immune response in HIT is peculiar in that Ag been reported for syndecan-4 expressed on lymphocytes and formation results from noncovalent interaction of a specific protein monocytes (33). Therefore, direct signaling through proteoglycans (PF4) with a specific drug (heparin) (20). In normal subjects, pep- cannot be ruled out for PF4. It should be considered that PF4 could also affect T cell function indirectly by induction of immunosup- tides derived from PF4 are undoubtedly presented regularly to T pressive cytokines. In line with previous observations (17), we cells without triggering an immune response, either because T cells found that isolated CD4ϩCD25ϩ T cells produced TGF-␤1, but reactive with such peptides have been deleted in the thymus or not IL-10. The levels of both TGF-␤1 and IL-10 were unchanged because they have been tolerized by exposure to PF4 peptides in in the presence of PF4 (Table I), suggesting that neither IL-10 nor the context of class II MHC in the absence of costimulatory mol- ␤ ecules. We propose that in HIT a transient impairment of the sup- TGF- 1 mediates PF4 effects on Tr cells. Future investigation into ϩ ϩ how PF4 signaling is initiated is necessary to fully characterize the pressive capacity of CD4 CD25 Tr cells primes naive T cells, mechanism of its effects. triggering this unusual immune response. Therefore, we hypothe- In our experimental conditions, the effects of PF4 were seen size that in HIT, PF4 is not only the target for the Ab (when using concentrations of PF4 Ͼ1 ␮g/ml (Fig. 1). Subsequent ex- complexed with heparin), but is also a modulator of T cell periments on regulatory and nonregulatory CD4ϩ T cells were activation. performed using PF4 at a concentration of 5 ␮g/ml. This is a high Our observation that PF4 modulates, in an opposite manner, concentration, considering that in physiological conditions only regulatory vs nonregulatory T cells, thus affecting their function, minute quantities (ϳ14 ng/ml) of PF4 are found in human plasma provides new insights into a previously unexpected role for this (34). However, activated platelets, from 1 ml of whole blood, rap- chemokine in regulation of the human immune response. There- idly release PF4 levels in the range of 10 ␮g/ml (35). Moreover, fore, our findings could be relevant to immune complex-mediated significant amounts of PF4 are normally associated with endothe- inflammatory and autoimmune disorders associated with platelet 2686 MODULATORY EFFECT OF PF4 ON T CELLS activation and subsequent release of PF4, among those HIT, sys- 21. Lyons, A. B. 2000. 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