Characterization of human platelet binding of recombinant T cell receptor ligand
© Itakura et al; licensee BioMed Central Ltd. 2010
Received: 15 July 2010
Accepted: 8 November 2010
Published: 8 November 2010
Recombinant T cell receptor ligands (RTLs) are bio-engineered molecules that may serve as novel therapeutic agents for the treatment of neuroinflammatory conditions such as multiple sclerosis (MS). RTLs contain membrane distal α1 plus β1 domains of class II major histocompatibility complex linked covalently to specific peptides that can be used to regulate T cell responses and inhibit experimental autoimmune encephalomyelitis (EAE). The mechanisms by which RTLs impede local recruitment and retention of inflammatory cells in the CNS, however, are not completely understood.
We have recently shown that RTLs bind strongly to B cells, macrophages, and dendritic cells, but not to T cells, in an antigenic-independent manner, raising the question whether peripheral blood cells express a distinct RTL-receptor. Our study was designed to characterize the molecular mechanisms by which RTLs bind human blood platelets, and the ability of RTL to modulate platelet function.
Our data demonstrate that human blood platelets support binding of RTL. Immobilized RTL initiated platelet intracellular calcium mobilization and lamellipodia formation through a pathway dependent upon Src and PI3 kinases signaling. The presence of RTL in solution reduced platelet aggregation by collagen, while treatment of whole blood with RTL prolonged occlusive thrombus formation on collagen.
Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing a major role in inflammation and immunity. This study provides the first evidence that blood platelets express a functional RTL-receptor with a putative role in modulating pathways of neuroinflammation.
Recombinant T cell receptor ligands (RTLs) represent a novel, bio-engineered therapeutic drugs for T cell-mediated autoimmune diseases. RTL molecules consist of the membrane distal α1 plus β1 domains of class II major histocompatibility complex molecules and contain covalently linked peptide antigen to induce immunosuppression by crosslinking to T cell receptor (TCR) in the absence of co-stimulatory signals. By inhibiting autoreactive T cell responses, RTLs have been shown to reverse the clinical and histological signs in experimental autoimmune encephalomyelitis (EAE), although the molecular mechanisms by which RTLs inhibit T cell proliferation and cytokine secretion are still poorly defined. While RTLs displayed preferential binding to murine antigen presenting cells (APCs) such as B cells, macrophages and dendritic cells, but not to T cells, the binding targets expressed on APCs are currently unknown.
Blood platelets are classically considered as key regulators of hemostasis. Platelets, however, are also emerging as modulators in immune responses as well as in the etiology of neuropathologies. Platelets possess a wide array of adhesion receptors and secretory products, consisting of chemokines and cytokines. It has been proposed that platelets partner with leukocytes to amplify the immune response at sites of tissue repair or inflammation[6, 7]. Along these lines, in a murine model of pulmonary acute lung injury, blockade of platelet-derived thromboxane reversed disease progression, while pharmacological inhibition of platelet-leukocyte interactions with P-selectin antibodies reduced pulmonary inflammation[8, 9]. Accordingly, the presence of platelet-specific markers such as P-selectin and platelet microparticles in MS patients[10, 11] suggests that platelets may contribute to the pathophysiology of MS[4, 12]. Thus, pharmacological regulation of platelet function may represent a potential therapeutic strategy for the treatment of neurovascular inflammation.
Materials and methods
Plasma-derived fibrinogen was from Enzyme Research Laboratories, Inc. (South Bend, IN, USA). RTL1000 and RTL551 was synthesized as previously described. Anti-factor XI mAb was generated and purified as described. All other reagents were from Sigma-Aldrich, Inc. (St. Louis, MO, USA) or previously named sources.
Preparation of purified platelets
Human venous blood was collected from healthy volunteers into sodium citrate (final concentration 0.38% vol/vol) and acid/citrate/dextrose (ACD, 10% vol/vol) to purify the platelets as previously described. Briefly, platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 200 g for 20 minutes. The platelets were isolated from PRP by centrifugation at 1000 g for 10 minutes in the presence of prostacyclin (0.1 μg/ml). After centrifugation, purified human platelets were resuspended in modified Tyrode's buffer (129 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCl2; pH 7.3). Mouse platelets were purified as previously described.
Static adhesion assays
Glass coverslips were incubated with a 50 μg/ml solution of RTL1000 or fibrinogen for 1 hour at room temperature. Surfaces were then blocked with denatured fatty acid-free bovine serum albumin (BSA, 5 mg/ml) for 1 hour and washed with phosphate-buffered saline (PBS). Purified human or mouse platelets (2 × 107/ml) were incubated on the protein-coated coverslips at 37°C for 45 minutes. Platelet spreading was imaged using Kohler illuminated Nomarski differential interference contrast (DIC) optics with a Zeiss 63× oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert 200 m microscope (Carl Zeiss). Images were collected and processed using Stallion 4.0 (Intelligent Imaging Innovations Inc, Denver, CO). The degree of platelet adhesion and surface area of bound platelets was quantified using Image J software as previously described.
Fluorescent binding assay
RTL1000 and RTL551 was processed using Zeba™ Desalt Spin Column (Thermo Fisher Scientific, Waltham, MA) for buffer-exchange, retained in 50 mM HEPES according to the manufacturer's instruction and labeled with Alexa Fluor® 488 using Alexa Fluor® 488 labeling kit (Invitrogen, Carlsbad, CA). Labeled RTL1000 and RTL551 (RTL1000-Alexa488 and RTL551-Alexa488, respectively) were processed using the spin column and recovered in 20 mM Tris (pH 8.4). The yield of the fluorescent protein was confirmed by measuring OD480 and OD290. Purified human or mouse platelets were incubated at 37°C for 30 min on fibrinogen-coated glass coverslips followed by washing with PBS. RTL1000-Alexa488 or RTL551-Alexa488 (20 μg/ml) in BSA (1 mg/ml) solution was then loaded over the adherent human or mouse platelets, respectively, and incubated at 37°C for 30 min. Fluorescence was recorded with a Zeiss Axiovert inverted fluorescent microscope.
Single platelet Ca2+measurements
Platelets were loaded with 15 μM Oregon Green BAPTA1-AM (Molecular Probes, Eugene, OR) as previously described. Loaded platelets were allowed to sediment onto RTL- or fibrinogen-coated coverslips over a period of 30 min at 37°C. Fluorescent intensities in single platelets were recorded and analyzed using a custom Matlab program.
Western blotting and phosphorylation studies
Purified human platelets were incubated for 45 minutes on protein-coated 24 well plates and lysed with an ice-cold lysis buffer (20 mM Tris, 300 mM NaCl, 2 mM EGTA, 2 mM EDTA, 2% NP-40 pH 7.5, containing Roche Complete Protease Inhibitor Cocktail). Lysates were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 1 hour prior to incubation with primary antibodies overnight at 4°C. Following washing with TBS-T, membranes were incubated with the appropriate secondary antibodies. Subsequent immunoblotting was carried out as previously described.
Platelet aggregation and clotting time measurements
Platelet aggregation was assayed using 300 μl of washed platelets (2 × 108/ml) in a Chrono-Log aggregometer (Chrono-Log, Havertown, PA) with continuous stirring at 1200 rpm at 37°C. Activated partial thromboplastin time (aPTT) of pooled human platelet-poor plasma (PPP) and recalcification time of PPP were measured with a KC4 Coagulation Analyzer (Trinity Biotech PLC, Co Wicklow, Ireland). Samples were pre-treated with either vehicle, RTL1000 (10 μg/ml), an anti-FXI mAb (14E11, 20 μg/ml) or tissue factor (TF, 1 pM) for 3 min prior to the addition of 16.6 mM CaCl2 for recalcification times or aPTT reagent (Helena Laboratories, Beaumont, TX) followed by 6.6 mM CaCl2 for aPTT tests. In each test, clotting time was measured following the addition of CaCl2 as previously described.
Capillary occlusion assay
Capillary tubes were coated with fibrillar collagen, aligned vertically and connected to a reservoir as previously described. Sodium-citrate (0.38% w/v) anticoagulated whole blood was sequentially supplemented with 7.5 mM Ca2+ and 3.75 mM Mg2+. Capillary flow was driven by the force of gravity, and the height of the sample reservoir was regulated in order to produce an initial shear rate of 300 s-1 as previously described.
Analysis of data
Data are shown as means ± SEM. Statistical significance of differences between means was determined by ANOVA. If means were shown to be significantly different, multiple comparisons were performed by the Tukey test. Probability values of P < 0.05 were selected to be statistically significant.
Characterization of RTL as a ligand for platelets
RTL supports platelet actin cytoskeletal reorganization and Ca2+mobilization
Characterization of the platelet signaling cascade downstream of RTL
Effects of inhibitors and antagonists on platelet surface area
Platelet adhesion (cells/mm2 × 102)
Surface area (μm2)
44.9 ± 1.1
52.8 ± 1.6
35.5 ± 3.1
31.8 ± 1.2
33.4 ± 0.5
34.3 ± 0.7
34.5 ± 3.9
32.1 ± 2.2
26.4 ± 0.8
7.9 ± 1.5
17.6 ± 1.8
16.7 ± 2.3
Src kinase inhibitor
26.4 ± 0.8
48.4 ± 1.9
23.7 ± 2.0
20.0 ± 1.5
Syk kinase inhibitor
36.1 ± 0.5
44.0 ± 0.4
19.5 ± 1.9
25.0 ± 1.4
PI3 kinase inhibitor
29.0 ± 0.5
30.8 ± 0.9
26.3 ± 1.7
17.3 ± 1.4
protein kinase C inhibitor
36.7 ± 0.5
53.7 ± 1.7
19.4 ± 2.3
27.3 ± 1.6
intracellular Ca2+ chelator
11.4 ± 1.1
13.2 ± 1.2
15.5 ± 2.1
17.2 ± 1.4
Effect of RTL on platelet aggregation, coagulation and occlusive thrombus formation
Next we determined the effect of RTL on the initiation of coagulation. RTL1000 did not affect the activated partial prothrombin time (aPTT) or the clotting time of platelet-poor plasma (PPP) initiated by the addition of excess molar Ca2+ (Figure 7A and 7B, respectively). In contrast, the addition of the anti-FXI mAb, 14E11, prolonged the aPTT (Figure 7A), while the addition of tissue factor (TF, 1 pM) drastically reduced clotting time in recalcified plasma (Figure 7B).
To assess the effect of RTL on platelet function, a solution of purified human platelets was treated with RTL under constant stirring in an aggregometer. Platelet aggregation induced by the platelet agonist, collagen, was inhibited by the addition of RTL1000 (29% aggregation vs. 20% aggregation after 2 min in the presence of vehicle or RTL1000, respectively; Figure 7C). Addition of RTL1000 alone failed to initiate either platelet shape changes or aggregation (data not shown).
Multiple sclerosis is regarded as a prototypical disease state resultant of neuroinflammation. RTLs have been engineered to augment antigen-driven immunosuppression of autoreactive T cells by presenting TCR ligands (self-antigen plus MHC molecule) in the absence of co-stimulatory signals that are normally supplemented by antigen presenting cells (APCs). RTLs have been shown to reduce T cell-driven encephalitogenicity and both clinical and histological signs of EAE. Previous studies suggested that RTLs confer a therapeutic benefit via the downregulation of T cell-mediated inflammation and by modifying the permeability of the blood-brain barrier in a murine model of EAE or ischemic stroke. RTLs were found to decrease the levels of systemic chemokines and adhesion molecules on the CNS endothelium as well as cytokines that switch on anti-inflammatory effectors rather than increasing antibody production by B cells. Although RTLs were originally thought to act as a partial agonist via TCR, our recent study demonstrated the inability of splenic CD3+ T cells to bind RTLs. Rather, we found that RTLs bound APC populations including B cells, macrophages, and dendritic cells, in an antigenic-independent manner. Perhaps RTL-armed splenic APCs suppress the transfer of EAE by antigen-stimulated T cells. Alternatively, peripheral blood cells may bind RTL through an unknown surface receptor distinct from TCR, indirectly inducing T cell tolerance against self-antigen. Our study was designed to determine whether peripheral blood cell populations, such as platelets, can bind to RTL, thus offering a level of redundancy to APCs.
Platelets, well-known regulators of hemostasis and thrombosis, have been implicated in playing an essential role in inflammation and immunity[5, 7]. Activated platelets shed granules and microparticles which contain a variety of adhesive molecules and immunomodulatory factors, resulting in the localized recruitment of immune cells under shear. Abnormal platelet activation (microparticle presence, P-selectin expression, and increased circulating aggregates) has been documented in MS patients. A recent gene microarray study revealed that the platelet αIIbβ3 receptor was transcriptionally upregulated in MS chronic lesions. Perhaps RTL may modulate the pathophysiological role that platelets play in neurodegenerative diseases such as MS.
A number of platelet ligands and pathways have been identified that negatively regulate platelet function. For instance, recent studies have shown negative regulatory roles for the platelet ligands semaphorin 3A, histone H1, thrombospondin-1, and low-density lipoprotein. Our current study provides evidence that RTL, in addition to serving as a platelet ligand, may serve to negatively regulate agonist-induced platelet function, as evidenced by the fact that RTL down-regulated platelet aggregation to collagen. Moreover, our study provides evidence that RTL inhibits occlusive thrombus formation on collagen-coated surfaces under physiologically relevant pressure gradients.
Among various platelet surface RTL-receptors, one potential candidate we identified was CD40, a co-stimulatory molecule that regulates lymphocyte signaling upon antigen presentation. CD40 is known to contribute to the initiation, activation, and amplification of immune responses. CD40-CD40 ligand (CD40L, or CD154) interactions are well described in MS, as CD40L+ T cells are known to be greatly increased in MS patients[29, 30]. Moreover, CD40 is expressed on platelets and APCs, but not T cells, in accord with the peripheral blood populations we have shown to bind RTL. We therefore tested the hypothesis that CD40 was the platelet receptor for RTL. Our preliminary findings demonstrated that purified platelets from CD40-/- mice bind to RTL at the same level as compared to platelets from wild type mice (unpublished observations, AI and OJTM), arguing against a role for CD40 as an RTL-receptor on mouse platelets. Our future efforts will be focused on identifying the putative RTL-receptor on peripheral blood platelets. The characterization of the molecular mechanisms by which RTL binds to peripheral blood cells may better the understanding of the pharmacokinetics of RTL drug delivery as well as the further development of RTL for therapy in autoimmune diseases.
The authors wish to thank J. Pang for experimental assistance. Dr. Sinha is a Postdoctoral Fellow of the National Multiple Sclerosis Society. This work was supported in part by the National Multiple Sclerosis Society (FG1749-A-1, RG3794-A-4 and RG3794-B-6), the National Institute of Health Grants T32 HL00778118, NS47661 and AI43960, the American Heart Association (09PRE2230117, 0910025G), and the Biomedical Laboratory R&D Service, Department of Veterans Affairs. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Multiple Sclerosis Society, American Heart Association, the National Institutes of Health, or the Department of Veterans Affairs.
- Offner H, Sinha S, Wang C, Burrows GG, Vandenbark AA: Recombinant T cell receptor ligands: immunomodulatory, neuroprotective and neuroregenerative effects suggest application as therapy for multiple sclerosis. Rev Neurosci. 2008, 19: 327-339.PubMed CentralView ArticlePubMed
- Sinha S, Subramanian S, Emerson-Webber A, Lindner M, Burrows GG, Grafe M, Linington C, Vandenbark AA, Bernard CC, Offner H: Recombinant TCR ligand reverses clinical signs and CNS damage of EAE induced by recombinant human MOG. J Neuroimmune Pharmacol. 2010, 5: 231-239. 10.1007/s11481-009-9175-1.PubMed CentralView ArticlePubMed
- Sinha S, Miller L, Subramanian S, McCarty OJ, Proctor T, Meza-Romero R, Huan J, Burrows GG, Vandenbark AA, Offner H: Binding of recombinant T cell receptor ligands (RTL) to antigen presenting cells prevents upregulation of CD11b and inhibits T cell activation and transfer of experimental autoimmune encephalomyelitis. J Neuroimmunol. 2010, 225: 52-61. 10.1016/j.jneuroim.2010.04.013.PubMed CentralView ArticlePubMed
- Horstman LL, Jy W, Ahn YS, Zivadinov R, Maghzi AH, Etemadifar M, Steven Alexander J, Minagar A: Role of platelets in neuroinflammation: a wide-angle perspective. J Neuroinflammation. 2010, 7: 10-10.1186/1742-2094-7-10.PubMed CentralView ArticlePubMed
- Smyth SS, McEver RP, Weyrich AS, Morrell CN, Hoffman MR, Arepally GM, French PA, Dauerman HL, Becker RC: Platelet functions beyond hemostasis. J Thromb Haemost. 2009, 7: 1759-1766. 10.1111/j.1538-7836.2009.03586.x.View ArticlePubMed
- Evangelista V, Manarini S, Coller BS, Smyth SS: Role of P-selectin, beta2-integrins, and Src tyrosine kinases in mouse neutrophil-platelet adhesion. J Thromb Haemost. 2003, 1: 1048-1054. 10.1046/j.1538-7836.2003.00214.x.View ArticlePubMed
- McNicol A, Israels SJ: Beyond hemostasis: the role of platelets in inflammation, malignancy and infection. Cardiovasc Hematol Disord Drug Targets. 2008, 8: 99-117. 10.2174/187152908784533739.View ArticlePubMed
- Zarbock A, Singbartl K, Ley K: Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation. J Clin Invest. 2006, 116: 3211-3219. 10.1172/JCI29499.PubMed CentralView ArticlePubMed
- Pitchford SC, Momi S, Giannini S, Casali L, Spina D, Page CP, Gresele P: Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation. Blood. 2005, 105: 2074-2081. 10.1182/blood-2004-06-2282.View ArticlePubMed
- Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, Langer-Gould A, Strober S, Cannella B, Allard J, et al: Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med. 2002, 8: 500-508. 10.1038/nm0502-500.View ArticlePubMed
- Sheremata WA, Jy W, Horstman LL, Ahn YS, Alexander JS, Minagar A: Evidence of platelet activation in multiple sclerosis. J Neuroinflammation. 2008, 5: 27-10.1186/1742-2094-5-27.PubMed CentralView ArticlePubMed
- Segal JB, Powe NR: Prevalence of immune thrombocytopenia: analyses of administrative data. J Thromb Haemost. 2006, 4: 2377-2383. 10.1111/j.1538-7836.2006.02147.x.View ArticlePubMed
- Burrows GG, Chang JW, Bachinger HP, Bourdette DN, Offner H, Vandenbark AA: Design, engineering and production of functional single-chain T cell receptor ligands. Protein Eng. 1999, 12: 771-778. 10.1093/protein/12.9.771.View ArticlePubMed
- White-Adams TC, Berny MA, Patel IA, Tucker EI, Gailani D, Gruber A, McCarty OJT: Laminin promotes coagulation and thrombus formation in a factor XII-dependent manner. Journal of Thrombosis and Haemostasis. 2010, 8: 1295-1301. 10.1111/j.1538-7836.2010.03850.x.PubMed CentralView ArticlePubMed
- Berny MA, White TC, Tucker EI, Bush-Pelc LA, Di Cera E, Gruber A, McCarty OJ: Thrombin mutant W215A/E217A acts as a platelet GPIb antagonist. Arterioscler Thromb Vasc Biol. 2008, 28: 329-334. 10.1161/ATVBAHA.107.156273.View ArticlePubMed
- McCarty OJ, Calaminus SD, Berndt MC, Machesky LM, Watson SP: von Willebrand factor mediates platelet spreading through glycoprotein Ib and alpha(IIb)beta3 in the presence of botrocetin and ristocetin, respectively. J Thromb Haemost. 2006, 4: 1367-1378. 10.1111/j.1538-7836.2006.01966.x.View ArticlePubMed
- White TC, Berny MA, Tucker EI, Urbanus RT, de Groot PG, Fernandez JA, Griffin JH, Gruber A, McCarty OJ: Protein C supports platelet binding and activation under flow: role of glycoprotein Ib and apolipoprotein E receptor 2. J Thromb Haemost. 2008, 6: 995-1002. 10.1111/j.1538-7836.2008.02979.x.View ArticlePubMed
- McCarty OJ, Zhao Y, Andrew N, Machesky LM, Staunton D, Frampton J, Watson SP: Evaluation of the role of platelet integrins in fibronectin-dependent spreading and adhesion. J Thromb Haemost. 2004, 2: 1823-1833. 10.1111/j.1538-7836.2004.00925.x.View ArticlePubMed
- Berny MA, Patel IA, White-Adams TC, Simonson P, Gruber A, Rugonyi S, McCarty OJ: Rational Design of an Ex Vivo Model of Thrombosis. Cellular and Molecular Bioengineering. 2010, 3: 187-189. 10.1007/s12195-010-0103-5.View Article
- Sinha S, Subramanian S, Miller L, Proctor TM, Roberts C, Burrows GG, Vandenbark AA, Offner H: Cytokine switch and bystander suppression of autoimmune responses to multiple antigens in experimental autoimmune encephalomyelitis by a single recombinant T-cell receptor ligand. J Neurosci. 2009, 29: 3816-3823. 10.1523/JNEUROSCI.5812-08.2009.PubMed CentralView ArticlePubMed
- Sinha S, Subramanian S, Proctor TM, Kaler LJ, Grafe M, Dahan R, Huan J, Vandenbark AA, Burrows GG, Offner H: A promising therapeutic approach for multiple sclerosis: recombinant T-cell receptor ligands modulate experimental autoimmune encephalomyelitis by reducing interleukin-17 production and inhibiting migration of encephalitogenic cells into the CNS. J Neurosci. 2007, 27: 12531-12539. 10.1523/JNEUROSCI.3599-07.2007.View ArticlePubMed
- Subramanian S, Zhang B, Kosaka Y, Burrows GG, Grafe MR, Vandenbark AA, Hurn PD, Offner H: Recombinant T cell receptor ligand treats experimental stroke. Stroke. 2009, 40: 2539-2545. 10.1161/STROKEAHA.108.543991.PubMed CentralView ArticlePubMed
- Wang C, Mooney JL, Meza-Romero R, Chou YK, Huan J, Vandenbark AA, Offner H, Burrows GG: Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation. J Immunol. 2003, 171: 1934-1940.View ArticlePubMed
- Kashiwagi H, Shiraga M, Kato H, Kamae T, Yamamoto N, Tadokoro S, Kurata Y, Tomiyama Y, Kanakura Y: Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A. Blood. 2005, 106: 913-921. 10.1182/blood-2004-10-4092.View ArticlePubMed
- Soslau G, Prest PJ, Class R, Jost M, Mathews L: Inhibition of gamma-thrombin-induced human platelet aggregation by histone H1subtypes and H1.3 fragments. Platelets. 2009, 20: 349-356. 10.1080/09537100903047745.View ArticlePubMed
- Zaslavsky A, Baek KH, Lynch RC, Short S, Grillo J, Folkman J, Italiano JE, Ryeom S: Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis. Blood. 2010, 115: 4605-4613. 10.1182/blood-2009-09-242065.PubMed CentralView ArticlePubMed
- Korporaal SJ, Koekman CA, Verhoef S, van der Wal DE, Bezemer M, Van Eck M, Akkerman JW: Downregulation of platelet responsiveness upon contact with LDL by the protein-tyrosine phosphatases SHP-1 and SHP-2. Arterioscler Thromb Vasc Biol. 2009, 29: 372-379. 10.1161/ATVBAHA.108.173278.View ArticlePubMed
- Danese S, de la Motte C, Reyes BM, Sans M, Levine AD, Fiocchi C: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification. J Immunol. 2004, 172: 2011-2015.View ArticlePubMed
- Chitnis T, Khoury SJ: Role of costimulatory pathways in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. J Allergy Clin Immunol. 2003, 112: 837-849. 10.1016/j.jaci.2003.08.025. quiz 850View ArticlePubMed
- Munroe ME: Functional roles for T cell CD40 in infection and autoimmune disease: the role of CD40 in lymphocyte homeostasis. Semin Immunol. 2009, 21: 283-288. 10.1016/j.smim.2009.05.008.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.