text:
Citrullination: A Specific Target for the Autoimmune Response in Rheumatoid Arthritis
David A. Fox
J Immunol July 1, 2015, 195 (1) 5-7; DOI: https://doi.org/10.4049/jimmunol.1501021
Division of Rheumatology and Clinical Autoimmunity Center of Excellence, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109
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Rheumatoid arthritis (RA) was one of the first diseases to be classified as autoimmune, based on the prevalence of an autoantibody, rheumatoid factor, which was first identified in the 1940s. However, the diagnostic value and pathogenic significance of rheumatoid factor have been constrained by its striking nonspecificity for RA, in view of its presence in many other chronic diseases and in large numbers of apparently healthy individuals, especially the elderly. Beginning in the 1960s, other autoantibody specificities were described in RA, primarily in immunofluorescence assays. These were termed "anti–perinuclear factor" or "anti-keratin" Abs, and a subset of these were found to react with fillagrin. These autoantibodies were proposed to be more specific for RA than was rheumatoid factor, but the nature of the epitope recognized was unclear.
In a landmark paper published in 1998 in the Journal of Clinical Investigation, Schellekens et al. (1) from the laboratory of Walther J. van Venrooij in Nijmegen showed that the unconventional amino acid citrulline was an essential component of the determinant recognized by anti-fillagrin autoantibodies in RA. This paper, thus far cited well over 500 times, has opened a new era in the diagnosis and classification of RA and has led to important new insights into RA pathogenesis and the role of posttranslational modifications of proteins in autoantigenicity. Citrullination of a variety of proteins is emerging as an essential component of inflammation in a variety of diseases; thus, both alteration of protein functions by citrullination and the interactions of anti-citrulline autoantibodies with these proteins now command increased attention in efforts to understand the etiology and pathogenesis of RA. Citrullination and autoantibodies to citrulline-containing proteins also provided early biomarkers of preclinical RA that now allow testing of strategies to prevent RA.
What is citrullination?
Citrulline is an amino acid that is not encoded in the genome, but is instead created by posttranslational modification of arginine residues in proteins, catalyzed by the enzyme peptidyl arginine deiminase (PAD), which exists in several isoforms (2). A wide variety of proteins can be citrullinated, including histones, cell surface receptors, and extracellular connective tissue components. Citrullination turns out to be essential for many important biological processes, for example neutrophil NETosis, which depends on citrullination of histones.
What did Schellekens and colleagues show in their 1998 Journal of Clinical Investigation paper?
Schellekens et al. (1) synthesized a variety of peptides corresponding to potential immunogenic epitopes of fillagrin, substituting citrulline for arginine in some of the peptides. Using immunoblotting, they showed that most of the anti-fillagrin reactivity in RA sera depended on the presence of citrulline instead of arginine. They then affinity purified citrulline-reactive autoantibodies from these RA sera and demonstrated that these autoantibodies were reactive in the immunofluorescence assays that had been used to describe "anti–perinuclear factor" and "anti-keratin" specificities. Moreover, they showed that anti-citrulline autoantibodies were present in 76% of RA sera but in <5% of sera from control groups, including patients with other autoimmune diseases (1). Multiple citrullinated peptide epitopes of fillagrin were recognized by the RA sera, foreshadowing subsequent demonstrations that a wide array of citrullinated proteins is recognized by RA-associated autoantibodies.
The impact of anti-citrullinated protein Abs on the diagnosis and management of RA
ACPA and anti-CCP are abbreviations commonly used to refer to anti-citrullinated protein Abs that are measured in widely available clinical laboratory assays. ACPAs can react with a variety of citrullinated proteins, such as vimentin, fibrinogen, α enolase, fillagrin, and type II collagen (3, 4). Most clinical assays currently in use employ cyclic citrullinated peptides as the target Ag, which may lead to a small percentage of false-negative results (5, 6). The level of sensitivity and specificity of anti-citrulline positivity reported in the 1998 Journal of Clinical Investigation paper (1) has held up well during the subsequent 17 years. Occasionally, tests for ACPAs are positive in patients with tuberculosis (7), but the specificity for RA has led to the incorporation of ACPAs into criteria for the classification of RA, which can also be used for early diagnosis of RA (8). ACPA positivity predicts more severe disease as defined by greater destruction of cartilage and bone (9–12), and RA patients are now commonly subdivided into those who are ACPA+ versus ACPA−. ACPA is superior to rheumatoid factor as a predictor of RA severity in the joints, but rheumatoid factor is more tightly associated with extra-articular manifestations such as rheumatoid nodules (13–15). Increased mortality in RA is most prominent in patients with both rheumatoid factor and ACPAs (16). Given all of this information, the presence of ACPAs is viewed as an indication for more aggressive management of patients with RA (17).
Citrullination and ACPAs in the pathogenesis of RA
Citrullination of proteins is widespread in inflamed tissue, for example in synovial tissue in both RA and other arthritides (18). Thus, proinflammatory effects of citrullination that are independent of ACPAs could influence RA and many other diseases. Indeed, in the mouse RA model of collagen-induced arthritis, inhibition of PAD reduces disease severity even though collagen-induced arthritis is not dependent on ACPAs (19). Inhibition of PAD also reduces vascular damage in a murine model of systemic lupus (20).
In another experimental system, it was shown that the chemokine CXCL5, when citrullinated, acquires the ability to bind to CXCR1 and CXCR2, which enables it to attract monocytes and enhance joint inflammation (21). Additionally, citrullination of calreticulin enhances the ability of this molecule to function as a receptor for the RA-associated MHC allele that is termed the shared epitope. This binding interaction is a novel mechanism for the shared epitope to enhance joint inflammation and damage that is distinct from the Ag-presenting function of MHC molecules (22).
Whereas ACPAs contribute to joint inflammation by forming immune complexes and engaging Fc receptors (23), citrullination can also contribute specifically to the pathogenesis of RA through unique mechanisms that depend on ACPAs binding to their citrullinated targets. For example, ACPAs that bind to citrullinated vimentin on the surface of osteoclasts activate the osteoclasts to promote bone resorption (24). This mechanism may explain the observation that osteopenia is present in ACPA+ individuals who do not yet have clinical RA (25). ACPAs also affect NETosis of neutrophils, a process that is enhanced in RA. NETs contain citrullinated Ags, and ACPAs not only bind to NETs but also enhance the rate of NETosis. NETs can directly activate synovial fibroblasts to secrete proinflammatory cytokines (26). Patients with RA can also have autoantibodies to PAD, which, remarkably, activate the PAD enzyme to augment citrullination (27). Taken together, these and other observations reveal a complex and interacting series of mechanisms through which targeted citrullination of proteins enhances inflammatory pathways, and the presence of ACPAs confers unique additional augmentation of proinflammatory and tissue-destructive processes.
ACPAs in the earliest stages of RA: clues to etiology and prevention?
In patients with early undifferentiated arthritis, ACPAs are a powerful predictor of progression to frank RA (28). Moreover, ACPAs are found in serum up to 10 years before the onset of RA (29), at a time when synovitis and citrullination of synovial proteins are not evident (30). In pre-RA, ACPAs already exhibit a pattern of decreased galactosylation that has been found in total IgG in active RA (31). The presence of ACPAs is associated with the shared epitope HLA-DR allele that is the most important genetic risk factor for RA (32), and is also associated with two other established risk factors for RA, smoking and periodontitis (33). These observations have led to hypotheses that the formation of ACPAs provides a link between the risk factors for RA and the pathway that ultimately leads to clinical arthritis. Notably, smoking can lead to formation of citrullinated proteins in the lung, the presence of ACPAs is associated with lung inflammation in RA, and ACPAs can be measured in sputum not only in those with RA, but even in individuals at risk for RA who do not yet have ACPAs in their serum (34–38). Periodontitis, which is bidirectionally associated with RA, is driven by the bacterium Porphyromonas gingivalis, which uniquely among oral flora expresses PAD and thus generates local protein citrullination, leading to formation of ACPAs (33, 39–41). These findings give important momentum to the notion that RA begins in organs and tissues other than the joints, such as the lung or periodontal tissue, with citrullination and ACPA formation being early and critical events in its pathogenesis. Given that ACPAs can be a very early biomarker for pre-RA and are present in some first-degree relatives of RA patients (who are known to have an increased risk for developing RA) (42), trials for prevention of RA can now be designed around the measurement of ACPAs and ACPA-triggered inflammatory pathways.
Concluding perspectives
The discovery of ACPAs by van Venrooij's group was a landmark event that has changed our approach to the diagnosis, classification, and management of RA, an autoimmune disease that has enormous impact on millions of people. It also has opened up new understanding of the pathogenesis of RA and may be getting us closer to grasping the etiology of RA and strategies for prevention. Moreover, conversion of arginine to citrulline turns out not to be the only posttranslational protein modification that engenders autoimmunity: carbamylation (43) and formation of malondialdehyde–acetaldehyde adducts (44) are other examples of posttranslational modifications that are recognized by recently defined RA-associated autoantibodies. Important questions remain for immunologists regarding how posttranslationally modified proteins are handled and recognized during immune repertoire selection and at peripheral checkpoints for establishment or maintenance of tolerance to these determinants.
The author has no financial conflicts of interest.
ACPA
anti-citrullinated protein Ab
peptidyl arginine deiminase
rheumatoid arthritis.
Schellekens G. A.,
B. A. de Jong,
F. H. van den Hoogen,
L. B. van de Putte,
W. J. van Venrooij
. 1998. Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies. J. Clin. Invest. 101: 273–281.
van Boekel M. A. M.,
. 2003. Modifications of arginines and their role in autoimmunity. Autoimmun. Rev. 2: 57–62.
van Beers J. J.,
A. Willemze,
J. J. Jansen,
G. H. Engbers,
M. Salden,
J. Raats,
J. W. Drijfhout,
A. H. van der Helm-van Mil,
R. E. Toes,
G. J. Pruijn
. 2013. ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression. Arthritis Res. Ther. 15: R140.
Szarka E.,
F. Babos,
A. Magyar,
K. Huber,
Z. Szittner,
K. Papp,
J. Prechl,
J. Pozsgay,
Z. Neer,
M. Ádori,
. 2014. Recognition of new citrulline-containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients. Immunology 141: 181–191.
Wagner C. A.,
J. Sokolove,
L. J. Lahey,
C. Bengtsson,
S. Saevarsdottir,
L. Alfredsson,
M. Delanoy,
T. M. Lindstrom,
R. P. Walker,
R. Bromberg,
. 2015. Identification of anticitrullinated protein antibody reactivities in a subset of anti-CCP-negative rheumatoid arthritis: association with cigarette smoking and HLA-DRB1 "shared epitope" alleles. Ann. Rheum. Dis. 74: 579–586.
Montgomery A. B.,
P. J. Venables,
B. A. Fisher
. 2013. The case for measuring antibodies to specific citrullinated antigens. Expert Rev. Clin. Immunol. 9: 1185–1192.
Lima I.,
R. C. Oliveira,
A. Atta,
S. Marchi,
L. Barbosa,
E. Reis,
M. G. Reis,
M. B. Santiago
. 2013. Antibodies to citrullinated peptides in tuberculosis. Clin. Rheumatol. 32: 685–687.
Aletaha D.,
T. Neogi,
A. J. Silman,
J. Funovits,
D. T. Felson,
C. O. Bingham III.,
N. S. Birnbaum,
G. R. Burmester,
V. P. Bykerk,
M. D. Cohen,
. 2010. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann. Rheum. Dis. 69: 1580–1588.
Kroot E. J.,
M. A. van Leeuwen,
H. Swinkels,
M. van't Hof,
M. H. van Rijswijk,
W. J. van Venrooij,
P. L. van Riel
. 2000. The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis. Arthritis Rheum. 43: 1831–1835.
van der Linden M. P.,
D. van der Woude,
A. Ioan-Facsinay,
E. W. Levarht,
G. Stoeken-Rijsbergen,
T. W. Huizinga,
A. H. van der Helm-van Mil
. 2009. Value of anti-modified citrullinated vimentin and third-generation anti-cyclic citrullinated peptide compared with second-generation anti-cyclic citrullinated peptide and rheumatoid factor in predicting disease outcome in undifferentiated arthritis and rheumatoid arthritis. Arthritis Rheum. 60: 2232–2241.
Renner N.,
G. Krönke,
J. Rech,
M. Uder,
R. Janka,
L. Lauer,
D. Paul,
B. Herz,
P. Schlechtweg,
F. F. Hennig,
. 2014. Anti-citrullinated protein antibody positivity correlates with cartilage damage and proteoglycan levels in patients with rheumatoid arthritis in the hand joints. Arthritis Rheum. (Munch) 66: 3283–3288.
Lindqvist E.,
K. Eberhardt,
D. Heinegård,
T. Saxne
. 2005. Prognostic laboratory markers of joint damage in rheumatoid arthritis. Ann. Rheum. Dis. 64: 196–201.
Kastbom A.,
G. Strandberg,
A. Lindroos,
T. Skogh
. 2004. Anti-CCP antibody test predicts the disease course during 3 years in early rheumatoid arthritis (the Swedish TIRA project). Ann. Rheum. Dis. 63: 1085–1089.
De Rycke L.,
I. Peene,
I. E. Hoffman,
E. Kruithof,
A. Union,
L. Meheus,
K. Lebeer,
B. Wyns,
C. Vincent,
H. Mielants,
. 2004. Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations. Ann. Rheum. Dis. 63: 1587–1593.
Hecht C.,
M. Englbrecht,
S. Schmidt,
E. Araujo,
K. Engelke,
S. Finzel,
. 2014. Additive effect of anti-citrullinated protein antibodies and rheumatoid factor on bone erosions in patients with RA. Ann. Rheum. Dis. DOI: 10.1136/annrheumdis-2014-205428.
Humphreys J. H.,
J. A. van Nies,
J. Chipping,
T. Marshall,
D. P. Symmons,
S. M. Verstappen
. 2014. Rheumatoid factor and anti-citrullinated protein antibody positivity, but not level, are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts. Arthritis Res. Ther. 16: 483.
Sakkas L. I.,
D. P. Bogdanos,
C. Katsiari,
C. D. Platsoucas
. 2014. Anti-citrullinated peptides as autoantigens in rheumatoid arthritis-relevance to treatment. Autoimmun. Rev. 13: 1114–1120.
Vossenaar E. R.,
T. J. Smeets,
M. C. Kraan,
J. M. Raats,
P. P. Tak
. 2004. The presence of citrullinated proteins is not specific for rheumatoid synovial tissue. Arthritis Rheum. 50: 3485–3494.
Willis V. C.,
A. M. Gizinski,
N. K. Banda,
C. P. Causey,
B. Knuckley,
K. N. Cordova,
Y. Luo,
B. Levitt,
M. Glogowska,
P. Chandra,
. 2011. N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-l-ornithine amide, a protein arginine deiminase inhibitor, reduces the severity of murine collagen-induced arthritis. J. Immunol. 186: 4396–4404.
Knight J. S.,
W. Zhao,
W. Luo,
V. Subramanian,
A. A. O'Dell,
S. Yalavarthi,
J. B. Hodgin,
D. T. Eitzman,
P. R. Thompson,
M. J. Kaplan
. 2013. Peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus. J. Clin. Invest. 123: 2981–2993.
Yoshida K.,
O. Korchynskyi,
P. P. Tak,
T. Isozaki,
J. H. Ruth,
P. L. Campbell,
D. L. Baeten,
D. M. Gerlag,
M. A. Amin,
A. E. Koch
. 2014. Citrullination of epithelial neutrophil-activating peptide 78/CXCL5 results in conversion from a non-monocyte-recruiting chemokine to a monocyte-recruiting chemokine. Arthritis Rheum. (Munch) 66: 2716–2727.
Ling S.,
E. N. Cline,
T. S. Haug,
D. A. Fox,
J. Holoshitz
. 2013. Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling. Arthritis Rheum. 65: 618–626.
van Venrooij W. J.,
J. J. van Beers,
. 2011. Anti-CCP antibodies: the past, the present and the future. Nat Rev Rheumatol 7: 391–398.
Harre U.,
D. Georgess,
H. Bang,
A. Bozec,
R. Axmann,
E. Ossipova,
P. J. Jakobsson,
W. Baum,
F. Nimmerjahn,
E. Szarka,
. 2012. Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin. J. Clin. Invest. 122: 1791–1802.
Kleyer A.,
B. Manger,
M. Krieter,
F. Faustini,
A. J. Hueber,
U. Harre,
. 2014. Bone loss before the clinical onset of rheumatoid arthritis in subjects with anticitrullinated protein antibodies. Ann. Rheum. Dis. 73: 854–860.
Khandpur R.,
C. Carmona-Rivera,
A. Vivekanandan-Giri,
A. Gizinski,
J. S. Knight,
S. Friday,
S. Li,
R. M. Patel,
. 2013. NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci. Transl. Med. 5: 178ra140.
Darrah E.,
J. T. Giles,
M. L. Ols,
H. G. Bull,
F. Andrade,
A. Rosen
. 2013. Erosive rheumatoid arthritis is associated with antibodies that activate PAD4 by increasing calcium sensitivity. Sci. Transl. Med. 5: 186ra165.
van Gaalen F. A.,
S. P. Linn-Rasker,
C. L. Verweij,
T. W. Huizinga
. 2004. Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid arthritis in patients with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum. 50: 709–715.
Arkema E. V.,
B. L. Goldstein,
W. Robinson,
C. A. Wagner,
S. Malspeis,
B. Rosner,
F. Grodstein,
E. W. Karlson,
K. H. Costenbader
. 2013. Anti-citrullinated peptide autoantibodies, human leukocyte antigen shared epitope and risk of future rheumatoid arthritis: a nested case-control study. Arthritis Res. Ther. 15: R159.
de Hair M. J.,
M. G. van de Sande,
T. H. Ramwadhdoebe,
M. Hansson,
R. Landewé,
C. van der Leij,
M. Maas,
G. Serre,
D. van Schaardenburg,
L. Klareskog,
. 2014. Features of the synovium of individuals at risk of developing rheumatoid arthritis: implications for understanding preclinical rheumatoid arthritis. Arthritis Rheum. (Munch) 66: 513–522.
Rombouts Y.,
E. Ewing,
L. A. van de Stadt,
M. H. Selman,
L. A. Trouw,
A. M. Deelder,
M. Wuhrer,
H. U. Scherer
. 2015. Anti-citrullinated protein antibodies acquire a pro-inflammatory Fc glycosylation phenotype prior to the onset of rheumatoid arthritis. Ann. Rheum. Dis. 74: 234–241.
J. van Aken,
G. M. Schreuder,
E. Zanelli,
R. R. de Vries
. 2004. Association between HLA class II genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum. 50: 2113–2121.
Lappin D. F.,
D. Apatzidou,
A. M. Quirke,
J. Oliver-Bell,
J. P. Butcher,
D. F. Kinane,
M. P. Riggio,
P. Venables,
I. B. McInnes,
S. Culshaw
. 2013. Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres. J. Clin. Periodontol. 40: 907–915.
Gizinski A. M.,
M. Mascolo,
J. L. Loucks,
A. Kervitsky,
R. T. Meehan,
K. K. Brown,
V. M. Holers,
K. D. Deane
. 2009. Rheumatoid arthritis (RA)-specific autoantibodies in patients with interstitial lung disease and absence of clinically apparent articular RA. Clin. Rheumatol. 28: 611–613.
M. K. Demoruelle,
L. A. Derber,
C. J. Chartier-Logan,
M. C. Parish,
I. F. Pedraza,
M. H. Weisman,
J. M. Norris,
. 2013. Sputum autoantibodies in patients with established rheumatoid arthritis and subjects at risk of future clinically apparent disease. Arthritis Rheum. 65: 2545–2554.
Reynisdottir G.,
R. Karimi,
V. Joshua,
H. Olsen,
A. H. Hensvold,
A. Harju,
M. Engström,
J. Grunewald,
S. Nyren,
A. Eklund,
. 2014. Structural changes and antibody enrichment in the lungs are early features of anti-citrullinated protein antibody-positive rheumatoid arthritis. Arthritis Rheumatol. 66: 31–39.
Perry E.,
C. Kelly,
P. Eggleton,
A. De Soyza,
D. Hutchinson
. 2014. The lung in ACPA-positive rheumatoid arthritis: an initiating site of injury? Rheumatology (Oxford) 53: 1940–1950.
Giles J. T.,
S. K. Danoff,
R. Winchester,
D. A. Pappas,
S. Siegelman,
G. Connors,
J. M. Bathon
. 2014. Association of fine specificity and repertoire expansion of anticitrullinated peptide antibodies with rheumatoid arthritis associated interstitial lung disease. Ann. Rheum. Dis. 73: 1487–1494.
Mikuls T. R.,
J. B. Payne,
F. Yu,
G. M. Thiele,
R. J. Reynolds,
G. W. Cannon,
J. Markt,
D. McGowan,
G. S. Kerr,
R. S. Redman,
. 2014. Periodontitis and Porphyromonas gingivalis in patients with rheumatoid arthritis. Arthritis Rheum. (Munch) 66: 1090–1100.
Gully N.,
R. Bright,
V. Marino,
C. Marchant,
M. Cantley,
D. Haynes,
C. Butler,
S. Dashper,
E. Reynolds,
M. Bartold
. 2014. Porphyromonas gingivalis peptidylarginine deiminase, a key contributor in the pathogenesis of experimental periodontal disease and experimental arthritis. PLoS ONE 9: e100838.
Gonzalez S. M.,
A. R. Erickson,
P. G. Johnson,
M. J. Schmid,
A. M. Reimold,
. 2015. Alveolar bone loss is associated with circulating anti-citrullinated protein antibody (ACPA) in patients with rheumatoid arthritis. J. Periodontol. 86: 222–231.
Young K. A.,
K. D. Deane,
J. M. Hughes-Austin,
J. H. Buckner,
T. R. Mikuls,
J. R. O'Dell,
. 2013. Relatives without rheumatoid arthritis show reactivity to anti-citrullinated protein/peptide antibodies that are associated with arthritis-related traits: studies of the etiology of rheumatoid arthritis. Arthritis Rheum. 65: 1995–2004.
Jiang X.,
T. J. van Wesemael,
H. Källberg,
V. Malmström,
L. Israelsson,
H. Hreggvidsdottir,
W. Verduijn,
. 2014. Anti-CarP antibodies in two large cohorts of patients with rheumatoid arthritis and their relationship to genetic risk factors, cigarette smoking and other autoantibodies. Ann. Rheum. Dis. 73: 1761–1768.
Thiele G. M.,
M. J. Duryee,
D. R. Anderson,
L. W. Klassen,
S. M. Mohring,
K. A. Young,
D. Benissan-Messan,
H. Sayles,
A. Dusad,
C. D. Hunter,
. 2015. Malondialdehyde-acetaldehyde adducts and anti-malondialdehyde-acetaldehyde antibodies in rheumatoid arthritis. Arthritis Rheum. (Munch) 67: 645–655.
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redpajama_set_name: RedPajamaGithub


John Carew kept Aston Villa in the hunt for a UEFA Cup spot with a late header in a dramatic 2-2 draw against Everton at Goodison Park on Sunday.
Norwegian striker Carew secured a hard-earned point with his 86th minute goal after Joseph Yobo looked to have won the game for Everton just a minute earlier.
Victory for fifth-placed Everton would have all but guaranteed a UEFA Cup spot, but both teams now go into the final two games of the season harbouring ambitions of a top five finish.
"It's disappointing but you are always at your most vulnerable when you score, so they say, and that was the case here," Everton manager David Moyes said.
Portsmouth's defeat at home to Blackburn earlier in the day had provided a boost to both Everton and Villa's hopes of securing a European place, with the home side knowing that victory here would guarantee them a place in the InterToto Cup at the very least.
Moyes's team have shown signs of fatigue in recent weeks, however, collecting just five points from their last six Premier League games.
In contrast, Villa arrived at Goodison Park having scored 15 goals in their three games ahead of this clash, so the Birmingham club had momentum behind them as they attempted to move level on points with the Merseysiders.
But on a rain-soaked pitch, both sides struggled to gain the upper hand in a dismal first-half that generated more yellow cards — three — than goalscoring opportunities.
Everton's Nigerian forward Ayegbeni Yakubu showed a glimpse of his 11 million pounds transfer value on 19 minutes, though, when he escaped Villa midfielder Nigel Reo-Coker before unleashing a powerful strike at goal.
Villa keeper Scott Carson could only palm the shot away, but Portuguese midfielder Manuel Fernandes scuffed his shot from the rebound.
Villa could hardly be described as wilting under the pressure and O'Neill's men were comfortably containing Everton when they were given their first sight on goal on the half hour after referee Phil Dowd had awarded a free-kick following Phil Jagielka's foul on Olof Mellberg.
Just 20 yards from goal, both Gareth Barry and Ashley Young lurked with intent at the set-piece and England winger Young rattled the crossbar with his powerfully struck effort.
Villa looked the stronger, fresher team, but Everton found an extra level in the second-half as they chased the victory that would all but secure a UEFA Cup spot.
And the breakthrough came on 55 minutes when Phil Neville put Everton ahead with a deflected strike following good work by Yakubu down the left flank.
The former Middlesbrough man broke free after latching onto Leon Osman's pass and his pull-back picked out Neville on the far edge of the penalty area. Neville was left with a difficult chance, but his powerful right foot shot beat Carson via a deflection off the chest of defender Martin Laursen.
With his team facing a six-point deficit on Everton with just two games to play, O'Neill went for broke by replacing defenders Wilfred Bouma and Zat Knight with forwards Marlon Harewood and Patrik Berger and the gamble paid off on 80 minutes when Gabriel Agbonlahor snatched an equaliser with a close range strike from Barry's corner.
Everton regained the lead four minutes later, though, when the Villa defence lost concentration at the far post to allow Yobo to volley Lee Carsley's cross into the roof of the net.
Goodison erupted, but the home fans were still celebrating when Carew netted Villa's second equaliser less then sixty seconds later with a pinpoint header from Barry's cross.


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text:
Гиздавац је насељено место у саставу општине Мућ, Сплитско-далматинска жупанија, Република Хрватска.

Историја

До територијалне реорганизације у Хрватској налазио се у саставу старе општине Солин.

Становништво

На попису становништва 2011. године, Гиздавац је имао 127 становника.

Напомена: У 1991. смањено издвајањем дела подручја које је припојено насељу Пругово (општина Клис), за које и садржи део података од 1857. до 1961.

Попис 1991.

На попису становништва 1991. године, насељено место Гиздавац је имало 109 становника, следећег националног састава:

Извори
  Савезни завод за статистику и евиденцију ФНРЈ и СФРЈ, попис становништва 1948, 1953, 1961, 1971, 1981. и 1991. године

Референце

Спољашње везе

Мапа

Фотографије

Насељена места у Хрватској
Насељена места у Сплитско-далматинској жупанији
Википројект географија/Насеља у Хрватској
Општина Мућ

redpajama_set_name: RedPajamaWikipedia


text:
Why Are You Still Doing Bikram Yoga?
in Yoga
In the southwestern Spanish city of Murcia, a few 60 yogis spent nine weeks closing spring in sweltering warmth learning the 26 postures and a couple of respiration practices of Bikram yoga so that they could move domestic and educate it. The guru himself sat onstage,
broadcasting commands with his microphone headset. Bikram Choudhury, often sporting nothing except a black Speedo and a gold
Rolex is notoriously brutal at his instructor training, but his dedicated fans embrace and fee his methods ― so much so that all of them paid between $12,500 and $sixteen,600 to be there.
This fall, dozens more will pay the identical amount for a trainer schooling in Acapulco, Mexico. Choudhury and his students ― most of the people of whom are girls ― will spend 9 weeks at a Sheraton inn on Mexico's Pacific coast, ready with an 18-hole golfing direction, pools, bars, restaurants, and "a wonderful wellbeing place," in step with the program description.
Acapulco is a convenient place for Choudhury ― he decamped there following numerous court cases, an arrest warrant, and allegations of rape and harassment against him. This increases the query of why such a lot of global humans retain to provide him their cash. For many, it appears to be the highlight of their yoga careers nonetheless. For others, those nine weeks with Bikram Choudhury were the maximum horrific studies of their lives.
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Jill Lawler, a former pupil of Choudhury's from a 2012 training, filed a civil suit in opposition to him for rape and harassment in July after her first grievance in 2016 became held up in Choudhury's company's financial disaster lawsuit. Lawler and her legal team are going straight for the jugular in a civil suit, looking for punitive damages from Choudhury himself.
"Teacher Training becomes severe and worrying," the lawsuit says. "Unbeknownst to [Lawler], Bikram Choudhury noted them as 'one massive brainwashing consultation.'"
While a few in the yoga community ― specifically the Bikram yoga community ― have taken lengths to distance their practices from the person himself, others have disregarded the allegations altogether or shrugged them off and given him hundreds of dollars. In fact, most of his students are ladies, and the bulk of his wealth has come from them, despite his alleged predation.
"It's weird to me that people nonetheless go to this training. I find it very hard to recognize how a person ought to appearance [at the allegations of rape] and feature questions about what passed off," said Jessamyn Stanley, a yoga teacher whose presence in the enterprise has inspired greater various bodies to choose up the practice. "We, as a network, need to get over this and observe this."
The Money That Flows To The Guru
Choudhury evolved what he has given that referred to as his personal exercise of doing yoga within the overdue Nineteen Sixties ― the now-notorious 26 postures and two respiration practices that must be achieved in a carpeted, one hundred and five-degree room. He delivered it to San Francisco's North Beach neighborhood in 1973. Inside the many years, because America experienced what Stanley referred to as "the Bikram craze" ― a global franchise led via teachers who needed to learn by way of Choudhury himself to acquire
certification. This recertification cost is between $750 and $1,2 hundred, and it must be repeated every 3 years. The places and training became increasingly lavish inside the 2000s as celebrities and public figures started to flock to his studios. At the height of the Bikram craze, there have been 650 studios in America by myself, and because the franchise elevated, so did Choudhury's wallet.
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Love Your Yoga? Here's How It All Started

redpajama_set_name: RedPajamaCommonCrawl


Fourteen years ago, Peter Roberts was given six months to live. He was fifty years old and had just been diagnosed with Stage IV prostate cancer.
While his doctor performed a DRE each year, he'd never before heard of the PSA test until he was referred to a urologist following some unusual back pain. At the time of his diagnosis, his PSA came back at 1,000. Afterward, he read everything he could about the disease, finally sitting both his urologist and oncologist down together to discuss his treatment plan. He first began to treat the disease with chemo, and his numbers started going down.
I insisted my urologist and oncologist sit down together to discuss my options. It was a tough meeting to arrange, but worth every second.
He decided then and there to share his story with others so that more men would become aware of the disease. He volunteered with local organizations and spread the word in his community in any way he could. Throughout his entire journey his positive attitude never wavered, despite the many side effects of his cancer. In the past 14 years he's had kidney, brain, and jaw surgery as a result of his disease and treatment, along with detached retinas, and other serious ailments. He hasn't let that get him down either. His positive attitude and strength have inspired many men on their journeys as well.
When my doctors need someone to talk to a man who is newly diagnosed, they refer him to me. I help make heads or tails of what he just heard from the doctor.
In addition to volunteering his time to raise awareness and talking to newly diagnosed men, Peter also shares information on his website, www.saveyourmales.org. He speaks to local support groups and has attended ZERO's Prostate Cancer Summit to lobby his elected officials on Capitol Hill for increased research funding.
Despite everything, I'm a happy guy, because I'm here. I have a mission to do. My advice to other men? Keep on going.
Read more about Peter in The Greenville News.


text:
She Trained for a Marathon in the Middle of Chemo
Her Doctor Told Her She'd Be Lucky to Run a Mile During Chemo. Instead, She Trained for a Marathon
"I just wanted to be normal. I figured if I could keep running, I could fight cancer."
By Hailey Middlebrook
Melissa Medina
At first, Melissa Medina thought the pain was just a muscle she had pulled in her chest. It was October 2014, and the 43-year-old mother of two was in the thick of half marathon training, preparing for the Route 66 Half in Tulsa, Oklahoma later in November. When she rolled over in bed that night, she felt it again: a sharp tenderness in her breast.
"I didn't think anything of it," Medina, who lives in Edmond, Oklahoma, told Runner's World. "I was training for a half marathon, for goodness sake! I was healthy."
When the pain continued the next day, the possibility of breast cancer briefly crossed her mind—she had an aunt who had suffered from the disease—but she figured she was safe, since she had been up-to-date on her mammogram screenings. Still, she checked in with her doctor that day.
Immediately, she was referred to a radiologist to have a mammogram done. It showed something suspicious, so Medina was instructed to get an ultrasound and biopsy of her breast.
"I told them I had to get to class," said Medina, who works as a pharmacy professor at Oklahoma University. She underwent the procedures, then booked it to her lecture hall. A little while later, the results came in: Medina had stage 0 breast cancer, meaning she had abnormal cells that could potentially grow into cancer.
"After I got the results, I started thinking about getting a mastectomy," she said.
Learning of a more serious diagnosis
Before preparing for surgery, though, she sent her medical scans to an oncologist friend at Rutgers University for a second opinion. Her friend thought something on the X-ray seemed amiss, and recommended Medina get another biopsy.
The second biopsy showed Medina actually had stage 3 breast cancer. To treat the disease, she would need months of chemotherapy and radiation, along with a mastectomy.
"My husband and I were both shocked," said Medina, whose husband, also a runner, works as an oncology pharmacist. Her doctors recommended that she start chemotherapy in November, just two weeks before the half marathon. The treatment would continue into the New Year, when Medina was planning to start training for her annual spring marathon in Oklahoma City.
"I wanted to keep working and training," she said. "I asked them if I could delay the chemotherapy until after the half marathon, but they told me no.
What It's Like to Run Again After a Mastectomy
"Then I asked if I could still train for my marathon during the treatment," Medina continued. "My doctor had this shocked look on his face. He told me I was safe to run, but that I would be lucky to be able to run a mile, let alone 10-mile long runs."
So Medina set out to prove him wrong—not out of spite, but because she, like so many runners, needed running in her everyday life in order to feel normal.
"I was so worried about how my kids would react when I told them I had cancer," said Medina, whose daughter was eight and son was four at the time. "Running is a big thing in our family, so I thought that if they saw that I was still training as normal, they wouldn't worry so much about me."
Melissa Medina, now cancer-free at 47, runs with her husband and their two children in Edmund, Oklahoma.
Training during treatment
Medina's family and friends wholeheartedly supported her decision to keep running after her diagnosis. That November, she finished the Route 66 Half Marathon in 2:06, just five minutes off her personal best, despite having to run with a painful chemotherapy port (a device that helped doctors access her bloodstream) implanted in her arm. "I couldn't move my arm much, but my legs felt fine," she said.
In the weeks that followed, Medina went in for cancer treatment while continuing to teach and run 20 miles per week to prepare for the April marathon. During treatment weeks, she would receive chemo on Thursday, then she'd do her long run with friends on Saturday.
While she kept a tough exterior, Medina admitted that training was extremely tough.
"My legs felt like cinder blocks, " Medina said. "No matter how hard I tried, I couldn't pick up the pace. My daughter would joke with me that I was running too slow."
She's Back to the Chicago Marathon, Cancer Free
On top of being wiped out from the treatment, she explained that she was anemic during this time, and the decreased hemoglobin levels in her blood left her feeling even more fatigued than usual. "After I ran on Saturdays, all I could do was go home and sit," she said.
As spring arrived, the Oklahoma City Marathon date neared, as did another event: Medina's scheduled mastectomy. Originally, she was supposed to have the surgery done earlier in April, but she convinced her doctors to postpone it until two days after the marathon.
"I didn't want to worry about it," she said. On race day, Medina powered through the 26.2 miles with a large group of friends and family members. At this point, she had lost all of her hair from chemo, and ran with a baseball cap. She wrapped her chest with Saran wrap to help prevent chafing, which was worsened by the radiation treatment.
Running cancer-free
When Medina crossed the line in 4:38, she was on top of the world. "I felt good running," she said. "People thought I had lost my mind for doing it. But when people have cancer, they should keep doing whatever it is they love. I didn't want to just sit and be sad."
After beating breast cancer earlier in 2015, Melissa Medina and her family ran the Race for the Cure 5K in Oklahoma City.
Later in October of 2015, Medina won the cancer survivors' race at Oklahoma City's Race for the Cure 5K. Finally cancer-free, she felt strong and capable.
Then, just a couple of months later in December when she was about to start training for the Oklahoma City Marathon again, she was at a doctor's visit when she received more bad news: She had cancer again. This time, it was of the thyroid.
"I knew what to do this time around," she said. She got out the baseball hat. Slipped on the running shoes. She put in the marathon training, ran the race, underwent surgery, and beat the cancer. And throughout it all, she never lost her love for the sport.
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"I really believe running helped me through it," said Medina, who's now 47 and cancer-free. This past October, she won the Race for the Cure 5K for the fourth year in a row, running 24:13.
"People are scared to talk to you when you have cancer, but running gave me something to talk about. I wasn't isolated. And it made me feel stronger and more confident that I could keep fighting."
Hailey Middlebrook Digital Editor Hailey first got hooked on running news as an intern with Running Times, and now she reports on elite runners and cyclists, feel-good stories, and training pieces for Runner's World and Bicycling magazines.
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text:
General / Judaism / Morality
First Person Singular
by Sarah Shapiro · Published November 1, 2020 · Updated November 3, 2020
It's par for the course, in society today, to publicize ourselves uninhibitedly. Check out my blog! Visit my website! Self-promoting "shout-outs" are the way to go.
But once upon a time, not so long ago, the opposite code of behavior prevailed. Self- interest was camouflaged. Self-promotion was a no-no.
Those who wax nostalgic for the good old days, and who aren't entirely comfortable with the contemporary ethos of brazenness, are most likely survivors of the ancient 20th century. That's when drawing attention to oneself immodestly was seen as crass and ungracious, impolite and unseemly.
In fact, the preceding paragraphs, that you've just read, contain fossilized evidence of that bygone era's cultural indoctrination. For in an earlier, discarded draft of this essay, the opening went like this:
When I was growing up in mid-20th century America, I learned in school to avoid the word "I" in my written compositions. I was given to understand, somewhere along the line, that "the personal" isn't as reliable as "impersonal" as a source of information.
Please note that I (excuse me) got rid of the I's, and of "me," too, thereby affecting a supposedly "objective" air, and perhaps leading you to assume that the author had engaged in historical research into 20th century America's secular educational policies, and thus knows what she's talking about.
In fact, however, the "facts" that strike me as factual, are those that corroborate my previously formed opinions, and it's those I'll be more inclined to report. Whether I reveal my "I" or conceal it, whatever I know–on this subject or any subject– is based on my personal experience.
Why were me, myself, and I relegated to the back of the bus? The name of the pronoun itself hints subtly at the reason. For while the phrase, "in my personal opinion," is associated with humble fallibility, the words "first person singular" suggest ego and self-centeredness. I– the first person– am The. Most. Important. Person.
Erasing the first-person pronoun, though, did not succeed in erasing us. We children were all I's, and remained I's. While the general drift of America's educational agenda was to guide its young citizens up the ladder to what's still known, until today, as success, successful social climbing requires putting oneself first (even at one's neighbor's expense.) As good citizens, therefore, we were obliged to mask the big, unattractive egos inside our lovely-looking, genteel, well-spoken selves.
So the explicit and subliminal lessons were replete with all sorts of contradictory messages, not only in relation to the written word but also in relation to social–and by extension, moral—issues. I gathered that I shouldn't talk too much (about me, myself, and I) not only because people would get bored, but because focusing on myself was seen as inherently wrong. For mankind possesses the universal awareness that egos are for taming, and most human beings know intuitively that it's wrong to be selfish, to care only about one's own benefit. The word "self-centered" probably didn't show up on any spelling tests. But like many of the other devastating put downs that loomed over that era's cultural landscape, crushing children's sense of self-worth and destroying their self-esteem (for example, "Who do you think you are?!" or "She thinks the whole world revolves around her!") golden nuggets such as these are not without their hard, unpopped popcorn kernels of wisdom. Indeed, if you turn some of those putdowns inside out, they drag along in their wake a whole wagonload of wisdom.
As a Jewish child growing up in non-Jewish society, it was my good fortune to have been unable–by virtue of my inborn Jewish nature– to squeeze into the straightjacket of the surrounding society's behavioral etiquette. And alas, thanks in part to that discomfort…I found myself on a different path
"Good manners" are good, and essential to everyone's wellbeing. But if courteous interpersonal behavior does not have as its underlying foundation the awareness that we are created in G-d's Image, then the rules of social etiquette foster a way of life devoid of connection; one conceals one's thoughts and feelings not only from others, but above all, from oneself.
Concealing the writer's "I" may succeed in creating an air of objectivity, but between the lines, our selves endure. Prevailing over the yetzer ha ra isn't achieved by concealing our egocentricity but rather, by scrupulously revealing it, thereby heightening our awareness of being error- prone, self-centered individuals, yet endowed by Hashem with the power to transcend that narrowness. How hard we each have to work, from childhood on through senescence, simply to understand that other people (who serve as props in our dramas, as do we in theirs) are as real as we, and have also been given the starring role.
It is written that had Adam been faithful to his tzelem Elokim, and had obeyed his Creator, and emulated Him, he could have fulfilled G-d's whole purpose in having created the world. The same is true, literally, of us. Torah teaches that we vastly underestimate our role in the universe, and our impact as individuals upon events supposedly beyond our control. How grossly we underestimate our potential—in our private communications with our Creator– to affect the course of world history. All the supposedly unimportant, minor, apparently foolish personal struggles that take place between a person's birth and death have significance beyond mortal perception.
Like identical-looking snowflakes in a storm, formed and crafted and produced in the same factory, each of us is absolutely unique as we travel along our solitary, often lonely, paths…until eventually…one way or another…vanishing from one another's sight…we must part from our worldly companions.
Then, once again, we will each be in the Garden, alone, and will know that for me, b'emet, bishvili nivra haolam.
And at this moment I have to admit that the impulse to say all this comes from a futile attempt to stay here, chasing after the illusory permanence of the typewritten page. I want the starring role, to engrave my name in Olam Ha Ze and give names to everything in this world…like Adam HaRishon…the first person…to say "I."
Originally published in Ami Magazine
The Sderot Menorah And The Hobnailed Sandal
by Yitzchok Adlerstein · Published December 5, 2012
True Independence
by Jonathan Rosenblum · Published April 6, 2012
Exercising the Empathy Muscle
by Avi Shafran · Published November 15, 2011
Bob Miller says:
See https://www.aish.com/tp/i/gl/155622505.html on the worth of the individual.
Next story When Innocence Really Isn't
Previous story Can Only a Black Person Explain the Black Experience


text:
T Cell Co-Stimulatory Pathways
Targeting the B7–CD28/CTLA-4 Pathway
Targeting the CD40/CD40 Ligand Pathway
Targeting the ICOS/ICOS-L Pathway
Novel Strategies for Targeting the Co-Stimulatory Pathway
Future Perspectives
Front. Microbiol., 28 September 2011
Sec. Microbial Immunology
Targeting co-stimulatory pathways in gene therapy
Xiaopei Huang1 and Yiping Yang1,2*
1 Department of Medicine, Duke University Medical Center, Durham, NC, USA
2 Department of Immunology, Duke University Medical Center, Durham, NC, USA
Gene therapy with recombinant viral vectors such as adenovirus and adenovirus-associated virus holds great promise in treating a wide range of diseases because of the high efficiency with which the viruses transfer their genomes into host cells in vivo. However, the activation of the host immune responses remains a major hurdle to successful gene therapy. Studies in the past two decades have elucidated the important role co-stimulation plays in the activation of both T and B cells. This review summarizes our current understanding of T cell co-stimulatory pathways, and strategies targeting these co-stimulatory pathways in gene therapy applications as well as potential future directions.
Virus-based vectors such as adenovirus and adenovirus-associated virus (AAV) have been widely used in gene therapy applications due to their high efficiency of transduction into a variety of cells in vivo. Indeed, several impressive results using viral vector-based gene therapy have been reported in humans (Ashtari et al., 2011; Fischer et al., 2011). However, one major barrier to successful gene therapy with these viral vectors is the host immune responses to both viral vectors and the transgenes (Huang and Yang, 2009). Our understanding of the mechanisms of immune responses to viral vectors has greatly improved over the past two decades, and as a result, strategies have been developed to manipulate various immune pathways in order to regulate the immune responses to viral vectors. One promising therapeutic strategy is targeting the co-stimulatory pathways to modulate the function of T and B cells.
The original two-signal hypothesis (Lafferty et al., 1974, 1980) proposes that full T cell activation requires two signals: antigen-specific T cell recognition (signal 1) provided by the engagement of the TCR–CD3 complex with a processed antigenic peptide bound to the major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell (APC), and an antigen-non-specific co-stimulatory signal (signal 2) provided by the interactions between cell surface molecules on the APC and the T cell. Indeed, early work by Jenkins and Schwartz showed that in the absence of the co-stimulatory signal, T cells are rendered anergic, a state marked by the inability of T cells to respond to subsequent antigenic stimulation, leading to a model that co-stimulation plays a critical role in controlling the fate of T cell responses: activation or anergy (Schwartz et al., 1989; Jenkins et al., 1990). Since then, these seminal studies have stimulated tremendous growth of the co-stimulation field. In addition to signals 1 and 2, we now appreciate that other signals also participate in determining the fate of T cell activation: some of these signals are stimulatory and others are inhibitory (Sharpe, 2009). Furthermore, some other signals are required to promote the differentiation into different T helper cell subsets (Pipkin and Rao, 2009; Simpson et al., 2010).
Because of the importance of co-stimulation in determining the outcome of the immune response, manipulation of various co-stimulatory pathways to regulate host immune responses is of therapeutic interest. In this review, we first summarize the current knowledge of T cell co-stimulatory pathways. We then focus on strategies targeting co-stimulatory pathways and their potential implications in viral vector-mediated gene therapy.
Several co-stimulatory pathways have been characterized including both activating and inhibitory pathways (Figure 1). The positive activating signals are balanced by the negative inhibitory signals to achieve optimal control of T and B cell activation. The activating pathways such as B7 and CD28, CD40 ligand (CD40L) and CD40, inducible co-stimulatory molecule (ICOS) and ICOS-ligand (ICOS-L), and OX40 (CD134) and its ligand, OX40L, are critical for the activation of T and B cells, whereas inhibitory pathways such as B7 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and programmed cell death-1 (PD-1) and PD ligand (PD-L) downregulate T cell activation. Here we mainly focus on the activating co-stimulatory pathways.
Figure 1. Co-stimulatory pathways. Co-stimulations either enhance or down-regulate T cell activation following the initial TCR and peptide-MHC ligation. Positive co-stimulatory pathways include B7–CD28, CD40L–CD40, ICOS–ICOS-L, and OX40–OX40L. Negative co-stimulatory pathways include B7–CTLA-4 and PD-1–PD-L.
B7-CD28/CTLA-4 Pathway
The CD28 glycoprotein, a member of the immunoglobulin superfamily, is expressed on the T cell membrane as disulfide-linked homodimers. It is expressed on the surface of 90% of human CD4+ T cells and 50% of human CD8+ T cells, and nearly 100% of murine T cells. The ligands for CD28 are B7-1 (CD80) and B7-2 (CD86), also members of the immunoglobulin superfamily, which are expressed by APCs such as DCs, macrophages, and activated B cells. Although B7-1 and B7-2 are co-expressed and share similar overall structure, they differ in their temporal responses to stimuli, with B7-2 induction occurring earlier and usually at much higher level than B7-1 in activated B cells and DCs (Hathcock et al., 1994). The expression of B7-1 and B7-2 is controlled by cytokines and cell–cell interactions. IL-4 is a potent inducer of B7-2 on B cells, whereas IFN-γ and IL-10 differentially regulate B7 expression, depending on cell types (Valle et al., 1991; Stack et al., 1994). Studies have shown that signals transmitted through the MHC class II cytoplasmic domain, which is required for effective antigen presentation, induce B7 expression on B cells (Nabavi et al., 1992).
Mice deficient in CD28 or treated with antagonists of CD28 exhibit substantially reduced proliferative responses (Green et al., 1994). Similarly, mice deficient in both B7-1 and B7-2 have compromised T cell-mediated responses (Borriello et al., 1997; Mcadam et al., 1998), whereas addition of B7 transfectants could augment T cell proliferation and IL-2 production induced with anti-CD3 or PMA in a CD28-dependent manner (Gimmi et al., 1991; Linsley et al., 1991). B7/CD28-mediated signaling enhances the production of IL-2 and IL-2 receptor in a nuclear factor (NF)-κB dependent manner, and upregulates the expression of other cytokines such as IL-1, IL-4, IL-5, TNF, and IFN-γ (reviewed in Lenschow et al., 1996; Kane et al., 2002); accelerates resting T cell entry into and progression through the cell cycle; plays a critical role in the development and differentiation of Th1 and Th2 T cell subsets (Van Der Pouw-Kraan et al., 1992; Seder et al., 1994; King et al., 1995); prevents anergy, and promotes T cell survival by enhancing the expression of the anti-apoptotic proteins such as BCL-xL. Recent studies have reported chromatin structural changes within minutes following T cell activation, and induction of DNA demethylation of IL-2 gene as early as 20 min after TCR/CD28 stimulation, suggesting a role for CD28 signaling in these early nuclear events, which are critical to the ensuing processes of cell proliferation and differentiation (Zhao et al., 1998; Acuto and Michel, 2003; Bruniquel and Schwartz, 2003).
Cytotoxic T lymphocyte antigen-4, also known as CD152, is expressed on the T cell membrane as disulfide-linked homodimers. Like CD28 and B7, they are also members of the immunoglobulin superfamily. The ligands for CTLA-4 are also B7-1 and B7-2, but its binding affinity for B7 molecules is about 20-fold greater than that of CD28. Although CTLA-4 and CD28 share sequence homology, CTLA-4 acts antagonistically of CD28, and delivers an inhibitory signal that down-regulates T cell activation. CTLA-4 knockout mice manifest lymphoproliferative disorder which is lethal by 3–4 weeks after birth, supporting the notion that CTLA-4 acts as a negative regulator of T cell activation and is vital for the control of lymphocyte homeostasis (Waterhouse et al., 1995). While CD28 is expressed by both resting and activated T cells, CTLA-4 is only expressed on activated T cells at much lower levels. CTLA-4 induction can be detected 24 h after T cell stimulation, with cell surface expression peaking around 2 days post activation, and returning to background levels by day 4 (Linsley et al., 1992; Walunas et al., 1994). Interestingly, CD28/B7-mediated signaling upregulates the expression of CTLA-4 which effectively competes with CD28 for B7-1/B7-2 at later stages of the immune response to counter T cell activation, suggesting a mechanism for achieving balance between positive and negative stimulation.
CD40L–CD40 Pathway
The CD40 glycoprotein is a member of the tumor necrosis factor-receptor (TNFR) superfamily. It is expressed on APCs such as B cells, DCs, activated macrophages as well as Langerhan cells, endothelial cells, and thymic epithelial cells (Buhlmann and Noelle, 1996). The ligand for CD40 (CD40L), a member of the TNF family, is preferentially expressed on the surface of activated CD4+ T cells in both humans and mice. Early studies have established an important role for CD40L–CD40 interactions in B cell activation and proliferation, as well as immunoglobulin (Ig) class switching (Allen et al., 1993; Disanto et al., 1993). For example, the underlying cause for a severe form of immunodeficiency, X-linked hyper IgM syndrome (HIGM) is a mutation in CD40L (Allen et al., 1993; Disanto et al., 1993). Patients with this disorder have elevated levels of IgM, but little to none IgG or IgA, and absent germinal centers.
In addition to the role in the humoral response, two seminal reports also demonstrate a critical role for the CD40L–CD40 pathway in the activation of T cell responses (Grewal et al., 1996; Yang and Wilson, 1996). In a murine model of experimental allergic encephalomyelitis (EAE), CD4+ T cell priming is defective in CD40L-deficient mice. These mice failed to develop EAE after priming with antigen and produced little to no IL-4 and IFN-γ (Grewal et al., 1996). Similarly, in a murine model of liver-directed gene transfer with adenoviral vectors, CD40L–CD40 signaling on APCs is essential for the development of CD8+ T cell response to adenoviral vector (Yang and Wilson, 1996). This is mediated by upregulating the expression of B7 molecules on APCs, which in turn promote the B7–CD28 pathway, leading to T cell activation (Yang and Wilson, 1996). Indeed, subsequent studies have further delineated that CD40–CD40L interactions between DCs and CD4+ T cells provide DCs with "licensing" in the activation of CD8+ T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998).
The broad spectrum of cells that express CD40 indicates that CD40L–CD40 interactions may exert regulatory effects at multiple levels. CD40L–CD40 interactions also result in the production of IL-12, which promotes the differentiation of Th1 immunity (Trinchieri, 1994). Furthermore, the interactions between CD40L on activated T cells and CD40 on vascular endothelial cells can stimulate the vasculature at sites of inflammation and may play a role in inflammatory responses (Hollenbaugh et al., 1995).
ICOS Pathway
ICOS, a third member of the CD28/CTLA-4 family, is expressed on activated T cells, but very little on resting naïve T cells. ICOS binds specifically to its ligand (ICOS-L), also known as B7-related protein-1 (B7RP-1), which is expressed constitutively on B cells. In vivo, the interaction of ICOS with ICOS-L is critical for T cell-dependent B cell responses (Mcadam et al., 2001; Tafuri et al., 2001). In the absence of ICOS, germinal center formation is impaired and immunoglobulin class switching is defective.
In addition to providing help for B cells, ICOS plays an important role in the differentiation of unpolarized CD4+ T cells into Th1, Th2, Th17, and Treg lineages (Simpson et al., 2010). It has been shown that ICOS can promote both Th1 and Th2 responses. However, ICOS and ICOS-L interaction during the early stages of T cell differentiation favors Th2 response (Nurieva et al., 2003). Although ICOS is not required for Th17 differentiation during primary responses, it plays a pivotal role in promoting Th17 compartment by upregulating IL-23R during secondary responses (Bauquet et al., 2009). Recent studies also demonstrate a critical role of ICOS in maintaining the homeostasis of CD4 + Foxp3 + Treg (Burmeister et al., 2008; Ito et al., 2008).
PD-1 Pathway
Programmed cell death-1, an inhibitory receptor in the CD28 family, is expressed on CD4 and CD8 T cells, B cells, NKT cells, and some DC subsets upon activation (Keir et al., 2008). PD-1 has two ligands, PD-L1 and PD-L2. Upon ligation to either PD-L1 or PD-L2, PD-1 attenuates TCR signaling, leading to suppression of T cell responses. Indeed, in a model of chronic LCMV infection, PD-1 is overexpressed on the exhausted T cells and blockade of PD-1 and PD-L1 interaction led to the functional restoration of these exhausted T cells (Barber et al., 2006). Furthermore, the PD-1 pathway plays an important role in regulating immune responses to acute infections (Brown et al., 2010). In addition to providing inhibitory signals to T cells, a recent study has also revealed an essential role for PD-1 signaling in germinal center B cell survival and the formation and affinity of long-lived plasma cells (Good-Jacobson et al., 2010).
Early studies in transplant setting have demonstrated that blockade of the CD28/B7 co-stimulation with CTLA-4Ig, a soluble fusion protein consisting of the extracellular domains of CTLA-4 and the constant region of the IgG1 heavy chain, could cause host T cells directed against the grafted tissue anergic, therefore leading to the long-term survival of the graft (Lenschow et al., 1993). CTLA-4Ig acts through competition with CD28 for its ligands, B7-1 and B7-2, disrupting the co-stimulation required for complete T cell activation. In addition, studies using a combination of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) in an allogeneic pancreatic islet transplant setting showed this approach selectively delayed CD4+ T cell infiltration into the graft, leading to inhibition of transplant rejection (Lenschow et al., 1995). Similarly, CTLA-4Ig has been used successfully to suppress T cell responses in animal models of autoimmunity (Dall'era and Davis, 2004). These studies provide evidence that blocking co-stimulatory pathway is a viable strategy in inducing T cell anergy.
A number of studies have also investigated the effect of blocking the B7–CD28 co-stimulatory pathway in viral vector-mediated gene therapy. Administration of CTLA-4Ig blocked the formation of anti-β-galactosidase antibodies following retrovirus-mediated gene transfer to the liver (Puppi et al., 2004). Similarly, treatment with CTLA-4Ig resulted in more stable transgene expression after retrovirus-mediated gene therapy in mucopolysaccharidosis I mice (Ma et al., 2007). Furthermore, potent immunosuppression has been observed with a high-affinity variant of CTLA-4Ig, LEA29Y (belatacept; Larsen et al., 2005). In a mouse model of Duchenne muscular dystrophy (DMD), adenovirus vectors carrying murine CTLA-4Ig (AdmCTLA-4Ig) was coadministered with an adenoviral vector carrying a full-length murine dystrophin cDNA (AdmDys) into skeletal muscle (Jiang et al., 2004a). Stable expression of dystrophin in skeletal muscle up to 8 weeks was achieved, in contrast to the control group without CTLA-4Ig where dystrophin expression was significantly diminished within 8 weeks. CTL response to adenoviral vector was markedly suppressed in the group receiving co-administration of AdmCTLA-4Ig at 2-week time point, and cytokine production such as IFN-γ, IL-4, and IL-6 were much lower than the control group as well. No dystrophin-specific CTL response was observed in either group during the 2-week period. Neutralizing antibody against adenoviral vector was detected in both groups, with highest levels found in the control group, indicating that CTLA-4Ig partially blocked the humoral response against adenoviral vectors.
Similar strategies employing CTLA-4Ig to block the B7–CD28 co-stimulatory pathway have been investigated in a murine model of type II collagen-induced arthritis (Ijima et al., 2001). However, in other model systems, the effect of AdmCTLA-4Ig has been modest (Laumonier et al., 2003), suggesting other strategies in combination with CTLA-4Ig may be needed to achieve successful therapeutic effect.
The critical role of the CD40L–CD40 pathway in activation of both T and B cells suggests interfering with this pathway may prevent both cellular and humoral immune responses to virus, which is critical for stable transgene expression and the repeated administration of the viral vectors. Indeed, initial studies revealed that a transient blockade of CD40 signaling with an antibody to CD40L infused at the time of vector administration resulted in stable transgene expression and diminished production of neutralizing antibodies in a murine model of adenovirus-mediated gene transfer to the liver and the lungs (Yang et al., 1996). Later, anti-mouse CD40L mAb in combination with CTLA-4Ig has been shown to further prolong adenovirus-mediated transgene expression after both primary and secondary vector delivery (Kay et al., 1995). Similarly, the combined CD40L blockade and CTLA-4Ig approach also proved effective in a murine model of retrovirus-mediated gene therapy for mucopolysaccharidosis (Ma et al., 2007). More recently, the dual blockade strategy has been shown to act synergistically to prevent antigen-specific immune responses in non-human primates, leading to stable expression of transgene and diminished neutralizing antibody production, and in some cases allowing for repeated administration of adenovirus (Haegel-Kronenberger et al., 2004).
The effectiveness of the combined antibody blockade strategy led to the evaluation for the effect of systemic administration of AdmCTLA-4Ig and AdmCD40Ig (adenoviral vector carrying murine CD40Ig) in murine models of gene delivery to the muscle (Jiang et al., 2004b) and the liver (Schowalter et al., 1997). These studies showed that systemic delivery of both AdmCTLA-4Ig and AdmCD40Ig was required to inhibit the production of anti-adenoviral vector neutralizing antibody, and that AdmCTLA-4Ig alone was insufficient. This may be due to insufficient circulating level of mCTLA-4Ig to prevent anti-adenoviral capsid protein neutralizing antibody production, and that addition of circulating levels of mCD40Ig decreases B cell activation by T cells hence leading to diminished neutralizing antibody production.
Taken together, these data lend support to the effectiveness of the combined CD40 and CD28 blockade strategy in viral vector-mediated gene therapy in animal models. However, the efficacy of this approach in human gene therapy requires further exploration in clinical trials.
T cell-dependent B cell responses are critically dependent on the interaction of ICOS with ICOS-L, which promotes germinal center formation and immunoglobulin class switching in vivo (Mcadam et al., 2001; Tafuri et al., 2001). It has been shown that anti-ICOS alone or in combination with CD40Ig or anti-CD40L can induce tolerance to islet allografts (Nanji et al., 2006) and inhibit allograft rejection in murine models of transplantation (Guillonneau et al., 2005; Taylor et al., 2005). In a murine model of non-viral mediated gene therapy for hemophilia A, blockade of the ICOS–ICOS-L pathway by anti-ICOS antibody alone was effective in inhibiting the formation of anti-factor VIII antibodies following plasmid DNA-mediated gene transfer (Peng et al., 2008). In this study, anti-ICOS treatment also resulted in elevation of Treg numbers and their suppressive activity (Peng et al., 2008). However, the efficacy of ICOS blockade has not been tested in animal models of viral vector-mediated gene transfer.
Recent advances in our understanding of the role of the innate immunity in adaptive immune responses suggest potential novel strategies for targeting the co-stimulatory pathways. The innate immune system is the first line of defense against invading pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by a set of receptors called pattern recognition receptors (PRRs; Huang and Yang, 2009). Among the PRRs identified to date, the best studied is the toll-like receptor (TLR) family. Thirteen TLRs have been identified in mammals, with each recognizing a unique set of PAMPs. Most TLRs signal through the myeloid differentiating factor 88 (MyD88)-dependent pathway, initiating the downstream signaling cascade leading to the induction of proinflammatory response characterized by the production of cytokines and chemokines (Akira et al., 2006). In addition, TLR pathways are critical for the ensuing adaptive immune responses by promoting DC maturation and function through upregulation of co-stimulatory molecules (Iwasaki and Medzhitov, 2004; Huang and Yang, 2010).
Recent studies have demonstrated that the innate immune response to AAV is mediated by TLR9 (Zhu et al., 2009; Martino et al., 2011). AAV mainly activates the TLR9–MyD88 pathway in pDCs, leading to the production of type I IFNs (Zhu et al., 2009). In vivo, the TLR9–MyD88 pathway is crucial to the activation of CD8 T cell response to both the transgene product and the AAV vector, leading to the loss of transgene expression and the generation of neutralizing antibodies to transgene product and AAV (Zhu et al., 2009). Thus, the TLR9–MyD88-type I IFN pathway plays a crucial role in promoting the adaptive immune responses to AAV.
Stimulation of TLR9 with its ligand, CpG-DNA leads to upregulation of surface expression of co-stimulatory molecules, such as CD40, B7-1/B7-2, and MHC class II on cDCs and macrophages (Hemmi et al., 2003). Therefore, DC maturation and Th1-like cytokine induced by TLR9 stimulation result in efficient and robust activation of Th1 and CD8 cytotoxic T lymphocytes (CTL; Lipford et al., 2000; Horkheimer et al., 2009). Type I IFNs, in addition to their direct anti-viral effects (Vaidya and Cheng, 2003), have also been shown to induce DC maturation by upregulating the expression of co-stimulatory molecules, which in turn promotes T cell responses (Hoebe et al., 2003). DCs matured by type I IFNs promote cross-priming of virus-specific CD8 T cells (Le Bon et al., 2003). Type I IFN can also promote effector function of virus-specific T cells (Cousens et al., 1999), and enhance the survival of activated T cells (Marrack et al., 1999). Furthermore, type I IFN signaling on DCs is important for the production of virus-specific IgM, whereas the generation of protective neutralizing antibodies to adenovirus critically depends on the type I IFN signaling on both CD4 T cells and B cells (Zhu et al., 2007).
A critical role for the TLR9 signaling pathway in the upregulation of co-stimulatory molecules and the induction of adaptive immune response to AAV suggests that strategies targeted to interfere with this signaling pathway may diminish T cell responses to the AAV vector and the transgene product, leading to prolonged transgene expression and reduction in inflammation, improving the safety and efficacy of AAV vectors for gene therapy in humans. Furthermore, blockade of the TLR9 signaling pathway will also likely inhibit antibody responses to the transgene product as well as the AAV vector.
One way to interfere with the TLR9–MyD88-type I IFN signaling pathway is to use antagonists specific to TLR9 such as inhibitory ODN to block the TLR9–MyD88 pathway (Zhu et al., 2009; Martino et al., 2011). Alternatively, blockade of type I IFNs by administering neutralizing antibodies to IFN-α and IFN-β may also prove effective as type I IFNs are required for adaptive immune responses to AAV (Zhu et al., 2009).
In this review, we have summarized the role of various T cell co-stimulatory pathways in the activation of both T and B cells and strategies targeting these pathways to improve the outcome of viral vector-mediated gene therapy. The majority of the studies so far are focused on targeting the B7–CD28 and the CD40L–CD40 pathways. It may be beneficial to investigate the effect of blocking other positive co-stimulatory pathways such as ICOS and ICOS-L, and OX40 and OX40L. In addition, strategies for stimulating the negative co-stimulatory pathways such as PD-1 and PD-L may prove effective. Given that the combined blockade of CD40 and CD28 pathways is more effective than the blockade of a single pathway alone, future studies should focus on targeting multiple pathways including both the positive and negative co-stimulatory pathways. In this regard, since the innate immunity is critical in regulating multiple co-stimulatory pathways, strategies targeting innate immune pathways may warrant further investigation. Ultimately, the utility of these strategies in improving the outcome of gene therapy with viral vectors will need to be tested in human clinical trials.
Acuto, O., and Michel, F. (2003). CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 3, 939–951.
Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783–801.
Allen, R. C., Armitage, R. J., Conley, M. E., Rosenblatt, H., Jenkins, N. A., Copeland, N. G., Bedell, M. A., Edelhoff, S., Disteche, C. M., Simoneaux, D. K., Fanslow, W. C., Belmont, S., and Spriggs, M. K. (1993). CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science 259, 990–993.
Ashtari, M., Cyckowski, L. L., Monroe, J. F., Marshall, K. A., Chung, D. C., Auricchio, A., Simonelli, F., Leroy, B. P., Maguire, A. M., Shindler, K. S., and Bennett, J. (2011). The human visual cortex responds to gene therapy-mediated recovery of retinal function. J. Clin. Invest. 121, 2160–2168.
Barber, D. L., Wherry, E. J., Masopust, D., Zhu, B., Allison, J. P., Sharpe, A. H., Freeman, G. J., and Ahmed, R. (2006). Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682–687.
Bauquet, A. T., Jin, H., Paterson, A. M., Mitsdoerffer, M., Ho, I. C., Sharpe, A. H., and Kuchroo, V. K. (2009). The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells. Nat. Immunol. 10, 167–175.
Bennett, S. R., Carbone, F. R., Karamalis, F., Flavell, R. A., Miller, J. F., and Heath, W. R. (1998). Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393, 478–480.
Borriello, F., Sethna, M. P., Boyd, S. D., Schweitzer, A. N., Tivol, E. A., Jacoby, D., Strom, T. B., Simpson, E. M., Freeman, G. J., and Sharpe, A. H. (1997). B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6, 303–313.
Brown, K. E., Freeman, G. J., Wherry, E. J., and Sharpe, A. H. (2010). Role of PD-1 in regulating acute infections. Curr. Opin. Immunol. 22, 397–401.
Bruniquel, D., and Schwartz, R. H. (2003). Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat. Immunol. 4, 235–240.
Buhlmann, J. E., and Noelle, R. J. (1996). Therapeutic potential for blockade of the CD40 ligand, gp39. J. Clin. Immunol. 16, 83–89.
Burmeister, Y., Lischke, T., Dahler, A. C., Mages, H. W., Lam, K. P., Coyle, A. J., Kroczek, R. A., and Hutloff, A. (2008). ICOS controls the pool size of effector-memory and regulatory T cells. J. Immunol. 180, 774–782.
Cousens, L. P., Peterson, R., Hsu, S., Dorner, A., Altman, J. D., Ahmed, R., and Biron, C. A. (1999). Two roads diverged: interferon alpha/beta- and interleukin 12-mediated pathways in promoting T cell interferon gamma responses during viral infection. J. Exp. Med. 189, 1315–1328.
Dall'era, M., and Davis, J. (2004). CTLA4Ig: a novel inhibitor of costimulation. Lupus 13, 372–376.
Disanto, J. P., Bonnefoy, J. Y., Gauchat, J. F., Fischer, A., and De Saint Basile, G. (1993). CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM. Nature 361, 541–543.
Fischer, A., Hacein-Bey-Abina, S., and Cavazzana-Calvo, M. (2011). Gene therapy for primary adaptive immune deficiencies. J. Allergy Clin. Immunol. 127, 1356–1359.
Gimmi, C. D., Freeman, G. J., Gribben, J. G., Sugita, K., Freedman, A. S., Morimoto, C., and Nadler, L. M. (1991). B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2. Proc. Natl. Acad. Sci. U.S.A. 88, 6575–6579.
Good-Jacobson, K. L., Szumilas, C. G., Chen, L., Sharpe, A. H., Tomayko, M. M., and Shlomchik, M. J. (2010). PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells. Nat. Immunol. 11, 535–542.
Green, J. M., Noel, P. J., Sperling, A. I., Walunas, T. L., Gray, G. S., Bluestone, J. A., and Thompson, C. B. (1994). Absence of B7-dependent responses in CD28-deficient mice. Immunity 1, 501–508.
Grewal, I. S., Foellmer, H. G., Grewal, K. D., Xu, J., Hardardottir, F., Baron, J. L., Janeway, C. A. Jr., and Flavell, R. A. (1996). Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273, 1864–1867.
Guillonneau, C., Aubry, V., Renaudin, K., Seveno, C., Usal, C., Tezuka, K., and Anegon, I. (2005). Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade. Transplantation 80, 546–554.
Haegel-Kronenberger, H., Haanstra, K., Ziller-Remy, C., Ortiz Buijsse, A. P., Vermeiren, J., Stoeckel, F., Van Gool, S. W., Ceuppens, J. L., Mehtali, M., De Boer, M., Jonker, M., and Boon, L. (2004). Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys. Gene Ther. 11, 241–252.
Hathcock, K. S., Laszlo, G., Pucillo, C., Linsley, P., and Hodes, R. J. (1994). Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and function. J. Exp. Med. 180, 631–640.
Hemmi, H., Kaisho, T., Takeda, K., and Akira, S. (2003). The roles of toll-like receptor 9, MyD88, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J. Immunol. 170, 3059–3064.
Hoebe, K., Janssen, E. M., Kim, S. O., Alexopoulou, L., Flavell, R. A., Han, J., and Beutler, B. (2003). Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229.
Hollenbaugh, D., Mischel-Petty, N., Edwards, C. P., Simon, J. C., Denfeld, R. W., Kiener, P. A., and Aruffo, A. (1995). Expression of functional CD40 by vascular endothelial cells. J. Exp. Med. 182, 33–40.
Horkheimer, I., Quigley, M., Zhu, J., Huang, X., Chao, N. J., and Yang, Y. (2009). Induction of type I IFN is required for overcoming tumor-specific T-cell tolerance after stem cell transplantation. Blood 113, 5330–5339.
Huang, X., and Yang, Y. (2009). Innate immune recognition of viruses and viral vectors. Hum. Gene Ther. 20, 293–301.
Huang, X., and Yang, Y. (2010). Targeting the TLR9-MyD88 pathway in the regulation of adaptive immune responses. Expert Opin. Ther. Targets 14, 787–796.
Ijima, K., Murakami, M., Okamoto, H., Inobe, M., Chikuma, S., Saito, I., Kanegae, Y., Kawaguchi, Y., Kitabatake, A., and Uede, T. (2001). Successful gene therapy via intraarticular injection of adenovirus vector containing CTLA4IgG in a murine model of type II collagen-induced arthritis. Hum. Gene Ther. 12, 1063–1077.
Ito, T., Hanabuchi, S., Wang, Y. H., Park, W. R., Arima, K., Bover, L., Qin, F. X., Gilliet, M., and Liu, Y. J. (2008). Two functional subsets of FOXP3+ regulatory T cells in human thymus and periphery. Immunity 28, 870–880.
Iwasaki, A., and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, 987–995.
Jenkins, M. K., Chen, C. A., Jung, G., Mueller, D. L., and Schwartz, R. H. (1990). Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J. Immunol. 144, 16–22.
Jiang, Z., Schiedner, G., Gilchrist, S. C., Kochanek, S., and Clemens, P. R. (2004a). CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle. Gene Ther. 11, 1453–1461.
Jiang, Z., Schiedner, G., Van Rooijen, N., Liu, C. C., Kochanek, S., and Clemens, P. R. (2004b). Sustained muscle expression of dystrophin from a high-capacity adenoviral vector with systemic gene transfer of T cell costimulatory blockade. Mol. Ther. 10, 688–696.
Kane, L. P., Lin, J., and Weiss, A. (2002). It's all relative: NF-kappaB and CD28 costimulation of T-cell activation. Trends Immunol. 23, 413–420.
Kay, M. A., Holterman, A. X., Meuse, L., Gown, A., Ochs, H. D., Linsley, P. S., and Wilson, C. B. (1995). Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat. Genet. 11, 191–197.
Keir, M. E., Butte, M. J., Freeman, G. J., and Sharpe, A. H. (2008). PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26, 677–704.
King, C. L., Stupi, R. J., Craighead, N., June, C. H., and Thyphronitis, G. (1995). CD28 activation promotes Th2 subset differentiation by human CD4+ cells. Eur. J. Immunol. 25, 587–595.
Lafferty, K. J., Andrus, L., and Prowse, S. J. (1980). Role of lymphokine and antigen in the control of specific T cell responses. Immunol. Rev. 51, 279–314.
Lafferty, K. J., Misko, I. S., and Cooley, M. A. (1974). Allogeneic stimulation modulates the in vitro response of T cells to transplantation antigen. Nature 249, 275–276.
Larsen, C. P., Pearson, T. C., Adams, A. B., Tso, P., Shirasugi, N., Strobert, E., Anderson, D., Cowan, S., Price, K., Naemura, J., Emswiler, J., Greene, J., Turk, L. A., Bajorath, J., Townsend, R., Hagerty, D., Linsley, P. S., and Peach, R. J. (2005). Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent immunosuppressive properties. Am. J. Transplant. 5, 443–453.
Laumonier, T., Potiron, N., Boeffard, F., Chagneau, C., Brouard, S., Guillot, C., Soulillou, J. P., Anegon, I., and Le Mauff, B. (2003). CTLA4Ig adenoviral gene transfer induces long-term islet rat allograft survival, without tolerance, after systemic but not local intragraft expression. Hum. Gene Ther. 14, 561–575.
Le Bon, A., Etchart, N., Rossmann, C., Ashton, M., Hou, S., Gewert, D., Borrow, P., and Tough, D. F. (2003). Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat. Immunol. 4, 1009–1015.
Lenschow, D. J., Su, G. H., Zuckerman, L. A., Nabavi, N., Jellis, C. L., Gray, G. S., Miller, J., and Bluestone, J. A. (1993). Expression and functional significance of an additional ligand for CTLA-4. Proc. Natl. Acad. Sci. U.S.A. 90, 11054–11058.
Lenschow, D. J., Walunas, T. L., and Bluestone, J. A. (1996). CD28/B7 system of T cell costimulation. Annu. Rev. Immunol. 14, 233–258.
Lenschow, D. J., Zeng, Y., Hathcock, K. S., Zuckerman, L. A., Freeman, G., Thistlethwaite, J. R., Gray, G. S., Hodes, R. J., and Bluestone, J. A. (1995). Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation 60, 1171–1178.
Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K., and Ledbetter, J. A. (1991). Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation. J. Exp. Med. 173, 721–730.
Linsley, P. S., Greene, J. L., Tan, P., Bradshaw, J., Ledbetter, J. A., Anasetti, C., and Damle, N. K. (1992). Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. J. Exp. Med. 176, 1595–1604.
Lipford, G. B., Sparwasser, T., Zimmermann, S., Heeg, K., and Wagner, H. (2000). CpG-DNA-mediated transient lymphadenopathy is associated with a state of Th1 predisposition to antigen-driven responses. J. Immunol. 165, 1228–1235.
Ma, X., Liu, Y., Tittiger, M., Hennig, A., Kovacs, A., Popelka, S., Wang, B., Herati, R., Bigg, M., and Ponder, K. P. (2007). Improvements in mucopolysaccharidosis I mice after adult retroviral vector-mediated gene therapy with immunomodulation. Mol. Ther. 15, 889–902.
Marrack, P., Kappler, J., and Mitchell, T. (1999). Type I interferons keep activated T cells alive. J. Exp. Med. 189, 521–530.
Martino, A. T., Suzuki, M., Markusic, D. M., Zolotukhin, I., Ryals, R. C., Moghimi, B., Ertl, H. C., Muruve, D. A., Lee, B., and Herzog, R. W. (2011). The genome of self-complementary adeno-associated viral vectors increases toll-like receptor 9-dependent innate immune responses in the liver. Blood 117, 6459–6468.
Mcadam, A. J., Greenwald, R. J., Levin, M. A., Chernova, T., Malenkovich, N., Ling, V., Freeman, G. J., and Sharpe, A. H. (2001). ICOS is critical for CD40-mediated antibody class switching. Nature 409, 102–105.
Mcadam, A. J., Schweitzer, A. N., and Sharpe, A. H. (1998). The role of B7 co-stimulation in activation and differentiation of CD4+ and CD8+ T cells. Immunol. Rev. 165, 231–247.
Nabavi, N., Freeman, G. J., Gault, A., Godfrey, D., Nadler, L. M., and Glimcher, L. H. (1992). Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression. Nature 360, 266–268.
Nanji, S. A., Hancock, W. W., Luo, B., Schur, C. D., Pawlick, R. L., Zhu, L. F., Anderson, C. C., and Shapiro, A. M. (2006). Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse. Diabetes 55, 27–33.
Nurieva, R. I., Duong, J., Kishikawa, H., Dianzani, U., Rojo, J. M., Ho, I., Flavell, R. A., and Dong, C. (2003). Transcriptional regulation of th2 differentiation by inducible costimulator. Immunity 18, 801–811.
Peng, B., Ye, P., Blazar, B. R., Freeman, G. J., Rawlings, D. J., Ochs, H. D., and Miao, C. H. (2008). Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice. Blood 112, 1662–1672.
Pipkin, M. E., and Rao, A. (2009). Snapshot: effector and memory T cell differentiation. Cell 138, e601–e602.
Puppi, J., Guillonneau, C., Pichard, V., Bellodi-Privato, M., Cuturi, M. C., Anegon, I., and Ferry, N. (2004). Long term transgene expression by hepatocytes transduced with retroviral vectors requires induction of immune tolerance to the transgene. J. Hepatol. 41, 222–228.
Ridge, J. P., Di Rosa, F., and Matzinger, P. (1998). A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393, 474–478.
Schoenberger, S. P., Toes, R. E., Van Der Voort, E. I., Offringa, R., and Melief, C. J. (1998). T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393, 480–483.
Schowalter, D. B., Meuse, L., Wilson, C. B., Linsley, P. S., and Kay, M. A. (1997). Constitutive expression of murine CTLA4Ig from a recombinant adenovirus vector results in prolonged transgene expression. Gene Ther. 4, 853–860.
Schwartz, R. H., Mueller, D. L., Jenkins, M. K., and Quill, H. (1989). T-cell clonal anergy. Cold Spring Harb. Symp. Quant. Biol. 54(Pt 2), 605–610.
Seder, R. A., Germain, R. N., Linsley, P. S., and Paul, W. E. (1994). CD28-mediated costimulation of interleukin 2 (IL-2) production plays a critical role in T cell priming for IL-4 and interferon gamma production. J. Exp. Med. 179, 299–304.
Sharpe, A. H. (2009). Mechanisms of costimulation. Immunol. Rev. 229, 5–11.
Simpson, T. R., Quezada, S. A., and Allison, J. P. (2010). Regulation of CD4 T cell activation and effector function by inducible costimulator (ICOS). Curr. Opin. Immunol. 22, 326–332.
Stack, R. M., Lenschow, D. J., Gray, G. S., Bluestone, J. A., and Fitch, F. W. (1994). IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2. J. Immunol. 152, 5723–5733.
Tafuri, A., Shahinian, A., Bladt, F., Yoshinaga, S. K., Jordana, M., Wakeham, A., Boucher, L. M., Bouchard, D., Chan, V. S., Duncan, G., Odermatt, B., Ho, A., Itie, A., Horan, T., Whoriskey, J. S., Pawson, T., Penninger, J. M., Ohashi, P. S., and Mak, T. W. (2001). ICOS is essential for effective T-helper-cell responses. Nature 409, 105–109.
Taylor, P. A., Panoskaltsis-Mortari, A., Freeman, G. J., Sharpe, A. H., Noelle, R. J., Rudensky, A. Y., Mak, T. W., Serody, J. S., and Blazar, B. R. (2005). Targeting of inducible costimulator (ICOS) expressed on alloreactive T cells down-regulates graft-versus-host disease (GVHD) and facilitates engraftment of allogeneic bone marrow (BM). Blood 105, 3372–3380.
Trinchieri, G. (1994). Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84, 4008–4027.
Vaidya, S. A., and Cheng, G. (2003). Toll-like receptors and innate antiviral responses. Curr. Opin. Immunol. 15, 402–407.
Valle, A., Aubry, J. P., Durand, I., and Banchereau, J. (1991). IL-4 and IL-2 upregulate the expression of antigen B7, the B cell counter structure to T cell CD28: an amplification mechanism for T-B cell interactions. Int. Immunol. 3, 229–235.
Van Der Pouw-Kraan, T., Van Kooten, C., Rensink, I., and Aarden, L. (1992). Interleukin (IL)-4 production by human T cells: differential regulation of IL-4 vs. IL-2 production. Eur. J. Immunol. 22, 1237–1241.
Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., Thompson, C. B., and Bluestone, J. A. (1994). CTLA-4 can function as a negative regulator of T cell activation. Immunity 1, 405–413.
Waterhouse, P., Penninger, J. M., Timms, E., Wakeham, A., Shahinian, A., Lee, K. P., Thompson, C. B., Griesser, H., and Mak, T. W. (1995). Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270, 985–988.
Yang, Y., Su, Q., Grewal, I. S., Schilz, R., Flavell, R. A., and Wilson, J. M. (1996). Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues. J. Virol. 70, 6370–6377.
Yang, Y., and Wilson, J. M. (1996). CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40. Science 273, 1862–1864.
Zhao, K., Wang, W., Rando, O. J., Xue, Y., Swiderek, K., Kuo, A., and Crabtree, G. R. (1998). Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 95, 625–636.
Zhu, J., Huang, X., and Yang, Y. (2007). Innate immune response to adenoviral vectors is mediated by both toll-like receptor-dependent and -independent pathways. J. Virol. 81, 3170–3180.
Zhu, J., Huang, X., and Yang, Y. (2009). The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice. J. Clin. Invest. 119, 2388–2398.
Keywords: co-stimulation, viral vectors, gene therapy
Citation: Huang X and Yang Y (2011) Targeting co-stimulatory pathways in gene therapy. Front. Microbio. 2:202. doi: 10.3389/fmicb.2011.00202
Received: 28 June 2011; Accepted: 07 September 2011;
Published online: 28 September 2011.
Roland W. Herzog, University of Florida, USA
Hildegund C. Ertl, Wistar Institute, USA
Ashley T. Martino, St John's University, USA
Copyright: © 2011 Huang and Yang. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.
*Correspondence: Yiping Yang, Departments of Medicine and Immunology, Duke University Medical Center, Box 103005, 106 Research Drive, Durham, NC 27710, USA. e-mail: yang0029@mc.duke.edu
Approaches to Blocking the Immune Response to Gene Transfer with Viral Vectors


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