Pathogenic features of CD4+CD28–

T cells in immune disordersBieke Broux1, Silva Markovic-Plese2,3, Piet Stinissen1 and Niels Hellings1

1 Hasselt University, Biomedical Research Institute and Transnationale Universiteit Limburg, School of Life Sciences,

3590 Diepenbeek, Belgium2 Department of Neurology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA3 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA

Review

Glossary

Compensatory autoproliferation: owing to thymic involution, the thymic

output decreases with advancing age. To compensate for that decrease,

proliferation of peripheral memory T cells is driven by the cytokines IL-7 and IL-

15. In collaboration with other mechanisms (e.g., latent infections), this

compensatory autoproliferation may lead to the expansion of end-differen-

tiated T cells.

Fractalkine and receptor (CX3CR1): fractalkine is the only member of the CX3C-

chemokine family and consists of an N-terminal chemokine domain attached to

a mucin-like stalk. Fractalkine is a chemokine when secreted and an adhesion

molecule when membrane bound. Both fractalkine and its receptor are

constitutively expressed in normal brain and upregulated in cerebrospinal

fluid, serum, and brain lesions of MS patients.

Granuloma: an organized collection of macrophages. It is formed when the

immune system is unable to eliminate a foreign substance, including

pathogens. Granulomata can therefore exist in many different immune

diseases, including granulomatosis with polyangiitis (GPA) and Crohn’s

disease (CD).

Inflammatory bowel disease (IBD): a group of inflammatory disorders of the

colon and small intestine. Crohn’s disease (CD) and ulcerative colitis (UC) are

the main types of IBD, respectively exhibiting inflammation of the entire

gastrointestinal tract and inflammation of the colon and rectum. Microscopic

differences lie in the fact that in CD the entire bowel wall is affected, whereas in

UC inflammation is restricted to the mucosa.

Myopathies: diseases of the muscles, caused by dysfunctional muscle fibers.

There are three types of myopathies caused by autoimmunity: dermatomyo-

sitis, polymyositis and inclusion body myositis. Muscle weakness is a common

symptom of these myopathies, but the mechanism and affected muscles are

different.

Oligoclonality: a healthy T cell population consists of T cells reactive to a broad

range of antigens; this population is called polyclonal. When T cells are

repeatedly stimulated by the same antigen, contraction of this diversity of T

cell specificities occurs because of the formation of memory T cells specific to

the antigen. When this process is repeated several times, oligoclonal T cell

populations will arise, taking up most of the T cell repertoire.

Synoviocytes: the synovial tissue consists mainly of synoviocytes, which are

fibroblast-like cells. Through cell–cell contacts, they form the synovial lining

layer and produce synovial fluid components. In rheumatoid arthritis (RA),

these cells secrete proinflammatory cytokines and proteases, thereby con-

tributing to disease pathogenesis

Thymopoiesis: T cell progenitors migrate from the bone marrow to the

thymus, where they mature through the following stages: double negative

(CD4–CD8–), pre-T cell, double positive (CD4+CD8+), single positive immature

(CD4+ or CD8+), and, finally, mature naive T cell. Mature T cells are then

released into the periphery. During the maturation process, only those T cells

Aging of the immune system contributes to the in-creased morbidity and mortality of the elderly popula-tion and may occur prematurely in patients with immunedisorders. One of the main characteristics of immuno-senescence is the expansion of CD4+CD28– T cells in theblood. These cells are effector memory T cells withcytotoxic capacity, and have been recently describedto have pathogenic potential in a variety of immunedisorders. Interestingly, CD4+CD28– T cells have nowbeen found to infiltrate target tissues of patients withmultiple sclerosis, rheumatoid arthritis, myopathies,acute coronary syndromes, and other immune-relateddiseases. In this review, we discuss potential factors andmechanisms that may induce the expansion of thesecells, as well as their putative pathogenic mechanisms inimmune disorders.

The emergence of CD4+CD28– T cells and their role inimmune disordersImmunosenescence, or aging of the immune system, has atremendous impact on the health of the elderly population,contributing to their increased susceptibility to infectionsand a suboptimal response to vaccination [1]. During agingthe immune system undergoes alterations in size, compo-sition, and function. For example, the humoral immuneresponse and associated antibody titers decrease with age,leading to an ineffective response to vaccination [2]. Bothhumoral and cellular immune responses require help fromCD4+ T cells, but these helper T cells are also affected byincreasing age due to thymic involution and chronic anti-genic stimulation [by e.g., latent viruses such as cytomeg-alovirus (CMV) and autoantigens], shifting the balancefrom naive to memory T cells within the CD4 compart-ment. Phenotypically, CD4+ T cells gradually lose expres-sion of the co-stimulatory molecule CD28, which isassociated with the loss of a CD28-specific initiator com-plex [3]. These CD4+CD28– T cells have distinct phenotyp-ical and functional characteristics. For instance, they haveshortened telomeres and reduced T cell receptor (TCR)diversity and cytotoxic capacity (for an extensive reviewon the alterations in CD28– T cells during aging, see [4]).

Several reports have described the expansion ofCD4+CD28– T cells in the peripheral circulation of patientswith various immune disorders, including autoimmune

Corresponding author: Hellings, N. (niels.hellings@uhasselt.be)

446 1471-4914/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org

diseases [5], chronic inflammatory diseases [6,7], and im-mune deficiency [8]. Patients suffering from these disordersoften show an age-inappropriate expansion of CD4+CD28–Tcells, indicating premature aging of their immune system[9]. Although this early immunosenescence likely reflectsthe chronic immune responses in these patients, it can alsorepresent pathogenic mechanisms that contribute to thedevelopment of the indicated disease groups. In the past

that are able to bind to self-MHC and have low affinity to self-antigens will

ultimately survive.

/10.1016/j.molmed.2012.06.003 Trends in Molecular Medicine, August 2012, Vol. 18, No. 8

Review Trends in Molecular Medicine August 2012, Vol. 18, No. 8

5 years, CD4+CD28– T cells were identified in target tissuesin several immune disorders [9–11]. Given their cytotoxicfeatures, it is hypothesized that CD4+CD28– T cells mightcontribute to disease pathogenesis.

In this review, we provide a detailed analysis of thefactors and mechanisms leading to CD4+CD28– T cellemergence in normal aging and the premature expansionof these cells in the context of immune disorders. Wediscuss the pathogenic role of CD4+CD28– T cells in thedevelopment and the progression of these diseases. Morespecifically, we review the possible mechanisms by whichthese cells exert their pathogenic function and discusstherapeutic approaches to target these cells.

Immunosenescence: normal versus premature aging ofthe immune systemThe thymus is a vital organ for the T cell maturation orthymopoiesis (see Glossary). With age, the thymus under-goes profound atrophy (also known as involution), in whichthe thymic epithelial tissue is replaced by fat. The migra-tion of naive T cells from the thymus to the periphery, orthymic output, starts to decline from the first year of life[12]. By the age of 70, thymopoiesis and thymic output arenegligible (for a recent review on thymic involution, see[13]). As a result of this gradual decline in the number ofnewly produced naive T cells, the peripheral immunebalance shifts towards the memory T cells. These memoryT cells undergo compensatory autoproliferation, driven bythe common g chain cytokines interleukin (IL)-7 and IL-15[14]. However, this T cell renewal seems to be independentof thymic involution in adult humans, in contrast with mice[15]. Nonetheless, phenotypically and functionally alteredT cells are found in the elderly, accounting for the in-creased risk for infection in the elderly population [16].

In addition to normal aging, premature immunosenes-cence may also occur in younger individuals. Chronicinflammation or latent infections induce repeated antigen-ic stimulation of T cells, causing them to proliferate andage more rapidly [16], and senescent T cells are present inapparently healthy individuals with latent CMV infections[17]. It is well established that CMV infection has a pro-found impact on the immune system, leading to the accu-mulation of late differentiated CD4+ and CD8+ T cells [18],expansion of CD4+CD28– T cells with cytotoxic properties[19], and differentiation of macrophages to the proinflam-matory M1 phenotype [20]. Together, these immunealterations suggest that CMV has a detrimental effecton health, which is not apparent in early life. Severalrecent studies have confirmed the hypothesis that CMVinfection correlates with increased mortality [21–23].Thus, even though CMV infection is typically asymptom-atic, it may affect the capacity of the immune system todevelop appropriate responses to pathogens throughoutthe entire life.

In summary, immunosenescence is a normal feature ofaging, and is due to thymic involution and decreasedthymic output. However, premature aging can occur inyounger individuals when caused by chronic inflammationand latent infections. In the latter case, aging of theimmune system can have severe consequences on theimmunological responses in these individuals.

CD4+CD28– T cells: loss of CD28 and gain of pathogenicfeaturesIn the late 1980s, the group of Hansen and Martin de-scribed a subset of CD4+ T cells that lack CD28 [24–26]. Itwas not until the late 1990s that the function of these cellswas better characterized. The group of Weyand and Gor-onzy was the first to identify CD4+CD28– T cells in thecontext of rheumatoid arthritis (RA), a chronic inflamma-tory joint disease characterized by massive inflammatoryinfiltrates and bone destruction [27]. Downregulation ofCD28 is caused by the loss of a CD28-specific initiatorcomplex [3,28–30]. CD28 is progressively lost after repli-cative senescence [29], but the loss can also occur underproinflammatory conditions, for example in the presence oftumor necrosis factor (TNF)-a [31].

The first functional characteristic of CD4+CD28– T cellsdiscovered was their co-stimulation independent nature.Although CD28 is absent on these cells, they are not anergicand respond to stimulation [27]. Soon thereafter, research-ers observed that CD4+CD28– T cells are oligoclonal andshow a restricted TCR diversity [27,32]. This propertyimplied that CD4+CD28– T cells are repeatedly stimulatedby the same antigens, thereby forming a population ofoligoclonal memory T cells. Functionally, these cells exhibitcytotoxic features including the expression of perforin, gran-zymes [33], and natural killer (NK) cell receptors [34]. Also,CD4+CD28– T cells are resistant to apoptosis after IL-2deprivation and activation [35,36]. This resistance is asso-ciated with the increased expression of the antiapoptoticmolecules Bcl-2 [35] and Fas-associated death domain-likeIL-1-converting enzyme inhibitory protein (FLIP) [36]. Fur-thermore, CD4+CD28– T cells infiltrate target tissues, asfirst described for RA by the group of Weyand and Goronzy[33,37]. This finding was however countered by Fasth et al.,who observed that CD4+CD28– T cells were scarce in thesynovial fluid and membrane [38]. More recently, it wasshown that CD4+CD28– T cells are less susceptible to sup-pression by CD4+CD25high regulatory T cells than conven-tional CD4+T cells [39]. The cytotoxic capacity, resistance toapoptosis, and tissue infiltration by CD4+CD28– T cellssuggested that these cells play a pathogenic role in diseasedevelopment. In RA, where this involvement was firstdescribed, the expansion of CD4+CD28– T cells waspositively correlated with the presence of extra-articularmanifestations [40].

The second disease for which the expansion ofCD4+CD28– T cells was described is multiple sclerosis(MS), a chronic inflammatory demyelinating disease of thecentral nervous system, where immune cells infiltrate thebrain and spinal cord and destroy the myelin sheaths sur-rounding axons [41,42]. CD4+CD28– T cells were reported tobe at least partly autoreactive because they showed in-creased proliferation against myelin basic protein (MBP, asuspected autoantigen in MS) as compared to CD4+CD28+ Tcells [43]. In the same study, CD4+CD28– T cells producedhigher levels of the proinflammatory cytokine interferon(IFN)-g, in comparison with their CD28+ counterparts.

Taken together, these early reports show thatCD4+CD28–T cells have pathogenic features, and suggestedthat they might play a role in the pathogenesis of immunedisorders.

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Review Trends in Molecular Medicine August 2012, Vol. 18, No. 8

CD4+CD28– T cells in immune disorders: bystanders orcontributors?After the discovery of this unique population of CD4+ T cells,they were characterized in several immune disorders.After their detection in RA and MS, both chronic inflamma-tory and, presumably, autoimmune diseases (Box 1),CD4+CD28– T cells were next identified in granulomatosiswith polyangiitis (GPA; formerly Wegener’s granulomatosis),Graves’ disease (GD), systemic lupus erythematosus (SLE)and autoimmune myopathies, among others. Recently,CD4+CD28– T cells have been described in cardiovasculardisease (acute coronary syndromes, stable angina, athero-sclerosis, coronary artery disease), inflammatory bowel dis-ease (Crohn’s disease and ulcerative colitis), and viralinfections (HIV, hepatitis B). A summary is provided inTable 1.

Autoimmune diseases

RA is considered an autoimmune disease, characterized bythe presence of autoantibodies (rheumatoid factor and anti-citrullinated peptide antibodies) and autoreactive T cells.Early reports describe the pathogenic characteristics ofthese cells, such as their cytotoxic potential [33,34], resis-tance to apoptosis [35,36], and tissue-infiltrating capacity[33,37]. Later studies have focused on the interactions ofCD4+CD28– T cells with other immune cells. Michel et al.reported that upon loss of CD28, CD4+ T cells gain expres-sion of CD56, and CD56+ T cells were found in lung infil-trates of RA patients suffering from RA-related interstitialpneumonitis. Moreover, cross linking of CD56 inducedproduction of IL-2, TNF-a, and macrophage inflammatoryprotein (MIP)-1b by these cells [44]. CD4+CD28– T cells

Box 1. RA and MS: common features of two chronic

inflammatory/autoimmune diseases

Although the target tissue of each disease differs, being the synovial

membrane of the joints in RA and the cells of the central nervous

system in MS, there are many common features shared between the

two (for an extended review on RA pathogenesis, see [78] and for

MS, see [79]). First, RA and MS both have a genetic component, with

HLA-DRB1 polymorphisms being the strongest contributors to the

risk of developing RA or MS. Both are chronic inflammatory

disorders, with an alleged autoimmune component, and in both

diseases immune infiltrates consisting of macrophages, T cells, and

B cells are found in the affected tissues. After migration to these

tissues, immune cells produce a variety of cytokines and chemo-

kines that perpetuate the immune response and contribute to tissue

destruction. This destruction leads to irreversible damage and loss

of function of the target organ. In RA, the joints are irreversibly

damaged, leading to progressive disability of patients. In MS, not

only myelin sheaths, but also neurons are lost, leading to

irreversible neurological deficits.

One might anticipate that these shared features regarding

pathogenesis might translate into common therapeutic options for

both patient populations. Nevertheless, there are substantial

differences concerning treatment in these cases. For example,

blockade of TNF-a is the standard treatment for RA patients,

whereas this mechanism has no effect on the inflammation in MS

patients and can even exacerbate disease [76]. This observation

suggests that, at first sight, there are general similarities between

both diseases, but when investigating these processes in more

detail, small but significant differences will be encountered. There-

fore, key processes need to be identified in both diseases to develop

specific therapeutic applications.

448

received costimulation by activating NK cell receptors uponTCR triggering [34,45]. The expression by synoviocytes offractalkine, which can be secreted as a chemokine and mayact as an adhesion molecule when membrane bound, alsoprovides costimulation to CD4+CD28– T cells through ex-pression of the fractalkine receptor (CX3CR1) [46]. The samegroup further reported that TNF-a production byCD4+CD28– T cells was induced by fractalkine–CX3CR1ligation, which in turn provided growth stimulation tosynoviocytes [47]. In summary, several studies have provid-ed substantial evidence for a contributing role ofCD4+CD28– T cells in the RA pathogenesis.

The reports on roles for CD4+CD28– T cells in MS arefew but compelling. The first identification of these cells inMS came in 1998, by two independent groups [41,42]. Theyfound that proliferation of MBP-reactive T cells in MSpatients is independent of CD28 costimulation. Markovic-Plese et al. further described CD4+CD28– T cells as Thelper 1 cells producing IFN-g and exhibiting increasedsurvival after activation [43]. For a long time, these werethe only reports on CD4+CD28– T cells in MS. In the pastfew years, it has been reported that CD4+CD28– T cellsexpress cytotoxic molecules, including perforin, granzyme,and IFN-g [48], and adhesion molecules that enhance theircapacity to infiltrate inflamed tissues [39]. More recently,Miyazaki et al. confirmed the capacity of these cells toproduce high amounts of IFN-g, especially in untreated MSpatients [49]. Because no reports were available on thepresence of CD4+CD28– T cells in MS brain lesions, weinvestigated the migratory capacity of this cell populationin the context of MS [10]. First, the selective expression ofthe fractalkine receptor CX3CR1 on CD4+CD28– T cellswas confirmed. This receptor mediated the in vitro migra-tion of CD4+CD28– T cells along a fractalkine gradient.Fractalkine is upregulated in serum and cerebrospinalfluid (CSF) of MS patients [50], and this finding wasconfirmed at the lesion site. Next, the exclusive CX3CR1expression on CD4+CD28– T cells was utilized to identifythese cells in the MS brain lesions, and CD4+CX3CR1

+ Tcells were found in the perivascular cuffs of a subgroup ofMS patients. Given their cytotoxic potential and the abilityto infiltrate brain tissue, CD4+CD28– T cells may contributeto the pathogenesis of MS.

Expansion of CD4+CD28– T cells was also reported inGPA, GD, SLE and autoimmune myopathy [5,9,11,51,52].In GPA, a disease characterized by granuloma formationand autoimmune vasculitis, these cells preferentiallyexpressed NKG2D, an activating NK cell receptor andwere found in GPA granulomata. In GD, a thyroid glandautoimmune disease, CD4+CD28– T cells were character-ized as memory T cells producing large amounts of IFN-g,in line with reports in other autoimmune diseases. In SLE,a multi-organ autoimmune disease characterized by thedeposition of immune complexes, an increased percentageof CD4+NKG2D+ T cells has been reported. BecauseNKG2D expression is limited to CD4+CD28– T cells, wepropose that this finding represents an expansion ofCD4+CD28– T cells. Furthermore, SLE-derived monocytesinduced expression of NKG2D on CD4+ T cells and NKG2Dligation activated these cells. Finally, in autoimmune my-opathies (dermatomyositis, polymyositis, and sporadic

Table 1. Characteristics of CD4+CD28– T cells in different immune disordersa

Disease Characteristics Correlation to disease? Refs

Rheumatoid arthritis Oligoclonal

Cytotoxic

Resistant to apoptosis

Tissue-infiltrating

Autoreactive (antigen unknown)

Proinflammatory (IFN-g, IL-2)

Restricted TCR diversity

Extra-articular manifestations

Decreased T cells after anti-TNF treatment

[27,31,33,34,37,39,40,44,

45,48,67,68,77]

Multiple sclerosis Oligoclonal

Cytotoxic

Tissue-infiltrating

Autoreactive (MBP, MOG)

Proinflammatory (IFN-g)

Restricted TCR diversity

ND [10,39,41–43,48,49]

Granulomatosis with polyangiitis Tissue-infiltrating ND [5]

Graves’ disease Proinflammatory (IFN-g) Graves’ ophthalmopathy

Anti-thyrotropin receptor antibodies

[51]

Systemic lupus erythematosus Activation via NKG2D ligation ND [52]

Autoimmune myopathies Proinflammatory (TNF, IFN-g)

Cytotoxic

Tissue-infiltrating

Restricted TCR diversity

% T cells decreases with increasing

disease duration

[9,11]

Acute coronary syndrome Proinflammatory (IFN-g)

Oligoclonal

Restricted TCR diversity

Cytotoxic

Autoreactive (hHSP60)

More severe atherosclerosis

Decreased after statin treatment

[7,53,54,56,57,72,73]

Inflammatory bowel disease Tissue-infiltrating

Cytotoxic

Proinflammatory (TNF, IFN-g, IL-2)

Activation via NKG2D ligation

Penetrating/stricturing phenotype

% T cells decreases after surgery

[6,64]

Chronic Hepatitis B Cytotoxic High viral load

Elevated aminotransferase levels

[65]

Human Immunodeficiency virus Proinflammatory (IFN-g) ND [8]

aAbbreviations: ND, not determined; TCR, T cell receptor; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; hHSP60, human heat shock protein 60.

Review Trends in Molecular Medicine August 2012, Vol. 18, No. 8

inclusion body myositis), CD4+CD28– T cells were reportedto be proinflammatory and cytotoxic, as well as beingidentified as the predominant infiltrating T cell populationin muscle tissue [9,11]. Both the expansion of CD4+CD28–

T cells and their presence in the target tissues suggest thatthis cell population may contribute to the pathogenesis ofautoimmune diseases.

Cardiovascular disease

Cardiovascular disease is a term encompassing a range ofdisorders from atherosclerosis to acute coronary syndromes(ACS). Thrombosis, associated with plaque rupture, is acommon finding in most of these diseases (Box 2), and inmany of these conditions CD4+CD28– T cell expansion cor-relates with the disease activity. CD4+CD28– T cells werefirst identified in patients with unstable angina (UA) [53], anACS. The frequency of CD4+CD28– T cells was increased incomparison with patients experiencing stable angina [53].Liuzzo et al. described CD4+CD28– T cells in UA patientsthat produced large amounts of IFN-g, which could stimu-late macrophages to secrete metalloproteases and degradethe fibrous cap surrounding the plaque [53]. In two otherstudies, the same group found that CD4+CD28–T cells in UApatients were clonally expanded [54] and could lyse endo-thelial cells via granule exocytosis in the absence of TCRstimulation [55]. Zal et al. studied the antigen specificity ofCD4+CD28–T cells in ACS patients and found that half of thepatients had CD4+CD28– T cells reactive to human heat

shock protein (hHSP) 60, a molecule present on endothelialcells and upregulated in plaques [56]. Furthermore, TCRstimulation with hHSP60 was not sufficient to induce cyto-toxicity. Instead, the interaction of KIR2DS2, an activatingNK cell receptor, with major histocompatibility complex(MHC) class I presenting hHSP60 induced a specific cyto-toxic response [57]. These findings suggest that killing byCD4+CD28–T cells is not TCR-dependent, but can be antigenspecific. Concerning costimulation, Dumitriu et al. showedthat OX40 and 4-1BB might be important factors becauseblocking these costimulatory receptors inhibits inflammato-ry and cytotoxic functions of CD4+CD28– T cells [58].

In addition to coronary artery disease on its own, recentstudies discuss the risk for cardiovascular disease inpatients with immune disorders. In patients with RA[59], end-stage renal disease (ESRD) [60], chronic kidneydisease [61], type 2 diabetes [62], or human immunodefi-ciency virus (HIV) infection [63], CD4+CD28– T cells werefound to be associated with atherosclerosis, suggesting thatCD4+CD28– T cells take part in vascular plaque destabili-zation and rupture in patients with inflammatory disorders.

Inflammatory bowel disease

IBD is a group of inflammatory conditions of the gastroin-testinal tract, with the most common types being Crohn’sdisease (CD) and ulcerative colitis (UC). An increasednumber of CD4+NKG2D+ T cells have been found in theperipheral blood and the lamina propria of CD patients [6].

449

Box 2. Thrombosis: mechanism of plaque rupture

Atherosclerosis has long been considered to be entirely due to

accumulation of lipids in the arterial wall. However, it is now clear

that many inflammatory reactions take place in atherosclerotic

plaques (for a recent update on atherosclerosis, see [80]). It is

important to note that atherosclerosis lesions can be subcategorized

into three stages. The first stage consists of a so-called fatty streak

that is a small lesion consisting of foamy (lipid-laden) macrophages

and T cells, which can already be found in early life. Environmental

factors, such as high cholesterol intake, smoking, hypertension, and

diabetes can lead to endothelial dysfunction. This means that the

endothelium will increase its permeability and its adhesiveness

towards leukocytes and platelets, perpetuating the immune re-

sponse within the lesion, which will progress to the stage of fibrous

plaque. In this stage, the lesion is covered by a fibrous cap

consisting of connective tissue, smooth muscle cells, and macro-

phages. Continued inflammation will cause more leukocytes to

migrate into this lesion, secreting cytokines and chemokines that

exacerbate the immune response. Eventually, the core of the lesion

will consist of lipids and necrotic tissue, which is very thrombogenic

(clot producing). At this stage, the lesion is called a complicated

lesion.

Thrombosis will occur when a complicated lesion ruptures, which

happens as a result of erosion or thinning of the fibrous cap.

Metalloproteases are believed to be involved in the degradation of

this cap, leading to rupture. Activated T cells inside the lesion may

stimulate macrophages to produce these molecules. In addition,

CD4+CD28– T cells have been shown to directly and indirectly

contribute to plaque destabilization. They can directly lyse endothe-

lial cells via granule exocytosis [55] or indirectly contribute to

rupture by producing IFN-g, which stimulates macrophages to

produce metalloproteases [54]. When this lesion ruptures, the

highly thrombogenic core is exposed to the blood and coagulation

of platelets occurs. This will ultimately lead to blood flow disruption,

causing myocardial infarction, stroke, and sudden death.

Review Trends in Molecular Medicine August 2012, Vol. 18, No. 8

As mentioned above, NKG2D expression on CD4+ T cells ismainly limited to the CD28– subset, suggesting that theseare the same population. Indeed, CD4+NKG2D+ T cells ofCD patients were CD28–, expressed perforin, and producedlarge amounts of IFN-g and TNF-a [6]. In the same report,Allez et al. described that ligand binding of NKG2D in-duced IFN-g production and cytotoxicity towards targetcells. UC patients also exhibited expansion of circulatingCD4+CD28– T cells [64]. Taken together, these resultssuggest that CD4+CD28– T cells play a role in the chronicinflammatory response in IBD patients.

Viral infections

In addition to CMV infection, CD4+CD28– T cells have beendescribed in the context of two other viral infections, chronichepatitis B (CHB) and HIV. In CHB patients, CD4+CD28– Tcell expansion correlated with the severity of the infection,marked by a high viral load and elevated aminotransferaselevels [65]. In HIV patients, an increased percentage ofcirculating CD4+CD28– T cells positively correlated withIFN-g secretion after TCR stimulation [8]. The authors ofboth reports did not provide evidence for the origin ofCD4+CD28– T cells (CMV-induced or hepatitis B/HIV-in-duced), but nevertheless suggested that these cells mightcontribute to the pathogenesis of both infectious diseases.

Can CD4+CD28– T cells be targeted by therapeuticintervention?Many researchers agree that CD4+CD28– T cells may con-tribute to the pathogenesis of several immune disorders,

450

justifying the development of therapeutic intervention tar-geting CD4+CD28–T cells. One way of manipulating this cellpopulation is to induce re-expression of CD28. The firstreport using this approach came from Warrington et al.,who discovered that in vitro activation of CD4+CD28–T cellsin the presence of IL-12 restored CD28 expression [66].Functionally, costimulation through CD28 was restored,resulting in the expression of CD25 and CD40L. Brylet al. targeted one of the putative causes of CD28 loss,TNF-a. RA patients receiving anti-TNF-a treatmentshowed increased expression of CD28 on CD4+ T cells[67], an observation confirmed by Rizzello et al., who showedthat in vitro treatment of whole blood taken from UApatients increased CD28 expression on CD4+ T cells [68].In both reports, no data were available on the functionalityof CD28 re-expressing cells, such as cytotoxicity or proin-flammatory behavior. However, Scarsi et al. observed thatthe effect of anti-TNF-a treatment positively correlated withthe clinical response [69]. The same group also showed thatthe baseline numbers of CD4+CD28– T cells in the blood ofRA patients can predict the response to TNF-a inhibition[70]. These observations were countered by Pierer et al., whofound no effect of TNF-a inhibitors on the percentage ofCD4+CD28– T cells in RA patients [71]; however, theydescribed a decline in the number of expanded clonotypes,indicating that anti-TNF-a treatment has an effect on T cellhomeostasis.

Another approach to target CD4+CD28– T cells is toreduce the number of these cells. Brugaletta et al. de-scribed that UA patients treated with statins had a lowerpercentage of circulating CD4+CD28– T cells in comparisonwith untreated patients [72]. It was not clear from thisreport whether CD4+CD28– T cells were being reduced orwhether they re-expressed CD28. However, a recent paperby Link et al. confirmed that rosuvastatin decreased thepercentage of CD4+CD28– T cells by inducing apoptosis[73].

A very recent paper by Xu et al. suggested that K+

channel blocking might be successful in targetingCD4+CD28– T cells in ACS patients [74]. Kv1.3 blockinglowered the production of IFN-g and perforin byCD4+CD28– T cells, both being important effector mole-cules in the cytotoxic pathways of CD4+CD28– T cells.Therefore, channel blocking might selectively target thepathogenic function of CD4+CD28– T cells in patients withimmune disorders.

The third approach proposed is to identify and target acell specific molecule on CD4+CD28– T cells. CD4+CD28– Tcells from healthy individuals, as well as patients with MSand RA, selectively express CX3CR1 [10], and one mightconsider blocking the fractalkine system to preventCD4+CD28– T cells from entering target tissues. However,this approach will also inhibit the fractalkine system in theother cells, such as NK cells, neurons, and microglia. Theseverity of experimental autoimmune encephalomyelitis(EAE), the animal model for MS, is exacerbated in CX3CR1knockout mice as compared with wild-type mice [75]. Therecruitment of NK cells to the CNS was impaired in thesemice, leading to a defective regulation of the local inflam-matory response. Therefore, the selective expression of thespecific target molecule on CD4+CD28– T cells must be

Compensatory autoprolifera�onenvironment (e.g. cytokines)

CD4CD28 CCR7 CD4 NKG2D

CX3CR1

CD56

Chronic s�mula�on by latentvirus or autoan�gen Perforin/

granzyme B

IFN-γ

Migra�on to target �ssue

Mul�plesclerosis

Granulomatosiswith polyangii�s

Rheumatoidarthri�s

Autoimmunemyopathy

Inflammatorybowel disease

Possibili�es for therapeu�c interven�on:stop conversion to CD4+CD28− T cells,block effector func�ons, orprevent migra�on to target �ssue

TRENDS in Molecular Medicine

Figure 1. Expansion of CD4+CD28– T cells in immune disorders. CD4+CD28+ T cells can become CD4+CD28– T cells by compensatory autoproliferation, environmental factors

(cytokines), and/or chronic antigen stimulation. Expansion of these cells has been found in target tissues of multiple sclerosis, rheumatoid arthritis, granulomatosis with

polyangiitis, autoimmune myopathy, and inflammatory bowel disease. Possible therapeutic interventions to target these cells include blocking the conversion to

CD4+CD28– T cells, inhibiting effector mechanisms, and blocking migration to target tissues.

Box 3. Outstanding questions

� What is the origin of CD4+CD28– T cells?

� Does CMV drive expansion of these cells, or does disease-related

immune activation?

� Which therapeutic intervention can selectively target CD4+CD28– T

cells in a safe manner?

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studied in detail before using this approach as a putativetherapy.

Concluding remarks and future perspectivesAlthough their role has not yet been fully defined,CD4+CD28– T cell numbers have been shown to be in-creased in several immune disorders (Figure 1), and inautoimmune diseases they accumulate in the affectedtissues. In cardiovascular diseases, their expansion hasbeen associated with plaque rupture and subsequentthrombosis. In other immune disorders, the relation withdisease pathogenesis is less clear, but the expansion ofCD4+CD28– T cells is often observed. Of the questionsremaining to be answered (Box 3), the most importantwould be ‘what is the origin of CD4+CD28– T cells?’ Forexample, does CMV drive expansion of these cells, andwhen present in patients with immune disorders, do thesecells contribute to the pathogenesis of the disease? In otherwords, does the disease cause expansion of CD4+CD28– Tcells, through repeated antigenic stimulation and immune

system activation? Clearly, future research is needed toresolve this question, which might provide more insightinto ways to target these cells through therapeutic inter-vention. Although some research has already been doneregarding modulation of this cell population, not all thera-peutic approaches will work equally well in every immunedisorder. For example, TNF-a inhibition is a standardtreatment for RA patients, but can have detrimental con-sequences for MS patients [76]. Therefore, a safe andwidely usable modulatory agent should be sought tospecifically and effectively target CD4+CD28– T cells in abroad range of immune disorders.

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AcknowledgmentsThis work was supported by Hasselt University, Belgian CharcotFoundation, and FWO Flanders. The authors have no conflictingfinancial interests.

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