Immunology and Cell Biology (2002) 80, 1–13

Review Article

CD8+CD28– T cells: Certainties and uncertainties of a prevalent human T-cell subset

F E R N A N D O A A R O S A

Laboratory of Molecular Immunology, Institute for Molecular and Cell Biology (IBMC) and Molecular Immunology and Pathology, Abel Salazar Institute for the Biomedical Sciences (ICBAS), Porto, Portugal

Summary Human peripheral blood CD8+ T cells comprise cells that are in different states of differentiation andunder the control of complex homeostatic processes. In a number of situations ranging from chronic inflammatoryconditions and infectious diseases to ageing, immunodeficiency, iron overload and heavy alcohol intake, majorphenotypic changes, usually associated with an increase in CD8+ T cells lacking CD28 expression, take place.CD8+CD28– T cells are characterized by a low proliferative capacity to conventional stimulation in vitro and bymorphological and functional features of activated/memory T cells. Although the nature of the signals that giveorigin to this T-cell subset is uncertain, growing evidence argues for the existence of an interplay between epithelialcells, molecules with the MHC-class I fold and CD8+ T cells. The possibility that the generation of CD8+CD28–

T cells is the combination of TCR/CD3ζ- and regulatory factor-mediated signals as a result of the sensing ofmodifications of the internal environment is discussed.

Key words: CD8+ T cells, CD28, downmodulation, epithelial cells, homeostasis, major histocompatibility complex-class I.

Introduction

One of the fundamental assets of the T-lymphocyte system, inaddition to its capacity to circulate, is its capacity to undergohomeostatic changes in response to alterations of the internalenvironment. Whether the origin of these alterations is fromoutside or from inside, physical contact between cells andmolecules of the immunological system on the one hand, andof the altered internal milieu on the other, are necessary toelicit activation signals that will ultimately result in qualita-tive and quantitative changes within the peripheral T-cellpool. The signalling events following the primary (e.g. TCR/MHC) and secondary (e.g. CD2/LFA-3, CD28/B7) inter-actions play key roles in the process of T-cell activation andfunction and have been well reviewed elsewhere.1–4 Never-theless, despite the fact that professional antigen-presentingcells (APC), such as dendritic cells, tissue macrophages,monocytes and activated B cells, can initiate an immuno-logical response by primarily activating CD4+ T cells,accumulating evidence indicates that the alterations of theT-lymphocyte system in response to signals of the internalmilieu result in phenotypic changes mostly affecting theCD8+ T-cell pool. The most conspicuous phenotypic changesunder these situations are downmodulation of the CD3ζ chainand complete loss of CD28, which are counteracted by denovo expression of non-specific T-cell markers such asCD11b, CD56, CD57 and CD161. The molecular basis for,

and physiological significance of, the increased tendency ofCD8+ T cells to undergo shifts in response to alterations of theinternal milieu is unclear. However, recent data indicate thatnon-haematopoietic cells, such as epithelial cells, are capableof activating CD8+ T cells. In turn, the activated CD8+ T cellsmay be involved in the regulation of basic physiologicalprocesses within the organ and/or tissue they populate. Toelucidate the mechanisms involved in the activation of CD8+

T cells, loss of CD28 expression and the generation ofdifferentiated CD8+CD28– T cells with regulatory functions isof foremost importance. The purpose of this review is to bringup to date the certainties and uncertainties of this prevalenthuman CD8+ T-cell subset.

CD8+CD28– T cells: From phenotype to function

Although CD28 provides a critical costimulus for T-cellactivation,1–4 a large fraction of human peripheral blood Tcells lack CD28, which is due to the different expression ofCD28 by the two major T-cell subsets. While in healthyhumans CD28 is expressed by the majority of CD4+ T cells,the percentage of CD8+ T cells coexpressing CD28 is highlyvariable.5–7 Peripheral blood CD8+CD28– T cells are enrichedfor large, granular lymphocytes (Fig. 1), express high levelsof LFA-1 and variable amounts of CD11b and CD57, aphenotype associated with memory/effector cells.5–7 Freshlyisolated peripheral blood CD8+CD28– T cells from healthyhumans do not express markers of early (e.g. CD69) orchronic (e.g. HLA-DR) activation and proliferate poorly inresponse to conventional mitogenic stimulation in vitro.7 ACD4+ T-cell subset with similar phenotypic and functionalcharacteristics also exists, at very low frequencies, in theperipheral blood of healthy humans.8,9

Correspondence: Dr FA Arosa, Laboratory of Molecular Immu-nology, Institute for Molecular and Cell Biology, Rua do CampoAlegre, 823, 4150-180 Porto, Portugal. Email: farosa@ibmc.up.pt

Received 19 February 2001; accepted 3 September 2001.

2 FA Arosa

Although the existence of a pool of circulating CD8+CD28–

T cells in humans has been known to exist for more than15 years, specific interest in this CD8+ T-cell subset aroseafter finding increased numbers during infections.10–14

CD8+CD28– T cells were first described as suppressors of B-and T-cell function, but subsequent studies have shown thatCD8+ T cells bearing the CD28– phenotype (CD11b+ and/orCD57+) may also mediate cytotoxicity.15–18 Studies withfreshly isolated peripheral blood lymphocytes demonstratedthat CD8+CD11b+ T lymphocytes contain cells capable ofsuppressing proliferation and IgG synthesis of B cells, andcells that express the IL-2β receptor chain (CD8+CD122+

T cells) and develop into cytotoxic T lympho-cytes (CTL)after culturing with IL-2.18–21 Importantly, IL-2-induced CD8+

T-cell proliferation results in CD11b loss and the generationof CD8+CD28–CD11b– T cells,21 a non-cytotoxic subsetexpanded in the blood of HIV-1-infected patients.22 Recentwork by Suciu-Foca and coworkers has disclosed some of themolecular mechanisms that account for the suppressor activ-ity of CD8+CD28– T cells.23–25 By inhibiting the nuclear factorkappa B (NF-κB) mediated transcription of the CD86 gene,CD8+CD28– T cells hamper the ability of classical APC toelicit CD28-driven CD4+ T activation and proliferation, asuppressor activity that is MHC-class I-restricted.23–25

These data clearly indicate that CD8+CD28– T cells are aheterogeneous subset that contains both memory and effectorcells.26 The most established view, however, considersCD8+CD28– T cells as terminally differentiated or end-stageCTL; a belief based on phenotypic and functional studies.First, CD8+CD28– T cells express CD11b, a member of theβ2-integrin family expressed by monocytes, neutrophils andnatural killer (NK) cells,27 which is considered to be a markerof CD8+ cytotoxic T-cell activation in response to viral

infection in mice.28,29 Yet, together with LFA-1, another β2-integrin, CD11b is believed to play an important role inextravasation of lymphocytes after adhesion to the endothe-lium via intercellular adhesion molecule-1 (ICAM-1).27,30 Inaddition, along with peripheral blood CD8+ T cells, CD11b isexpressed by a significant fraction of CD3+ T cells in the liverand spleen of healthy subjects.31 Recent studies suggest thatthe acquisition of CD11b represents an early event duringhuman CD8+ T-cell differentiation, which may allow extrava-sation to peripheral tissues.32 Second, CD8+CD28– T cellsexpress CD57, a molecule containing the epitope humannatural killer cell carbohydrate antigen-1 (HNK-1), which ispresent on human NK, CD4+ and CD8+ T cells.33,34 CD57, ina manner analogous to CD11b, is a marker associated withCTL effector function due to its increased expression amongCD8+ T cells during immune responses to viral infections.35–38

However, the HNK-1 epitope is also present on glycolipidsand glycoproteins expressed in the central nervous system(e.g. MOG and NCAM) and involved in cell–cell interac-tions.39 In this context it is important to note that CD8+ T cellsexpressing CD57, but not CD11b, are present at low levels innormal bone marrow.40,41 Finally, CD8+CD28– T cells areenriched for large granular lymphocytes that contain per-forin,7,26 a protein involved in target cell apoptosis.42 Thus,these data reveal that CD8+CD28– T cells are a multifacetedpopulation capable of performing many functions, with cyto-toxicity the most extensively studied. Indeed, a recent studyon circulating CD8+CD28– T cells has shown that CTLeffector function correlates with the expression of CD56,43 atypical NK marker also containing the HNK-1 epitope.39

Although the loss of CD28 and gain of CD11b and/orCD57 by peripheral blood CD8+ T cells is the result of adifferentiation process that generates cells with cytotoxic

Figure 1 Human CD8+CD28– Tcells are enriched for large, gran-ular lymphocytes. Freshly col-lected human peripheral bloodlymphocytes from a healthy blooddonor were stained for CD8/CD28and acquired in a FACScaliburaccording to forward and sidescatter characteristics (FSC/SSC,left dot blot). After magnification,two electronic gates were createdaround small/agranular (gate 1)and large/granular (gate 2) lym-phocytes. CD28 expression among(CD3+)CD8bright T cells withineach electronic gate is shown onthe right dot blots. The percent-age of (a) CD8+CD28+ and (b)CD8+CD28– T cells are indicated.

Human CD8+CD28– T cells 3

potential, to consider CD8+CD28– T cells as end-stage CTLeffectors constrains the study of other functions that thisCD8+ T-cell subset may perform. Thus, the increase inCD8+CD28– T cells in peripheral blood could be the result ofa viral infection,10–13,44–49 but may also be a response toalterations of the internal environment,50–52 or even of homeo-static changes within the peripheral T-lymphocyte systemitself. In addition, the CTL effector function detected amongexpanded CD8+CD28– T cells during viral infection43–45 hasbeen difficult to detect by some authors,47–49 unless the bulk ofthe CD8+ T cells were first incubated with IL-2, a cytokinethat upregulates CD3ζ and restores their cytotoxic potential.48

The recently developed peptide-HLA tetramer technology hasallowed researchers to isolate and study in more detailpeptide-specific CD8+ T cells from peripheral blood duringprimary and secondary viral infections.53 Surprisingly, thesestudies have shown that tetramer-specific CD8+ T cells are acomplex population, which in most cases show impairedcytotoxicity activity.54–59 Although the CD8+CD28– T-cellphenotype has been associated with terminal differentiation,unresponsiveness and cytotoxicity it is important to note thatincreased numbers of circulating CD8+CD28– T cells in anumber of disease conditions and situations have beenreported. As shown in Table 1, despite being the most stud-ied, viral infections are only one among the many possiblesituations that could lead to the generation of CD8+CD28–

T cells in humans.

Signals and pathways for CD28 downmodulation

T-lymphocyte activation in vitro and in vivo results inincreased internalization and degradation of components ofthe TCR/CD3ζ complex, namely the CD3ζ chain, and this

results in the downmodulation of cell surface CD3.60 T-cellactivation by MHC-peptide complexes, B7-expressing cellsor mitogens, such as phytohaemagglutinin, also results inCD28 downmodulation by peripheral blood T cells.61–63 Incontrast to CD3ζ, modulation of cell surface CD28 initiatedafter TCR-engagement results in the complete loss of CD28after several cycles of cell division in the presence of γ-chainreceptor signalling cytokines such as IL-2, IL-7 and IL-15.64–67

Hence, CD28 downmodulation initiated after TCR triggeringappears to be an irreversible process that is modulated byvarious factors and cytokines and leads to the generation ofCD8+CD28– T cells in humans. In an elegant study usingCD8+ T cells, Fiorentini et al. showed that after prolongedin vitro stimulation in the presence of IL-2, CD8+CD28+

T cells showed a gradual decrease in CD28 cell surface andmRNA levels and matured into a stable CD8+CD28– pheno-type.66 Similar results were obtained by Labalette et al.which, in addition, showed that IL-4 inhibited this process.68

Thus, loss of CD28 is a hallmark of T cells that haveundergone many cycles of cell division. Indeed, loss oftelomeric DNA resulting from cell division69,70 is observed inCD8+CD28– T cells from healthy and diseased subjects,71,72 anindication of their higher replicative past when comparedwith their CD8+CD28+ T-cell precursors. Telomeric shorten-ing is only evident among CD8+ T cells that have completelylost CD28 expression,73–75 thus, suggesting a link betweenCD28 downmodulation and telomerase activity. Two recentstudies have revealed that telomere shortening in peripheralblood lymphocytes also takes place during the first years oflife,76,77 indicating that T cells undergo a high turnover duringearly childhood, which is a time when internal changes (e.g.organ and tissue remodelling) are still taking place.

To date, CD28 is the only T-cell surface molecule, whichhas been described, that undergoes complete downmodula-tion, a process that is more apparent on CD8+ T cells.Considering the phenotypic changes associated with CD28downmodulation,38 identification and characterization of theactivation signals and factors that regulate CD28 is of greatimportance. In this context, recent studies by Goronzy andcoworkers have shown that the differences in CD28 expres-sion between CD4+ and CD8+ T cells correlate with differ-ences in nuclear protein-binding activities to two motifs, αand β, located within the CD28 promoter.78 In vitro activationvia the TCR/CD3ζ complex correlates with loss of α/β-binding activities both in CD4+ and CD8+ T cells and partialCD28 downmodulation. On the contrary, repeated activationand long-term culture in the presence of IL-2 results in loss ofβ- but not α-binding activities and correlates with a markedmodulation of CD28 expression solely on CD8+ T cells, apattern of expression that mirrors what happens in vivo.78 It isimportant to note that freshly isolated CD4+CD28– andCD8+CD28– T cells lack α/β-binding activities. Further char-acterization of these lymphoid factors may turn out to be ofparamount importance in elucidating the nature of theenvironmental signals and factors that lead to the loss ofCD28 by CD8+ T cells.

Despite the recent advances in our understanding of howCD28 expression is regulated, it is difficult to determinewhich cells provide the original stimulus and the context inwhich CD8+CD28– T cells develop. Still, the well-knownMHC-restriction of CD8+ T cells argues in favour of a

Table 1 Human conditions and disorders associated with anincrease in the percentage and/or number of peripheral bloodCD8+CD28– T cells (defined either as CD28–, CD11b+or CD57+)

Condition/disorder References

CD4+ lymphopenia 132Chagas disease 14Chemotherapy 147Chronic lymphocytic leukaemia 124CMV infection 36, 37, 44, 46, 48, 49, 65Immunodeficiency 122, 123Crohn’s disease 114EBV infection 13, 65Healthy heavy alcohol drinkers 89, 106Healthy humans (aged, including

centenarians)31, 64, 79, 82, 85, 86, 91–93

Healthy humans (with a highpercentage of CD8+ T cells)

87, 89

Hematological tumours 124, 125Hemochromatosis 87Hemodialysis patients 121Hemophilia A 120HIV infection 10, 12, 57, 65, 72Melanoma patients 88Myeloma patients 122, 126Rheumatoid arthritis 115, 127Systemic lupus erythromatosus 73, 117Transplants 50–52Wegener’s granulomatosis 118, 119

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predominance of MHC-class I+ versus MHC-class II+ cells indisplaying alterations of the internal environment. Further-more, in the case of continuous encountering with the MHC-class I+ cells, this prevalence would have an immediateoutput: the accumulation within the peripheral CD8+ T-cellpool of CD28– T cells carrying similar TCRαβ specificities.The reported oligoclonality of CD8+(CD57+)CD28– cellsboth in health and in disease is in accordance with thisscenario.79–83 The fact that CD8+CD28– T cells are oligoclonaland have short telomeres,71,83 together with the existence ofidentical clones in CD8+CD28+ and CD8+CD28– Τ cells84

stresses the MHC-class I driven mechanism. CD8+CD28–

T-cell oligoclonality may also suggest that a restricted pool ofMHC-class I/peptide structures account for most of theCD8+CD28– T-cell expansions. Alternatively, conservedstructures in physical association or combination with MHC-class I molecules may also be involved. Finally, the possiblerole played by non-classical MHC-class I molecules indriving the CD28+ to CD28– transition cannot be ruled out. Inany case, and contrary to current beliefs, the preferentialmodulation of CD28 observed in CD8+ T cells may beadvantageous rather than deleterious.

CD8+CD28– T-cell origin and function: Clues from health and disease

The presence of CD8+CD28– Τ cells in peripheral blood ofhealthy individuals is a common event. In some subjectsCD28– T cells may represent more than half of the peripheralCD8+ T-cell pool.6,7 Among the factors implicated in thegeneration of peripheral blood CD8+CD28– T cells in healthysubjects, age is the most important and most studied(Table 1). Posnett et al. first described expanded pools ofoligoclonal CD28– T cells among freshly isolated peripheralblood CD8+ T cells of aged humans.79 In another study, Effroset al. reported that centenarians, and long-term T-cell cul-tures, showed a decline in CD28+ T cells.64 Herein after,Fagnoni et al. studying a group of healthy people thatincluded centenarians showed a direct correlation betweenage and the numbers of peripheral blood CD8+CD28–

T cells.85 Although some groups have reported an increase inCD8+CD28– T cells with age;31,85,86 others have not been ableto find such a correlation.87–89 The basis for these discrepan-cies may result from the study of cohorts with differentgenetic backgrounds or from the unforeseen influence ofenvironmental factors89 on the percentage of CD8+ T cells.The only reliable correlation found with age is a decline in(CD8+)CD28+ T cells.31,64,85–90

The reduction in peripheral blood CD8+CD28+ T cells thattakes place with age, along with the accumulation of a pool ofunresponsive CD8+CD28– T cells with shortened telomeres,has led to the concept of replicative senescence.91–94 However,the weak responsiveness of CD8+CD28– T cells to mitogenicstimulation rather than being a feature of their senescencemay be an indication that they are prepared to receive adifferent stimulus. The recent evidence that telomerase activ-ity is intact in T cells from aged people reinforces thisassumption.95 Thus, study of costimulatory moleculesexpressed on activated/memory T cells, such as ICOS, PD-1,LFA-1, 4-1BB,96–98 may give important clues to the functionalstatus of CD8+CD28– T cells. In this context, special attention

should be given to de novo receptors expressed byCD8+CD28– T cells, such as the family of natural killerreceptors.88,99

Studies of diseases in which increased numbers ofCD8+CD28– T cells have been reported may provide insightsinto the mechanisms that drive the emergence of these CD8+

T cells in peripheral blood and into their physiologicalfunction (Table 1). In haemochromatosis (HFE), a geneticdisorder characterized by an inappropriately high iron absorp-tion in the small intestine and its subsequent accumulation inparenchymal cells of the liver, pancreas and other organs,increased numbers of CD8+CD28– T cells are a hallmark ofthe disease.87 Interestingly, follow-up studies showed thatCD8+CD28– T-cell expansions persist over time and affordprotection from development of hepatocellular carcinoma.87,100

Although HFE, the gene product responsible for the disease,is a non-classical MHC-class I molecule,101,102 there is noformal proof for its direct involvement in the activation ofCD8+ T cells. However, the presence of activated and oligo-clonal CD8+ T cells in these patients87,103 is an indication oftheir continuous priming under an iron-overloaded environ-ment. Furthermore, increased numbers of CD8+CD28– T cells,containing expansions of oligoclonal T cells, have also beenreported in the peripheral blood of heavy alcohol drinkerswithout liver disease.89 Interestingly, high CD8+CD28– T-cellnumbers were associated with low levels of liver injury.These data are in accordance with previous studies in heavyalcohol drinkers without liver disease, which showed thatCD8+ T cells have a typical profile of activated/memory cells,such as expression of CD57, CD45RO and HLA-DR.104–106

Together with the data from haemochromatosis patients theseresults suggest that CD8+CD28– T cells may play a protectiveregulatory role under tissue injury caused either by ethanol oriron, and point to an epithelial-driven CD8+CD28– T-cellgeneration. Through the production of reactive oxygenspecies iron and ethanol mediate protein damage and lipidperoxidation in parenchymal hepatocytes and result in epithe-lial cell injury.107–111 From an immunological point of viewthe stressed and/or injured epithelial cells may modulate theexpression of molecules involved, directly or indirectly, insignalling to either resident or passenger CD8+ T lympho-cytes112 (see next section). Repeated CD8+ T-cell activation inthe presence of γchain signalling cytokines may lead to thegeneration of a progeny of CD8+CD28– T cells with protec-tive regulatory functions as discussed elsewhere.113

Increased numbers of peripheral blood CD8+CD28– T cellshave also been reported in chronic diseases (e.g. Crohn’sdisease, rheumatoid arthritis, Felty’s syndrome, systemiclupus erythromatosus and Wegener’s granulomatosis),114–119

in haemophilic and haemodialysis patients,120,121 and in patientswith haematological tumours and immuno-deficiency122–126

(see Table 1). In some of these disease conditions increasednumbers of CD8+CD28– T cells are concomitant with chronicstimulation of CD4+ T cells. For example, in rheumatoidarthritis the increased number of circulating CD8+CD28–

T cells is paralleled by an increase in oligoclonal CD4+CD28–

T cells.127,128 The CD4+CD28– T-cell subset is a rare humanT-cell subset characterized by autoreactivity and resistance toapoptosis,8,9,128–130 which may preferentially originate in thecontext of certain HLA-DR alleles.131 As mentioned previ-ously, the possibility that increased numbers of circulating

Human CD8+CD28– T cells 5

CD8+CD28– T cells in these diseases may result in part fromhomeostatic changes within the peripheral T-cell pool cannotbe ruled out.

Longitudinal studies of peripheral T-cell populations inhealth and in disease have indeed unveiled the existence of aclose relationship between CD8+CD28– T-cell developmentand CD4+ and CD8+ T-cell numbers in peripheral blood. Inother words, it is very likely that a variable fraction of thecirculating pool of CD8+CD28– T cells may, under certaincircumstances, arise without the need of TCR triggering.First, human CD4+ T-cell lymphopenia due to Lck deficiencyresults in the replenishment of the peripheral T-cell pool withCD8+CD28– T cells.132 Second, low CD4/CD8 T-cell ratiosanticipate high percentages of CD8+CD28– T cells in periph-eral blood regardless of age.87,89 Third, the progressive loss ofCD4+ T cells, and naive CD8+ T cells observed in HIV-infected subjects is counteracted by an increase in effector/memory CD8+(CD28–) T cells,133–135 an increase also observedin centenarians in which reductions in naive CD8+ T cells takeplace.136 In all, these data suggest that CD8+CD28– T-celldevelopment is constrained by the size of the peripheral CD8+

T-cell compartment, in relation to the CD4+, and by thenumber of naive CD8+ T cells within this compartment. Thisconstraint is in accord with the genetic control of the periph-eral CD4/CD8 T-cell ratio,137,138 its constancy over time inhumans139,140 and the close relationship observed betweenboth pools in reconstituted mice.141 In this scenario, CD8+

CD28– T cells will expand when a decrease in total CD4+

T cells, or naive CD8+ T cells, takes place within the periph-eral T-cell compartment, regardless of the origin and nature ofthe cause. This increase could be modulated in a positive or anegative manner by the presence or absence, respectively, ofsignalling cytokines both in humans142 and in mice,143–145 orby additional stimuli such as viral infection, iron overloador ethanol consumption. This setting fits well with currentdata indicating that CD8+CD28– T cells divide faster and livelonger than CD8+CD28+ and CD4+ T cells, features likely tobe related to a shorter cell division cycle, a higher resistanceto apoptosis and a different response to regulatorycytokines.65–68,146,147

Are epithelial cells involved in the generation and/or maintenance of CD8+CD28– T cells?

Cells expressing high levels of MHC-class II molecules, suchas dendritic cells, activated monocytes and B cells and tissuemacrophages, are considered the classical APC. However,evidence accumulated during the past decade indicates thatintestinal epithelial cells (iEC) are also capable of activatingT cells.148 Early in vitro studies demonstrated that ex vivo iECfrom rats and humans were capable of activating peripheralblood T cells. Paradoxically, the T cells driven into prolifera-tion by iEC were CD8+ T cells.149,150 It is now known that bothex vivo iEC and iEC tumour cell lines are capable of generat-ing CD8+CD28– T cells in vitro, although using differentmechanisms.150,151 While CD8+CD28– T-cell generation byex vivo iEC involves activation and proliferation of CD8+

T cells, generation by iEC lines entails death of CD4+CD28+

and CD8+CD28+ T cells, thus strengthening the resistance ofCD8+CD28– T cells to apoptosis.151

Evidence gathered during the last few years suggests thatCD1d and gp180 are involved in the activation of humanCD8+ T cells.152–155 CD1d is a non-classical MHC-class Imolecule that is expressed in iEC, parenchymal hepatocytesand stromal cells.156,157 Dendritic cells, monocytes, B cells andactivated T cells express low levels of CD1d.158–160 In con-trast, gp180 is a heavy glycosylated protein that is expressedby the intestinal, thymic and airway epithelium, and introphoblasts.161 While CD1d is thought to interact with theTCR of the CD8+ T cell, gp180 interacts with the CD8molecule and activates its associated tyrosine kinaseLck.152,153 The CD8/gp180 interaction plays an important rolenot only in Lck activation, but also in the adhesion of CD8+

T cells to iEC.151 In this scenario, MHC-class II moleculesappear to modulate the epithelial-driven CD8+ T-cell activa-tion.152 The capability of iEC to activate human CD8+ T cellsin vitro has also been observed with hepatic parenchymalcells,162,163 airway epithelial cells,164 endothelial cells165 andfibroblasts.166 Whether the CD1d/gp180 pair is involved andwhether the proliferating CD8+ T cells become CD28– isunknown, but cocultures of epithelial cell lines from differentorigins with peripheral blood T cells results in accumulationof CD8+CD28– T cells.151

The generation of CD8+CD28– T cells by epithelial cellscould be envisaged as the result of the existence of communi-cation between epithelial tissues and CD8+ T cells. This cross-talk constitutes a unique feature that may help to understandthe phenotypic changes that take place within the peripheralT-lymphocyte system under a variety of situations (Table 1).Thus, altered expression of CD1d by epithelial cells has beenimplicated in the pathogenesis of certain autoimmune dis-orders.170–172 Other studies, however, have suggested thatfaulty expression of gp180, rather than CD1d overexpression,may be responsible for the pathogenesis of certain inflamma-tory diseases.173 On the contrary, when expressed by dendriticcells CD1d seems to preferentially activate CD4+ T cells andCD4–CD8– T cells bearing a conserved Vα chain.174–176 Thepotential of human CD1d to interact and activate oppositeT-cell subsets may reflect presentation of different tissue-specific ligands, as shown in mice,177 or a different physicalassociation with conserved structures. In this context, CD1dhas three distinctive features: (i) a hydrophobic bindinggroove that binds lipid compounds instead of classical pep-tides, in mouse CD1d1;178,179 (ii) the presence of the YQGVmotif in its cytoplasmic tail, a tyrosine-based internalizationsignal that targets the molecule to endosomal compart-ments;180 and (iii) the capability to be expressed in the cellsurface in a β2m-independent manner.181,182 Nevertheless,even though CD1d is considered to be an important signallingmolecule involved in the activation of distinct subsets of Tcells,148,161,167–169,174–176 it must be noted that cell surfaceexpression of CD1d by untransfected human cells is veryweak or absent, and is mainly cytoplasmic. This pattern ofexpression does not meet the classical requirements for anantigen-presenting molecule. Therefore, human CD1d mayhold a specialized biological function that may be intracellu-lar rather than extracellular (for a recent review of CD1d seeJoyce183).

Molecules having the MHC-fold, aside from CD1d, aloneor in combination with conserved monomorphic structuresand/or oxidatively modified proteins are candidate molecules

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whose expression on epithelial cells may allow a preferentialCD8+ T-cell activation. Human MICA and MICB molecules(for MHC class I-chain related molecules) are expressed onthe cell surface of stressed iEC and fibroblasts, and have thecapability to activate γδCD8+ T cells.184–186 MICA is alsoexpressed by human keratinocytes, endothelial cells andmonocytes and seems to be expressed in the cell surface in aβ2m-independent manner.187 Although detailed studies ofMICA and MICB expression in the conditions described inTable 1 have not been reported, it is anticipated that theirexpression, along with the expression of other signallingmolecules, could be altered. In this context, increased expres-sion of MHC-class I and ICAM-1 on hepatocytes of patientswith haemochromatosis has been reported.112 By analogy withstudies in humans and mice,188–193 altered expression of con-served molecules and molecules with the MHC-class I fold byepithelial cells under stressed conditions, such as during ironoverload, may activate passenger and/or resident CD8+ T cellsand generate a pool of CD8+CD28– T cells that may act on theinjured epithelial cell immediately or after recirculation. Thecytokines and factors secreted may directly or indirectlyregulate stress/injury and restore normal hepatic function.Although speculative at present, this scenario is not unlikely.Clinical and experimental data accumulated during the past10 years have established the impact that low numbers ofCD8+ T cells, both in peripheral blood and in the liver, havein the clinical setting of haemochromatosis.100,140,194–196

Studies of iron-rich body fluids, such as breast milk and thesynovial fluid of rheumatoid arthritis patients197,198 and of amodel of hepatic injury199 appear to support the assumptionthat CD8+CD28– T cells play a protective role under stressedconditions and thus regulate epithelial physiology.

The majority of T cells populating epithelial tissues inhumans (e.g. small intestine and liver parenchyma) are, likeCD8+CD28– T lymphocytes, phenotypically and functionallymemory CD8+ T cells.31,200–205 Recent work by O’Farrelly andcollaborators203,204 has shown that a large fraction of humanhepatic CD8+ T cells express activating and inhibitory NKreceptors.206 These CD8+ T cells, called NKT cells, are alsopresent in peripheral blood and are characterized by a lack ofCD28 expression.43,88,207,208 Although evidence for the expres-sion of NK receptors by human intestinal intraepitheliallymphocytes (iIEL) is absent, a recent study showed thatBY55, a glycosylphosphatidylinositol-linked protein, isexpressed by both circulating CD8+CD28– T cells and iIEL.209

First described in circulating NK cells, BY55 is expressed bycord blood and bone marrow CD8+ T cells, and in a largefraction of circulating CD8+ T cells in HIV patients.210–212

Therefore, this molecule links the circulating CD8+CD28–

T-cell pool and the mucosal CD8+ T-cell pool, which is alsoCD28–.202,213 In addition, both iIEL and hepatic NK CD8+

T cells lack cytotoxic activity, but gain cytotoxicity afterculture in IL-2,204,214,215 a feature also shared by CD8+CD28–

T cells.19 These phenotypic and functional similarities suggestthat circulating CD8+CD28– T cells may originate fromCD8+CD28+ T-cell precursors that have acquired typical NKreceptors during the differentiation process in epithelial tis-sues, thus, becoming NKT cells, as observed in mice.216,217

It is important to note that NK receptors have also beendescribed in circulating CD4+CD28– T cells from rheumatoidarthritis patients, thus, indicating that induction/acquisition of

NK receptors is linked to the differentiation process of the Tcell.218–220 The likelihood that thymus- and/or bone marrow-derived CD8+ T cells, in the context of signals provided byenvironments such as the liver parenchyma or the intestine,differentiate, lose expression of CD28, transcribe and expresscell surface NK receptors, such as CD161,221 is an intriguing,but as yet, unexplored question.

Conclusions and future prospects

Studies in mice have shown that the maintenance of naive andmemory CD8+ T cells in the periphery requires TCR–MHCinteractions as well as cytokines and growth factors.222

Although similar studies in humans are unfeasible, the nega-tive impact of the absence of MHC-class I molecules orcytokine receptors, such as the γchain, on peripheral T-cellhomeostasis is well known.223,224 Nevertheless, the nature ofthe MHC-class I+ cells that interact with CD8+ T cells in theperipheral tissues and organs and assure their survival isuncertain. Likely candidates are epithelial cells of the intes-tine and liver, two peripheral tissues usually populated byCD8+ T cells. Other organs and tissues, such as bone marrow,can also play an important role. By communicating withepithelial cells, circulating CD8+ T cells could secure not onlytheir own survival but participate in the regulation of basicbiological processes, from modulation of epithelial physio-logy199,225–230 and removal of apoptotic cells231 to the regula-tion of local cell homeostasis.232,233

The primary signals delivered by epithelial tissues to CD8+

T cells can only be envisaged within the context of thecytokines produced in situ;234–237 the nutrients for which CD8+

T cells compete,222 soluble factors and receptors influencingT-cell function and receptivity,238,239 and novel modulators ofT-cell survival.240 Together with the signals delivered whenthe T lymphocyte has changed to the CD28– phenotype,including those provided by engagement of NK receptorssuch as NKG2D via MICA,241–243 this myriad of environmen-tal signals is likely to shape the peripheral T-lymphocytesystem. Classical and non-classical MHC-class I molecules,together with the variety of the NK receptors that recognizethose molecules, are thus central to T-lymphocyte homeo-stasis, namely of the CD8+ T-cell pool.244 These signals areexpected to be delivered in normal conditions (e.g. duringepithelial cell renewal) and in a number of situations duringdegeneration (e.g. senescence), injury (e.g. excess of iron andalcohol, viral infection) or modification (e.g. organ transplan-tation) of the internal environment. In these latter situations,however, a hastened generation of CD8+CD28– T cells wouldtake place. Given their functional heterogeneity and proper-ties, CD8+CD28– T cells are better positioned than other T-cell subsets to counteract these environmental changes. Thisprevalent human CD8+ T-cell subset, contrary to the currentbelief, may encompass highly specialized T cells with thecapability to control and regulate tissue remodelling andrepair within the microenvironment they populate on a day-to-day basis.

Acknowledgements

I thank EMP Cardoso for invaluable suggestions and helpduring the writing of this manuscript, G Porto and M Santos

Human CD8+CD28– T cells 7

for helpful discussions, AM Carmo for his unwavering beliefin the immune system and AM Fonseca for providing the datafor Fig. 1. Last, but not least, I want to thank M de Sousa forher continuous support and for teaching me to look at theimmunological system in a different way. Some of the workdescribed in this review was supported by the PortugueseScience Funding Agency (FCT) and by the American Portu-guese Biomedical Research Fund (APBRF). Regretfully, anddue to space constraints, some original work was notreferenced.

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