the ability to develop and maintain highly functional memory t cells after infection or immunization is a hallmark of the adaptive immune response . however , the human memory t - cell pool is heterogeneous , and comprises distinct populations that can be distinguished based on homing markers and effector functions .
cytokines have been shown to be essential for the long - term maintenance of functional cd8 and cd4 memory t cells . yet , cytokine - induced signaling can modulate phenotypic and functional characteristics of memory t cells [ 47 ] .
in addition , chronic viral infections and autoimmune diseases have been shown to induce distinct phenotypic and functional changes within the memory t - cell pool .
thus , antigens and cytokines dynamically shape the memory t - cell pool throughout life .
so far , the role of the transmembrane glycoprotein cd5 in the process of memory t - cell differentiation and maintenance has not been addressed .
cd5 surface expression is tightly regulated during t - cell development , with low levels expressed on immature , double negative cd4cd8 thymocytes , followed by an approximately sixfold increase in cd5 at the double positive cd4cd8 stage and a further three- to fivefold increase in cd5 expression on mature , single positive cd4 and cd8 thymocytes .
all mature t lymphocytes and a subset of b cells , b-1a cells , express cd5 .
data from cd5/ mice indicate that cd5 acts as a negative regulator of cellular activation by being recruited into lipid rafts and dephosphorylating signaling molecules [ 1316 ] .
recently , it has been demonstrated in mice that the level of cd5 on naive t cells does not correlate only with the strength of tcr - mediated signaling but also with the strength of responsiveness to cytokines , such as interleukin ( il)15 .
furthermore , the level of the inhibitory molecule cd5 on naive t cells could be downregulated by cytokines , such as il-7 and il-21 , which rendered the cytokine - primed naive t cells more responsive to antigenic stimulation . in this study
we demonstrate that the expression of the inhibitory molecule cd5 is decreased during human memory t - cell differentiation and is associated with the strength of responsiveness to il-15 .
in addition , we show which environmental cues may be involved in regulating cd5 expression on post - thymic human memory t cells .
all participants had given their informed written consent , and the study was approved by the ethics committee of the medical university innsbruck .
cytomegalovirus ( cmv ) seropositivity was determined by enzyme - linked immunosorbent assay ( elisa ) using enzygnost ( dade behring , vienna , austria ) .
peripheral blood mononuclear cells ( pbmc ) were isolated by ficoll - hypaque density gradient centrifugation ( amersham biosciences ) . paired blood and bone marrow ( bm ) samples were obtained from individuals who had given informed written consent and the study was approved by the ethics committee of the medical university innsbruck .
bone from the iliac crest , which otherwise would have been discarded based on necessary bone molding / recontouring before insertion into other areas of the body , in particular facial regions was harvested at the department of cranio - maxillofacial and oral surgery at the medical university , innsbruck .
exclusively bone biopsies from systemically healthy individuals who did not receive immunomodulatory drugs or who had diseases known to influence the immune system , including autoimmune diseases and cancer , were used .
the bone biopsy samples were washed with complete mem ( invitrogen , lofer , austria ) , fragmented , and treated with highly purified collagenase ( worthington , lakewood , nj ; 20 u / ml ) for 1 h at 37c .
thereafter , collagenase - treated cells were centrifuged and bm mononuclear cells ( bmmc ) were purified by density gradient centrifugation ( ficoll - hypaque ; amersham biosciences , vienna , austria ) .
peripheral blood samples were obtained from 10 individuals who fulfilled the american college of rheumatology ( acr ) classification criteria for ra .
median disease duration was 18 years ( range 525 years ) , and 80% of the patients were positive for rheumatoid factor .
the ra patients had moderate clinical disease activity ( median disease activity score ( das)-28 4.3 ; range 1.76.6 ) and were treated with methotrexate (
n = 5 ) , other conventional disease - modifying antirheumatic drugs ( dmards ) ( n = 3 ) , or tumor necrosis factor ( tnf) blocking agents ( n = 2 ) .
immunofluorescence surface staining was performed by adding a panel of directly conjugated mabs to freshly prepared pbmc and bmmc .
the antibodies used were cd3 ( pe - cy7 and apc - cy7 ) , cd4 ( percp ) , cd5 ( pe and apc ) , cd28 ( apc ) , cd45ra ( apc ) , cd62l ( apc - cy7 ) , cd69 ( pe ) , cd122 ( pe ) , cd132 ( pe ) , and tcr ( pe ) ( all bd pharmingen , san diego , ca ) , cd8 ( percp ) ( biolegend , san diego , ca ) , and ccr7 ( fitc ) ( r&d systems , minneapolis , mn ) . the labeled cells were measured by a facscanto ii ( bd biosciences , san diego , ca ) , and the data were analyzed using facsdiva software ( bd biosciences , san diego , ca ) .
cell culture experiments were performed with rpmi 1640 ( invitrogen , lofer , austria ) supplemented with 10% fcs ( sigma - aldrich , vienna , austria ) and 1% kanamycin sulfate ( invitrogen , lofer , austria ) . to analyze the responsiveness of memory t - cell subsets to il-15 ,
peripheral blood t cells were stimulated with il-15 ( 50 ng / ml ; sigma - aldrich , vienna , austria ) for 4 days and cd69 was measured on cd8 and cd4 memory t - cell subsets .
low expression of cd5 was defined by gating on the lower third of cd5-expressing t cells and the gating was confirmed by additional cd45ra and cd28 staining .
thus , cd5 t cells had a cd45racd28 phenotype . to determine the regulation of cd5 expression , peripheral blood cd8 and cd4 t cells were labeled with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester ( cfse ) as described previously   and stimulated with il-15 , 50 ng / ml for 7 days in the presence of irradiated autologous pbmc .
by using flow - cytometric analysis , we could demonstrate that cd5 was highly expressed on central - memory cd8 t cells , whereas its expression was downregulated in cd45raccr7 and cd45raccr7 effector - memory cd8 t cells ( fig .
similarly , when using cd45ra and cd28 to characterize memory t - cell subsets , cd45racd28 effector - memory t cells displayed the lowest level of cd5 expression ( figs .
s1a and s1b ) . however , the expression of cd3 and tcr were not downregulated during cd8 memory t - cell differentiation ( fig .
the expression of cd5 was also downregulated during post - thymic memory t - cell differentiation ( fig .
s1c and s1d ) , whereas the expression of cd3 and tcr was not ( fig .
these results indicate that the expression of cd5 is not only regulated during thymic development , but highlight a role for cd5 during post - thymic cd8 and cd4 memory t - cell differentiation in human beings .
cd5 has been shown to act as a negative regulator of cellular activation by dephosphorylating signaling molecules   and to modulate cytokine responsiveness of naive cd8 t cells .
we thus analyzed whether memory t cells with distinct cd5 levels differ in their responsiveness to il-15 , which is known to be an important cytokine for memory t - cell survival and proliferative renewal .
although memory t - cell subsets did not express cd69 in the absence of il-15 ( data not shown ) , il-15 led to the upregulation of the activation molecule cd69 on cd8 and cd4 memory t cells and was inversely associated with the level of cd5 ( figs .
one explanation for the hyper - responsiveness of cd5 cd8 and cd4 memory t cells to il-15 could be that these cells have a higher number of cytokine receptors . however , this is unlikely , because there were no marked differences in the expression of the signaling chains cd122 ( il-2r ) and cd132 ( c ) between memory t cells with different levels of cd5 ( figs .
interestingly , a high expression of monosialotetrahexosylganglioside ( gm ) 1containing lipid rafts was associated with a high responsiveness to il-2 and il-15 in naive cd8 t cells and was directly correlated with the expression of cd5 .
accordingly , a recent study in human subjects demonstrated that central - memory cd4 t cells expressed a high membrane lipid order ( gm1 ) , whereas effector - memory cd4 t cells expressed an intermediate membrane lipid order ( gm1 ) .
this indicates that the gm1 content directly correlates with the responsiveness to il-15 in memory t - cell subsets but inversely correlates with the level of cd5 .
we next analyzed which factors may contribute to the downregulation of cd5 on human t cells .
we therefore stimulated purified , cfse - labeled cd8 and cd4 t cells with il-15 for 7 days .
although cd8 and cd4 t cells did not proliferate in the absence of il-15 ( data not shown ) , cd5 was progressively downregulated in proliferating cd8 and cd4 t cells stimulated with il-15 ( fig .
comparison of cd5 levels between cd8 t cells that have not proliferated ( cfse ) and cells that have undergone four or more cell divisions ( cfse ) demonstrated that il-15mediated signaling decreased the cd5 mean fluorescence intensity ( mfi ) by 21% ( fig .
similarly , cd5 was downregulated in proliferating cd4 t cells stimulated with il-15 and il-15mediated signaling decreased the cd5 mfi by 36% ( fig .
the bm has been shown to be a preferred site for il-15mediated proliferation of memory t cells   and a higher expression of il-15 was identified in bmmc compared with pbmc .
( moreover , cd4 and cd8 t cells were in close contact with il-15producing cells in the human bm .
we therefore determined the expression of cd5 on memory t cells in the human bm .
our results demonstrate that a higher number of cd5 cd8 and cd4 memory t cells accumulated in the human bm compared with peripheral blood ( fig .
, our results highlight a so far unappreciated role for il-15mediated signaling in modulating the expression of the inhibitory molecule cd5 , which is associated with the strength of responsiveness to il-15 .
intriguingly , as memory t - cell homeostasis is critically dependent on il-15dependent signaling , increased sensitivity of cd5 memory t cells to il-15 may explain the selective survival and/or expansion of cd5 memory t cells in the human bm .
we next examined whether persistent infection with cmv , which is known to drive the accumulation of highly differentiated t cells , may alter the frequency of cd5 memory t cells in human beings . indeed
, the number of cd5 cd8 and cd4 memory t cells was increased in cmv seropositive compared with cmv seronegative persons ( fig .
, a higher percentage of cd5 cd4 memory t cells was identified in the peripheral blood of patients with ra compared with age- and cmv - matched healthy individuals ( fig .
our results indicate that persistent infection with cmv and pro - inflammatory autoimmune diseases , such as ra , characteristically shape the functional composition of the human memory t - cell pool by driving the accumulation of cd5 memory t cells
. the lower expression level of cd5 on these highly differentiated memory t cells is also associated with an increased responsiveness to the homeostatic cytokine il-15 , which may represent a survival advantage and may drive their accumulation in the bm . in the case of ra , il-15 is increased in the synovium and bm   and may decrease the activation threshold of highly differentiated , il-15sensitive cd5 memory t cells , thereby contributing to increased t - cell activation and inflammation .
 immunologic memory is a critical feature of the adaptive immune system to fight recurrent infections . 
 however , the mechanisms that shape the composition and function of the human memory t - cell pool remain incompletely understood . 
 we here demonstrate that post - thymic human t - cell differentiation was associated with the downregulation , but not loss , of the inhibitory molecule cd5 . the sensitivity of human cd8 + and cd4 + memory t cells to interleukin ( il)15 was inversely associated with the level of cd5 expression . 
 cd5 expression was downregulated by il-15mediated signaling in vitro and cd5lo memory t cells accumulated in the bone marrow . persistent antigenic stimulation , as in the case of cytomegalovirus infection and rheumatoid arthritis ( ra ) 
 , was also associated with an increased number of cd5lo memory t cells . in conclusion 
 , cd5 may be a useful marker to identify memory t - cell subsets with distinct responsiveness to the homeostatic cytokine il-15 . 
Introduction
Subjects and methods
Results and discussion
Supplementary data