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T cell development in human cytomegalovirus infection

Gamadia, L.E.

Publication date2003

Link to publication

Citation for published version (APA):Gamadia, L. E. (2003). T cell development in human cytomegalovirus infection.

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C H A P T E R 5 THE SIZE AND PHENOTYPE OF VIRUS-

S P E C I F I C T CELL POPULATIONS IS

DETERMINED BY REPETITIVE

ANTIGENIC STIMULATION AND

ENVIRONMENTAL CYTOKINES

Laila E. Gamadia1,2, Ester M.M. van Leeuwen1'2,

Ester B.M. Remmerswaal2, Si-La Yong2,

Sugianto Surachno', Pauline M.E. Wertheim-van

Dillen3, Ineke J.M. ten Berge1'4 and René A.W.

van Lier2.

'Renal Transplant Unit, Department of Internal

Medicine, laboratory for Experimental

Immunology, 'Department of Virology and

Division of Clinical Immunology and

Rheumatology, Department of Internal Medicine,

Academic Medical Centre, University of

Amsterdam, Amsterdam, the Netherlands.

Submitted

ABSTRACT

Based on the expression of the TNF-R SFP CD27 two antigen-primed CD8 T cell subsets

can be discerned in the circulation of healthy individuals: CD27~ T cells that secrete a variety

of cytokines but do not display immediate cytolytic activity and CD27", cytotoxic T cells with

limited cytokine production potential. The mechanism that controls the generation of these

different phenotypes is unknown. We show that CMV reactivation not only increases the

number of virus-specific T cells but induces their transition from a CD27' to a CD27"

phcnotype. In support of a relation between pool-size and phenotype in a cohort of latently

infected individuals, the number of antigen-specific CD27" CD8+ T cells was found to be

linearly related to the total number of CMV specific CD8+ T cells. In vitro studies revealed

that the acquisition of the CD27" phenotype on CMV-specific T cells depended on the

interaction of CD27 with its cellular ligand, CD70. Expression of CD70 was proportional to

the amount of antigenic stimulation and blocked by the CD4 T cell derived cytokine IL-21.

Thus, induction of CD70, which may vary in distinct viral infections, appears to be a key

factor in determining the size and phenotype of the CMV-specific T cell population in latently

infected individuals.

48

INTRODUCTION

T cell memory is established after clearance of acute infection and maintained lifelong. The

generation of CD8~ memory T cells is supposedly by the survival of a number of antigen-

stimulated effector T cells that are generated early in the primary adaptive immune response

to the causative pathogen. What determines a primary induced CD8+ T lymphocyte to survive

into a long-lived memory cell is as yet unclear1. Primed T cells in the peripheral blood of

healthy individuals show considerable phenotypic and functional heterogeneity. At one side

of the spectrum are CD45R(TCD28+CD27+ T cells that produce a large variety of cytokines,

but are unable to execute cytolysis without prior in vitro culture2. These cells have been

defined as "memory-type" T cells because of their ability to clonally expand in response to

recall antigens in the absence of helper T cell-derived cytokines. Based on the expression of

the chemokine receptor CCR7, this population has been subdivided by Sallusto et al. into

CCR7T "central-memory" T cells that have the ability to migrate to secondary lymphoid

organs and CCR7" "effector-memory" T cells that do not have this ability. Although it was

originally reported that CCR7+ and CCR7" T cells differ in immediate effector functions, this

has been disputed by others4:5. Distinct from these "memory-type" cells is the population of

CD45RA+CCR7-CD28-CD27" T cells (further referred to as CD45RA+CD27" T cells). Based

on the expression of CD95-ligand, perforin and granzyme B and the ability to execute

cytotoxicity directly ex vivo these T cells have been designated "effector-type" T cells. This

population, that is also CD 11a'8 and CD57pos increases with age and has a restricted

diversity of TCR-Vp genes6"8.

T cells with specificities for different persistent viruses have been reported to vary in

phenotype and function. For instance, EBV-specific T cells are predominantly

CD45R(TCD28'CD27+ and in asymptomatic HIV-carriers, HIV-specific T cells are mostly

CD45R0+CD28"CD27*, contrasting the phenotype of CMV-reactive T cells that are

predominantly CD45RA"CD27"9. From these findings it was postulated that viruses might

induce phenotypically distinct T cells . However, it is clear that the selection for phenotypes

is not absolute since e.g. in healthy CMV carriers during latency virus-specific T cells with

either CD45R0+CD27^ and CD45RA'CD27" surface profiles can be found 9: 10. Analysis of

virus-specific T cells during acute infections with EBV, HIV or CMV revealed that early

effector T cells are clearly distinct from the "effector-type" CD45RACD27" T cells found in

49

latently infected persons. T cells expanding early in immune responses have CD45R0, CD28,

CD27 surface expression, express the Gl phase-associated nuclear antigen K.i-67 and contain

perforin and granzyme B " . These observations raised questions about the generation of

CD45RA~CD27" T cells and their role in protective immunity9.

Although it was originally proposed that CD45RAXD28CD27" T cells had entered a state of

senescence , telomere length analyses revealed similar replicative histories in vivo of

CD45R(fCD27T and CD45RACD27" CD8' T cells7. Moreover, it was shown that

CD45RA CD27" T cells were unable to divide when stimulatory monoclonal antibodies to

CD3 and CD28 are combined to activate these cells". Still, CMV-specific T cells with this

phenotype potently expanded when stimulated by specific MHC/peptide ligands in the

presence of helper T cell derived cytokinesl5; '6. These latter experiments implied that

although CD45RA+CD27~ T cells are fully differentiated T cells where it concerns CTL

effector functions they are not terminally differentiated in the sense of having lost the ability

to yield progeny.

Here, we studied patients with well-documented CMV reactivation to investigate the

generation of CD27" T cells in vivo and their role in maintaining CMV latency. We show that

"effector-type" T cells can expand in vivo when viral replication occurs. Moreover, antigenic

load and helper T cell derived cytokines are key regulators of the phenotype of virus-specific

T cells during latency, likely through regulation of the CD27 ligand, CD70.

MATERIALS AND METHODS

5 HLA-A2 positive and 1 HLA-B7 positive CMV-seropositive renal transplant recipients of

either a kidney of a CMV-seropositive donor (2) or a CMV-seronegative donor (4) were

longitudinally studied. Basic immunosuppressive therapy consisted of cyclosporin A, blood

trough level aimed at 150 ng/mL. mycophenolate mofetil (lOOOmgs twice daily) and

prednisone 10 mgs daily. Viral replication was monitored longitudinally by quantitative PCR

from the timepoint of transplantation, and reactivation was determined by detectable viral

loads above the cut-off point of 80 copies/mL. All subjects experienced asymptomatic CMV-

reactivation and no anti-viral treatment was given. Heparinized peripheral blood samples were

collected before transplantation and Meekly during 17 weeks after transplantation, whereafter

samples were collected once a month. PBMC were isolated using standard density gradient

50

centrifugation techniques and subsequently cryopreserved. In addition CMV-specific T cells

were characterised in 11 healthy individuals. All patients gave written informed consent and

the local medical ethical committee approved the study.

Peptides

The HLA-A2 binding CMV pp65 derived peptide NLVPMVATV and the HLA-B7 binding

CMV pp65 derived peptide TPRVTGGGAM were purchased from the IHB-LUMC peptide

synthesis library facility (Leiden, The Netherlands).

Generation of tetrameric complexes

Tetrameric complexes were manufactured at the tetramer facility of Sanquin, and generated

essentially as described by Altman et al.18. In brief, purified HLA-A2.1 heavy chain or HLA-

B7.2 heavy chain and p2 micro-globulin were synthesized using a prokaryotic expression

system (pET; Novagen, Milwaukee, WI, USA). The heavy chain was modified by deletion of

the transmembrane/cytosolic tail and COOH-terminal addition of a sequence containing the

BirA enzymatic biotinylation site. The HLA-A2.1 binding CMV pp65 derived peptide

NLVPMVATV and the HLA-B7.2 derived peptide TPRVTGGGAM were used for refolding.

Monomeric complexes were concentrated, biotinylated by BirA (expressed using the pET

expression system, purified using Clontech cobalt beads) in the presence of biotin (Molecular

Probes), ATP (Sigma Chemical) and MgCl2. The biotinylated product was separated from

free biotin by FPLC using a Superdex 200 ITR16 / 60 column (Amcrsham Pharmacia, Little

Chalfont, UK). Streptavidin-allophycocyanin (APC) conjugate (Molecular Probes) was added

in a 1:4 molar ratio and subsequently tetramers were FPLC purified using the same column.

CFSE labelling

PBMCs were pelleted and resuspended in PBS at a final concentration of 5-10 x 106 cells/ml.

PBMCs were labeled with 2.5 |iM (final concentration) of 5-(and-6)-Carboxyfluorescein

Diacetate Succinimidyl Ester (CFSE; Molecular Probes) in PBS for 8 minutes shaking at

37°C. Cells were washed and subsequently resuspended in IMDM supplemented with 10%

Human Pool Serum (HPS), antibiotics and 3.57 x 10"*% (v/v) (3-mercapto-ethanol (Merck)

(culture medium).

51

Culture and stimulation of cells

CFSE labeled cells were cultured in culture medium for five days in 24 wells plates at a

concentration of 0.5-1 x 106 cells/ml. CMVpp65-derived peptide was added at a final

concentration of 1.25 |ig/ml. CMV-antigen (inactivated whole virus, 10 jil/'ml; Microbix) was

used to stimulate cells. Furthermore, for stimulation IL-2 (50 U/ml; Biotest Ag, Dreieich,

Germany), IL-15 (3 ng/ml; R&D systems, Abingdon, United Kingdom), IL-21 (50 ng/ml;

Zymogenetics, Seattle, WA), were added. For blocking experiments we used CD70 antibody

(clone2F2) at concentrations of 40(i.gr/mL. For dose dependent analysis of CD70 expression,

1.25 jig peptide and 10 |iL CMV-antigen/ mL were titrated. Flow cytometric analysis was

performed before culture and after five days.

Immunofluorescent staining and flowcytometry of CMV-specific CD8+ T cells

Thawed PBMC were resuspended in RPMI, containing 10% FCS and antibiotics. 200.000

PBMC were incubated with fluorescent label conjugated mAbs (concentrations according to

manufacturer's instructions) and an appropriate concentration of tetrameric complexes.

Negative controls to validate specificity of the CMV-peptidc-tetrameric complexes consisted

of HLA-A2.1/HLAB7.2 negative CMV-seropositive or HLA-A2.1/HLA-B7.2 positive CMV-

seronegative healthy individuals and renal transplant recipients. Negative controls always

showed tetramer staining of less than 0.01% of total lymphocytes (data not shown). For

staining with the mouse anti-human CCR7 monoclonal antibody (mAb), a three step staining

protocol was performed consisting of incubation with the CCR7 antibody (Pharmingen), for

30 minutes, washing, incubation with biotinylated goat anti mouse IgM (Pharmingen) for 30

minutes, incubation with 10% (v/v) normal mouse serum (Sanquin, Amsterdam, The

Netherlands) followed by incubation with streptavidin-PE and directly conjugated mAbs and

tetrameric complexes for 30 minutes. For staining with the mouse anti-human monoclonal

antibody CD70 (clone 2F2) a two step staining protocol was performed consisting of

incubation with the CD70 antibody for 30 minutes, washing, incubation with FITC

conjugated anti-mouse IgGl antibodies, washing, incubation with 10% (v/v) normal mouse

serum followed by incubation of directly conjugated mAbs. Analyses consisted of APC-

conjugated tetramers and CD8-PerCP (BectonDickinson, San Jose, CA) in combination with

either CD28 (Sanquin, the Netherlands) and CD27 (BD), CCR7 and CD45RA, CD27 and

52

CD45RA (BD) and CD45RA and CD45R0 (BD), CD38 (BD) and CD70 all combinations in

FITC and PE, and additional stainings for CD70 expression were done with CD70 in

combination with CD4-PE, CD8-APC and CD19-PercP.

CMV-PCR

Quantitative PCR was performed in EDTA whole blood samples as described for plasma or 19

serum .

Statistical analyses

Linear regression analysis using a mixed model was performed for the absolute numbers of

total CMV-specific CD8+ T cells in relation with total CD27"CMV-specific CD8+ T cells, and

for the percentage CMV-specific CD8+ T cells and the percentage CD27" of CMV-specific

CD8+ T cells taken as a logit. Subsequently, the regression coefficient was calculated and

tested for significance. Repeated observations were analysed by paired Students t-test or a

repeated measures Anova, p-values, < 0.05 were considered statistically significant.

RESULTS

CMV-reactivation induces quantitative and qualitative changes in the CD8+ virus-

specific T cell compartment

In healthy individuals and renal transplant recipients during latency, CMV-specific CD8+ T

cells may have different predominant phenotypes ranging from CD45R0+CD28+CD27+

("memory-type", patient 1, figure la, t=0) to CD45RAXD45R0duU'"CD27" ("effector-type"

patient 2, figure Id, t=0)9: 1(). The fate of these distinct CMV-specific T cell populations was

followed during CMV reactivation. In the patients with a CD45RA"CD28fCD27+ CD8^ T cell

phenotype prior to reactivation a substantial number of these cells lost expression of CD28

and CD27 (Fig 1, row a). Longitudinal analysis of CMV-specific CD8+CD27" T cells in these

individuals showed that this subset increased immediately when replication of virus occurs,

(Fig. 2a: CD27"tetramer' T cells in relation to viral load in one representative individual,

Figure 2 b: CD27" tetramer+ T cells in all individuals; median before reactivation 26.5%,

range 0.0 to 91.1 % median at the timepoint of reactivation 45.22%), and keeps on doing so

long after viral replication has ceased (median at the first timepoint after reactivation 66.35 %,

53

median at the last time-point measured, > 6 months after reactivation, 72.73%, p = 0.039). In

the individuals with a predominant effector. CD45RA"CD28"CD27" CD8^ T cell phenotype,

there was an increase in the number of CMV-specific T cells but no phenotypic change of

virus-specific CD8' T cells with regard to the expression of CD28 and CD27 (Fig 1, row d).

Patient 1 Patient 2

rr>4SRft

CD38 — •

Figure 1: Quantitative and qualitative changes of CMV-specific CD8* memory cells during reactivation Figure la-c: Differentiation of CMV-specific CD8' T cells in one patient with a starting population of CD45R(fCD27XD28* CMV-specific cells. Figure ld-f: Differentiation of CMV-specific CD8* T cells in one patient with a starting population of CD45RA CD27CD28" cells. Time defined as t=0. before reactivation. t=l. during reactivation at peak PCR value and t=2. first timepoint after reactivation. All plots gated on CD8' T cells. CMV-specific CD8* T cells as defined by specific tetramer staining plotted in black, total CD84" T cells plotted in gray. Figure la and d: x-axis log fluorescence CD27-Fitc. y-axis log fluorescence CD28-PE; figure lb and e: x-axis log fluorescence CD45R0-PE, y-axis log fluorescence CD45RA-Fitc; figure lc and f: x-axis log fluorescence 38-PE, y-axis log fluorescence HLA-DR-Fitc.

Irrespective of the pre-reactivation phenotype, the activation markers CD38 and HLA-DR

were upregulated on CMV-specific cells in all patients at the time of viral replication (Fig. 1,

rows c and f), as was the expression of CD45R0, reflecting recent proliferation of these cells

(Fig. 1 rows b and e). CCR7 expression, documented to be upregulated upon activation161

did not change in any of the patients during reactivation (data not shown), although re-

expression of this marker could redirect subjected cells to secondary lymphoid organs

rendering them undetectable in peripheral blood.

These data suggest that irrespective of their phenotype CMV-specific T cells can confer

protective immunity in situations of virus reactivation. Moreover, virus-reactivation appears

54

to not only increase the size of the virus-specific T cell pool but also, in individuals that start

off with "memory-type" CMV-specific T cells, induces a change from a predominant CD27+

to a CD27' phenotype.

a _ 0.0075-, a S-C 0.0050-> « S Ö O o S j l " 0.0025-O co w ü

0.0000-

0 100 200 300 400 500 600 0 1 2 3

time (days after transplantation)

Figure 2: Ongoing downmodulation of CD27 on CMV-specific CD8+ T cells after reactivation Figure 2a: Longitudinal analysis of CD27 expression on CMV-specific CD8+ T cells in relation to CMV-viral load in one representative patient with a CD28CD27TD45R0* phenotype of CMV-specific cells prior to CMV-reactivation. 2b: Percentage of CD27" CMV-tetramer' T cells in all patients during follow-up of reactivation. Time defined as t=0, before reactivation, t=l, during reactivation at peak PCR value, t=2, first timepoint after reactivation and t=3, > 6 months after reactivation

The CD27" phenotype is correlated with the magnitude of the CD8+ T cell response

during latency

The above findings suggested a connection between the number of virus-specific T cells in

latency and their phenotype which would accord with the expansion of CMV-specific

CD8+CD27" T cells in immuno-compromised patients'0120, B-CLL patients21 and the elderly2" 23. Indeed, close analysis of the total percentage of CD8+ CMV-specific cells as determined

by tetramer staining and the expression of CD27 by these cells shows a strong correlation

between the loss of CD27 and a higher percentage of CMV-specific CD84 T cells as

determined by tetramer staining, both for renal transplant recipients and healthy control

individuals. The correlation of the percentage of CMV-tetramer+ CD8+ T cells and the

percentage of CD27" of these cells is depicted in figure 3a, where it is shown that after the

percentage of tetramer4" cells reaches approximately 1 %, > 50 % of these cells are CD27"

whereas virtually all CMV-specific cells are CD27" when a percentage of 2 % or higher is

reached (p=0.005). Also when depicted in absolute numbers, the amount of CMV-specific

cells is linearly correlated to the amount of CD27" cells, figure 3b).

55

a b

- 5 r i —

8 *s 5 005°

£ > u

5 10 15 20 % of CMV-tctramcrt cells

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07

number of CMV-tetramer* cells (10''mL)

Figure 3: High frequencies of CMV-specific CD27' effector ceils are correlated to the total amount of CMV-specific CD8* T cells. Figure 3a: Correlation of the percentage of total CMV-tetramer" T cells and the percentage CD27" of CMV-specific T cells. Closed circles: values in renal transplant recipients before or after reactivation; open circles: values in healthy control individuals, curve: functional relationship determined by:

e**(0.226 + 0.86*ln(%tetramer» % CD27- = 100 * 1 + e**(0.226 + 0.86*ln(%tetramer)) p=0.005 Figure 3b: Correlation of the absolute number of total CMV-tetramer T cells and the absolute number CD27" CMV-specific T cells. Closed circles: values in renal transplant recipients, curve: linear relationship determined by: Absolute number of CMV-tetramer' cells = 0.001 + (1.46 * Absolute number CMV- tetramer'CD27") p=0.0012.

Generation of the CD27" phenotype of virus-specific T cells is dependent on interaction

of CD27 with its cellular ligand, CD70

T cells downmodulate CD27 after interaction with its cellular ligand CD70 both in vitro and

24; 25 24: 25 , In this process T cells receive co-stimulatory signals for expansion ' ~ and ,26 acquisition of effector functions" . To analyse if virus-induced CD70 expression could

account for the down-modulation of CD27 on virus-specific T cells after CMV reactivation,

CD70 expression was measured in the reactivating patients. Indeed, whereas during latency

most CMV tetramer-binding T cells were CD70 negative (Figure 4 top panel left), CD70

transiently increased during reactivation episodes (Figure 4 middle and right panel).

Accordingly, viral antigen-induced CD70 expression could be induced in vitro and the

magnitude of CD70 expression showed a dose-response relation with the virus-derived

stimulus (Figure 4, bottom panel).To test if viral antigen-induced upregulation of CD70

dictated CD27 expression, T cells from donors with a CD45R0CD27' phenotype (Figure

5a) were cultured for five days with specific peptide and CMV-antigen to activate helper T

cells in the absence and presence of blocking CD70 mAb. In these cultures, CMV-specific T

cells rapidly downregulated CD27 (Figure 5b). However, addition of CD70 mAb to these

56

" ifl

t = 0

2.1

t = l t = 2

Tetramer

Figure 4: CD70 expression is induced by antigen

and is dose dependent Figure 4a:CD70 is expressed on CD8 ' and CMV-specific CD8* T lymphocytes during viral replication in vivo. All plots gated on C D 8 ' T cells. Time defined as t=0, before reactivation, t= l , during reactivation t=2, after reactivation, quadrant percentages depicted as percentage CD70' of tetramcr* cells.Figure 4b: Left panel: Upon in vitro stimulation with CMV-antigen and CMV-peptide, CD70 expression is induced on CD8* T cells; total CD8+ T cells plotted in gray, CMV-specific CD8 ' T cells plotted in black. Right panel: Expression levelsof CD70 on CD8* T cells in various doses of CMV-antigen and peptide stimulation. X-axis: Log fluorescence CD70; Filled histogram: no stimulus, closed histogram: stimulation with 0.15625 uL/ mL CMV-antigen and 0.0195 ugr/mL CMV-peptidc, dotted histogram: stimulation with 0.625 uL/ mL CMV-antigen and 0.0781 ugr/mL CMV-peptide, bold histogram: stimulation with 2.5 uL/ mL CMV-antigen and 0.3125 ugr/mL CMV-peptide.

CFSF. CD70

Ji4r^ Mr'

CD27

Lymphocytes

C CD70

CD70 block Peptide +

CMV-antigen

Tetramer CD27

Figure 5: CD70 induces differentiation of CD27* CMV-specific CDS* T cells Figure 5a: phenotype of CMV-specific CD8' T cells before stimulation, x-axis log fluorescence CD27, y-axis log fluorescence CD45RA. Figure 5b: Left panel: expression of CD70 on total lymphocytes after stimulation with CMV-peptide and CMV-antigen (open histogram) and no stimulus (filled histogram). X-axis: log fluorescence CD70. Right panel: Dot plots of CD27 expression on tetramer* CD8" T cells after 5 days of stimulation with peptide and CMV-antigen with or without blocking of CD70. Plots gated on CD8' T cells x-axis: log fluorescence CMV-tetramer y-axis: log fluorescence CD27; Histogram overlay of CD27 expression on CMV-specific CD8~ T cells without blocking of CD70 (filled histogram) and with blocking of CD70 (open histogram).

cultures reversed this blocking effect and yielded CD27+ CMV-specific T cells at day 5 of

culture. Helper CD4f T cells can directly support CD8+ T cell expansion through secretion of

stimulatory cytokines and provision of membrane-bound helper factors"7. Furthermore, the

homeostatic cytokine IL-15 was shown to be involved in maintenance and

activation/functional differentiation of memory CD8T T cells28"30. To analyse a possible

qualitative role of different cytokines on CD8+ T cell differentiation, the effects of cytokines,

57

Lymphocytes Peptide

CD70 block

CD70 Tetramer CD27

Figure 6: CD7Ö expression is induced by IL-2 and IL-15 but not by IL-21. Figure 6a-c: Left panels: expression of CD70 on total lymphocytes after stimulation with CMV-peptide and IL-2, IL-15 or 11-21 respectively (open histogram) and no stimulus (filled histogram). X-axis: log fluorescence CD70. Right panels: Dot plots of CD27 expression on tetramer" CD8~ T cells after 5 days of stimulation with peptide and IL-2, IL-15 or IL-21 respectively with or without blocking of CD70. Plots gated on CD8^ T cells x-axis: log fluorescence CMV-tctramer; y-axis: log fluorescence CD27: Histogram overlays of CD27 expression on CMV-specific CD8' T cells with different stimulations without blocking of CD70 (filled histogram) and with blocking of CD70 (open histogram).

which previously were found to support proliferation of CMV-specific T cells16, on CD70 and

CD27 expression were analysed. In conjunction with peptide, IL-2 and IL-15 induced CD70

expression on the CMV-specific T cells and CD70-dependent down-modulation of CD27 (Fig

6 a and b). Though IL-21 was reported to only stimulate CD45RA' thymocytes31, in our

experiments, in an antigen-specific setting, also CD45R0+CD8+ T cells were responsive to IL-

21. Interestingly, IL-21, a cytokine presumed to limit effector cell expansion32, did not induce

CD70 expression and supported propagation of CD27+ CMV-specific T cells without

differentiation Fig 6c).

DISCUSSION

Our results show that, in persistent viral infection, reactivation increases the number of

memory cells and skews these cells to a CD27" phenotype. In primary infection, the amount

58

of antigen determines the size of the murine CD8+ T cell response. Subsequently, the

magnitude of this CD8+ T cell response sets the size of the antigen-specific T cell pool during

the memory phase33'34. Recently it was demonstrated that the contraction phase of the primary

immuneresponse is independent of the clearance of antigen35. However, both cytokines

present during initial priming and the amount of antigen present after the contraction phase

are likely to have an impact on the quality and quantity of the memory cells rendered

thereafter34"36. It was proposed that different viruses elicit different human CD8+ T cell

responses with respect to their surface phenotype9. EBV and HIV predominantly elicit CD28" +CD27+CD8+ responses. The analysis of antigen-specific memory CD8! T cells directed

against influenza virus37; 38, which is completely cleared from the host, reveals that these cells

are all CD28+CD27^ and present at low percentages compared to the frequency of CD8+ T

cells directed at persistent viruses. CMV-specific responses in both healthy individuals and

renal transplant recipients vary in magnitude and phenotype among individuals ' . The

strong correlation between the percentage of virus-specific cells in the total CD8+ T cell

population and the percentage of CD27* cells of the virus-specific cells indicates that a

uniform mechanism takes place that drives both the expansion of virus-specific memory cells

and their differentiation to cytotoxic "effector-type" cells.

In this study, replication of CMV, measured by PCR, could be pinpointed to a very limited

period shortly after transplantation, whereas the increase in total CMV-specific cells and the

down-modulation of CD27 on these cells was an ongoing process for upto more than a year

after transplantation. The ongoing rise in CMV-specific CD8+ T cells therefore is possibly

generated by an increase in the number of infected host cells that persistently present antigen

to immune cells. The only known mechanism through which CD27 is downregulated is after

interaction with its ligand CD70. CD70 belongs to the TNF family and its expression on B

and T cells can be induced by antigenic stimulation in vitro 39 and in vivo (this study) and

appears to be modulated by the cytokine milieu. Indeed, our study showed that TCR-

triggering in combination with IL-2 and IL-15 induced CD70, whereas IL-21 did not give

CD70 expression. Accordingly, IL2 and IL-15 induced expansion of CD27" virus-specific T

cells whereas IL-21 promoted outgrowth of CD27+ cells. Though both IL-2 and IL-21 are

helper cell derived cytokines, the differentiation of CMV-specific memory CD8~ T cells to a

CD27" phenotype upon activation with whole CMV-antigen in combination with peptide

59

shows that the role of IL-21 production by CMV-specific CD4' memory T cells, reported to

have a Thl profile , is limited in accordance with previous reports where IL-21 was shown to

be a Th2 cytokine41. Whereas production of helper-cell derived cytokines as IL-21 and IL-2 is

limited to the antigenic activation phase, IL-15 can be produced by a number of lymphoid and

non-lymphoid cells42. It is well feasible that viruses elicit distinct helper cell cytokine profiles

by differential infection of APC and by different target cell tropism, thereby also determining

the number and phenotype of CD8' T cells during the memory phase43"45.

CD27-deficient mice have a reduced ability to form adequate numbers of antigen-specific T

cells upon viral infection46. In contrast, mice that constitutively express CD70 on B cells have

a strongly enhanced capacity to form effector T cells in response to viruses, tumours and

protein antigens47 (R. Arens, manuscript in preparation). Thus these mouse models strongly

infer that the CD27-CD70 interaction is a major determinant in setting the size of the antigen-

specific pool after immunization. The way in which CD27 signalling regulates these

processes is incompletely unravelled but CD27"'" T cells show diminished survival after

stimulation46. An effect of CD27 on T-cell survival would be in line with proposed functions

of related TNF-R family molecules such as OX40 and 4-IBB that increase expression of anti-

apoptotic molecules such as Bcl-2 and Bcl-xL48o°. Indeed, recently we found strong

upregulation of Bcl-xL expression by CD27 triggering (M. van Oosterwij k. manuscript in

preparation). On basis of these observations, it can be postulated that through pathogen-

induced expression of CD70, and subsequent triggering of CD27, upregulation of Bcl-xL and

increased survival of antigen-specific T cells, the contraction phase after initial expansion is

altered leading to a higher setpoint in memory cell numbers. Simultaneously, through

prolonged survival, T cells may receive differentiation signals for a longer period. This

proposed link between survival and differentiation may explain the clear correlation we found

between CMV-specific T cell numbers and their phenotype. In our in vitro studies we did not

find an effect of CD70 blockade on numbers of antigen-specific T cells although these

antibodies were capable to block CD27 signalling (figure 5 and 6). The lack of effect might

be explained by the fact that upregulation of Bcl-xL may protect T cells from death by

neglect, i.e. cytokine shortage, which may be essential in vivo but irrelevant in our cultures

where ample cytokines are present.

60

Taken together, our data imply that the induction and expansion of CD8 virus specific T cells

and their differentiation to the CD27" effector phenotype is driven both by antigenic load and

helper T cell-derived or homeostatic cytokines. We submit that regulation of CD70

expression could be central in this process. Elucidating the differentials inducing and

sustaining the CD27" effector phenotype could have profound impact on immunotherapeutic

strategies, especially in disease situations where strong and persistent cytolytic responses are

warranted.

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