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Cytomegalovirus-specific T-cell dynamics in HIV infection

Bronke, C.

Publication date2005

Link to publication

Citation for published version (APA):Bronke, C. (2005). Cytomegalovirus-specific T-cell dynamics in HIV infection.

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Download date:24 Apr 2021

Chapterr 2

Dynamicss of CMV-specifïc T cells in HIV-1-infected individuals progressingg to AIDS with CMV end-organ disease

Corinee Bronke1, Nanette M. Palmer1, Christine A. Jansen1, Geertje HA . Westerlaken1'*,, Abeltje M. Polstra2, Peter Reiss3, Margreet Bakker2, Frank Miedema1,2'*,, Kiki Tesselaar1'*, and Debbie van Baarlel,#

'Departmentt of Clinical Viro-Immunology, Sanquin Research and Landsteiner Laboratory, Academicc Medical Centre, University of Amsterdam, Amsterdam, the Netherlands departmentt of Human Retrovirology, Academic Medical Centre, University of Amsterdam, Amsterdam,, the Netherlands departmentt of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Universityy of Amsterdam, Amsterdam, the Netherlands #Currentt address: Department of Immunology, University Medical Centre Utrecht, Lundlaan 6,, 3584 EA Utrecht, the Netherlands

TheThe Journal of Infectious Diseases, in press

ChapterChapter 2

asymptomaticss with more than 7 years of asymptomatic follow-up and CD4 counts > 400/nll (classification of the Centres for Disease Control 1993). None of the individualss received anti-(retro)viral therapy during the study period. For the AIDS-CMVV group, time points were studied from just after HIV seroconversion or study entryy up to CMV diagnosis. The earliest time point studied in the AIDS-OI and the long-termm asymptomatics groups was between 1 and 2 years after HIV seroconversion.. The latest time point studied for AIDS-OI was on average 5 years (aroundd the time of AIDS diagnosis) and for long-term asymptomatics 10 years after HIVV seroconversion. Characteristics of the study participants are summarised in Table 1. .

HLA-classs I tetramer staining in parallel with intracellular IFNy staining after antigen-specificc stimulation Itt has been well characterised that the immune response to CMV in HLA-A 2 individualss is dominated by the NLVPMVATV (NLV) epitope of the pp65 lower matrixx protein [11,12]. HLA-A2 NLVPMVATV peptide-tetrameric complexes were produced,, and four-colour fluorescence analysis was performed as described previouslyy [13]. Briefly, PBMC were stained with HLA-A2-NLV tetramers. After fixationn and permeabilisation (Becton Dickinson reagents, hereafter BD, San José, California,, USA), cells were stained (intracellularly) with fluorochrome-conjugated monoclonall antibodies (mAb) against CD8, perforin (BD) and granzyme B (Sanquin Reagents,, Amsterdam, the Netherlands). In parallel, PBMC were stimulated either withh 10 u.g/ml of NLVPMVATV peptide, or with CMV lysate (BioWhittaker, Walkersville,, USA; 60 \il/m\ or Microbix Biosystems, Toronto, Canada; 10 u-l/ml) in thee presence of 2 ng/ml anti-CD28 (Sanquin Reagents) and CD49d (BD) mAb at 37°CC for 6 hours. After 1.5 hours, 5% (w/v) monensin was added [13]. As a positive controll for the capacity of PBMC to produce IFNy, phorbol myristate acetate (PMA) andd ionomycin (Sigma-Aldrich, Zwijndrecht, the Netherlands; 5 ng/ml and 1 u.g/ml respectively)) were added. Unstimulated cells were used as a negative control. Cells weree placed at 4°C overnight, after which they were stained as described above with mAbb against CD8, and IFNy (BD), or CD4, CD3 (BD), and IFNy. Cells were fixed in Cellfixx (BD), and up to 400,000 events acquired using a FACSCalibur flow cytometer (BD).. Lymphocytes were gated by forward-sideward scatter, and data were analysed usingg the software programme CELL Quest 3.3 (BD). In case of IFNy measurements, thee number of responding T cells was calculated after subtracting negative control values.. Data are expressed in absolute numbers per u.1 blood, to take into account that duringg HIV infection and progression to AIDS, total CD4+ T-cell numbers drop dramatically. .

Vira ll load determination CMVV viral load was determined in PBMC with the COBAS AMPLICOR™ CMV MONITORR KIT (Roche Diagnostics, Almere, the Netherlands) as described previouslyy [14]. In short, PBMC in lysis buffer were added to the lysis reagent that

20 0

DynamicsDynamics of CMV-specific T cells in HIV infection

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ChapterChapter 2

containedd a quantitation standard. DNA was precipitated with isopropanol, and pelletedd by centrifugation. The pellet was washed, and resuspended in specimen diluent.. The extracted sample was added to PCR Master Mix and placed in the COBASS AMPLICOR instrument for automated amplification, detection and quantificationn of CMV DNA. The detection limit is 100 copies. Copy numbers betweenn 1 and 100 are considered to be positive, but cannot be quantitated.

Statisticall analysis Wilcoxonn signed rank tests were performed to compare values at early and late time points,, within patient groups. Differences between the patient groups were analysed usingg the Mann-Whitney test. Correlations were tested using the Spearman's non-parametricc correlation test. All statistical analyses were performed using the software programmee SPPS 10.00 (SPSS Inc., Chicago, Illinois, USA).

RESULTS S

Numberss of CMV-specific CD8+ T cells increase over time prior to onset of CMV-EO D D HLA-A2-NL VV tetramers were used to determine CMV-specific CD8+ T-cell numbers.. Figure 1 shows the results of all time points in the AIDS-CMV group. To be ablee to analyse the data, early time points as defined as the earliest time point after HIVV seroconversion or study entry, and late time points, on average 10 years after HIVV seroconversion for long-term asymptomatics, 5 years after HIV seroconversion forr progressors to AIDS-OI, and time points between 5 and 11 months before CMV end-organn disease for progressors to AIDS-CMV were analysed (Figure 2).

Inn long-term asymptomatics (Figure 2a), numbers of A2-NLV-tetramer+CD8+

TT cells were readily detected, which increased significantly during follow-up (median 8.511 to 49.33/ul; p=0.043*, Wilcoxon). Progressors to AIDS-OI (Figure 2a) had relativelyy low and stable numbers of A2-NLV-tetramer+CD8+ T cells (median 0.73 to 1.39/(il;; p=1.000, Wilcoxon). Interestingly, in progressors towards AIDS-CMV (Figuress la and 2a), like in long-term asymptomatics, A2-NLV-tetramer+CD8+ T cells weree detected at high and increasing numbers (median 8.77 to 21.58/ul; p=0.093, Wilcoxon).. Both long-term asymptomatics and progressors to AIDS-CMV contained moree A2-NLV-tetramer+CD8+ T cells than the progressors to AIDS-OI early (p=0.050** and p=0.024* respectively, Mann-Whitney) and late (p=0.014* and p=0.005** respectively, Mann-Whitney) in HIV infection.

Lackk of IFNy-producing CMV-specific CD8+ T cells within progressors to AIDS-CMV V Too investigate responsiveness of the CMV-specific CD8+ T cells, the number of IFNy-producingg CD8+ T cells was measured using intracellular IFNy staining, after stimulationn with NLV peptide. In long-term asymptomatics (Figure 2b), IFNy+CD8+

22 2

DynamicsDynamics of CMV-specific T cells in HIV infection

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Figur ee 1. Dynamic s of CMV-specifi cc CD8+ T cells , CD4+ T cell ss and CMV load in the cours e off HIV-1 infectio n in progressor s too AIDS-CMV. T-cell responses weree analysed in progressors to AIDS-CMV.. Patient samples were analysedd for a) numbers of A2-NLV-tetramer+CD8++ T cells/u I blood;; b) numbers of IFNy+CD8+ T cells/nll blood after stimulation with NLVV peptide; c) percentage of perforin*granzymee B+ cells within A2-NLV-tetramer+CD8++ T cells; d) numberss of IFNY*CD4* T cells/nl bloodd after stimulation with CMV lysate;; and e) CMV load in copies/1066 PBMC (dotted line representss detection limit). X-axis indicatess time in months. Due to the variationn with respect to the time to CMVV end-organ disease (e.g. light bluee versus green line in Figure 1a), thee data is plotted retrospectively fromm the time of onset of CMV end-organn disease. Each colour representss the same patient in differentt analyses.

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23 3

ChapterChapter 2

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Figuree 2. Comparison of early and late time points of CMV-specific T-cell responses betweenn groups. Early and late time points from the longitudinal data of long-term asymptomaticss (left column), progressors to AIDS-OI (middle column) and progressors to AIDS-CMVV (right column) were compared using the Wilcoxon test. The two-tailed p-values aree depicted in the upper right or left corner of the graphs. Depicted are a) numbers of A2-NLV-tetramer+CD8++ T cells/nl blood; b) numbers of IFNy+CD8+ T cells/(il blood after stimulationn with NLV peptide; c) percentage of IFNy* cells within A2-NLV-tetramer+CD8+ T

24 4

DynamicsDynamics ofCMV-specific T cells in HIV infection

cells ;; d) percentag e of perforin +granzym e B+ cell s withi n A2-NLV-tetramer +CD8* T cells ; and e)) number s of IFNy+CD4+ T cells/^ l bloo d after stimulatio n with CMV lysate . In long-ter m asymptomatics ,, the upper graph is depicte d with a scale from 0 to 80 cells/ji l bloo d and the lowerr graph with a scale from 0 to 6 cells/|il , like the other graph s of AIDS-OI and AIDS-CMV, forr bette r comparison . Dots represen t individua l patient s and the median is show n as a bar.

TT cells could be detected (median 2.82 to 17.22/ul; p=0.138, Wilcoxon), whereas IFNY +CD8++ T cells in progressors to AIDS-OI (Figure 2b) were present at low numberss (median 0 to 0.68/u,l; p= 0.109, Wilcoxon). In progressors to AIDS-CMV (Figuress lb and 2b), IFNY +CD8+ T-cell numbers increased towards, but dropped beforee the time of CMV event (median 0 to 2.25/u.l; p=0.327, Wilcoxon). In the cumulativee data from all individuals, correlations were found between the number of A2-NLV-tetramer+CD8++ T cells and IFNY +CD8+ T cells both early (p=0,826, p<0.01*, Spearman's)) and late (p=0.787, pO.01*, Spearman's; data not shown).

Ass depicted in Figure 2c, percentages of IFNy-producing cells within A2-NLV-tetramer+CD8++ T cells were lowest at the late time point in progressors to AIDS-CMVV (median 6%) compared to long-term asymptomatics (median 28%) or progressorss to AIDS-OI (median 58%), and differed at the late time point between long-termm asymptomatics and progressors to AIDS-CMV (p=0.019\ Mann-Whitney). Thus,, despite high numbers of CMV-specific A2-NLV-tetramer+CD8+ T cells before onsett of CMV-EOD, a relatively low proportion of these cells was able to produce IFNyy after stimulation with peptide.

Percentagee of perfori n and granzyme B expressing CMV-specific CD8+ T cells increasess to high levels in progressors to AIDS-CMV Too investigate potential cytotoxic capacity of the CMV-specific A2-NLV-tetramer+CD8++ T cells, perforin and granzyme B expression within these cells was analysed.. In all patient groups, percentages of single perforin+ T cells were low, whereass single granzyme B+ T cells were detected abundantly in the CMV-specific CD8++ T cells (data not shown). In addition, perforingranzyme B~ T cells could be detectedd in all groups. In long-term asymptomatics (Figure 2d), high percentages of T cellss expressing both perforin and granzyme B were detected (median 24.40 to 43.75%;; p=0.138, Wilcoxon). In progressors to AIDS-OI (Figure 2d) these percentagess were lower and stable (median 19.45 to 19.89%; p=1.000, Wilcoxon). Forr progressors to AIDS-CMV (Figures lc and 2d), the fraction of these cells started att similar percentages as the progressors to AIDS-OI, and increased towards onset of CMV-EODD (median 22.75 to 54.73%; p=0.080, Wilcoxon). Within all individuals, thee number of A2-NLV-tetramer+CD8+ T cells and perforin+granzyme B+ A2-NLV-tetramer++ T cells correlated significantly (early, p=0,990, p<0.01*, Spearman's; late, p=0.884,, p<0.01 ; data not shown). Interestingly, at the late time point, progressors to AIDS-OII had significantly lower percentages of perforin+granzyme B+ CMV-specific CD8++ T cells compared to progressors to AIDS-CMV (p=0.050*, Mann-Whitney). Thus,, even though in progressors to CMV-EOD a relatively low fraction of CMV-

25 5

ChapterChapter 2

specificc CD8+ T cells was capable of producing IFNy, expression of the cytotoxic moleculess perforin and granzyme B was not impaired.

IFNy-producin gg CMV-specific CD4+ T cells are lost in the year before the first CMV-associatedd clinical event CMV-specificc CD4+ T cells were studied using intracellular IFNy expression after stimulationn with CMV lysate. In long-term asymptomatics (Figure 2e), IFNY+CD4+ T cellss were readily detected (range 0 to 75.58/^1), and stable (median 0.88 to 0.11/|J.1, p=0.893,, Wilcoxon). In progressors to AIDS-OI (Figure 2e), IFNy+CD4+ T cells couldd be detected though at lower numbers (median 0.08 to 0.51/uJ, p=0.285, Wilcoxon).. Remarkably, in progressors to AIDS-CMV (Figures Id and 2e), even thoughh IFNy+CD4+ T-cell numbers could be detected early in infection (range 0 to 2.71/|xl),, they dropped significantly over time from a median of 0.30 to 0/ul (p=0.0288 , Wilcoxon). However, no correlation could be found in the HIV1" data betweenn numbers of IFNy+CD8+ T cells and IFNy+CD4+ T cells. Also, absolute CD4+

T-celll number and number of IFNy+CD8+ T cells did not correlate. Thee disappearance of IFNy+CD4+ T cells in progressors to AIDS-CMV could

nott be explained by an absolute lack of responsiveness of CD4+ T cells, since stimulationn with PMA and ionomycin readily induced IFNy expression by CD4+ T cellss both in absolute numbers and percentages (data not shown). Thus, the disappearancee of IFNy-producing CMV-specific CD4+ T cells in progressors to AIDS-CMVV was associated specifically with the development of CMV disease.

CMVV load can be detected a few months prior to the onset of CMV-EOD Too investigate the role of CMV replication, CMV load was measured in a large numberr of PBMC samples. No CMV load was detectable in long-term asymptomatics.. In progressors to AIDS-OI, CMV load was detected in a few samples att the later time points, but too low to be quantified (data not shown). In all progressorss to AIDS-CMV, CMV load was detectable. In 8 out of 10 individuals, CMVV load could be quantified in samples obtained in the year before the onset of CMV-EOD,, and ranged from 148 to 18184 copies per 106 PBMC (median 439 copies perr 106 PBMC; Figure le). Although CMV load did not correlate with the CMV-specificc CD8 or CD4 response, it did relate to disease progression, since only in progressorss to AIDS-CMV, load could be detected in quantifiable amounts.

DISCUSSION N

Inn this study, longitudinal samples from long-term asymptomatics, progressors to AIDS-OI,, and progressors to AIDS-CMV were analysed in terms of CMV-specific CD8++ and CD4+ T-cell responses in parallel to CMV load in PBMC in order to investigatee which factors might contribute to CMV-associated disease progression.

26 6

DynamicsDynamics ofCMV-specific T cells in HIV infection

Stainingg with HLA-A2-NLV tetramers clearly showed that CMV-specific A2-NLV-tetramer+CD8++ T cells were present in all individuals, and increased over time bothh in long-term asymptomatics and progressors to AIDS-CMV. These increasing numberss of CMV-specific T cells may be induced by episodes of CMV reactivation. CMVV load was indeed detectable and much increased in the AIDS-CMV group prior too CMV diagnosis. This may suggest that long-term asymptomatics are successful at keepingg the CMV load below detection limits during follow-up, whereas progressors too AIDS-CMV fail to control CMV replication. Progressors to AIDS-OI were shown too have low numbers of CMV-specific T cells, which fits the low numbers of virus-specificc T cells in general [15,16]. This may be explained by a lower capacity to proliferatee in response to viral antigens as has been shown for HIV-specific CD8+ T cellss [17]. As very low levels of CMV DNA were detectable at some of the later time points,, and no clinical CMV-related symptoms were observed, this suggests that CMVV reactivation may simply be less severe despite the low numbers of CMV-specificc CD8+ T cells.

Wee used IFNy as a functional read-out, and showed in progressors to AIDS-CMVV much lower percentages of IFNY +CD8+ T cells within A2-NLV-tetramer+CD8+

TT cells compared to progressors to AIDS-OI and long-term asymptomatics, suggestingg CMV-specific CD8+ T-cell dysfunction. Indeed, in three HIV-1-infected individualss with low or no CD4+ T cells at all, also dysfunction of HIV and CMV-specificc CD8+ T cells was shown [18]. Likewise, this has been described for HIV-specificc CD8+ T-cells in individuals progressing to AIDS [19], and for EBV-specific CD8++ T-cells in HIV-1-infected individuals progressing to AIDS-related non-Hodgkinn lymphoma [15]. In contrast, the percentages of perforin and granzyme B doublee positive CMV-specific CD8+ T cells were highest in progressors to AIDS-CMV,, indicating that these individuals do have a substantial number of CTLs with cytotoxicc capacity [5].

Loww numbers of IFNy-producing CMV-specific CD8+ T cells in combination withh high numbers of both perforin and granzyme B expression, indicates that in CMV-specificc T cells, IFNy release and perforin and granzyme B expression are not necessarilyy linked [20]. As CMV is a typical cytopathic virus [1], that has been shown too depend more on the antiviral effect of cytokines like IFNy and TNFa, than on the perforin-dependentt granule exocytosis pathway [21], the decreased IFNy-producing capacityy of the CMV-specific cells may well be crucial.

Thee most conspicuous observation in this study was that in the progressors to AIDS-CMV,, CMV-specific IFNy+CD4+ T cells disappeared in the year before onset off CMV-EOD suggesting that CMV-specific IFNY +CD4+ T cells could play an importantt role in protection from CMV-EOD. Dysfunction of CTL (in terms of IFNy) andd memory B cells was shown to be associated with SIV-induced impairment of CMV-specificc CD4+ T helper cells in rhesus macaques [22], and CMV-specific IFNy hass been shown to act as an immunological predictor of CMV control [23]. However, noo correlation was found between numbers of IFNy-producing CD8+ and CD4+ T cellss as has been shown in healthy donors, but not in asymptomatic CMV-seropositive

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renall transplant recipients who are on basic immunosuppressive drug therapy [24]. Possibly,, the IFNy produced by the CMV-specific CD4+ T cells may well have a role inn controlling CMV directly, like IFNv produced by the CD8+ T cells.

Inn conclusion, our data show that progressors to AIDS-CMV lose CMV-specificc IFNy+CD4+ T cells in the year before onset of CMV-EOD, in parallel with a sharpp increase in load. CMV-specific CD8+ T cells remain in high numbers, with a highh proportion of these cells expressing perforin and granzyme B, and a low proportionn producing IFNy. The decrease in IFNy production in the CD 8 compartmentt may be due to insufficient CD4 Thl cells, and may allow for CMV dissemination.. High viral load leads to high numbers of virus-infected cells and inducess high numbers of perforin+granzyme B+A2-NLV-tetramer+CD8+ T cells. Thesee high numbers of cytotoxic T cells in combination with the large numbers of infectedd cells, may lead ultimately to disease in the organ(s) infected, irrespective of thee type of CMV-EOD (data not shown). Our data suggests that the balance between CD4++ T cells, CD8+ T cells, IFNy and perforin/granzyme B is very important to controll CMV replication and prevent virus-associated immune pathology.

ACKNOWLEDGEMENTS S

Thiss study is part of the Amsterdam Cohort Studies on HIV-1 infection and AIDS amongg homosexual men, a collaboration of the Municipal Health Service, the Academicc Medical Centre and Sanquin Research at CLB. We thank Dr. M.Th.L. Rooss and collaborators for the T lymphocyte immunophenotyping and processing of patientt samples. This study was supported by the Landsteiner Foundation for Blood Transfusionn Research (grant number LSBR0007)

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