The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation. Despite this, EBV load remained unaltered, even after 5 years of therapy, although a decline in both CD4+ and CD8+ T cells specific for the lytic EBV protein BZLF1 suggested a decreased EBV reactivation rate. In contrast, latent EBV Ag EBNA1-specific CD4+ and CD8+ T cell responses were restored after 5 years of treatment to levels comparable to healthy individuals. In two individuals who were treated by HAART late during HIV progression, a lymphoma developed shortly after initiation of HAART, despite restoration of EBV-specific CD4+ and CD8+ T cells. In conclusion, long-term HAART does not alter the EBV DNA load, but does lead to a restoration of EBNA1-specific T cell responses, which might allow better control of EBV-infected cells when applied early enough during HIV infection.

The EBV is a widespread human gammaherpesvirus (1) that persists for life in a latent form in resting memory B cells after primary infection in the oropharynx (2, 3). EBV-infected B cells persist in a quiescent form in the peripheral blood, and can remigrate to the tonsils or lymph nodes, where they can either be driven into proliferating lymphoblasts when the so-called growth program is induced (involving EBV latent genes), or differentiate into plasma cells, which induces the expression of lytic replication of EBV and ultimately virus release by a fraction of these reactivated cells (1). Control of EBV-infected B cells is achieved mainly by CD8+ T cells (4, 5). HIV infection is associated with an alteration of the EBV viral set point, reflected by an elevated EBV load early in infection which is paralleled by increased numbers of EBV lytic Ag-specific CD8+ T cells. This may be related to more frequent EBV reactivation (6, 7). After this increase early in HIV infection, EBV load increases only slowly during chronic infection (6). This results in the commonly observed elevated and fluctuating EBV load in untreated HIV infection (8), which may be the reason why the absolute level of EBV DNA in PBMC is not predictive of EBV-related non-Hodgkin lymphoma (NHL) 4 (8, 9). Persistent high EBV burden might, however, lead to EBV-related NHL when EBV-specific CD8+ T cell function is lost. An association of this loss of CD8+ T cell function with a decrease in total CD4+ T cell numbers (10) suggested that it may be due to a lack of CD4+ T cell help, which is in accordance with many human (11, 12, 13, 14, 15) and animal studies (16, 17, 18, 19, 20, 21, 22).

In untreated HIV-infected individuals, the incidence of NHL is 60- to 250-fold increased compared with healthy individuals, a majority being systemic or primary CNS lymphomas, which in 70–80% of cases express latent EBV proteins (23, 24, 25). The incidence of both types of lymphomas has decreased since the introduction of highly active antiretroviral therapy (HAART) (26, 27, 28, 29, 30), while their prognosis has greatly improved (27, 31, 32, 33).

The long-term effects of HAART on EBV viral load and EBV-specific immunity have not been studied. No consistent changes in EBV DNA load were found in either short-term longitudinal (34, 35, 36) or cross-sectional studies (9), while IFN-γ production by EBV-specific CD8+ T cells was increased shortly after initiation of HAART (34, 37). However, it remains unclear whether on the long term, HAART will lead to a restoration of the EBV load to levels measured in healthy EBV carriers, by a combination of decreased immune activation and possibly improved immune responses. Alternatively, it may be that HIV seroconversion irreversibly alters the individual EBV viral set point. The aim of this study was to investigate the long-term effects of HAART on EBV viral load and EBV-specific CD4+ and CD8+ T cell responses. To this end, we studied 10 HIV-infected subjects early (2 years postseroconversion) and late during untreated HIV infection (7 years) and at an early (7 mo) and later time point after HAART (5 years). All these individuals responded to HAART by both a decrease in HIV RNA load and a restoration of total CD4+ T cell numbers. Both CD4+ and CD8+ T cells to an EBV latent (EBNA1) and lytic protein (BZLF1) were studied, using an in vitro expansion method that was recently developed (38), next to the quantification of specific CD8+ T cells by direct staining with HLA-peptide tetramers, and in relation to EBV load.

All HIV seropositive subjects were participants of the Amsterdam Cohort studies on AIDS and HIV-1 infection. Blood samples from these homosexual men at risk for HIV-1 infection were collected every 3 mo for HIV-1 serology and immunological studies. In addition, at all time points PBMC were cryopreserved. Ten patients were selected based on HLA class I typing and availability of cryopreserved PBMC at several time points before and after HAART (Table I). None of these 10 subjects was diagnosed with a NHL or other AIDS-defining event. Samples were studied early (73 mo initiation of HAART, range 88–41) and later during HIV infection (11 mo pre-HAART, 16–1), and early (7 mo, 1–15) and late (56 mo, 43–81) after initiation of HAART. The median age of the patients at the first time point studied was 39.5 years (27.5–50). Start of HAART was defined by a drug regimen consisting of at least two nucleotide or nucleoside reverse transcriptase inhibitors and 1 protease inhibitor or 1 nucleoside reverse transcriptase inhibitor + 2 protease inhibitor. Time from HIV seroconversion, HLA class I and II typing, and medication of the HIV-infected individuals are indicated in Table I.

Table I.

Patient data: time from HIV seroconversion, HLA typing, treatment

SEQt1at2at3at4aHLA AHLA BHLA DRHLA DQTreatmentb
36 33 63 75 122 A1, A32 B8, B44 DRB1*03011/1201 DQB1*0201/0301 3TC, Ind, Saq 
156 76 146 161 206 A11, A28 B8, B38 DR3, DR52 DQ2 3TC, Ind, Saq 
164 85 111 147 A1, A24 B8, B44 DRB1*0305/1100 DQB1*0201 zdv, 3TC, ddC, Ind, Rit 
188 80 96 142 A2 B7 DRB1*1302/1501 DQB1*0602/0604 Rit, ddC, Ind, 3TC 
523 12 49 68 105 A1, A33 B8, B44 DRB1*03011/0701 DQB1*0201 3TC, Ind, ddC, Saq 
545 17 90 107 154 A1, A24 B8 DRB1*0300/0400 DQB1*0201/0302 zdv, 3TC, ddC, Ind, Saq 
1194 21 83 95 167 A11, A24 B8, B62 DRB1*03011/1301 DB1*0201/0603 zdv, 3TC, Saq 
1113 19 77 88 158 A1, A11 B8, B57 DRB1*03011/0400 DQB1*0201/0302 3TC, Ind, Saq 
1140 42 108 138 185 A2, A11 B40, B52 DRB1*1300/1500 DQB1*0604/0607 zdv, 3TC, ddC, Ind, Saq 
1186 39 105 128 191 A1, A24 B7 DRB1*15011 DQB1*0602 zdv, 3TC, ddC, Ind, Saq 
Median 20 84 101 156      
Minimum 49 68 105      
Maximum 76 146 161 206      
SEQt1at2at3at4aHLA AHLA BHLA DRHLA DQTreatmentb
36 33 63 75 122 A1, A32 B8, B44 DRB1*03011/1201 DQB1*0201/0301 3TC, Ind, Saq 
156 76 146 161 206 A11, A28 B8, B38 DR3, DR52 DQ2 3TC, Ind, Saq 
164 85 111 147 A1, A24 B8, B44 DRB1*0305/1100 DQB1*0201 zdv, 3TC, ddC, Ind, Rit 
188 80 96 142 A2 B7 DRB1*1302/1501 DQB1*0602/0604 Rit, ddC, Ind, 3TC 
523 12 49 68 105 A1, A33 B8, B44 DRB1*03011/0701 DQB1*0201 3TC, Ind, ddC, Saq 
545 17 90 107 154 A1, A24 B8 DRB1*0300/0400 DQB1*0201/0302 zdv, 3TC, ddC, Ind, Saq 
1194 21 83 95 167 A11, A24 B8, B62 DRB1*03011/1301 DB1*0201/0603 zdv, 3TC, Saq 
1113 19 77 88 158 A1, A11 B8, B57 DRB1*03011/0400 DQB1*0201/0302 3TC, Ind, Saq 
1140 42 108 138 185 A2, A11 B40, B52 DRB1*1300/1500 DQB1*0604/0607 zdv, 3TC, ddC, Ind, Saq 
1186 39 105 128 191 A1, A24 B7 DRB1*15011 DQB1*0602 zdv, 3TC, ddC, Ind, Saq 
Median 20 84 101 156      
Minimum 49 68 105      
Maximum 76 146 161 206      
a

Time in months from HIV seroconversion.

b

Ind = Indinavir; Saq = Saquinavir; Rit = Ritonavir.

In addition, we studied two patients progressing to EBV-related AIDS NHL shortly after start of HAART, at the time points indicated in Fig. 4 (described in Results). Furthermore, a cross-sectional analysis of EBV-specific T cell responses was performed in PBMC from 14 healthy EBV-seropositive blood bank donors.

FIGURE 4.

Longitudinal follow-up of two patients progressing to EBV-related NHL shortly after start of HAART. HIV and EBV viral load (A and E); total CD4+ and CD8+ T cell numbers (B and F); EBV-specific T cell responses after 12 days of specific expansion (C and G); ex vivo CD8+ T cell responses measured by ELISPOT for IFN-γ (D), were studied in subject 68 (A–D) and 434 (E–G), who were both diagnosed a non-Hodgkin lymphoma shortly after initiation of antiretroviral therapy (4 and 24 mo, respectively). The dotted line (ART) represents initiation of therapy, whereas the x-axis indicates the number of days from NHL diagnosis.

FIGURE 4.

Longitudinal follow-up of two patients progressing to EBV-related NHL shortly after start of HAART. HIV and EBV viral load (A and E); total CD4+ and CD8+ T cell numbers (B and F); EBV-specific T cell responses after 12 days of specific expansion (C and G); ex vivo CD8+ T cell responses measured by ELISPOT for IFN-γ (D), were studied in subject 68 (A–D) and 434 (E–G), who were both diagnosed a non-Hodgkin lymphoma shortly after initiation of antiretroviral therapy (4 and 24 mo, respectively). The dotted line (ART) represents initiation of therapy, whereas the x-axis indicates the number of days from NHL diagnosis.

Close modal

MHC class I tetramers complexed to EBV peptides were produced as previously described (10, 39). The immunodominant epitopes were derived from both EBV lytic cycle proteins (A2-GLCTLVAML from BMLF1; B8-RAKFKQLL from BZLF1), and EBV latent Ags (A11-AVFDRKSDAK and A11-IVTDFSVIK from EBNA3B; B7-RPPIFIRRL from EBNA3A; B8-FLRGRAYGL from EBNA3A) (5). HIV-specific CD8+ T cells were studied using tetramers containing several epitopes, depending on the HLA type of the subject: A2-SLYNVATL, B8-EIYKRWII and B57-KAFSPEVIPMF from Gag; A2-ILKEPVHGV from Pol; B8-FLKEKKGL from Nef.

Four-color fluorescence analysis was performed. Briefly, PBMC were thawed and 1 to 1.5 × 106 cells were stained with PerCP-conjugated mAb CD8 (BD Biosciences), CD27 FITC (Sanquin Reagents) and two different HLA peptide tetramers, conjugated with PE and allophycocyanin, respectively. Immune activation on CD4+ and CD8+ T cells was measured by staining of 3 × 105 PBMC with CD4, CD8, HLA-DR (BD Biosciences), and CD38 (Sanquin Reagents). A total of 200,000 events were acquired using a FACSCalibur flow cytometer (BD Biosciences). Lymphocytes were gated by forward and sideward scatter and data were analyzed using the software program CellQuest (BD Biosciences).

EBV-specific CD4+ T cells were stimulated using 15-mer peptides with an 11-aa overlap spanning the immunogenic C-terminal region of EBNA1 (57 peptides) and the entire BZLF1 protein (59 peptides), which were synthesized by Jerini. Purity and sequences were verified by HPLC and mass spectrometry. Peptides were dissolved in DMSO and pooled at a final concentration of 1 mg/ml of each peptide.

As a negative control, PBMC were stimulated with medium in the presence of costimuli. Stimulations with peptide pools and medium were performed in the presence of costimuli, as indicated in the paragraph about detection of EBV-specific T cells. As a positive control PBMC were stimulated with 10 ng/ml PMA and 2 μg/ml ionomycin.

To expand EBV-specific T cells, PBMC were cultured for 12 days in the presence of the EBNA1 or the BZLF1 peptide pool (38). The culture medium consisted of RPMI 1640 (Invitrogen Life Technologies) supplemented with Penicillin/Streptomycin and 10% human pool serum. Cells were cultured at 2 × 105 PBMC/well in 100 μl medium in 96 round-bottom plates, at 37°C and 5% CO2. The peptide pool (at 2 μg/ml of each peptide) was added on days 0 and 6. IL-2 was added at 10 U/ml on days 3, 6, and 9. On day 12 cells were pooled, washed in RPMI 1640, and rested overnight in complete medium. On day 13 cells were restimulated for 6 h using the protocol indicated below. The results of this assay were expressed as the number of specific T cells recovered out of 106 PBMC put into culture, as previously described (38).

IFN-γ-producing cells after stimulation with overlapping peptide pools were enumerated by intracellular cytokine staining (10, 38, 39, 40). Briefly, 106 PBMC were stimulated in 500 μl of medium containing 10% human pool serum for 18 h ex vivo (or 6 h after expansion in culture) with EBNA1 or BZLF1 peptide pools (at 2 μg/ml of each peptide) and both anti-CD28 (2 μg/ml) and anti-CD49d (1 μg/ml) as costimuli, in the presence of 1/1000 brefeldin A (Golgiplug; BD Biosciences) after 1 h to allow accumulation of cytokines in the cytosol. After stimulation, cells were washed in PBS + 0.5% BSA, permeabilized (FACS Permeabilizing Solution; BD Biosciences), washed again and stained with Abs specific for CD3 PerCP, CD4 allophycocyanin, CD8 PE, IFN-γ FITC (BD Biosciences). Cells were washed again, fixed (Cellfix; BD Biosciences) and 200,000 events were acquired on a FACSCalibur flow cytometer (BD Biosciences). Lymphocytes were gated by forward and sideward scatter and data analyzed using the software program CellQuest (BD Biosciences). Responses were scored as positive when they were two times above the medium control value.

HIV RNA load was measured in plasma by several assays. The NASBA HIV-1 QT assay (Organon Teknika) and Amplicor HIV monitor (Roche Diagnostic) had a detection limit of 1000 and 400 copies/ml, respectively. After August 1999, load was determined by the more sensitive Quantiplex bDNA 3.0 assay (Bayer), with a detection limit of 50 copies/ml. Values of 1000, 400, and 50 (Table II) indicate that the load was undetectable by the method used, the cut-off values corresponding to the assay which was used.

Table II.

Cell counts/viral loads

t1t2t3t4p t1–t2ap t1–t3ap t1–t4ap t2–t3ap t2–t4a
CD4b Med. 0.46 0.4 0.46 0.55 0.022e 0.721 0.123 0.059 0.107 
 Range 0.30–0.85 0.25–0.63 0.21–0.88 0.32–0.94      
CD8b Med. 0.95 1.57 1.22 1.16 0.028e 0.185 0.208 0.285 0.401 
 Range 0.60–2.00 0.73–2.69 0.44–4.88 0.76–3.21      
CD19b Med. 0.095 0.18 0.19 0.205 0.012e 0.005e 0.018e 0.058 0.183 
 Range 0.04–0.20 0.07–0.24 0.14–0.40 0.10–0.38      
HIV RNAc Med. 34,500 57,000 400 50 0.374 0.005e 0.008e 0.007e 0.008e 
 Range 1,000–110,000 271–580,000 50–1,000 50–1,073      
EBV DNAd Med. 557 393 421 759 0.114 0.508 0.508 0.878 0.445 
 Range 24–11,144 25–51,684 0–29,876 19–23,820      
t1t2t3t4p t1–t2ap t1–t3ap t1–t4ap t2–t3ap t2–t4a
CD4b Med. 0.46 0.4 0.46 0.55 0.022e 0.721 0.123 0.059 0.107 
 Range 0.30–0.85 0.25–0.63 0.21–0.88 0.32–0.94      
CD8b Med. 0.95 1.57 1.22 1.16 0.028e 0.185 0.208 0.285 0.401 
 Range 0.60–2.00 0.73–2.69 0.44–4.88 0.76–3.21      
CD19b Med. 0.095 0.18 0.19 0.205 0.012e 0.005e 0.018e 0.058 0.183 
 Range 0.04–0.20 0.07–0.24 0.14–0.40 0.10–0.38      
HIV RNAc Med. 34,500 57,000 400 50 0.374 0.005e 0.008e 0.007e 0.008e 
 Range 1,000–110,000 271–580,000 50–1,000 50–1,073      
EBV DNAd Med. 557 393 421 759 0.114 0.508 0.508 0.878 0.445 
 Range 24–11,144 25–51,684 0–29,876 19–23,820      
a

Value of p of the Wilcoxon signed ranks test for the indicated time points.

b

Cell numbers × 109 per liter.

c

HIV RNA copies per milliliter of plasma.

d

EBV DNA copies per 106 PBMC.

e

Significant differences according to Wilcoxon signed rank test.

EBV load was measured in duplicate in DNA from 2 × 105 cells. Real-time PCR amplification was performed as previously described (8, 41), using PCR primers specific for the nonglycosylated membrane protein BNRF1 p143 (42) and a fluorogenic probe (Applied Biosystems) to detect the 74-bp product. As a control for input DNA the amount of β-albumin DNA, a household gene present at 2 copies/cell, was also determined, using primers and probes as described before (43).

For calculation of longitudinal changes, the Wilcoxon signed rank test was used. Correlations were calculated using Spearman’s correlation test. Data from different groups were compared using Mann-Whitney U tests. All statistics were calculated using the software program SPSS 11.5 for Windows (SPSS).

All individuals studied responded to HAART by a reduction in HIV plasma RNA concentration, from a median of 57,000 RNA copies/ml plasma 1 year before treatment to 400 early after treatment (p = 0.007) and 50 at 5 years after treatment (p = 0.008) (Fig. 1,A). Total CD4+ T cell numbers tended to increase from 400/μl to 460/μl at 7 mo (p = 0.059) and 550/μl at 5 years after HAART (p = 0.107), while CD8+ T cell numbers were not altered by treatment. B cell numbers increased during untreated HIV infection (from 100 to 180 cells/μl, p = 0.012), but did not change after short-term (190/μl) or long-term HAART (210/μl) (Table II).

FIGURE 1.

Effects of HAART on HIV RNA, EBV DNA, and T cell immune activation. A, HIV RNA concentration in plasma and, B, EBV DNA concentration in PBMC were measured at the indicated time points before and after of HAART. Viral load data were indexed to 100 at the first measurement, indicating a clear decline in HIV RNA after start of HAART, whereas EBV DNA is fluctuating. Decline in (C) CD4+ and (D) CD8+ T cell activation after initiation of therapy, studied by measurement of CD38 and HLA-DR expression on these subsets. The dotted line indicates initiation of HAART.

FIGURE 1.

Effects of HAART on HIV RNA, EBV DNA, and T cell immune activation. A, HIV RNA concentration in plasma and, B, EBV DNA concentration in PBMC were measured at the indicated time points before and after of HAART. Viral load data were indexed to 100 at the first measurement, indicating a clear decline in HIV RNA after start of HAART, whereas EBV DNA is fluctuating. Decline in (C) CD4+ and (D) CD8+ T cell activation after initiation of therapy, studied by measurement of CD38 and HLA-DR expression on these subsets. The dotted line indicates initiation of HAART.

Close modal

In accordance with earlier data, we found a high and fluctuating EBV DNA load in PBMC (6), and EBV load did not increase during untreated HIV infection (from 557 early to 393 copies/106 PBMC late in untreated infection, p = 0.114). Initiation of HAART did not lead to a reduction in the number of EBV DNA copies measured in PBMC on the short term (421 copies/106 PBMC at 7 mo after initiation of HAART, p = 0.878 compared with pretreatment value). Even long-term antiretroviral treatment did not alter the EBV load (759 copies/106 PBMC at 56 mo post-HAART; p = 0.445 compared with pretreatment value, Table II). The contrast between EBV and HIV load became more pronounced when the load data were related to the first time point. HIV RNA load clearly decreased in each individual studied (Fig. 1,A), whereas the median EBV load remained stable (Fig. 1 B).

T cell immune activation, as an indication of the general state of immune activation, which may induce an elevation of the EBV load, decreased significantly after HAART. The percentage of CD38+HLA-DR+ CD4+ T cells, which had increased from 4.39% at 73 mo to 11.21% at 11 mo before initiation of therapy (p = 0.028), decreased to 3.76% at 7 mo (p = 0.037) and 3.87% at 56 mo (p = 0.037, Fig. 1,C). The percentage of CD38+HLA-DR+ CD8+ T cells changed from 19.50% at 11 mo before to 7.55% shortly after (p = 0.013) and 2.93% at 56 mo after initiation of therapy (p = 0.009, Fig. 1 D).

To study EBV-specific CD4+ T cell responses, we used a recently developed method, enabling specific and reproducible in vitro expansion and restimulation of specific T cells with EBNA1 or BZLF1 peptide pools (38). Specific CD4+ central memory T cells capable of both proliferation and IFN-γ production in response to Ag are measured by this method, and were shown to correlate with protection against hepatitis C virus and malaria (13, 44). For EBV we have shown that results from this assay correlated with EBV viral load and thus may be a good indication of an individuals’ ability to mount an effective EBV-specific CD4+ memory T cell response (38).

As shown in representative FACS plots, EBNA1-specific CD4+ T cell responses tended to decline during untreated HIV infection, and were restored by antiretroviral treatment (Fig. 2,A). In contrast, BZLF1-specific CD4+ T cell responses were maintained before treatment, but decreased after initiation of HAART (Fig. 2,B). To better compare the changes within the whole group of individuals, we indexed the responses to the first time point measured for each subject. EBNA1-specific CD4+ T cell responses tended to decrease during untreated HIV infection in a majority (7 of 9) of the subjects studied (Fig. 2,C, p = 0.086), whereas no changes in BZFL1-specific CD4+ T cell responses were observed before initiation of HAART (Fig. 2,D, p = 0.374). Interestingly, we observed a significant restoration (in 8 of 9 individuals) of EBNA1-specific CD4+ T cells after long-term HAART (p = 0.021, from 11 mo pre- to 56 mo post-HAART, Fig. 2,C), whereas BZLF1-specific CD4+ T cells decreased significantly (in 8 of 10 individuals) after initiation of HAART (p = 0.038, from 7 to 56 mo post-HAART, Fig. 2 D).

FIGURE 2.

Effects of HAART on EBV-specific CD4+ T cells. Representative FACS plots showing changes in CD4+ T cell responses to EBNA1 (A) and BZLF1 (B) during follow-up. C (EBNA1) and D (BZLF1), CD4+ T cell responses indexed to the first time point studied, showing a restoration of EBNA1-specific CD4+ T cell responses after 5 years of therapy, whereas BZLF1-specific CD4+ T cell responses declined. The dotted line represents the height of the first measurement. E (EBNA1) and F (BZLF1), Comparison of CD4+ T cell responses of the 10 subjects treated with HAART for 5 years (HIV+) with healthy EBV+ donors (HIV). The y-axes indicate the number of specific T cells measured after 12 days of in vitro expansion.

FIGURE 2.

Effects of HAART on EBV-specific CD4+ T cells. Representative FACS plots showing changes in CD4+ T cell responses to EBNA1 (A) and BZLF1 (B) during follow-up. C (EBNA1) and D (BZLF1), CD4+ T cell responses indexed to the first time point studied, showing a restoration of EBNA1-specific CD4+ T cell responses after 5 years of therapy, whereas BZLF1-specific CD4+ T cell responses declined. The dotted line represents the height of the first measurement. E (EBNA1) and F (BZLF1), Comparison of CD4+ T cell responses of the 10 subjects treated with HAART for 5 years (HIV+) with healthy EBV+ donors (HIV). The y-axes indicate the number of specific T cells measured after 12 days of in vitro expansion.

Close modal

To determine the level of restoration of EBNA1 and BZLF1-specific CD4+ T cells, we compared the responses after long-term HAART with those measured in healthy EBV carriers. In accordance with the restoration observed for EBNA1-specific T cells after initiation of therapy, numbers of CD4+ T cells specific for EBNA1 after long-term HAART were comparable to values measured in healthy EBV carriers (3431 in healthy vs 1319 after long-term HAART, p = 0.109, Fig. 2,E). In contrast, numbers of BZLF1-specific T cells were lower than in healthy donors (254 vs 54, p = 0.03, Fig. 2 F).

Along with CD4+ T cells, CD8+ T cell responses were also measured after 12 days of expansion with EBV peptide pools (Fig. 3, A and B). Both EBNA1- and BZLF1-specific CD8+ T cells followed approximately the same kinetics as the respective CD4+ T cells (Fig. 3, C and E), although EBNA1-specific CD8+ T cell numbers were restored sooner after initiation of HAART than the CD4+ T cells, and the decrease in BZLF1-specific CD8+ T cells was not significant (p = 0.953, 11 mo pre- vs 7 mo post-HAART, p = 0.314, 11 mo pre- vs 5 years post-HAART). Thus, antiretroviral treatment tended to lead to a restoration of latent Ag- (EBNA1, EBNA3A) specific T cells, and a decrease in lytic Ag- (BZLF1) specific T cells. Similar to the CD4+ T cell response after long-term HAART, EBNA1-specific CD8+ T cells were restored to levels observed in healthy individuals (761 in healthy vs 744 in HAART-treated, p = 0.557, Fig. 2,D), and BZFL1-specific CD8+ T cells were lower than in healthy donors (7754 vs 254, p = 0.001, Fig. 2 F).

FIGURE 3.

Effects of HAART on EBV-specific CD8+ T cells. Along with CD4+ T cells, CD8+ T cells were expanded after 12 days of culture with EBV peptides, as shown in A (EBNA1) and B (BZFL1). C (EBNA1) and E (BZLF1), CD8+ T cell responses indexed to the first time point studied, showing a restoration of EBNA1-specific CD4+ T cell responses after initiation of therapy, whereas BZLF1-specific CD4+ T cell responses tended to decline. The dotted line represents the height of the first measurement. D (EBNA1) and F (BZLF1), Comparison of CD4+ T cell responses of the 10 subjects treated with HAART for 5 years (HIV+) with healthy EBV+ donors (HIV). The y-axes indicate the number of specific T cells measured after 12 days of in vitro expansion. Kinetics of FLR (G) and RAK (H) -specific CD8+ T cells in seven HLA B8-positive individuals, detected by tetramer staining, indexed to the first time point studied.

FIGURE 3.

Effects of HAART on EBV-specific CD8+ T cells. Along with CD4+ T cells, CD8+ T cells were expanded after 12 days of culture with EBV peptides, as shown in A (EBNA1) and B (BZFL1). C (EBNA1) and E (BZLF1), CD8+ T cell responses indexed to the first time point studied, showing a restoration of EBNA1-specific CD4+ T cell responses after initiation of therapy, whereas BZLF1-specific CD4+ T cell responses tended to decline. The dotted line represents the height of the first measurement. D (EBNA1) and F (BZLF1), Comparison of CD4+ T cell responses of the 10 subjects treated with HAART for 5 years (HIV+) with healthy EBV+ donors (HIV). The y-axes indicate the number of specific T cells measured after 12 days of in vitro expansion. Kinetics of FLR (G) and RAK (H) -specific CD8+ T cells in seven HLA B8-positive individuals, detected by tetramer staining, indexed to the first time point studied.

Close modal

Interestingly, CD4+ and CD8+ T cell response to EBNA1 were positively correlated (0.706, p < 0.001), which indicates a possible role for CD4+ T cells in helping the CD8+ T cell response. In contrast, the CD4+ and CD8+ T cell responses to BZLF1 were not correlated (0.049, p = 0.769).

EBV- (and HIV) -specific CD8+ T cells were also enumerated directly by staining with HLA-peptide tetrameric complexes. HIV-specific CD8+ T cells declined in response to a reduction in HIV load (from a median of 11.2/μl at 11 mo pre-HAART to 4.6/μl at 7 and 6.7/μl at 56 mo post-HAART, p = 0.038 and p = 0.017, respectively, data not shown). In contrast, no consistent pattern could be distinguished in the kinetics of the sum of EBV-specific CD8+ T cells (14.3/μl at 11 mo pre-HAART, to 15.3/μl at 7 and 14.0/μl at 56 mo post-HAART, p = 0.260 and p = 0.767, respectively, data not shown). To study latent and lytic epitope EBV-specific CD8+ T cells in more detail, we focused on seven individuals with an HLA B8 genotype, in whom we could study cells recognizing a latent epitope FLRGRAYGL (from EBNA3A) and a lytic epitope RAKFKQLL (from BZLF1). To enable comparison between individuals, we indexed the numbers of tetramer-positive CD8+ T cells to the first time point studied. As shown in Fig. 3, G (FLR) and H (RAK), a wide array of patterns was observed, but for the group as a whole, no changes where observed over therapy.

Interestingly, we were able to study two individuals who were diagnosed with an EBV-positive diffuse large B cell lymphoma shortly after initiation of antiretroviral therapy. Antiretroviral treatment consisted of Lamuvidine and Indinavir starting at 4 mo before NHL for subject 68; patient 434 received Zidovudine and Lamivudine from 24 mo and, in addition, Saquinavir from 21 mo before diagnosis. Both patients responded to HAART by a decline in HIV load (Fig. 4, A and E) and an initial increase in total CD4+ T cell numbers (Fig. 4, B and F). EBV load was elevated in patient 68 during the whole follow-up, whereas in patient 434 an important increase from 2,181 to 10,958 copies per 106 PBMC occurred ∼2 years before NHL diagnosis (Fig. 4, A and E).

EBV-specific CD4+ T cell numbers had decreased already >5-fold, 54 (patient 68) and 37 (patient 434) before diagnosis (data not shown). In both patients restoration of the EBNA1-specific CD4+ and CD8+ T cell response was already observed before the start of HAART, which might have been driven by Ag from a developing malignancy, and continued to increase after HAART in subject 434 (Fig. 4, C and G). In subject 68, we were also able to enumerate IFN-γ-producing CD8+ T cells specific for epitopes derived from EBNA3A (A30-AYSSWMYSY), EBNA3B (B44-VEITPYKPTW), and EBNA3C (B44-KEHVIQNAF), respectively. These responses were clearly restored rapidly after initiation of therapy (Fig. 4 D). Thus, these data show that, despite a restoration of EBV-specific CD4+ and CD8+ T cell responses, the occurrence of an EBV-related NHL could not be prevented.

In this study, we investigated whether long-term highly active antiretroviral therapy would lead to a lower EBV viral set point, which at least in part could explain the decreased incidence of AIDS NHL since the introduction of HAART. In 10 successful responders to antiretroviral therapy (both increase in CD4+ T cell numbers and a decrease in HIV load), no alterations in EBV viral load were found, despite a clear decrease in immune activation, and a restoration of EBNA1-specific central memory CD4+ and CD8+ T cell responses. Interestingly, while a restoration of latent Ag-specific T cells occurred, lytic Ag-specific responses decreased, suggesting a reduction in the rate of reactivation of EBV after initiation of HAART.

Earlier studies did not report changes in EBV load shortly (up to 1 year) after initiation of HAART (34, 35), although higher numbers of IFN-γ-producing EBV-specific CD8+ T cells (34, 37), and an increased concentration of EBV-specific Abs, were measured (36). We hypothesized that either 1) EBV viral load would decline after long-term antiretroviral treatment, due to a decline in immune activation and a restoration of EBV-specific T cell immunity or 2) EBV viral load would remain high as a consequence of a definitive alteration of the equilibrium between EBV and immunity after HIV seroconversion. EBV load was not altered after 5 years of antiretroviral therapy, which indicates that an individuals’ EBV viral set point is irreversibly altered after HIV seroconversion (6, 45). This may be explained by a number of factors. First, while T cell activation diminishes quickly after initiation of therapy, perturbations of the B cell compartment remain for years after start of therapy, as evidenced by a lack of recovery of the memory B cell subset and persistence of elevated IgG levels (46, 47, 48). In line with this, it may be that, although chronic activation of T cells is normalized in a few years, their ability to provide help to B cells is restored (49), which might help to maintain reactivation of EBV-carrying memory B cells (50). However, a reduction in lytic Ag-specific T cells does not support this explanation. Alternatively, while a decrease in EBV reactivation rate is suggested by the kinetics of EBV lytic Ag-specific T cells, it might still take a long time to reduce the pool of latently EBV-infected B cells. Furthermore, in untreated HIV infection, a slow increase in EBV load over years is usually observed (6, 51). Thus, it appears that long-term HAART does not influence the altered EBV viral set point initiated after HIV seroconversion (but may lead to a stabilization). Finally, one could argue that redistribution of EBV-infected B cells could explain our findings, but the relatively stable number of B cells after HAART in the individuals studied here argues this.

Interestingly, different patterns of recovery of EBNA1 and BZLF1-specific T cells were observed. Although no EBV reactivation is measurable by RT PCR in the blood of HIV-infected EBV carriers (9), the oropharynx is known as a major site of EBV replication (52), which is accompanied by an increase in the frequency of EBV lytic Ag-specific CD8+ T cells (7). Our data is compatible with a diminished rate of EBV reactivation, followed by a decreased lytic Ag-specific T cell response. At the same time, the general immune restoration associated with HAART (53) resulted in increased numbers of EBNA1-specific CD4+ T cells, possibly because of the restoration of the central memory CD4+ T cell pool specific for latent Ag EBNA1. The increase in CD8+ T cell function observed in earlier studies (34) might well be associated with the recovery of specific CD4+ Th cells (37). In addition, the CD8+ T cell response to EBNA1 was improved, possibly also through an improved CD4+ T cell helper function, as suggested by a correlation between the EBNA1-specific CD4+ and CD8+ T cell response. This is particularly interesting in light of recent papers showing that, in contrast to earlier reports (54, 55, 56), EBNA1-specific CD8+ T cells are able to recognize EBV-infected B cells (57, 58, 59, 60), and, next to EBNA1-specific CD4+ T cells (61), might be an important factor in controlling outgrowth of EBV-positive tumors (60). It will be interesting to investigate whether the difference between EBNA1 and BZLF1-specific T cell responses is a general feature of latent vs lytic EBV Ags, or something specific for these two proteins.

Most recent data indicate a clear reduction in the incidence of NHL since the introduction of HAART (26, 27, 62), although it is still a matter of debate whether so-called “virological failers” will not be at higher risk on the longer term (29, 30). The 10 patients selected for our study had relatively preserved CD4+ T cell numbers, and responded to therapy by a clear reduction in HIV RNA load. They are thus likely to represent subjects who will have a decreased risk of developing NHL. A decrease in general immune activation and EBV reactivation, together with a restoration of EBV latent Ag-specific responses, may create a much “safer” equilibrium between EBV and its host. The development of lymphoma is known to be a multistep process, which can cover a period of several years, starting with alterations in immune control together with chronic antigenic stimulation and cytokine deregulation, followed by a phase of accumulation of genetic lesions, which can ultimately lead to the uncontrolled proliferation of a clonal B cell population (63, 64). EBV-specific CTL may be most efficient in controlling the early stages of EBV-associated polyclonal B cell proliferation. The two patients who developed NHL after the start of HAART likely represent individuals in which evolution toward an EBV-related malignancy was too advanced to be stopped by the immunological improvements of antiretroviral treatment. It may thus be important to start antiretroviral therapy before irreversible genetic alterations in EBV-infected B cells have occurred, although other reports show that the prognosis of NHL has also clearly improved since the introduction of HAART (27, 31, 32), even when antiretroviral treatment is initiated after diagnosis (33). In addition, it is known that central memory CD4+ T cell responses are better restored when HAART is initiated before total CD4+ T cell numbers drop below 350/μl (53).

In conclusion, the long-term follow-up of subjects who were successfully treated by HAART shows that that despite an improved EBV-specific T cell response and a decrease in T cell immune activation, the EBV load remains high in these individuals. Interestingly, changes in the relative importance of latent and lytic Ag-specific T cell responses suggest a decrease in EBV reactivation, but this does not alter the EBV load in the peripheral blood. The data are thus in accordance with the idea that an elevated EBV load in the HIV setting does not in itself correlate with the incidence of EBV-related malignancies (8). Thus, early initiation of HAART might result in a new equilibrium, much more favorable for the host, consisting of a still elevated EBV load, but in the presence of sufficient CD4+ T help to preserve CD8+ T cells.

We thank Marijke Roos for cell subset counts and Suzanne Jurriaans for measurement of HIV RNA viral load. We also thank the participants in the Amsterdam Cohort Studies on HIV and AIDS.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was financially supported by AIDS Funds Netherlands, Grant 5005 and Grant CLBD2000-2164 of the Dutch Cancer Society.

4

Abbreviations used in this paper: NHL, non-Hodgkin lymphoma; HAART, highly active antiretroviral therapy.

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