A2 T-lymphocytes in HIV-patients

Immunology Letters 98 (2005) 73–81

Significance of the detection of HIV-1 gag- and/or pol-CD8/A2 T-lymphocytes in HIV-patients Ruihua Wua,∗ , Garrick C. Owenb , Tianmin Liuc , Guo-Qiu Shena , Robert I. Morrisa a b

RDL Reference Laboratory, 10755 Venice Blvd, Los Angeles, CA 90034, USA Specialty Laboratories, 2211 Michigan Avenue, Santa Monica, CA 90404, USA c University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

Received 29 September 2004; received in revised form 26 October 2004; accepted 26 October 2004 Available online 25 November 2004

Abstract Cytotoxic T-lymphocytes (CTL) play an important role in the immune system’s defense against human immunodeficiency virus (HIV) infection. The functional status of CTL closely relates to the progression of HIV disease. We have validated the characteristics of the assay for HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells from peripheral blood by flow cytometry. Sixty-nine healthy individuals and 38 HIVpatients with HLA-A2 antigen-positive subjects were included in the study. Neither HIV-1 gag- nor pol-specific-CD8/HLA-A2 T-cells were determined in these healthy subjects. HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells could be detected in HIV-patients. The frequency of specific CTL was 58% (22/38) in the patient group. There was a significantly inverse correlation (p < 0.05) between HIV-1 gag- and pol-specific-CD8/HLA-A2 T-cells and HIV plasma viremia in the patients. Conclusion: The HIV-1 gag- or pol-specific-CD8/HLA-A2 T-cells assay is sensitive and specific, being able to detect at the single T-cell level. This assay may provide a versatile tool for structured HIV treatment and for monitoring vaccination efficacy. © 2004 Elsevier B.V. All rights reserved. Keywords: HIV; Cytotoxic T-lymphocyte; HIV-1 gag-specific-CD8/HLA-A2 T-cells; HIV-1 pol-specific-CD8/HLA-A2 T-cells; Flow cytometry

1. Introduction Functional impairment of human immunodeficiency virus (HIV)-specific CD8 T-cells is a key indicator of immune status and disease progression in the HIV-infected individual [1]. A cytotoxic T-lymphocyte (CTL) response is important in protection from HIV-infection. The regulation of HIV level is mostly mediated through specific CTL responses [2]. Longitudinal studies during acute HIV-infection in both long-term, non-progressors and highly active, antiretroviral therapy (HAART)-treated individuals have demonstrated that viremia was decreased in association with increasing cytotoxic HIV-specific, CD8+ T-lymphocytes and a sustained increase in levels of HIV-specific CD4+ T∗ Corresponding author. Tel.: +1 800 3381918/+1 310 2535455; fax: +1 310 2535466. E-mail address: [email protected] (R. Wu).

0165-2478/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2004.10.016

lymphocytes. The CD4+ T-lymphocytes help the CD8+ Tlymphocytes eliminate HIV-infected cells [3–6]. However, in some HIV-infected individuals, plasma viremia is high while CD4+ T-lymphocytes are extremely low. CD8+ T-lymphocyte function is impaired and cannot effectively eliminate HIVinfected cells [5,7,8]. Accurate assessment of the functional status of CD8+ Tcells is an important tool for managing the clinical therapy of HIV-infected patients. Several current methods have limited usefulness. Lymphocyte proliferation assays primarily determine CD4+ cell responses and has a low sensitivity for detection of T-cell responses to general antigens. Limiting dilution assay for CTL and intra-cellular staining technique for detection of cytokine production can measure CD8+ Tcell function, but both have major disadvantages including complexity of performance, sterility concerns, and longer time requirements. Elispot assay is a highly sensitive technique, but it has poor reproducibility especially with higher

74

R. Wu et al. / Immunology Letters 98 (2005) 73–81

background. All the above assays require a cumbersome restimulation step. With the combined use of peptide–HLA tetrameric complexes and T-lymphocyte phenotype-specific fluorescent-labeled antibodies in flow cytometry, antigenspecific T-lymphocytes can be directly detected in peripheral blood [9–13]. To better understand an effective HIV-specific immune response, one of the interesting studies has been focused on MHC class I-restricted epitopes derived from the internal virus antigens. Both gag and pol peptides are internal viral antigens and conserve in viral genome. Gag is one of the group-specific antigens, which include matrix, capside, and nulceocapside. Pol belongs to a family of polymerases, which include protease, reverse transcriptase, and integrase. The virus-specific CD8+ T-cells typically focus on these conserved epitopes within the products of HIV gag and pol genes. These epitopes and MHC class I molecules may be involved in restricting viral replication in HIV-1 patients and especially the long-term non-progressors. However, the response in progressors is broader and includes responses restricted by other class I alleles carried by an individual patient [14–17]. Gag is a dominant, viral antigen that can elicit a strong CD8+ T-cell response. The cross-clade CTL responses against pol epitopes have been detected in both HIV-infected and exposed but uninfected individuals [18–20]. In this study, we have validated an assay for quantitative determination of HIV-1 gag- and pol-specific-CD8/HLA-A2 T-lymphocytes in peripheral blood by flow cytometry using the tetrameric technique. Our results indicate that the assay is highly sensitive and easy to perform. The result demonstrates that concentrations of HIV-CTL are inversely related to clinical disease progression in HIV-patients. This assay may provide a new, versatile tool for the quantitative determination of HIV-1 gag- and pol-specific-CD8+ /HLA-A2 T-cells as a monitor of disease progress or assessment of vaccination efficacy.

Park, CA) and anti-CD8 PC5 mAb (Beckman Coulter; Miami, FL) for 20 min at room temperature. After incubation, using the T Qprep system (Beckman Coulter; Miami, FL) the erythrocytes were lysed and the MHC HLA-A2 allele was analyzed using a XL flow cytometer (Beckman Coulter; Miami, FL).

2. Materials and methods

3. Results

A total of 452 samples were studied of which 318 samples were randomly selected from the remnant of specimens submitted for post-diagnostic HIV testing [21] to Specialty Laboratories between December 2000 and June 2001. The remaining134 samples were from control subjects, 84 samples were collected from Specialty Laboratories’ employeesand 50 samples were obtained from SeraCare Inc. (Oceanside, CA). Blood was drawn after obtaining informed consent. The specimens were stored at room temperature and tested within 24 h. The study was approved by Specialty Laboratories Review Board.

3.1. Instrument set up for CD8/HLA-A2 determination

2.2. HIV-1 gag- and pol-specific-CD8+ /HLA-A2 T-cells detection For HLA-A2-positive samples, 100 ␮L of peripheral blood was added to 10 ␮L of iTAgTM MHC tetramer HLAA*0201 HIV-1 gag-PE/CD8-FITC or iTAgTM MHC tetramer HLA-A*0201 HIV-1 pol-PE/CD8-FITC (Beckman Coulter; Miami, FL) and 5 ␮L of CD3-ECD mAb (Beckman Coulter; Miami, FL) and 5 ␮L CD45–PC5 mAb (Beckman Coulter; Miami, FL). After samples were gently mixed, the cells were incubated for 20 min at room temperature. In order to lyse the erythrocytes, the cells were incubated with 500 ␮L of OptiLyse® C (Beckman Coulter; Miami, FL) for 10 min at room temperature. The cells were washed one time with 4 mL of PBS and centrifuged at 500 × g for 5 min. After the supernatant was aspirated, the cells were re-suspended in 500 ␮L of PBS containing 0.5% formaldehyde. 10,000 events of CD8positive cells were counted and analyzed by COULTER® EPICS XLTM Flow Cytometer Serial Number AD38242 with COULTER EPICS XL-MCLTM Flow Cytometer SYSTEM IITM Software (Beckman Coulter; Miami, FL). 2.3. Statistical analysis Conventional methods were used to calculate the means and standard deviations. For skewed variables, nonparametric tests were employed for comparisons between the groups (Mann–Whitney U-test), whereas significance levels were put at a probability of <0.05.

Using a two-parameter side-scatter versus CD8–PC5 dot plot, CD8+ cells were identified using a region-gating strategy (Fig. 1a). From this CD8+ region, HLA-A2+ cells were then identified as double positive CD8+ PC5 and HLA-A2+ FITC. There were two blood samples stained by CD8–PC5/HLAA2/FITC to be shown in the figure, one of them indicates an HLA-A2-negative subject (Fig. 1b) and the other HLA-A2positive subject (Fig. 1c).

2.1. MHC HLA-A2 detection

3.2. Instrument set up for CD8/HIV-1 gag+ and pol+ T-cell determination

Briefly, 100 ␮L of peripheral blood was incubated with 5 ␮L of anti-HLA-A2 FITC mAb (One Lambda; Canoga

Using a two-parameter side-scatter versus CD45–PC5 set up dot plot A. CD45+ leukocytes were identified using a

R. Wu et al. / Immunology Letters 98 (2005) 73–81

75

region-gating strategy (Fig. 2a). From this CD45+ region, CD8+ cells were then identified as dot plot B dual positive CD3+ ECD and CD8+ FITC (Fig. 2b). From further gating on the CD3+ /CD8+ region, CD8+ HIV-1 gag+ cells were identified as double positive CD8+ FITC and HIV-1 gag+ PE. Similar gating strategies were used for identifying CD8+ HIV-1 pol+ cells. Two individual specimens were stained by iTAgTM MHC tetramer HLA-A*0201 HIV-1 gag-PE/CD8-FITC and CD3-ECD mAb and CD45–PC5 mAb. One of them indicates negative in CD8/HIV-1 gag− (Fig. 2c). Another of the subjects was positive in CD8/HIV-1 gag+ (Fig. 2d). 3.3. Interpretation of results and reference interval A total of 134 specimens from healthy donors were tested for the CD8+ /HLA-A2+ phenotype. All of them were negative in serum HIV antibody tests. From the cohort, 69 specimens were positive with CD8+ /HLA-A2+ (51.5%). These specimens were then further tested for CD8/HIV-1 gag+ and pol+ T-cells. In determining the reference interval, three results, one from CD8/HIV-1 gag test and two from CD8/HIV-1 pol were excluded as outliers. A “rank and order” analysis was used to determine outliers, where the difference between the outlier sample (n) and the previous sample (n − 1) was greater than the imprecision of the assay (we considered that three cases were non-specific staining). In order to avoid the non-specific staining, the median channel fluorescence (MCF) of CD8/HIV-1 gag- or pol-positive population was combined to judge the reliability of the result. MCF less than 5.0 of stained cell population was identified as an unclear determination (Fig. 2e). Normal ranges of the HIV-1 gag- or pol-specific-CD8/HLA-A2 T-cells tests were established by mean + 2S.D. from the healthy group. Samples with CD8+ /HIV-1 gag+ T-cell results less than 0.15% or pol+ T-cell results less than 0.18% of the total CD8+ cells were reported as “not detected” (ND, see Fig. 2c). Samples with CD8+ /HIV-1 gag+ cells greater than or equal to 0.15% and with MCF greater than or equal to 5.0, as well as samples with CD8+ /HIV-1 pol+ cells greater than or equal to 0.18% and with MCF greater than or equal to 5.0 were reported as “detected” (Fig. 2f). Samples with CD8+ /HIV-1 gag+ or pol+ cells greater than the normal range and MCF less than 5.0 (Fig. 2e) were reported as “indeterminate”. 3.4. Specimen integrity Fig. 1. Using a two-parameter, side scatter vs. CD8–PC5 dot plot, CD8+ T-lymphocytes (gate A) were identified using a region-gating strategy (a). Data acquisition from gate A of part (a) was further analyzed by quad-stat. The percentage of double stained CD8+ and HLA-A2+ cells was counted by data analysis software of flow cytometry in parameter histogram with quad-stat 2. One subject with HLA-A2− detection is shown in part (b); one subject with HLA-A2+ determination is shown in part (c).

Assessment of sample viability is crucial for evaluating specific receptor expression on the surface of CD8 T-cells. Anti-coagulants, storage environment, and time can all affect the membrane integrity of lymphocytes and introduce variation into the assay. The choice of collection anti-coagulant was investigated. An HLA-A2-positive and an HLA-A2negative individual specimen were each collected in ACD, EDTA, and heparin anti-coagulants. The samples were stored at room temperature and HLA-A2 typing was performed

76

R. Wu et al. / Immunology Letters 98 (2005) 73–81

Fig. 2. Using a two-parameter, side scatter vs. CD45–PC5 dot plot (A), CD45+ leukocytes were identified using a region-gating strategy in (a). From this CD45+ region, CD8+ cells were then identified as (B) dual positive CD3+ -ECD and CD8+ -FITC in (b). CD3+ CD8+ region from (b) were further identified as double positive CD8+ -FITC and HIV-1 gag+ -PE cells. Similar gating strategies were used for identifying CD8+ /HIV-1 pol+ cells. The percent of CD8+ /HIV-1 gag or pol cells of all CD8+ cells was determined and calculated from gated parameters established using flow cytometry data analysis software. Data acquisition was established at 10,000 events of CD8+ cells, and included a percentage of the positive HIV-1 CTL in total events counted and median channel fluorescence (MCF) of positive cell population. One example of a CD8+ /HIV-1 gag− T-cell case is shown in (c); one of case of a CD8+ /HIV-1 gag+ T-cell case is shown in (d). In order to avoid the detection of false-positives, the MCF was introduced into final identification of reporting results. The lower MCF of the double CD8 and tetramers stained cells shows a random distribution that cannot be identified as a positive result (e). The true-positive population is always accompanied with a higher MCF and a uniform distribution of the detected cell population (f).

R. Wu et al. / Immunology Letters 98 (2005) 73–81 Table 1 Verifiability of specimens for HLA-A2/CD8 cells phenotyping detection in different anti-coagulated blood Day

0 1 2 3 4 5 Mean S.D. CV%

Specimen 1 EDTA

Heparin

100 99.4 100 99.7 100 99.6

99.8 99.3 100 99.5 99.9 98.5

99.9 99.4 100 96.6 99.7 98

99.78 0.26 0.3

99.5 0.55 0.6

Table 3 Intra-assay for CD8/HIV-1 gag and pol cells phenotyping detection Replicate

Specimen 2

ACD

98.93 1.36 1.4

ACD

EDTA

0.4 0.13 0.33 0.3 0.2 0.2

0.7 0.07 1.6 3 9.4 2

0.5 0.21 0.8 0.9 0.2 0.2

0.26 0.1 38.5

2.8 3.39 121.1

0.47 0.32 68.1

77

Heparin

All values are percentages.

Specimen 1

Specimen 2

CD8+

CD8+

CD8+

gag+

pol+

gag+

(%)

(%)

CD8+ pol+ (%)

(%)

1 2 3 4 5

3.39 2.64 2.75 2.93 2.91

0.79 0.61 0.61 0.6 0.64

0.03 0 0 0.01 0

0 0.01 0 0.02 0

Mean S.D. CV%

2.92 0.29 9.8

0.65 0.08 12.3

0.01 0.01 163.0

0.01 0.01 149.1

cates in one run were 0.3% for the positive specimen and 16.1% for the negative specimen. The intra-assay imprecision of flow cytometry staining for CD8+ /HIV-1 gag+ or pol+ T-cell phenotyping was determined using a pair of CD8+ /HLA-A2+ HIV-1 gag/pol-positive and -negative specimens. The CVs of five replicates in one run were 9.8 and 12.3%, respectively for CD8+ /HIV-1 gag+ or pol+ T-cell phenotyping. The mean of 0.01% was obtained with the negative specimen (Table 3).

daily for 5 days by flow cytometry (Table 1). The results demonstrated that the ACD anti-coagulant provided the best sample integrity over time with the least typing variation for flow cytometric staining. The coefficient of variation (CV) was 0.3% for the HLA-A2+ specimen and 38.5% for the HLA-A2− specimen. Using ACD as the anti-coagulant, three HLA-A2+ specimens from HIV-infected patients were obtained and stored at room temperature. CD8/HIV-1 gag- and pol-tetramer staining was performed daily for up to 3 days. The CVs ranged from 0.3 to 8.5% for detection of CD8+ /HIV-1 gag+ or pol+ T-cell phenotyping in specimens tested over 2 days. There was an obvious decrease when the same samples were tested for CD8+ /HIV-1 gag+ or pol+ T-cells on the third day. These results suggest that samples can be tested within 48 h after blood draw (Table 2). The accuracy of the assay was analyzed to compare between using flow cytometry technique and using phenotyping technique to detect HLA-A2 typing in 32 individuals. The results of HLA-A2 typing expression detected by the two techniques were in complete agreement (data not shown).

3.6. Clinical studies Three hundred and eighteen blinded peripheral blood specimens, which were remnant specimens submitted for CD4 and CD8 T-cell phenotyping and quantification of plasma HIV, were tested for HLA-A2 phenotype. From the cohort of 318 specimens, 168 were HLA-A2+ (52.8%). The specimens were then checked for HIV-1 status by determining the inverse ratio of CD4 cells to CD8 cells and assessing HIV plasma viremia. Thirty-eight specimens were clearly identified as HIV-patients. Then, the specimens were further tested for CD8/HIV-1 gag and pol T-cells. The 38 patients’ characteristics are shown in Table 4. Based on the results of CD8/HIV-1 gag- or/and polspecific T-cells, the 38 samples were divided into a CD8/HIV1 CTL-positive group and a negative group. Plasma viremia levels were compared for both groups (samples with HIV viral loads less than 50 copies/mL were arbitrarily designated as 40 copies/mL for the purposes of calculation). The HIV-1 viral loads were significantly lower in the CD8/HIV-1 gag-

3.5. Precision of assay The intra-assay imprecision of the flow cytometric staining for HLA-A2 phenotyping was determined using a pair of HLA-A2+ and HLA-A2− specimens. CVs of the five repliTable 2 Verifiability of specimens for CD8/HIV-1 gag and pol cells phenotyping detection Day

Specimen 1

Specimen 2

CD8+ gag+ (%)

CD8+ pol+ (%)

CD8+ gag+ (%)

CD8+ pol+ (%)

1 2 3

0.95 1.02 0.13a

0.75 0.66 0.88a

2.94 2.92 2.69a

0.67 0.65 0.45a

Mean S.D. CV%

0.98 0.05 5.1

0.71 0.06 8.5

2.93 0.01 0.3

0.66 0.01 1.5

a

Data not included in calculation due to a significant variation shown between the day and previous days.

Specimen 3 CD8+ gag+ (%) 0.01 0 0a 0.01 0.01 141.42

CD8+ pol+ (%) 1.46 1.3 1.11a 1.38 0.11 8.0

78

R. Wu et al. / Immunology Letters 98 (2005) 73–81

Table 4 HIV-1 patients’ individual characteristics Patient number

Age

Sex

HIV-1-UQ (copies/mL)

CD8+ gag+ (%) (MCF)

Interpretation

CD8+ pol+ (%) (MCF)

Interpretation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

33 45 33 21 44 43 32 32 24 34 51 45 36 33 44 39 44 31 42 40 39 22 44 19 24 42 26 54 26 56 44 35 44 32 46 34 23 36

M M M M F F F F M F F F M M F F M M F F M F M M M M M F F M M M M F M M F M

<50 65 120 120 122 320 1488 1835 2961 5080 5311 6754 10150 11350 13650 14607 23321 28312 33165 76774 83473 85146 93269 100085 113195 114365 120082 146397 204981 500000 500545 <50 <50 <50 <50 <50 <50 <50

0.1 0.35 (6.5) 2.12 (7.4) 0.43 (5.4) 0.86 (7.0) 0.01 0.02 0 0.08 0.05 0.1 0.07 0.35 (20) 0.11 0 0.62 (8.7) 0.04 0.30 (5.9) 0 0.54 (10.6) 0 0.1 0.16 0 0.50 (5.5) 0.04 0.19 0.22 (2.7) 0 0.50 (5.2) 0.01 0.24 (2.6) 0.29 (8.3)a 2.94 (10.2)a 0.3 (2.8) 0.67 (9.4)a 0.64 (11)a 0.3 (9.9)a

Not detected Detected Detected Detected Detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Detected Not detected Not detected Detected Not detected Detected Not detected Detected Not detected Not detected Not detected Not detected Detected Not detected Not detected Indeterminate Not detected Detected Not detected Indeterminate Detected Detected Indeterminate Detected Detected Detected

0 0.12 1.12 (8.2) 0 1.00 (5.5) 0.45 (5.8) 0.01 0 0.21 (5.1) 0.1 0.1 0 0.06 1.46 (8.2) 0 0.1 0 0 0.20 (3.2) 0 0.93 (10.2) 1.50 (8.2) 1.16 (6.94) 0.08 0.1 0 0 0.04 0 0.1 0.05 0.25 (5.6)a 0.38 (8.9)a 0.67 (6.2)a 0.2 (2.4) 0 0 0.1

Not detected Not detected Detected Not detected Detected Detected Not detected Not detected Detected Not detected Not detected Not detected Not detected Detected Not detected Not detected Not detected Not detected Indeterminate Not detected Detected Detected Detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Not detected Detected Detected Detected Indeterminate Not detected Not detected Not detected

a

HIV-1 CTL-positive individual who is HIV viral loads to be less than 50 copies/mL.

and/or pol-specific CTL-positive group than in the CD8/HIV1 gag- and/or pol-specific CTL-negative group (p < 0.05; 46,374 ± 108,137 versus 88,854 ± 136,386). The copies of HIV-1 viral load were significantly lower in CD8/HIV-1 gag-CTL-positive group than in the CD8/HIV-1 gag-specific CTL-negative group (p < 0.05; 49,578 ± 1,128,995 versus 70,362 ± 116,088). There was no significant difference in viral load for the CD8/HIV-1 pol-CTL-positive group versus the CD8/HIV-1 pol-specific CTL-negative group (p > 0.05; 25,171 ± 40,106 versus 79,491 ± 139,073). The dot plot of HIV-1 viral load in each group is shown in Fig. 3. Eight samples had plasma viral loads less than 50 copies/mL and six out of eight samples were positive for CD8/HIV-1 gagand/or pol-specific CTL. The HIV progression of six patients who were detected CD8/HIV-1 gag- and/or pol-specific CTL were reviewed by checking HIV plasma viral loads during the past 2 years. All of them showed that plasma viral loads clearly decreased during the prior 2 years. The variations of

Fig. 3. Dot plots showed individual levels of plasma HIV in positive group of HIV-1-specific CTL and negative group of HIV-1-specific CTL.

R. Wu et al. / Immunology Letters 98 (2005) 73–81

Fig. 4. Variation of plasma HIV viral loads in six positive individuals of HIV-1 gag- or/and pol-CTL during the past 2 years.

viral loads in those individuals during the 2 years are shown in Fig. 4.

4. Discussion We have validated the assay for quantification of HIV1 gag- or pol-specific-CD8/HLA-A2 T-cells in peripheral blood for assessing specific CTL recognizing function of patients with HIV-infection. The advantage of this method is its high sensitivity and specificity enabling detection of HIV-1 CTL at the signal cell level. The test is easy to perform without a re-stimulation step. The weakness of the test is that it is limited by MHC-HLA typing status and certain determinants (gag and pol) of HIV-antigen. Several potential problems should be avoided in performance of the test. One potential interfering factor is natural killer (NK) cells, which may be dimly stained by multiple antibody reagents (CD8/tetramer HIV-1 gag/tetramer HIV-1 pol) to induce a false-positive result. In order to avoid NK interference, we have adopted two parameters: (1) percentage of double-stained cells and (2) median channel fluorescence to identify the HIV-1 gag- or pol-specific CTL (see Fig. 2e and f). Dimly stained NK cells may be differentiated from the specific CTL. The number of the HIV-CTL is very low in most positive individuals (<0.5%); non-specific staining of a few cells or particles can induce an erroneous result. Crucial to preventing this problem is careful calibration of instruments and performing isotope staining in pairs to accurately setup the regions. A protocol (http://www.niaid.nih.gov/reposit/tetramer/protocol.html) for soluble MHC/peptide tetramer use is recommended. With experienced users, as few as 0.1% of CD8-positive T-cells in peripheral blood lymphocytes can be detected. In order to detect rare cell populations, it is necessary to analyze a large number of events. After reviewing the protocol and test performance, we determined that 10,000 CD8+ cell events must be collected as the lowest limit for obtaining a reliable result.

79

HIV viremia was well controlled in six samples out of eight patients with plasma viremia <50 copies/mL and CD8/HIV-1 gag- and/or pol-CTL-positive. An extremely interesting result of this study is that specific CD8+ /HIV gagand/or pol-positive T-cells have a negative correlation between the CTL and the viremia copy counts. For example, 15 patients with CD8+ /HIV-1 gag-positive T-cells were divided into two groups depending on their viremia counts. One group with their viremia counts lower than 1000 copies/mL has a CTL counts at 0.96 ± 0.93% (mean ± S.D.); another group with their viremia counts higher than 1000 copies/mL has a CTL counts at 0.47 ± 0.12% (mean ± S.D.). In contrast, specific CTL was reduced in the group with plasma viremia over 1000 copies/mL. A shift in the virus/host balance that favors production of CTL is consistent with animal studies when removal of CD8+ cells in vivo results in a rapid increase in the steady-state level of viremia [22,23]. In addition, these results suggest that determination of the HIV-1 CTL status in HIV-patients is clinically important to evaluate treatments of HIV. The result conformed with Ogg et al.’ result [22] that an inverse correlation exists between viral load levels and HIV-1-specific CD8+ T-lymphocytes. Similar findings have also been reported in acute HIV-1-infected individuals treated with HAART, in whom decreased viral load levels were correlated with increased HIV-1-specific CD8+ T-lymphocytes [24–26]. In addition, the data showed that copies of plasma HIV in the patients with CD8/HIV-1 gag- and/or pol-positive group were significantly lower compared with CD8/HIV-1 gagand/or pol-CTL-negative group (p < 0.05), but the level of viremia have overlapping in the patients with CD8/HIV-1 gag- and/or pol-positive group. A few patients with lower copies of plasma HIV have not been detected as specific CD8/HIV-1 gag and/or pol T-cells in vivo. This may be corrected by presenting other HIV-1-specific CD8+ Tlymphocytes besides gag and pol in the patients. It has been reported [27] that five synthesized HIV-peptides can strongly bind to HLA-A2 including at least env and vac in addition to gag and pol peptides. HIV-infected persons might show a cytotoxic response against those peptide-labeled target cells. However, cellular immune responses to HIV are very complex and the real mechanism of the CTL is unclear. The application of the procedure for quantification of HIV1 gag- or pol-specific-CTL in peripheral blood is limited by the necessity for a specific HLA-A2 allele. About 50% of the whole human population is positive for HLA-A2. Thus, other major HLA alleles should be evaluated for the possibility of determining CTL status in HLA-A2-negative populations. It should also be noted that the tetramer staining provides quantitative information about a T-lymphocyte population based on its specificity, not its function. Some studies [28,29] failed to find an inverse correlation between specific CTL and HIV progression. Immune functional status in patients is complicated by the fact that in some circumstances, tetramer-binding CTL may be functionally quiescent, that cells are unable to “kill” HIV [4,26,30]. A possible expla-

80

R. Wu et al. / Immunology Letters 98 (2005) 73–81

nation may be that many of these viruses may have already mutated to escape CTL [31]. Another explanation may be that although the majority of HIV-specific CTL secrete antiviral cytokines, they can lack perforin; HIV-specific CD8+ perforin-expressing cells may have direct antiviral consequences [32]. Alternatively, the inability of a CTL response to control viremia is due to decreased absolute numbers of CD4 cells [22,33,34]. It has been suggested that CTL response cannot be maintained and eventually wanes when CD4 cells are absent [35]. If the helper T-cell count is very low or the integrity of antigen presenting cells has been impaired by HIV [3–6], CTL response is compromised. Another possible explanation is that an impaired extra-cellular release of IFNgamma, perforin, MIP-1, and other active factors occurs even though these proteins are produced intra-cellularly [36]. It has been shown that the immune system can provide a control mechanism against HIV progression, and that the immune response to HIV can be boosted in vivo to increase effectiveness [1]. In studies of long-term non-progressors with HIV-1 infection, CD8+ CTL have been shown to participate in the control of the progression of both of HIV-1 infection in humans and simian immunodeficiency virus (SIV) infection in macaques [11,12]. Use of the HIV-1-specific T-lymphocyte assay to quantitative HIV-1 gag- and pol-specific-CD8+ /A2+ T-cells in whole blood of HIV-1-infected individuals along with viral load levels will provide a measure for assessing and monitoring individual immune systems in the fight against HIV-1 infection [37]. An important notation must be made the tetramer-staining test provides quantitative information based on the specificity of HIV-1 CTL, but this does not indicate functional status. It is also important to develop a reliable test to add functional information. A test using intra-cellular staining for detection of IFN-gamma production in CD8 and CD4 cells has been validated (unpublished data). The intracellular staining test combined with the tetramer-staining test may provide a more comprehensive assessment of the HIV1-specific CTL. This assay can also provide a versatile tool for monitoring the effectiveness of immune therapy and HIV vaccines. Additional tests of HIV-specific CTL matched with other HLA alleles should be developed for detection of CTLs in HLA-A2− subjects. Techniques for measurement of CTL functional status should be further explored.

Acknowledgments We thank Phillip Stepanik and John Sama for their critical review of the manuscript.

References [1] Altfeld M, Walker BD. Less is more? STI in acute and chronic HIV-1 infection. Nat Med 2001;7:881–4. [2] McMichael AJ, Rowland-Jones SL. Cellular immune responses to HIV. Nature 2001;410:980–7.

[3] Rosenberg ES, Billingsley JM, Caliendo AM, Boswell SL, Sax PE, Kalams SA, Walker BD. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science 1997;278: 1447–50. [4] Gray CM, Lawrence J, Schapiro JM, Altman JD, Winters MA, Crompton M, Loi M, Kundu SK, Davis MM, Merigan TC. Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). J Immunol 1999;162:1780–8. [5] Kalams SA, Goulder PJ, Shea AK, Jones NG, Trocha AK, Ogg GS, Walker BD. Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy. J Virol 1999;73:6721–8. [6] Oxenius A, Price DA, Easterbrook PJ, O’Callaghan CA, Kelleher AD, Whelan JA, Sontag G, Sewell AK, Phillips RE. Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes. Proc Natl Acad Sci USA 2000;97:3382–7. [7] Sepkowitz KA. AIDS—the first 20 years. N Engl J Med 2001;344: 1764–72. [8] Ogg GS, Jin X, Bonhoeffer S, Moss P, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Hurley A, Markowitz M, Ho DD, McMichael AJ, Nixon DF. Decay kinetics of human immunodeficiency virus-specific effector cytotoxic T lymphocytes after combination antiretroviral therapy. J Virol 1999;73:797–800. [9] Garboczi DN, Hung DT, Wiley DC. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci USA 1992;89:3429–33. [10] O’Callaghan CA, Byford MF, Wyer JR, Willcox BE, Jakobsen BK, McMichael AJ, Bell JI. BirA enzyme: production and application in the study of membrane receptor–ligand interactions by site-specific biotinylation. Anal Biochem 1999;266:9–15. [11] Paliard X, Liu Y, Wagner R, Wolf H, Baenziger J, Walker CM. Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques. AIDS Res Hum Retroviruses 2000;16:273–82. [12] Jordan HL, Kuroda MJ, Schmitz JE, Steenbeke T, Forman MA, Letvin NL. Detection of simian immunodeficiency virus gag-specific CD8(+) T lymphocytes in semen of chronically infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J Virol 1999;73:4508–12. [13] Bousso P. Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses. Microbes Infect 2000;2:425–9. [14] Gillespie GM, Kaul R, Dong T, Yang HB, Rostron T, Bwayo JJ, Kiama P, Peto T, Plummer FA, McMichael AJ, Rowland-Jones SL. Cross-reactive cytotoxic T lymphocytes against a HIV-1 p24 epitope in slow progressors with B*57. Aids 2002;16:961–72. [15] Evans DT, O’Connor DH, Jing P, Dzuris JL, Sidney J, da Silva J, Allen TM, Horton H, Venham JE, Rudersdorf RA, Vogel T, Pauza CD, Bontrop RE, DeMars R, Sette A, Hughes AL, Watkins DI. Virus-specific cytotoxic T-lymphocyte responses select for aminoacid variation in simian immunodeficiency virus Env and Nef. Nat Med 1999;5:1270–6. [16] Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, Nelson JA, Gairin JE, Hahn BH, Oldstone MB, Shaw GM. Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med 1997;3:205–11. [17] Goulder PJ, Phillips RE, Colbert RA, McAdam S, Ogg G, Nowak MA, Giangrande P, Luzzi G, Morgan B, Edwards A, McMichael AJ, Rowland-Jones S. Late escape from an immunodominant cytotoxic T-lymphocyte response associated with progression to AIDS. Nat Med 1997;3:212–7.

R. Wu et al. / Immunology Letters 98 (2005) 73–81 [18] Betts MR, Krowka J, Santamaria C, Balsamo K, Gao F, Mulundu G, Luo C, N’Gandu N, Sheppard H, Hahn BH, Allen S, Frelinger JA. Cross-clade human immunodeficiency virus (HIV)-specific cytotoxic T-lymphocyte responses in HIV-infected Zambians. J Virol 1997;71:8908–11. [19] Betts MR, Krowka JF, Kepler TB, Davidian M, Christopherson C, Kwok S, Louie L, Eron J, Sheppard H, Frelinger JA. Human immunodeficiency virus type 1-specific cytotoxic T lymphocyte activity is inversely correlated with HIV type 1 viral load in HIV type 1-infected long-term survivors. AIDS Res Hum Retroviruses 1999;15:1219–28. [20] Rowland-Jones SL, Dong T, Fowke KR, Kimani J, Krausa P, Newell H, Blanchard T, Ariyoshi K, Oyugi J, Ngugi E, Bwayo J, MacDonald KS, McMichael AJ, Plummer FA. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J Clin Invest 1998;102:1758–65. [21] Peter JB, Blum RA. Monitoring HIV viral loads in the United States: recent trends and methodologies. J Acquir Immune Defic Syndr 2002;30:261–2. [22] Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon MA, Lifton MA, Racz P, Tenner-Racz K, Dalesandro M, Scallon BJ, Ghrayeb J, Forman MA, Montefiori DC, Rieber EP, Letvin NL, Reimann KA. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 1999;283:857–60. [23] Jin X, Bauer DE, Tuttleton SE, Lewin S, Gettie A, Blanchard J, Irwin CE, Safrit JT, Mittler J, Weinberger L, Kostrikis LG, Zhang L, Perelson AS, Ho DD. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 1999;189:991–8. [24] Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, Hurley A, Markowitz M, Ho DD, Nixon DF, McMichael AJ. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998;279:2103–6. [25] Altman DJ, Moss PA, Goulder PJ, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996;274:94–6. [26] Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman JD, Ahmed R. Viral immune evasion due to persistence of activated T cells without effector function. J Exp Med 1998;188:2205–13. [27] Brander C, Pichler WJ, Corradin G. Identification of HIV proteinderived cytotoxic T lymphocyte (CTL) epitopes for their possible use as synthetic vaccine. Clin Exp Immunol 1995;101:107–13.

81

[28] Klein MR, van Baalen CA, Holwerda AM, Kerkhof Garde SR, Bende RJ, Keet IP, Eeftinck-Schattenkerk JK, Osterhaus AD, Schuitemaker H, Miedema F. Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J Exp Med 1995;181:1365–72. [29] Pontesilli O, Klein MR, Kerkhof-Garde SR, Pakker NG, de Wolf F, Schuitemaker H, Miedema F. Longitudinal analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocyte responses: a predominant gag-specific response is associated with nonprogressive infection. J Infect Dis 1998;178:1008–18. [30] Lee PP, Yee C, Savage PA, Fong L, Brockstedt D, Weber JS, Johnson D, Swetter S, Thompson J, Greenberg PD, Roederer M, Davis MM. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nat Med 1999;5:677–85. [31] Allen TM, O’Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, Dunphy E, Liebl ME, Emerson C, Wilson N, Kunstman KJ, Wang X, Allison DB, Hughes AL, Desrosiers RC, Altman JD, Wolinsky SM, Sette A, Watkins DI. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000;407:386–90. [32] Moss RB, Giermakowska WK, Wallace MR, Jensen FC, Maigetter RZ, Carlo DJ. Expression of perforin on HIV-1-specific CD8+ lymphocytes after immunization with a gp120-depleted, whole-killed HIV-1 immunogen. Clin Exp Immunol 2001;124:248–54. [33] Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O’Callaghan CA, Steven N, McMichael AJ, Rickinson AB. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein–Barr virus In vivo. J Exp Med 1998;187:1395–402. [34] Reddehase MJ, Koszinowski UH. Significance of herpesvirus immediate early gene expression in cellular immunity to cytomegalovirus infection. Nature 1984;312:369–71. [35] Matloubian M, Concepcion RJ, Ahmed R. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection. J Virol 1994;68:8056–63. [36] Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, Ogg GS, Spiegel HM, Conlon C, Spina CA, Havlir DV, Richman DD, Waters A, Easterbrook P, McMichael AJ, Rowland-Jones SL. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J Exp Med 2000;192:63–75. [37] Rosenberg ES, Altfeld M, Poon SH, Phillips MN, Wilkes BM, Eldridge RL, Robbins GK, D’Aquila RT, Goulder PJ, Walker BD. Immune control of HIV-1 after early treatment of acute infection. Nature 2000;407:523–6.