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HIV-Specific Cytotoxic Cell Frequencies Measured Directly Ex Vivo by the Lysispot Assay Can Be Higher or Lower Than the Frequencies of IFN--Secreting Cells: Anti-HIV Cytotoxicity Is Not Generally Impaired Relative to Other Chronic Virus Responses¹

Jennifer E. Snyder-Cappione^*, Anagha A. Divekar^*, Genny M. Maupin^*, Xia Jin, Lisa M. Demeter and Tim R. Mosmann^2,*

^* David H. Smith Center for Vaccine Biology and Immunology, and Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642; and Infectious Diseases Unit, Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD8⁺ T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8⁺ T cells, as measured by IFN- secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN--secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN--secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-. These results indicate that measurement of IFN- production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN- ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of "preterminal differentiation" of HIV-specific CD8⁺ T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.

	Introduction

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The CD8⁺ T cell is believed to be an integral component of effective anti-lentivirus immune responses. Depletion of CD8⁺ cells from macaques results in a dramatic increase of SIV viral load, either during acute (1) or chronic (2) (3) infection. In humans, CD8 epitope escape mutants emerge during the course of HIV infection within one individual, and the original epitopes can re-emerge on transfer of the infection to a new individual (4). These data suggest that effective CD8 immune responses exert selective pressure on the virus (5, 6). Higher frequencies of HIV-specific CD8⁺ T cells, as measured by cytotoxic cell precursor frequencies, are associated with decreased levels of viremia during acute infection (7).

Because of the antiviral and immunopotentiating activities of IFN-, several studies have examined whether IFN- synthesis is a major determinant of the effectiveness of CD8⁺ T cells against HIV. The magnitude of IFN- CD8⁺ T cell responses to the Gag and p24 HIV proteins in chronically infected patients has been reported to correlate inversely with viral loads and directly with absolute CD4⁺ T cell counts (8). However, others have reported that the frequencies of HIV-specific IFN--secreting cells correlate positively with viral loads (9, 10, 11), or that measures of disease progression do not correlate with the magnitude of HIV-specific IFN- responses (12, 13, 14). Thus, the secretion of IFN- ex vivo may not reflect the effector function of CD8⁺ T cells that contributes most strongly to the containment of HIV during chronic infection.

Cytotoxicity, which is important in clearing many viral infections (15, 16, 17, 18), may be a more significant effector function of HIV-specific CD8⁺ T cells in controlling HIV pathogenesis. Quantifying IFN--secreting CD8⁺ T cells does not provide an estimate of cytotoxic potential, as these two functions can be independently exerted by individual CD8⁺ T cells (19). Populations of virus-specific CD8⁺ T cells vary in their ability to secrete IFN- and/or lyse target cells within 5 h ex vivo (20) or after in vitro stimulation (21).

Results from previous studies measuring ex vivo cytolytic function during HIV infection are conflicting. HIV-specific CD8⁺ T cells mediating ex vivo cytotoxicity have been reported in acute (22) or chronically infected patients (23, 24, 25, 26, 27, 28), whereas others report that ex vivo CTL activity is not detectable (29, 30). When HIV-specific cytotoxic cells have been detected ex vivo, it has been suggested that they have selectively impaired cytotoxicity compared with CD8 responses specific for other chronic infections within the same patient (27). The above studies measured ex vivo cytolysis using either bulk lysis assays (measuring the cytotoxicity of whole populations), such as the chromium release assay (31), or by measuring changes in the caspase activation of target cells undergoing lysis (32).

To help resolve this diverse information, and to directly compare the frequencies of IFN--secreting cells and cytolytic cells specific for the same peptide pools, we have used the Lysispot assay (19) to measure the frequencies of cytotoxic cells in HIV-infected patients at different stages of chronic infection. In contrast to previous results using bulk cytotoxicity assays, simultaneous measurement of cytotoxicity by Lysispot and IFN- in a short-term ELISPOT assay allowed us to directly compare the cytotoxic and IFN--secreting cell frequencies ex vivo. Of the nine HIV⁺ patients analyzed, eight contained ex vivo cytotoxic cells specific for at least one of the Env, Gag, Nef, or Pol peptide pools tested. The ratio of IFN--secreting to cytotoxic cells was variable between different HIV peptide pools, even within individual patients. This emphasizes the importance of measuring other effector functions in addition to IFN- secretion to assess the overall frequencies of HIV-specific T cells. The IFN- to cytotoxicity ratio for HIV could be higher or lower than the corresponding ratios for EBV or CMV responses in the same individuals. Thus, in contrast to previous reports (27), we did not observe selectively reduced ex vivo cytotoxicity specific for HIV relative to other chronic viral infections.

	Materials and Methods

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Study individuals

PBMC were analyzed from nine HIV-1-infected and six healthy volunteers who were recruited at the University of Rochester Medical Center. All samples were obtained according to protocols approved by the Research Subjects Review Board of the University of Rochester Medical Center, and informed consent was obtained from all study individuals.

HIV peptides

Overlapping 15-mer peptide sets spanning the entire Gag, Pol, Nef, and Env consensus sequences of HIV-1 subtype B were obtained from the National Institutes of Health AIDS Research and Reference Program (Rockville, MD). Each peptide overlapped the next by 11 aa. Peptides were pooled into 11 Env, 5 Gag, 8 Pol, and 2 Nef pools and used at a concentration of 1 µg/ml for each peptide as Ags in the IFN- ELISPOT and Lysispot assays. Although these peptides could potentially elicit both CD4⁺ and CD8⁺ T cell responses, intracellular cytokine staining of four HIV-infected patients showed that the responses were mostly due to CD8⁺ T cells (data not shown), consistent with findings that CD8⁺ cells were responsible for >95% of the T cell responses to similar overlapping HIV peptides (13).

CMV and EBV peptides

Six immunodominant CMV or EBV peptides (33) were selected to measure anti-CMV and anti-EBV responses. Because these peptides are presented by common HLA alleles, a high percentage of subjects should respond to at least one of these peptides. Three immunodominant CMV peptides were pooled: NLVPMVATV (from pp65, HLA-A2-restricted), TPRVTGGGAM (HLA-B7-restricted), and EFFWDANDIY (HLA-B44-restricted). Three immunodominant EBV peptides were pooled: RAKFKQLL (HLA-B8-restricted), RPPIFIRRL (HLA-B7-restricted), and GLCTLVAML (HLA-A2-restricted). These peptides were used at a concentration of 1 µg/ml for each peptide in the ELISPOT and Lysispot assays.

Human lymphocyte/target cell preparation

Blood samples were drawn into heparinized tubes. PBMC were separated by Ficoll-Hypaque and frozen in FBS containing 10% DMSO at a concentration of 1–10 x 10⁶/ml and stored in liquid nitrogen until use. B cell lines were grown from each subject by stimulation with CD40L and IL-4 (34) to be used as targets in the spot assays. These were also stored in liquid nitrogen until use.

Generation of lymphokine-activated killer (LAK)³ cells

In each experiment, HSV-Lac transfected autologous B cells were incubated with LAK cells to ensure that the target B cells could generate Lysispots when killed by cytotoxic cells. LAK cells were generated by adding 5 x 10⁶ PBMC to individual wells of a 24-well plate in 2 ml of complete medium (RPMI 1640 with 8% FBS) with 5 ng/ml IL-2 for a minimum of 3 days before use.

Human Lysispot and IFN- ELISPOT assays

ELISPOT plates (Millipore no. S2ER097M8 or MAIPN4550) were coated with either anti-IFN- Ab (5 µg/ml, 50 µl/well in PBS; Mabtech no. mAb 1-D1K) or anti--galactosidase Ab (diluted 1/1000, 50 µl/well in PBS; Sigma-Aldrich G-8021). Plates were coated for a minimum of 1 h at room temperature, and washed twice with PBS before cells were added. Target cells were added to non-tissue culture-treated dishes and transfected with the HSV-Lac amplicon (35) at a multiplicity of infection of five. After 1 h at 37°C, cells were removed and added to a 15-ml conical tube and washed twice with complete medium. B cells were resuspended at a concentration of 1 x 10⁶/ml, pulsed with peptides for 90 min and then washed twice with warm complete medium. Typically 15–30,000 APCs, and 7 x 10⁴ to 5.3 x 10⁵ PBMCs were added per well. The total volume in each well was 100 µl. The plates were incubated at 37°C for 3–5 h (the IFN- and Lysispot assays performed for each patient were cultured for identical amounts of time). The frequencies of IFN--producing cells for each peptide pool in a 40-h assay (see below), without additional APC, were similar to the frequencies obtained in the shorter assay using the peptide-pulsed autologous B cells as APCs (data not shown). Thirty minutes before the end of the assay, biotinylated anti--gal (diluted 1/1000; Sigma-Aldrich no. B-0271) or biotinylated anti-IFN- (1 µg/ml; Mabtech no. mAb7-B6-1) Abs were added in complete medium as an additional 50 µl/well. At the end of the assay, plates were washed three times with PBST, and streptavidin-alkaline phosphatase diluted 1/1000 in PBSTB was added overnight at 4°C. Plates were again washed with PBST, and substrate mix (Vector Laboratories catalog no. SK5300) was added. The plates were rinsed with tap water when spots were clearly visible. An aliquot of the transfected APCs were cultured separately for the duration of the Lysispot assay. These cells were then fixed and stained with a X-gal substrate to determine the transfection frequency, which was consistently >95%.

Spot counting

All ELISPOT and Lysispot plates were counted using the ImmunoSpot Series 1 Analyzer (Cellular Technology). The operator was blinded to the subject characteristics of the samples throughout experimentation and data analysis. Fluorispots were counted manually.

Validation of the Lysispot assay

We have previously described the validation of the Lysispot assay for enumerating individual CTLs (19). Briefly, CD8⁺ T cells normally kill one target each during the 4-h Lysispot assay (note that migration is required to find each target), and the number of spots is a valid estimate of the number of cytotoxic cells. Due to the relatively small numbers of spots in each well for most subjects in our current study, the spots were well-separated, and clustering of spots within the migration radius of a single CD8⁺ T cell would have been apparent. This did not occur, confirming that the spot counts were an accurate measure of the cytotoxic T cell number. Similarly, the short duration of the parallel IFN- ELISPOT assay also minimized the possibility that one cell could give multiple spots.

Calculation of Lysispot results

Three backgrounds were subtracted from the Lysispot values: 1) the spontaneous lysis of the HSV-Lac-transfected B cell targets; 2) the increased spontaneous lysis of the B cells due to pulsing with particular peptide pools; and 3) the nonpeptide-specific lysis of the B cells by the PBMCs. The third background could potentially be due to the ability of PBMC to lyse target cells due to EBV reactivity, HSV reactivity, a cytotoxic response to serum proteins, etc. To accurately deduct these nonpeptide-specific Lysispots, the frequency of peptide-specific cytolytic cells was calculated from the control Lysipsot frequencies using the equation D – (B – (C – A)), where A = unpulsed B cells, B = B cells pulsed with a particular peptide pool, C = PBMC + unpulsed B cells, D = PBMC + B cells pulsed with a particular peptide pool. Samples were assayed in triplicate, except for some samples the PBMC + unpulsed B cell control wells were increased to six replicates and located on different portions of the plate to ensure changes in spot number were not due to the location of the wells.

The 40-h human IFN- Fluorispot assay

This assay was modified from the method described by Gazagne et al. (36). Cells and Ags were set up as described above for the ELISPOT, except that anti-human CD19, anti-human IL-2 (BD Biosciences), and anti-human IFN- (Mabtech) coating Abs were included together in each well. PBMC were added (2 x 10⁵) to individual ELISPOT wells coated with the anti-human IFN- Abs (Mabtech). The HIV peptide pools were also added at a concentration of 1 µg/ml. After a 40-h incubation, the Fluorispot plates were washed and developed using a biotinylated anti-human IL-2 Ab (1/100; BD Biosciences) and streptavidin-PE (1 µg/ml; BD Biosciences). IFN- spots were developed using biotinylated anti-human IFN- Ab (1 µg/ml; Mabtech) followed by an Alexa 488-conjugated streptavidin (1 µg/ml; Molecular Probes). The plates were dried and individual wells were photographed under a fluorescent microscope.

Statistical analysis

The one-tailed t test was used to compare net Lysispot frequencies between HIV-uninfected and -infected individuals. The Mann-Whitney U test was used to discern significant IFN- and cytotoxic frequencies in comparison to wells containing PBMC + B cells without peptide. The Pearson product moment correlation coefficient was used to compare the net IFN- and CTL frequencies per HIV⁺ subject with CD4 counts or viral load.

	Results

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Study subject characteristics

The individual characteristics of nine HIV-infected and six control subjects are shown (Table I). In the HIV⁺ group, the median time since HIV diagnosis was 10 (range 3–15) years. PBMC prepared from each patient were screened in an initial IFN- and IL-2 Fluorispot assay for reactivity against 26 peptide pools comprising overlapping peptide sets for Env, Gag, Pol, and Nef proteins. Strong IFN- reactivity against one or more HIV peptide pools was detected in all of the HIV-infected subjects (data not shown). IL-2 responses were low or undetectable, and were not analyzed further.

To conserve PBMC and autologous B cell targets during the analysis of cytotoxicity, a limited set of peptide pools was then analyzed in simultaneous IFN- ELISPOT and Lysispot assays using autologous B cells from each patient pulsed with the selected peptide pools. For subjects responding in the preliminary screen, the majority of selected pools included those that induced significant IFN- responses from that subject. Subjects with no significant IFN- responses to any HIV peptide pools (HIV uninfected subjects) were stimulated with peptide pools selected from the pools that stimulated significant cytotoxic cell frequencies from responding subjects. Although the Lysispot has similar sensitivity to traditional bulk cytotoxicity assays for detecting cytotoxic populations (19), this assay has the advantage that it measures the frequency of lytic cells, allowing direct comparison with the frequency of IFN--secreting cells.

To accurately measure the frequencies of peptide-specific cytolytic cells ex vivo using the Lysispot assay, three potential background contributions were considered: the spontaneous lysis of the HSV-Lac transfected B cell targets; increased spontaneous lysis of the B cells due to pulsing with particular peptide pools; and nonpeptide-specific lysis of the B cells by the PBMCs. These backgrounds were each subtracted (see Materials and Methods) from the total Lysispot number in the wells with PBMC and target cells pulsed with peptides.

Lysispot assays were performed directly ex vivo on PBMC from nine HIV-infected and six uninfected individuals. Fig. 1 (top panel) shows an example of PBMC from an uninfected individual in whom no HIV peptide-specific Lysispot responses were elevated significantly above the background (unpulsed B cell control). In contrast, PBMC from an HIV-infected patient contained statistically significant frequencies of cytotoxic cells when stimulated with the Pol 3 peptide pool (Fig. 1, middle panel). This patient also showed statistically significant IFN- responses against the Pol 3 peptide pool, as well as two other HIV peptide pools (Fig. 1, bottom panel). Comparing the results obtained from all the peptide pools tested in all subjects by the one-tailed t test, there was a highly significant increase in HIV-specific cytotoxicity in the HIV-infected group vs the uninfected group (p < 0.007).

View larger version (18K): [in this window] [in a new window]   FIGURE 1. The frequencies of HIV-specific cytotoxic cells can be measured directly ex vivo from chronically infected subjects using the Lysispot assay. Frequencies of cytotoxic and/or IFN--secreting cells were measured by Lysispot and ELISPOT assays from the PBMC of one HIV– subject (PT4, upper panel) and one HIV+ subject (PT11, middle and lower panels). Background responses were subtracted as described in Materials and Methods. Statistically significant responses (p < 0.05) are denoted with an asterisk (*).

Within an individual, the ratios of cytotoxic to IFN--secreting cells vary between different HIV peptide pools

Of the nine HIV-infected patients tested, all except PT12 yielded HIV peptide-specific ex vivo cytotoxic cell frequencies significantly higher than the control wells without peptide (Fig. 2). In most cases, more than one peptide pool induced significant cytotoxic cell frequencies for each patient. The ratios of cytotoxic to IFN--producing cells varied widely between different peptide pools, even within the same patient. These ratios ranged from peptide pools inducing cytotoxic cell frequencies in the absence of significant IFN- secretion, to others showing significant IFN- secretion without cytotoxicity (Fig. 2). Thus, the frequencies of HIV-specific T cell responses with distinct qualitative properties can differ dramatically within individual patients.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. The frequencies of IFN--secreting to cytotoxic cells vary among HIV-specific T cell responses within individual patients. PBMC from chronically HIV-infected subjects were assayed in simultaneous IFN- and Lysispot assays containing peptides to HIV peptide pools. The net frequencies of cytotoxic and IFN--secreting cells in response to the HIV viral peptide pools were calculated. Statistically significant responses are denoted by shading in the upper left portion of the symbols (IFN-) or the lower right portion (cytotoxic cells). Each panel represents one patient.

Some HIV-specific CTL can kill peptide-loaded targets directly ex vivo without secreting IFN-

Overall, cytotoxicity was detected in 22 of 61 HIV peptide pool/subject combinations that had been chosen on the basis of a significant IFN- response in the initial screen. Surprisingly, statistically significant cytotoxic responses were also detected in two subjects (PT2 and PT13) against peptide pools that were initially chosen as negative for IFN- production (as confirmed in the second analysis, Fig. 2, arrows). As only eight of the IFN--negative pools were selected for the cytotoxic analysis, these two responses suggest that there may be a substantial frequency of IFN-^– CTL⁺ responses. One CMV response was also detected with significant frequencies of cytotoxic but not IFN--secreting cells (PT13) (Fig. 3). These results confirm the existence of Ag-specific cells that kill target cells without releasing IFN-, and suggest that measuring only IFN- production may significantly underestimate both the magnitude and the epitope specificity of the immune response to chronic infections.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. The ratio of cytotoxic to IFN--secreting cells is not impaired in HIV responses compared with CMV and/or EBV responses within individual patients. PBMC from chronically HIV-infected subjects were assayed in simultaneous IFN- and Lysispot assays for reactivity against HIV, CMV, and EBV peptide pools. Statistically significant CMV or EBV responses are denoted by shading in the upper left portion of the symbols (IFN-) or the lower right portion (cytotoxic cells). HIV responses (small triangles, results from Fig. 2) are shown for comparison. Each panel represents one patient. Note that the cytotoxicity scales are different between the left and right panels. The individual HIV peptide pools are denoted as follows: Gag 3 = g3, Nef 2 = n2, etc.

The cytotoxic:IFN- ratios of HIV-specific T cells are not lower than the ratios for CMV and EBV responses within the same subject

To compare the functions of HIV-specific cytotoxic cells with cytotoxic cells specific for other chronic infections, responses to CMV and EBV peptide pools, each containing three immunodominant CD8 epitopes, were measured in several of the HIV⁺ patients. Both the magnitude of the cytotoxic response and the CTL:IFN- ratio for individual peptide pools varied among the responses to the different chronic viral infections. HIV-specific CTL:IFN- ratios could be similar to (e.g., subject PT10) or higher than (e.g., subjects PT2, PT8) responses against one or both of the other two chronic viral infections (Fig. 3). These results demonstrate that the cytotoxic function of HIV-specific T cells is not generally impaired compared with other chronic infections within the same subject. There was no significant correlation between the ratio of IFN- to cytotoxic cells and the viral protein from which the peptide pools were derived (data not shown).

Correlation between the frequencies of ex vivo HIV-specific cytotoxic cells and parameters of disease progression

Two important parameters describing the clinical status of an HIV-infected patient are viral load and CD4⁺ T cell counts. We compared the frequencies of ex vivo HIV-specific Lysispots with these parameters to determine whether the frequencies of circulating cytotoxic cells correlate with these parameters of disease progression (Fig. 4). Although not significant, there was a trend toward significance suggesting that the frequencies of HIV-specific cytotoxic cells may correlate positively with viral load (p = 0.10), and inversely with CD4 counts (p = 0.16), whereas the total IFN--secreting cell frequencies did not correlate with either parameter (p = 0.93 and p = 0.90).

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Association between the frequencies of cytotoxic cells with parameters of HIV disease progression. The total frequencies (the sum of responses against all peptides tested) of IFN--secreting and cytotoxic cells measured from each HIV-infected subject were compared with viral load (copies per milliliter of plasma) and CD4 T cell counts (cells per microliter of blood). A, IFN--secreting cells vs CD4 T cells, r = 0.05, p = 0.90; B, cytotoxic cells vs CD4 T cells, r = –0.51, p = 0.16; C, IFN--secreting cells vs viral loads, r = –0.04, p = 0.93; D, cytotoxic cells vs viral loads, r = +0.58, p = 0.10.

	Discussion

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Our data indicate, in agreement with a previous study (25), that the IFN- ELISPOT is not a reliable surrogate measurement of cytotoxic T cell frequencies in HIV-infected individuals. Measurement of IFN- to evaluate HIV-specific T cell immune responses may be misleading in two ways. First, many CD8⁺ T cells can respond to Ag by secreting IFN- without lysing the target cells. Second, some CD8⁺ T cells may be lytic but not secrete IFN-, as shown in our study. This is also suggested by the description of ex vivo populations of HIV-specific (37) or CMV-specific (38) T cells from the PBMC of HIV-infected patients that express proteins to facilitate lysis (perforin, granzymes) yet do not secrete IFN-. Chronically infected subjects contain CD8⁺ T cells that bind HIV tetramers but do not produce IFN- ex vivo (39). Although some of these cells may simply be unresponsive, it is also possible that some may mediate lysis without producing IFN-. Such cells are particularly relevant to studies following changes in epitope recognition longitudinally in individual patients; if IFN- secretion is used as a correlate of "CTL frequencies," many may be missed.

Our findings indicate HIV-specific cytotoxic cells are readily detected directly ex vivo from the PBMC of chronically infected patients, in agreement with other reports using bulk lysis assays (23, 24, 26, 40), and in contrast to others reporting ex vivo cytolysis is impaired or not detectable (27, 29, 30). This discrepancy may be due to the clinical status of the patient groups analyzed in the different studies. For example, our data suggest that the patients with lower levels of viremia contain lower frequencies of cytotoxic cells ex vivo. Also, the Lysispot method may offer slightly higher sensitivity than the bulk lysis assays, and the FACS-based analysis of degranulation (41) may not always correlate with cytotoxicity (42).

The data obtained by our Lysispot assays do not support the model that reduced cytotoxic function contributes to a state of "altered differentiation" of HIV-specific CTL (43). We observed no consistent trend in the net frequencies of cytotoxic cells or the ratio of IFN-:CTLs between HIV-specific T cells and those specific for other chronic infections within individual patients. In particular, the HIV-specific IFN-:CTL ratio could be either higher than, or similar to, the corresponding ratios for EBV or CMV. The amount of CMV and EBV viremia is not known in these patients; therefore, any differential relationship between viral load and ex vivo CTL frequencies among these three chronic infections is unknown.

There was no obvious association between surrogates of HIV disease progression and the frequencies of ex vivo CTL specific for any one of the viral proteins tested (Gag, Nef, Env, Pol). However, it is important to note the majority of cytotoxic T cell frequencies measured was from those peptide pools that gave an initial IFN- response during the first set of spot assays; therefore, most potential peptide-specific CTL responses without IFN- secretion were not considered.

Cytotoxicity could be related to viral load in two opposite ways: first, strong cytotoxicity may reduce viral load, and second, after an effective (cytotoxic) immune response has reduced viral load, immediate cytotoxic ability may be reduced due to low Ag concentration and hence conversion of the CD8⁺ T cell response to a noncytotoxic, resting memory phenotype. The correlative trend between increased viral load and higher HIV-specific cytotoxic cell frequencies found in our study agrees with previous reports that measured target cell lysis using the ⁵¹Cr release assay (26) or granzyme A expression (44). Thus, it appears likely that when HIV is not effectively contained, the resulting rise in viral load induces a higher frequency of cytotoxic cells detectable ex vivo. Therefore, during chronic HIV infection, ex vivo cytotoxicity may indicate immune activity rather than immune containment. Longitudinal studies of changes in ex vivo cytotoxicity, or in the ratio of IFN- secretion to cytotoxicity, would help to identify the type of effector functions exerted at each stage of the infection, and may help to predict the progress of disease.

The wide variations in cytotoxic to IFN--secreting cell ratios during HIV infection emphasize the importance of measuring both parameters independently for assessing epitope recognition, effector function, and therapeutic effects. The Lysispot provides a sensitive tool for this purpose. The correlative trend between disease progression and ex vivo frequencies of cytolytic but not IFN--secreting cells suggests that direct measurement of cytotoxic cell frequencies may provide useful information about the ongoing immune response during chronic HIV infection, as well as during vaccine responses.

	Acknowledgments

 
We thank Dacheng Liu and Jason Barbour for their help with the statistical analysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
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 work was supported by Grant T32 AI49815 (to J.E.S.-C.).

² Address correspondence and reprint requests to Dr. Tim R. Mosmann, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail address: Tim_Mosmann{at}urmc.rochester.edu

³ Abbreviation used in this paper: LAK, lymphokine-activated killer.

Received for publication July 14, 2005. Accepted for publication December 6, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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