The mechanisms underlying the impairment of CD4 T cell responses during the course of HIV-1 infection are complex. Quantitative reduction in CD4 T-cell precursor numbers partially explains the impairment, but selective infection of CD4 subsets [1,2], altered CD4 memory phenotype [3,4], thymic dysfunction [5,6] and many other abnormalities seem to be involved. A role for antigen presenting cell dysfunction has long been postulated [7,8], with evidence both for HIV-1 infection and modulation of dendritic cells (DC) [9,10]. Decreased plasmacytoid and myeloid DC numbers in peripheral blood has been correlated with high HIV-1 viral load . The diminished responses to recall antigens are at least partially reversed during successful HAART [12,13], correlating with both a clinical improvement and a fall in HIV-1 viral load, but CD4 T-cell responses to HIV-1 itself do not usually recover spontaneously .
In this cross-sectional study, we tested the hypothesis that impaired CD4 antigen-specific ex vivo responses in HIV-1 infected individuals could be restored by stimulating the T cells with purified monocyte derived DC. We first examined the function of DC isolated from individuals at different stages of HIV-1 infection in patients who remained immunocompetent, patients who were immunosuppressed before starting treatment, and those on treatment with undetectable viral load. These DC showed normal cell surface phenotype and no impairment in ability to stimulate proliferative or cytokine response of allogeneic T cells. The function of the DC in stimulating recall antigen specific T-cell responses was then investigated. The responses to both HIV-1 p24 antigen and third party recall antigens were improved when purified DC acted as the APC. Optimization of antigen presentation may therefore be important in both vaccination and immunotherapeutic strategies for HIV-1 infected individuals.
Materials and methods
All HIV-1 infected patients and healthy volunteers who participated in this research provided written informed consent prior to venesection. The study protocols were approved by Camden and Islington Community Health Services NHS Trust Local Research Ethics Committee.
HIV-1 infected patients (n = 33) were recruited from the Bloomsbury Clinic, Mortimer Market Centre, Camden Primary Care NHS Trust, London. They had documented HIV-1 infection, which was of probable clade B (all but one were homosexual men, and the majority were Caucasian). Three groups of patients were studied. (i) An immunocompetent group (IC; six patients, two patients sampled twice): all individuals were HIV-1 antibody positive for at least 5 years, had CD4 T lymphocyte counts > 400/μl (median, 735/μl; range, 400–930/μl) and a low level of plasma HIV-1 viraemia (< 1000 copies/ml). These criteria were chosen so as to maximize immunocompetence. Three patients were long-term non-progressors, defined as antiretroviral naive individuals with asymptomatic HIV-1 infection for at least 7 years and stable CD4 T lymphocyte cell count of > 600/μl . (ii) An immunosuppressed pretreatment (IS) group (15 patients, four of them sampled twice): all individuals were HIV positive and antiretroviral naive, with CD4 T-lymphocyte cell counts between 50 and 400/μl (median, 245/μl). A lower cut-off was included to ensure that individuals had sufficient T cells to perform the cell proliferation assays. Plasma HIV levels were between 30 900 and 4 659 100 copies/ml. All individuals fulfilled the British HIV association guidelines for antiretroviral treatment . The objective was to recruit immunosuppressed individuals before they entered therapy, but recruitment of individuals with advanced HIV-1 infection was limited because of very low CD4 counts and because they often commenced HAART before it was possible to obtain a study sample. (iii) A HAART treated (T) group with undetectable virus load (12 patients, five sampled twice): all individuals had been continuously on antiretroviral therapy for at least 18 months previously, and had an HIV-1 viral load < 50 copies/ml (Chiron 3.0 assay, Bayer UK, Newbury, UK). As a significant proportion of individuals in clinical practice fail to effectively control virus in response to HAART, this group are selected for good response to therapy. CD4 counts were varied (median, 380/μl; range, 200–900/μl) although generally higher than in the IS group, and lower than in the IC group. Healthy volunteers (n = 16) were recruited from staff and students in the Department of Virology and the Department of Immunology and Molecular Pathology, UCL, London.
All participants in this study had either been vaccinated with Bacillus Calmette–Guerin vaccination or had a documented history of tuberculosis. All participants had also completed a course of tetanus toxoid (TT) vaccination. The median age and range (in years) at the time of study entry were IC: 46 (31–49), IS: 38 (21–56), T: 41 (32–59) and healthy volunteers 34 (24–59).
All tissue culture media and sera were obtained from GibcoBRL Limited (Paisley, UK) unless otherwise stated.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMC) were isolated as described previously .
Generation of DC from PBMC
The protocols for DC culture followed established granulocyte macrophage–colony stimulating factor (GM–CSF)/ interleukin (IL)-4 methods, with some modifications which were found to give lower background in proliferation assays . Specifically, DC were cultured in AIM-V media, a serum free medium containing human serum albumin. AIM-V medium was supplemented with 0.075% sodium bicarbonate, 50 μM 2-β-mercaptoethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2.4 mM L-glutamine (combined medium was denoted as AIM-V CM). The initial 2.5-h adherence step for DC isolation was carried out in RPMI with 5% v/v foetal calf serum and the resultant monocytes cultured in AIM-V CM. All subsequent experimental steps were performed in AIM-V CM. The non-adherent cells were stored in liquid nitrogen for future T cell isolation. On day 7 of culture, DC were purified by negative depletion using monoclonal antibodies directed against CD2, CD3 and CD19 .
Purification of T cells
T cells were retrieved from the non-adherent cells stored in liquid nitrogen and purified by magnetic bead immunodepletion using monoclonal antibodies directed against HLA-DR, CD14 and CD19 . Purity was > 98% CD3.
Immunophenotyping of DC
Day 7 purified DC were phenotyped using phycoerythrin-conjugated antibodies with a panel of antibodies as described previously . Flow cytometry was performed on a Becton Dickinson (Mountain View, California, USA) fluorescence activated cell analyser (FACScan) using Cellquest software. The log10 median fluorescent intensity (MFI) was linearized using the formula: linearized MFI = anti-log [log MFI]/254 in order to enable statistical analysis.
Cell proliferation assays
Cultures were generally performed in triplicate, in 200-μl flat-bottomed wells. Cell proliferation was determined by thymidine incorporation as described . To inactivate residual virus, 1% Nonidet P40 was added to all wells prior to harvesting (modified from ) and counted using standard methods . The threshold for a positive cell proliferation result was defined as a cell proliferation result that was greater than the sum of the mean thymidine incorporation for the highest background counts within the same assay twice the SE of the mean for the background counts.
Cultures were in 2-ml rather than 200-μl volume. PBMC were used at 106/ml. For the DC/T cell assays 105/ml DC and 1 × 106/ml T cells were used. Supernatants were collected on day 6, inactivated with 1% Nonidet P40, aliquoted and frozen in liquid nitrogen until needed.
Mixed leukocyte responses (MLR)
DC were generated from the study individuals as described above. Allogeneic T cells were obtained from four healthy volunteers not participating in this study. The maximal DC number per well was 104 and seven 1: 2 dilutions were made. Allogeneic T cells (105) were added to each well. Controls included DC alone and T cells alone. The cells were pulsed on day 6 and harvested 16–18 h later. Alternatively, supernatants were collected on day 6 and stored in liquid nitrogen for use in the cytokine assays. In some cultures, the protease inhibitor saquinavir (Roche Diagnostics, Welwyn Garden City, UK) was added to the in vitro culture system to a final concentration of 625 nM as recommended by the manufacturer for media containing human albumin.
Antigen specific autologous T-cell responses
The PBMC antigen specific proliferation assays were set up using 105 and 2 × 105 cells/well. The DC/T cell autologous assays were set up using 2.5 × 103, 5 × 103 and 104 DC, and 105 T cells/well. These numbers were selected on the basis of preliminary experiments showing that PBMC and DC/T cell assays gave an equivalent range of proliferative responses to a panel of recall antigens in healthy volunteers. The maximal cell proliferation response obtained in the PBMC and DC/T cell assays for the antigen concentration tested was used for statistical analysis. Tuberculin purified protein derivative (PPD, Evans Medical Limited, Leatherhead, UK) was used at 125 U/ml and 500 U/ml. Tetanus toxoid (TT, Pasteur Merieux Connaught, France) was used at 2 μg/ml and 10 μg/ml. HIV-1 p24 antigen (EVA620 HIV-1IIIB gag p24; Centralised Facility for AIDS Reagents, NISBC, Potters Bar, Herts, UK) was used at 2 μg/ml and 10 μg/ml. The assays were pulsed on day 6.
For cytokine responses culture supernatants were collected on day 6 and frozen in liquid nitrogen.
IL-12 p40 and interferon (IFN)-γ were measured using kits purchased from E Bioscience, or Pharmingen. The protocol was performed according to the manufacturer's instructions.
p24 levels in cultures were measured using an ELISA .
A sample size of 15 individuals in each group was chosen as a target to provide 80% power for comparisons between any two groups, based on simulating data and applying the Mann–Whiney test, assuming a difference in the distribution of responses felt important. We were unable, however, to recruit sufficient individuals meeting the inclusion criteria for IC or T groups within the timescale of the study. The magnitudes of a response observed in each of two study groups were compared using the Mann–Whitney Test. The responses in the PBMC versus the DC/T cell assays were compared using Wilcoxon matched-pairs signed rank test. In the figures results of all individual assays are shown, but for testing of difference the two values obtained from some patients were averaged, because of likely correlation between such samples. Correlation coefficients presented are Pearson correlations. Two-tailed statistical testing is used throughout, and a 5% significance level.
Antigen specific CD4 responses are impaired in IS and T HIV infected individuals
Conventional PBMC proliferative T-cell responses to HIV p24, to TT and PPD are shown in Fig. 1. A HIV-1 p24 antigen response was seen in almost all members of the IC group. In contrast, only one of 14 cultures from the IS group responded to HIV-1 p24 antigen (10 μg), and the median was significantly lower (P < 0.01) than the IC group in agreement with several previous studies . A response to HIV-1 p24 antigen (10 μg) was seen in about one-third of the individuals in the T group, but the median value was still lower than for the IC group (P < 0.01).
The PBMC recall response to both PPD and TT in the IS group demonstrated a lower median proliferation compared to the healthy volunteers (P < 0.01) or compared to the IC group (P < 0.02) (Fig. 1). PBMC responses in the T group were intermediate, remaining below the controls (P < 0.01), but above the IS cohort (P < 0.04). As reported previously, therefore, the fall in viral load following antiretroviral therapy correlates with a partial restoration of immunity. The overall hierarchy in antigen specific responses (healthy volunteers ≥ IC > T > IS) in the three groups of HIV infected individuals and healthy volunteers therefore reflect their clinical status [12,13] and validate the selection criteria on which this study was based.
DC from HIV-1 infected individuals show normal phenotype
DC were successfully isolated from all individuals participating in the study with no differences in yield between study groups. DC were > 95% pure, and had the characteristic cell surface phenotype shown in Fig. 2a. HIV-1 replication in the DC cultures was not measured. No evidence of syncitia formation was seen in any DC culture, but the possibility that a proportion of DC from HIV-infected individuals were infected with virus cannot be excluded. The expression of a panel of phenotypic markers was examined (Fig. 2b). Some markers (especially CD1a and CD40) varied widely between individuals, but no significant differences were seen between the four groups.
DC from HIV-1 infected individuals show normal function in allogeneic MLR
The functional ability of the DC was assessed in allogeneic MLR using responder T cells from healthy volunteers. Preliminary experiments showed that the MLR was abolished by depletion of CD4 T cells from the responder population (data not shown). The allogeneic MLR were performed with a range of DC stimulator numbers, and the number of DC giving maximum response (DC potency, left column), as well as the maximum T-cell proliferation observed (right column) was determined for each individual. The frequency distribution of both these parameters in each group are shown in Fig. 3a. DC and responder T cells were not HLA typed, so the degree of mismatch between stimulator and responder cells was not known. DC from the majority of individuals in all groups stimulated an allogeneic proliferative response, but the magnitude was very variable. In some cases, including all the individuals who showed no or low MLR response in the first assay, DC were tested a second time against T cells isolated from a second unrelated donor (e.g. Fig. 3b). All individuals in all groups responded to at least one set of T cells tested. There was no significant difference in proliferation between any of the groups (P > 0.1 for all comparisons). There was also no significant correlation between maximum proliferation and HIV-1 load in patients from the IS group (Pearson correlation = −0.21; P > 0.05).
The ability of the DC to stimulate a TH1-type T-cell response was investigated by measuring levels of IFNγ and IL-12p40 in the supernatants of allogeneic MLR cultures (Fig. 3c). There was no significant difference in the levels of cytokine between the four experimental groups, and no association between cytokine levels and HIV load within the IS group.
HIV replication in DC cultures may cause DC activation or influence the MLR directly . MLR reactions using DC from the IS group were therefore carried out in the presence of the HIV proteinase inhibitor saquinavir. No differences were observed in the presence or absence of saquinavir (data not shown).
DC stimulation of autologous antigen specific responses
Having established that DC from all groups were functionally competent to stimulate allogeneic uninfected T cells, we examined if DC could ‘correct’ the impaired responses illustrated in Fig. 1. We first examined the autologous CD4 response to HIV p24, as there is increasing evidence that this plays a key role in determining disease progression. The number of responders (left panels) and the maximum proliferation obtained for each individual (right panels) is shown in Fig. 4. The presence of DC resulted in a marked increase in the number of individuals responding to p24 in the IS group, from < 10% to over 50% (IS, left panel). The median HIV-1 p24 antigen (10 μg) responses in this group (Fig. 4a) was also enhanced compared to PBMC although this did not quite reach 5% significance (P = 0.08; P = 0.04 if duplicate measurements were included). In contrast, the response to HIV-1 p24 antigen in the IC and T groups was not greatly different in the PBMC assay and the DC/T cell assays. The full DC dose responses for six responders in the IC and IS groups are shown in Fig. 4b. The qualitative pattern of response was different between the two groups. In particular, the responses seen in the IS group were much more strongly dependent on the number of DC added to the cultures. No responders were identified in either IS or T group, using either PBMC or DC, using 2 μg/ml p24. The responses obtained from IS samples therefore required higher numbers of DC and higher antigen concentration than those from the IC group.
An additional blood sample was obtained from a subgroup of individuals (four from the IC and six from the IS group who remained within the criteria for inclusion in their respective groups and gave consent) and we were able to investigate the release of IFNγ, a major TH1 effector cytokine, present in the culture supernatants (Fig. 4c) in the presence of p24 antigen (10 μg/ml). The qualitative pattern of IFNγ response was similar to that seen for proliferation, although numbers were too small for statistical analysis.
As for p24 responses, DC increased the proportion of responders to both PPD and TT, although the effects were less striking (Fig. 5). Enhanced median proliferation (comparing DC to PBMC) was consistently observed in the IS group, using both TT and PPD. This reached statistical significance for TT at the higher antigen dose (P < 0.01). The stimulating effects of DC were quite selective for this IS group, and had little consistent effect on the antigen specific responses in any of the other groups.
The presence of replicative HIV-1 virus was a possible cause for functional impairment in the antigen-stimulated cultures from the IS groups. Low levels (< 400 pg/ml) of p24 were indeed detected in supernatants from DC/T cell cultures from this group in the presence of TT (4/6 supernatants with p24 > 50 pg/ml) and PPD (5/6 supernatants with p24 > 50pg/ml). However, the lower responses in PBMC cultures could not be due to higher viral replication, as the median p24 levels in PBMC cultures were lower than in DC/T cell cultures (59 versus 101 pg/ml for TT; and 71 versus 98 for PPD). Furthermore, there was no obvious correlation between proliferative response and HIV-1 p24 antigen release (data not shown).
Many previous studies have examined CD4 T-cell antigen-specific responses at various stages of HIV-1 infection (reviewed in [13,21]). In general, responses to third-party recall antigens (e.g. PPD, and TT) are impaired later in the progression of HIV-1 infection, while HIV-specific T-cell responses (at least in terms of proliferation) can be detected in the majority of early asymptomatic individuals [22,23] but disappear rapidly well before significant decreases in CD4 T-cell counts or increase in HIV-1 viral load measurements. This decrease correlates with disease outcome . Responses are at least partially restored following successful control of HIV-1 viraemia by antiretroviral chemotherapy [12,25]. HIV-specific responses, however, remain very weak or absent, long after virus has become undetectable .
Most published studies measure responses using PBMC cultures, in which the antigen presenting cell component is often poorly defined. In this study, in contrast, we measure T-cell responses using a purified well characterized population of DC to present the test antigens. The functional characteristics of DC isolated from HIV-1 infected individuals have received quite a lot of attention [10,26,27]. However, few previous studies have examined autologous antigen-specific responses using DC. Furthermore, previous studies have not compared the functional characteristics of DC isolated from well characterized groups of patients at different stages of HIV infection.
Both phenotype (especially of CD1a and CD40) and allostimulatory potential (measured as proliferative, IFNγ and IL-12 responses) varied widely, in both the controls and the HIV-1 infected groups. Overall, the DC cultures from all groups tested behaved indistinguishably, with normal phenotype, alloproliferative and cytokine responses, and with no correlation to HIV-1 viral load or CD4 T cell numbers. The variability in phenotype and function observed within groups may obscure subtle differences between patient groups. However, the results are in agreement with two previous studies showing normal allostimulation by monocyte derived DC from HIV infected individuals [10,26]. In contrast, a recent study found profound impairment in allogeneic as well as autologous responses using monocyte derived DC from HIV infected individuals . The reasons for this discrepancy remains obscure, although findings must be interpreted with caution, in the light of the very significant natural variation in DC function and phenotype we have observed.
Several studies [11,28–31] have reported changes in numbers and phenotype of various DC subsets when isolated directly ex vivo, and these cells may also have altered immunostimulatory function [32,33]. It is important to emphasise, therefore, that our studies have focused exclusively on DC generated from monocytes cultured in GM–CSF and IL-4 and the in vitro culture period may favour the emergence of ‘normal’ DC. The ability to generate functionally intact DC from HIV infected individuals is nevertheless important in the context of attempts to use autologous DC-based vaccines for HIV therapy .
We tested the DC for their ability to present recall antigens to autologous T cells, and compared this to the standard PBMC assays. DC did not enhance responses to any of the antigens tested in either IC HIV-infected individuals or controls (healthy volunteers), consistent with the ability of other cell types (monocytes, B cells etc.) to present to previously primed cells. However, DC enhanced antigen specific proliferative responses in the IS group, namely in that group in which responses in PBMC showed the most striking deficit. In particular, DC stimulated significant HIV-1 p24 responses in over half the IS group, in which PBMC HIV-1 p24 responses were very rare. Indeed this enhancement may be an underestimate, as the current study was performed using ‘immature’ DC (since these are believed to show the best combination of uptake, processing and presentation). Future studies using DC ‘matured’ by various stimuli after antigen uptake may reveal even more ‘hidden’ T-cell responses.
The observations reported above reflect the special efficiency in antigen presenting activity of DC. The results are in agreement with a study which demonstrated HIV-specific responses in the majority of infected individuals, by including anti-CD28 and anti-CD49d antibodies in the cultures to provide the strongest possible co-stimulation of T cells . Massive loss of antigen specific memory T cells is known to occur very early in infection , and the nature of the residual T cells which are being stimulated by DC in this study therefore requires careful inspection. The requirements for DC or exogenous co-stimulation are suggestive that the residual T cell repertoire is of ‘resting’ or ‘central’ memory phenotype. Although these cells are precisely those carrying the highest viral load , their low activation level may paradoxically help them to survive in vivo despite their high viral load. Further phenotypic and functional studies will be necessary to assess this possibility.
Irrespective of the mechanism, the stimulation of T-cell responses in HIV-infected individuals revealed by DC has important implications in terms of immunotherapy of HIV infection. In particular, immunisation strategies that target DC may therefore offer significant advantages in the ability to stimulate HIV-specific protective immune responses.
We thank the following: the HIV-1 positive patients and healthy volunteers who participated in this study, D. Aldam and D. Cornforth, J. Dodds and M. Moreno (The Mortimer Market Centre), for identifying potential study patients, P. Balfe (Centre for Virology, UCL) for assistance in adapting the experimental protocols for use in category III containment facilities, U. Ayliffe and S. Rice (Centre for Virology,UCL), for continuing assistance and advice regarding safety aspects related to the category III containment facility, U. Ayliffe, S. Rice, S. Kaye, P. Balfe, C. Perrons, J. Garson and K. Ward (Centre for Virology, UCL) for acting as keyholders for the category III containment facility, A. McKnight and S. Neil (Wohl Virion Centre, UCL). for assisting with HIV-1 p24 Ag ELISA and providing associated consumables, Harvey Holmes (Centralised Facility for AIDS Reagents, NIBSC, Potters Bar, Herts, UK) for providing HIV-1 p24 Ag and C. Craig (Roche Discovery, Welwyn Garden City, UK) for gifting the saquinavir.
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