HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vβ repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-γ responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vβ repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia. HIV infection, if left untreated, leads to the progressive disruption of the immune system, the destruction of the CD4+ T cell population, and the occurrence of multiple opportunistic infections. However, a small fraction of HIV-infected individuals (less than 1%) avoid these deleterious effects by spontaneously controlling HIV replication to very low levels in the absence of antiretroviral therapy. Emerging evidence indicates that these rare patients, named HIV controllers, contain HIV through a very active T cell-mediated immune response. In this study, we found that memory CD4+ T cells from HIV controllers had the capacity to respond to minimal amounts of antigen derived from the viral protein Gag. This property was intrinsic to controller CD4+ T cells, and resulted from the expression of T cell receptors (TCRs) with high avidity for a particular Gag peptide. The presence of high avidity CD4+ T cells may explain how HIV controllers maintain the antiviral immune response in constant alert, even though the amount of virus inducing this response is minimal. Based on this study, we propose that future candidate vaccines against HIV should induce high avidity memory CD4+ T cells, to mimic the rapid and persistent antiviral response characteristic of HIV controllers. HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment Emerging evidence indicates that controllers suppress HIV replication through a very active immunological process. HIV controllers harbor effector memory CD8+ T cells capable of rapidly killing infected autologous CD4+ T cells through a cytotoxic mechanism involving the upregulation of perforin and Granzyme B One element contributing to the persistence of an active immune response may be the quality of the HIV-specific central memory (CM) compartment. CM T cells are thought to be responsible for the long-term maintenance of immune memory, due to their long half-life, high proliferative potential, and capacity to replenish the pool of effector and effector memory (EM) T cells that directly control pathogens in vitro, comparing primary CD4+ T cell lines derived from HIV controllers and efficiently treated patients with equivalent duration of infection. We found that HIV controller harbored a pool of memory CD4+ T cells able to differentiate into effector cells with high functional avidity for an immunodominant Gag epitope. This heightened sensitivity to Gag antigen could be explained by a high avidity interaction between the TCR and the peptide/MHC complex, as measured by class II tetramer binding. The capacity to mount a CD4 recall response in the presence of minimal amounts of Gag antigen may help explain how HIV controllers maintain a continuously activated antiviral response in spite of very low viremia.We have previously shown that signs of CD4+ T cell immune activation could be detected in HIV controllers who nevertheless had an intact CM CD4+ T cell compartment, with preserved IL-2 secretion capacity and efficient proliferative responses Memory CD4+ T cell responses were compared in patients who spontaneously controlled HIV replication  and in patients who achieved viral control following successful antiretroviral therapy . Patients in both groups had viral loads <40 HIV RNA copies/ml plasma. The duration of infection and the CD4+ T cell count did not differ significantly between the two groups .Ex vivo IFN-γ ELISPOT responses to the 3 Gag peptides were low, as expected for CD4 responses directed to single HIV peptides (ex vivo response to Gag293 while only 1/13 Controller responded to Gag161 (P<0.05). This finding supported the notion of a higher frequency of Gag293-specific than Gag161-specific CD4+ T cells in Controller patients. Taken together, these observations suggested that the HIV controller status may be associated with a change in the immunodominance pattern of Gag CD4 epitopes.We analyzed the properties of memory CD4+ T cell precursors by determining their capacity to generate CD4+ T cell lines specific for three immunodominant HIV-1 Gag peptides . The peppeptides . HoweverAs controls, we generated CD4+ T cell lines specific for the CMV pp65 protein. Since the immunodominance pattern of CMV responses proved more variable than that observed for HIV-1, we optimized the generation of CMV-specific CD4+ T cell lines by choosing the pp65 peptide in function of the HLA DR genotype of the patients  [31]–[33CD4+ T cell lines typically showed an initial loss of cells due to apoptosis, followed by growth due to multiplication of HIV- or CMV-specific cells. Measurement of the growth ratio at day 7 showed that viable CD4+ T cells lines from HIV controllers had a faster growth kinetics than those from treated patients, with a significant difference in response to the 3 Gag peptides but not in response to CMV peptides . Analysi6 cells = 7,716), while most cell lines from treated patients remained negative. However, the fact that cell lines from treated patients had not yet entered the exponential growth phase could account for these differences. To compare CD4+ T cell lines at equivalent growth stages, all following measurements were made at doubling time . In these conditions, IFN-γ production remained higher in the HIC group compared to the HAART group in response to the immunodominant Gag293 peptide . However, we observed that the shapes of the response curves differed, with a marked trailing end in the HIC group, suggesting the presence of a high avidity component within the responding CD4+ T cell population . In contrast, functional avidities measured for Gag263, Gag161, and CMV peptides did not differ significantly between groups. These results pointed to a particular efficiency of CD4+ T cells specific for the immunodominant Gag293 in HIV controllers. Interestingly, the functional avidity of these cells correlated with the level of the IFN-γ response measured by ELISPOT assay at high peptide dose . Thus, CD4+ T cells responses to Gag293 appeared both sensitive and potent in the group of controller patients.This analysis revealed that the functional avidity of CD4+ T cells recognizing the immunodominant Gag293 peptide was higher in HIV controllers . In the Patients were genotyped for the HLA DRB1 gene at 4 digit resolution. 6 cells = 2458, n = 6) than DRB1*0701-negative individuals . Taken together, these findings suggest that the DRB1*0701 allele may confer an increased chance to acquire a controller phenotype upon HIV infection, but that once the controller phenotype is established, the presence of the DRB1*0701 allele does not confer further benefit in terms of CD4+ T cell function.To further explore the possibility that DRB7*0701 conferred an advantage in CD4+ T cell memory function, we compared functional parameters in DRB1*0701 positive versus DRB1*0701-negative individuals included in the study. We did not detect significant differences within the HIC group in terms of growth ratio, IFN-γ response, or functional avidity of CD4+ T cells following Gag293 stimulation. Within the HAART group, DRB1*0701-positive individuals showed lower IFN-γ responses  cells for all the patients tested, confirming the antigen specificity of the cell lines (representative examples in lo) was comparably high for the 3 HIC and 3 HAART cell lines analyzed  did not show significant differences between the HIC and HAART groups. However, the shape of the binding curves differed between groups, with a persistence of detectable binding at low tetramer concentrations in the HIC group . One should note that the growth ratio of CD4+ T cell lines depended on the intrinsic proliferative capacity of specific cells but also on the frequency of these specific cells at the initiation of culture. We have previously shown that the frequency of p24 Gag-specific cells measured ex vivo by intracellular cytokine staining was approximately 3 fold higher in HIV controllers than in efficiently treated patients et al.The genetic background may play a role in conferring a better ability to mount high avidity CD4+ T cell responses. The increased frequency of HLA DRB1*0701 in the controller group could suggest a beneficial effect of this allele on the development of anti-HIV CD4 responses. However, since we did not detect an association between the presence of HLA DRB1*0701 and the level or avidity of the CD4 response within the controller group, we speculate that this allele may play a role in initially facilitating viral control, rather than in maintaining high avidity CD4+ T cells. Alternatively, HLA DRB1*0701 may be in linkage disequilibrium with a protective MHC class I allele associated with viral control. A beneficial effect of the HLA DRB1*13 alleles on CD4 responses has also been suggested The selection of high avidity gag-specific CD4+ T cells may result from a lower exposure to HIV antigens during the acute infection stage, when the repertoire of responding T cells is initially shaped. The few reported cases of acute HIV-1 infection followed by spontaneous viral control support the notion of a lower viral peak in patients who acquire a controller status On the other hand, we cannot rule out that high avidity Gag-specific CD4+ T cells are selected but subsequently lost in progressor patients. Since high avidity CD4+ T cells are the first to respond in the presence of low HIV antigen amounts, they may be the first to get activated in the presence of replicating HIV, and may represent the initial wave of target cells available to the virus. HIV is known to preferentially infect HIV-specific cells In conclusion, this study provides evidence for the presence of high avidity CD4+ T cells directed against Gag in HIV controllers. It is remarkable that the distinctive properties of HIV-specific T cells in Controllers, including high proliferative potential HIV controllers  were recruited through the French “Observatoire National des HIV Controllers” established by ANRS. HIV controllers were defined as HIV-1 infected patients who had been seropositive for >10 years, had received no antiretroviral treatment, and for whom >90% of plasma viral load measurements were <400 copies of HIV RNA/ml. All HIV controllers included in the present study had current viral loads <40 copies/ml. Control groups included: (1) HAART group (n = 20): HIV-1 infected patients successfully treated with antiretroviral therapy for more than 5 years and with a viral load <40 copies of HIV RNA/ml; (2) VIR group (n = 10): viremic patients with viral loads >10,000 copies HIV RNA/ml. Viremic patients had been infected with HIV-1 for more than 1 year and had not received antiretroviral therapy. Patients from the HAART and VIR groups were recruited through the SEROCO-HEMOCO cohort and the Bicêtre hospital.The study was promoted by ANRS under number EP36 and approved by the Comité de Protection des Personnes IDF VII under number 05–22. All participants gave written informed consent prior to blood sampling.6 cells per well in 24-well plates in the presence of one HIV-1 Gag or pp65 CMV peptide (10 µM) in RPMI 1640 supplemented with 10% human AB serum, 2 mM L-glutamine, 10 mM HEPES, 100 ug/ml penicillin/streptomycin, 0.5 µM AZT, 5 nM Saquinavir and 5 ng/ml recombinant IL-7 (Cytheris). The peptides used to stimulate the culture were highly purified 20-mers . Recombinant IL-2 was added after 2 days to a final concentration 100 U/ml. Cell lines were restimulated with IL-2 every 2 days until the end of culture. Starting from day 7, cells were counted every day by trypan blue exclusion to determine the growth ratio (GR: observed number of cells/number of input cells at day 0). The CD8+ T cell population represented a median of 5.9% (range: 0–22.7%) of the CD3+ population. CD8+ T cells were depleted with magnetic beads  at doubling time (GR = 2), before performing functional assays. Less than 1% CD8+ T cells remained after CD8 depletion , according to the manufacturer's instructions. The kit covered approximately 70% of human Vβ specificities. The Vβ nomenclature is that of Wei Data are expressed as medians and range. Analyses were performed with the GraphPad Prism 5.0 software, using nonparametric statistical tests in all cases. Differences in variables between groups were analyzed with the Mann-Whitney U Test. Differences in percentages of response were analyzed with the Fisher's exact test. Correlations were analyzed with Spearman's coefficient R. All significant differences between groups (P<0.05) were reported on data plots.Text S1Supplementary methods and figure legends.(0.03 MB DOC)Click here for additional data file.Figure S1ex vivo IFN-γ ELISPOT responses in PBMC from HIV controllers and efficiently treated patients. Production of IFN-γ by PBMC stimulated with Gag293, Gag263, and Gag161 20-mer peptides was measured in HIV controllers  and treated patients . IFN-γ production was measured by ELISPOT assay on PBMC plated at 105 cells/well and stimulated with 4 µM peptide for 24 h. IFN-γ production was expressed as the number of spot forming cells (SFC) per million cells. Horizontal bars indicate medians. All statistically significant differences (P<0.05) evaluated by the Mann-Whitney U test are reported on graphs.Analysis of (0.31 MB EPS)Click here for additional data file.Figure S2Control experiments demonstrating that IFN-γ production is mediated by CD4+ T cells. (A) Representative examples of the efficacy of CD8+ T cell depletion: The expression of CD4 and CD8 within the CD3+ T cell population was evaluated in Gag293-specific cell lines from two HIV controllers (HIC1 and HIC2). CD8+ T cells present in cell lines at doubling time (top row) were depleted with anti-CD8 coated magnetic beads. Less than 1% CD8+ T cells remained in the CD3+ population after depletion (bottom row). (B) Comparison of ELISPOT responses before and after CD8 of CD4 depletion: Gag293-specific CD4+ T cells lines from one HIV controller (HIC) and one treated patient (HAART) were evaluated at doubling time for IFN-γ production by ELISPOT assay. ELISPOT responses were measured on cell lines before depletion (No depletion), after depletion of CD8+ cells (CD8−), or after depletion of CD4+ cells (CD4−). The cellular concentration was normalized to 30,000 cells per well prior to ELISPOT analysis. IFN-γ production was expressed as the number of spot forming cells (SFC) per million cells. (C) Additional examples demonstrating that CD4 depletion abrogated the ELISPOT response: Gag293-specific cells lines from 3 treated patients were depleted either for CD8 or for CD4 prior to evaluation of IFN-γ production by ELISPOT assay.(0.49 MB EPS)Click here for additional data file.Figure S3−6 M to 10−11 M). For each peptide dilution, IFN-γ production was expressed as the number of spot forming cells (SFC) per million cells. The functional avidity was defined as the last peptide dilution that gave a positive IFN-γ response at least 2 fold above background level. (B) Representative examples of functional avidity measurements in response to Gag263, Gag161 and CMV peptides. Cell lines from one HIV controller and one treated patient were analyzed in each case.Representative examples of functional avidity measurements in response to Gag and CMV peptides. (A) Functional avidity in Gag293-specific cell lines was evaluated at doubling time by ELISPOT assay. Representative examples are shown for 3 HIV controllers (HIC) and 3 treated patients (HAART). Functional avidity was measured by IFN-γ ELISPOT assay in the presence of decreasing peptide concentrations Click here for additional data file.Figure S4Analysis of the proliferative capacity of Gag293-specific cells. Gag293-specific CD4+ T cell lines from 3 treated patients (HAART) and 3 HIV controllers (HIC) were labeled at doubling time with CFSE. The Gag293-specific Tetramer+ CD4+ CD3+ population was analyzed 3 days later by flow cytometry. The number of generations was evaluated by the decrease in CFSE labeling. CFSE histograms were fitted with models that separated the populations into individual generations, using the FlowJo v8.8 software. The modeled generations are represented in dark blue, and the resulting curve fit is represented in red. “% divided” is the percentage of cells in the original sample which divided. “Proliferation index” is the average number of divisions undergone by the cells which divided (it does not take into account generation 0). Also reported in red is the percentage of cells that have undergone 5 or more divisions. This parameter highlights the presence of a population of Gag293-specific cells with high proliferative capacity in HIV controller cell lines.(0.99 MB EPS)Click here for additional data file.Table S1Reproducibility of functional avidity measurements.(0.04 MB XLS)Click here for additional data file.Table S2Frequencies of amplified TCR Vβ in CD4+ T cell lines specific for the Gag293 peptide.(0.03 MB XLS)Click here for additional data file.


The mortality in patients with persistent low CD4 count despite several years of HAART with sustained viral suppression is poorly documented. We aimed to identify predictors for inadequate CD4 cell recovery and estimate mortality in patients with low CD4 count but otherwise successful HAART.In a nationwide cohort of HIV patients we identified all individuals who started HAART before 1 January 2005 with CD4 cell count ≤ 200 cells/μL and experienced three years with sustained viral suppression. Patients were categorized according to CD4 cell count after the three years suppressed period , >200 cells/μL; immunological responders (IRs)). We used logistic regression and Kaplan-Meier analysis to estimated risk factors and mortality for INRs compared to IRs.We identified 55 INRs and 236 IRs. In adjusted analysis age > 40 years and > one year from first CD4 cell count ≤ 200 cells/μL to start of the virologically suppressed period were associated with increased risk of INR. INRs had substantially higher mortality compared to IRs. The excess mortality was mainly seen in the INR group with > one year of immunological suppression prior to viral suppression and injection drug users (IDUs).Age and prolonged periods of immune deficiency prior to successful HAART are risk factors for incomplete CD4 cell recovery. INRs have substantially increased long-term mortality mainly associated with prolonged immunological suppression prior to viral suppression and IDU. The introduction of HAART has decreased morbidity and mortality in HIV patients due to viral load (VL) suppression and immunological recovery . Still, Within a nationwide cohort of HIV infected patients we identified all individuals who were on stable HAART, had been virally suppressed for more than three years and had a CD4 cell count ≤ 200 cells/μL prior to the virally suppressed period. In this population we identified immunologic non-responders who did not rise to a CD4 cell count > 200 cells/μL after the three years of sustained VL suppression. We aimed to identify predictors for inadequate CD4 cell recovery and to determine the mortality in the immunological non-responders compared to those with an adequate CD4 cell response.The study was performed as a nationwide cohort study. In the first part of the study we estimated risk factors for immunological non-response (CD4 cell count ≤ 200 cells/μL after three years of VL suppression). In the second part of the study we estimated mortality in immunological non-responders (INRs) versus immunological responders (IRs).Denmark had a population of 5.5 million as of 31 December 2008, with an estimated HIV prevalence of approximately 0.07% in the adult population ,11. PatiThe Danish HIV Cohort study (DHCS), described in details elsewhere, is a population-based prospective nationwide cohort study of all HIV-infected individuals 16 years or older at diagnosis and who are treated at Danish HIV centres after 1 January 1995 . DecembePrimary causes of death were obtained from The Danish Civil Registration System and The Danish Register of Causes of Death . Data onFrom DHCS we identified all HIV-1 positive patients, who 1) started HAART before 1 January 2005, 2) had a CD4 cell count ≤ 200 cells/μl at start of HAART, 3) had a VL < 50 copies/ml for more than three consecutive years before 1 January 2008, 4) had no intervals of more than seven months between VL tests in the suppressed period, and 5) had a CD4 cell count ≤ 200 cells/μl at start of the virally suppressed period.Patients were defined as IRs in case the first CD4 cell count measured after 3 years of viral suppression was > 200 cells/μL and INRs in case the CD4 count was ≤ 200 cells/μL. Mortality was also stratified for more differentiated CD4 cell strata after 3 years of viral suppression  but the prognosis differed little for all strata above 200 cells/μL why we chose to pool all CD4 responses above 200 cells/μL in one group (data not shown).2 test, Fisher's exact test and Kruskal-Wallis Test when appropriate.Differences in characteristics between groups were evaluated by the χWe used binary logistic regression in order to identify predictors for immunological non-response. The following covariates were included in the model: Gender, age at start of the suppressed period (≤ 40 years vs. > 40 years)  and INRs; 42.6 years ), race (Caucasian vs. non-Caucasian), route of HIV infection (men who have sex with men (MSM) vs. heterosexually vs. injection drug user (IDU) vs. other), chronic HCV infection (positive vs. negative PCR for HCV RNA), cancer prior to start of the suppressed period, one or more AIDS defining event before start of suppressed period, HIV diagnose before 1 January 1995, nadir CD4 cell count < 50 cells/μL and > one year from first CD4 ≤ 200 cells/μL to start of the virologically suppressed period  and INRs; 1.5 years ). Selection of potential confounders was performed using the "change in estimate" method with age and gender forced into the model .Index date was defined as date of first CD4 cell count measurement after three years of sustained viral suppression. We calculated person-years at risk from index date until death, 1 January 2008, emigration or lost to follow-up, whichever came first. We used Kaplan Meier analysis to construct survival curves for INRs vs. IRs and further stratified these by time from first CD4 measurement ≤ 200 cells/μL to start of the virologically suppressed period (≤ one year vs. > one year). Cox regression analyses were used to estimate mortality rate ratios (MRR). We further calculated MRRs stratified by cancer (no cancer diagnosed prior to index date vs. cancer diagnosed prior to index date), previous AIDS defining event (none vs. > one or more AIDS defining events prior to index date) and time from first CD4 measurement ≤ 200 cells/μL to start of the virologically suppressed period (≤ one year vs. > one year). The estimates were adjusted for gender and age.In a robustness analysis we used a cut-off value of 500 copies/ml in the virologically suppressed period to test the impact of VL cut-off on our estimates.Using a previously described method  we groupThe study was approved by the Danish Data Protection Agency . Data in the database is not publicly available. SPSS statistical software, Version 15.0  and R software, version 2.8.1, was used for data analysis.In The Danish HIV Cohort Study 3165 patients started HAART before 1 January 2005 (figure After three years of sustained VL 236 81.1%) patients reached a CD4 cell count above 200 cells/μl (IRs) and 55 (18.9%) of the HIV infected patients did not (INRs). Characteristics of IRs and INRs are shown in table .1% patieIn un-adjusted analysis age, Caucasian race and time from first CD4 cell count ≤ 200 cells/μL to start of the virologically suppressed period were associated with immunological non-response  patients died in the observation period, 11 (20.0%) in the INR group and 11 (4.7%) in the IR group. As shown in figure 128 patients (44.0%) had more than one year from first CD4 measurement ≤ 200 cells/μL to start of the virologically suppressed period. The un-adjusted MRR among these 128 patients was 3.9  and after adjustment for age and gender it was 3.6 . When excluding these patients from the analysis the unadjusted MRR was reduced to 3.1  and after adjustment for age and gender it was 1.8 . 90 (70.3%) of the 128 patients who had more than one year of immunological suppression prior to the virologically suppressed period were diagnosed before 1995. When excluding IDUs from our analysis the unadjusted MRR was 2.5  and the adjusted MRR was reduced to 1.8 . The un-adjusted MRR calculated exclusively for IDUs was 12.7  and 9.8  after adjustment for age and gender. Exclusion of patients with previous cancer or AIDS defining event, respectively, did not change the estimates substantially. Only one death in each group  appeared to be a classical HIV related cause of death. Other causes of death were: cancer , sudden death , liver related , suicide , opiate overdose , pneumonia  and cachexia (not HIV related) .80% of the INRs achieved a CD4 cell count > 200 cells/μL after six years of observation (i.e. more than nine years after starting HAART). However, only 20 (35.1%) of the INRs were still under observation six years after study inclusion. After six years of observation, 95.5% of the whole study population who were still alive had fully suppressed VL.When using a cut-off value of 500 copies/ml for viral suppression, 445 study subjects (372 IRs and 73 INRs) were identified, of whom 18 (4.5%) patients in the IR group and 15 in the INR group (20.5%) died during follow-up. In the un-adjusted analysis age, Caucasian race, IDU, time from first CD4 cell count ≤ 200 cells/μL to start of the virologically suppressed period and CD4 cell count at start of the suppressed period were associated with immunological non-response. However, after having adjusted for potential confounders only age, IDU and CD4 cell count at start of the suppressed period remained associated with increased risk of being INR. MRRs were 4.5  and 3.69  when adjusted for gender and age.In a population of HIV patients with initial low CD4 count we found that almost one out of five was INR after three years of successful HAART. Older age and more than one year of severe immune deficiency prior to start of sustained VL were associated with INR. The patients with insufficient immunological response had substantially increased long term mortality. The increased mortality was mainly seen in patients with more than one year of immunological deficiency prior to the period of sustained VL suppression and IDUs.The major strength of this study is the identification of a homogeneous group with stringent viral suppression, essentially eliminating the possibility that incomplete VL suppression explains our findings. The study had a nationwide design with long and almost complete follow-up including data on cancer as well as HCV status.A limitation of the study is the small study population (in part caused by the stringent definitions of INRs and IRs) and the small number of outcomes, which did not allow us to extend the logistic regressions analysis or to calculate cause specific rate ratios of death. The study population encompasses a highly selected group of patients surviving more than three years in spite of low initial CD4 cell count, and a healthy survivor effect may lead to underestimation of the excess mortality in the INRs. Still, by using an extended virally suppressed period we allowed the patients to reach a steadier CD4 plateau without the interference of CD4 cell redistribution as seen after HAART start. HCV testing in the cohort is "intended" and may only be performed yearly on known high risk groups in the cohort e.g IDU why chronic HCV may be underestimated in the cohort.19% of the study patients did not have an adequate immunological response to HAART. Our results corresponds to the findings of other studies who found a negative impact of low initial CD4 cell count on long-term CD4 recovery ,6,17-19.INRs were significantly older than the IRs and age predicted inadequate CD4 cell recovery which corresponds to the findings of several other studies ,9,19,20.The excess mortality seen among the INRs was mainly related to prolonged immunological suppression prior to successful HAART and IDU. The median time from start of HAART to start of the virologically suppressed period was less than half a year. A longer period of immunological suppression prior to the three years of sustained VL suppression is therefore explained by delayed initiation of HAART and not poor compliance. This is supported by the fact that most of the patients with more than one year of immunological suppression before start of the virologically suppressed period were diagnosed with HIV before 1995 when treatment options were scarce. As many of the INRs who died were patients who initiated HAART in the mid-nineties it could be hypothesized that resistance played a role, but the stringent design with full VL suppression eliminates that possibility. We find it unlikely that the late death of these patients were directly related to the toxicity of the antiretroviral agents, but cannot rule out that toxicity should interact with the immune reconstitution. The increased mortality in patients with delayed initiation of HAART is well documented  and the We only encountered two AIDS related deaths suggesting that these are rare after prolonged HAART even without adequate CD4 recovery. Thus, it seems that the INRs are not immune-suppressed in the classical sense where a low CD4 count leads to AIDS defining diseases and eventually death. Still, we cannot rule out that there is a biological cause of the increased mortality in immunological non-responders related to immunodeficiency and that our study simply do not have power enough to demonstrate an association.The risk factors for incomplete CD4 cell recovery and increased mortality points toward earlier diagnosis of HIV as the main prophylactic measure. Also, the increased mortality in these patients calls for increased concern in terms of treatment and screening for co-morbidity.We conclude that age and prolonged immunological suppression are risk factors for incomplete CD4 cell recovery in patients with otherwise successful HAART. Patients with insufficient immunological response have substantially increased long-term mortality compared to IRs, but the increased mortality is mainly associated with prolonged immunological suppression prior to VL suppression and IDU. We therefore presume that in the modern HAART era where patients are started early on HAART the prevalence of insufficient CD4 recovery will decrease substantially.Potential competing of interest: N Obel has received research funding from Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, GlaxoSmithKline, Abbott, Boehringer Ingelheim, Janssen-Cilag and Swedish Orphan. F Engsig has received research funding from Merck Sharp & Dohme. J Gerstoft has received research funding from Abbott, Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, Pharmasia, GlaxoSmithKline, Swedish Orphan and Boehringer Ingelheim.The study was financed by The Research Foundation at Copenhagen University Hospital, Rigshospitalet and Faculty of Health Science, Copenhagen University. The funders had no role in the study design; in the collection, management, analysis, and interpretation of data; in the preparation, review, or approval of the manuscript, or in the decision to submit the article for publication. The researchers are independent from the funders.The authors contributions are the following: FNE (MD) contributed with study design, data collection, data analysis, interpretation of findings and writing of the manuscript. JG  contributed with study design, data collection, interpretation of findings and critical edit of the manuscript. GK , CSL , GP (MD), BR , JJ (MD) and LNN (MD)contributed with data collection, study design, interpretation of findings and critical edit of the manuscript. NO  contributed with data collection, study design, critical review of data analyses, interpretation of findings and critical edit of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/10/318/prepubTable S1. Drugs included in the last HAART regimen prior to index date.Click here for file


T-cell responses were observed in the majority (78%) of samples. The frequency of CD4+ responders was far lower among those with progressive disease, indicating that the CD4+ T-cell response might be important in HPV clearance. CD8+ reactivity to E6 peptides was dominant across all disease grades, inferring that E6-specific CD8+ T cells are not vitally involved in disease clearance. T-cell responses were demonstrated in the majority (80%) of cervical cancer patients, but are obviously ineffective. Our study reveals significant differences in HPV16 immunity during progressive CIN. We conclude that the HPV-specific CD4+ T-cell response should be an important consideration in immunotherapy design, which should aim to target preinvasive disease.Infection with high-risk genital human papillomavirus (HPV) types is a major risk factor for the development of cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma. The design of effective immunotherapies requires a greater understanding of how HPV-specific T-cell responses are involved in disease clearance and/or progression. Here, we have investigated T-cell responses to five HPV16 proteins  in women with CIN or cervical carcinoma directly  Cervical carcinoma is the second most common female cancer worldwide, with 400 000 new cases diagnosed each year , CD4 responses to HPV16 E6  in mediating regression of HPV-related disease has not been proven. Using a variety of restimulation protocols, HPV16 E2-, E6- and E7-specific CTLs can be detected in patients with previous  from a cohort of 41 women with varying grades of cervical disease. The frequency, magnitude and antigen specificity of the responses obtained are discussed in relation to disease severity.The link between HPV16 and cervical carcinoma opens up the possibility of immune T-cell intervention, either against the preinvasive lesions from which tumours arise, or against the virus antigen-positive tumour cells themselves. To effect such strategies will require a better understanding of the spectrum of T-cell responses induced by HPV16 antigens during the course of natural infection, and of their role in disease clearance and/or progression. Much of the work to date has understandably focused on responses to the E6 and E7 transforming proteins, since these are the only viral antigens constitutively expressed in HPV-positive tumour cells. By contrast, there has been no concerted effort to look at the totality of responses to a single agent, including responses to viral antigens that are expressed later in the replication cycle that may be useful targets for the treatment of preinvasive lesions. In this study, we have used CD4- and CD8-depleted populations of responder cells in an ELISPOT assay of IFN-A total of 41 women attending colposcopy clinics or undergoing surgery for cervical disease  were investigated. Ethical approval for this study was obtained from the South Birmingham Research Ethics Committee (Study Number 5147) and informed consent was obtained from all subjects. Abnormal cervical cytology was diagnosed and confirmed by histopathology. Patients were assigned to one of four groups according to the histology reports obtained from cervical tissue taken at the time they were bled: those reported to have no evidence of CIN, those with low-grade disease (CIN I), high-grade disease (CIN II/III) and those with cervical carcinoma. The clinical details are shown in The HPV genotype of patient biopsies obtained during the course of this study could not be established. However, the transient nature of genital HPV infections questions the validity of HPV typing at a single time point, since this may not necessarily reflect a past history of HPV infection in a significant proportion of women, especially those with low-grade disease  cells were separated from 40–60 ml heparinised blood samples by isopycnic centrifugation on lymphocyte separation medium  and cryopreserved in liquid nitrogen. CD4+ and CD8+ T-cell depletions were carried out on UM cells using antibody-coated magnetic beads  according to the method previously described . HPV16 VLPs were produced following the infection of SF9 insect cells, and were purified on caesium chloride gradients according to published methods . The ELISPOT assay procedure has been published previously (5 cells per well (in duplicate) and peptides were added to a final concentration of 10 μg ml−1. A negative control well with no peptide or VLP and a positive control well containing cells and 0.2% PHA-P (Difco Laboratories) instead of peptide were included for every sample. The plates were counted using an automated system  and background counts obtained in the absence of peptide or VLP were subtracted. An ELISPOT response among the patient group was only considered positive if the number of spots obtained fell above a negative cutoff value. These were calculated for every peptide and the VLP preparation, and taken as 2 standard deviations (s.d.) above the mean of the counts obtained using the negative control group comprising nine age-matched female virgins.Overlapping synthetic peptides  covering the entire primary sequences of the HPV16 E6, E7 and E4 proteins  were used in an ELISA to determine HPV16-specific antibody levels in plasma samples obtained from each subject. This source of VLPs was unsuitable for use in the ELISPOT assay due to problems with toxicity. An ELISA method for detection of HPV1 VLP-specific antibodies has been published by us recently . The results of the ELISPOT assays are shown in To determine whether responding IFN-The majority of the control samples showed little or no T-cell reactivity, with the exception of numbers 5 and 9, both of which demonstrated responses to a few peptides, particularly those covering HPV16 E6 . AlthougThe results of the ELISPOT assays showed that CD8+ T-cell reactivity occurred at almost twice the frequency of CD4+ reactivity . CD4 res6 CD4+ T cells) was obtained from a woman with high-grade disease (patient 12), using an E6 peptide . In a CD8 response, the highest frequency of spots (1538 spots per 1 × 106 CD8+ T cells) was obtained from a patient with no CIN (patient 27), again in response to an E6 peptide .There was no significant difference in the magnitude of CD4 or CD8 responses obtained among patient groups . The greDifferences in the antigen specificities of the CD4 and CD8 responses were also observed in the ELISPOT assays, although this was not related to disease grade . A similP-values all greater than 0.187; Mann–Whitney test). All of the patient groups had significantly higher titres than those obtained from the negative control group of female virgins , with the exception of those women with low-grade disease (P=0.091).The serological status of all subjects in the study was assessed in order to obtain additional information about HPV16 immune status. Antibodies to HPV16 VLPs were detected in 20 out of the 41 (48.7%) plasma samples overall . In womeOut of the 20 seropositives obtained in this study, 18 (90%) showed either CD4+ or CD8+ T-cell reactivity to HPV16 peptides in the ELISPOT assays. In all, 15 samples (71%) out of the 21 with negative serology also gave a T-cell response. These samples were from patients who fell predominantly into the no CIN or low-grade disease groups. Interestingly, the patients who demonstrated T-cell responses to multiple peptides and proteins   were serγ release, to HPV16 E4, E6, E7, L1 and L2. Significantly, in this study, cells were used directly ex vivo in the ELISPOT assay, without undergoing any kind of in vitro restimulation. We demonstrated either CD4+ or CD8+ T-cell reactivity in the majority of the patient samples tested (78%), with 34% showing both CD4 and CD8 responses. There was little difference in the magnitude of the responses obtained between the CD4- or CD8-enriched populations  were seronegative, whereas those showing far fewer T-cell reactivities had the highest antibody titres . One explanation for these results is that the antibody response is slower than the T-cell response. Perhaps, the women showing multiple T-cell reactivities have only recently encountered the virus, and have large numbers of effector T cells, but the antibody response is not yet established. Conversely, in the other group of women with low T-cell reactivity and high antibody titres, the primary effector T-cell response may have subsided, but the serological response is still present and helping to prevent re-infection. It is difficult to define precisely the relationship between T-cell reactivity and antibody status; more knowledge is required about the relative timescales and roles of the T- and B-cell responses to HPV during natural infection.γ release are capable of revealing T-cell reactivities to HPV16 antigens in women with cervical dysplasia and can be used to establish the spectrum of T-cell responses induced during natural infection. The ELISPOT assay is becoming established as a good method for charting HPV-specific immunity, both during natural infection and following vaccination (ex vivo, which is more likely to be representative of the in vivo situation. The majority of the other studies employed an in vitro stimulation step prior to ELISPOT analysis. Secondly, we established negative cutoff values using a valid negative control group that obviously minimises the problems associated with nonspecificity. Thirdly, many of the published studies have concentrated on HPV-specific responses in normal individuals. By looking at the healthy population, these studies are likely to be detecting predominantly memory T-cell reactivity, whereas we were studying women with recent or current disease, and are therefore likely to be detecting effector populations as well as memory.In summary, we have shown that ELISPOT assays of IFN-The results from the current study suggest that there are differences in how viral antigens are handled by the immune system during progressive disease of the cervix. In order to define this more precisely, more noninterventional prospective studies will be required, although there are obvious ethical problems associated with this. The vast majority of women in this cohort underwent surgical excision that is likely to have an effect on HPV16 immunity, and so a prospective study would not be possible. Defining the role played by HPV16-specific T cells in the natural history of cervical disease will aid the development of immunological assays to determine the risk of CIN progression, and therefore the management of premalignant and malignant HPV-associated neoplasia.


Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications.T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes  were assessed by quantitative flow cytometry.CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75.A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome. Sepsis has been defined as the systemic inflammatory response syndrome that occurs in response to bacterial infection . AlthougThe T cell compartment plays a pivotal role in regulating the effector stage of the immune response. CD3+CD4+ T cells are mainly involved in the regulation of the immune response, and CD3+CD8+ T cells are critical in the cytotoxic response . SeveralA role for T lymphocytes in severe systemic bacterial infections has been described in several studies -16 , andFifty-two consecutive patients admitted to the ICU of the University Hospital Príncipe de Asturias, Madrid, Spain, with septic shock, diagnosed according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine , were enThirty-six age-matched and sex-matched healthy blood donors were studied in parallel with the patients (0 and 28 days of the follow up). They were studied to control the adequacy of the cytometric techniques as well as to characterise the normal range of the T lymphocyte compartment parameters analysed.® LH instrument .Blood was collected from the patients at baseline (ICU admission) and at 3, 7, 14 and 28 days of follow up, and at baseline and at 28 days in healthy controls. White blood cell differential counts were conducted in a COULTERPeripheral blood mononuclear cells (PBMC) were obtained from heparinised venous blood by Ficoll-Hypaque  density gradient centrifugation. Cells were resuspended (1 × 106 cells/ml) in RPMI-1640  supplemented with 10% heat-inactivated FBS , 25 mM Hepes  and 1% penicillin streptomycin .T cells were phenotypically analysed in PBMC by four-colour flow cytometry in a FACScalibur cytometer using CellQuest-3.3 software . PBMC were incubated with combinations of fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin-cyanine 5.5 (PE-cy5.5), peridinin chlorophyll protein (PerCP) and allophycocyanin (APC)-labelled monoclonal antibodies. The monoclonal antibodies were CD3-PerCP, CD3-FITC, CD45RA FITC, CD56-PE, CD28-PE, CD62L-PE , CD19-PE-CY5, CD8-APC, CD45RO PE .®. Next, we obtained the absolute number of CD4+ and CD8+ T lymphocytes by multiplying the total number of T lymphocytes previously calculated by the percentage of positive cells for each one of both antigens in CD3+ T cells. We simultaneously stained PBMC with CD3, CD4 and CD8 antibodies to obtain this data. Finally, we calculated the absolute number of the CD3+CD4+ and CD3+CD8+ T cells subsets defined by the expression of CD45 isoforms CD45RA+CD45RO-, CD45RO+CD45RA-, CD45RA+CD45RO+ and the expression of the antigens CD28+ and CD62L+. To calculate these numbers, we multiplied the percentage obtained of each subset in the parents' CD3+CD4+ or CD3+CD8+ populations by the absolute count of CD3+CD4+ and CD3+CD8+ T cells, respectively. All absolute numbers are expressed as cells/ml.The absolute numbers of T lymphocyte subsets were calculated according to standard flow cytometry criteria for lymphocyte subset identification and the lymphocytes obtained in conventional haemogram. First, we calculated the percentage of cells expressing CD3 in the total lymphocytes gate defined by forward and side scatter in PBMC. The absolute number of circulatory T lymphocytes was calculated by the percentage of CD3+ cells in peripheral blood lymphocytes multiplied by the total number of lymphocytes per microlitre measured by a CoulterP < 0.05.Analyses were performed using SPSS-11.0 software . Most variables did not fulfill the normality hypothesis, so the Mann Whitney U-test for non-parametric data was used to analyse differences between the groups, and analysis of variance followed by Wilcoxon Signed Ranks tests were used for within group analyses. The level of significance was set at There were ninety-two ICU admissions with a diagnosis of septic shock during the study period. Forty patients were excluded from the study: three patients were HIV-positive, 18 patients were taking corticosteroids on admission, 12 were on or had received chemotherapy, five patients suffered neoplasia, one had rheumatoid arthritis and one had anaphylactic shock in response to antibiotic therapy for sepsis. Mortality was 34.6%. The mean (± standard error) Acute Physiology and Chronic Health Evaluation (APACHE) II  score waCounts and distributions of the main circulating T lymphocyte subsets were systematically examined in 52 patients with septic shock at admission to the ICU and at 3, 7, 14 and 28 days of follow up in the ICU. Furthermore, patients were classified as survivors or nonsurvivors according to their clinical outcome of sepsis during the four weeks of follow up. Thirty-six healthy blood donors who were age-matched and sex-matched  were studied in parallel with the patients as controls of the adequacy of the cytometric technique procedure as well as for characterisation of the normal range of the T lymphocyte compartment parameters analysed.Both surviving and nonsurviving patients showed significantly lower absolute CD3+ T lymphocyte numbers  than controls (1394 ± 36 cells/μl) on admission and during the first 14 days of follow up . In survivors, CD3+ counts had significantly returned to normal by day 28 (1191 ± 172 cells/μl). The CD3+CD4+ T cell subset was also reduced in surviving and nonsurviving patients with respect to healthy controls at baseline and during the first week of follow up. However, this dramatically decreased CD3+CD4+ T lymphocyte count had normalised in survivors by day 14 Figure . On admiThe activation stage of the CD3+CD4+ and CD3+CD8+ T lymphocytes was determined by examining the expression of the CD45 RA and RO isoforms. The retraction in circulating CD3+CD4+ and CD3+CD8+ T lymphocytes observed in the patients could be mainly explained by a decrease in the noneffector CD45RA+CD45RO- subset Figures . InteresWe also analysed the expression of CD28 and CD62L antigens on CD3+CD4+ and CD3+CD8+ T lymphocytes. When CD3+CD8+ T lymphocytes are activated, CD28 expression is lost ,20. The A prediction receptor operative curve (ROC) was then used to estimate the value of CD3+CD8+CD28+ and CD3+CD8+CD62L+ T cell counts for predicting death in the patients with septic shock at admission and days 3 and 7. We found that a cutoff value of 136 CD3+CD8+CD28+ T cells/ml on admission to the ICU of a patient with septic shock showed a sensitivity of 70% and 100% specificity for predicting the risk of death, and the area under the ROC curve was 0.84. For the CD3+CD8+CD62L+ T cells, the cut off on admission was 141 cells/ml, with a 60% sensitivity and 100% specificity for predicting the risk of death and an area under the curve of 0.75. The sensitivity and specificity of the data obtained at days 3 and 7 were worse than those found at admission (data not shown).The number of circulating CD3+CD4+CD28+ T cells was significantly lower in surviving and nonsurviving patients with septic shock compared with healthy controls on admission and on day 3 of follow-up Figure . In survIn this study, we show that surviving and nonsurviving patients with septic shock have different patterns of involvement in circulating T lymphocyte compartment. A drop in circulating CD3+CD4+ and CD3+CD8+ T cells has been described in patients with severe sepsis or septic shock at admission to the ICU . In our Plasmodium berghei develop a syndrome similar to septic shock and the depletion of CD3+CD8+ T cells also significantly ameliorates the complications that induce shock [In a mouse sepsis model of caecal ligation and puncture, the depletion of CD3+CD8+ T and natural killer cells was associated with a survival benefit with decreased blood bacterial concentrations, improved physiological function and an attenuated proinflammatory response . It has ce shock . In addice shock . In agreCD45 is a phosphatase that is essential in T cell development and antigen receptor signalling . The expCD28 is a costimulatory molecule that plays a key role in regulating the activation and survival of T lymphocytes . ActivatThe migration of circulating T lymphocytes to peripheral lymph nodes depends on the expression of the CD62L homing receptor . We founOur T lymphocyte phenotype data show a time difference, or shift, in the recirculation of T lymphocytes between patients who survive septic shock and those who do not. Taken together and analysing the phenotype of the circulating T cells according to the activation criteria (CD45RA+ and CD45RO+) related to CD28 (activation and co-stimulation) and CD62L (activation and migration) expression point to a slower migration of naive and effector cells in nonsurviving patients. This different T lymphocyte kinetics would mean a delayed tissue response that could determine the failure of the immune system and the fatal prognosis of the patient. In particular, the delay in the disappearance of CD45RA+, CD45RA+CD45RO+, CD28+, CD62L+, T CD4+ and CD8+ lymphocytes observed between days 3 and 7 of follow up in the nonsurvivors appears to be crucial to the final outcome. Accordingly, in surviving patients, effector cells would migrate more rapidly to tissues and this would in turn trigger the quick action of the immune system in combating the infection and thus determine the survival of the patient. It is known that cellular immune responses play a critical role in the defense against viral infections and strong T-cell responses have been reported in patients who clear infection . If the Not surprisingly, the survivor group had lower APACHE II, MODS and SOFA scores than nonsurvivors. An increasing APACHE II score reflects an increasing severity of illness and escalating risk of hospital death for multidiagnostic ICU patient groups. However, an APACHE II score cannot be directly equated with a specific risk of lower mortality than the same score for a patients with septic shock . In thisSeptic shock patients show a severe redistribution of circulating T lymphocyte subsets. We found that CD62L and CD28 expression on circulating T cells at ICU admission are good markers to predict the outcome of shock septic patients. T lymphocyte phenotype data show a time difference in the recirculation of T cells between survivors and nonsurvivors that might provoke a delayed tissue response of the immune system.• Septic shock patients show a severe redistribution of circulating T lymphocyte subsets.• CD62L and CD28 expression on circulating T cells at ICU admission are good markers for predicting the outcome of shock septic patients.• T lymphocyte phenotype data show in nonsurviving patients a slower migration of naive and effector cells that might provoke a delayed immune response.APACHE: Acute Physiology and Chronic Health Evaluation; APC: allophycocyanin; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; ICU: intensive care unit; MODS: multiple organ dysfunction syndrome; PBMC: peripheral blood mononuclear cells; PE: phycoerythrin; PerCP: peridinin chlorophyll protein; PE-cy5.5: phycoerythrin-cyanine 5.5; ROC: receptor operative curve; SOFA: Sequential Organ Failure Assessment.The authors declare that they have no competing interests.JM and RP are joint authors and contributed equally to this manuscript. AP, MAM, JM and RP contributed to the design of the study and drafted the manuscript. JM, DD and HB obtained the data. JM, RP, MÁ, AH, MRZ and ER participated in data analysis and interpretation of the results.


The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults.We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag–specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28−) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27−CD28−), low activation phenotype. Participants with the highest fraction of late memory (CD27−CD28−) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts . In turn, those with the highest fraction of intermediate memory (CD27+ CD28−) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation , indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment.A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile. Elevated CD8+ T cell activation is established early in HIV-1 infection On encountering antigen CD8+ T cells differentiate from the least differentiated (naïve or early memory) stage to the most mature (memory/effector) stage. In this process, cell surface receptors are progressively down-regulated  as CD8+ T cells differentiate and up-regulated or re-expressed . While there is broad general agreement on how to define a naïve T cell, there is not yet a unified model that describes the process of human T cell differentiation  are shown in The magnitude of IFN-γ and IFN-γ/IL-2 CD8+ T cell responses to HIV-1 Gag were lower than the response to CMV pp65 at visit 1 (prior to therapy), and subsequent study time points during and after anti-retroviral therapy . The magAt visit 1, prior to anti-retroviral therapy, the naïve and early memory CD8+ T cell subset (EM:CD27+CD28+) was most frequent (median 43.3% Interquartile range (IQR) 41.4, 49.7) followed by the intermediate memory ) and late memory (LM:CD27−CD28−) median 16.3% ) subsets . FollowiWe observed that the maturation profiles of antigen-specific T cells differed from the profiles of total T cell populations. HIV-1 Gag-specific IFN-γ expressing CD8+ T cells were predominantly CD27+CD28−, IM ), while the majority of CMV pp65-specific CD8+ T cells had a LM CD27−CD28− phenotype ) . HIV-1 GTypical of early HIV-1 infection, the proportion of activated CD8+ T cells was high  than CMV pp65-specific CD8+ T cells; this was also true for each of the maturation subsets  . Of the At study visit 1, during early untreated HIV-1 infection, we examined the relationship between the CD8+ T cell activation level and maturation profile of total and HIV-1 Gag specific T cell responses . Among uThe size of any differentiation stage of the HIV-1 Gag IFN-γ+ CD8+ T cell response was not associated with viral load (not shown). A higher fraction of HIV-1 Gag specific IFN-γ expressing CD8+ T cells in the LM pool was found to associate with higher CD4+ T cell counts .Although there was a sharp decline in the level of activation (CD38 MFI) on HIV-1 Gag specific CD8+ T cells, there was no significant drop in the fractional size of the CD8+ T cell HIV-1 Gag specific IM CD27+CD28− population , or incrIn an effort to understand the relationship of two key features of the early T cell response to HIV-1 we examined the simultaneous expression of both activation and differentiation markers on total and antigen-specific T cells from treatment naïve adults in early HIV-1 infection before, during and after anti-retroviral therapy. Our data suggests that there may be a link between the activation and maturation stage of CD8+ T cells. The skewed, less differentiated maturation profile of HIV-1-specific T cells was more pronounced at higher CD8+ T cell activation levels. We also found that increased numbers of fully differentiated HIV-1-specific CD8+ T cells associated with higher CD4+ T cell counts suggesting that the presence of mature Gag-specific CD8+ T cells may be protective. However the HIV-1 Gag-specific T cell differentiation profile was not readily altered by suppression of T cell activation by anti-retroviral treatment.We compared CMV- and HIV-1-specific responses in early infection. Previous studies of differences between CMV- and HIV-specific-responses have suggested that the failure of HIV-specific cells to reach a more differentiated phenotype may explain their inability to fully control HIV replication It has been suggested that distinct T cell maturation profiles, associated with responses to different viruses, are established for the purpose of generating memory or maintaining latency We found that patients with the highest numbers of EM Gag-Specific CD4+ T cells had the lowest levels of T cell activation, suggesting that – in contrast to our findings on CD8+ T cell responses – that a less differentiated CD4+ T cell response may be associated with better clinical status. Our data is consistent with a report suggesting a less differentiated CD4+ T cell response marks long-term non-progression in HIV disease, a state of low CD8+ T cell activation Repeated stimulation of Gag-specific T cells, by persistent HIV-1 replication, may induce high levels of activation and block maturation into a mature effector phenotype. Based on our model linking activation to maturation impairment, we reasoned that in the absence of repeated antigen stimulation the proportion of Gag-specific T cells with a low activation, LM phenotype may increase. We therefore examined maturation and activation following anti-retroviral therapy and reduction of circulating virus to undetectable levels. While a significant decline in activation levels on all CD8+ T cell subsets was apparent, reduced antigen load had different effects on total, CMV- and HIV-1-specific T cell differentiation profiles. Following therapy, the CMV-specific CD4+ IM T cell pool did expand as the LM pool shrunk. This suggests that HIV-1 viremia, or generalized immune activation may influence the differentiation profile of CMV specific CD4+ T cell responses. Among total CD8+ T cells there was a significant reduction in the IM and a corresponding increase in the LM fraction. As activation declines the total IM CD8+ T cell fraction may mature, or die by apoptosis, shifting the population to an LM profile. In contrast, despite a significant reduction of activation levels on Gag-specific CD8+ T cells, there was no corresponding shift in the maturation profile of Gag specific CD8+ T cells. Taken together, these data suggest that high activation and viral load does not prevent the maturation of Gag-specific cells, although these effects may be apparent on T cells of other specificities. The Gag-specific T cells that we measured may have already become replicatively senescent due to repeated stimulation The mechanism by which maturation and activation are associated prior to treatment is not clear from our results. The relative paucity of LM HIV-specific CD8+ T cells may be due to increased susceptibility of these to apoptosis, even after ART suppression of activation. That said, the failure of CD8+ T cells to mature may result from manipulation of signaling pathways in responding HIV-1 specific CD8+ T cells by a viral product, such as secreted Nef, which has been shown to upregulate PD-1 We observed that HIV-specific T cells are predominantly immature and highly activated at early stages of infection. Although activation is reduced by anti-retroviral therapy immature Gag-specific cells continue to predominate. In a previous report we demonstrated that initiation of anti-retroviral therapy within the first month of HIV-1 infection associated with improved viral control and clinical outcomes once therapy was halted, compared to those who start anti-retroviral therapy one month or later into infection Thirteen treatment naïve adults in early HIV infection were studied for T cell responses and phenotypes at 1) study entry (within 2–4 months of acquiring infection), 2) after having reached complete virologic suppression on a first anti-retroviral regimen  and 3) several months after having halted anti-retroviral therapy.All specimens were drawn from persons enrolled in the Options study of early HIV-1 infection conducted in a university based research clinic. Among those enrolled in OPTIONS, approximately 90% are within 6 months of acquiring HIV-1 infection 6–2×106 cells/mL in the same medium, and rested overnight in slanted 15-mL conical tubes with loosened caps, in a CO2 incubator at 37°C. For phenotyping assays cells were washed in FACS buffer  for staining the same day.PBMC were isolated, cyropreserved and stored by the UCSF/ARI AIDS Specimen Bank, then transported to the Core Immunology Laboratory for analysis. Cryopreserved PBMCS were rapidly thawed in to warm RPMI 1640 with 10% fetal bovine serum (UCSF Cell Culture Facility) and counted using the Viacount assay on a Guava Personal Cell Analysis system (Guava Technologies). For Cytokine Flow Cytometry (CFC) assays PBMC were re-suspended at 1×10Thawed PBMCS were surfaced stained with the following combination of fluorescently labeled antibodies: CD3 Pacific Blue, CD4-AmCyan, CD8-Alexa 700, CD38-PE-CY7, CD27-APC, CD28-APC-CY7, and HLADR FITC  in the presence of 5 µg/ml ethidium monoazide bromide . After a 50 minute incubation in the dark at 4°C cells were exposed to a 40-W fluorescent light for 10 min at room temperature to cross-link the EMA. FMO (Fluorescent minus-one) controls were run with each experiment to ensure that breakdown of tandem conjugates (e.g. APC-CY7 to APC) was not occurring and to assist with setting gates. Following staining, PBMCs were washed in FACS buffer, fixed in 0.5% formaldehyde (Polyscience), stored at 4°C until analysis.Thawed and rested PBMC were stimulated with overlapping peptide pools  in the presence of 10 µg/ml Brefeldin A (Sigma Aldrich) for 18 hours at 37°C. Peptide pools were HIV-1 SF2 GAG  and CMV pp65 . Unstimulated cells were run in parallel as a negative control for each subject. Following stimulation PBMCs were treated with 2 mmol/L EDTA, washed in PBS, then stained with anti-CD4, CD8, CD38, CD27, and CD28 in the presence of EMA, as described above. Cells were then washed in FACS buffer, fixed and permeabilized by a 10 minute incubation in FACS lyse, and a 10 minute incubation in FACS Perm (both from BD Biosciences) before staining with fluorescently conjugated antibodies: IFN-γ-FITC, IL-2-PE and CD3-Pacific Blue . Cells were washed, re-suspended in FACS buffer, stored at 4°C until analysis.All samples were run on a customized BD LSR II Flow cytometer within 18 hours of staining. Rainbow beads (Spherotec) were used to standardize instrument settings between runs. Between 900,000 and 1 million lymphocytes were collected for each sample. Data was compensated and analyzed by using Flowjo Software (Treestar Inc). The gating strategies used to define CD4+ and CD8+ T cell maturation and activation populations, to define antigen-specific T cells, and to subset these cells for activation and maturation markers are shown in All statistical analyses were performed in the SAS System Version 9.1 for Windows XP. Non-parametric statistical tests were employed in all cases. Spearman rank correlations were generated for all correlation tests. Signed Rank tests were used to test significance of change in values between study time-points. The Wilcoxon Two Sample test was used to compare different values at a given study time-points.


HIV DNA and RNA levels in the gut exceed those in the blood up to 10-fold. The ileum and rectum differ in HIV levels and responses to intensification, suggesting that mechanisms of persistence may vary between sites. HIV persistence could be impacted by T cell activation, absolute CD4 levels, or composition of memory T cell subsets.Mucosal biopsies were obtained from the ileums and rectums of eight HIV-negative controls and 10 ART-suppressed HIV-positive patients. Isolated cells were analyzed for T cell composition , activation , and memory subpopulations  using flow cytometry. CD4 numbers were also measured by immunohistochemistry. In two patients, HIV DNA was measured in sorted subpopulations of memory CD4+ T cells.CD4 reconstitution appears to reach normal levels in the rectum , but not the ileum . Ileal CD4 numbers appear normal in the lymphoid aggregates, but not the lamina propria. In both the ileum and rectum, T cell activation remains higher than in HIV-negative controls  CD4+ T cells (mean 41.6% of CD4+ T cells) than the PBMC (15.4%), but similar numbers of transitional memory (TM) CD4+ T cells . HIV DNA concentrations in ileal EM (1 copy/2100 cells) were 25-fold higher than in blood EM .T cell reconstitution and activation differ between gut sites. The higher concentration of HIV DNA in the gut is not due to increased TM cell%, but could reflect differences in the rate of infection of memory subtypes. The abnormal immune activation and lack of CD4 reconstitution in the ileum could reflect ongoing replication or chronic virus production.


However, the cause-effect relationship between HIV-specific cellular immunity and disease progression is unknown. We investigated in a large prospective cohort study involving 96 individuals of the Amsterdam Cohort Studies with a known date of seroconversion whether the presence of cytokine-producing HIV-specific CD8+ T cells early in infection was associated with AIDS-free survival time.T-cell immunity is thought to play an important role in controlling HIV infection, and is a main target for HIV vaccine development. HIV-specific central memory CD8+ T cells was measured after in vitro stimulation with an overlapping Gag-peptide pool in T cells sampled approximately one year after seroconversion. Kaplan-Meier survival analysis and Cox proportional hazard models showed that frequencies of cytokine-producing Gag-specific CD8+ T cells  shortly after seroconversion were neither associated with time to AIDS nor with the rate of CD4+ T-cell decline.The number and percentage of IFNγ and IL-2 producing CD8+ T cells can be found early in HIV infection, irrespective of subsequent clinical outcome. The fact that both progressors and long-term non-progressors have abundant T cell immunity of the specificity associated with low viral load shortly after seroconversion suggests that the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression.These data show that high numbers of functional HIV-specific CD8 With an estimated 3 million human immunodeficiency virus (HIV)-related deaths each year, an effective treatment for HIV is urgently needed. Highly active antiretroviral therapy (HAART) results in significant suppression of HIV viral load, but is unable to eradicate the virus from the body, has severe side effects and may lead to resistance. An alternative option to prevent HIV infection and progression to acquired immunodeficiency syndrome (AIDS) might be therapeutic or prophylactic vaccination.+ T cells play an important role in controlling HIV replication. The fact that i) the decline in viral load in acute infection coincides with the peak in HIV-specific CD8+ T-cell numbers + T cells in macaques results in higher viral load + T cells may be unrealistic, a T cell-based vaccine might limit early viral dissemination and immune damage by controlling HIV more effectively during initial infection, thereby leading to slower progression to AIDS It is generally accepted that CD8+ T cells control HIV, the mechanism by which these cells can do so is still poorly understood. Consequently, it is not known what functions will be required of vaccine-induced CD8+ T cells to protect against progression to AIDS. At present, virus-specific CD8+ T cells are mostly quantified using the IFNγ ELISpot assay when evaluating potential therapeutic and prophylactic vaccines + T cells upon recognition of viral peptides presented on HLA class I molecules. It has been shown that IFNγ production by HIV-specific CD8+ T cells correlates with tetramer staining  and anti-viral cytotoxicity as measured by chromium release assays + T cells has been associated with low viral load and sustained CD4+ T-cell counts + T cells are able to produce multiple cytokines  and are able to proliferate and degranulate upon in vitro peptide stimulation + T cells is necessary for proliferation and differentiation into effector T cells. In contrast to long-term non-progressors, central memory CD8+ T cells capable of producing IL-2 are only rarely detected in viremic individuals progressing rapidly to AIDS + T cell function. Interestingly Gag, but not Env, specific responses were reported to correlate with low viral load Although there is evidence that CD8+ T cells and low viremia + T cells early in infection, suggesting that these cells are deleted or become functionally impaired as HIV infection progresses Cross-sectional studies have shown a striking relation between functional CD8+ T cells in HIV infection thus far are often limited by small numbers of patients and/or a cross-sectional nature. To reveal the effect of HIV-specific CD8+ T cells on progression to AIDS, we therefore investigated in a large prospective cohort study whether the frequency of HIV-specific cytokine-producing CD8+ T cells approximately one year after seroconversion, when the viral set point has been established Studies evaluating the role of CD8st 1996, ensuring that none of the subjects received highly active anti-retroviral therapy (HAART). Median follow-up to AIDS diagnosis or censoring was 82.7 months (range 14.9–153.9). Forty of the 96 participants (42%) progressed to AIDS during follow-up. Details on inclusion criteria, enrolment and baseline characteristics of ACS participants can be obtained at www.amsterdamcohortstudies.org. Informed written consent was obtained from all participants and the study was approved by the Medical Ethical Committee of the Academic Medical Center.Ninety-six participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS with a known date of seroconversion were selected based on availability of cryopreserved PBMC approximately one year after seroconversion . Follow-up time was censored at July 16 cells per ml in round bottom tubes and stimulated with a Gag-peptide pool  in the presence of co-stimulation  and 2 µg/ml αCD49d ) for 6 hours at 37°C, 5% CO2. As a positive control, cells were stimulated with a combination of PMA and Ionomycin . After one hour, Brefeldin A (Becton Dickinson (BD), San José, California, United States) was added. After fixation and permeabilisation  cells were stained for 20 minutes at 4°C with αCD3-PerCP, αCD4-APC, αIL-2-PE and αIFNγ-FITC monoclonal antibodies (BD), after which cells were fixed in Cellfix (BD) and flow cytometry was performed. First, lymphocytes were gated based on forward scatter and side scatter. Next, cytokine production  was measured in CD3+ CD4− T cells. At least 300.000 events were acquired and the data was analysed using the software program CELL Quest (BD). Frequencies of IFNγ and IL-2 producing cells were reported after subtraction of the frequencies in medium controls. Absolute numbers per volume blood were calculated by multiplication of the fraction of cytokine-positive CD8+ T cells with the absolute CD8+ T-cell count per microliter of blood.Intracellular cytokine staining was performed as previously described + T cells for progression to AIDS was tested using Cox proportional hazards analyses and Kaplan-Meier survival curves. Individual markers were first tested univariately. In multivariate analyses, each marker was adjusted for plasma HIV-1 RNA and CD4+ T-cell counts + T cells, or co-expression of CD38 and HLA-DR on CD8+ T cells, all markers known to be predictive for progression to AIDS The effect of HIV-specific cytokine-producing CD8+ T cells early after HIV infection, we analysed the capability of Gag-specific CD8+ T cells to produce cytokines upon in vitro stimulation of T cells sampled approximately one year after seroconversion in each individual , when the viral set point has been established. HLA class I typing was performed for all study participants. This revealed no selection of the HLA alleles known to be associated with either faster (e.g. HLA-B35) or slower  progression to AIDS . Forty of the 96 participants (42%) progressed to AIDS during follow-up. In this prospective cohort study, 96 participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS (ACS) with a known date of HIV-1 seroconversion were included. Since we aimed to investigate the potential prognostic effect of functional CD8 to AIDS . Detaile+ T cells approximately one year after seroconversion was associated with AIDS-free survival, we measured the number and percentage of IFNγ and/or IL-2 secreting CD8+ T cells after in vitro stimulation with an overlapping Gag-peptide pool  were comparable to previously reported results by us and others in treatment-naïve patients To test if the presence of HIV-specific cytokine-producing CD8ide pool . We exam+ T cells was plotted against the AIDS-free survival time of the study participants. No correlation could be found for any of the three cytokine profiles with AIDS-free survival time . Furthermore, no correlation was observed between IL-2 and/or IFNγ producing Gag-specific CD8+ T cells and set point HIV RNA load  we first categorized each study participant in one of three equally-sized groups corresponding to the level of production of the specific cytokine  relative to the overall study population. One year after seroconversion, neither the absolute number of single IFNγ producing .To investigate the potential prognostic value of cytokine-producing CD8roducing , nor of roducing  nor of droducing  Gag-spec+ T cells, we also performed multivariate analyses using Cox proportional hazard analysis. When adjusted, respectively, for HIV-1 RNA load, total CD4+ T-cell counts, Ki67 expression on CD4+ T cells, and/or the activation status of CD4+ or CD8+ T cells, high numbers of cytokine-producing Gag-specific CD8+ T cells one year after seroconversion were again not associated with slow disease progression .Alternatively we analyzed CD4+ and high CD8+ T-cell responses one year after seroconversion was associated with slow progression to AIDS. This revealed that individuals whose CD4+and CD8+ Gag-specific T-cell response were in the highest tertile had a tendency for prolonged AIDS-free survival. This result was not significant, however, which might be due to the low number of individuals who met these criteria .Finally, we analyzed whether a combination of relatively high CD4+ T cells measured approximately one year after seroconversion for progression towards AIDS, although at least two previous studies have suggested that high frequencies of HIV-specific T cells are associated with subsequent long-term non-progression It has been suggested that strong CTL responses might be capable of controlling HIV replication in individuals with a long-term non-progressive disease course. In the present study we did not find any prognostic value of cytokine-producing HIV-specific CD8+ T cells to investigate their prognostic value for AIDS-free survival. The individuals included in this study were infected in the late nineteen-eighties in Amsterdam, The Netherlands, therefore, we decided to use an overlapping peptide pool based on Gag sequences from the in 1983 isolated reference strain HXB2. There is always a risk of missing responses when using peptides that are not based on the autologous sequence. However, it has been shown that a large proportion of all T cell responses detected using autologous peptides was detected using consensus sequences as well + T-cell responses are most important in containing HIV infection. Kiepiela et al have shown a clear negative correlation between the number of epitopes targeted in Gag and HIV viral load, which was not seen for other HIV proteins et al have shown that the latter HLA alleles have an intrinsic preference to present Gag P24-derived epitopes, in contrast to other HLA alleles not associated with low viral load and slow disease progression + T-cell responses are implicated and would be the first to show an effect. Even using this potentially important protein we did not observe an association with AIDS-free survival. We cannot rule out the possibility, however that, in the early phase of chronic HIV infection, responses to early HIV-1 proteins dominate and might in part determine viral set point We quantified Gag-specific CD8+ T-cell responses one year after seroconversion might not be early enough to reveal a possible cause-effect relationship between HIV-specific cellular immunity and disease progression. However, since i) at that time all individuals have established their viral load set point and the immune system has largely rebounded from the initial effects of acute infection, ii) measuring CD8+ T cell responses too early after seroconversion might lead to false negative responses since it has been shown that the magnitude of the HIV-specific CD8+ T cell response is much lower in primary infection than in the chronic phase + T cell capable of secreting cytokines , we did not find any correlation what so ever . Therefore, it appears that the exact time of sampling after seroconversion was not critical.One could argue that measuring CD8+ T cells one year after seroconversion has no prognostic value for the rate of disease progression does not imply that HIV-specific CD8+ T cells do not contribute to control during HIV infection. Their role in controlling viral load in both acute and chronic HIV infection has been firmly established + T cells can have on the virus + T cells can be found shortly after seroconversion.Our finding that the frequency of cytokine-producing HIV-specific CD8+ T cells producing IFNγ, IL-2, or both, one year after seroconversion +and CD8+ Gag-specific T-cell response are particularly high have a tendency for longer AIDS-free survival. In the present study this holds true for only a small fraction of HIV-infected individuals, so it cannot explain the wide spectrum in HIV disease progression rates observed in untreated HIV infection. If neither CD4+ nor CD8+ HIV-specific T cells are associated with the rate of HIV-disease progression, the question remains what distinguishes individuals who progress rapidly or slowly to AIDS, and why typical progressors lose their functional HIV-specific CD8+ T cells faster compared to individuals with a long-term non-progressive disease course. It cannot be ruled out that other functions of CD8+ T cells than IFNγ or IL-2 production + T-cell repertoire may determine the clinical outcome of HIV infection. Since both progressors and long-term non-progressors have abundant T cell immunity of the type associated with low viral load early in infection, the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression, for which systemic immune activation might be a likely candidate We have previously reported a lack of prognostic value of the number of HIV-specific CD4+ T cell-inducing vaccine has failed in a phase IIB clinical trial + T-cell responses to the vaccine components  after vaccination. Although disappointing, these data are in line with our finding that the presence of high numbers of cytokine-producing HIV-specific CD8+ T cells does not guarantee a better clinical outcome.Recently, it became apparent that a CD8