In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and –B alleles and against heterologous MHC-Ineg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56neg/CD16pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24pos blasts derived from primary T cells.Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research.  In vitro studies with exogenously infected CD4+ T cell blasts from healthy donors have demonstrated that NK cells can kill autologous HIV-1-infected target cells. However, the ability of NK cells from HIV-1-infected viremic patients to kill autologous, endogenously infected CD4+ T cells had never been examined and remains uncertain. Given the reported abnormalities in phenotype and functions of NK cells from HIV-infected viremic individuals, we determined the function of NK cells in killing HIV-1-infected target cells under conditions that more closely mimic the in vivo environment in HIV-infected individuals. We show that NK cells from HIV-1-infected viremic patients display a variable although generally low ability to selectively eliminate autologous and endogenously HIV-1-infected CD4+ T cell blasts expanded ex vivo from peripheral blood. Various factors, including the markedly defective engagement of important NK cell activation pathways and high frequencies of the pathologic CD56neg/CD16pos NK cell subset in HIV-1-infected viremic patients, influenced NK cell–mediated cytolysis of endogenously infected CD4+ T cell blasts.Natural killer (NK) cells represent an important line of defense against viral infections.  Natural killer (NK) cells are important effectors of innate immune responses and are capable of providing cellular immunity against tumor-transformed and virally-infected cells, without prior antigen sensitization neg/CD16pos (CD56neg) NK cell subset that is preferentially expanded in HIV-1 infected viremic patients Several studies have already described numerous aberrancies of NK cell phenotype and function in chronically HIV-1 infected patients with high levels of ongoing viral replication. These abnormalities include aberrant expression and function of several iNKRs and natural cytotoxicity receptors (NCRs), markedly impaired cytolytic activity against tumor cell targets, defective production of important antiviral cytokines Because the frequency of peripheral blood CD4+ T cells that harbor replication-competent virus is extremely low in HIV-1 infected patients in vitro infection with different HIV-1 strains have been developed. Through these experimental methods, several reports show that HIV-1 selectively down-modulates HLA-A and -B alleles in both cell lines and CD4+ T cell-derived blasts in vitro infection have significantly contributed to understanding the cellular interactions between NK cells and autologous HIV-1 infected CD4+ T cell blasts, it is still unclear what role, if any, NK cells obtained from HIV-1 infected viremic patients play in the clearance of endogenously infected autologous CD4+ T cells ex vivo.In order to further understand the direct effects of HIV-1 on CD4+ T cells and other cell types, several models of In the present study, we describe the killing of endogenously infected CD4+ T cell blasts by autologous NK cells from HIV-1 infected viremic individuals. In addition, we describe several mechanisms involved in the regulation of this NK cell-mediated killing. Finally, we characterize phenotypically the endogenously HIV-1 infected CD4+ T cell blasts used as targets in this experimental system.in vitro with different stimuli of either total PBMCs or purified CD4+ T cells from HIV-1 infected individuals induces viral expression and replication in vitro  and started to decrease progressively after this time point  with a simultaneous down-modulation of cell surface CD4 . In contrast, the expression of HLA-C and HLA-E molecules did not substantially differ between p24neg and p24neg blasts   . To furts blasts .pos/CD4neg cell blasts was still relatively low and variable from patient to patient . Even when interactions between iNKRs and MHC-I were completely blocked, NK cells only partially eliminated both p24pos and p24neg autologous CD4+ T cell blasts   . In orde: 38.4%) . A possiIf the dominant and negative effect of interactions between iNKRs and MHC-I molecules is partially overcome by the HIV-1 induced selective down-modulation of HLA-A and –B alleles, NK cell killing of autologous and endogenously infected CD4+ T cell blasts must occur through activating NK cell receptors. In order to identify those NK receptor(s) that trigger this NK cell-mediated lysis, we analyzed the phenotype and functions of NCRs and NKG2D that, under physiological conditions, represent the major activating receptors that regulate NK cell cytotoxicity.pos/CD4neg cell blasts, indicating that the NCRs were not playing a substantial role in this killing . These data raise the question of whether soluble human Ig fusion proteins may be sensitive enough in detecting all ligands for NCRs.We also analyzed the expression of cellular ligands for NCRs using soluble human NCR-Ig fusion proteins. We found that the surface levels of NCR ligands on p24 ligands . The facneg subset of NK cells in which surface expression of NKp46 and NKp30 and cytolytic activitiy against tumor cell line targets were found to be very low pos blasts is inversely correlated with the frequencies of anergic CD56neg NK cells   plays an important role in the NK cell killing of endogenously HIV-1 infected CD4+ T cell blasts As previously demonstrated for NCRs, we then performed masking experiments to understand the extent of the contribution of NKG2D to the NK cell-mediated killing of infected p24<0.0001) . Moreovel blasts . Consistpos CD4+ T cell blasts   .pos blasts become partially susceptible to lysis by rIL-2 activated NK cells, while p24neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, decreased NK cell expressions of NCRs contribute to the low level of NK cell cytolytic activity. In addition, the unusually high frequency of dysfunctional CD56neg NK cell subsets among HIV-1 infected viremic patients was shown to strongly correlate with the low degree of NK cell cytolytic responses against infected p24pos cell blasts.In the present study we demonstrate the ability of NK cells from HIV-1 infected viremic patients to kill endogenously HIV-1-infected autologous CD4+ T cell blasts derived from viremic patients. We show that, subsequent to the selective down-modulation of MHC-I molecules, infected p24in vitro with exogenously infected CD4+ T cell blasts from healthy donors in vivo situation in HIV-infected individuals. Our experimental system relied on rIL-2 activated NK cells instead of freshly purified NK cells. We previously reported that prolonged activation with rIL-2 did not restore phenotype and function of highly dysfunctional NK cells from HIV-1 infected viremic individuals. Only CD56 expression was recovered upon activation with rIL-2 after 3 weeks of culture. Despite the reversion of the CD56neg to a CD56pos phenotype, rIL-2 stimulated CD56neg-derived NK cell populations still expressed very high percentages of iNKRs and extremely low levels of NCRs, similar to results from experiments performed with freshly purified CD56neg NK cell subsets. Even after 21 days of activation with rIL-2 the cytolytic potential of these highly dysfunctional NK cells from HIV-1 infected viremic patients did not improve and remained significantly lower compared to that of NK cells from uninfected individualsThe ability of NK cells to kill autologous HIV-1 infected target cells mainly through the NKG2D pathway has been previously demonstrated in vitro with several and multiple stimuli enhanced viral replication in CD4+ T cells from HIV-1 infected patients. The in vitro induced replication of HIV-1 was measured by the release of p24 HIV-1 core antigen in culture-supernatant pos cell blasts differed among samples from the 30 HIV-1 infected viremic patients analyzed in the present study. The reasons for such heterogeneous results are unclear and many variables such as cellular or soluble suppressive factors, different numbers of circulating latently HIV-1 infected cells, different viral strains, culture conditions or other factors could contribute to this variability. Further investigations are needed to extensively characterize the kinetics of HIV-1 in endogenously infected cells and the replication cycle of these p24pos/CD4neg cell blasts. Our aim in the present study was restricted to an examination of the interactions between endogenously HIV-1 infected CD4+ T cell blasts and autologous NK cells from HIV-1 infected viremic patients.Circulating CD4+ T cells from HIV-infected individuals harbor very low frequencies of replication-competent virus pos from uninfected p24neg T cell blasts.In the preparation of target cells, we separated infected from uninfected cells on the basis of the lack of expression of CD4 together with intracellular expression of p24 on certain cells (infected) and the expression of CD4 and lack of intracellular expression of p24 on other cells (uninfected). It has been reported that HIV-1 is able to down-modulate the expression of CD4 on T cell surfaces, a phenomenon induced either by Nef, which enhances the internalization and degradation of CD4, or by Vpu and Env, which interfere with the transport of newly synthesized CD4 to cell surface in vitropos/CD4neg cell blasts susceptible to NK cell-mediated killing. In fact, although the surface levels of HLA-C and -E may still protect infected cell blasts from the cytolysis exerted by autologous NK cells expressing iNKRs specific for these conserved alleles of MHC-I pos/CD4neg blasts was significantly higher compared with that of p24neg/CD4pos blasts. Moreover, masking experiments highlighted the important role of the selective down-modulation of HLA-I molecules, because only the complete blocking of all MHC-I alleles rendered infected p24pos and uninfected p24neg cell blasts equally susceptible to NK cell-mediated lysis.In line with results previously reported with HIV-1 infection pos NK cell subset is greatly decreased in chronic HIV-1 infected viremic patients compared to that of healthy donors pos blasts (data not shown). The reason for the discrepancy in the role of NKG2A/HLA-E interactions between these previous studies and our data may be the fact that the effector cells used in those previous studies were heterologous NK cell lines expressing high levels of NKG2A against HLA-E transfected target cell lines.Other studies reported that conserved or even up-regulated levels of HLA-E on HIV-1 infected cells are able to inhibit NK cell-mediated cytolysis of HIV-1 infected cells through binding to its specific inhibitory receptor NKG2A pos/CD4neg cell blasts was found to be mainly NKG2D-dependent. These results are in line with the highly conserved expression of NKG2D on NK cells from HIV-1 infected viremic individuals and with the relatively high percentages of p24pos blasts expressing NKG2D ligands. The direct effect of HIV-1 on the positive or negative modulation of NKG2D ligands on the surfaces of primary CD4+ T cells infected in vitro with HIV-1 is controversial pos/CD4neg blasts, play an important role in NK cell-mediated killing of autologous infected cells. Several groups previously reported that HIV-1 viremia affects several functions of NK cells and dramatically influences their phenotype As mentioned above, the engagement of activating NK cell receptors should be able to trigger the cytolytic activity in NK cells expressing iNKRs specific for HLA-A and -B and lacking iNKRs for HLA-C and-E. In this regard, NK cell-mediated killing of infected p24pos/CD4neg blasts remain still poorly sensitive to killing exerted by autologous NK cells. This is partly the result of inhibitory interactions between iNKRs and conserved HLA-C molecules, as demonstrated by the relatively low levels of killing of both infected and uninfected autologous blasts even in the presence of anti-MHC-I mAbs which completely block the interactions between MHC-I molecules and iNKRs. The relatively low degree of NK cell cytolytic activity against endogenously HIV-1 infected CD4+ T cell blasts might be secondary to aberrancies in NK cell triggering through important activating receptors other than NKG2D. This concept is further supported by the finding that NK cells from HIV-1 infected viremic patients were markedly impaired, compared to that from healthy donors, in their ability to kill highly susceptible target cells such as K562 and 221 tumor cell lines that do not express MHC-I molecules. Moreover, if we compare these experimental data with our previously reported results obtained with HIV-1 infection in vitroneg K562 and 221 cell lines (data not shown) and of endogenously HIV-1 infected autologous CD4+ T cell blasts. These data suggest that the negative effect of HIV-1 viremia on NKp46 and NKp30 expression interfere with the NK cell lysis of endogenously HIV-1 infected autologous CD4+ T cell blasts. HIV-1 infected autologous CD4+ T cell blasts. Our finding of the negative contribution of NCRs in the killing of endogenously infected targets in HIV-infected viremic individuals differs from the findings of a previous study in which we described that NKG2D was important in NK lysis of infected targets, but that NCRs played no demonstrable role in vitro with several viral strains, whereas the present study employed NK cells from HIV-infected viremic individuals and endogenously infected target cells.Although defective in HLA-A and –B expression, p24in vitroex vivo  and the median viremia was 32,677 HIV-1 RNA copies  per ml of plasma as detected by an ultrasensitive branched DNA (bDNA) assay (Chiron) with a lower limit of detection of 50 copies per ml. Patients were either naïve to antiretroviral therpay (ART) or had formerly been receiving ART, but were not receiving therapy at the time of the study. Leukapheresis was conducted in accordance with protocols approved by the Institutional Review Boards (IRBs) of the University of Toronto, Ontario, Canada and the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA. Each patient signed a consent form that was approved by the above IRBs. As negative controls, cells from 30 healthy donors seronegative for HIV-1 were obtained by apheresis generously provided by the Transfusion Medicine Department of the Mark O. Hatfied Clinical Research Center of the NIH as a part of IRB approved clinical studies.PBMCs were obtained from leukapheresis packs by Ficoll-Hypaque density gradient centrigugation . CD4+ T cells and NK cells were freshly isolated by negative selection (Stem Cell Technologies) according to the protocol provided by the manufacturer. The purity of CD3+/CD4+ T cells was ≥97%. Purified NK cells contained ≤ 3% contamination with other PBMC subsets, as determined by expression of CD3, TCR-a/b, TCR-g/d, CD19 or CD14.6/ml) with different stimuli, as shown in 6/ml.In order to expand CD4+ T cell blasts productively and endogenously infected with HIV-1, we activated freshly purified CD4+ T cells  were used in this study: mAbs 289 (IgG2a anti-CD3), C218 and A6-220 , KD1 (IgG2a anti-CD16), AZZ20 and F252 , BAB281 and KL247 , Z231 and KS38 , ON72 and Bat221 (IgG1 anti-NKG2D), MA127 and ON56 , pp35 and Co54 , Ma152 and CER1 , KRA236 and F5 , L14 (IgG2a anti-Nectin 2), L95 (IgG1 anti-poliovirus receptor), Z270 (IgG1 anti-NKG2A), Y9 (IgM anti-CD94), EB6 (IgG1 anti-p58.1/KIR2DL1), Gl183 (IgG1 anti p58.2/KIR2DL2), Z276 (IgG1 anti-p70/KIR3DL1), F278 (IgG1 anti-LIR-1ILT2) and A6.136 . FITC-, PE- or APC-labeled anti-CD3, anti-CD4, anti-CD8, anti-TCRα/β, anti-TCRγ/δ, anti-CD14, anti-CD19, anti-CD56, anti-MICA/B and anti-CD48 mAbs were purchased from BD Biosciences. Soluble fusion proteins for NKp30, NKp46, NKp44 and NKG2D with the Fc portion of human IgG and anti-human ULBP-1,-2 and -3 mAbs were purchased from R&D Systems. PE-labeled anti-human Fc fragment mAb was purchased from Jackson ImmunoResearch Laboratories. FITC- and PE- anti human anti-HIV-1 p24 mAb (clone KC57) used for intracellular flow cytometry staining was purchased from Coulter Clone. PE-labeled anti-HLA-A mAbs were purchased from Lab Vision Corporation. Anti-human HLA-C mAb (clone L31) was kindly provided by Dr. Patrizio Giacomini  and used in flow cytometry as previously describedFor one-, two- or three-color cytofluorimetric analysis , cells were stained with the appropriate FITC-, PE- or APC-labeled mAbs. For indirect staining, cells were stained with appropriate unlabeled mAbs followed by FITC- or PE-conjugated isotype-specific goat anti-mouse second reagent (Southern Biotechnology Associates). Second appropriate anti-isotypic mAbs stained with FITC and/or PE and/or APC were used as negative controls. For intracellular staining, samples were fixed and permeabilized by cytofix/cytoperm solution and washed with perm-wash solution 1X (BD-Pharmigen) according to the protocol provided from the manufacturer. The data were analyzed using FlowJo software (Tree Star Inc.).The percentages of HIV-1 infected CD4+ T cell blasts were detected by intracellular flow cytometry with an anti-p24 core virus antigen mAb.Cells undergo cell cycling were evaluated by detecting the intra-nuclear expression of Ki67 (BD-Pharmigen).3[H]thymidine uptake assay (16 hours). Cellular proliferation was also evaluated by dilution of the vital dye CFSE (Molecular Probes) according to the supplier's instructions.CD4-derived T cell blast proliferation was detected by ) followed by a biotin conjugated mouse anti R-PE mAb (BD). Infected p24pos CD4+ T cell blasts were detected by a Pacific Blue fluorescent-dye conjugate of streptavidin (Molecular probes) according to the supplier's instructions. Fluorescent cells were then washed in PBS, suspended in medium, and sealed on the slides with cover slips. Images were collected on a Leica TCS-NT/SP confocal microscope (Leica) using a 63x oil immersion objective NA 1.32. Pacific Blue was excited using an Argon laser at 364 nm. DIC  images were collected simultaneously with the fluorescence images using the transmitted light detector. Images were processed using Leica TCS-NT/SP software (version 1.6.587), Imaris 3.3.2 (Bitplane AG), and Adobe Photoshop 7.0 (Adobe systems).After 10–12 days of activation with PHA and rIL-2, unfractionated CD4+ T cell blasts were permeabilized by cytofix/cytoperm solution and stained with an PE-labeled anti-p24 HIV-1 core antigen mAb . As a result, we obtained a highly purified population of CD4+ T cell-derived blasts containing ≤5% contamination of other lymphocyte subsets . HIV-1 infected CD4+ T cell blasts were then separated from uninfected blasts on the basis of CD4 surface expression through magnetic microbeads conjugated with an anti-human CD4 mAb , according to the protocol provided by the manufacturer. The purities of fractions of uninfected CD4pos and infected CD4neg blasts , as assessed by intracellular staining with HIV-1 p24 core antigens, was ≥97% and ≥70%, respectively. p24neg/CD4pos and p24pos/CD4neg cell blasts were then used as target cells against autologous rIL-2 activated NK cells in a 4-hour 51Cr release assay as described previouslyIn line with the timeframe of maximal expansion of p2451Cr release assay against MHC-Ineg erythroleukemia K562 and MHC-Ineg B-EBV cell line 721.221 (thereafter termed 221). E/T ratios are indicated in the figures.Polyclonal NK cells were also tested in a 4-hour 51Cr release cytolytic assay were performed on cells from 15 HIV-1 infected viremic patients.pos and p24neg blasts from HIV-1 infected individuals were evaluated using the Wilcoxon signed ranks test. The functional differences between NK cell-mediated baseline lysis and lysis in masking experiments were evaluated using the Wilcoxon signed ranks test. All p-values are 2-sided and unadjusted. All statistical associations between different immune parameters were determined by the Spearman rank test for correlation. To estimate the time of maximal infection, the mean outcome of infected p24pos over time was modeled as a polynomial function of time and estimated using least squares. The maximum of this function was then determined and a bootstrap procedure was used to provide a confidence interval ≥95% for the maximum.Immune response distributions between healthy donors and HIV-1 infected viremic patients were compared using the Mann-Whitney test. The phenotypic and functional differences between p24Figure S1pos blasts were expanded from total PBMCs obtained from HIV-1 infected viremic patients and used as targets for autologous rIL-2 activated NK cellsMethodology. p24(0.05 MB PDF)Click here for additional data file.Figure S2ex vivo of HIV-1 infected CD4+ T cell-derived blasts by using different stimuli. Percentages of p24pos blasts (open squares) expanded at day 12 after activation with rIL-2 or rIL7 alone, with PHA plus rIL-7 or rIL-2±rIL-7 and with anti-CD3 plus anti CD28 mAbs from a representative HIV-1 infected viremic patient.Expansion (0.04 MB PDF)Click here for additional data file.Figure S3pos from p24neg blasts through magnetic microbeads conjugated with an anti-CD4 mAb. Purities of sorted uninfected CD4pos and infected CD4neg T cell blast fractions were assessed by intracellular staining with HIV-1 p24 core antigens in a double color flow cytometric analysis.Sorting of HIV-1 infected and uninfected CD4+ T cell-derived blasts. Representative example of separation of p24(0.04 MB PDF)Click here for additional data file.Figure S4neg tumor target cell lines. Spontaneous killing of K562 (A) and 221 (B) tumor cell lines by rIL-2 activated NK cells-. Data are presented as the average of experiments conducted on 15 healthy donors (black squares) and 15 HIV-1 infected viremic patients (red diamonds).Cytolytic Activity of NK cells against HLA-I(0.01 MB PDF)Click here for additional data file.Figure S5neg (upper left quadrants of dot plot graphs and green lines in histogram graphs) and p24pos (upper right quadrants of dot plot graphs and red lines in histogram graphs) blasts derived from a representative HIV-1 infected viremic patient. Data are indicated as percentage of expression (A) and as MFI (b). (C) Cytolysis (in triplicate ±SD) of autologous p24neg/CD4pos blasts exerted by rIL-2 activated NK cells purified from a representative HIV-1 infected viremic patient. Cells were incubated either in the absence (baseline lysis) or in the presence of specific mAbs masking 2B4 and NTBA. We used an anti-human CD56 IgM mAb as an isotype control for masking experiments. The NK cell:CD4-derived blast ratio in all experiments was 10∶1.NK cell-mediated killing of autologous HIV-1 infected CD4+ T cell-derived blasts: role of 2B4 and NTBA and expression of their ligands on cell targets. (A-B) Surface expression of CD48 and NTBA in p24(0.04 MB PDF)Click here for additional data file.


Expression levels of cell surface antigens such as CD38 and HLA-DR are related to HIV disease stages. To date, the immunophenotyping of cell surface antigens relies on flow cytometry, allowing estimation of 3–6 markers at a time. The recently described DotScan antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens. This new technology provides new opportunities to identify novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient.Using this new technology, we compared cell surface antigen expression on purified CD4+ and CD8+ T cells between 3 HIV disease groups  with HIV plasma viral loads <50 copies/ml; and HIV+ patients with viremia during HAART) and uninfected controls. Pairwise comparisons identified 17 statistically differential cell surface antigens including 5 novel ones , not previously reported. Notably, changes in activation marker expression were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells.Our study not only confirmed cell surface antigens previously reported to be related to HIV disease stages, but also identified 5 novel ones. Of these five, three markers point to major changes in responsiveness to certain cytokines, which are involved in Th1 responses. For the first time our study shows how density of cell surface antigens could be efficiently exploited in an array manner in relation to HIV disease stages. This new platform of identifying disease markers can be further extended to study other diseases. Pneumocystis carinii pneumonia [HIV infection leads to characteristic alterations in the subset composition of circulating CD4+ and CD8+ T lymphocytes. The activation marker CD38, in particular, and its level of expression on CD8+ T cells is a marker that is strongly associated with immune activation, particularly during primary HIV-1 infection and progression to AIDS, respectively . The enuneumonia . Howeveret al. [To date, the immunophenotyping of CD antigens relies on flow cytometry. Although very reliable, the flow cytometry only allows estimation of 3–6 markers in a given assay. The recently developed antibody microarray technology enables the simultaneous analysis of a large number of cell surface antigens on a single chip. This new technology may permit the identification of novel differential markers expressed or co-expressed on CD4+ and CD8+ T cells, which could aid in defining the stage of evolution of HIV infection and the immune status of the patient . This anet al. , who useHere, we have used an antibody microarray constructed on the surface of a nitrocellulose coated slide to simultaneously analyze 135 different cell surface antigens (128 cluster of differentiation antigens plus 7 other surface antigens) on peripheral blood CD4+ and CD8+ T cell subsets from HIV+ and HIV- individuals. A comparison of CD4+ and CD8+ T cells purified from peripheral blood of three different HIV-infected patient groups . LTNP-CD4 differed from BDL and/or VIR groups in that CD71, HLA-DR, CD38, CD3epsilon and CD183 were significantly upregulated.The signature pattern for each group and representative dot pattern from which the raw data were derived are shown in Figure To date the analysis of cell surface antigens on peripheral blood lymphocytes of HIV infected individuals has been largely carried out with whole PBMC using flow cytometry. In this study we used DotScan antibody microarray technology  to simultaneously analyze CD4+ and CD8+ T cell subsets obtained from HIV+ individuals at different stages of HIV disease for the expression of 135 different cell surface antigens. For the first time, our study shows how the immunophenotype of diverse blood cell types (in this case CD4+ and CD8+ T cells) can be exploited to study various HIV disease stages using antibody microarray technology. Our analyses identified 17 statistically significant (p < 0.05) differentiating CD antigens distributed between CD4+ and CD8+ T cells. Of these, 11  were differential for CD4+ T cells, while 10  for CD8+ T cells. Among these, CD212b1, CD218a, CD183, CD3epsilon and CD9 have not been previously described in relation to HIV disease.For the activation markers, the results are in accordance with previous studies using flow cytometry, confirming the utility of this new technology. As previously reported ,14, HLA-In addition, CD27 was significantly downregulated on CD8+ T cells in the LTNP group compared to the NEG group. It has been suggested in one study that HIV-specific CD8+ T cells that have differentiated to the CD27- stage are related to the delayed disease progression , while aWe also observed significantly increased CD95 expression on both CD4+ and CD8+ T cells in the VIR group. Although partially elevated CD95 levels were also observed in BDL on HAART and LTNP groups, the increases were not significant, which is consistent with reduced immune activation in patients with reduced viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes has previously been demonstrated in a large group of HIV-1-infected patients when compared against normal controls , and eviThe most notable feature for the activation markers was significant downregulation of CD9 expression on CD8+ T cells in the VIR group compared to the NEG group. CD9 belongs to a transmembrane protein family known as the tetraspanin family. It has been proposed that these proteins act as scaffolding proteins by laterally organizing cellular membranes via specific associations with each other and distinct integrins. A recent study has shown that the tetraspanin-enriched microdomains on the cell membrane can function as gateways for HIV egress . The oveIt has been suggested that CD57 is a marker for replicative senescence ,22. We fThree cytokine receptors  were found to be significantly upregulated on CD4+ T cells in LTNP group, indicating a polarized Th1 cell immune response in LTNP group in HIV infection. The CD4+ T cells of the LTNP group expressed higher levels of CD183 (CXCR3) than those of the BDL and VIR groups. Since CD183 is reported to be preferentially expressed on Th1 versus Th2 cells in peripheral blood ,31 and tWith regard to cell signaling, CD3epsilon expression on CD4+ T cells was found to be significantly lower in the VIR group than in LTNP, which indicates that the expression level of CD3epsilon can be used to differentiate VIR and LTNP groups. A previous study on HIV+ patients has shown that the expression of CD3 complex  is downregulated on T cells compared to healthy control , while oSignificant group-specific differences were also found for 3 cell adhesion molecules , which may have significant implications for the pathogenesis of HIV disease, since adhesion molecules can affect cell distribution, migration and immune response. CD11b was significantly upregulated on CD4+ T cells in the VIR group compared with the NEG group. This increase was also seen in the BDL and LTNP groups, but was not statistically significant. Although there is evidence for increases in CD8+/CD11b+ T cells during progression of HIV infection in asymptomatic patients , we are Interestingly, the expression levels of two NK associated receptors on CD8+ T cells, CD16 and CD56 were significantly higher for LTNP than for BDL and VIR groups, though not all detected differences reached statistical significance (p = 0.0093 to 0.0871). CD56 is expressed on a subset of CD8+ T cells (mature cytolytic effector cells) and it has previously been suggested that the defective expression of CD56 on these cells in HIV-infected individuals could contribute to the decreased peripheral blood T-cell cytotoxicity found in HIV infection . These fDotScan antibody microarray technology enabled the identification of 3 distinct HIV disease groups based on an extensive immunophenotypic characterization of the patients' CD4+ and CD8+ peripheral blood T cells. This research not only confirmed previously reported findings from flow cytometric investigations, but also demonstrated the power of the antibody microarray technology, by identifying 5 new cell surface antigens that may potentially be associated with HIV disease stages. Simultaneous screening for a large number of cell surface antigens revealed that changes in the expression of activation markers were more pronounced in CD8+ T cells, whereas changes in the expression of cell membrane receptors for cytokines and chemokines were more pronounced in CD4+ T cells.Since these changes were shown to be related to the disease status, we suggest that the use of this technology will facilitate further investigation of the causes and control of HIV disease progression and eventually lead to a better understanding of the pathogenesis of the disease. Our study is the first to demonstrate how density of cell surface antigens can be efficiently exploited in an array manner in relation to disease stages. This new platform of identifying disease markers can be further extended to study other diseases. Increasing patient group size should correspondingly improve the statistical significance of the observed differences in antigen expression associated with disease stage. A simplified protocol of direct purification of CD4+ or CD8+ T cells from whole blood may also allow a broader diagnostic utility. Serial time course studies of patients during their disease progression should also provide useful information on the modulation of cell surface antigens over time and could potentially identify new prognostic and therapeutic markers relevant to HIV disease, enabling prediction of patient responsiveness to therapy.1) Healthy HIV- individuals ; (2) HIV+ individuals on HAART with "below detectable levels" of plasma viremia and classed as patients controlling viremia with HAART ; (3) HIV+ individuals on HAART with detectable plasma viremia ; (4) Treatment naïve HIV+ long-term non-progressors , who have maintained high CD4+ T cell counts (>500 cells/μl), with the average infection time of >20 years and natural control of plasma viremia to below detectable levels. One of the 5 LTNP patients (patient 26) in this category did show very low plasma viremia (128 HIV RNA copies/ml of plasma), but was included because this patient met all the other selection criteria.Blood (20 ml EDTA) was obtained from 26 HIV+ individuals attending HIV clinic at the Westmead Hospital Table  and 5 HIvice versa further confirmed that cross contamination was negligible and would not compromise assay specificity.A single blood sample (20 ml) was obtained from each patient. After separation of plasma, PBMC were isolated by Ficoll-gradient centrifugation and then purified. CD4+ and CD8+ T cells, respectively, were obtained by positive isolation with antibody-conjugated magnetic beads according to the manufacturer's instructions . Flow-cytometric analysis performed on separated CD4+ and CD8+ T cell populations demonstrated that in CD4+ T cell isolations 99.2% ± 0.165% (mean ± SD) of cells were single positive for CD4 marker, while 99.1% ± 0.128 (mean ± SD) of purified CD8+ cells were single positive for CD8 marker . AbsenceTM microarrays, prepared as previously described [Medsaic Pty. Ltd.  provided the DotScanescribed . Monoclo6 cells) was incubated for 30 min on the microarray chip, after which unbound cells were removed by gentle immersion in PBS. Captured cells were fixed and imaged using a Medsaic DotReader™ and dot intensities were quantified for each antigen in duplicate using Dotscan data analysis software on an 8-bit pixel grey scale from 0–255 that reflects the level of expression of a particular antigen as well as the proportion of cells expressing that antigen [Purified CD4+ and CD8+ T cell populations were tested on antibody microarrays using DotScan technology as previously described . Briefly antigen . The limThe main strength of antibody microarray is its capacity to rapidly screen for a large number of antigens, producing an extensive immunophenotype using a relatively small number of cells in a single assay. However, it would not provide all of the information obtained by flow cytometry such as multiparameter analysis on single cells and level of antigen expression per cell. When the same sample is tested by the same operator on 3 different arrays (unpublished data), the coefficient of variation (CV) for binding densities tends to be low (8.3%) for dots of high density (>50 pixels), but higher (33.3%) for dots of low density (2–50 pixels). Reproducible dot binding patterns can be achieved if a technically consistent standard assay protocol is followed for all assays.a priori. No fewer than 5 samples per group were included for statistical analysis, as required for the application of Medsaic's standard bioinformatics tools used in this study. Data were log transformed and log transformed antibody bindings have a symmetrical distribution which shows reasonable stability of variance with mean expression. Following transformation, the distributional properties for individual antibodies were examined using box plots and kernel density estimators. Differential expression was analysed on an antibody-by-antibody basis. Individual intra-group comparisons were first carried out, followed by pairwise inter-group comparisons between the four study groups. The "Antibody Ranking" analysis of the array binding results was carried out as a one way analysis of variance, which provided p values and adjusted p values for each antibody and ranked the antibodies in order of significance. P values were adjusted using Holm's method, a conservative approach to maintain strong control of an inflated type I error rate [The objective of this analysis was to identify antibody dots showing differential levels of binding of cells derived from different disease categories, where the sample categories had been established ror rate . DiffereAbbreviations used in this paper: BDL, below detection level;HAART, highly active antiretroviral therapy;   LTNP, long-term non-progressor;NEG, HIV seronegative individuals; VIR, viremic patients.The author(s) declare that they have no competing interests.JQW fully performed the work, analyzed data and wrote the paper. BW contributed to the writing. LB and JC analyzed data, did statistical evaluation, contributed to the technology and the writing. JL and DED contributed to vital patient samples and immunological interpretation of findings. WBD, JZ, ALC, NKS designed the research project, supervised this work and contributed to the writing. All authors read and approved the final manuscript.


The mucosal pathogenesis of HIV has been shown to be an important feature of infection and disease progression. HIV-1 infection causes depletion of intestinal lamina propria CD4+ T cells (LPL), therefore, intestinal CD4+ T cell preservation may be a useful correlate of protection in evaluating vaccine candidates. Vaccine studies employing the cat/FIV and macaque/SIV models frequently use high doses of parenterally administered challenge virus to ensure high plasma viremia in control animals. However, it is unclear if loss of mucosal T cells would occur regardless of initial viral inoculum dose. The objective of this study was to determine the acute effect of viral dose on mucosal leukocytes and associated innate and adaptive immune responses.Cats were vaginally inoculated with a high, middle or low dose of cell-associated and cell-free FIV. PBMC, serum and plasma were assessed every two weeks with tissues assessed eight weeks following infection. We found that irrespective of mucosally administered viral dose, FIV infection was induced in all cats. However, viremia was present in only half of the cats, and viral dose was unrelated to the development of viremia. Importantly, regardless of viral dose, all cats experienced significant losses of intestinal CD4+ LPL and CD8+ intraepithelial lymphocytes (IEL). Innate immune responses by CD56+CD3- NK cells correlated with aviremia and apparent occult infection but did not protect mucosal T cells. CD4+ and CD8+ T cells in viremic cats were more likely to produce cytokines in response to Gag stimulation, whereas aviremic cats T cells tended to produce cytokines in response to Env stimulation. However, while cell-mediated immune responses in aviremic cats may have helped reduce viral replication, they could not be correlated to the levels of viremia. Robust production of anti-FIV antibodies was positively correlated with the magnitude of viremia.Our results indicate that mucosal immune pathogenesis could be used as a rapid indicator of vaccine success or failure when combined with a physiologically relevant low dose mucosal challenge. We also show that innate immune responses may play an important role in controlling viral replication following acute mucosal infection, which has not been previously identified. Identification of appropriate and reliable correlates of protection has been elusive in pathogenesis and vaccine studies. Many potential immunologic correlates have been suggested including cytotoxic CD8+ T cells, neutralizing antibodies, and preservation of memory and effector lymphocyte populations in the gastrointestinal mucosa [The recent failure of the STEP clinical trial of the MRKAd5 HIV-1 gag/pol/nef vaccine has raised important questions about vaccine development for HIV-1. Particil mucosa . Howeverl mucosa -12, havel mucosa -16.Collectively, these observations raise new questions about defining correlates of protection and how they could be more clearly distinguished in the context of future vaccine trials ,18. FurtVaccine studies using animal models often employ high doses of challenge virus to ensure a high viral set point in control animals so that a reduction of viral burden in vaccinated animals can be used as an indicator of efficacy. Unfortunately, high challenge doses do not mimic natural infection and could lead to flawed conclusions about the true efficacy of a vaccine . The majIn the present study, we employed the well-described cat/feline immunodeficiency virus (FIV) model -39 to in4 to 2.0 × 103 copies/ml plasma), 3/6 middle  and 2/5 low  dose inoculated cats. Provirus in PBMC was detected in 3/6 high, 1/6 middle and 2/5 low dose inoculated cats. Virus was isolated from unfractionated bone marrow in 5/6 high, 5/6 middle, and 5/5 low dose inoculated cats. By at least one of these measures, each cat, regardless of inoculum dose, was infected with FIV. Interestingly, although shown to be infected, eight cats did not have detectible plasma viremia.Cats vaginally infected with high, middle, or low doses of cell-associated and cell-free virus were evaluated for viral load by PCR and virus isolation. Figure Two weeks post-infection, absolute CD4+ T cell numbers in PBMC Figure  were dec7 vs. FIV-infected LPL yield of 3.94 × 107, p = 0.00037). Furthermore, a significant decrease in the percentage of CD8α+ and CD8β+ T cells was found in IEL from all dose groups as compared to controls , classic NK cells (CD56+CD3-) and NKT cells (CD56+CD3+) in blood, draining lymph node (data not shown), spleen, and IEL. Total CD56+ NK cell expression was significantly decreased in FIV-infected cats as compared to control cats in each site at eight weeks post infection Figure. . Figure Anti-Gag and anti-Env specific CD4+ and CD8+ T cell responses were assessed in PBMC, peripheral and draining lymph nodes, spleen, IEL and LPL  and Env peptides. Significant differences were noted in viremic cats when comparing the production of IFNγ by CD4+ and CD8+ T cells from the lamina propria in response to Gag versus Env peptides. This trend was also evident in CD4+ and CD8+ LPL, which produced both IL-2 and IFNγ against Gag peptides, but not Env peptides.FIV-infected, non-viremic cats were more likely to produce cytokines in response to Env peptide stimulation as compared to viremic cats. This was evident in CD4+ and CD8+ IFNγ specific responses in LPL, IL-2 responses in the draining lymph node (ILN), and IL-2+IFNγ+ producing cells in the ILN. Compared to viremia positive cats, non-viremic cats also showed significant differences in anti-Env responses in CD4+ IL-2 producing PBMC. CD8+ IL-2 producing PBMC also showed a marked difference (p = 0.06). The trend of anti-Env responses was also identified in CD4+IL-2+IFNγ+ LPL and CD8+IL-2+IFNγ+ splenocytes.To understand the contribution humoral immunity may have played in control of viremia, we assessed anti-Gag and anti-Env responses in serum and vaginal wash samples using a highly sensitive chemiluminescent ELISA assay. Two cats did not produce antibodies against either Gag or Env, three cats produced antibodies only to Env, and six cats produced anti-Gag antibodies at levels that would not be detectable using a commercial diagnostic test for FIV. The remaining six cats produced substantial titers to Gag and/or Env Table . Almost Given the trends identified for innate, cell-mediated and humoral responses, we next determined if any of these responses correlated with control of viremia. In Figure Significant inverse correlations to viremia were identified in both PBMC and Spleen CD56+CD3- NK cells, with r = -0.51 and r = -0.52 respectively Figure . Cell me4 to 1.0 × 108, resulting in a potentially significant number of HIV-infected leukocytes in seminal fluid [Parameters of viral challenge are an important consideration in animal model pathogenesis and vaccine studies. The majority of human HIV-1 infections occur via the reproductive mucosa and frequently involve cell-associated and cell-free forms of virus -43. Furtal fluid -46. The al fluid . A goal For obvious reasons there is great interest in individuals with transient or controlled HIV-1 infection (thoroughly reviewed by Shacklett). Some stOf course, determination of virologic status depends on the sensitivity of methods used and tissue compartments that are evaluated. Clinically measurable seroconversion was only evident in 1/8 non-viremic cats, suggesting that these cats have occult infection. In this study, we used whole bone marrow, which has been shown to be a site of latency in FIV and other retroviral infections -51, to iin vitro NK cell function of elite controllers suggested a more limited role for NK cells in control of viral replication [This study also comprehensively evaluated innate, cell-mediated and humoral immunity. NK cells are an important innate immune defense, particularly against intracellular pathogens , as theylication . A limitAs numerous studies have shown an important role for cell-mediated immunity in HIV-1 control, we anticipated T cell function would be correlated with reduced viremia. However, correlation of IFN-γ and IL-2 production by CD4+ and CD8+ T cells yielded surprising results. A positive correlation to viremia was found for LPL CD4+ T cell production of IL-2 in response to Gag stimulation, with a similar trend observed for CD8+ LPL producing IL-2. In addition, while trends for inverse correlation with viremia were present for draining lymph node and LPL responses to Env stimulation, none were significant. This is in spite of significant differences in cytokine production when comparing viremic and non-viremic cats. Further, tissue specific cytokine responses in LPL were evident. Viremic cats were more likely to produce IFNγ and IL-2 against Gag peptides, whereas non-viremic cats produced IFNγ and IL-2 against Env peptides. However, these responses were insufficient to prevent the loss of CD4+ LPL and CD8+ IEL in non-viremic cats. However, we cannot dismiss the possibility that these cytokine responses may have helped reduce mucosal viral reservoirs, preventing widespread viral dissemination and viremia. As has been previously reported, control of viremia or lower viral set points are typically associated with the production of IFNγ and IL-2 within the same cell, or are typically associated with the continued ability to produce IL-2 upon stimulation ,61,62. OAnother surprising result was the strong positive correlation between anti-Env antibody production and the level of viremia. Studies of HIV-1 infected patients and SIV-infected non-human primates have divergent results with respect to anti-Env antibody responses and their role in altering progression of disease course. Some have shown that individuals with profound antibody responses progressed rapidly to AIDS -69. HoweSeveral studies of mucosal pathogenesis in HIV-1 infected humans and SIV infected macaques have focused on depletion of CD4+ LPL, as they are a primary target of infection -78. HoweA key observation in this study was that all cats, regardless of initial viral dose, experienced profound acute losses in mucosal lymphocyte populations. In a previous study we demonstrated that protected vaccinated cats had mucosal immune populations similar to control cats one year after FIV infection, while unvaccinated cats that were non-viremic had disruptions in their mucosal lymphoid compartment . In addiIn summary, this study provides valuable insight into the immune responses associated with early viral control in FIV infection. We found that NK cells may play a greater role in acute viral control than previously believed; however, other immune responses were associated with the ability of some of these cats to control viremia, and prevent seroconversion. We also show that more attention must be directed to dissecting immune responses not previously addressed in acute pathogenesis, with particular attention to innate immunity. Further, we identified the intestinal mucosa as a very sensitive indicator of retroviral infection that is independent of viral dose and seroconversion. Collectively, our data suggest that low dose challenge may be sufficient to test vaccine efficacy when considering mucosal immune integrity as a primary correlate of protection.1, a FIV pathogenic sub-group A virus [5 FIV positive cells and 9.75 × 104 TCID50 cell-free FIV (high dose), 1.88 × 105, FIV positive cells and 4.87 × 104 TCID50 cell-free FIV (middle dose) or 9.3 × 104 FIV positive cells and 2.43 × 104 TCID50 cell-free inoculum in RPMI, or were unexposed controls. Briefly, cats were sedated and placed in sternal recumbency with a small rolled towel placed under their caudal abdomen to elevate the reproductive tract. Cell-associated and cell-free viral inoculums were combined immediately prior to administration in a sterile microcentrifuge tube. The inoculum was atraumatically deposited on the mucosal surface of the vaginal vault using using a pipettor with a blunt polypropylene pipette tip and was completely absorbed in approximately five minutes.Twenty-three specific pathogen free (SPF) cats were obtained from Liberty Labs , group housed and cared for in accordance with AAALAC standards and IACUC guidelines. FIV-infected female cats included high dose (n = 6), middle dose (n = 6) and low dose (n = 5); control cats (n = 6) included 4 female and 2 neutered males. Age was six to eighteen months at euthanasia.Cats were infected with cell-associated and cell-free NCSU A virus . Cell-as4 FIV positive cells and 7.5 × 104 TCID50 cell-free and infected 6/10 control cats [1 showed that all cats vaginally inoculated with 104 - 106 FIV-positive cells became FIV-positive, whereas those infected with 103 - 102 FIV-positive cells were either negative or latently positive [5 but not 2.0 × 103 FIV-positive cells were infected three weeks post-inoculation [Inoculum dose was determined based upon the results of several mucosal inoculation studies. We have previously used a combined dose of 7.5 × 10rol cats . Other spositive . Anotherculation . Given tPlasma, serum and vaginal wash fluids were collected at weeks 0, 2, 4, 6, and 8 post-infection and processed . PeripheReal-time PCR to detect viral RNA in plasma was performed as previously described , with miReal time PCR was performed to quantify FIV provirus  using pr5 co-cultured with 3.0 × 105 FCD4-Ecells or Mya-1 cells, both of which are FIV susceptible cell lines, in triplicate in LBT medium supplemented with 100U/ml recombinant human IL-2 . Supernatants were analyzed for FIV p24 by antigen capture ELISA at 16 and 20 days of culture [Cryopreserved bone marrow samples were thawed, washed, counted and 1.5 × 10 culture . In addiMonoclonal antibodies (mAb) used for cell surface staining were directly conjugated to FITC, PE, biotin, PerCP, APC, PECy7, or Alexa 700. Previously described antibodies included anti-CD4 (3-4F4), anti-CD8α (3.357), Streptavidin-PerCP, and Streptavidin-APC . Other aPBMC, spleen, PLN, ILN, IEL and LPL were cultured overnight in LBT medium without stimulation. Cells were then stimulated with either Gag or Env peptide pools, Con A, or media alone for six hours, with 1.5 μl/ml of 1x Monensin  present for the final five hours. Following culture, cells were washed with PBS and stained with antibodies to CD4 and CD8, washed, and fixed with 4% paraformaldehyde (PFA) for ten minutes, then washed and resuspended in PBS/2%FBS, covered and stored at -4°C. Within 24-48 hours of PFA fixation, cells were washed with Perm/Wash Buffer (BD Cytofix/Cytoperm), stained with antibodies to IFNγ, IL-2, and TNFα for 30 minutes, washed and analyzed immediately, collecting 100,000 gated events whenever possible. For analysis, cell populations were initially gated to identify the viable leukocyte population on FSC and SSC. Using this gate, CD4+ and CD8+ T cells were individually gated and CD4+ and CD8+ T cells producing IL-2, IFNγ and TNFα in response to Gag or Env peptide stimulation identified  were coated with 1.0 μg/ml p24-GST fusion protein  and ELISAnti-envelope responses were measured using a previously described envelope fusion protein (gp95-FC) . Anti-EnComparison of viremic versus non-viremic FIV inoculated cats was completed using an unpaired t-test. Statistical analysis of differentially FIV-inoculated groups and control cats utilized a one-way analysis of variance (ANOVA) with a Tukey-Kramer multiple comparison post-test or nonparametric ANOVA  with Dunn's multiple comparison post-test. The choice of test was dictated by testing the assumptions necessary for parametric methods, such that if a parametric method was not appropriate, non-parametric testing was used. Significance was defined as p ≤ 0.05. Analyses were completed using GraphPad InStat version 3.05 .The authors declare that they have no competing interests.KH designed and coordinated the study, collected and processed biological samples, performed FACS assays and virus isolation, analyzed data, performed statistical analysis and drafted the manuscript. SR helped design the study, collected and processed biological samples, performed FACS assays and assisted in data analysis. EE collected and processed biological samples, performed PCR and ELISA assays and assisted in data analysis. GD helped design the study, critically reviewed the results and helped to draft the manuscript. All authors read and approved the final manuscript.Supplemental Figure 1. Gating strategy for intracellular cytokine assessment. Samples stained for surface and intracellular antigens were gated based on forward and side scatter (A). The gated population was used to identify CD4+ and CD8+ T cells (B). Using either CD4+ or CD8+ T cells as the parent gate, specific staining for IFNγ (y-axis) and IL-2 (x-axis) are shown for unstimulated (C) and stimulated samples (D) from the same medial iliac lymph node. For analysis, the percent of cells staining positively in unstimulated samples was subtracted from stimulated samples to determine net cytokine production reported in Figure Click here for file


Timely access to antiretroviral therapy is a key to controlling HIV infection. Late diagnosis and presentation to care diminish the benefits of antiretrovirals and increase risk of transmission. We aimed to identify late presenters in patients sent for first CD4 T cell count after HIV diagnosis, for therapy initiation evaluation. Further we aimed at identifying patient factors associated with higher risk of late presentation.Retrospective data collection and analysis was done for 3680 subjects visiting the laboratory for CD4 T cell counts between 2001 and 2007. We segregated the patients on basis of their CD4 T cell counts after first HIV diagnosis. Factors associated with risk of late presentation to CD4 T cell counts after HIV diagnosis were identified using univariate analysis, and the strength of association of individual factor was assessed by calculation of odds ratios.p < 0.001), and older age groups of 45 years and above (p = 0.0004) to late presentation. Female sex, children below 14 years of age and sexual contact with HIV positive spouse were associated with significantly lower risks to presenting late. Intravenous drug users were also associated with lower risks of late presentation, in comparison to heterosexual transmission route.Of 3680 subjects, 2936 (83.37%) were defined as late presenters. Late testing varied among age groups, transmission categories, and gender. Males were twice as likely to present late as compared to females. We found significant positive association of heterosexual transmission route (The study identifies HIV infected population groups at a higher risk of late presentation to care and treatment. The risk factors identified to be associated with late presentation should be utilised in formulating targeted public health interventions in order to improve early HIV diagnosis. AIDS is a dreaded disease, as even with recent advances in the field of medicine and public health, no effective vaccine or drug therapy is available to completely eliminate the infection. During the natural course of HIV infection, there is a progressive loss of CD4 T cells; the rate of this loss being variable in patients, but averaging around 60-100 cells/uL per year . This drIntroduction of highly effective antiretroviral therapy (HAART) has substantially improved patient prognosis over the past decade ,6. TestiRecent data indicates that early initiation of HAART is advantageous when opportunistic infections are present , as the India, with 2.27 million people living with HIV/AIDS and a disease prevalence of 0.29% (2008-09), accounts for roughly half of Asia's HIV prevalence. New Delhi, the area where the study was conducted, is a low prevalence state. However, being the nation's capital, the state sees a huge influx of immigrants and truckers, thereby increasing the high risk population. Most of the infections occur through the heterosexual route, though the major transmission route in north-eastern India is injection drug use. The first phase National AIDS Control Programme (NACP) was launched in 1992 with the objective of slowing down the HIV infections in order to reduce the morbidity, mortality and impact of HIV epidemic in the country. Since then, the third phase of NACP has been planned and implemented in 2007 with the objective of halting and reversing the HIV epidemic in India over a period of five years. The guiding principle of the NACP - III objectives is provision of universal access to HIV prevention, care, and support and treatment services. The current HIV testing guidelines states client initiated testing, except in case of pregnant females in which case HIV testing is provider initiated. HIV treatment and monitoring is universally accessible and free of cost at NACO designated clinics, and one such clinic is housed in the AIIMS, which is the study's focus. The institute's HIV division consists of an Integrated Counselling and Testing Centre, in addition to a CD4 monitoring laboratory and treatment clinic, all funded and supported by the national program and NACO. The CD4 monitoring laboratory at AIIMS, is housed in the Microbiology department, and provides the monitoring services to all patients referred to it through various OPDs, wards, and specialty clinics at AIIMS providing HIV treatment and care.This is a retrospective cross sectional study of all individuals coming for CD4 testing for the first time after HIV diagnosis , to the CD4 testing laboratory at the Department of Microbiology, All India Institute of Medical Sciences. Subjects included 3,680 HAART naïve individuals with a confirmed diagnosis of HIV with or without clinical symptoms, coming to the laboratory between 2001 and 2007. Subjects on antiretroviral therapy at the time of first visit to the laboratory were excluded from the present study analysis. Similarly, subjects with a positive HIV diagnosis more than one month before entering the HIV care services at AIIMS were also excluded from the study in order to control for the time since first HIV diagnosis. The All India Institute of Medical Sciences Institute Ethics Committee (AIIMS IEC) approved routine HIV diagnosis and CD4 testing.For data analysis, as made classically all subjects with CD4 T+ cell count below 200 cells/uL were classified as 'late presenters to care". Subjects were also stratified into late and very late presenters depending on their CD4 T cell counts being below 200 cells/uL and below 50 cells/uL respectively. Further, subjects with CD4 T cell counts above 200 cells/uL but with chronic clinical symptoms like fever, diarrhoea, weight loss etc, were also included in the category of late presenters to care and in need of immediate start of HAART.Laboratory CD4 test subject database was accessed to obtain following patient characteristics: age, sex, date of CD4 testing, HIV transmission route as reported by the patient, and clinical classification of patients on presence or absence of symptoms. Presence of TB, both pulmonary and extrapulmonary, and Sexually Transmitted Diseases (STDs) were also recorded.Medians were calculated for study variable: age, CD4 T cell counts, etc. Univariate regression analysis was performed to identify patient factors which varied among late presenters and non-late presenters. Multinomial logistic regression analysis was performed to identify independently associated patient factors with risk of presenting late for HIV related treatment and care. Confidence intervals at 95% level of confidence were calculated for each associated factor. A p value of 0.05 or less was considered significant.During 2001-2007, a total of 4,260 patients were tested for CD4 T cell counts at the laboratory. Of these subjects, 3,680 subjects coming for HAART initiation evaluation were retrospectively included in the study, excluding subjects coming for follow-up visit or subjects already on antiretroviral therapy at the time of first visit to laboratory. Patient characteristics according to CD4 T cell counts is summarized in Table Majority of subjects presented with one or more AIDS related and/or AIDS defining illnesses ,12 at thThe study group had a median CD4 T cell count of 242 cells/uL. Males had a lower median CD4 T cell count (207 cells/uL) as compared to females (243 cells/uL) with this difference being statistically significant (p < 0.001). Of the total study population, 2,113 (57.4%) had CD4 T cell counts more than 200 cells/uL, 1,214 (33%) had CD4 T cell count below 200 cells/uL, and 353 (9.6%) had less than 50 CD4 T cells/uL. Patient characteristics according to CD4 T cell counts are summarised in Table Subjects with less than 200 CD4 T cells/uL with (n = 1086) or without clinical symptoms (n = 120), and subjects with clinical HIV infection irrespective of CD4 T cell counts (1509), were together termed as "late presenters to care". Further, subjects with less than 50 CD4 T cells/uL were termed "very late presenters", of which 340 subjects were symptomatic and 13 subjects did not have any clinical symptoms. Four hundred and seventy three (473) late presenters (17.4% of 2723 late presenters) and 91 very late presenters  had clinically defined AIDS at the time of first presenting to the laboratory.Univariate analysis was performed for association between patient characteristics and late presentation, summarised in Table Multinomial logistic regression analysis was carried out to identify patient factors independently associated with late and very late presentation to care. In this analysis we found that females were at a significantly lower risk to present late or very late for CD4 testing as compared to their male counterparts . Similarly age groups 35-44 years , and age group of 45 years and above  were associated with higher frequency of late presentation.On analysing various transmission routes reported by subjects for their association with propensity for late presentation to care, transfusion of blood and blood products, homosexual transmission, occupational exposure and those unaware or unwilling to disclose probable route of HIV acquisition, were all found to increase the risk of late presentation. Further we observed that female sex, age at presentation less than 25, mother to child route of transmission, HIV transmission through sexual contact with infected spouse, and intravenous drug abuse, were all associated with decreased chances of delayed or late presentation to care with most associations statistically significant Table .Late presentation with low baseline CD4 T cell counts and presence of AIDS defining and/or AIDS associated symptoms is the strongest prognostic marker for early mortality in both low and high income countries. Generally late testing and presentation to HIV care centres and programs is a result of diagnostic testing as a consequence of symptoms, while earlier diagnosis is more typical of patient self-request for testing, self-perceived risk, or universal screening . An alarIn the present retrospective study, we set out to examine the level of late presentation to care of HIV infected Indian individuals in a tertiary health care setting and to identify the extent of immunosuppression in these subjects along with presence of opportunistic infections. The study population was mostly male of the reproductive age group of 25-44 years thereby highlighting the most affected population. The significantly low number of females coming to the laboratory for CD4 T cell count underscores the vast gap in health care uptake between males and females. Heterosexual route of transmission was the most common transmission route reported by patients as a possible mode of disease acquisition as is true for majority of HIV cases reported throughout India.Pneumocystis carinii pneumonia, along with patients having less than 200 CD4 T cells/uL of blood at the time of first CD4 T cell count, were classified as late presenters to care. We argued that the presence of chronic illnesses like fever and diarrhoea etc though not AIDS defining, are major pointers to raise suspicion of a possible HIV infection. Further such symptoms would highlight disease chronicity and thus should be labelled as a missed opportunity for early diagnosis and care. Nearly 80% of subjects were classified as late testers based on above criteria. This proportion is very high compared to other studies. As the setting for the study was one of the nation's largest tertiary health care facilities, majority of patients are referred here after efforts to treat them at primary and secondary health care levels and also at private settings. Therefore the proportion of sick and very sick patients is usually higher at the laboratory as compared to the clientele in private or secondary healthcare settings, and can partly explain the huge proportions of late presenters in our study. Also, our inclusion of chronic symptoms associated with HIV infection in addition to AIDS defining events and low CD4 T cell counts further increased this already large number of late presenters. A sizeable number of patients (15.4%) had CDC stage C HIV infection, ie clinical AIDS. This high number of AIDS cases at time of first presentation to care marks an alarming trend, clearly showing the lack of knowledge, and low risk perception of being possibly HIV infected and warrants further and renewed emphasis on health literacy.Several previous studies have reported various ways of characterising late presenters to care. In our study, patients having AIDS associated symptoms like chronic diarrhoea, chronic fever, unexplained weight loss, oral ulcers, etc and AIDS defining illnesses like pulmonary and extra-pulmonary Tuberculosis (TB), STDs, Bacterial diarrhoea, Of all subjects studied, 14.4% had a pulmonary or extrapulmonary TB event at first time presentation to care. Management of TB in late presenters with CD4 T cell counts less than 200 cells/mL is a challenge due to an increased risk of developing IRIS and additive drug toxicities and interactions resulting from antimycobacterial therapy. Treatment in case of suspected IRIS involves close monitoring and symptomatic administration of steroidsAt the time of first CD4 T cell count, 33% of subjects had a CD4 T cell count below 200 cells/uL with 9.5% subjects having CD4 T cell count below 50 cells/uL. This huge number of immunocompromised patients at first visit comprises subjects in need of immediate HAART initiation. Further when segregated on symptoms, 120 patients with CD4 T cell counts less that 200 cells/uL were found to be asymptomatic. As a sizeable part of the study population were ones who visited the laboratory before government of India roll out of free public sector ART and free CD4 testing facilities for HIV infected patients, many subjects without any apparent HIV related symptoms would have been missed for initiation of HAART. Further we observed in our study population, presence of patients with CD4 T cell counts well above 200 cells/uL and with chronic HIV associated symptoms without any AIDS defining illness. Treatment strategy for these patients is not clear in the currently available HIV-1 infection treatment guidelines, and thus most of them just get symptomatic treatment instead of initiation of HAART, thereby limiting the beneficial aspect of early HAART initiation. Men had a lower CD4 T cell count at first presentation to care for HAART evaluation as compared to females, which is consistent with previous study results. This can be attributed to the proportionately low utilization of health care facilities by men as compared to women.In order to identify patient factors associated with late presentation to care, we performed multinomial logistic regression analysis to check the strength of association of each factor independently with late and very late presentation events. The factors associated with a risk of delayed presentation to care as uncovered in present study are consistent with results in other such studies -19. HeteAssociation of higher age groups of 35-44 years ages 45 years and above seen in our study with late and very late presentation to care has been emphasized in literature ,22,23. HIn view of the results, there is a need for expanded routine testing of HIV infection with a stronger focus on men who are observed as most vulnerable population for late presentation. Also, emphasis should be given to testing for HIV in higher age group subjects as they are also more likely to be late presenters. The increased proportion of late presenters highlights the delay in treatment to these individuals, indicating the need to develop national level strategies effective in promoting early diagnosis and of HIV infection in order to increase the impact of universal access HAART. In view of the public health implication of early HIV diagnosis for reducing HIV transmission and preventing late presentation, HIV testing should be more frequently prescribed in all health care settings.The authors declare that they have no competing interests.KM and MV contributed to study inception. MV supervises the laboratory involved in HIV diagnosis and testing. KM, MV, NKC, and SM participated in data analysis, and interpretation. MV provided data analysis inputs. KM and MV drafted the manuscript. All authors read and approved the final manuscript. MV takes responsibility for the paper as a whole.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/416/prepub


Many HIV-infected patients only access health care once they have developed advanced symptomatic disease resulting from AIDS Defining Conditions (ADCs). We carried out a study to establish the effect of ADCs on immunological recovery among patients initiated on antiretroviral therapy (ART).A retrospective cohort of 427 HIV-1 patients who were initiated on ART between January 2002 and December 2006 was studied. Data on ADCs was retrieved from Joint Clinical Research Centre (JCRC) data base and backed up by chart reviews. We employed Kaplan-Meier survival curves to estimate median time to 50 CD4 cells/μl from the baseline value to indicate a good immunological recovery process. Cox proportional hazard models were used at multivariate analysis.3 . A low baseline CD4 count of ≤ 200 cells/μl  was associated with a longer time to immunological recovery. There was no interaction between low CD4 counts and ADC group.The median time to gaining 50 CD4 cells/μl from the baseline value after ART initiation was longer in the ADC (9.3 months) compared to the non-ADC group (6.9 months) . At multivariate analysis after adjusting for age, sex, baseline CD4 count, baseline HIV viral load, total lymphocyte count and adherence level, factors that shortened the median time to immunological recovery after ART initiation were belonging to the non-ADC group , adherence to ART of ≥ 95%  and a total lymphocyte count ≥ 1200 cells/mmPatients with ADCs take longer to regain their CD4 counts due to the defect in the immune system. This may prolong their risk of morbidity and mortality. During 2005, the World Health Organization (WHO) had estimated that there were over 1.3 million people receiving anti retroviral therapy (ART) in low and middle-income countries, representing 20% of 6.5 million estimated to need it [As the number of individuals able to access treatment is increasing, one of the challenges facing ART services in sub-Saharan Africa is that many HIV-infected patients only access healthcare once they have developed advanced symptomatic disease resulting from AIDS Defining Conditions (ADCs). The medThis study was a retrospective cohort. JCRC is one of the established centers providing ART and is located in Kampala, the capital city of Uganda. It was founded in 1991 and has been providing anti-retroviral therapy since1996. Combination ART (cART) became available in 1998 but was quite expensive at that time. Generic Fixed Drug cART which was much cheaper(about $50/month) and more accessible by 2002 under the UNAIDS/Ministry of Health (Uganda) HIV Drug Access Initiative Programme . We retrDiagnosis was done and documented in the charts by the attending physician. This depended on history from the patient, clinical examination, and backed up by laboratory results. HIV wasting syndrome was defined as more than 10% documented loss of body weight and either unexplained chronic diarrhea (> one month), or chronic weakness and unexplained prolonged fever (> one month) with pyrexia recorded on at least on one occasion . CryptosLaboratory measurements included a complete blood cell count, CD4 lymphocyte count, and quantitative measurement of HIV load. Quality control assurance was done in reference to international accredited laboratories; UK National External Quality Assessment Service (UKNEQAS) and College of American Pathologists (CAP).Viral load count was done with use of Amplicor monitor standard assay, version 1.5 (Roche Molecular Systems), with a minimum detection limit of 400 copies/ml. In 2006, an assay with a detection limit of 50 copies/ml was acquired by the unit. Viral load count was not consistently done.CD4 lymphocytes were analyzed by flow cytometry . This was periodically done every 6 months during the routine visit to the out-patient clinic. Frequently, this schedule was not routinely followed and depended upon the discretion of the attending physician during the interim visits when patients were sick. The time to immunological recovery was estimated to the nearest clinic visit and CD4 count. We right censored patients if they were lost to follow-up after a period exceeding 12 weeks or when failed to gain 50 CD4 cells/μl above the baseline value. A patient was considered lost to follow-up if did not make contact with JCRC out-patient clinic for a period exceeding 90 days during the study period. Adherence to ART was determined according to the patient's previous four days recall when visited the JCRC out-patient clinic and was considered when the patient took ≥ 95% of the prescribed medications .We aimed to achieve a sample size with a power of 90% to detect the differences between ADC and non-ADC group, and was estimated using formula for survival analysis . We set st 2002 and December 31st 2006, at JCRC out-patient clinic. This number excludes patients who are currently enrolled in ongoing clinical trials and patients who were monitored by physicians outside the JCRC facility but receiving ART at JCRC pharmacy, whose data is stored else where. A total of 182 records were excluded because the patients were seen once at ART initiation and lacked follow-up visits or had previous exposure to dual ART. The remaining 427 patients whose records were used for analysis, had baseline CD4 load count and had been seen on one or more subsequent visits within a period of 12 months follow-up  patients attained immunological recovery during the study period, giving an overall immunological recovery rate of 2.5 per 1000 patient-days. The proportions of patients achieving immunological recovery were 13.0% (18/138), 39.9% (55/138), 56.5% (78/138), 71.7% (99/138) in the ADC group at 3, 6, 9 and 12 months respectively. For the non-ADC group, the proportion of patients achieving immunological recovery were 13.1% (38/289), 40.8% (118/289), 64.0% (185/289), 77.2% (223/289) at 3, 6, 9 and 12 months respectively. The median time to immunological recovery in the ADC group was longer than that of the non-ADC group (271 vs 208 days),   were associated with reduced time to attain immunological recovery. A low baseline CD4 count (≤ 200 cells/μl) and a high viral load count (≥ 6.0 log copies/ml) were associated with a longer time to attain immunological recovery (Table Old age (> 40 years), having no ADC at ART initiation, a baseline total lymphocyte count of ≥ 1200 cells/mm3 and those with adherence to ART was equal or greater than 95%. A low baseline CD4 count of ≤ 200 cells/μl at ART initiation was associated with a longer time to immunological recovery  in the ADC group compared to 208 days (6.9 months) in the non-ADC group. The ADC group status was associated with a longer median time to immunological recovery after ART initiation in comparison to the non-ADC group. Factors that were independently associated with time to immunological recovery were group status (presence or absence of ADC), baseline CD4 count, baseline total lymphocyte count and adherence to ART. Although, immunological responses to ART have been estimated to be at least an increase of 50–100 CD4 cells/μl/year after ART initiation, the median time to attain this CD4 cell threshold has not been documented ,16. We tThe differences in median times to immunological recovery between the ADC and non-ADC group found in our study may be a result of profound pre-treatment immuno-suppression which is a common feature of ADCs. The ADCs cause a loss of CD4 memory cells that represent a component of the T-cell repertoire that is specific to most ADC infections. The defect in the CD4 memory cells as a result of ADCs accounts for the inability of patients with ADCs to respond to recall antigens and evoke immunological responses leading to a slower immunological recovery process compared to patients without ADCs . Another3) and adherence to ART (≥ 95%). The hazard of attaining immunological recovery reduced when comparing a patient with ≤ 200 CD4 cells/μl to a patient with > 200 cells/μl by 50%. Our results are consistent with other studies where immunological recovery is largely dependent on baseline CD4 count and thus the timing of ART initiation is important in order to maximize the CD4+ T-cell response to therapy [Other determinants that favored a good immunological recovery process after ART initiation were a baseline CD4 count (> 200 cells/μl), baseline total lymphocytes count  where increasing age was associated with a slower and lower immunological recovery due to age-related decreases in thymopoiesis . The 2 cWe also found no effect of sex on median time to immunological recovery. Our findings are similar to other findings else where that show that there are no gender differences between males and females regarding response to ART . On the We recognize our study had the following limitations;2 for men and < 18.5 kg/m2 for women is predictive of poor immunological recovery, increased morbidity and mortality [Our study design did not allow for the control of all possible confounders as it was retrospective cohort. We relied on measurements taken in the past and where a possible confounder was not measured we could not control for it. For example, we were not able to determine the effect of Body Mass Index (BMI) as height measurements were only available in about 35 patients. Although the baseline body weights were available, subsequent measurements during the follow-up visits were missing and only available in 40 patients. A baseline BMI of < 20.3 kg/mortality ,30. We wThe time taken to attain 50 CD4 cells/μl after ART initiation was estimated according nearest date of visit to JCRC outpatient clinic when the CD4 count was done. This estimation could have over or underestimated the true estimate. To minimize errors in estimation of time to attain 50 CD4 cells/μl both the principal investigator and data entrant independently calculated the time, and where differences arose an average was taken. Overall inter-observer kappa statistic was 0.68.Another bias could have resulted from under diagnosis of ADCs because of lack of diagnostic equipments, a common finding in resource limited settings. It is surprising that some of the ADCs were not diagnosed during 2002–2006 when we reviewed records, whereas had been found to be prevalent by other study sites. For example in a nearby main referral hospital, 18% of hospitalized patients presented with non-typhoid salmonella, as an ADC in blood stream . We miniSome of the immune responses after ART initiation were likely to have been evoked by immune reconstitution syndrome (IRS) and diagnosed as ADCs. This is an inflammatory response against infectious and non-infectious antigens and usually occurs within 12 weeks after ART initiation . HoweverWe could also not explain a big loss to follow-up of patients who had enrolled for ART as it was inadequately documented. Of 609 who were eligible and enrolled for ART, 182 (29.9%) could not come for second or subsequent review visits. We could explain the loss follow-up could have resulted from deaths due to profound immuno-suppression at ART initiation. Our findings may have overestimated individuals achieving immunological recovery, as those who died from an AIDS-defining disease shortly after starting ART were excluded from the analysis. It is also possible that these patients sought treatment in other treatment centers elsewhere. Since 2003, Uganda has benefited from roll-out program for ART services where more centers have been accredited to offer HIV services and treatment has been either subsided or become free. The loss to follow-up of 29.9% could have resulted into affecting the generalizability of the study findings. This was minimized by taking precautions to prevent under or over diagnose ADCs by chart review, and correct estimation of time to end points from baseline value by taking independent measurements. Secondly, the baseline characteristics of the patients who were lost to follow-up were similar to the patients who had a subsequent follow-up after the baseline visit . Lastly, findings of this study are strengthened by the relatively homogeneous study population receiving treatment at a single health facility using the national treatment protocols. Patients were all ART-naïve and received a standard triple-drug regimen.We can draw the following conclusions from the study;Patients with ADCs take longer to regain their CD4 counts due to the defect in immune system. This may prolong their risk to morbidity and mortality as a result of opportunistic infections. This paper provides a very strong argument for earlier ART initiation in a Ugandan setting. A key challenge is how to identify and initiate ART among patients with less advanced disease. Early Voluntary Counseling and Testing (VCT), the promotion of VCT within community settings so that people know their HIV sero-status and those who are HIV positive are encouraged so seek early health interventions is one option. Another option would be starting ART at above the threshold of 200 CD4 cells/μl. Recently, the Ugandan ART guidelines has recommended initiating ART at 250 and to 350 cells/μl among pregnant women [The authors declare that they have no competing interests.BKK: Study concept and design, data analysis, interpretation of the study findings and wrote-up the manuscript. SS, MP, BN, CK, EK: Assisted in the interpretation of study findings and the critical revision the manuscript. JK: assisted in data analysis, interpretation of study findings and the critical revision of the final manuscript. MO: Queried and extracted data from JCRC database. PM: assisted in critical revision and the final approval of manuscript. FS: Assisted in the study concept and design, interpretation of the study findings and the critical revision of the manuscript.