Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins  and three immunoglobulin isotypes .Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant.The data obtained indicated that TIL of NPC patients produced significantly more IL-2 , IL-10 , IgM  and IgG  than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 , IL-10  and IgM  than those of controls. No significant differences for IL-6 and IgA were observed.Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC. Undifferentiated nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor characterized by a heavy infiltration of non malignant lymphocytes and most of these tumor infiltrating lymphocytes (TIL) have been shown to be T cells .The Epstein-Barr virus (EBV) is causally associated with this malignancy since viral DNA is regularly present in the malignant epithelial cells but not in the neighbouring normal tissues. In addition, NPC patients show a specific pattern of humoral responses against EBV antigens .Viral proteins known to be expressed in NPC tumor cells are the EBV-encoded nuclear antigen 1 (EBNA-1) and the latent membrane proteins LMP-1 in 35 to 65% of cases, and LMP-2 . The latRecently, CD8 positive EBV-specific cytotoxic T cell clones were isolated from the peripheral blood and tumors of NPC patients . The majThe expression of IL-10 in NPC has been controversial. While it has been reported that IL-10 is not expressed by NPC cells as detected by RNA in situ hybridisation , some reIn this report, we investigated the ability of both peripheral blood lymphocytes (PBL) and TIL of undifferentiated NPC patients to express three interleukins  and three immunoglobulin isotypes  following pokeweed mitogen (PWM) stimulation in vitro.The data obtained indicated some significant differences between NPC patients and controls in interleukin and immunoglobulin production. However, further investigations are needed to establish the relevance of these differences to the discrepancy between humoral and cellular immune responses characteristic of NPC.17 untreated Tunisian NPC patients were included in this study. Informed consent was obtained from all patients before collection of blood and biopsy samples. These patients presented for treatment at the National Cancer Institute Salah Azaiez, Tunis or at the Farhat Hached hospital, Sousse, Tunisia. Their mean age was 47,5 years (range 25–70). All patients were diagnosed histopathologically as undifferentiated NPC.17 healthy EBV carriers who presented to donate blood at the National Blood Transfusion Centre, Tunis, Tunisia, were included as controls in this study.Sera of all patients and controls were titrated for antibodies directed against EBV antigens by indirect immunofluorescence .6 cells/ml.Peripheral blood and biopsy samples were taken on the same day from each of the 17 NPC patients. Peripheral blood was collected from patients and healthy donors in heparin. Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Pharmacia) according to Boyum . Followi6 cells/ml.For the preparation of tumor-infiltrating lymphocytes, biopsies were collected from NPC tumors under sterile conditions and immediately transferred to RPMI-1640 medium supplemented with 50 μg/ml Gentamicin. The biopsies were washed several times in this medium and then minced into small pieces in order to extract lymphocytes. The extracted cells were then washed by centrifugation in the same medium and then resuspended in complete medium at a final concentration of 2 × 102 fragment. The labelled cells were counted under a fluorescence microscope.Samples from each lymphocyte preparation were taken for immunophenotyping by indirect membrane immunofluorescence using mouse monoclonal antibodies directed against CD3, CD4, CD8 or CD19 and FITC-conjugated goat anti-mouse F(ab')6 cells/ml in complete medium were distributed at 1 ml / well in 24-well Costar plates. One hundred microliters of a predetermined optimal dilution of PWM (Gibco), for treated wells, or complete medium alone, for untreated controls, were added in corresponding wells. The cells were then cultured for 12 days at 37°C, 5% CO2 and 98% relative humidity in a CO2 incubator. At the end of this incubation period, culture supernatants were harvested and used for cytokine and immunoglobulin determination as described below.PBL from healthy donors or NPC patients and TIL from the same patients suspended at 2 × 10Culture supernatants of PBL and TIL stimulated by PWM under optimal conditions were used for both cytokine and immunoglobulin determination by enzyme-linked immunosorbent assays (ELISA).IL-2, IL-6 and IL-10 concentrations were measured using commercial sandwich-type ELISA kits  according to the procedures described by the manufacturer.IgM, IgG and IgA concentrations were measured using sandwich-type ELISA assays prepared in our laboratory. Briefly, 96 well-microplates  were coated for 2 hours at 37°C and overnight at 4°C with 150 μl/well of isotype-specific mouse monoclonal antibody  appropriately diluted in 0,1 M carbonate buffer, pH 9.6. The microplates were washed four times with phosphate-buffered saline pH 7.4 containing 0,1 % Tween 20 (PBS-Tween), then blocked with 300 μl/well of PBS containing 1% FCS for 30 minutes at 37°C. For the assay, 100 μl /well of appropriately diluted culture supernatants in PBS-Tween containing 10% FCS were incubated in coated microplates for 2 hours at 37°C. The microplates were then washed four times with PBS-Tween and 100 μl/well of the appropriate alkaline phosphatase-labeled conjugate  were added at a 1:20000 dilution in PBS containing 1% FCS. After a two-hour incubation at 37°C, the microplates were washed and a p-nitrophenylphosphate substrate solution was added. The microplates were incubated for 1 hour at 37°C and the reaction was stopped with 50 μl/well of 1 N NaOH. Immunoglobulin standards were generated using purified human immunoglobulins of each isotype . The microplates were read at 405 nm on a microplate reader . Data were represented as the mean immunoglobulin concentration of triplicate cultures. Immunoglobulin values were obtained by interpolation from standard curves.Data were analysed using "Student's t-test". Probability values below 5% were considered significant.All patients showed a typical serological profile characteristic of NPC as determined by indirect immunofluorescence. Levels of IgG antibodies to VCA were considerably higher than those in healthy donors. Their titers varied from 640 to 2560 (mean titer = 1065), whereas in healthy donors such titers fluctuated between 40 and 160 (mean titer = 70). IgG antibodies to EA were present in NPC patients only, with titers ranging from 20 to 320 (mean titer = 50).Anti-VCA IgA antibodies were detected in all patients  and anti-EA IgA antibodies were detected in only 4 patients . None of the controls showed IgA antibodies against these antigens.Immunophenotypic analysis of the lymphocyte preparations obtained from patients and controls indicated that CD3+ lymphocytes constituted the major subpopulation with a mean frequency of (60 ± 8)% in PBL of both patients and controls and (55 ± 9)% in TIL. CD4+ lymphocytes represented (35 ± 5)% in PBL of both patients and controls and (20 ± 3)% in TIL. CD8+ cells showed mean values of (40 ± 6)% in PBL of patients, (36 ± 4)% in TIL, and (28 ± 5)% in PBL of controls.On the other hand, CD19+ B lymphocytes represented in average (25 ± 5)% in PBL of patients, (23 ± 6)% in TIL, and (22 ± 3)% in PBL of controls.As illustrated in Figures Unstimulated lymphocytes cultured in the absence of PWM did not show any detectable cytokine production (data not shown).The results of immunoglobulin determination showed that the PBL of NPC patients produced significantly higher levels of IgM (8262 ± 5315 ng/ml) than the PBL of controls .Previous reports indicate the absence of EBV-specific cytotoxic T lymphocytes detectable by the standard chromium release assay in both the peripheral and intratumoral compartments -10,21 inThe expression of IL-10 in NPC has been controversial. While some authors have reported that NPC tumor cells do not express IL-10 , others The expression of cytokines such as IL-2 , IL-6 1 and IL-1In this report, we investigated the ability of both peripheral blood and tumor infiltrating lymphocytes of 17 undifferentiated NPC patients to produce cytokines following mitogenic stimulation in culture. Since immunoglobulin isotypes are determined by cytokine patterns, we also looked for possible correlations between measured cytokine levels and immunoglobulin isotypes produced. PWM was chosen to stimulate the lymphocyte cultures because it is known to activate both T and B lymphocytes in humans ,30. In aThe data obtained indicate that the highest levels of IL-2 were produced by TIL cultures followed by patients'PBL. This is in line with a report by Lakhdar et al  showing IL-10, a representative of the Th2 pattern of cytokines, is generally considered immunosuppressive. It inhibits IL-6 secretion by activated macrophages but not by Th2 lymphocytes . In the Cultures of tumor infiltrating lymphocytes secreted significantly higher amounts of IgM and IgG than the PBL of either patients or controls in good agreement with the increased levels of IL-2 and IL-10, since these cytokines are involved in B cell activation and enhance immunoglobulin synthesis ,25. No sIn an attempt to see whether the observed differences in cytokine and immunoglobulin production between patients and controls or between PBL and TIL of patients could be due to differences in the composition of individual lymphocyte preparations, we performed an immunophenotypic analysis of each lymphocyte preparation. As shown in the results, the only significant changes in the proportions of lymphocyte subsets between patients and controls were observed in the CD8 subpopulation which showed an increase in patients PBL and TIL. On the other hand, the CD4 subset showed a significant decrease in TIL. Such differences in lymphocyte subpopulations are not expected to produce the changes in cytokine and immunoglobulin production observed here, since CD4 lymphocytes are well known for being the main source of IL-2 and IL-10, and for their ability to stimulate immunoglobulin synthesis .Recently, CD4+ lymphocyte populations have been shown to be more heterogeneous and complex than previously thought  and new In conclusion, our data point to the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. Their lymphocytes, especially those infiltrating the tumors, showed in particular a high propensity to produce IL-10 following mitogenic stimulation in vitro. Overproduction of this pleiotropic cytokine may lead to a discrepancy between humoral and cellular immune responses similar to that seen in NPC. The relevance of the present observations to the in vivo situation is not yet established and warrants further investigations.The authors declare that they have no competing interests.LFJ designed and carried out the experimental work, performed the statistical analysis and drafted the manuscript. NK maintained the cell cultures and provided technical help. RHK and KB participated in the design and coordination of the study. RK directed the research and finalized the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The Easy CD4 assay showed 100% sensitivity for estimating the CD4+ T cell counts < 200 cells/mm3 and < 350 cells/mm3 and 97% sensitivity to estimate CD4+ T cell count < 500 cells/mm3. The specificity ranged from 82 to 100%. The Kappa factor ranged from 0.735 for the CD4+ T cell counts < 350 cells/mm3 to 0.771 for < 500 cells/mm3 CD4+ T cell counts. The system works with a simple protocol, is easy to maintain and has low running cost. The system is compact and generates minimum amount of waste. Hence the EasyCD4 System could be applied for estimation of CD4+ T cell counts in resource poor settings.The CD4+ T cell count estimation is an important monitoring tool for HIV disease progression and efficacy of anti-retroviral treatment (ART). Due to availability of ART at low cost in developing countries, quest for reliable cost effective alternative methods for CD4+ T cell count estimation has gained importance. A simple capillary-based microflurometric assay  was compared with the conventional flow cytometric assay for estimation of CD4+ T cell counts in 79 HIV infected individuals. CD4+ T cell count estimation by both the assays showed strong correlation . The Bland Altman plot analysis showed that the limits of variation were within agreeable limits of ± 2SD (-161 to 129 cells/mm Three by five initiative by World Health Organization (WHO) has accelerated efforts to provide anti-retroviral treatment (ART) to all those who need it even in the developing world . The ARTFlow cytometry has been used as a method of choice for CD4+ T cell measurements since the beginning of HIV epidemic ,4. AlthoHence, alternative methods for CD4+ T cell count estimation with lower costs and simplicity in techniques are being explored worldwide ,6. The EWe compared the CD4+ T cell estimations in HIV infected individuals using EasyCD4 System with the conventional flow cytometry.Thirty-one of the 79 study subjects were females and 48 were males with mean age of 29 and 36 years respectively.The CD4+ T cell counts estimated using EasyCD4 System and flow cytometry showed strong correlation   .A limited number of samples (N = 10) were additionally processed by FACSCount , a single platform system. The mean CD4 counts obtained by FACSCount and Guava EasyCD4 system were 218 and 221 cells/mmThe performance of EasyCD4 assay on five stabilized blood samples showed satisfactory performance within acceptable range of ± 2SD figure .The intra-sample variation in the EasyCD4 assay was assessed in four samples for the CD4 counts obtained on the same day and after 24 hours. The mean % coefficient of variation (CV) was found to be 6.75% in the range of 1 to 18% while the mean % CV of duplicate testing was found to be 5.17 % (range: 1 to 13%).Flow cytometry has been accepted as a gold standard for estimating CD4+ T cell count. However, it has high initial as well as running cost and requires frequent maintenance. The frequent power failures, unavailability of temperature controlled laboratories make the maintenance of flow cytometer difficult. Hence the use of flow cytometry is constrained in resource-poor settings. A number of alternative assays have been developed and some are commercially available, i.e., Dynal CD4 assay using magnetic beads and Coulter Cytoshpere assay. These two microscope-based assays were found to be highly comparable with the Flow cytometry -9. HowevThe EasyCD4 System from Guava Technologies evaluated in the present study is a modified flow cytometer that uses simple volumetric micro capillary-based technology instead of conventional flow cell using sheath fluid for carrying the cells. In conventional flow cytometry, the sheath fluid carries the cells through the LASER path to maintain a single cell suspension so that only one-cell passes through laser beam at one time. In Guava PCA the cells pass through a micro capillary eliminating need of sheath fluid for maintaining single cell suspension. Due to this modification, the volume of accumulated waste is 20 times less than the conventional flow cytometry. Additionally, the conventional flow cytometry in the present study uses the dual side scatter gating depending upon cell morphology (size (FSC) and granularity (SSC)), where as the guava Easy CD4 System uses T cell gating strategy to gate CD3+ cells and estimates CD3+CD4+ cells. The gating on CD3+ T cells removes the monocytes (expressing CD4 but not CD3 on their cell surface) from the gate. The Easy CD4 System is a single platform volumetric system that calculates absolute CD4+ T cell counts on the basis of volume of sample acquired. This eliminates the variability introduced by using Absolute Lymphocyte Count (ALC) from the other analyzer as in dual platform system used in conventional flow cytometry.3, < 350 cells/mm3 and < 200 cells/mm3. The degree of agreement is high as showed by kappa factor. The Bland-Altman analysis, which is a more reliable method to assess variation, also showed that the variation was within agreeable limits. Hence the method is reliable for making decisions on starting ART or prophylaxis for opportunistic infections. The assay has shown very good agreement with single platform technology like FACSCount or multiTest assay using flowcytometer (13–16) and in the present study although on limited sample size.The CD4+ T cell counts estimated by EasyCD4 System showed high correlation with the CD4+ T cell counts obtained by conventional flow Cytometry and showed high sensitivity and specificity to identify patients with CD4 + T cell count < 500 cells/mmThe intra-sample %CV in Easy CD4 assay as 6.75% (1 to 18 %), which was comparable with the values (5 to 13%) reported in previous studies  and less than the %CV reported for "double-platform" systems ranging form 14.5 to 43.4%  (17). Hence, the Easy CD4 assay could be better than the double platform system.The assay showed satisfactory performance when five stabilized blood samples were assessed for CD4+ T cell counts.As compared to the conventional flow cytometry the EasyCD4 System was found to be simple to operate, easy to maintain and the equipment requires less space. Since it requires only 10 μl of sample it is possible to explore its application using finger prick blood samples. Hence, it can be adopted for CD4+ T cell count estimation in HIV infected individuals. The running cost of the CD4+ T cell estimation by EasyCD4 System is 5 times lower than the conventional flowcytometry at the current costing. However, the pricing of the reagents and instruments might be subjected to change due to higher demand and wider choice of technologies available to the customer.EasyCD4 assay, although operationally simple, the users need training in gating of the CD3+ T lymphocytes. The use of minimum quantity of antibodies (1 μl of antibody cocktail) requires precision in technique of reverse pipetting used in case of minute quantities of reagents using the air displacement pipettes as described (18). Also, the EasyCD4 System does not prescribe validity criteria for assessing the formation of the gate. This was found to be extremely critical for reliable gating for accurate estimation of CD4+ T cell counts, to minimize the variations in CD3+ T cell counts and to overcome the acquisition of debris causing high event rate. The laboratory using the equipment needs to set up such criteria locally such as use of commercially available controls or use of healthy individuals sample for gating the CD3+ T cells. This essentially highlights the need of development of laboratory based quality control check on the equipment.In conclusion, the availability of EasyCD4 System enhances the options for reliable and valid CD4+ T cell count estimation technology for HIV infected individuals. The validation of the system on finger prick samples could be taken up to assess the simplicity of sample collection and cost reduction.79 HIV infected individuals attending the referral clinic of National AIDS Research Institute (NARI), Pune were enrolled in the study after obtaining written informed consent from 5 February to 21 April 2004. The blood sample was collected in a vacutainer containing K3 EDTA to avoid clotting of blood.The CD4 + T cell counts were estimated by dual platform Flow cytometry  as a part of routine investigations using IMK Plus kit . The kit included a panel of monoclonal antibodies of CD45-Fluorescein Isothiocynate (FITC)/CD14-Phycoerythrin (PE), CD3-FITC/CD19-PE, CD4-FITC/CD8-PE, CD3-FITC/CD3 HLA-DR-PE and CD3-FITC/CD16+56-PE and SimulSET software. Hundred μl of noncoagulated blood was stained with 20 μl of each of the antibody pair. After 30 minutes incubation the red blood cells were lysed using freshly diluted (1:10) FACS lysing solution . The samples were then acquired in the instrument and the lymphocytes were gated on the basis of size (Forward scatter: FSC) and relative granularity (Side scatter: SSC) for further analysis. Two-colour analysis for each antibody subset was performed to obtain percentages of each lymphocyte subset. The run was considered valid only if more than 95% lymphocytes were in the gate.3. These ALCs were obtained on the hematology analyzer .The absolute CD4+ T cell counts were computed by feeding the ALC in the SimulSET software and were expressed as cells/mmAn aliquot of the same blood samples were coded to blind the technician and processed by Guava Easy CD4 assay  on the same day of sample collection. The coding was carried out only when at least three samples were available on a given day to ensure blinding of the laboratory technician.Ten μl of noncoagulated blood was stained with 1:10 diluted antibody cocktail of CD3-PE-Cy5 and CD4-PE or CD3-PE-Cy5 and CD8-PE separately for 15 minutes at room temperature in dark and the RBCs were lysed using the freshly prepared lysing solution for 15 minutes. The instrument was calibrated daily using Guava check beads (cat# 42000070) in triplicate and the instrument was considered to be ready for use if the average CV% for FSC intensity and PM1 and PM2 mean fluorescence Index (MFI) of all three replicates is within 1–5. The sample was acquired within five hours of staining using Cytosoft software. The gate was automatically set around the CD3+ T lymphocytes using the scatter plot showing cells stained with antibodies against CD3 conjugated to PECy5 and the size of the cells as shown in figure 3 = (tCD4 × df)/v cells.Absolute CD4+ T cell count/mmDuring the preliminary experiments, it was observed that in few cases there was variation in the CD3+ T cell counts obtained in both, sample tubes estimating CD4+ and CD8+ T cell counts and also there was variations in the event rate (no of cells acquired per second). The high event rate was found to occur in case of the samples having a high proportion of non-lymphocyte population in the sample. So the run was considered valid if the variation between the CD3+ T cell counts in both sample tubes (used to estimate CD4 and CD8 T cell counts) was less than 10%, the event rate of acquisition of the sample was less than 700/μl and the CD3+ cells acquired in the gate ranged from 1000 to 2000 cells/μl.At the end of the study, the CD4+ T cell counts obtained by the EasyCD4 System were decoded and compared with the CD4+ T cell counts obtained by the flowcytometry. The correlation between the two methods was assessed using Pearson's correlation test and the degree of agreement was estimated by calculating the kappa factor. The Bland-Altman plots were generated for assessment of the variation between the two methods. The sensitivity and specificity of the Easy CD4 assay was estimated for different ranges of CD4+ T cell counts. Data analysis was carried out using SPSS statistical package (version 12.0) to calculate degree of agreement, and to calculate correlation coefficient. The percent coefficient of variation (%CV) was calculated to assess intra-sample variation by using Microsoft Excel software.The author(s) declare that they have no competing interests.MT conceived of the study, and participated in its design, coordination and drafted the manuscript. BM carried out the CD4+ T cell count estimation assays. BK participated in the design of the study and performed the statistical analysis. SM and RP participated in design of the study, monitored the progress and reviewed the draft of the manuscript. All authors read and approved the final manuscript.


IRIS usually occurs in individuals with a rapidly rising CD4 T-cell count or percentage upon initiation of ART, who develop a deregulated immune response to infection with or without reactivation of opportunistic organisms. Here, we evaluated rises in absolute CD4 T-cells, and specific CD4 T-cell responses in 4 HIV-1in vitro proliferative responses. Results were compared with those observed in 11 matched, successfully treated asymptomatic clinical progressors (CP) with no evidence of opportunistic infections, and uninfected controls.We assessed CD4 T-cell counts, HIV-1 RNA loads, phenotype for naïve and activation markers, and Median CD4 T-cell counts in IRIS patients rose from 22 cells/μl before initiation of ART, to 70 cells/μl after 8 months of therapy (median 6.5 fold increase). This coincided with IRIS diagnosis, lower levels of naïve CD4 T-cells, increased expression of immune activation markers, and weak CD4 T-cell responses. In contrast, CP had a median CD4 T-cell counts of 76 cells/μl at baseline, which rose to 249 cells/μl 6 months post ART, when strong T-cell responses were seen in > 80% of patients. Higher levels of expression of immune activation markers were seen in IRIS patients compared to CP and UC (IRIS > CP > UC). Immunotherapy with IL-2 and GM-CSF paralleled clinical recovery.These data suggest that mycobacterial IRIS is associated with inadequate immune reconstitution rather than vigorous specific T-cell responses, and concomitant administration of IL-2 and GM-CSF immunotherapy with effective ART may correct/augment T-cell immunity in such setting resulting in clinical benefit. Results are expressed as stimulation indices (SI) with a positive response defined as an SI of 3 or more and Δ counts per minute (CPM) > 600 as described previously thymidine, and cells were harvested as described above. Results are expressed as the mean cpm for triplicate cultures, with an error of the mean of ± 15%. A positive result is defined as significant proliferation above the background activity and detection threshold. In all experiments, a standard titration of indicator cell proliferation to a range of recombinant IL-4 from 0.01 to 100 U/ml was included. Control wells for calculation of background activity contained indicator cells only.Fifty μl of supernatant from proliferative cultures was transferred to 96-well round-bottomed plates in triplicate for quantification of IL-4 on the indicator cell line CT.h4S  as previously described -22. BriePMBC were incubated with a panel of murine anti-human mAbs , for 30 minutes at 4°C. Directly conjugated antibodies used were: Fluorescein isothiocyanate (FITC)-CD8, CD45RA; Phycoerytherin (PE)-CD38, HLA-DR, CD27, and CD45RA; PE-cyanine (PC-5)-CD4, all used according to the manufacturer's instructions. Cells were washed and fixed in PBS containing 2% paraformaldehyde (Sigma). On acquisition, a gate was set around the lymphocyte population on a forward scatter versus side scatter dot plot, and 10,000 gated events collected for each sample. Data analysis was performed using CELLQuest™ Software . Appropriate isotype matched controls were run in parallel for each sample.P values below 0.05 were considered significant.Computer software  was used for all statistical calculations. Data are presented as median (inter-quartile range IQR). Analysis of data between the different groups was performed using a Mann Whitney-U-test and intra-group variations were compared using the Wilcoxon signed rank test. We studied both IRIS and CP patients who had been receiving effective ART for similar periods. IRIS was diagnosed at a median 10 months (IQR 8–13) after initiation of ART (Table + T-cell counts of 70 cells/μl (IQR 63–123) after a median 10 months on ART, with persistent MAC infection, received IL-2 and GM-CSF (see Table + T-cell counts to 171 cells/μl (IQR 128–302)  who were unable to achieve remission after receiving ART and anti-mycobacterial therapy. Four patients with median CD4l IQR 63–23 after l IQR 63–23 after 2) Table , and wertion Fig . The partion Fig . We alsol IQR 63–23 after tion Fig .In 3/4 IRIS patients we carried out IL-4 bioassays in culture supernatants, rather than ELISA, in order to assess the levels of bioactive cytokine being produced. We were able to detect production of IL-4 in cultures with antigens to which the patients had been previously exposed including anti-PPD responses in 2/3 patients  (Table We assessed T-cell proliferation in CP and uninfected controls (UC) and compared these with the responses seen in IRIS patients. CP presented a median 3.9 fold increase in CD4 T-cell counts 6 months post initiation of ART from 76 cells/μl (IQR 22.5–90) to 249 cells/μl (IQR 187.5–303.5) (+HLA-DR+ T lymphocytes (p < 0.005), and significantly higher percentages of CD8+CD38+ T cells (p < 0.05) , as demonstrated by analysing the mean fluorescent intensity levels of CD38 expression (Table +CD45RA+CD27+ T cells in IRIS patients was significantly lower than in CP and UC (p < 0.005). These observations are not surprising as IRIS patients were more immuno-compromised when therapy was initiated suggesting that IRIS may be associated with persistent hyperactivation of both CD4 and CD8 T lymphocytes, and associated with a lack of naïve CD4 T cells possibly due to absence of thymic function.Compared to CP, IRIS patients showed significantly higher percentages of CD45) Table . When acin vitro immune function of T lymphocytes in peripheral blood and direct comparison with asymptomatic HIV-1+ subjects as well as uninfected controls. Some reports have shown the lack of correlation between these two assays [in vitro functional data and clinical evidence.Immune reconstitution after initiation of ART may be concurrent with both an increase in immuno-pathological responses against opportunistic pathogens and with the induction of IRIS -19,24. To assays , as funco assays . TherefoThere were two important findings in this study. Firstly, patients admitted with IRIS lacked antigen-specific T-cell responses. Secondly, administration of IL-2 and GM-CSF appeared to rapidly introduce these responses and was associated with clinical recovery in patients with advanced HIV-1 infection.Data from uninfected controls and treated asymptomatic clinical progressors, revealed the presence of lymphoproliferative responses to recall/viral antigens, compared to IRIS patients, confirming that functional specific T lymphocytes are associated with the control of opportunistic infections. Thus, the clinical picture presented by our cohort of IRIS patients is likely to be associated with the lack of lymphoproliferation and IL-2 production rather than with robust antigen-specific T cell responses. This suggests an alternative/additional mechanism for IRIS, distinct from previous hypotheses which suggest that IRIS is caused by pathogen-specific responses induced by successful ART.Of note, are the generally weak proliferative responses observed in response to other pathogens such as CMV, which recover after initiation of ART ,28. AlthThe mechanisms behind IRIS still remain elusive. Through cell-to-cell contact, various complex molecular interactions and the production of cytokines, CD4 T helper lymphocytes modulate the activity of all cells involved in both innate and acquired immunity, including virus-specific cytotoxic CD8 T lymphocytes -33. AlthThe use of immunotherapy in severely immuno-compromised patients has been shown to have beneficial effects in partially reversing the CD4 T-cell defects exerted by HIV-1, when administered concomitantly with ART ,21. Suchet al describing the occasional lack of correlation between the percentage of T cells expressing activation markers and thymidine incorporation [During HIV-1 infection immune hyperactivation is accompanied by up-regulation of surface expression of CD38 and HLA-DR on CD4 and CD8 T cells . Higher poration . It is sporation . Furtherporation ,45. Thisporation . The lowOur data suggests that the degree of immune reconstitution achieved with potent ART alone is dependent on the clinical stage of the patient when therapy was initiated. Furthermore, we hypothesise that in some late-stage patients ART may elicit the expansion of abnormal/anergic T cell clones responsible for an erratic immune response. Concomitant administration of IL-2 and GM-CSF may be associated with provision of proliferative signals to immature thymocytes and/or rescue of anergic CD4 T cells to generate fully functional T-cell responses. GM-CSF acts directly on antigen presenting cells, including macrophages, which may induce and contribute to a more rapid remission from intracellular infection -48.Mycobacterium avium. Such responses may be associated with a better outcome and, possibly quicker recovery, in immuno-compromised patients who fail to achieve immune reconstitution with ART alone, indicating that this therapeutic approach as salvage immuno-therapy may have an impact on short-term mortality. The small number of patients is noteworthy- this is nonetheless an interesting and provocative finding that deserves further prospective exploration in larger numbers of similar patients.In conclusion, ART induced immune reconstitution restores specific responses, which likely help in the clearance of opportunistic pathogens. However, inadequate immune responses observed in treated late-stage disease, in addition to other possible confounders, may be causative of IRIS. Administration of IL-2 and GM-CSF concomitantly with ART in late-stage patients may result in proliferation of pathogen-specific CD4 T-lymphocytes, which likely enable a more rapid clearance of intracellular pathogens such as The author(s) declare that they have no competing interests.AP carried out the proliferation assays, phenotypic analysis, data analysis, participated in the study design and wrote the manuscript. MN, ALP, MF and BG recruited patients and participated in the study design. FG participated in the study design and participated in the drafting of the manuscript. NI conceived the study, carried out proliferation assays and bioassays, data analysis and the design, coordination, the draft and finalisation of the manuscript. All authors read and approved the final manuscript.


As part of the Houston Vanguard study, a subset of 10 patients randomized to receive IL-2 therapy were compared to 4 patients randomized to not receive IL-2, for markers of T cell activation and death during the first three cycles of IL-2. All patients were treated with combination antiretroviral therapy (ART) and were virally suppressed. The purpose of the study was to examine the role of CD8+ T cell death in responses to ART and IL-2 therapy.Lymphocytes were examined at Day 0, 5 and 30 days during three cycles of IL-2 therapy. CD25, CD38, HLA-DR expression and annexin (cell death) were examined on CD4 and CD8 subpopulations. Follow up studies examined CD4 levels and CD4:CD8 reconstitution after 6 years using both univariant and multivariate analyses.+ T cells, leading to an increase in CD4 cell counts. CD8+ T cells did not increase CD25 expression, but upregulated activation antigens (CD38 and DR) and had increased death. At baseline, 7 of the 14 patients had high CD8+ T cell apoptosis (mean 17.0% ± 6.0). We did an exploratory analysis of immune status after six years, and found that baseline CD8+ T cell apoptosis was correlated with CD4 cell count gain beginning two years post enrollment. Patients with low levels of CD8+ T cell apoptosis at baseline (mean 2.2% ± 2.1) had significantly higher CD4 cell counts and more normalized CD4:CD8 ratios than patients with high CD8+ T cell apoptosis .Human lymphocytes responded to IL-2 therapy by upregulation of CD25 on CD4+ T cell apoptosis may reflect inherent activation status, which continues in some patients even though viral replication is suppressed which influences the ability of CD4+ T cells to rebound. Levels of CD8+ T cell apoptosis may therefore be an independent predictor of immune status, which should be shown in a prospective study.We postulate that CD8 Recent data suggests that the expansion of CD25 expressing cells after IL-2 administration is associated with less proliferation of T cells in the long-term (12 months) as measured by both Ki67 expression [+ CD25+ T cells, which might limit subsequent CD4 T cell activation [IL-2 therapy leads to dose-dependent, sustained CD4 cell count increases in most HIV-infected patients treated with antiretroviral therapy (ART) ,2. Earlyong-term ,4. The mpression ,4 and af+ T cells return after IL-2 therapy. Likewise, the older the IL-2 treated patient, the less likely they are to respond well to IL-2, especially after three cycles [Other predictors of the amount of CD4 restoration after IL-2 include the CD4 nadir and the age of the patient ,7. The le cycles ,7.+ T cell return after IL-2 therapy was being non-white; this group of individuals had almost a 200 cells/mm3 larger increase in CD4 cell counts than the white group studied from a cohort of more than 250 patients [In another study, a predictor of the amount of CD4patients .+ and CD8+ T cells that is commonly seen in HIV-infected patients. Increased CD4+ T cell death could be due to HIV infection directly or bystander CD4+ T cells may die via multiple mechanisms (8–10). CD8+ T cell death is likely due to increased activation [Another feature of HIV pathogenesis that might influence T cell normalization after immune reconstitution is the increased cell death of both CD4tivation -13. In ttivation -16.+ T cell apoptosis, was associated with recovery of CD4+ T cell numbers after ART, so that higher levels of CD8+ T cell apoptosis was associated with fewer CD4+ T cells after therapy [+ T cells also had more CD8+ T cell apoptosis, whether or not they were on ART therapy [+ T cell apoptosis was reduced after four weeks with or without IL-2 therapy, but reduction of CD8+ T cell apoptosis levels took 24 weeks to develop [+ T cell apoptosis remained after ART, which were reflective of continuing low levels of viral replication [One study showed that the levels of T cell apoptosis, especially CD8 therapy . This is therapy . Another develop . However develop ,20. Whenlication .+ T cell apoptosis at baseline before commencing scIL-2 therapy was associated with both CD4 cell counts as well as CD4:CD8 ratio normalization after six years. These differences in T cell numbers based on initial CD8+ T cell apoptosis levels appeared after two years of scIL-2 therapy and were maintained until the current analysis.The present Houston Vanguard sub-study examines a small group of subcutaneously IL-2 (scIL-2) treated patients. Patients were randomized from October 1998 to May 1999. An exploratory analysis of immunologic status was assessed an average of six years after randomization. We found that, in this small group of patients, the level of spontaneous CD83 at screening. The Karnofsky performance status had to be ≥ 80 and the patients had to be receiving antiretroviral therapy for at least 7 days prior to starting scIL-2 therapy. Women of childbearing potential were required to have a negative pregnancy test before initiation of scIL-2 therapy. At baseline, all but one of the individuals had plasma HIV RNA levels below 500 copies/mL. That single individual had a viral load of 2,007 copies/mL. The main exclusion criteria for this study included any concurrent or prior history of AIDS-defining illnesses, malignancy requiring systemic therapy within the prior 5 years, concomitant use of systemic corticosteroids, chemotherapy or experimental cytotoxic drugs, or current or prior history of autoimmune and/or inflammatory diseases and breastfeeding. This study was approved by the institutional review board (IRB) at The University of Texas Health Science Center at Houston and the National Institute of Allergy and Infectious Disease (NIAID) IRB. All subjects provided written informed consent.To participate in the Houston Vanguard study, subjects had to be at least 18 years old, have documented HIV-1 infection and a CD4 cell count ≥ 350 cells/mmThe Houston Vanguard study was an open-label, randomized trial designed with a target sample size of 72 subjects with CD4 cell count change from baseline as the primary endpoint. Subjects were sequentially randomized to three groups of 24 . The first group of 24 subjects received 1.5 Million International units (MIU) per dose of scIL-2 plus antiretroviral therapy or antiretroviral therapy alone (control); the second group of 24 subjects was randomized to 4.5 MIU scIL-2 or control; and the third group was randomized to 7.5 MIU of scIL-2 or control. For the purpose of this immunology sub-study of the Houston Vanguard, consecutive patients who volunteered to participate in the sub-study were enrolled: five patients from the 1.5 MIU per dose cohort (low dose), five patients from the 7.5 MIU per dose cohort (high dose) and four from the no IL-2 cohort (control group).Subcutaneous IL-2  was administered for 5 days every 8 weeks for a minimum of three cycles in addition to continued antiretroviral therapy. Dosing with scIL-2 was begun at 1.5 MIU. This dose was escalated to 4.5 MIU and then to 7.5 MIU when at least 9 of the 12 subjects had completed all doses of the first cycle with scIL-2 at a lower dose level in the absence of dose-limiting toxicities. Dose reductions of scIL-2 were allowed for dose-limiting toxicities defined as grade IV toxicities , investigator's assessment or subject tolerance.® in a Randomized International Trial). Additional cycles of scIL-2 were encouraged to maintain CD4 cell counts more than twice baseline or greater than 1,000 cells/mm3. For subjects randomized to 1.5 and 4.5 MIU, dose escalation was allowed by increments of 1.5 MIU per dose up to a maximum of 7.5 MIU. Subjects receiving less than 4.5 MIU were permitted to dose escalate by increments of 1.5 to 3.0 MIU upon the investigator's discretion. Dosage could be reduced by 1.5 MIU decrements (to a minimum dose of 1.5 MIU) if the assigned dose was not tolerated. During the extension phase and roll-over ESPRIT no control subjects were treated with scIL-2 at any dose. The cumulative amount of IL-2 given to the 10 IL-2 treated patients in this study ranged between 165–570 MIU, with a mean of 302 ± 132.After completion of three cycles, subjects were enrolled in an extension phase (months 6 to 12) and, thereafter, rolled-over into the ESPRIT study . CD4 and CD8 counts were done by commercial flow cytometry laboratories using CAP defined criteria.Peripheral blood was separated using Ficoll gradients. The cells were washed in PBS, counted and then stained for cell surface markers and Annexin V to determine levels of apoptotic cells. CD4, CD8, CD25, CD38, and DR antibodies were purchased from BD Pharmingen  and Annexin V was obtained from Biosource International . The cells were analyzed using a Beckman Coulter XL2 flow cytometer. Twenty-thousand events were collected, gated on "viable" light scatter, and fluorescence distributions collected and analyzed and expressed as percentages or mean fluorescence intensity .Group means and standard deviations of study variables: CD4, CD8, and CD4:CD8 ratios were obtained for both low and high annexin groups. Tests for normality were performed. Normally distributed data were analyzed using the t-test and one way repeated measures analysis of variance. A difference of p < 0.05 was considered significant. For missing data, the last value carried forward (LVCF) principle was applied to the analyses. A multivariate analysis of the data was also performed using generalizing estimating equations (GEE) a technique appropriate for longitudinal data with a small number of subjects . VariablInitial studies conducted after randomization examined five scIL-2 treated patients at high dose (7.5 MIU per dose) and five on low dose (1.5 MIU per dose) for three cycles of scIL-2 over a six month period. These groups were compared to four patients treated with ART who did not receive scIL-2 therapy (controls). Results show that the absolute CD4 cell counts increased in all three groups and that the ending CD4:CD8 ratios were significantly different between groups  and 7 patients had low levels CD8+ T cell apoptosis (2.2% ± 2.1). Four and 6 patients from each group, respectively, received scIL-2. After five days of scIL-2 therapy there was an increase in CD8 apoptosis which mirrored the amount of the CD38+ DR+ increase, as shown in a representative patient in Figure + T cell death was seen. We also observed increases in CD38 and DR expression on the CD4+ T cells at 5 days (data not shown). The levels of CD38 and DR returned to normal on both CD4+ and CD8+ T cells after each cycle. There was variability in the amounts of cell death and CD38 and DR increases over the cycles, depending on the person and cycle number with no discernible pattern. In sum, our initial data is characteristic of the data in a number of published papers with larger groups of patients, except that we saw little CD4+ T cell death [Next, we examined whether cells were dying at increased levels both before and after IL-2 therapy by Annexin V staining. At baseline, 7 patients had high levels of CD8ll death .Six years after our initial study, we examined the patients for their immune status using an exploratory analysis. We compared their CD4 and CD8 cell counts and the CD4:CD8 ratios to their baseline parameters. At baseline, only 3 of the 14 patients had CD4:CD8 ratios over 1; five patients had ratios below 0.5. Examination after six years showed that 9 of 12 patients had CD4:CD8 ratios over 1 and only 2 patients had ratios below 0.5.+ CD8+ T cells at baseline and the final CD4 cell count and CD4:CD8 ratio, which indicates the extent of T cell normalization. Patients with high levels of CD8+ T cell apoptosis at baseline (mean 17.0% ± 6.0) did not normalize their CD4 cell counts or CD4:CD8 ratios after six years of follow-up; whereas, patients with low levels of CD8+ T cell apoptosis (mean 2.2% ± 2.1) normalized their CD4 cell counts and CD4:CD8 ratio. The mean CD4 cell counts were 1,209 ± 164 vs 754 ± 320 cells/mm3 and the CD4:CD8 ratios 1.55 vs. 0.70, for the low and high baseline CD8+ T cell apoptosis level groups, respectively  .We next used GEE modeling to exam the outcomes over time. Baseline annexin had a significant inverse relationship with CD4 T cell count and ratio over time (p ≥ 0.02). When age at randomization was entered into the model, baseline annexin remained a significant predictor of both CD4 T cell count over time (p ≥ 0.04) and CD4:CD8 ratio (p ≥ .0.05). A similar finding occurred for age at HIV diagnosis, so that baseline annexin remained a significant predictor of CD4+ cell count over time (p ≥ 0.03) and CD4:CD8 ratio (p ≥ .0.04). When race was entered into the model, annexin was no longer independently related to CD4 T cell and CD4:CD8 ratio over time. When annexin, age and race were entered into the model, age at diagnosis was significant predictor of CD4 T cell number over time (p ≥ 0.03). Thus, this data is similar to previously reported trends with age as a predictor.+ T cell apoptosis was a characteristic of about 70% of previously studied patients where we found a negative correlation between high CD8+ T cell apoptosis and low CD4+ T cell percentages [+ T cells in HIV-infected patients is an independent predictor of immune status, separable from viral load and CD4 cell count [+ T cell apoptosis also may independently predict response to therapies, including scIL-2, by not only increasing the absolute number CD4 cells, but perhaps by regulating the levels of CD8+ T cell expansion.These results, in a small group of scIL-2 treated, virally suppressed HIV-infected patients suggest that the amount of CD8 cellular death at baseline may correlate with the ability to restore CD4 cell counts and CD4:CD8 ratios after immune reconstitution involving ART with or without additional scIL-2 therapy. These differences were detectable after two years of study enrollment. Increased CD8centages . Others ll count ,25. Our + and CD8+ T cells tend to be reduced after ART (14–16). However, in multiple studies, the levels of apoptosis still remains higher than in controls, which likely indicates the presence of continued immune activation which was previously demonstrated in the lymph nodes [+ T cells was also seen by another group at 5 days post scIL-2 therapy similar to our observations [+ T cell apoptosis levels with time, suggesting that IL-2 did not change the inherent level of CD8+ T cell apoptosis.The role of lymphocytic apoptosis in the response to ART with or without IL-2 has been studied by several groups. Most suggest apoptosis levels of both CD4ph nodes . IL-2 thrvations . However+ CD8+ T Cells and CD4+ T cells over two years of ART therapy. Another difference in our work is that the patients were already virally suppressed when baseline apoptosis was measured. Our data, with a small group of patients indicates that the starting level of apoptosis reflects long term outcome to therapy and that the level of CD8 T cell death is independent of viral load.The work that most closely approximates the work reported here followed 17 patients during the first 2–3 years of ART and showed that lymphocyte apoptosis was reduced most in those who had the best response to therapy . Althoug+ DR+ population, which dies after acute administration of IL-2, is indicative of this type of response to IL-2 in vivo. Since CD8+ T cells do not upregulate CD25, it is likely they respond to IL-2 via binding to CD122, (B chain IL-2R), which at the time of these studies was not commercially available. Of importance is that we saw no increase in CD4+T cell death, even though there was CD38+ DR+ activation marker upregulation in the treated people at Day 5 of the scIL-2 cycle. This is in contrast to the conclusions of another report; however, the data in that manuscript also clearly show more apoptosis in the activated CD8+ T cells than the CD4+ T cells after scIL-2 administration at Day 5 [+ vs. CD8+ T cell subpopulations. Recent data, which examined DNA turnover in CD4+ and CD8+ T cell subsets of HIV-infected people after long-term scIL-2 therapy, are supportive of this idea because IL-2 therapy enhanced CD4+ T cell survival, but not CD8+ T cell survival [IL-2 is known as the consummate T cell growth factor; however, IL-2 not only enhances proliferation and survival of T cells, it can also signal death in susceptible cell populations. We suggest that the activated CD38at Day 5 . We thin+ T cell death is unclear. It may be associated with a better overall CD4 cell count at the beginning point in the low CD8+ T cell apoptosis group. The CD4 cell count nadir in this group was about 100 cells/mm3 higher and the median was higher than in the high CD8+ T cell apoptosis group, which is consistent with our previous observations that a high level of CD8+ T cell apoptosis was correlated with fewer CD4+ T cells [The mechanism responsible for enhanced normalization of CD4 cell counts in those with less CD8 T cells .+ T cell apoptosis at baseline indicates existing immune activation to HIV antigens, which can continue in some patients, even without detectable viremia [+ T cells, therefore, get activated and die at an increased level in some individuals. Even though CD4 cell count increases in IL-2 treated patients, the amount of proliferation in the long term actually becomes reduced as shown in two studies [2M phase of the cell cycle, is similar to measurements of activation using CD38 and DR expression [+ and CD8+ T cells remained high, even after 22 cycles of scIL-2, indicating that chronic activation in some people is not relieved by IL-2 therapy. Of interest is that this patient also had about a log10 more HIV RNA  than other patients. However, we saw no correlation in our small study with viral load, which remained undetectable or barely detectable throughout the study [+ T cell death. The underlying reason for this is not clear, but could be related to inherent host differences or a "set point" of CD4 cell count, below which an enhanced CD8+ T cell apoptosis is triggered.Our interpretation of this data is that the amount of CD8 viremia -16. CD8+ studies ,4. It hapression . Hence, pression ,29. In the study . We did + T cell apoptosis relates to what drives the proliferation of T cells. That is, homeostatic mechanisms, like that driven by IL-7, are one way that T cell proliferation is driven. By contrast, activation-induced proliferation is another mechanism whereby T cells divide. We think it likely that the predominant form of CD8 proliferation occurring in those with high CD8+ T cell apoptosis is activation-induced, rather than homeostatic-driven proliferation, because death occurs mainly with activation induced proliferation, not homeostatic driven proliferation [Another possible mechanism to account for the role of CD8feration . This ma+ T cell apoptosis might serve as an indirect measure of the ability to respond to immunoreconstitution. Our longitudinal univariate analyses showed that both age and annexin were significant predictors of CD4 T cell numbers over time. However, entry of race into the model, showed that annexin lost it's independent relationship to CD4+ T cell number over time. Examination of possible ethnic differences in response to immune reconstitution is therefore also warranted.These data suggest that examination of CD8Interleukin-2 IL-2Antiretroviral therapy ARTMillion International Units MIUGeneralizing estimating equations GEEThe author(s) declare that they have no competing interests.DEL wrote the paper, RAC conducted the clinical aspects dealing with patients, CAK performed the multivariate analysis, KLG did the initial data analysis of the flow cytometry data, other's  helped with patient accrual and data management.


All CD4+ TCCs of patients and all control TCCs secreted IFNγ and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNγ, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNγ lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNγ, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients ( Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a wide spectrum of multi-organ manifestations, and genetic, hormonal, environmental and immunoregulatory factors are known to contribute to expression of the disease . HoweverAbout 15 years ago, the heterogeneous nuclear ribonucleoprotein (hnRNP-)A2, another component of the spliceosome, was characterized as a novel target of autoAbs in systemic autoimmune diseases . AlthougMost of the autoAbs in SLE patients have undergone immunoglobulin class switching and affinity maturation. This and the association of HLA-DR subtypes with the presence of certain autoAbs indicates an antigen-driven immune response, emphasizing the role of T cells in SLE ,19. The Recently, we were able to characterize the cellular response against hnRNP-A2 in patients with RA . We obseTo gain more insight into the role of autoantigen specific T cells in SLE, we investigated spontaneous T cell responses to hnRNP-A2 in patients with SLE and in healthy control subjects and characterized TCCs specific for this antigen. The data obtained suggest that hnRNP-A2 may constitute an important T cell autoantigen in patients with SLE, indicating a potential role for it in the pathogenesis of this disorder.n = 21) and/or low-dose glucocorticoids (n = 15). Nine patients did not receive any medication. Disease activity was determined by European Consensus Lupus Activity Measurement (ECLAM) score TdR  was added and the incorporated radioactivity was measured by scinitillation counting. Results were expressed as stimulation index (SI) defined as the ratio of mean counts per minute (cpm) obtained in cultures with antigen to mean cpm obtained in cultures incubated in the absence of antigen. An SI ≥3.0 and a Δcpm >1,000 (mean cpm obtained in cultures with antigen minus mean cpm obtained in cultures incubated in the absence of antigen) was regarded as a positive response.Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood of SLE patients and controls by centrifugation on Ficoll Hypaque (Pharmacia). After washing and counting cells were either immediately used or frozen in RPMI medium containing 10% dimethylsulfoxide and 20% fetal calf serum. Cells were cultured in the presence of the antigens for 5 days at 37°C in triplicate in 96-well plates  in a total volume of 200 μl  in 96-wells plates. T cells were cultured in the presence of 1 × 105 irradiated  allogeneic PBMCs as 'feeder cells', 0.5 μg/ml phytohemagglutinin and 20 U/ml IL-2 in medium containing 1% heat-inactivated human AB serum. Growing microcultures were then expanded at weekly intervals with fresh feeder cells in the presence of IL-2. The specificity of TCCs was assessed by proliferation assays incubating 2 × 104 T cells with 0.35 μg/ml hnRNP-A2 in the presence of 105 autologous irradiated PBMCs. After 48 hours incubation and pulsing with [3H]TdR for an additional 16 hours, cells were harvested and the incorporated radioactivity was measured by scintillation counting. Production of IL-4, IFNγ and IL-10 was measured by ELISA in supernatants collected after 24 hours of incubation as described TdR and proliferation was measured after anadditional 16 hours. Incubation with IL-10 alone  was used as control.To investigate a possible functional difference between the CD4+CD28+ and CD8+CD28- TCCs, purified CD4+ T cells from two SLE patients and two healthy controls  were observed in 66% of the SLE patients and 24% of the controls , the difference was not significant. Of note, while in controls the stimulation elicited by tetanus toxoid was considerably higher than the one induced by hnRNP-A2, the response of SLE patients to tetanus toxoid was slightly but significantly lower than the response to hnRNP-A2  could be raised. Twenty-two TCCs were derived from SLE patients: 13 (59%) of them were CD4+/CD8- and thus belonged to the helper T cell subset, while 9 were CD4-/CD8+. The TCCs showed high variability in their degree of proliferation, with SIs ranging from 3.0 to 26.0 and a mean SI of 6.2 ± 1.2. Figure . Analysin = 8; 67%) were CD4+/CD8-, while 3 clones (25%) were CD4-/CD8+ and one TCC was CD4-/CD8-. In contrast to SLE TCCs, all clones secreted IFNγ, though in highly varying amounts , the difference between patient and control TCCs (39.5 ± 29.4 pg/ml versus 376 ± 312 pg/ml) did not reach statistical significance.Proliferation of the 12 TCCs derived from control individuals was comparable to patient-derived clones, with SIs ranging from 3.6 to 37 and a mean SI of 9.0 ± 2.8 Figure . Phenotys Figure . Whereass Figure . Additios Figure ; howeverExpression of the costimulatory molecule CD28 was measured in six CD4+ and seven CD8+ TCCs from SLE patients and in five TCCs from healthy subjects Figure .As pronounced differences in the cytokine secretion pattern of SLE patient derived CD4+CD28+ and CD8+CD28- TCCs had been observed, we were interested in the functional properties of these two populations. To address this question, CD4+ T cells  were stimulated with anti-CD3 and anti-CD28 mAbs and subsequently incubated with supernatants from two CD8+CD28- and two CD4+CD28+ TCCs derived from SLE patients Figure . All supIn previous investigations we characterized the humoral response to hnRNP-A2 in SLE patients and some clinical implications thereof ,10,31,32The occurrence of autoreactive T cells in healthy individuals is a common finding that has previously been reported for antigens associated with SLE  as well Interestingly, no correlation of cellular reactivity to hnRNP-A2 with the appearance of the respective autoAbs in SLE patients could be observed. Thus, hnRNP-A2 appears to be a predominant T cell antigen, while the generation of autoAbs might represent a bystander phenomenon occurring in a subgroup of patients. However, recent data indicate that in SLE patients the humoral response to hnRNP-A2 fluctuates considerably and increases during flares. Therefore, the prevalence of these autoAbs may be considerably higher than previously assumed (unpublished observation).in vitro from CD8+CD28- T cells [Although the cellular reactivity to hnRNP-A2 appeared to be primarily a Th1 response, we observed a relatively high percentage of CD8+ TCCs in SLE patients. Of particular interest, most of these clones lacked CD28 expression and produced neither IFNγ nor IL-4, but did produce IL-10. This may indicate a special pathological role of this T cell subset because this phenotype appeared to be restricted to SLE patient derived TCCs. In previous reports, CD4+ T cells in SLE were shown to be of predominantly Th1 or Th0 subtypes ,24. Furt T cells . This su T cells -40. An iSurprisingly, though, the supernatants of the hnRNP-A2 specific CD8+CD28- TCCs derived from SLE patients did not inhibit proliferation of CD4+ T cells, but instead enhanced anti-CD3/anti-CD28 induced stimulation, similar to supernatants derived from CD4+CD28+ TCCs. As IL-10 alone had an antiproliferative effect, the increased content of IL-10 in the supernatants of CD8+CD28- TCCs was obviously counteracted by other yet unknown stimulatory components of the supernatant, which remain to be identified. Of note, the lack of CD28 expression is also a hallmark of senescent T cells, which are known to be increased in various autoimmune diseases and chronic inflammatory conditions and may show a rather aggressive phenotype . TherefoElevated IL-10 serum levels have been reported in SLE patients and correlated with clinical and serological disease activity, especially anti-DNA antibody titres . SeveralFinally, the question remains why hnRNP-A2 is such a preferred target of the T cell response in SLE, while autoAbs occur less frequently and may represent an epiphenomenon of ongoing T cell autoreactivity. On the other hand, autoAb titers increase during disease flares (unpublished observation) and, importantly, autoAbs to hnRNP-A2 are, together with anti-Sm antibodies, among the earliest detectable autoAbs in MRL/lpr mice, where they were found to precede anti-dsDNA autoAbs . HnRNP-AOur data reveal a pronounced T cell response against the autoantigen hnRNP-A2 in the majority of SLE patients in contrast to a scarcer and lower response in healthy subjects. Although this autoreactivity seemed to be mainly conferred by CD4+ T cells showing a Th1 phenotype, a newly defined hnRNP-A2 specific CD8+CD28- T cell-subset was observed in SLE patients. This subpopulation showed a predominant IL-10 secretion and a lack of IFNγ or IL-4 production. Our data suggest involvement of both populations in the complex and still incompletely understood pathogenesis of SLE, and warrant further investigations into the cellular aspects of this autoimmune response.autoAb = autoantibody; cpm = counts per minute; ds = double stranded; ECLAM = European Consensus Lupus Activity Measurement; ELISA = enzyme-linked immunosorbent assay; FITC = fluorescein isothiocyanate; hnRNP = heterogeneous nuclear ribonucleoprotein; IFN = interferon; IL = interleukin; mAb = monoclonal antibody; PBMC = peripheral blood mononuclear cell; RA = rheumatoid arthritis; SI = stimulation index; SEM = standard error of the mean; SLE = systemic lupus erythematosus; TCC = T cell clone.They authors declare that they have no competing interests.RF participated in the design of the study, worked with the T cell cultures and drafted the manuscript. DM performed the T cell cloning and T cell assays. KS prepared the recombinant antigen. BJS participated in the design of the study. JS and GS conceived of the study, participated in its design and coordination and helped to write the manuscript. All authors read and approved the final manuscript.


HIV-associated immune reconstitution disease (IRD) is the clinical presentation or deterioration of opportunistic infections that results from enhancement of pathogen-specific immune responses among patients responding to antiretroviral treatment (ART). The vast majority of reported cases of IRD have been associated with mycobacterial, chronic viral and invasive fungal infections; such cases result from dysregulated augmentation of cell-mediated type 1 cytokine-secreting host immune responses. However, the spectrum of infections now recognized as associated with IRD is expanding and includes a number of parasitic infections, which may be mediated by different immunopathological mechanisms. These include leishmaniasis , schistosomiasis and strongyloidiasis. Since the major burden of HIV lies in resource-limited countries where access to ART is now rapidly expanding, increased awareness and knowledge of these phenomena is important. Here we review the clinical spectrum and pathogenesis of IRD associated with parasitic infections. HIV-associated morbidity and mortality has dramatically decreased in many high-income countries since the advent of antiretroviral treatment (ART) in the mid 1990s . Robust IRD is not a new phenomenon, but has long been recognized as a complication occurring among patients with severe immunosuppression in whom immune function is rapidly restored. Thus, for example, IRD may occur following withdrawal or rapid dosage reduction of corticosteroid treatment and among patients in whom the blood neutrophil count recovers following cytotoxic chemotherapy or bone marrow transplantation . The patMycobacterium tuberculosis, Mycobacterium avium complex and other non-tuberculous mycobacteria); (b) viruses ; and (c) fungi  (vide infra).The vast majority of cases of IRD associated with ART have been reported from high-income countries and have been associated with a wide range of non-parasitic opportunistic infections, including: (a) bacteria (ma spp.) . HoweverThe majority of people with HIV/AIDS live in resource-limited countries and in June 2005 WHO estimated that 6·5 million people living in such countries were in urgent need of ART . DespiteIRD can be defined as the clinical presentation or deterioration of opportunistic infections that results from enhancement of pathogen-specific immune responses among patients responding to ART. However, diagnosis in practice is not straightforward, being one of clinical judgement based on various lines of indirect evidence. These may include: (a) the clinical manifestation or pattern of progression of an opportunistic infection that is unusual; (b) a temporal relationship with ART initiation; (c) exclusion of alternative explanations; (d) demonstrated efficacy of ART  evidence of improved CD4 cell function (e.g. development of a positive tuberculin skin test); and (f) histopathology consistent with the diagnosis. Competing explanations for these clinical manifestations that should be excluded are the occurrence of opportunistic infections as a result of residual immunodeficiency and inadequate treatment of an opportunistic infection, including that resulting from drug resistance.+CD45RO+ memory cells previously sequestered in lymphoid tissue and a reduction in apoptotic cell death (+CD62L+ cells (Successful ART is associated with a rapid (> 90%) reduction in plasma viral load within the first weeks of ART. The most characteristic immunological feature of HIV infection is depletion of CD4 T cell numbers, and restoration of the CD4 cell subset during ART appears to occur in two principal phases. The initial rapid phase of CD4 cell recovery can usually be detected within the first 1–2 weeks of starting treatment and extends over 2–3 months . Data sull death ,17. Thosll death –21. A slL+ cells .The increase in circulating CD4 cell numbers is also associated with an improvement in effector function, the extent of which is directly related to the degree of viral load suppression and the CD4 cell counts in the longer term . IL-2-meAlthough HIV is principally characterized by CD4 cell depletion, functional deficits in other cells of the innate and acquired immune systems also occur. However, the literature concerning effects of ART on other cell types is much less comprehensive than that concerning CD4 cell reconstitution. Moreover, many of these effects are CD4 cell-dependent, so that attributing ART-induced improvements in immune function to either CD4 cells or to other cell types is difficult.+ T cell counts are also associated with decreased circulating CD3−/CD16+/CD56+ NK cells  decreases during ART ,16,22, p+ CD25+ IL-10-secreting T regulatory cells  cytokine-secreting immune responses  is the clinical form of leishmaniasis that has the most substantial geographical overlap with HIV and typically occurs as an opportunistic infection among HIV-infected individuals with CD4 cell counts < 200 cells/µL . This oveficient .L. infantum VL in southern France describe asymptomatic patients who developed fever and hepatosplenomegaly due to VL within the first 2 weeks of initiating ART  are associated with an intense granulomatous response, causing tissue pathology via a CD4 cell-dependent process ,73. FromLeishmania braziliensis were established in both patients and both responded well to treatment with either amphotericin B or pentavalent antimony . On. On74). Two cases of PKDL presenting as IRD have been reported from Israel  and ItalLeishmania donovani during ART has been reported from the Netherlands .,84.83,84et al. , schistosomiasis and strongyloidiasis. No studies have yet examined the mechanisms of IRD associated with helminth infections, but these may be due to augmentation of appropriate type 2 immune responses. More research is needed to increase our understanding of these emerging phenomena.


CD4+ T lymphocyte (CD4) cell count testing is the standard method for determining eligibility for antiretroviral therapy (ART), but is not widely available in sub-Saharan Africa. Total lymphocyte counts (TLCs) have not proven sufficiently accurate in identifying subjects with low CD4 counts. We developed clinical algorithms using TLCs, hemoglobin (Hb), and body mass index (BMI) to identify patients who require ART.We conducted a cross-sectional study of HIV-infected adults in Uganda, who presented for assessment for ART-eligibility with WHO clinical stages I, II or III. Two by two tables were constructed to examine TLC thresholds, which maximized sensitivity for CD4 cell counts ≤ 200 cells μL, while minimizing the number offered ART with counts > 350 cells μL. Hb and BMI values were then examined to try to improve model performance.1787 subjects were available for analysis. Median CD4 cell counts and TLCs, were 239 cells/μL and 1830 cells/μL, respectively. Offering ART to all subjects with a TLCs ≤ 2250 cells/μL produced a sensitivity of 0.88 and a false positive ratio of 0.21. Algorithms that treated all patients with a TLC <2000 cells/μL, excluded all patients with a TLC >3000 cells/μL, and used Hb and/or BMI values to determine eligibility for those with TLC values between 2000 and 3000 cells/μL, marginally improved accuracy.TLCs appear useful in predicting who would be eligible for ART based on CD4 cell count criteria. Hb and BMI values may be useful in prioritizing patients for ART, but did not improve model accuracy. Guidelines developed by the World Health Organization (WHO) for the use of antiretroviral therapy (ART) in low-income countries state that HIV-infected individuals should commence ART if they have WHO stage IV disease, stage III disease and a CD4+ T lymphocyte (CD4) cell count of ≤350 cells/μL, or stage I or II disease with CD4 cell counts ≤200 cells/μL . RecentlIf CD4 cell counting is not available, as is the case in most of sub-Saharan Africa, the WHO guidelines recommend using clinical staging alone, or in combination with total lymphocyte counts (TLCs) of < 1200/μL in order to determine ART eligibility-5. StudiWe designed an analysis among HIV-infected adults in rural Uganda being screened for ART eligibility. We examined the clinical utility of TLCs, Hb, and body mass index (BMI) to maximize the sensitivity to detect individuals with CD4 cell counts below 200 cells/μL, and limit the proportion of individuals who would be offered ART with CD4 cell counts above 350 cells/μL.The Home-Based AIDS Care Program (HBAC) is a clinical trial of three different monitoring strategies for patients receiving ART in rural Eastern Uganda. Registered clients of The AIDS Support Organization (TASO), a local HIV/AIDS care and support organization in Tororo and Busia districts, were invited to be screened for ART eligibility. The study includes participants from a prior diarrhea prevention and cotrimoxazole study described elsewhere , as wellAll subjects in this analysis were HIV-infected adults aged ≥ 18 years. Clinical and laboratory assessments at baseline included a complete blood counts (CBC), viral load, and CD4 cell counts. Those who had a CD4 count ≤250 or had WHO stage III (excluding pulmonary TB) or IV were offered ART. Blood samples for TLC and CD4 testing were collected in the same vacutainer tube containing EDTA at the study clinic and transported to the CDC laboratory in Entebbe. CD4 cell counts were measured by a dual-platform protocol using a FACScan instrument and Tritest reagents . TLCs were measured both using a hematology analyzer  and the FACScan instrument. We have previously found that delays in transport of up to 5 days do not affect the accuracy of the FACScan results for CD4 cell counts  and therefore used the TLC results obtained on the FACScan in order to determine if transport time to the lab significantly affected the difference between the two methods. TLCs results from the hematology analyzer were used for the analysis. Clinical information during screening was collected using standardized instruments completed by study physicians. Subjects with WHO stage IV disease were excluded for this analysis.Bivariate correlations between TLC results obtained on the FACscan and hematology analyzer were conducted. Differences between the two results were calculated and compared with results stratified by time between blood draw to testing of ≤ 1 day and > 1 day, using the Wilcoxan Rank Sum test. Distributions of TLCs, Hb and BMI across CD4 cell count strata were compared in a pair-wise fashion using the Wilcoxan Rank Sum test. Two by two tables were constructed to examine the association between different strata of TLCs, Hb and BMI with CD4 cell counts ≤ 200/μL or ≤350/μL. Sensitivity, specificity, positive and negative predictive values and accuracy  of the models were then calculated. We examined different TLC thresholds in terms of their ability to maximize sensitivity in detecting subjects with CD4 cell counts ≤ 200 cells/μL and minimize the proportion of subjects offered ART with cell counts > 350 cells/μL . Hb and BMI thresholds, alone and in combination, were then used to classify subjects with intermediate TLCs as qualifying for therapy. 95% confidence intervals for sensitivity and false positive ratios were calculated for the final models according to the Wald method . Final mThe effects of tuberculosis, malaria parasitemia, or diarrhea at the time of screening were examined by conducting sub-group analyses which excluded subjects with these illnesses. All statistical analyses were conducted in SAS version 9.0 2 = 0.85, p < 0.001) with the analyzer consistently giving greater values . Differences were slightly greater for subjects whose blood samples were tested 2 days after blood draw in comparison with those whose blood was tested ≤ 1 day after draw .Between May 2003 and June 2005, 1944 HIV-infected adults presented for assessment of ART eligibility. Of these, 104 (5.2%) were excluded from this analysis as they were found to have WHO clinical stage IV disease. Another 53 individuals were excluded because of missing TLC or CD4 cell count values leaving 1787 subjects available for analysis. 75.2% were women and 27.8% were men. Median baseline CD4 cell counts and total lymphocyte counts, were 239 (inter-quartile range [IQR] = 119–411) and 1830 (IQR = 1420–2360) cells/μL, respectively. The mean time between blood sample collection and TLC and CD4 testing was 1.4 days; 56% of subjects had blood tested on the same day or one day after blood draw and 43% were tested 2 days after blood draw. One patient had blood tested 8 days after blood draw and was excluded from the analysis. TLC results between the hematology analyzer and the FACScan instrument were highly correlated (Pearson's r2 (IQR = 18.-21.9). TLCs, Hb and BMI were distributed differently between the CD4 cell count strata , with higher values for all parameters measured in the higher CD4 cell count strata.In total, 763 (42.7%) subjects had baseline CD4 cell counts ≤ 200 cells/μL, 459(25.7%) had cell counts of 201–350 cells/μL and 565 (31.6%) had CD4 counts > 350 cells/μL (Table A TLC threshold of 2250 cells/μL was the most accurate (0.73) predictor of CD4 cell counts ≤ 350 cells/μL, yielding a sensitivity of 0.81 and a specificity of 0.54 (Table Results for women were unchanged from that of the whole group (data not shown). Both algorithms from the whole group analysis performed well for men separately, with accuracies of 0.72 and 0.73 for the models including TLC and Hb; and TLC, Hb and BMI, respectively Table . HoweverOffering ART to all subjects with WHO stage III disease and using the algorithms to determine ART-eligibility for those with stage I or II disease would have resulted in treatment being offered to 1282 or 1328 subjects with similar levels of sensitivity and false positives. A total of 19 subjects had malaria parasitemia, 60 subjects were on TB treatment or diagnosed with TB at screening and 96 subjects had diarrhea at screening. However, excluding any of these subjects did not improve the predictive value of the final models.We have demonstrated that using a TLC of 2250 cells/μL to determine ART eligibility in subjects with WHO clinical stages I, II or III, could identify 88% of subjects with CD4 cell counts ≤ 200 cells/μL, while 21% of subjects offered ART would have CD4 cell counts > 350 cells/μL. Using a TLC threshold of 2000 cells/μL to offer ART and an upper threshold of 3000 cells/μL to exclude subjects from treatment with Hb and BMI thresholds to determine ART eligibility for those with intermediate TLCs only marginally improved the accuracy of TLCs alone.Despite the failure to improve accuracy of TLCs in predicting CD4 cell counts, using Hb and BMI may still be of value in determining who should initiate ART in resource-limited settings. Recent studies have shown that low Hb values and low BMI are independent risk factors for early mortality on ART in African settings ,16. TherThe number of patients offered therapy under our TLC/Hb/BMI criteria were similar to the number offered treatment had ART been offered to all patients with CD4 cell counts ≤ 350 cells/μL, however, the same patients would not be treated under both scenarios. Between 17 and 19% of subjects with CD4 cell counts ≤350 cells/μL would not be offered ART using the TLC/Hb/BMI algorithms, however, as they would not have low BMIs or low Hb values, unless they also had TLCs >3000 cells/μL, it is unlikely that they would be at risk for early mortality without treatment.More than 75% of the participants in this study were women. While the proportion of women aged 15 – 59 with HIV in Uganda is about 28% greater than men (7.3 versus 5.2%), it is lIf TLCs are to prove to be a viable alternative to using CD4 cell counting in managing HIV-infected individuals in resource-limited settings, they will need to be shown to be useful not only in determining when individuals should initiate ART, but also in monitoring patients on ART. There have been few studies on the use of TLCs in monitoring individuals on ART, but all have concluded that TLCs correlate well with changes in CD4 cell counts on ART,20. HoweThe proportion of subjects in our study with CD4 cell counts ≤ 200 cells/μL and ≤ 350 cells/μL was very high , resulting in a large number of subjects being ART-eligible. It is likely that our algorithm may not function as well in settings where the proportion of ART eligible subjects is not so high. However, uptake of voluntary counseling and testing is quite low in Uganda in that only 13% of women and 11% of men have ever been tested for HIV and is aThis study has two potential limitations. Firstly, TLCs and CD4 cell counts were not conducted at the site of blood drawing, but were transported to the CDC laboratory in Entebbe, which may have resulted in some deterioration of the blood samples. However, we had previously shown that CD4 cell count results from the FACScan instrument are stable up to 5 days after blood draw and the variability between the TLC values obtained on the FACScan were only a median of 40 cells/μL less for subjects whose blood was tested 2 days after draw compared to those whose samples were tested after ≤ 1 day delay between blood collection and testing. These observations suggest that transport time may not have caused significant inaccuracy in our laboratory test results. Secondly, the gold standard for determining inclusion or exclusion criteria for ART used in this study, that of CD4+ T lymphocyte counts, are also not perfect predictors of morbidity and mortality among people with HIV. While CD4 cell counting has proven useful in allowing stratification of risk for disease progression on ART individual variation of disease progression within CD4 cell count strata is large . TLCs haThus, while TLCs may not correlate perfectly with CD4 cell counts, they may be as useful in predicting disease progression and therefore can be equally useful in determining when ART should be initiated. While evaluating how well ART-eligibility criteria based on TLCs perform in comparison to CD4 cell count – based criteria can only be answered by conducting clinical trials, this study has again demonstrated that TLCs are useful proxy measures for CD4 cell counts in determining ART eligibility.