Reconstitution in the peripheral blood during therapy with highly active antiretroviral therapy (HAART) is well established. However, the extent of immune reconstitution in the GI tract is unknown.During acute and early HIV-1 infection (AEI), up to 60% of CD4+ HLA-DR+, returned to levels seen in the uninfected control participants in the peripheral blood, but were elevated in the GI tract of patients with persistent CD4+ T cell depletion despite therapy. Rare HIV-1 RNA–expressing cells were detected by in situ hybridization.Fifty-four AEI patients and 18 uninfected control participants underwent colonic biopsy. Forty of the 54 AEI patients were followed after initiation of antiretroviral therapy . Lymphocyte subsets, markers of immune activation and memory in the peripheral blood and GI tract were determined by flow cytometry and immunohistochemistry. In situ hybridization was performed in order to identify persistent HIV-1 RNA expression. Of the patients studied, 70% maintained, on average, a 50%–60% depletion of lamina propria lymphocytes despite 1–7 y of HAART. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45ROApparently suppressive treatment with HAART during acute and early infection does not lead to complete immune reconstitution in the GI mucosa in the majority of patients studied, despite immune reconstitution in the peripheral blood. Though the mechanism remains obscure, the data suggest that there is either viral or immune-mediated accelerated T cell destruction or, possibly, alterations in T cell homing to the GI tract. Although clinically silent over the short term, the long-term consequences of the persistence of this lesion may emerge as the HIV-1–infected population survives longer owing to the benefits of HAART. Despite early initiation of anti-HIV therapy, loss of T cells in the gastrointestinal mucosa persisted for years in most members of a clinical cohort identified early after HIV-1 infection. AIDS causes disease by inactivating the body's immune response against infection. The AIDS virus (HIV) is most active against the white blood cells called T lymphocytes, particularly the CD4 T lymphocytes, which recognize infection and activate other cells of the immune system to fight it. In what was formerly believed to be a gradual process, HIV infection is now known to deplete a subset of the body's CD4 lymphocytes, called memory cells, quite rapidly—over only a few days—within a few weeks after a person becomes infected with the AIDS virus. This was not known until recently because researchers were counting CD4 cells only in blood, while a majority of the memory lymphocytes are located in and around the digestive system. It is these intestinal memory lymphocytes that are rapidly wiped out, while those in the blood fall much more gradually, usually over several years. Few studies of mucosal lymphocytes have been done in humans because such studies require biopsies of the intestinal lining (mucosa).Although CD4 cells in the blood can return and remain at normal levels when HIV infection is treated with antiviral drugs, it has been unclear as to whether the mucosal CD4 cells return as well. People who begin treatment as soon as possible after becoming infected with HIV might seem to have the best chance of regaining their mucosal immunity, compared to those who wait until the CD4 cells in their blood have fallen, which is a generally accepted reason to start medication for HIV. Therefore, the researchers wanted to see whether people who start treatment early after becoming infected with HIV might experience restoration of their mucosal immunity over time and, if so, what kinds of lymphocytes would return.The researchers studied people who started treatment for HIV within a few weeks to months of becoming infected and who then remained on treatment. Some volunteers underwent biopsies of the intestinal mucosa before starting treatment and then at various points from 1 y until as long as 3 y after infection. Others volunteered for biopsy only one time, anywhere from less than 1 y to 7 y following treatment. The biopsy specimens were examined under the microscope and with a technique called flow cytometry using specific staining methods to assess their structure and functional characteristics. Results were compared to biopsies from a group of HIV-uninfected volunteers.The researchers found that the percentage of CD4 lymphocytes dropped much lower in the intestinal mucosa than in blood during early infection and then, unlike in blood, remained low even after several years of treatment for HIV. In the microscope images, they found that mucosal CD4 cells were lost mostly from regions of active battle against invading germs, rather than from “training sites” for new CD4 cells. Over time, only 30% of the volunteers showed return of CD4 cells to normal levels in these active sites.Unlike T lymphocytes in the blood, which tend to return to a resting state after HIV is treated, the T lymphocytes in the intestinal mucosa tended to persist in an activated state despite HIV treatment, even though only a tiny fraction of these cells were found to be infected with HIV. A high level of activation of mucosal lymphocytes soon after infection was found to predict poor restoration of mucosal CD4 cells over time.These experiments confirm that studying easily obtained blood lymphocytes provides only a limited view of how HIV affects the immune system as a whole. The finding that immune cells of the intestinal mucosa remain depleted and over-activated for years despite antiretroviral treatment raises the concern that over time this will result in clinical problems. Fortunately, this does not appear to be the case in most people currently being treated for HIV, some for as long as 10 y, but the results of this study suggest that we should remain vigilant for gastrointestinal problems resulting from impaired immunity over time. The finding that mucosal lymphocytes do appear to return to normal levels in a minority of volunteers is of interest, and suggests that early interventions to reduce activation of intestinal T cells  might be worth investigating in those recently infected with HIV. Finally, these results suggest that a vaccine to prevent HIV may need to stimulate immune responses that can act very quickly following infection, before the bulk of lymphocytes capable of countering the infection are lost, perhaps irreversibly.http://dx.doi.org/10.1371/journal.pmed.0030484.Please access these Web sites via the online version of this summary at UCSF HIV InSite includes resources on HIV immunology and vaccine developmentAIDS fact sheets and brochures from the US National Institute of Allergy and Infectious DiseasesMedline Plus article on acute HIV infection from the US National Library of Medicine Over the past 25 y, more than 25 million individuals have succumbed to the complications of HIV-1 infection . Recent + T cells in the gastrointestinal (GI) lamina propria (LP) compared with levels measured in the peripheral blood ), the sections were incubated with anti-CD4 or CD8 antibodies overnight as described above. Immunodetection was performed either with the StreptABComplex/HRP  using 3-amino-9-ethylcarbazole (Sigma-Aldrich) as the substrate or with APAAP (DakoCytomaton) and Fast Blue as chromogen. The sections were then heat-treated again for 5 min with 0.01 M buffered sodium citrate solution (pH 6.0). This was followed by an overnight incubation with MIB-1 . For the second antibody, either the APAAP (DakoCytomaton) or the StreptABComplex/HRP (DakoCytomaton) visualization system was applied.35S-labeled, single-stranded anti-sense RNA probe of HIV-1 , composed of fragments of 1.4–2.7 kb, which collectively represent approximately 90% of the HIV-1 genome [The in situ hybridization was performed on paraffin sections as described previously . A 35S-l1 genome , was use2 of tissue was calculated. To evaluate the number and distribution of T cell subsets, transmission light was used. Using a 40× objective, a standard area was set by the image analyzer. The number of positive cells within this unit area was determined by manual counting. For the LP, a total of 10–15 consecutive nonoverlapping fields were analyzed for each staining. For the OLT, between two and five representative areas were chosen. The individual values obtained with each T cell marker were then pooled, and the mean numbers of positive cells per unit area of LP or OLT were determined separately.The autoradiographs were examined with a microscope equipped with epiluminescent illumination , a 3CD camera, and a PC-based image-analysis system  as described previously . Brieflyt-test. Statistical comparisons were made between HIV-1–infected patients and control participants using a two-sample, unequal variance t-test. All reported p-values were two-sided at the 0.05 significance level using SPSS 11.0 for Windows software .Values are expressed as mean ± standard deviation. Statistical comparisons were made between PBMCs and MMCs from individuals using a paired n = 5), 2 y (n = 9), and 3 y (n = 4). An additional 22 patients were studied cross sectionally after initiation of HAART for up to 1 y (n = 7), 2 y (n = 7), and 3–7 y (n = 8)  . On presThe 18 HIV-1–uninfected control participants were recruited from a population undergoing screening colonoscopy at the time of study recruitment. This group comprised ten men and eight women. None of the HIV-1–infected patients or HIV-1–uninfected control participants were found to have macroscopic evidence of GI mucosal disease, nor were any concomitant pathological processes found on histological examination. All enrolled patients and control participants signed an informed-consent form that was approved by the institutional review boards of the Rockefeller University, Bellevue Hospital Center, and Manhattan Veteran's Administration Hospital Center.+ T cells, we utilized flow cytometry to determine the percentage of CD4+ T cells in the GI tract and peripheral blood (n = 32) and uninfected control participants (n = 18). In the uninfected control participants, the mean PBMC CD4+ T cell percentage was 59.6% ± 14.3% and the mean MMC CD4+ T cell percentage was 56.4% ± 8.8%. In AEI patients, the mean PBMC CD4+ T cell percentage was 41.5% ± 12.9% and the mean MMC CD4+ T cell percentage was 19.3% ± 8.8%. In patients treated for up to 1 y (n = 7), the mean PBMC CD4+ T cell percentage was 46.9% ± 10.1% and the mean MMC CD4+ T cell percentage was 27.8% ± 14.5%. In patients treated for 1–3 y (n = 7), the mean PBMC CD4+ T cell percentage was 58.1% ± 12.3% and the mean MMC CD4+ T cell percentage was 42.3% ± 3.1%. In patients treated for 3–7 y (n = 8), the mean PBMC CD4+ T cell percentage was 59.9% ± 12.0% and the mean MMC CD4+ T cell percentage was 42.5% ± 13.6% , and remained low after treatment for up to 1 y (p < 0.001), 1–3 y (p = 0.003), and 3–7 y (p = 0.02).In order to study the effect of antiretroviral therapy on the reconstitution of CD4al blood A. Twenty ± 13.6% B. Thus, + T cells remained significantly lower in the GI tract compared with the peripheral blood , 2 y , and 3 y  of suppressive antiviral therapy , 6.5 ± 1.6 (p < 0.001), and 7.8 ± 2.6 cells/unit area (p = 0.004)—in the three treatment groups, respectively. On examination of individual patients, nine showed “normalization” (defined as mean CD4+ T cells per unit area in the LP of 18 HIV-uninfected control participants—11.0 ± 3.3 cells/unit area). The mean LP CD4+ T cell count in these nine patients was 9.5 ± 1.4 cells (p = 0.13 compared with HIV-uninfected), the mean LP CD8+ T cell count was 8.4 ± 2.7 cells (p = 0.3), and the mean CD4:CD8 ratio in the LP was 1.2 ± 0.6 (p = 0.07).To corroborate flow cytometry–derived data and to assess for the absolute numbers of mucosal CD4n the LP , we exam+ T cells was noted in the LP, with a mean CD4+ T cell count of 5.5 ± 1.1 cells (p < 0.001). The mean CD8 T cell count in these 21 “non-reconstitutors” was 8.0 ± 1.7 cells (p = 0.3) and the mean CD4:CD8 ratio was 0.7 ± 0.1 (p < 0.001). Thus, significant depletion of the GI CD4+ T cell count persisted in 21 out of 30 (70%) of patients despite uninterrupted, apparently suppressive, combination antiretroviral therapy. As a corollary, in nine out of 30 (30%) of patients, the absolute CD4+ T cell count per unit area reached HIV-uninfected levels with treatment.In the other 21 patients, persistent depletion of CD4+ T cells expressing chemokine receptors. Using four-color flow cytometry, we quantified the percentage of CD3+/CD4+ MMCs that expressed CCR5 or CXCR4, or co-expressed CCR5 and CXCR4 (n = 40). Comparisons were made between HIV-uninfected control participants, AEI, AEI treated for up to 1 y, AEI treated for 1–3 y, and AEI treated for 3–7 y. In the HIV-uninfected patients, 78.8% ± 10.3% CD4+ T cells expressed CXCR4, 68.4% ± 18.9% expressed CCR5, and 52.4% ± 12.4% co-expressed CXCR4/CCR5. As reported previously [+ MMCs expressing CXCR4 , more so in CD4+CCR5+ T cells , and the most significantly in CD4+ MMCs dually expressing both CCR5 and CXCR4 chemokine receptors .Since a majority of the viruses during AEI are CCR5-tropic , and a snd CXCR4  in all peviously , depleti+CCR5+ and CD4+ dual-positive MMCs, when compared with HIV-uninfected patients. Specifically, CD4+CCR5+ MMCs in patients treated for up to 1 y , 1–3 y , and 3–7 y  remained depleted. Similarly, though more prominently, levels of CD4+CCR5+/CXCR4+ MMCs were lower in patients treated for up to 1 y , 1–3 y , and 3–7 y , respectively.Despite antiretroviral therapy, significant depletion persisted in the CD4+ T cell decline without therapy [+ and CD8+ T cells in the GI tract and peripheral blood. Using four-color flow cytometry, we assessed CD3+CD4+ and CD3+CD8+ lymphocytes for the expression of CD45RO, a marker of immunological memory, and HLA-DR, a marker of immunological activation.HIV-1 infection results in a significant increase in cellular activation, which has been shown to be of prognostic value in predicting the rate of CD4 therapy –24. We w+) phenotype [+ T cells were CD45RO+ in HIV-uninfected control participants. In patients with untreated AEI, the percentage of GI CD4+CD45RO+ T cells showed only a modest reduction to 86.3% ± 12%, p = 0.17 despite a well-documented, profound depletion of the total number of GI CD4+ T cells. Thus, the relative percentage of memory cells in the GI tract remained stable despite an overall reduction in the total number of CD4+ T cells. To examine the levels of cellular activation, we examined memory (CD45RO+) lymphocytes for the expression of HLA-DR.It is well established that a majority of mucosal cells (>90%) have a memory  and CD8+CD45RO+HLA-DR+ MMCs  was noted. After 1 y of treatment, the percentage of CD8+CD45RO+HLA-DR+ PBMCs remained elevated  but approximated HIV-uninfected levels in the 1–3 y treated  and 3–7 y treated groups . In contrast, in the GI tract, the percentage of CD8+CD45RO+HLA-DR+ MMCs remained elevated despite HAART in all groups—1 y treated , 1–3 y treated , and 3–7 y treated   A.+ T cell subsets. CD3+CD4+ PBMCs co-expressing CD45RO/HLA-DR were 5.4% ± 5.0% of the CD4 population in the HIV-uninfected control participants. During AEI, there was a significant increase in this population . Post-HAART, activated memory CD4+ PBMCs approximated HIV-uninfected levels in all groups . In the GI tract, however, activated memory CD4+ T cells persisted despite therapy. When compared with HIV-uninfected CD4+CD45RO+HLA-DR+ MMCs (11.3% ± 5.0%), an increase in activated memory CD4+ T cells that developed during AEI  persisted despite potent antiretroviral therapy in the 1 y treated group  and 1–3 y treated group , but not in the 3–7 y treated group   B.+ T cells and CD8+ T cells by immunohistochemistry (+CD4+ T cells and 1.5% ± 1.3% Ki67+CD8+ T cells), during untreated AEI, a significant increase was noted in the percentage of Ki67+CD4+ T cells  and CD8+ T cells . This increase persisted in the group treated for 1 y  and 1–3 y . In the 3–7 y treated group, the increase in the percentage of Ki67+ cells persisted , although it was not statistically significant.To study a more specific marker of cellular dynamics, we examined the expression of Ki67 on GI mucosal CD4hemistry C. Compar+ and CD8+ lymphocyte activation reverses with treatment in the peripheral blood, it remains elevated in the GI tract. In addition, there is a persistent increase in proliferating CD4+ and CD8+ T cells in the GI tract during antiretroviral therapy.Thus, while CD4+ T cells (and the CD4:CD8 ratio) in the GI LP persists in the majority of individuals despite therapy, we sought to identify virological and immunological factors at baseline and posttreatment that could account for inter-individual differences. Patients whose CD4+ T cell numbers reconstituted to HIV-uninfected levels  were compared with patients with persistent CD4+ T cell depletion in the LP .Having established that depletion of CD4+ T cell count, duration of infection prior to biopsy, timing of initiation of antiretroviral therapy, and duration of therapy (+ T cells were significantly higher at baseline (54.4% ± 17.8%) and posttreatment (29.7% ± 16.2%) in group B patients than in group A patient at baseline  and posttreatment , respectively (−CD62L− phenotype [−CD62L− CD4+ T cells (90.4% ± 5.4%) and CCR7−CD62L− CD8+ T cells (95.9% ± 2.2%) in group A patients was significantly higher than in group B patients (80.1% ± 12.3% and 91.4% ± 2.5%), respectively  cells wectively . Thus, cGI biopsy sections were hybridized using a radiolabeled probe  to detec+ T cells do not reconstitute in the LP of the GI tract despite uninterrupted, apparently suppressive, antiretroviral therapy for up to 5–7 y. Based on immunohistochemistry, we estimate that approximately 50%–60% of LP CD4+ T cells remain depleted when compared with HIV-uninfected control participants. CD4+ T cells expressing the CCR5 receptor or dually expressing the CCR5/CXCR4 receptors are the cell populations most severely affected. Accompanying this persistent lesion are increased levels of activated CD4+ and CD8+ T cells within the GI tract. In contrast to the degree of CD4+ T cell depletion, only rare HIV-1 RNA–expressing cells were detected by in-situ hybridization.The present study established that in a majority of patients, 70% of our cohort, CD4+ T cells residing in the LP are selectively depleted early in the course of acute HIV-1 infection [Recent studies of the role of the GI tract have demonstrated that the CD4nfection –12. Crosnfection . To defi+ T cell reconstitution within the GI tract. Our patients were closely followed, demonstrated excellent adherence to medication regimen, and maintained undetectable plasma viral loads for the study duration. Despite this, a majority of our patients show significant mucosal CD4+ T cell depletion. In this regard, our results are in contrast to two prior short-term follow-up studies in HIV-1–infected humans [+ T cell reconstitution in the GI tract is equivalent to that in peripheral blood.This is, to our knowledge, the largest study describing the effects of antiretroviral therapy initiated during AEI on CD4d humans  and SIV-d humans  which in+ T cell percentage in the GI tract, 30% of the studied patients did show “normalization” of absolute numbers of CD4+ T cells within the GI LP. This distinction needs to be taken into consideration so as not to over-interpret suggestions of “a universal lack of mucosal reconstitution.”It is important to recognize that although flow cytometry permits the characterization of cellular phenotype for a large number of cells, the data obtained thus are based on relative cellular percentages, such that an increase in one cell population can affect the percentage of the other. To overcome this potential confounder and to examine absolute numbers of cells, immunohistochemistry was employed. It is noteworthy that, while none of the patients examined by flow cytometry showed “normalization” of CD4+ T cell reconstitution, we sought to understand clinical, immunological, and virological differences between patients. While we did not observe significant clinical differences between the “reconstitutors” and “non-reconstitutors” in terms of baseline plasma viral loads, peripheral blood CD4+ T cell count, CD4:CD8 ratio, duration of antiretroviral therapy, or the timing of onset of antiretroviral therapy, we did observe immunological differences within the GI tract: the level of activated memory CD8+ T cells at baseline and posttreatment inversely correlated with CD4+ T cell reconstitution. In other words, the higher the level of CD8+ T cell activation, the less likely that reconstitution would be observed in the GI tract. These data echo the previously observed inverse correlation between levels of activated CD8+ T cells, as measured by CD38 expression, and total CD4+ T cell counts in the peripheral blood [+ T cell reconstitution. Perhaps an even larger study could confirm whether or not correlates of mucosal immune reconstitution exist among clinical parameters such as plasma viral load, duration of therapy, and timing of onset of therapy, among others.Based on the variation in CD4al blood –24. We h+ T cell depletion within the effector compartment may be due to reductions in the education of gut-homing lymphocytes, to altered cellular recruitment or homing, or to increased cell death due to either ongoing viral replication and/or immune activation in the effector compartment.Within the GI tract, there appears to be sub-compartmental variation: immune-inductive sites reconstitute better than immune-effector sites and instead resemble peripheral blood lymphocyte subsets in their reconstitution. It is currently believed that naïve lymphocytes enter inductive sites through high endothelial venules expressing mucosal addressin cell adhesion molecule-1 (MAdCAM-1) interacting with cell surface receptors, particularly α4β7 integrin . Exposurl-selectin (CD62L) expression into cells with lymphoid tissue–homing potential (CD62L+) and non-lymphoid tissue–homing potential (CD62L−) [+ T cells expressing α4β7 integrin during primary HIV-1 infection and partial recovery following 48 wk of antiretroviral therapy [+ T cell depletion. Indirect support for our hypothesis was provided by the observation that patients who reconstituted CD4+ T cells in the GI tract had a significantly greater percentage of GI effector memory cells (CD62L−CCR7−) compared with the “non-reconstitutors” (Memory cells can be classified on the basis of (CD62L−) . GI lymp(CD62L−) . Krzysie therapy . Our datitutors” . These d+ T cells expressing CCR5 and CCR5/CXCR4 (+ and CD8+ T cells, despite normalization in the peripheral blood (+ T cell depletion observed. Low sensitivity of in-situ hybridization, owing to the relatively small number of cells that can be examined, may possibly be one of the factors involved. Further studies using PCR-based techniques are underway to address this issue.It has been suggested that there may be ongoing viral replication during antiretroviral therapy ,36. PersR5/CXCR4 , the celal blood A and 4B,al blood . However+ T cell depletion in the GI tract are unknown at this point. It is clear that the largest reservoir of immune cells [+ T cell reconstitution during HAART. In the pre-HAART era, opportunistic mucosal infections were a common feature of disease progression. It is evident that, with treatment, patients do not demonstrate any short-term effects of having a 50% loss of CD4+ T cells in the GI LP. It is possible that sufficient redundancy in the immune system prevents adverse outcome in chronically HIV-1–infected patients. However, it is important to recognize that, given the projected long-term survival of HIV-1–infected patients, significant loss of mucosal CD4+ T cells could accelerate immune senescence with attendant consequences. In recent clinical studies, an increased incidence of polyps and colorectal malignancies has been observed in HIV-1–infected individuals independent of antiretroviral therapy [+ T cell depletion become evident, modalities such as IL-15 may be considered as adjuncts to HAART in mucosal immune restoration.The consequences of persistent CD4ne cells  shows a  therapy ,39. Thes therapy . If longWhile previous studies have focused on the role of the GI tract during AEI, the present study establishes that there is a delay in the majority of patients in mucosal immune reconstitution despite years of apparently successful antiretroviral therapy. Taken together, these data highlight the susceptibility of mucosal sites to HIV-1 and underscore the critical need to develop better mucosal-protection strategies. Recent work in the SIV-macaque model has demonstrated that mucosal delivery of a vaccine may confer better protection against SIV challenge when compared with subcutaneous administration of the same vaccine . Other s+ T cell depletion in the GI tract, there were inherent limitations in a study of this nature. The patient population was selected and not random—particularly those who are willing to undergo repeated biopsy. Furthermore, the group of control participants was limited in size and was not matched to the HIV-1–infected study patients. Our study did not conclusively establish mechanisms of persistent CD4+ T cell depletion in the GI tract, and we were therefore unable to definitively identify factors associated with reconstitution—an issue that we believe to be important. It is likely that a larger sample size is needed to address this issue. Finally, the consequences of the persistence of the identified lesion remain obscure. Ongoing research to better understand both the mechanisms of persistent depletion and its implications is in progress.Even though we believe this to be the largest study to date assessing the effect of antiretroviral therapy on the reconstitution of CD4In summary, we have shown that despite the early initiation of HAART, the marked depletion suffered by the GI mucosal immune system during acute infection does not completely reconstitute in the majority of patients. Though not clinically apparent in the short term, careful observation is warranted as the long-term consequences of this lesion may become evident as the HIV-1–infected population ages.


Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection.Chronically infected patients with low CD4 counts T-cell counts who commenced antiretroviral therapy (ART) were subsequently treated with combined interleukin-2 and therapeutic vaccination.Thirty six anti-retroviral naive patients were recruited and initiated on combination ART for 17 weeks before randomization to: A) ongoing ART alone; B) ART with IL-2 twice daily for 5 days every four weeks starting at week 17 for 3 cycles; C) ART with IL-2 as in group B and Remune HIV-1 vaccine administered once every 3 months, starting at week 17; and D) ART with Remune vaccine as in group C. Patients were studied for 65 weeks following commencement of ART, with an additional prior 6 week lead-in observation period. CD4 and CD8 T-cell counts, evaluations of HIV-1 RNA levels and proliferative responses to recall and HIV-1 antigens were complemented with assessment of IL-4-secretion alongside quantification of anti-HIV-1 CD8 T-cell responses and neutralizing antibody titres.Neither IL-2 nor Remune™ vaccination induced sustained HIV-1-specific T-cell responses. However, we report an inverse relationship between HIV-1-specific proliferative responses and IL-4 production which continuously increased in patients receiving immunotherapy, but not patients receiving ART alone.Induction of HIV-1-specific cell-mediated responses is a major challenge in chronically HIV-1-infected patients even when combining immunisation with IL-2 therapy. An antigen-specific IL-4-associated suppressive response may play a role in attenuating HIV-specific responses. Immune recovery subsequent to antiretroviral therapy (ART) often appears to be partial and does not comprise the HIV-1-specific CD4 or CD8 T-cell proliferative and IL-2-producing responses that are associated with protection from disease progression . These pSub-cutaneous (S/C) interleukin (IL)-2, administered with ART, increases CD4 T-cell numbers -21 and rWe investigated the ability of Remune™ and IL-2, combined and separately, to induce HIV-1-specific CD4 and CD8 T-cell responses in chronically HIV-1-infected patients on ART in an observational, open-label, randomized, pilot study. We also assessed antigen-specific IL-4 release as this cytokine plays a role in balance and/or suppression of cell-mediated responses ,27, We r6U) was administered S/C, twice daily, for 5 days at weeks 17, 21 and 25. 100 μg Remune™ was administered I/M at weeks 17, 29, 41 and 53. Laboratory analysis was conducted at weeks- -6, -3 and 0 before ART, and weeks 1, 2, 4, 8, 16, 17, 21, 25, 29, 41, 53 and at study completion at week 65. The primary outcome was induction of positive changes in lymphocyte proliferative responses to HIV-1 antigens. In addition to the main study time points, a further sub-study of viral loads and lymphocyte subset numbers was conducted in a sub-set of patients (n = 15) receiving IL-2 in groups B and C on the 5th day of each IL-2 cycle, i.e. at weeks 18, 22 and 26. This sub-study was initiated after the main study had begun and included all patients receiving IL-2 in groups B and C from the date of its inception. Appropriate regulatory approval was granted by Riverside Ethics Committee and patients gave written informed consent.In this observational, phase I, pilot study conducted at Chelsea and Westminster Hospital, London, 36 antiretroviral-naive patients were initiated on ART at week 0, which was continued for the duration of the study. ART comprised 2 nucleoside analogues and one protease inhibitor or non-nucleoside reverse transcriptase inhibitor. At week 17 patients were randomized to receive immunotherapy with IL-2 and/or therapeutic immunisation with a gp120-depleted whole inactivated HIV-1 immunogen. Sufficient Remune™ was donated for use in 20 patients by Immune Response Corporation (IRC), Carlsbad, CA, USA. Patients were randomized at week 17 only if their viral load was <50 copies ml/plasma and CD4 T-cell count was ≥ 300 cells/μl blood at week 16. Treatment groups for randomization were as follows: A) ART alone (n = 9); B) ART plus IL-2 (Proleukin™) (n = 11); C) ART plus IL-2 and Remune™ (n = 7); and D) ART plus Remune™ (n = 9). IL-2  and 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine supplemented with 10% human AB plasma .PBMC were separated from whole blood by density gradient centrifugation and cultured in RPMI-1640 with NaCOPlasma viral load was measured using the Bayer HIV-1 RNA 3.0 assay (bDNA) (lower limit 50 copies/ml) . Whole blood lymphocytes were counted with monoclonal antibodies to: CD3, CD4, CD8, and CD45  on an Epics XL-MCL flow cytometer (Beckman Coulter).Recombinant HIV-1 and recall antigens  were used as described previously , at a fiProliferation assays and supernatant collection for IL-4 assessment were conducted as previously described . StimulaIL-4 bioassays were carried out as previously described  using ILIn vivo delayed-type hypersensitivity (DTH) skin tests to Remune™ antigen were performed to assess HIV-1-specific cell-mediated immune responses as described elsewhere [lsewhere  in all pHLA haplotypes of patients were assessed by PCR-SSP .5 PBMC from individual patients who had been tissue typed were cultured with or without 10 μg/ml of appropriate HLA-restricted peptides or PHA (positive control) in 96-well anti-IFN-γ  coated PVDF-backed plates . After overnight incubation IFN-γ spot-forming cells (SFC) were detected according to the manufacturer's instructions (Mabtech).IFN-γ ELIspots were conducted as described previously . BrieflySF162 for 1 hr at 37°C and then plated onto NP2/CD4+/CCR5+ cells. Infection was detected after 48 hrs by p24-immunostaining, as detailed before [Immunoglobulin (Ig)G was purified from patient and HIV-1 negative plasma using the MAbTrap kit , and quantified by Protein Assay . Two-fold serial dilutions of purified IgG, from 1 mg/ml, were incubated with 100 focus forming units (FFU) of HIV-1d before . The perChanges in lymphocyte subsets and viral load between week 0 and 16, weeks 0 and 65 and weeks 16 and 65, were assessed using the Wilcoxon Signed Rank test. Proliferation and IL-4 data were assessed with repeated measures analysis of variance using MIXED procedure in SAS statistical software. For log-transformed antigen-specific proliferative stimulation indices, between and within subject weeks 0 to 17, weeks 17 to 65 and weeks 0 to 65, adjusted separate slopes were estimated for each study arm with 95% confidence interval. Differences in overall viral load from week 18–26, between groups B and C were assessed using the Pearson Chi-squared test.Fifty two anti-retroviral naïve patients were screened for this study which was carried out at the Chelsea and Westminster Hospital, London over a six year period. Of the screened patients 16 dropped out prior to randomisation at week 17. All the remaining 36 patients who were randomised completed the study. The mean age of patients at study entry was 38.75 years. There was one female patient (2.78%) and 100% of patients were of white European ethnicity.Median viral loads and CD4 T-cell counts for each group are depicted in Figure By week 17 the median viral load in all groups was less than 50 copies. All patients, except 5 , maintained viral suppression at the main study time points from week 17 onwards while receiving ongoing ART. One patient in Group C (patient 11) elected to discontinue ART at week 47 causing subsequent rebound in viraemia. At week 17 the median CD4 T-cell count in each complete group was as follows: Group A, 456 cells/μl (range 261–673) Figure ; Group Bp = 0.005) was observed  and whole Remune™ antigen (p = 0.005) from week 0 to 65. . Patient 8 in this group demonstrated a very large response to Remune™ (SI = 261) and np24 (SI = 144) at week 29 following breakthrough of resistant virus at week 25  (data not shown). No other substantial increases in responses occurred between week 17 and 65 for any group or at any time point between the groups.HIV-1-specific proliferative responses increased transiently for many patients. Regression analysis of these responses revealed no significant changes in group A Fig. . In grouved Fig. . In grouved Fig. . Of note65. Fig. . The onlInduration size of hypersensitivity reactions to Remune™ did not become positive (>9 mm diameter) in any patient at any of the time points tested .persistent recall antigens: For group A ; for group B , CMV responses increased p = 0.002 and Candida responses increased (p = 0.0006); in group C ; in group D . Responses to transient antigens were less apparent. In group A PPD and tetanus responses showed significant positive regressions over the 65 weeks (p = 0.018 and p = 0.018 respectively); in group B only the PPD response regression curve was significant (p = 0.044); and in group C only the tetanus response was significant (p = 0.0003). There were no significant differences in recall responses between treatment groups.Baseline to week 16 T-cell responses have been described in detail previously [A Figure  responseB Figure  HSV respC Figure  CMV and D Figure  HSV and p = 0.023), and group C (p = 0.032) and in response to gp120 in group D (p = 0.037)  Fig. . HIV-1-sWe observed an apparent relationship between IL-4 production and loss/lack of proliferation in 17 of 28 patients tested, distributed evenly between groups, evident from week 0 to 16 and week 17 to 65. Patient 11 (group C) is shown as an example, where HIV-1-specific proliferative responses to p24, gp120, nef and Remune™ are high at week 29 Fig.  with absWhere possible we investigated CD8 T-cell IFN-γ production in response to characterised HLA class I restricted peptides .Secondary to the main study protocol, further viral load and lymphocyte subset analysis was conducted at additional time points – week 18, week 22 and week 26 – for a sub-group of 15 patients receiving IL-2 in groups B (n = 9) and C (n = 6) on the last day of each IL-2 cycle. Small, transient elevations in HIV-1 RNA load were apparent on 12 occasions for 7 of 9 (78%) patients in group-B, and 2 of 6 patients (33%) in group-C. All these viral load elevations had spontaneously resolved by the next viral load test 3 weeks later at weeks 21, 25 and 29. The difference in occurrence of these viral load elevations remains evident when comparing the median viral loads for all the patients in groups B and C as shown in Fig. SF162 at weeks 0, 16 and 65. There was no effect of immunotherapy or ART on virus neutralization (data not shown).As Remune™ displays the transmembrane (gp41) envelope antigen with strong neutralization epitopes -35, it mIn this pilot study of combined immunotherapy with the therapeutic HIV-1 vaccine Remune™ and subcutaneous IL-2 in the context of ART, neither IL-2 nor Remune™ immunisation confered any substantial benefits to the effect of ART with regard to HIV-1-specific CD4 or CD8 T-cell responses, or neutralising antibodies. For the majority of these chronically HIV-1-infected patients responses to HIV-1 antigens remained negligible and there was little difference between the four immunotherapy arms. Possible reasons for this could be: the low CD4 T-cell counts of these patients when ART was initiated resulting in protracted immunosuppression and/or clonal dysfunction; a need for longer duration of ART before immunotherapeutic intervention, thus allowing greater immune recovery; the permanent depletion of HIV-1-specific CD4 T-cells by direct HIV-1 infection; and/or the timing of IL-2 therapy with immunisation which may have important consequences for induction or suppression of induced responses.Previous Remune™ studies which induced proliferative responses comprised patients with higher pre-ART mean CD4 T-cell counts of 617 cells/μl  and 700 The fate of missing HIV-1-specific CD4 T-cell responses is unclear. HIV-1 preferentially infects and deletes many HIV-1-specific CD4 T-cells , while spersistent antigens. These increases were largely evident between week 0 and 65 with no differences between groups, suggesting immunotherapy was ineffective in this respect. While some significant increases in proliferative responses to HIV-1 antigens were apparent from week 0 to 65 (group B and D), the same cannot be said regarding the immunotherapy period between weeks 17 and 65. Nor were there any significant differences in responses between groups. These results suggest that any improvements were singularly attributable to duration of ART.Despite the lack of induced HIV-1-specific T-cell responses in these patients we report significant increases in recall antigen proliferative responses, particularly for Of note is one patient in group B, vaccinated with tetanus 4 weeks before receiving IL-2. High tetanus-specific proliferation was enhanced and sustained by subsequent IL-2, as reported separately . In contWe observed low-levels of transient self-limiting viraemia resulting from IL-2 therapy as previously reported . Despitein vivo DTH responses these results imply that induction of renewed HIV-1-specific cell-mediated responses by therapeutic immunisation, even when supplemented with IL-2, is extremely problematic in patients who initiate ART with lower CD4 T-cell counts. Although a recently reported clinical trial of the vCP1433 canary pox-based therapeutic vaccine elicited p24-specific responses which were significantly associated with time off therapy in a subsequent treatment discontinuation protocol, these responses also remained transient, diminishing at study end [Combined Remune™ and IL-2 with ART in advanced HIV-1 infection conferred no immunological benefits to ART. Taking together the absence of induced HIV-1-specific lymphocyte proliferative responses, CD8 T-cell IFN-γ responses and tudy end . This untudy end  to whichtudy end . In our In these chronically infected treated patients, we found that immunotherapy was associated with increasing HIV-1-specific IL-4 production, which appears to negatively impact proliferative responses. HIV-1-specific IL-4 production may result from a general dysfunction of HIV-1-specific CD4 T-cells with pathological implications for induction of HIV-1-specific responses. We suggest that these results underscore the importance of early initiation of ART. As Remune™ may have positive effects in less advanced patients , we suggART , CMV , cpm (counts per minute), DNA (Deoxyribonucleic Acid), HIV-1 (Human Immunodeficiency Virus type-1), HLA (Human Leukocyte Antigen), HSV (Herpes Simplex Virus), IgG (Immunoglobulin G), IFN (Interferon), IL (Interleukin), I/M (Intramuscular), PBMC , PCR (Polymerase Chain Reaction), RNA (Ribonucleic Acid), S/C (subcutaneous), SI (Stimulation Index).The author(s) declare that they have no competing interests.All authors have read and approved the final version of this manuscript. GH, FG, NI and BG conceived the study and co-coordinated the design of the study. GH and BG participated in the application for government and ethical approval, and in co-ordination of the study; he conducted laboratory work and cellular immunological assays, conducted data analysis and Interpretation, and preparation and completion of the manuscript. NI had responsibility for overall management and coordination of the study, she conducted laboratory work and cellular immunological assays, coordinated data analysis and interpretation, and participated in preparation of the manuscript. FG and NI secured funding for the study. FG participated in data analysis and interpretation, and in preparation of the manuscript. RM provided Remune™ vaccine and Remune™ antigen and its native p24 antigen and participated in study design. MAA conducted virus neutralisation assays. BG participated in the design of the study, co-coordinated application for government and ethical approval, and coordinated patient management. AS undertook patient care and management and participated in study co-ordination, and data interpretation. MN participated in patient care and management and data interpretation.


Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease.neg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease.We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.The novel antigen-specific CD45RA+ IFN-gamma Increasing evidence in humans and in nonhuman primate models of HIV-1 infection indicates that CD8+ T cells play a direct role in controlling or limiting HIV-1 replication. CD8+ T-cell depletion during acute  SIV infeEMRA, and the precursor CD45RAneg memory CD8+ T cells, subdivided into central memory, TCM and effector memory, TEM. By applying this experimental setting, we identified a population of HIV-1-specific CD8+ T cells which is significantly associated with the NP cohort, completely absent in the cohort of ART-treated patients and not related to the levels of viral replication.In order to improve our understanding of the relationship between cellular immune response and nonprogressive HIV-1 infection, we analyzed the CD8+ T-cell response in the peripheral blood compartment of HIV-1 infected individuals with different histories of infection. Eleven NP were compared to 10 progressors (PR) with unrestricted control of viral replication. All NP and PR had not received ART before. In addition, we analyzed 23 ART-treated patients in whom HIV-1 replication is pharmacologically controlled and the role of the immune system is less relevant. Finally, we characterized the immune response of 6 ART-treated patients who interrupted the assumption of ART investigating the effect of rebounding virus replication on the HIV-1-specific CD8+ T cell responses. We focused on the role of specific CD8+ T cells with respect to the non-structural HIV-1 proteins Nef and Tat. Indeed, these two nonstructural proteins are known to strongly influence HIV-1 replication, pathogenicity and the host immune response ,18. SincNef- and Tat-specific CD8+ T-cell responses were analyzed by multicolor flow cytometry in a cohort of NP and compared to responses observed in PR and ART-treated patients Table . Followineg CD8+ T cells. The staining panel was originally designed as routine immune-assay to evaluate simultaneously CD4+ and CD8+ T cell responses, and for this reason includes the measurement of CD154 [neg CD8+ T cells expressing MIP-1β or MIP-1β and IFN-γ  Nef-specific CD45RA+ CD8+ T cells was significantly higher in NP  than in PR  or ART-treated individuals , whereas the proportion of polyfunctional CD45RAneg CD8+ T cells was significantly higher in ART-treated subjects  than in PR . On the other hand, monofunctional Nef-specific CD45RAneg IFN-γ+ IL-2neg MIP-1βneg CD8+ T cells were detected in significantly higher proportion in PR  than in NP  and ART-treated individuals . Surprisingly, the proportion of responding CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells in NP was significantly higher than in PR and ART-treated patients with extremely low p values  and 1 out of 10 PR (10%), whereas they were completely undetectable in the 22 ART-treated patients analyzed . Interestingly, Nef-specific CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells in NP, when detectable, represented a high proportion of the total response (range: 10.7 to 49.9%). The same population detected in one PR (PR05) represented only 6.8% of the total response. As shown in figures neg IL-2neg MIP-1β+ CD8+ T cells and the NP cohort was a distinctive feature of this MIP-1β+ cell population and was not shared with other MIP-1β+ T-cell populations. In fact, although a significant higher proportion of CD45RA+ IFN-γ+ IL-2neg MIP-1β+ responding CD8+ T cells was observed in NP in comparison to ART-treated individuals . In addition, NP showed detectable plasma viremia, although at low levels . As a consequence, antigen exposure could have played a direct role in generating CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells. However, the analysis of the relationship between plasma viremia and Nef-specific CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells expressed as percentage of the total Nef-specific response or as percentage of the total CD8+ T cells revealed no significant correlation (data not shown). Furthermore, only one PR (10%) showed detectable levels of Nef-specific CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells, supporting the idea that this novel CD8 T-cell population is not directly driven by antigen levels.We next explored the potential effect of differences in the level of viremia on the presence of CD45RA+ IFN-γin vivo HIV-1 replication in generating CD45RA+ IFN-γneg IL-2neg MIP-1β+ CD8+ T cells, we analyzed Nef- and Tat-specific CD8+ T-cell responses in a longitudinal set-up. Six ART-treated patients with highly suppressed viremia  underwent a single cycle of therapy interruption (TI). Viremia became detectable in all patients between day 5 and 21 after TI. ART was resumed between day 27 and 185 when viremia levels reached >100,000 HIV-1 RNA copies/ml. A significant expansion of the Nef-specific CD8+ T-cell responses was observed in all the subjects analyzed . This observation indicates that IL-2 expression represents neither an essential marker of nonprogressive HIV-1 infection nor a distinctive feature of CD45RA+ IFN-γneg MIP-1β+ CD8+ T cells in NP.Since in our cohorts of HIV-1 infected patients IL-2-producing cells were rarely detected, we reanalyzed the data shown in Figure neg MIP-1β+ CD8+ T cells derived from one HIV-1 infected individual that following treatment interruption showed a partial control of viral replication . In this patient, Nef-specific CD45RA+ IFN-γneg MIP-1β+ CD8+ T cells were previously characterized (unpublished data). As shown in Figure neg MIP-1β+ CD8+ T cells were CCR7neg. In the same experiment, we additionally measured TNF-α expression and observed that Nef-specific CD45RA+ IFN-γneg MIP-1β+ CD8+ T cells did not express TNF-α upon antigenic peptide stimulation  antigenic peptide stimulation. Thus, only experienced (memory or effector T-cells) can be detected by this assay, since the number of circulating naive T-cells carrying a T-cell receptor specific for a given peptide is too low to be detected by short term assays. Nevertheless, we characterized the expression of CCR7 in CD45RA+ IFN-γn Figure .neg MIP-1β+ CD8+ T cells did not express the chemokine receptor CCR7 and are therefore classified as effector CD8+ T-cells  CD8+ T cells potentially represent a valuable immune correlate of disease progression, since they were detected in response to Nef stimulation in 7 out of 11 NP, in only 1 out of 10 PR and were completely absent in 22 ART-treated patients. We further demonstrated on 6 ART-treated patients undergoing single cycle TI that the presence of MIRA CD8+ T cells is independent of viral load. These observations render this novel population particularly interesting as a potential surrogate clinical marker of immunological reconstitution or maintenance after immune-based interventions.A fundamental prerequisite for the development of immune-based therapies and an effective vaccine against HIV/AIDS is the identification of solid immune correlates of disease progression. In order to identify such correlates, we compared Nef and Tat specific CD8+ T-cell immune responses in three cohorts of HIV-1 infected individuals with different degree of HIV-1 control: NP, PR and ART-treated patients. With this setting, we identified a novel population of CD8+ T cells associated with nonprogressive HIV-1 infection. CD45RA+ IFN-γCM (CD45RAneg CCR7+), TEM (CD45RAneg CCR7neg) or TEMRA (CD45RA+ CCR7neg) [EMRA population. The absence of CCR7 expression by MIRA CD8+ T cells was demonstrated in one representative sample, further supporting the TEMRA phenotype. Several studies have suggested a possible role of the fully differentiated HIV-1-specific TEMRA CD8+ T cells in the effective control of HIV-1 replication: IFN-γ producing TEMRA CD8+ T cells have been associated with the control of virus replication in NP [EMRA CD8+ T cells were preferentially detected in acutely infected individuals who achieved control of viremia either spontaneously or after structured TI [EM cells) has been reported for HIV-1-specific CD8+ T cells in therapy-naive viremic patients [in vivo. Here we support and extend the link between HIV-1-specific TEMRA CD8+ T cells and slow disease progression by identification of a novel HIV-1-specific population of effector cells specific for nonprogressive HIV-infection.According to the expression of CD45RA and CCR7, antigen-experienced CD8+ T cells are classified as TCCR7neg) . Since Mon in NP  and in ion in NP . Furthertured TI . A pre-tpatients ,24. The neg CD8+ T cells in ART-treated individuals in comparison to PR  CD45RAneg CD8+ T cells in PR in comparison to NP and ART-treated individuals  showed also the highest total Nef-specific response with a large proportion of Nef-specific MIRA CD8+ T cells (36.5%). These observations suggest that MIRA CD8+ T cells are not the direct consequence of ongoing in vivo antigen exposure, but possibly may represent a correlate of HIV-1 disease progression. However, it can be suggested that the prolonged exposure to low levels of plasma viremia in NP is responsible for the appearance of MIRA CD8+ T cells, and that MIRA CD8+ T cells do not appear following the short term exposure to high level of viremia during TI. To address this hypothesis it would be helpful to analyze elite controllers, who are characterized by controlled HIV infection with undetectable viral load. In addition, more detailed longitudinal studies will be necessary to definitively demonstrate the role of MIRA CD8+ T cells in HIV-1 infection.In chronic viral infections, the main obstacle to the definition of a correlate of disease progression is the ability to discriminate between phenotypes responsible for the control of viral replication and phenotypes that are the consequence of a different infection history . MIRA CDnef and p24 genes in comparison to other HIV-1 genes [http://www.avip-eu.org. A genome-wide analysis of the HIV-1-specific response using our intracellular cytokine staining analysis will aid in understanding the role of MIRA CD8+ T cells specific to p24 and other HIV-1 antigens in the control of viral replication.Several reports showed an exceptional HLA class I associated sequence polymorphism in the -1 genes ,27, sugg-1 genes . Our stuOur study demonstrated that the sole measurement of the Nef-specific response might be sufficient to define MIRA CD8+ T cells as a correlate of nonprogression in HIV-1 disease. In addition, the presence of MIRA CD8+ T cells in 2 Tat-responding NP and the absence of the same population in the Tat-responding PR and ART-treated patients suggest that MIRA CD8+ T cells may represent a correlate of nonprogression independently of the targeted viral protein. Since, it has been described that the use of autologous peptides allows the detection of stronger and broader T-cell responses , we cannHIV-1 infection causes hyperactivation of the immune system leading to immune exhaustion and disease progression -32. SincSince the present study was observational, it was not our objective to clarify whether MIRA CD8+ T cells exert a direct protective function. However, it has been shown, that MIP-1β dominates HIV-1-specific CD8+ T-cell responses ,34 and tin vivo viral replication. MIRA CD8+ T cells may be useful to ameliorate the timing of ART initiation in HIV-1 infected individuals and represent a potential correlate to determine the efficacy of immune-based interventions.In conclusion, our study presents a novel population of Nef-specific effector CD8+ T cells associated with nonprogressive HIV-1 infection. This population, named MIRA, expresses CD45RA and produces MIP-1β but not IFN-γ. This T cell subset was shown to be independent of Eleven NP, 10 PR and 23 ART-treated patients were included in the present study. Of the 11 NP subjects, 10 matched the definition of long-term nonprogressors (LTNP), i.e. naive to ART with a documented HIV-1 infection of >9 years (median: 19.5 years), CD4+ T-cell counts ranging between 421 and 1,042 (median: 522 cells/μl). In comparison to the other NP, study subject NP11 had lower levels of CD4+ T-cell counts (274 cells/μl). Nevertheless he was included in the NP cohort because of 19 years of documented history of HIV-1 infection, with stable CD4+ T-cell counts over a one year follow-up post-sampling (range: 256-310 cells/μl). The median plasma viral load in the 11 NP was 900 HIV-1 RNA copies/ml (range: <50-10756). PR had poor restriction of viral replication (HIV-1 RNA copies/ml >99000) and declining CD4+ T-cell counts . All the PR were ART naive. PR05 was the only rapid progressor in the PR cohort. Already five month after putative HIV-1 infection (the timepoint of blood sampling) CD4+ T-cell counts dropped in this patient to 208 counts/ml without recovery and persistently high level viremia. The 23 ART-treated patients were on treatment for 2 or more years. Twenty ART-treated subjects had undetectable viral load, whereas subjects ART12, ART11 and ART23 had 36600, 3362 and 13965 HIV-1 RNA copies/ml, respectively. The median CD4+ T-cell count in the 23 ART-treated subjects was 488 cells/μl (range: 229-969). Study subjects ART12, ART14, ART15 and ART16 were previously classified as NP but initiated ART more than 4 years before blood sampling for the present study and therefore are here included in the ART-treated cohort. Six of the ART-treated individuals  underwent a single cycle of therapy interruption (TI). From these patients PBMC were obtained before TI . Written informed consent was obtained for all study participants.CD3-AmCyan, CD4-PerCP, CD45RA-PE-Cy7, CD154-FITC, IFNγ-Al700, IL2-APC and MIP-1β-PE were obtained from Becton Dickinson. CD8-PacB was obtained from DAKO. CCR7-FITC and TNFα-PacB were obtained from R&D and NatuTec, respectively.Nef- and Tat-specific cellular responses were identified by using two sets of overlapping peptides covering the HIV-1 encoded Tat (BH10 strain) and Nef (BRU strain) antigens. Nef peptides were divided into 2 pools: Nef N-term (amino acids 1 to 101) and Nef C-term (amino acids 97 to 206). All pools were used at the final concentration of 2 μg/ml of each peptide. The Tat- and Nef-specific sets of overlapping peptides have been designed using a strategy termed Variable Overlapping Peptide Scanning Design (VOPSD)  that hasBriefly, all peptides were biased at the C-terminus avoiding 7 amino-acid residues never found among optimal CTL epitopes and MHC class I binding motifs . Optimal epitopes were placed in the N- or C-terminus of the peptides. Finally, conserved and variable regions were segregated in different peptides. In order to fit all the above mentioned design features the derived peptides were conceived to be variable in length (10 to 19 amino-acids) and degree of overlap . Moreover, the degree of overlap and the overall peptide length were empirically tuned according to the frequency of known T-cell epitopes . Peptides were obtained through the Centre for AIDS Reagents, National Institute for Biological Standards and Control, Hertfordshire, UK.6 PBMC per well were incubated with 1.3 μg/ml of anti-CD28 mAb and with 1.3 μg/ml of anti-CD49d mAb (Becton Dickinson) together with defined peptide pools. For each individual donor, a sample without peptides was included to calculate the background staining. Following 60 min incubation, 10 μg/ml of Brefeldin A (Sigma) were added to the cell suspension and the incubation was carried out for additional 4 h. Stimulated cells were then resuspended in Stain Buffer  and incubated with the photoreactive fluorescent label ethidium monoazide  to assess their viability. After washing, cells were fixed and permeabilized using the BD Cytofix/Cytoperm™ Kit (Becton Dickinson). Then fluorochrome-conjugated Abs were added to the cell suspension. Incubation was carried out on ice for 30 min and after washing, cells were acquired using an LSRII flow cytometer (Becton Dickinson) equipped with a high throughput system. Sample analysis was performed using FlowJo version 8 (Tree Star). Lymphocytes were gated on a forward scatter area versus side scatter area pseudo-color dot plot and dead cells were removed according to EMA staining. Events were gated on CD3+ events versus IFN-γ, IL-2, MIP-1β and CD154 to account for down-regulation. CD3+ events were then combined together using the Boolean operator "OR". The same procedure was used to subsequently gate CD8+ events. CD4+ events were excluded before creating a gate for each function or phenotype as shown in Figure Cryopreserved samples were used throughout the study. The 9-color intracellular cytokine staining used in the present study has been previously described . Brieflyneg CD8+ T-cell populations were combined using SPICE software. SPICE version 4.1.5  and Prism version 5.01 (GraphPad Software) were used for graphical representation of the data and statistical analysis. To compare paired groups, the Wilcoxon signed-rank test was performed. To account for multiple comparisons, a closed testing procedure was applied in testing differences of all groups by the Kruskal-Wallis test followed by pair-wise comparisons with the Wilcoxon rank sum test. The SAS Version 9.1 was used to get exact P values for the statistical analysis.Since background levels varied between subpopulations, i.e. MIP-1β staining showed a higher background than IFN-γ, and the combination of 3 or more functions had extremely low background, we calculated a threshold level for each subpopulation. Following background subtraction, the threshold level was calculated using the 90th percentile of the negative values. Then, all the resulting negative values were set to zero and all values >0 were considered as positive responses. The frequency of the total CD8+ T cell response was calculated by summing each unique CD8+ T-cell population expressing at least one functional marker. Percentages of total responses were calculated by dividing the frequency of each subpopulation by the frequency of the total CD8+ T cell response. In order to analyze the quality of the response without considering the CD154 marker, the CD154+ and CD154AC, PL, GP, VE and MM are named as inventors on a provisional patent application based on this work. The patent has been filled by the Helmholtz Zentrum München - German Research Center for Environmental Health and the San Raffaele Scientific Institute.GP, VE, MM and AC conceived the study. CJD, SK, SA and AC established the flow cytometry based assays. CJD, SK, SH, SA and DH performed experiments. SG, EV, GT, JRB, HJS, ES, TV, FDG, RD, MT, GP and AC participated to the collection of patient samples. CJD, SH, SK, SA, PB, PR, MM and AC analyzed data. PL and UP contributed to research and critical discussion. AC wrote the paper. All authors read and approved the final manuscript.


The intestinal mucosa displays robust virus replication and pronounced CD4+ T-cell loss during acute human immunodeficiency virus type 1 (HIV-1) infection. The ability of HIV-specific CD8+ T-cells to modulate disease course has prompted intensive study, yet the significance of virus-specific CD8+ T-cells in mucosal sites remains unclear.We evaluated five distinct effector functions of HIVgag-specific CD8+ T-cells in rectal mucosa and blood, individually and in combination, in relationship to clinical status and antiretroviral therapy (ART). In subjects not on ART, the percentage of rectal Gag-specific CD8+ T-cells capable of 3, 4 or 5 simultaneous effector functions was significantly related to blood CD4 count and inversely related to plasma viral load (PVL) (p<0.05). Polyfunctional rectal CD8+ T-cells expressed higher levels of MIP-1β and CD107a on a per cell basis than mono- or bifunctional cells. The production of TNFα, IFN-γ, and CD107a by Gag-specific rectal CD8+ T-cells each correlated inversely (p<0.05) with PVL, and MIP-1β expression revealed a similar trend. CD107a and IFN-γ production were positively related to blood CD4 count (p<0.05), with MIP-1β showing a similar trend. IL-2 production by rectal CD8+ T-cells was highly variable and generally low, and showed no relationship to viral load or blood CD4 count.The polyfunctionality of rectal Gag-specific CD8+ T-cells appears to be related to blood CD4 count and inversely related to PVL. The extent to which these associations reflect causality remains to be determined; nevertheless, our data suggest a potentially important role for mucosal T-cells in limiting virus replication during chronic infection. A significant body of work has demonstrated the contribution of HIV-specific CD8+ T-cell responses to immunological control of HIV infection Leishmania major is accompanied by T-helper type 1 cells that are able to produce multiple cytokines Given the ambiguous relationship between HIV-specific T-cell response magnitude and clinical parameters, investigators have sought new combinations of immunological markers whose expression might more reproducibly correlate with clinical status. The advent of multiparameter flow cytometry, allowing the simultaneous measurement of multiple effector molecules, has led to an emphasis on T-cell response “quality” or “functional heterogeneity” rather than “quantity” or “magnitude” of IFNγ responses alone Most prior studies of HIV-specific cell-mediated immunity have assessed responses only in the peripheral blood compartment; little information is available concerning HIV-specific T-cell responses in mucosal tissues. In a previous study focused primarily on IFN-γ production, we reported that rectal CD8+ T-cell responses to Gag were immunodominant (as compared to Pol and Env) in chronically infected subjects not on antiretroviral therapy with a broad range of viral loads In the present work, we have analyzed paired blood and rectal biopsies from chronically infected patients for five distinct responses to HIVgag stimulation: production of the cytokines IFN-γ, IL-2, TNF-α, the chemokine MIP-1β, and the cytolytic granule constituent CD107a, which is an indirect marker for cytotoxic potential Using an expanded panel of response parameters coupled with a new and rigorous statistical approach to evaluating when a response is significant see , here wep>0.05). The subject pool was primarily Caucasian (56%) and African American (33%), and the gender distribution was approximately two-thirds male and one-third female. Among seropositive subjects not on ART, viral loads ranged from <50 to 89,700 copies per ml plasma, and blood CD4 counts ranged from 304 to 883 cells per µl blood  between rectal CD4 percentage and plasma viral load (data not shown).The dynamics of CD4+ T-cell restoration in mucosal tissues following initiation of ART is an area of current interest All Gag-specific responses reported in this study were first evaluated for significance, in relation to responses associated with exposure to costimulatory antibodies alone, using a statistical test rather than a simple cutoff value see . Responsp<0.05). Furthermore, CD107a and IFN-γ responses in rectal mucosa were positively correlated with blood CD4 count (p<0.05). In contrast, Gag-specific response magnitudes in PBMC were not significantly related to viral load or to blood CD4, and rectal CD8+ T-cell responses did not show a significant relationship to rectal CD4 percentage (data not shown). These findings suggest that, in individuals with chronic infection in the absence of ART, HIV-specific T-cell responses measured in rectal mucosa may be a stronger correlate of viral replication and immune status than the corresponding responses measured in peripheral blood.As shown in In the subject group not on ART, the enhanced HIVgag responsiveness of rectal CD8+ T-cells in comparison to PBMC, and the significant relationship of rectal responses with plasma viral load and blood CD4 count , led us The mean total percentage of rectal CD8+ T-cells responding to HIVgag stimulation in any way  was 5.9% for the group not on ART. The pie charts and bar graphs provide finer details illustrating that nearly one-half of the rectal response in patients not on ART was comprised of polyfunctional cells capable of 3, 4, or 5 responses simultaneously. In contrast, in the group on ART, the mean percentage of rectal CD8+ T-cells responding to HIVgag stimulation was 2.9%. Even more strikingly, cells producing 3, 4, or 5 responses accounted for less than 10% of the responding population, as shown in the pie chart. Thus, in this cross-sectional comparison of patients on and off ART, ART was associated with a striking absence of polyfunctionality in rectal mucosa.In PBMC of patients not on ART, the mean percentage of HIVgag-specific CD8+ T-cells was lower than in rectal mucosa (3.8% vs. 5.9%), and approximately 30% of this response was derived from cells capable of at least 3 responses. In the ART group, the mean percentage of responding cells was 1.1%, fewer than 20% of which were polyfunctional. The utility of Boolean gating analysis lies not only in the ability to assemble a detailed view of responding CD8+ T-cell populations , but alsp = .024) and negatively correlated with plasma viral load . In contrast, the summed 1- and 2-function responses showed no relationship to blood CD4 or plasma viral load (not shown). Additional significant correlations (p<.05) included mucosal 4 and 3-function CD8 responses in relation to blood CD4 count; mucosal 4-function CD8 responses in relation to plasma viral load; and the summed 1+ through 5+ mucosal Gag-specific CD8 response in relation to blood CD4 count and plasma viral load (data not shown). Although similar statistical analyses were performed for blood CD8+ T-cell responses, we did not identify any significant relationships between total blood CD8 responses or any subset of blood CD8 responses and either plasma VL or blood CD4 count (not shown). In addition, functionality of rectal CD8+ T-cell responses failed to exhibit any significant correlation with rectal CD4 frequency as a percent of CD3+ T-cells (not shown).Rectal, but not peripheral, Gag-specific CD8+ T-cell responses within certain functional categories showed significant correlations with clinical status. The strongest of these relationships was observed when the percent response was summed across the three predominant polyfunctional categories and the summed values were plotted against blood CD4 count or plasma viral load. Polyfunctional CD8+ T-cells have previously been demonstrated to produce more IFN-γ on a per-cell basis than cells of lower functionality Typical lymphocyte activation markers were generally expressed at higher levels by CD8+ T-cells in rectum than in PBMC . In agreAs previously reported, perforin expression was extremely low in rectal CD8+ T-cells as compared to PBMC In this study we introduce the use of a statistical approach to establish when an antigen-specific response is significantly different from the response elicited by costimulatory antibodies alone see . While tVirions in circulation are thought to originate from tissue sites of active virus replication. As the intestinal tract is a major lymphoid organ that supports high levels of virus replication during acute infection, it may also serve as a significant source of viremia during chronic infection. In a previous study of a more limited patient group, we observed that plasma viremia was inversely, though not significantly, associated with IFNγ production by Gag-specific rectal CD8+ T-cells, while blood CD4 count showed a significant positive correlation with Gag-specific rectal responses Others have observed that peripheral Gag-specific CD8+ T-cell responses have clinical relevance as suggested by high-magnitude and complex Gag-specific responses in HIV controllers Our finding of enhanced production of MIP-1β and CD107a on a per-cell basis as functionality increases is the first observation of this kind for mucosal CD8+ T-cells yet is consistent with other studies reporting increased IFN-γ production by polyfunctional T-cells in peripheral blood It has been known for some time that the magnitude of HIV-specific CD8+ T-cell responses in peripheral blood declines rapidly when individuals begin ART Immune reconstitution in the context of ART, particularly as defined by CD4 repopulation in the intestinal tract and peripheral blood, is of intense current interest. Previously we reported a significant positive correlation between CD4 percentages in these two compartments In summary, we have shown here that rectal HIVgag-specific CD8+ T-cells from chronically infected subjects not on ART are able to mount robust and diverse effector responses which correlate positively with blood CD4 count and inversely with plasma viral load. These associations were not evident for peripheral blood CD8+ T-cells, suggesting a unique status for mucosal HIV-specific CD8+ T-cells which remains to be fully defined. In the context of ART, a predictable drop in the total responding CD8+ T-cells in both tissues was accompanied by a marked loss of response complexity in rectal mucosa. The provision of ART was also linked to the observation that peripheral and rectal CD4+ T-cell percentages are tightly positively correlated.Although a role for mucosal T-cell responses in limiting viral dissemination during early/acute infection has been postulated, the contribution of mucosal effector cells to the immune control of viral replication during chronic infection has not been addressed. The results presented here suggest that, when individuals throughout a broad range of viral loads and blood CD4 counts are evaluated, there are significant relationships between antiviral mucosal effector responses , and both VL and CD4. However, a cause/effect relationship between mucosal CD8+ T-cells and clinical status cannot be definitively established due to the cross-sectional nature of this study; indeed, it is possible that polyfunctional responses are a consequence of an intact immune system in individuals with low viral replication, rather than the cause of low virus replication. Additional studies will be required to address the relationship between mucosal T-cell responses and tissue viral loads.Subjects were chronically HIV-infected patients and seronegative volunteers and at the Center for AIDS Research, Education and Services (CARES), Sacramento, CA. Patients taking and not taking antiretroviral drugs were enrolled in the study. This patient cohort has been described previously PBMC were isolated from blood using Ficoll-Paque™ . Isolation of mononuclear cells from rectal biopsies was done using an optimized collagenase digestion procedure as previously described Flow cytometry data were acquired on an LSRII  equipped with 405, 488, and 643 nm lasers and utilizing FACSDIVA software (BDIS). Analysis of cytometry data was done with FlowJo software ; for experiments measuring five functional responses, individual cytokine gates were evaluated alone and also processed through Boolean combinations to partition responding cells into one of 31 possible specific response categories. Cytometry data were biexponentially transformed in order to include all events. SPICE software  was used to graph response data.Blood CD4 count was determined by the clinical laboratory at University of California, Davis, Medical Center. Plasma viral load was measured by the California Department of Health Services, Viral and Rickettsial Disease Laboratory using the Roche COBAS HIV-1 Monitor v1.5 test. This method has a sensitivity cutoff of 50 HIV-1 RNA copies per ml plasma.Fluorochrome-labeled monoclonal antibodies to CD3 (clone UCHT1), CD107a (clone H4A3), IFNγ (clone B27), TNF-α (clone MAb11), IL-2 (clone 6344.111), MIP-1β (clone D21-1351), Granzyme B (clone GB11), and CD25 (clone M-A251) were purchased from Pharmingen , as were unlabeled CD28 (clone L293) and CD49d (clone L25), and 7-Amino-Actinomycin D (7-AAD). Fluorochrome-labeled aniti-CD8 (clone 3B5), CD4 (clone S3.5), HLA-DR (clone TU36), and CD69 (clone CH/4) were purchased from Invitrogen. Anti-CD4 (clone SFCI12T4D11) was obtained from Beckman Coulter  and anti-Human Perforin (clone PF-164) was obtained from MABTECH. The HIVgag peptide pool  consisted of 15-mers with an 11 amino acid overlap .2 prior to analysis. For ICS, cells were stimulated with pooled HIVgag peptides (3.5 µg/ml) plus costimulatory antibodies (CD28 and CD49d). Negative and positive controls were media containing peptide vehicle (DMSO) plus costimulatory antibodies, and staphylococcus enterotoxin B , respectively. The expression of CD107a, MIP-1β, IL-2, TNF-α and IFN-γ were measured using a modification of a previously described method 6 cells in 200 µl complete medium were treated with anti-CD28 (2.5 µg/ml), anti-CD49d (5 µg/ml), anti-CD107a, monensin , brefeldin A , and HIVgag peptide pool or DMSO. Following a 5 hour incubation, cells were incubated for 5 minutes in PBS/2% FCS/0.5 mM/EDTA, stained for surface markers and 7-AAD (Pharmingen) in PBS/2% FCS for 20 min at 4°C, fixed in 4% formaldehyde, then permeabilized using FACS Perm 2 (BD Biosciences). Cells were then washed in PBS/2% FCS, stained for intracellular markers and CD3, washed again, then stored at 4° C in PBS/1% formaldehyde until analysis (within 24 hours). Non-fluorescent Actinomycin D (2 µg/ml) was included in reagents after the 7-AAD staining step to saturate sites that could bind residual 7-AAD ICS assays and phenotypic staining were carried out on freshly isolated PBMC and RMC that were rested overnight at 37°C, 5% COPhenotypic staining of unstimulated peripheral and rectal CD8+ T-cells for surface and intracellular antigens was carried out using Caltag Fix and Perm (Invitrogen) according to the manufacturer's protocol. Stained cells were stored at 4° C in PBS/1% formaldehyde until analysis (within 24 hours). Flow cytometry gating was as shown in in vivo and frequently exhibit elevated cytokine production relative to PBMC 1X is the number of cells  responding to peptide plus costimulation within a total CD8+ T-cell population designated as 1n. Similarly, 0X is the number of events associated with costimulation alone within a total CD8+ T-cell population designated as 0n. The test was carried out as follows: (1) If one or both of 0X and 1X was equal to zero, then the value was set to 0.5; (2) the test statistic 3W was calculated; (3) the test statistic 3W was referred to a standard normal distribution and the p-value was calculated; (4) If the p-value was less than the significance level (0.05), this indicated that the background cytokine response and the Gag-stimulated cytokine response were statistically different. In this case, the background response was subtracted from the Gag-specific response to yield a statistically meaningful net response. If, on the other hand, the p-value was equal to or greater than 0.05, this indicated that the background cytokine response and the Gag-stimulated response were not statistically different. In this case, the net Gag-specific response was set to zero.Net antigen-specific responses were evaluated using a statistical test For comparisons of T-cell function between tissues within the same patients, paired T-tests (two-tailed) were used. Mann Whitney (two-tailed) tests were used to compare T-cell function data between subjects  within a given tissue type. Expression of phenotypic markers was compared between tissues within the same patients using a paired T-test, and between patient groups using a Kruskal-Wallis test followed by Dunn's Multiple Comparison Test. Correlations between CD8+ T-cell response magnitudes and plasma viral load, CD4+ T-cell count (blood) and CD4+ T-cell frequency  were determined using Spearman Correlation . Linear regression analysis was used to display a best fit line to the data.T-cell responses to antigenic stimulation were evaluated by Boolean partitioning of responses into 31 distinct responding populations


Most HIV-infected patients receiving virologically suppressive antiretroviral therapy continue to have abnormal, generalized T cell activation. We explored whether the degree of ongoing cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) replication was associated with higher virus-specific T cell activation and the failure to achieve normal absolute CD4+ T cell counts in the face of long-term suppressive antiretroviral therapy.Longitudinally collected PBMC and saliva specimens obtained from HIV-infected patients on effective antiretroviral therapy for at least one year (plasma HIV RNA <75 copies/mL) were examined using a multiplex CMV, EBV and KSHV DNA PCR assay. Eleven cases were chosen who had CD8+ T cell CD38+HLA-DR+ expression >10% and plateau absolute CD4+ T cell counts <500 cells/µL. Five controls from the same study had CD8+ T cell CD38 expression <10% and plateau absolute CD4+ T cell counts >500 cells/µL.Among all subjects combined, 18% of PMBC samples were positive for CMV DNA, and 27%, 73% and 24% of saliva samples were positive for CMV, EBV and KSHV DNA, respectively. No significant differences or trends were observed between cases and controls in proportions of all CMV, EBV or KSHV DNA positive specimens, proportions of subjects in each group that intermittently or continuously shed CMV, EBV or KSHV DNA in saliva, or the median number of genome copies of CMV, EBV and KSHV DNA in saliva. Overall, number of genome copies in saliva were lower for KSHV than for CMV and lower for CMV than for EBV. Although replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients, we observed no evidence in this pilot case-control study that the magnitude of such human herpesvirus replication is associated with abnormally increased CD8+ T cell activation and sub-normal plateau absolute CD4+ T cell counts following virologically suppressive antiretroviral therapy. Currently available highly active antiretroviral therapy (HAART) regimens are able to dramatically decrease plasma HIV RNA levels in the majority of patients with chronic HIV infection, leading to sustained increases in absolute CD4+ T cell counts and decreased risk of progression to AIDS and death. These antiretroviral regimens, however, have limitations in terms of immunologic efficacy The reasons underlying persistent T and B cell activation, in the face of effective antiretroviral therapy, are unknown. Potential causes include 1) persistent HIV replication, at levels below those detectable using currently available commercial plasma HIV RNA assays, 2) persistent systemic replication of a co-infecting virus such as human herpes or hepatitis viruses, 3) persistent microbial translocation We chose saliva as a compartment to monitor active CMV replication because of the expectation that this body fluid would provide a sensitive source of this virus in antiretroviral-suppressed HIV-infected patients Finally, given the fact that both EBV or KSHV are also shed in increased amounts in saliva in HIV-infected patients This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of California San Francisco. All patients provided written informed consent for the collection of samples and subsequent analysis.PMBC and saliva specimens were obtained from a prospective observational study of HAART (SCOPE). In SCOPE, subjects are evaluated by medical history and measurement of plasma HIV viral load and absolute CD4+ T cell count every 4 months. At each of these visits, samples of blood for plasma and PBMC separation and of unstimulated whole saliva are obtained and stored at −80°C. For this study we identified specimens from SCOPE participants who met the following criteria:On HAART with at least one year of sequential plasma HIV RNA levels consistently sustained at levels <75 copies/mL before the time the first specimens tested were obtained.Paired stored PBMC and saliva specimens were available from at least 2 separate visits spanning a minimum 6 month period while the subject remained on HAART with a HIV RNA level consistently <75 copies/mL and during which the percentage of circulating CD8+ T cells co-expressing CD38 and HLA-DR was measured.Among the specimens from these participants, we selected “cases” who had high CD8+ T cell activation and low absolute CD4+ T cell counts at the time specimens tested were obtained, and were defined as:Percentage of circulating CD8+ T cells co-expressing CD38 and HLA-DR was >10%.Absolute CD4+ T cell counts were consistently <500 cells/µL.In addition, since chronic hepatitis C infection has been reported to cause abnormally increased CD8+ T cell activation and low absolute CD4+ T cell count in HIV-coinfected patients We also identified a group of controls who had low CD8+ T cell activation and high absolute CD4+ T cell counts at the time specimens tested were obtained, defined as:Percentage of circulating CD8+ T co-expressing CD38 and HLA-DR was <10%Absolute CD4+ T cell counts were consistently >500 cells/µL.Freshly drawn EDTA anti-coagulated whole blood was stained, lysed and fixed as previously described 5 cell equivalents. Aliquots of 106 freshly separated PBMC were lysed, boiled for 5 min, cooled on ice, and stored at −80°C until use. DNA was isolated from samples using the QIAGEN DNeasy Tissue kit  according to the manufacturer's instructions. The initial amplification of DNA was with primers IEP2AII (5′ATGGAGTCCTCTGCCAAGAGAAAGATGGAC3′) and IEP4BII (5′CAATACACTTCATCTCCTCGAAAGG3′), followed by IEP3B (5′TCTGCCAGGACATCTTTCTC3′ and IEP3A (5′GTGACCAAGGCCACGACGTT3′). For the initial qualPCR, 5 µl  of extracted PBMC and PMN DNA or 50 ng EMB DNA was added to 45 µl reaction mixtures containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 200 µM of each deoxynucleotide triphosphate (Invitrogen Corporation), 1 U Taq polymerase (Roche Diagnostics Corporation), and 1 µM of each primer. For the second qualPCR, 5 µl out of the first reaction mixture was added to a 45 µl reaction mixture as used for the initial qualPCR. qualPCR mixtures were layered with 50 µl of mineral oil and subjected to one cycle of 94°C for 3 min, followed by 30 cycles of 1 min of denaturation at 94°C, 1 min of annealing at 62°C, and 2 min of extension at 72°C, and a final extension step of 7 min at 72°C. Positive and negative controls were included in each run, and, after qualPCR, each sample was electrophoresed through a 2% agarose gel containing 0.2 µg/ml ethidium bromide with appropriate DNA size markers. Amplicons were visualized on a UV transilluminator and photographed.As previously described 5 cells. Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System . Five µl of sample was added to 45 µl of reaction mix containing 25 µl SYBR green PCR Master Mix (Applied Biosystems) and 10 pmol of each primer. The primers used for the real-time PCR were also located in the gene encoding the IE1 protein, exon 2 . Conditions for the real-time PCR were established empirically and included incubation at 50°C for 2 min to enable uracil-DNA-glycosylase (in Master Mix) to act on samples and 94°C for 10 min to activate AmpliTaq Gold polymerase, followed by 40 cycles of 15 s of denaturation at 94°C and 1 min of annealing and extension at 60°C. A standard curve of serial dilutions of known amounts of plasmid DNA was used in each run as an internal control and to determine the copy number in the samples. In addition, a natural CMV DNA positive sample was run to assess consistency. The detection limit of this test was 5 CMV DNA copies/105 cells, and positive samples were quantitated as copies/5 µL of lysed cells. Each run included positive and negative samples, and specificity of the amplified products was assessed on each run by dissociation curve analysis to ensure that all products had an expected, uniform melting temperature.To quantify viral load in the qual-PCR-positive samples, we used SYBR green detection of real-time PCR products able to detect a range of 3 to 100,000 CMV genome copies/105′- AAGGTGCATTTACCCCACTG, EBNA3cR, 5′- AGCAGTAGCTTGGGAACACC5′- GGAAGAGCCCATAATCTTGC, LANA78r 5′- GCCTCATACGAACTCCAGGT, FOR HCMV GlyBcmvF 5′-AAGTACCCCTATCGCGTGTG, GlyBcmvR 5′-ATGATGCCCTGATCCAAGTCDNA was isolated from 250 µl saliva (diluted to 1000 µl with PBS) using the QIAGEN QIAamp UltraSens Virus Kit  according to the manufacturer's instructions. DNA was eluted in 80 µl 0.1× TE pH 7.8 (Fisher Inc.). A single QPCR reaction contained 14 µl 2×SYBR (Roche Inc.), 1 µl primer , 7 µl PCR grade water and 8 µl sample. Single plex reactions were set up on a RoboGo (MWG Inc.) pipetting station and analyzed on a MJR Opticon II machine. Cycle conditions were 56°C: 5 min, 95°C: 3 min, . The primers were for EBV: EBNA3cF 10 copies/ml (just below the lower limit of detection of 1.8 log10 copies/ml). Fisher's exact tests were used to compare the proportion of cases and controls with ≥1, ≥2, or all samples positive. The Spearman rank correlation test was used to test the association of subject's percent of activated CD8+ T cells with the amount of herpesvirus DNA in their saliva.For analyses including multiple observations per participant, we evaluated differences in categorical outcomes  between cases and controls with logistic regression for clustered data and differences in DNA copy number between cases and controls with generalized estimating equations. For analyses comparing DNA copy numbers between groups, undetectable levels were assigned a value of 1.79 logEleven SCOPE subjects meeting the criteria for inclusion were sampled as cases in the high CD8 T cell activation/low CD4 count group. The cases had 2–4 saliva and PMBC specimens assayed which spanned a 6–12 month period. The cases had a median (range) absolute CD4+ T cell count of 279 (112–450) cells/µL and percentage of activated (CD38+HLA-DR+) CD8+ T cells of 17.9% (11–38%) during this study period. Five subjects met the criteria for inclusion as controls in the lower CD8 T cell activation/higher CD4 count group, had 4 PMBC and saliva specimens assayed which spanned a 12 month period, and had a median (range) absolute CD4+ T cell count of 687 (519–858) cells/µL and percentage of activated (CD38+HLA-DR+) CD8+ T cells of 5.4% (4–7.6%) during this study period. Demographics and characteristics of the subjects studied are summarized in 5 cells in PBMC), as well as comparisons of the proportion of case and control subjects who had ≥1, ≥2 or all saliva specimens positive. In addition, salivary herpesvirus DNA levels (log10 copies/mL) were compared between cases and controls using generalized estimating equations.Among all subjects combined, 18% of PMBC samples were positive for CMV DNA and 27%, 73% and 24% of saliva samples were positive for CMV, EBV and KSHV DNA, respectively. However, all categorical comparisons between the case and control groups were non-significant with p values >0.5, and all comparisons of median DNA copy number between cases and controls were non-significant with p values >0.9.As there was no consistent difference between the cases and controls in terms of any of the herpesviruses measured, we combined the groups to determine if the level of CMV, EBV or KSHV DNA in saliva was associated with the level of CD8+ T cell activation. The time point of saliva collection temporally closest to that of a CD8+ T cell CD38/HLA-DR expression measurement was used. We found no correlation between the percentage of activated CD8+ T cells and the amount of CMV DNA , EBV DNA , or KSHV DNA  in saliva.In general, positive readings tended to occur within subjects, suggesting that the “steady-state” levels varied by subject. Of note, all positive PBMC samples had very low copy numbers, <3 CMV DNA copies per 5 µL of lysed cells. Also, the number of genome copies in saliva overall were lower for KSHV than for CMV and lower for CMV than for EBV.Replication of CMV, EBV and KSHV persists in many antiretroviral-suppressed, HIV-infected patients to a greater degree than in HIV-uninfected adults Miller, et al, previously reported results using a similar PCR assay to detect CMV, EBV and KSHV in similar unstimulated whole saliva specimens from 58 HIV-infected patients, most of whom had HIV replication suppressed by antiretroviral therapy Similar to the results from Miller et al, we noticed that median CMV levels were lower than median KSHV levels, which in turn were lower than median EBV levels . This suThis pilot study has several important limitations. First and foremost, the sample size was small, and thus the potential Type II error is likely substantial. However, we avoided confounding factors by requiring prolonged HIV RNA undetectability in plasma for study eligibility and excluding patients with chronic HCV infection. Second, we selected our cases and controls based on more than one factor . This study design would have been particularly problematic had we observed differences between the two groups. Since it is unlikely that low absolute CD4+ T cell counts may have blunted the impact of herpes viruses on immune activation, it is also unlikely that the negative results observed in this study were due to our study design. Third, we only measured human herpesvirus replication in two reservoirs, saliva and PBMC. Assaying specimens from other reservoirs  may have demonstrated a difference between the two groups. In addition, the saliva and PBMC specimens we assayed were obtained infrequently (only once every four months). Sampling more frequently, for example by daily oral swabs Given the strong association between inflammation/immune activation and disease outcomes in treated HIV disease, defining the mechanisms associated with HIV-associated inflammation is of high importance. Our preliminary data do not provide strong evidence for a role of persistent high level herpesvirus replication.Other possible mechanisms includes the following:Persistent microbial translocation. It is now well-appreciated that HIV causes irreversible damage to the gut mucosal integrity early in the course of disease, leading to translocation of gut microbial products into the system circulation Residual HIV viremia. Another factor leading to persistent abnormal immune activation in antiretroviral-suppressed patients could be persistent low level replication of HIV. Several studies have reported that by using an ultra-ultrasensitive HIV RNA PCR assay, low levels of plasma HIV viremia  can be detected in the majority of HIV-infected patients successfully treated with antiretroviral therapy Persistant immune dysregulation. Finally, the damage to the immune system done by uncontrolled HIV replication before antiretroviral therapy is initiated, for example in altering T cell diversity in adults with limited thymic reserve for repopulating diverse naïve T cells In summary, future studies to examine the effect of suppressing CMV, EBV and KSHV with drugs such as valganciclovir