Toll-like receptor 4 ablation in mdx mice reveals innate immunity as a therapeutic target in Duchenne muscular dystrophy.

Toll-like receptor 4 (TLR4) recognizes specific structural motifs associated with microbial pathogens and also responds to certain endogenous host molecules associated with tissue damage. In Duchenne muscular dystrophy (DMD), inflammation plays an important role in determining the ultimate fate of dystrophic muscle fibers. In this study, we used TLR4-deficient dystrophic mdx mice to assess the role of TLR4 in the pathogenesis of DMD. TLR4 expression was increased and showed enhanced activation following agonist stimulation in mdx diaphragm muscle. Genetic ablation of TLR4 led to significantly increased muscle force generation in dystrophic diaphragm muscle, which was associated with improved histopathology including decreased fibrosis, as well as reduced pro-inflammatory gene expression and macrophage infiltration. TLR4 ablation in mdx mice also altered the phenotype of muscle macrophages by inducing a shift toward a more anti-inflammatory (iNOS(neg) CD206(pos)) profile. In vitro experiments confirmed that lack of TLR4 is sufficient to influence macrophage activation status in response to classical polarizing stimuli such as IFN-gamma and IL-4. Finally, treatment of dystrophic mice with glycyrrhizin, an inhibitor of the endogenous TLR4 ligand, high mobility group box (HMGB1), also pointed to involvement of the HMGB1-TLR4 axis in promoting dystrophic diaphragm pathology. Taken together, our findings reveal TLR4 and the innate immune system as important players in the pathophysiology of DMD. Accordingly, targeting either TLR4 or its endogenous ligands may provide a new therapeutic strategy to slow disease progression.

Divergent impact of Toll-like receptor 2 deficiency on repair mechanisms in healthy muscle versus Duchenne muscular dystrophy.

Injury to skeletal muscle, whether acute or chronic, triggers macrophage-mediated innate immunity in a manner which can be either beneficial or harmful for subsequent repair. Endogenous ligands for Toll-like receptor 2 (TLR2) are released by damaged tissues and might play an important role in activating the innate immune system following muscle injury. To test this hypothesis, we compared macrophage behaviour and muscle repair mechanisms in mice lacking TLR2 under conditions of either acute (cardiotoxin-induced) or chronic (mdx mouse genetic model of Duchenne muscular dystrophy; DMD) muscle damage. In previously healthy muscle subjected to acute damage, TLR2 deficiency reduced macrophage numbers in the muscle post-injury but did not alter the expression pattern of the prototypical macrophage polarization markers iNOS and CD206. In addition, there was abnormal persistence of necrotic fibres and impaired regeneration in TLR2-/- muscles after acute injury. In contrast, TLR2 ablation in chronically diseased muscles of mdx mice not only resulted in significantly reduced macrophage numbers but additionally modified their phenotype by shifting from inflammatory (iNOS(pos) CD206(neg) ) to more anti-inflammatory (iNOS(neg) CD206(pos) ) characteristics. This decrease in macrophage-mediated inflammation was associated with ameliorated muscle histopathology and improved force-generating capacity of the dystrophic muscle. Our results suggest that the role of TLR2 in macrophage function and skeletal muscle repair depends greatly upon the muscle injury context, and raise the possibility that inhibition of TLR2 could serve as a useful therapeutic measure in DMD.

A protein expression atlas on tissue samples and cell lines from cancer patients provides insights into tumor heterogeneity and dependencies.

The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE) are foundational resources in cancer research, providing extensive molecular and phenotypic data. However, large-scale proteomic data across various cancer types for these cohorts remain limited. Here, we expand upon our previous work to generate high-quality protein expression data for approximately 8,000 TCGA patient samples and around 900 CCLE cell line samples, covering 447 clinically relevant proteins, using reverse-phase protein arrays. These protein expression profiles offer profound insights into intertumor heterogeneity and cancer dependency and serve as sensitive functional readouts for somatic alterations. We develop a systematic protein-centered strategy for identifying synthetic lethality pairs and experimentally validate an interaction between protein kinase A subunit α and epidermal growth factor receptor. We also identify metastasis-related protein markers with clinical relevance. This dataset represents a valuable resource for advancing our understanding of cancer mechanisms, discovering protein biomarkers and developing innovative therapeutic strategies.

Study of CD4+CD8+ double positive T-lymphocyte phenotype and function in Indian patients infected with HIV-1.

CD4+CD8+ double positive T cells represent a minor peripheral blood lymphocyte population. CD4+ expression on CD8+ T cells is induced following cellular activation, and as chronic HIV-1 infection is associated with generalized immune activation, double positive T cells studies have become necessary to understand the immunopathology of human immunodeficiency virus (HIV). The frequency of double positive T cells in persons infected with HIV was studied in comparison to uninfected controls. Further, the expression of CD38, HLA-DR, and programmed death (PD)-1 on these cells were ascertained. HIV-1 specific double positive T cells were also studied for their cytokine secretory ability and phenotype. A significantly higher double positive cell population was observed in the patients with advanced HIV disease (CD4+ T cell counts below 200 cells/µl), as compared to patients with CD4+ T cell counts above 500 cells/µl. Double positive T cells from patients with symptomatic HIV disease had a significantly increased activation and exhaustion levels, compared to asymptomatic subjects and to single positive T cells from the same subjects. HIV-1 specific double positive T cells showed further increase in CD38 and PD-1 expression levels. The proportion of CD38 and PD-1 expressing total and HIV-1 specific double positive T cells correlated positively with HIV-1 plasma viremia and negatively with CD4+ T cell counts. HIV infection results in a marked increase of double positive T cell population, and this cell population shows higher level of activation and exhaustion (increased PD-1 expression) compared to the single positive CD4+ and CD8+ T cells.

HIV-1 diseases progression associated with loss of Th17 cells in subtype 'C' infection.

Th17 cells play a crucial role in host immune response. We examined the role of Th17 cells in HIV-1 'subtype-C' infection and report that HIV-1 specific Th17 cells are induced in early infection and slow progressors but are significantly reduced at late stage of infection. There was a further decline in Th17 cells in late stage subjects with gastrointestinal infections. Additionally, we observed expanded population of IL-21 (needed for Th17 population expansion) producing CD4 T cells in early and slow progressors compared to subjects with late stage infection. A significant positive correlation existed between virus specific IL-17 and IL-21 producing CD4 T cells suggesting that HIV-1 infection induces a demand for Th17 cells. A significant negative correlation between virus specific Th17 cells and HIV-1 plasma viral load (pVL) was also observed, indicating a gradual loss of Th17 cells with HIV-1 disease progression.

Tumor-intrinsic SIRPA promotes sensitivity to checkpoint inhibition immunotherapy in melanoma.

Checkpoint inhibition immunotherapy has revolutionized cancer treatment, but many patients show resistance. Here we perform integrative transcriptomic and proteomic analyses on emerging immuno-oncology targets across multiple clinical cohorts of melanoma under anti-PD-1 treatment, on both bulk and single-cell levels. We reveal a surprising role of tumor-intrinsic SIRPA in enhancing antitumor immunity, in contrast to its well-established role as a major inhibitory immune modulator in macrophages. The loss of SIRPA expression is a marker of melanoma dedifferentiation, a key phenotype linked to immunotherapy efficacy. Inhibition of SIRPA in melanoma cells abrogates tumor killing by activated CD8+ T cells in a co-culture system. Mice bearing SIRPA-deficient melanoma tumors show no response to anti-PD-L1 treatment, whereas melanoma-specific SIRPA overexpression significantly enhances immunotherapy response. Mechanistically, SIRPA is regulated by its pseudogene, SIRPAP1. Our results suggest a complicated role of SIRPA in the tumor ecosystem, highlighting cell-type-dependent antagonistic effects of the same target on immunotherapy.

A functional genomic approach to actionable gene fusions for precision oncology.

Fusion genes represent a class of attractive therapeutic targets. Thousands of fusion genes have been identified in patients with cancer, but the functional consequences and therapeutic implications of most of these remain largely unknown. Here, we develop a functional genomic approach that consists of efficient fusion reconstruction and sensitive cell viability and drug response assays. Applying this approach, we characterize ~100 fusion genes detected in patient samples of The Cancer Genome Atlas, revealing a notable fraction of low-frequency fusions with activating effects on tumor growth. Focusing on those in the RTK-RAS pathway, we identify a number of activating fusions that can markedly affect sensitivity to relevant drugs. Last, we propose an integrated, level-of-evidence classification system to prioritize gene fusions systematically. Our study reiterates the urgent clinical need to incorporate similar functional genomic approaches to characterize gene fusions, thereby maximizing the utility of gene fusions for precision oncology.

Loss of CD127 & increased immunosenescence of T cell subsets in HIV infected individuals.

BACKGROUND & OBJECTIVES: HIV infection is characterized by a perturbation in T cell homeostasis, leading to alteration in T cell subsets. In addition to alteration in differentiation, HIV infection also leads to change in T cell survival and regenerative capacity, as suggested by differential expression of CD127 and CD57. We evaluated the expression patterns of CD127 and CD57 on CD4 and CD8 effector, memory and naïve T cell subsets in HIV-infected and uninfected individuals. METHODS: We characterized T cell subsets based on expression of these markers, and compared their expression pattern in HIV infected subjects and uninfected controls. We further assessed therapy generated changes in these subsets and expression of CD127 and CD57 on them. RESULTS: There was a generalized decrease in naïve CD4 and CD8 T cells in HIV infected subjects. These changes in T cell subset distribution were related to antigen load. CD127 expression was significantly reduced in T cells from HIV infected subject. In association to this, HIV infected subjects had higher percentage of T cell subsets expressing CD57. Increased CD57 and reduced CD127 expression correlated with plasma viraemia and CD8 T cell activation state. Incomplete restoration of T cell subset proportions was observed, despite suppression of viral replication and increase in CD4 T cell counts. Further, the improvement was more pronounced in CD127 expression. INTERPRETATION & CONCLUSIONS: HIV infected subjects have reduced T cell regenerative capacity along with increased senescence, highlighting decreased proliferation and effector activities.

Defects in blood dendritic cell subsets in HIV-1 subtype c infected Indians.

BACKGROUND & OBJECTIVES: DCs trigger both innate and adaptive immune responses to control HIV infection and represent a viral reservoir acting as target and HIV carriers for infection of permissive CD4(+) T-cells. DCs thus form a very attractive study subject to further our existing knowledge of HIV induced immunopathogenesis due to its diverse and crucial role in HIV infection establishment, viral dissemination, immune evasion, viral persistence, etc. We aimed to characterize the effect of HIV infection on myeloid and plasmacytoid dendritic cell subsets in a group of HIV-1 subtype C infected treated or untreated Indian individuals. METHODS: Blood DC subset numbers and immunophenotype were studied for 79 HIV infected subjects at various stages of disease and compared with 13 HIV-uninfected controls. Comparisons were also made between groups of subjects based on their CD4(+) T cell counts and also experience of antiretrovirals. RESULTS: Significant decreases were observed in blood DC counts and the two DC subsets in HIV infected individuals. Subjects with lowest CD4(+) T cell counts also had a drastically reduced DC subset pool which correlated positively with plasma viraemia and negatively with CD4(+) T cell counts. DC subsets from HIV infected subjects showed higher expression of co-stimulatory molecules CD40 and CD86, and HIV-1 co-receptors CXCR4 and CCR5 which correlated positively with HIV-1 plasma viraemia. The alterations in blood DCs were partly resolved in ART receiving study subjects. INTERPRETATION & CONCLUSIONS: Correlation between DC subset activation state and viraemia supports the role of DC activation on viral replication and CD4(+) T cell depletion.

Late presenters to HIV care and treatment, identification of associated risk factors in HIV-1 infected Indian population.

BACKGROUND: Timely access to antiretroviral therapy is a key to controlling HIV infection. Late diagnosis and presentation to care diminish the benefits of antiretrovirals and increase risk of transmission. We aimed to identify late presenters in patients sent for first CD4 T cell count after HIV diagnosis, for therapy initiation evaluation. Further we aimed at identifying patient factors associated with higher risk of late presentation. METHODS: Retrospective data collection and analysis was done for 3680 subjects visiting the laboratory for CD4 T cell counts between 2001 and 2007. We segregated the patients on basis of their CD4 T cell counts after first HIV diagnosis. Factors associated with risk of late presentation to CD4 T cell counts after HIV diagnosis were identified using univariate analysis, and the strength of association of individual factor was assessed by calculation of odds ratios. RESULTS: Of 3680 subjects, 2936 (83.37%) were defined as late presenters. Late testing varied among age groups, transmission categories, and gender. Males were twice as likely to present late as compared to females. We found significant positive association of heterosexual transmission route (p < 0.001), and older age groups of 45 years and above (p = 0.0004) to late presentation. Female sex, children below 14 years of age and sexual contact with HIV positive spouse were associated with significantly lower risks to presenting late. Intravenous drug users were also associated with lower risks of late presentation, in comparison to heterosexual transmission route. CONCLUSIONS: The study identifies HIV infected population groups at a higher risk of late presentation to care and treatment. The risk factors identified to be associated with late presentation should be utilised in formulating targeted public health interventions in order to improve early HIV diagnosis.

HIV voluntary counseling and testing: an experience from India.

Despite proof of voluntary counseling and testing (VCT) effectiveness in HIV disease prevention and management, there are limited reports on experience with pre- and post-HIV-test counseling in developing countries. In view of this, we aimed to bring to the fore the voluntary counseling and testing experience at a tertiary healthcare center. The present study was conducted at the voluntary counseling and testing center of a tertiary healthcare center and the National HIV Reference Center. Participants were 1169 men and 581 females attending the VCT clinic from February 2005 to March 2006. Odds ratios were calculated for each of the variable to analyze the strength of association with HIV sero-status. Out of 1750 patients, 322 (27.5%) males and 156 females (26.9%) tested HIV-positive. HIV-sero-positivity was observed to be associated to participant age (approximately 1.5 for 25-44 yrs age group), marital status (2.3 times in married patients), primary or lower education level (1.5 times), citing spouse death/HIV-infected spouse as the reason for seeking VCT (2.2 times) and reporting a history of risk behavior as reason for getting tested. This study aims to evaluate the effectiveness of existing client initiated voluntary counseling and testing facility in the light of a recent recommendation by WHO/UNAIDS for the implementation of provider initiated voluntary counseling services. Through this study, we could also highlight socio-demographic factors, like education and age, and reasons stated by participants for seeking VCT, which were associated with HIV-positive status and put an individual at a higher risk of HIV infection.

A-to-I-edited miRNA-379-5p inhibits cancer cell proliferation through CD97-induced apoptosis.

Both miRNAs and A-to-I RNA editing, a widespread nucleotide modification mechanism, have recently emerged as key players in cancer pathophysiology. However, the functional impact of RNA editing of miRNAs in cancer remains largely unexplored. Here, we focused on an ADAR2-catalyzed RNA editing site within the miR-379-5p seed region. This site was under-edited in tumors relative to normal tissues, with a high editing level being correlated with better patient survival times across cancer types. We demonstrated that in contrast to wild-type miRNA, edited miR-379-5p inhibited cell proliferation and promoted apoptosis in diverse tumor contexts in vitro, which was due to the ability of edited but not wild-type miR-379-5p to target CD97. Importantly, through nanoliposomal delivery, edited miR-379-5p mimics significantly inhibited tumor growth and extended survival of mice. Our study indicates a role of RNA editing in diversifying miRNA function during cancer progression and highlights the translational potential of edited miRNAs as a new class of cancer therapeutics.

Comparative Proliferation Capacity of Gag-C-Specific Naive and Memory CD4+ and CD8+ T Lymphocytes in Rapid, Viremic Slow, and Slow Progressors During Human Immunodeficiency Virus Infection.

The exact cause of altered dynamics in T cells compartment during HIV infection remains elusive to date. In this longitudinal study, the proliferation frequency of different T cell subsets was investigated in untreated HIV-1-infected Indian individuals stratified as rapid (R), viremic slow (VS), slow (S) progressors, and healthy controls. Ten healthy and 20 treatment-naive HIV-1-infected individuals were enrolled. Expression of Ki67 nuclear antigen was examined on HIV-specific T cell subsets in peripheral blood lymphocytes. Upon stimulation with HIV-1 Gag-C peptide pools, effector memory (EM) CD4 T cells (R vs. S, EM CD4, p < 0.05) of R progressors proliferated significantly compared with those of S progressors at baseline. However, central memory (CM) CD8 T cell subsets proliferated significantly in VS and S progressors compared with those in R progressors, wherein highest proliferation frequency of EM CD8 T cells was observed. At follow-up visit, the proliferation frequency of naive CD8 T cells was significantly higher in R progressors than S progressors (R vs. S naive CD8, p < 0.05). The findings suggest altered dynamics of different CD4+ and CD8+ T cell subsets in R, VS, and S progressors. The increase in CM T cell proliferation in VS and S progressors could be attributed to slower progression of the HIV infection. Hence, treatment strategies must be focused on restoring the homeostatic balance to restore T cell functionality.

A-to-I RNA Editing Contributes to Proteomic Diversity in Cancer.

Adenosine (A) to inosine (I) RNA editing introduces many nucleotide changes in cancer transcriptomes. However, due to the complexity of post-transcriptional regulation, the contribution of RNA editing to proteomic diversity in human cancers remains unclear. Here, we performed an integrated analysis of TCGA genomic data and CPTAC proteomic data. Despite limited site diversity, we demonstrate that A-to-I RNA editing contributes to proteomic diversity in breast cancer through changes in amino acid sequences. We validate the presence of editing events at both RNA and protein levels. The edited COPA protein increases proliferation, migration, and invasion of cancer cells in vitro. Our study suggests an important contribution of A-to-I RNA editing to protein diversity in cancer and highlights its translational potential.

Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer.

Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional "omic" data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.

Molecular Characterization and Clinical Relevance of Metabolic Expression Subtypes in Human Cancers.

Metabolic reprogramming provides critical information for clinical oncology. Using molecular data of 9,125 patient samples from The Cancer Genome Atlas, we identified tumor subtypes in 33 cancer types based on mRNA expression patterns of seven major metabolic processes and assessed their clinical relevance. Our metabolic expression subtypes correlated extensively with clinical outcome: subtypes with upregulated carbohydrate, nucleotide, and vitamin/cofactor metabolism most consistently correlated with worse prognosis, whereas subtypes with upregulated lipid metabolism showed the opposite. Metabolic subtypes correlated with diverse somatic drivers but exhibited effects convergent on cancer hallmark pathways and were modulated by highly recurrent master regulators across cancer types. As a proof-of-concept example, we demonstrated that knockdown of SNAI1 or RUNX1-master regulators of carbohydrate metabolic subtypes-modulates metabolic activity and drug sensitivity. Our study provides a system-level view of metabolic heterogeneity within and across cancer types and identifies pathway cross-talk, suggesting related prognostic, therapeutic, and predictive utility.

Antiretroviral treatment in resource-poor settings: a view from India.

CONTEXT: The introduction of highly effective generic antiretroviral drugs at reduced cost has transformed the face of HIV/AIDS epidemic in developing countries like India. However, there is an urgent emphasis on developing and implementing guidelines for antiretroviral treatment monitoring by laboratory methods utilizing the available technologies in resource-limited settings. AIM: We studied the efficacy of antiretroviral treatment, adherence to therapy and motivation of patients for regular treatment monitoring by CD4 counts. SETTINGS AND DESIGN: A longitudinal cohort study on an established cohort of 166 HIV-1-infected Indian individuals. MATERIALS AND METHODS: Study subjects were followed up for the period from January 2002 to November 2006. Their clinical status and treatment regimen were recorded and CD4 counts were performed at each visit. STATISTICAL ANALYSIS: Repeated-measures ANOVA was used to compute changes in median CD4 counts at each visit in the different treatment groups. RESULTS: We observed a growing awareness and motivation for regular HIV disease monitoring among patients, accompanied by a trend of increasing median CD4 counts at all subsequent follow-up visits after initiation of antiretroviral treatment. CONCLUSIONS: The study gives an insight into the institutional efforts for the establishment of cohorts for longitudinal studies, which will help in designing effective treatment guidelines, thus providing impetus to the free public sector antiretroviral therapy program in India. Such formative research aims to fill the lacunae in the limited available data for the formulation of treatment-monitoring guidelines in resource-poor settings of developing countries like India.

Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2.

Myofiber necrosis and fibrosis are hallmarks of Duchenne muscular dystrophy (DMD), leading to lethal weakness of the diaphragm. Macrophages (MPs) are required for successful muscle regeneration, but the role of inflammatory monocyte (MO)-derived MPs in either promoting or mitigating DMD is unclear. We show that DMD (mdx) mouse diaphragms exhibit greatly increased expression of CCR2 and its chemokine ligands, along with inflammatory (Ly6C(high)) MO recruitment and accumulation of CD11b(high) MO-derived MPs. Loss-of-function of CCR2 preferentially reduced this CD11b(high) MP population by impeding the release of Ly6C(high) MOs from the bone marrow but not the splenic reservoir. CCR2 deficiency also helped restore the MP polarization balance by preventing excessive skewing of MPs toward a proinflammatory phenotype. These effects were linked to amelioration of histopathological features and increased muscle strength in the diaphragm. Chronic inhibition of CCR2 signaling by mutated CCL2 secreted from implanted mesenchymal stem cells resulted in similar improvements. These data uncover a previously unrecognized role of inflammatory MOs in DMD pathogenesis and indicate that CCR2 inhibition could offer a novel strategy for DMD management.

Altered T cell differentiation associated with loss of CD27 and CD28 in HIV infected Indian individuals.

BACKGROUND: HIV-1 infection is associated with depletion of naïve T cell subsets and skewed T cell differentiation and maturation, leading to accumulation of T cells at intermediate and end stages of differentiation. CD27 and CD28 expression have been utilized in assessing these population subsets. METHODS: We characterized T cell subsets based on expression of CD45RA, CCR7, CD27, and CD28 and compared these subsets in HIV-1 infected Indian subjects and uninfected controls. RESULTS: HIV-1 infection was associated with an increase in effector and memory T cell subsets and a concomitant decrease in naïve T cells. HIV-1 infected subjects showed accumulation of intermediate CD8 T cell (CD27+CD28-) differentiation subsets, whereas CD4 T cells progressed to late stage differentiation (CD27-CD28-). These subsets were negatively associated with CD4 T cell counts and positively associated with plasma viremia. CD57, an immunosenescence marker, was also increased on T cell subsets from HIV-1 infected individuals. Antiretroviral therapy resulted in partial restoration of differentiation status. CONCLUSION: Persistent HIV-1 replication and chronic immune activation, along with altered cytokine secretion profile, lead to impaired T cell differentiation and maturation. Detailed understanding of factors associated with differentiation defects in HIV-1 infected Indian individuals will strongly assist in Indian HIV-1 vaccine efforts and add to our knowledge of HIV-1 subtype C pathogenesis.

Polyfunctional analysis of Gag and Nef specific CD8+ T-cell responses in HIV-1 infected Indian individuals.

Polyfunctional CD8+ T-cells have been described as most competent in controlling viral replication. We studied the impact of antigen persistence on the polyfunctional immune responses of CD8+ T-lymphocytes to HIV Gag and Nef peptides and polyclonal stimuli in 40 ART naïve HIV infected individuals and analyzed the alterations in T-cell functionality in early and late stages of infection. Significantly elevated level of global response and polyfunctional profile of CD8+ T-cells were observed to polyclonal stimulation, than HIV specific antigens in chronically infected individuals. However no key differences were observed in CD8+ T-cell functional profile in any of the 15 unique subsets for Gag and Nef specific antigens. The subjects in early stage of infection (defined as a gap of 6 months or less between seroconversion and enrolment and with no apparent clinical symptoms) had a higher degree of response functionality (4+ or 3+ different functions simultaneously) than in the late stage infection (defined as time duration since seroconversion greater than 6 months). The data suggest that persistence of antigen during chronic infection leads to functional impairment of HIV specific responses.