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1.  Targeting of Conserved Gag-Epitopes in Early HIV Infection Is Associated with Lower Plasma Viral Load and Slower CD4+ T Cell Depletion 
Abstract
We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8+ T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8+ T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8+ T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log10 difference). Furthermore, the rate of CD4+ T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome.
doi:10.1089/aid.2012.0171
PMCID: PMC3581067  PMID: 23140171
2.  CD56negCD16+ NK cells are activated mature NK cells with impaired effector function during HIV-1 infection 
Retrovirology  2013;10:158.
Background
A subset of CD3negCD56negCD16+ Natural Killer (NK) cells is highly expanded during chronic HIV-1 infection. The role of this subset in HIV-1 pathogenesis remains unclear. The lack of NK cell lineage-specific markers has complicated the study of minor NK cell subpopulations.
Results
Using CD7 as an additional NK cell marker, we found that CD3negCD56negCD16+ cells are a heterogeneous population comprised of CD7+ NK cells and CD7neg non-classical myeloid cells. CD7+CD56negCD16+ NK cells are significantly expanded in HIV-1 infection. CD7+CD56negCD16+ NK cells are mature and express KIRs, the C-type lectin-like receptors NKG2A and NKG2C, and natural cytotoxicity receptors similar to CD7+CD56+CD16+ NK cells. CD7+CD56neg NK cells in healthy donors produced minimal IFNγ following K562 target cell or IL-12 plus IL-18 stimulation; however, they degranulated in response to K562 stimulation similar to CD7+CD56+ NK cells. HIV-1 infection resulted in reduced IFNγ secretion following K562 or cytokine stimulation by both NK cell subsets compared to healthy donors. Decreased granzyme B and perforin expression and increased expression of CD107a in the absence of stimulation, particularly in HIV-1-infected subjects, suggest that CD7+CD56negCD16+ NK cells may have recently engaged target cells. Furthermore, CD7+CD56negCD16+ NK cells have significantly increased expression of CD95, a marker of NK cell activation.
Conclusions
Taken together, CD7+CD56negCD16+ NK cells are activated, mature NK cells that may have recently engaged target cells.
doi:10.1186/1742-4690-10-158
PMCID: PMC3892122  PMID: 24351015
Natural killer cells; NK cells; CD7; Human immunodeficiency virus; HIV-1; HIV pathogenesis; CD56neg NK cells
3.  CD57 Expression and Cytokine Production by T Cells in Lesional and Unaffected Skin from Patients with Psoriasis 
PLoS ONE  2013;8(2):e52144.
Background
The immunopathogenic mechanisms leading to psoriasis remain unresolved. CD57 is a marker of replicative inability and immunosenescence on CD8+ T cells and the proportion of CD57 expressing CD8+ T cells is increased in a number of inflammatory conditions.
Methodology
We examined the expression of CD57 on T cells in the skin of patients affected with psoriasis, comparing lesional and unaffected skin. We also assessed functionality of the T cells by evaluating the secretion of several inflammatory cytokines (IL-17A, IFN-gamma, IL-2, IL-33, TNF-alpha, IL-21, IL-22, and IL-27), from cell-sorted purified CD4+ and CD8+ T cells isolated from lesional and unaffected skin biopsies of psoriasis patients.
Principal Findings
We observed that the frequency of CD57+CD4+ and CD57+CD8+ T cells was significantly higher in unaffected skin of psoriasis patients compared to lesional skin. Sorted CD4+ T cells from psoriatic lesional skin produced higher levels of IL-17A, IL-22, and IFN-gamma compared to unaffected skin, while sorted CD8+ T cells from lesional skin produced higher levels of IL-17, IL-22, IFN-gamma, TNF-alpha, and IL-2 compared to unaffected skin.
Conclusions/Significance
These findings suggest that T cells in unaffected skin from psoriasis patients exhibit a phenotype compatible with replicative inability. As they have a lower replicative capacity, CD57+ T cells are less frequent in lesional tissue due to the high cellular turnover.
doi:10.1371/journal.pone.0052144
PMCID: PMC3585296  PMID: 23468834
4.  Early Detection of Simian Immunodeficiency Virus in the Central Nervous System Following Oral Administration to Rhesus Macaques 
The timing of HIV dissemination to the central nervous system (CNS) has the potential to have important implications regarding HIV disease progression and treatment. The earlier HIV enters the CNS the more difficult it might be to remove with antiretroviral therapy. Alternatively, HIV may only enter the CNS later in the course of disease as a result of disruption of the blood-brain-barrier. We utilized the simian immunodeficiency virus (SIV) infection of rhesus macaques to evaluate the oral route of infection and the subsequent spread of SIV to the CNS during the acute infection phase. A high dose oral SIV challenge was utilized to ensure a successful infection and permit the evaluation of CNS spread during the first 1–14 days post-infection. Ultrasensitive nested PCR was used to detect SIV gag DNA in the brains of macaques at 1–2 days post-infection and identified SIV gag DNA in the brain tissues from three of four macaques. This SIV DNA was also present following perfusion of the macaque brains, providing evidence that it was not residing in the circulating blood but in the brain tissue itself. The diversity of the viral envelope V1–V2 region at early times post-infection indicated that the brain viral variants were similar to variants obtained from lymph nodes. This genetic similarity between SIV obtained from lymphoid and brain tissues suggests that the founder population of viral species entered and subsequently spread without any evidence of brain-specific SIV selection. The relatively rapid appearance of SIV within the CNS tissue following oral transmission may also occur during HIV transmission where it may impact disease course as well as representing a challenge for long-term therapies and future viral eradication modalities.
doi:10.3389/fimmu.2013.00236
PMCID: PMC3743037  PMID: 23966995
SIV; oral transmission; CNS; acute infection; rhesus macaque; monkey model; HIV
5.  Differential Innate Immune Responses to Low or High Dose Oral SIV Challenge in Rhesus Macaques 
Current HIV Research  2011;9(5):276-288.
Mucosal transmission of HIV predominately occurs during sexual intercourse or breast-feeding and generally results in a successful infection from just one or few founder virions. Here we assessed the impact of viral inoculum size on both viral and immune events within two groups of Rhesus macaques that were non-traumatically, orally inoculated with either multiple low (1000 to 4000 TCID50) or high (100,000 TCID50) doses of SIV. In agreement with previous studies, more diverse SIV variants were observed in macaques following infection with high dose oral SIV compared to a low dose challenge. In peripheral blood cells, the immune gene transcript levels of CXCL9, IFNγ, TNFα and IL10 remained similar to uninfected macaques. In contrast, OAS and CXCL10 were upregulated following SIV infection in both the high and low dosed macaques, with a more rapid kinetics (detectable by 7 days) following the high SIV dose challenge. In peripheral lymph nodes, an increase in CXCL10 was observed irrespective of viral dose while CXCL9 and OAS were differentially regulated in the two SIV dosed groups. Magnetic bead sorting of CD3+, CD14+ and CD3−/CD14− cells from peripheral blood identified the increase in OAS expression primarily within CD14+ monocytes, whereas the CXCL10 expression was primarily in CD3+ T cells. These findings provide insights into the impact of SIV challenge dose on viral and innate immune factors, which has the potential to inform future SIV/HIV vaccine efficacy trials in which vaccinated hosts have the potential to be infected with a range of viral challenge doses.
PMCID: PMC3337698  PMID: 21861823
AIDS; HIV; Innate immune responses; Low Dose; Oral transmission; Rhesus macaques; SIV
6.  The Calm Mouse: An Animal Model of Stress Reduction 
Molecular Medicine  2012;18(1):606-617.
Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a “calm mouse model” with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.
doi:10.2119/molmed.2012.00053
PMCID: PMC3388136  PMID: 22398685
7.  Lack of clinical AIDS in SIV-infected sooty mangabeys with significant CD4+ T cell loss is associated with double-negative T cells  
The Journal of Clinical Investigation  2011;121(3):1102-1110.
SIV infection of natural host species such as sooty mangabeys results in high viral replication without clinical signs of simian AIDS. Studying such infections is useful for identifying immunologic parameters that lead to AIDS in HIV-infected patients. Here we have demonstrated that acute, SIV-induced CD4+ T cell depletion in sooty mangabeys does not result in immune dysfunction and progression to simian AIDS and that a population of CD3+CD4–CD8– T cells (double-negative T cells) partially compensates for CD4+ T cell function in these animals. Passaging plasma from an SIV-infected sooty mangabey with very few CD4+ T cells to SIV-negative animals resulted in rapid loss of CD4+ T cells. Nonetheless, all sooty mangabeys generated SIV-specific antibody and T cell responses and maintained normal levels of plasma lipopolysaccharide. Moreover, all CD4-low sooty mangabeys elicited a de novo immune response following influenza vaccination. Such preserved immune responses as well as the low levels of immune activation observed in these animals were associated with the presence of double-negative T cells capable of producing Th1, Th2, and Th17 cytokines. These studies indicate that SIV-infected sooty mangabeys do not appear to rely entirely on CD4+ T cells to maintain immunity and identify double-negative T cells as a potential subset of cells capable of performing CD4+ T cell–like helper functions upon SIV-induced CD4+ T cell depletion in this species.
doi:10.1172/JCI44876
PMCID: PMC3049370  PMID: 21317533
8.  Elevated Levels of Innate Immune Modulators in Lymph Nodes and Blood Are Associated with More-Rapid Disease Progression in Simian Immunodeficiency Virus-Infected Monkeys▿  
Journal of Virology  2009;83(23):12229-12240.
Cytokines and chemokines are critical for establishing tissue-specific immune responses and play key roles in modulating disease progression in simian immunodeficiency virus (SIV)-infected macaques and human immunodeficiency virus (HIV)-infected humans. The goal here was to characterize the innate immune response at different tissue sites and to correlate these responses to clinical outcome, initially focusing on rhesus macaques orally inoculated with SIV and monitored until onset of simian AIDS. Cytokine and chemokine mRNA transcripts were assessed at lymph nodes (LN) and peripheral blood cells utilizing quantitative real-time PCR at different time points postinfection. The mRNA expression of four immune modulators—alpha interferon (IFN-α), oligoadenylate synthetase (OAS), CXCL9, and CXCL10—was positively associated with disease progression within LN tissue. Elevated cytokine/chemokine expression in LN did not result in any observed beneficial outcome since the numbers of CXCR3+ cells were not increased, nor were the SIV RNA levels decreased. In peripheral blood, increased OAS and CXCL10 expression were elevated in SIV+ monkeys that progress the fastest to simian AIDS. Our results indicate that higher IFN-α, OAS, CXCL9, and CXCL10 mRNA expression in LN was associated with rapid disease progression and a LN environment that may favor SIV replication. Furthermore, higher expression of CXCL10 and OAS in peripheral blood could potentially serve as a diagnostic marker for hosts that are likely to progress to AIDS. Understanding the expression patterns of key innate immune modulators will be useful in assessing the disease state and potential rates of disease progression in HIV+ patients, which could lead to novel therapy and vaccine approaches.
doi:10.1128/JVI.01311-09
PMCID: PMC2786739  PMID: 19759147
9.  Gamma/Delta T Cell mRNA Levels Decrease at Mucosal Sites and Increase at Lymphoid Sites Following an Oral SIV Infection of Macaques 
Current HIV research  2008;6(6):520-530.
The oral and esophageal mucosa have been identified as possible sites of HIV/SIV entry following oral infection. Here, gamma/delta (γδ) T cells, a multi-functional T cell subset, were assessed at oral/esophageal mucosa and lymphoid sites at the earliest times (1–14 days) post-oral SIV inoculation utilizing quantitative RT-PCR. During these earliest times post-infection, decreased γδ TCR mRNA levels were observed at the oral gingiva and esophageal mucosa, while increased levels were observed within regional lymph nodes (cervical and retropharyngeal). Higher lymph node γδ TCR levels were associated with increased mRNA expression of the lymphoid homing chemokine/receptor (CCL21/CCR7) pair in these lymph nodes. In contrast to γδ TCR levels, CD4 mRNA expression remained relatively stable through 4 days post-infection, and depletion of CD4 T cells was only evident after 7 or 14 days post-infection. The decrease of γδ T cell mRNA from mucosal sites and the corresponding increase at lymphoid sites suggest a rapid redistribution of these immune cells at these earliest times post-SIV infection.
PMCID: PMC2777980  PMID: 18991617
gammadelta T cells; SIV; Mucosa; Acute
10.  Severe Depletion of Mucosal CD4+ T Cells in AIDS-Free Simian Immunodeficiency Virus-Infected Sooty Mangabeys1 
HIV-infected humans and SIV-infected rhesus macaques experience a rapid and dramatic loss of mucosal CD4+ T cells that is considered to be a key determinant of AIDS pathogenesis. In this study, we show that nonpathogenic SIV infection of sooty mangabeys (SMs), a natural host species for SIV, is also associated with an early, severe, and persistent depletion of memory CD4+ T cells from the intestinal and respiratory mucosa. Importantly, the kinetics of the loss of mucosal CD4+ T cells in SMs is similar to that of SIVmac239-infected rhesus macaques. Although the nonpathogenic SIV infection of SMs induces the same pattern of mucosal target cell depletion observed during pathogenic HIV/SIV infections, the depletion in SMs occurs in the context of limited local and systemic immune activation and can be reverted if virus replication is suppressed by antiretroviral treatment. These results indicate that a profound depletion of mucosal CD4+ T cells is not sufficient per se to induce loss of mucosal immunity and disease progression during a primate lentiviral infection. We propose that, in the disease-resistant SIV-infected SMs, evolutionary adaptation to both preserve immune function with fewer mucosal CD4+ T cells and attenuate the immune activation that follows acute viral infection protect these animals from progressing to AIDS.
PMCID: PMC2365740  PMID: 17709517
11.  Gamma/Delta T-Cell Functional Responses Differ after Pathogenic Human Immunodeficiency Virus and Nonpathogenic Simian Immunodeficiency Virus Infections▿  
Journal of Virology  2007;82(3):1155-1165.
The objective of this study was to functionally assess gamma/delta (γδ) T cells following pathogenic human immunodeficiency virus (HIV) infection of humans and nonpathogenic simian immunodeficiency virus (SIV) infection of sooty mangabeys. γδ T cells were obtained from peripheral blood samples from patients and sooty mangabeys that exhibited either a CD4-healthy (>200 CD4+ T cells/μl blood) or CD4-low (<200 CD4 cells/μl blood) phenotype. Cytokine flow cytometry was utilized to assess production of Th1 cytokines tumor necrosis factor alpha and gamma interferon following ex vivo stimulation with either phorbol myristate acetate/ionomycin or the Vδ2 γδ T-cell receptor agonist isopentenyl pyrophosphate. Sooty mangabeys were observed to have higher percentages of γδ T cells in their peripheral blood than humans did. Following stimulation, γδ T cells from SIV-positive (SIV+) mangabeys maintained or increased their ability to express the Th1 cytokines regardless of CD4+ T-cell levels. In contrast, HIV-positive (HIV+) patients exhibited a decreased percentage of γδ T cells expressing Th1 cytokines following stimulation. This dysfunction is primarily within the Vδ2+ γδ T-cell subset which incurred both a decreased overall level in the blood and a reduced Th1 cytokine production. Patients treated with highly active antiretroviral therapy exhibited a partial restoration in their γδ T-cell Th1 cytokine response that was intermediate between the responses of the uninfected and HIV+ patients. The SIV+ sooty mangabey natural hosts, which do not proceed to clinical AIDS, provide evidence that γδ T-cell dysfunction occurs in HIV+ patients and may contribute to HIV disease progression.
doi:10.1128/JVI.01275-07
PMCID: PMC2224435  PMID: 18045946
12.  Virus Subtype-Specific Features of Natural Simian Immunodeficiency Virus SIVsmm Infection in Sooty Mangabeys▿  
Journal of Virology  2007;81(15):7913-7923.
Simian immunodeficiency virus (SIV) SIVsmm naturally infects sooty mangabeys (SMs) and is the source virus of pathogenic infections with human immunodeficiency virus type 2 (HIV-2) and SIVmac of humans and macaques, respectively. In previous studies we characterized SIVsmm diversity in naturally SIV-infected SMs and identified nine different phylogenetic subtypes whose genetic distances are similar to those reported for the different HIV-1 group M subtypes. Here we report that, within the colony of SMs housed at the Yerkes National Primate Research Center, at least four SIVsmm subtypes cocirculate, with the vast majority of animals infected with SIVsmm subtype 1, 2, or 3, resulting in the emergence of occasional recombinant forms. While SIVsmm-infected SMs show a typically nonpathogenic course of infection, we have observed that different SIVsmm subtypes are in fact associated with specific immunologic features. Notably, while subtypes 1, 2, and 3 are associated with a very benign course of infection and preservation of normal CD4+ T-cell counts, three out of four SMs infected with subtype 5 show a significant depletion of CD4+ T cells. The fact that virus replication in SMs infected with subtype 5 is similar to that in SMs infected with other SIVsmm subtypes suggests that the subtype 5-associated CD4+ T-cell depletion is unlikely to simply reflect higher levels of virus-mediated direct killing of CD4+ T-cells. Taken together, this systematic analysis of the subtype-specific features of SIVsmm infection in natural SM hosts identifies subtype-specific differences in the pathogenicity of SIVsmm infection.
doi:10.1128/JVI.00281-07
PMCID: PMC1951324  PMID: 17507488
13.  Mucosal Innate Immune Response Associated with a Timely Humoral Immune Response and Slower Disease Progression after Oral Transmission of Simian Immunodeficiency Virus to Rhesus Macaques▿  
Journal of Virology  2007;81(12):6175-6186.
Mucosal transmission is the predominant mode of human immunodeficiency virus (HIV) infection worldwide, and the mucosal innate interferon response represents an important component of the earliest host response to the infection. Our goal here was to assess the changes in mRNA expression of innate mucosal genes after oral simian immunodeficiency virus (SIV) inoculation of rhesus macaques (Macaca mulatta) that were followed throughout their course of disease progression. The SIV plasma viral load was highest in the macaque that progressed rapidly to simian AIDS (99 days) and lowest in the macaque that progressed more slowly (>700 days). The mRNA levels of six innate/effector genes in the oral mucosa indicated that slower disease progression was associated with increased expression of these genes. This distinction was most evident when comparing the slowest-progressing macaque to the intermediate and rapid progressors. Expression levels of alpha and gamma interferons, the antiviral interferon-stimulated gene product 2′-5′ oligoadenylate synthetase (OAS), and the chemokines CXCL9 and CXCL10 in the slow progressor were elevated at each of the three oral mucosal biopsy time points examined (day 2 to 4, 14 to 21, and day 70 postinfection). In contrast, the more rapidly progressing macaques demonstrated elevated levels of these cytokine/chemokine mRNA at lymph nodes, coincident with decreased levels at the mucosal sites, and a decreased ability to elicit an effective anti-SIV antibody response. These data provide evidence that a robust mucosal innate/effector immune response is beneficial following lentiviral exposure; however, it is likely that the anatomical location and timing of the response need to be coordinated to permit an effective immune response able to delay progression to simian AIDS.
doi:10.1128/JVI.00042-07
PMCID: PMC1900075  PMID: 17428863
14.  Decreased Levels of Recent Thymic Emigrants in Peripheral Blood of Simian Immunodeficiency Virus-Infected Macaques Correlate with Alterations within the Thymus 
Journal of Virology  2002;76(19):9981-9990.
The thymus is responsible for de novo production of CD4+ and CD8+ T cells and therefore is essential for T-cell renewal. The goal of this study was to assess the impact of simian immunodeficiency virus (SIV) infection on the production of T cells by the thymus. Levels of recent thymic emigrants within the peripheral blood were assessed through quantification of macaque T-cell receptor excision circles (TREC). Comparison of SIV-infected macaques (n = 15) to uninfected macaques (n = 23) revealed stable or increased TREC levels at 20 to 34 weeks postinfection. Further assessment of SIV-infected macaques (n = 4) determined that TREC levels decreased between 24 and 48 weeks postinfection. Through the assessment of longitudinal time points in three additional SIVmac239-infected macaques, the SIV infection was divided into two distinct phases. During phase 1 (16 to 30 weeks), TREC levels remained stable or increased within both the CD4 and CD8 T-cell populations. During phase 2 (after 16 to 30 weeks), TREC levels declined in both T-cell populations. As has been described for human immunodeficiency virus (HIV)-infected patients, this decline in TREC levels did at times correlate with an increased level of T-cell proliferation (Ki67+ cells). However, not all TREC decreases could be attributed to increased T-cell proliferation. Further evidence for thymic dysfunction was observed directly in a SIVmac239-infected macaque that succumbed to simian AIDS at 65 weeks postinfection. The thymus of this macaque contained an increased number of memory/effector CD8+ T cells and an increased level of apoptotic cells. In summary, reduced levels of TREC can be observed beginning at 16 to 30 weeks post-SIV infection and correlate with changes indicative of dysfunction within the thymic tissue. SIV infection of macaques will be a useful model system to elucidate the mechanisms responsible for the thymic dysfunction observed in HIV-infected patients.
doi:10.1128/JVI.76.19.9981-9990.2002
PMCID: PMC136511  PMID: 12208974

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