Michelle Bailey

HIV disease progression despite suppression of viral replication is associated with exhaustion of lymphopoiesis

The mechanisms of CD4(+) T-cell count decline, the hallmark of HIV disease progression, and its relationship to elevated levels of immune activation are not fully understood. Massive depletion of CD4(+) T cells occurs during the course of HIV-1 infection, so that maintenance of adequate CD4(+) T-cell levels probably depends primarily on the capacity to renew depleted lymphocytes, that is, the lymphopoiesis. We performed here a comprehensive study of quantitative and qualitative attributes of CD34(+) hematopoietic progenitor cells directly from the blood of a large set of HIV-infected persons compared with uninfected donors, in particular the elderly. Our analyses underline a marked impairment of primary immune resources with the failure to maintain adequate lymphocyte counts. Systemic immune activation emerges as a major correlate of altered lymphopoiesis, which can be partially reversed with prolonged antiretroviral therapy. Importantly, HIV disease progression despite elite control of HIV replication or virologic success on antiretroviral treatment is associated with persistent damage to the lymphopoietic system or exhaustion of lymphopoiesis. These findings highlight the importance of primary hematopoietic resources in HIV pathogenesis and the response to antiretroviral treatments.

Simian Immunodeficiency Virus Infects Follicular Helper CD4 T Cells in Lymphoid Tissues during Pathogenic Infection of Pigtail Macaques

ABSTRACT T follicular helper (Tfh) cells are a specialized subset of memory CD4+ T cells that are found exclusively within the germinal centers of secondary lymphoid tissues and are important for adaptive antibody responses and B cell memory. Tfh cells do not express CCR5, the primary entry coreceptor for both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), and therefore, we hypothesized that these cells would avoid infection. We studied lymph nodes and spleens from pigtail macaques infected with pathogenic strain SIVmac239 or SIVmac251, to investigate the susceptibility of Tfh cells to SIV infection. Pigtail macaque PD-1high CD127low memory CD4+ T cells have a phenotype comparable to that of human Tfh cells, expressing high levels of CXCR5, interleukin-21 (IL-21), Bcl-6, and inducible T cell costimulator (ICOS). As judged by either proviral DNA or cell-associated viral RNA measurements, macaque Tfh cells were infected with SIV at levels comparable to those in other CD4+ memory T cells. Infection of macaque Tfh cells was evident within weeks of inoculation, yet we confirmed that Tfh cells do not express CCR5 or either of the well-known alternative SIV coreceptors, CXCR6 and GPR15. Mutations in the SIV envelope gp120 region occurred in chronically infected macaques but were uniform across each T cell subset investigated, indicating that the viruses used the same coreceptors to enter different cell subsets. Early infection of Tfh cells represents an unexpected focus of viral infection. Infection of Tfh cells does not interrupt antibody production but may be a factor that limits the quality of antibody responses and has implications for assessing the size of the viral reservoir.

High Levels of Human Antigen-Specific CD4+ T Cells in Peripheral Blood Revealed by Stimulated Coexpression of CD25 and CD134 (OX40)

Ag-specific human CD4+ memory T lymphocytes have mostly been studied using assays of proliferation in vitro. Intracellular cytokine and ELISPOT assays quantify effector cell populations but barely detect responses to certain recall Ags that elicit strong proliferative responses, e.g., tetanus toxoid, that comprise non-Th1 CD4+ cells. We have found that culturing whole blood with Ag for 40–48 h induces specific CD4+ T cells to simultaneously express CD25 and CD134. This new technique readily detects responses to well-described CD4+ T cell recall Ags, including preparations of mycobacteria, CMV, HSV-1, influenza, tetanus toxoid, Candida albicans, and streptokinase, as well as HIV-1 peptides, with high specificity. The assay detects much higher levels of Ag-specific cells than intracellular cytokine assays, plus the cells retain viability and can be sorted for in vitro expansion. Furthermore, current in vitro assays for human CD4+ memory T lymphocytes are too labor-intensive and difficult to standardize for routine diagnostic laboratories, whereas the whole-blood CD25+CD134+ assay combines simplicity of setup with a straightforward cell surface flow cytometry readout. In addition to revealing the true extent of Ag-specific human CD4+ memory T lymphocytes, its greatest use will be as a simple in vitro monitor of CD4+ T cell responses to Ags such as tuberculosis infection or vaccines.

Group 2 innate lymphoid cells (ILC2s) are increased in chronic rhinosinusitis with nasal polyps or eosinophilia

Chronic rhinosinusitis (CRS) is a heterogeneous disease with an uncertain pathogenesis. Group 2 innate lymphoid cells (ILC2s) represent a recently discovered cell population which has been implicated in driving Th2 inflammation in CRS; however, their relationship with clinical disease characteristics has yet to be investigated.

HIV DNA Subspecies Persist in both Activated and Resting Memory CD4+ T Cells during Antiretroviral Therapy

ABSTRACT The latent HIV reservoir is a major impediment to curing HIV infection. The contribution of CD4+ T cell activation status to the establishment and maintenance of the latent reservoir was investigated by enumerating viral DNA components in a cohort of 12 individuals commencing antiretroviral therapy (ART) containing raltegravir, an integrase inhibitor. Prior to ART, the levels of total HIV DNA were similar across HLA-DR+ and HLA-DR− (HLA-DR±) CD38± memory CD4+ T cell phenotypes; episomal two-long terminal repeat (2-LTR) HIV DNA levels were higher in resting (HLA-DR− CD38−) cells, and this phenotype exhibited a significantly higher ratio of 2-LTR to integrated HIV DNA (P = 0.002). After 1 year of ART, there were no significant differences across each of the memory phenotypes of any HIV DNA component. The decay dynamics of integrated HIV DNA were slow within each subset, and integrated HIV DNA in the resting HLA-DR− CD38− subset per mm3 of peripheral blood exhibited no significant decay (half-life of 25 years). Episomal 2-LTR HIV DNA decayed relative to integrated HIV DNA in resting cells with a half-life of 134 days. Surprisingly, from week 12 on, the decay rates of both total and episomal HIV DNA were lower in activated CD38+ cells. By weeks 24 and 52, HIV RNA levels in plasma were most significantly correlated with the numbers of resting cells containing integrated HIV DNA. On the other hand, total HIV DNA levels in all subsets were significantly correlated with the numbers of HLA-DR+ CD38− cells containing integrated HIV DNA. These results provide insights into the interrelatedness of cell activation and reservoir maintenance, with implications for the design of therapeutic strategies targeting HIV persistence. IMPORTANCE It is generally believed that HIV is not cleared by extensive antiretroviral therapy (ART) due to the difficulty in eradicating the latent reservoir in resting CD4+ T cells. New therapies that attempt to activate this reservoir so that immune or viral cytopathic mechanisms can remove those infected cells are currently being investigated. However, results obtained in this research indicate that activation, at least on some level, already occurs within this reservoir. Furthermore, we are the first to describe the dynamics of different HIV DNA species in resting and activated memory CD4+ T cell subsets that point to the role different levels of activation play in maintaining the HIV reservoir.

Integrated HIV DNA accumulates prior to treatment while episomal HIV DNA records ongoing transmission afterwards

Objective:We investigated the dynamics of HIV RNA and HIV DNA levels after the commencement of raltegravir-based antiretroviral therapy (ART) in primary (PHI) and chronically HIV-infected (CHI) individuals (the PINT study). Design:We recruited 8 PHI and 8 CHI ART-naive individuals who commenced a 1-year combination regimen of Truvada and the integrase inhibitor raltegravir. Methods:Nonlinear mixed effects modelling was used to determine multiphasic decay of plasma HIV RNA levels (pVL), as well as dynamics of total, episomal [2-long terminal repeats (LTR)] and integrated HIV DNA in CD4+ T cells from peripheral blood. Results:Although pVL decreased faster through first and second phase for PHI individuals there was no difference in the final level reaching a mean of 9 copies/ml by week 16 that was maintained thereafter. Total HIV DNA and integrated HIV DNA levels from CHI patients were significantly higher than from PHI patients. However, at no time did 2-LTR levels differ between groups. Of note, 2-LTR circles exhibited an initial increase peaking at week 3 followed by biphasic decay with a half-life of 29 days. Second phase integrated HIV DNA levels were significantly correlated with duration of infection and consistent with this form of infection occurring at approximately 100 000 integration events per day in the absence of ART, achieving its 50% level 2 years after infection. Conclusions:Integrated HIV DNA levels accumulate with duration of untreated HIV infection. The relatively short half-life and high levels of 2-LTR circles after 1 year support continued HIV transmission during ART.

A novel assay detecting recall response to Mycobacterium tuberculosis: Comparison with existing assays

A strategy to reduce the burden of active TB is isoniazid preventive therapy for latent TB infection (LTBI). However, current assays used to diagnose LTBI all have limitations. In these proof of concept studies, we compared the agreement of a novel flow cytometry assay detecting CD25/CD134 co-expression with QuantiFERON-TB Gold In-Tube (QFN-GIT) and Tuberculin skin test (TST) in the detection of recall immune response to TB. The CD25/CD134 assay, QFN-GIT and TST were performed on 74 participants referred for TB screening in Sydney and on 50 participants with advanced HIV infection (CD4 ≤ 350 × 10(6) cells/L) in Bangkok. The agreement between CD25/CD134 assay and QFN-GIT was 93.2% (Kappa 0.631 95% CI 0.336-0.926) in Sydney and 90% (Kappa 0.747 95% CI 0.541-0.954) in Bangkok. Discordant results occurred around the cut off of both tests. The agreement between CD25/CD134 assay and TST was 73.6% (Kappa 0.206 95% CI 0.004-0.409) in Sydney and 84% (Kappa 0.551 95% CI 0.296-0.806) in Bangkok. The CD25/CD134 assay showed good agreement with QFN-GIT in detecting recall response to TB both in well and less resourced setting as well as in persons with advanced HIV infection. Further study into the performance of this assay is thus warranted.

Cellular comparison of sinus mucosa vs polyp tissue from a single sinus cavity in chronic rhinosinusitis

Nasal polyposis is a common development in chronic rhinosinusitis (CRS), and sinus mucosa and polyp tissue have been used interchangeably in studies investigating CRS. However, potential differences may exist between these 2 tissue types, which have not been entirely characterized.

Serial study of lymph node cell subsets using fine needle aspiration in pigtail macaques

Lymphoid tissues are of intense interest for studies of the pathogenesis of human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques but are relatively difficult to sample non-invasively. Fine needle aspiration (FNA) cytology, conventionally a diagnostic procedure for lymphadenopathy, can be used for longitudinal study of tissue cell subsets during HIV/SIV infection. In this study, we serially sampled lymph node (LN) FNA from pigtail macaques and studied cell subsets in the aspect of absolute count, frequency, and functionality by flow cytometry. The median recovered lymphocyte count from FNA samples was 2.01×10(5) (3.0×10(3) to 2.25×10(6), n=38) and median CD4+ T cell subset recovered was 5.94×10(4) (277 to 6.17×10(5), n=38). Although we observed a relatively large variation in the frequencies of cell subsets of FNA samples taken from different time points, the cell subset composition of FNA samples, in particular T cell and CD4+ T cell frequencies, was broadly comparable to whole excised LNs (n=6) and distinct from peripheral blood. A subset of CD4+ T cells that is located almost exclusively in secondary lymphoid tissues, T follicular helper (TFH) cells, was readily identifiable in LN FNAs and the TFH cell frequencies were strongly correlated with B cell frequencies. In vitro functionality of FNA lymphocytes was demonstrated using polyclonal SEB stimulation, resulting in a median 6% of responding CD4+ T cells, comparable to circulating CD4+ T lymphocytes. We conclude that serial sampling of macaque LNs using FNA is a potentially useful method to study the immunopathogenesis of SIV infection and may be extended to HIV infection.

A Novel Chemokine-Receptor-5 (CCR5) Blocker, SCH532706, Has Differential Effects on CCR5+CD4+ and CCR5+CD8+ T Cell Numbers in Chronic HIV Infection

HIV treatment with CCR5 receptor blockers may impact CCR5(+) cell distribution. T cell subsets, plasmacytoid dendritic cells (PDC), and antigen-specific [Mycobacteria tuberculosis/avium (M.TB/MAI), cytomegalovirus (CMV), Herpes simplex (HSV), HIV-Gag] CD4(+) T cells were measured in untreated R5-tropic-HIV-infected adults receiving 10 days of SCH532706 (Phase 1), 15 days no therapy, then 10 days of cART (without SCH532706) (Phase 2). Ten males were enrolled with median cells/microl (range) of CD4(+) 310 (92-848), CCR5(+)CD4(+) 57 (17-118), CD8(+) 895 (459-1666), and CCR5(+)CD8(+) 392 (250-983), and median plasma HIV RNA of 4.6 log(10) copies/ml. At baseline, proportions of M.TB, MAI, CMV, HSV, and HIV-Gag-specific CD4(+) T cells were 0.3%, 3.0%, 6.0%, 2.0%, and 1.6%, respectively. Median log(10) HIV RNA copies/ml declines were 1.5 (Phase 1) and 1.75 (Phase 2) (p = 0.7). Median CD4(+) and CD8(+) changes, respectively, during Phases 1 (+16; +91) and 2 (+28; -71) were similar (p = 0.7 both). However, CCR5(+)CD8(+) T cell fluctuations were significantly different (p = 0.02) during Phase 1 (+147 cells) vs. Phase 2 (-35 cells). PDC increased significantly more during Phase 1 (p = 0.04). Declines in antigen-specific cells were similar except for M. avium, which declined significantly during Phase 2 (p = 0.04). Similar declines in activation and proliferation of T cell subsets were observed during both treatment phases. For equivalent HIV RNA declines, CCR5-receptor blockade differentially increased CD8(+) T cell and PDC numbers in the circulation. These results confirm that cell surface CCR5 expression on these cells constantly directs trafficking during HIV infection. The persistence and clinical meaning of these immunological changes during long-term exposure to this class of anti-HIV drugs are unknown, but may have implications for immunosurveillance of inflammation.