John Zaunders

Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells

Abnormalities in CD4+CD25+Foxp3+ regulatory T (T reg) cells have been implicated in susceptibility to allergic, autoimmune, and immunoinflammatory conditions. However, phenotypic and functional assessment of human T reg cells has been hampered by difficulty in distinguishing between CD25-expressing activated and regulatory T cells. Here, we show that expression of CD127, the α chain of the interleukin-7 receptor, allows an unambiguous flow cytometry–based distinction to be made between CD127lo T reg cells and CD127hi conventional T cells within the CD25+CD45RO+RA− effector/memory and CD45RA+RO− naive compartments in peripheral blood and lymph node. In healthy volunteers, peripheral blood CD25+CD127lo cells comprised 6.35 ± 0.26% of CD4+ T cells, of which 2.05 ± 0.14% expressed the naive subset marker CD45RA. Expression of FoxP3 protein and the CD127lo phenotype were highly correlated within the CD4+CD25+ population. Moreover, both effector/memory and naive CD25+CD127lo cells manifested suppressive activity in vitro, whereas CD25+CD127hi cells did not. Cell surface expression of CD127 therefore allows accurate estimation of T reg cell numbers and isolation of pure populations for in vitro studies and should contribute to our understanding of regulatory abnormalities in immunopathic diseases.

Characterization of CD4+ CTLs Ex Vivo

The cytotoxic potential of CD8+ T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4+ T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4+ T cells have been the subject of debate. Here we show that a population of CD4+ perforin+ T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4+ T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4+ T cells. The existence of CD4+ cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction

In progressive viral infection, antiviral T cell function is impaired by poorly understood mechanisms. Here we report that the inhibitory immunoregulatory receptor CTLA-4 was selectively upregulated in human immunodeficiency virus (HIV)–specific CD4+ T cells but not CD8+ T cells in all categories of HIV-infected subjects evaluated, with the exception of rare people able to control viremia in the absence of antiretroviral therapy. CTLA-4 expression correlated positively with disease progression and negatively with the capacity of CD4+ T cells to produce interleukin 2 in response to viral antigen. Most HIV-specific CD4+ T cells coexpressed CTLA-4 and another inhibitory immunoregulatory receptor, PD-1. In vitro blockade of CTLA-4 augmented HIV-specific CD4+ T cell function. These data, indicating a reversible immunoregulatory pathway selectively associated with CD4+ T cell dysfunction, provide a potential target for immunotherapy in HIV-infected patients.

The extent of HIV-1-related immunodeficiency and age predict the long-term CD4 T lymphocyte response to potent antiretroviral therapy.

Objective To study the long-term immunological recovery in HIV-1-infected individuals receiving potent antiretroviral therapy (ART). Design Prospective, observational study. Methods Plasma HIV-1 RNA, CD4 and CD8 T lymphocyte counts were determined at 3–6 monthly intervals in 95 HIV-1-infected subjects receiving ART who suppressed plasma HIV-1 RNA to levels below 400 copies/ml during a median observation period of 45 months. Results The median CD4 cell count rose from 325 to 624 cells/μl at 48 months, increasing by 22.6 cells/μl per month in the first 3 months, 8.1 cells/μl per month from months 3 to 12, 6.8 cells/μl per month in the second year, 3.3 cells/μl per month in the third, and 1.7 cells/μl per month in the fourth year. At 48 months, 98% of subjects reached CD4 cell counts > 200 cells/μl, 86% > 350 cells/μl, and 74% > 500 cells/μl. A higher nadir CD4 cell count and younger age were independently associated with greater increases in CD4 cell counts, and higher absolute CD4 cell counts at 48 months. Poor immunological responders who did not reach 500 CD4 lymphocytes/μl at 48 months showed lower nadir and baseline CD4 cell counts than good responders (99 versus 300 cells/μl and 160 versus 373 cells/μl, respectively). Conclusion The recovery of CD4 T lymphocytes occurs mainly in the first 2 years after the initiation of ART, and is associated with age and the pre-existing degree of HIV-1-related immunodeficiency, suggesting that the long-term exposure to HIV-1 infection has caused damage to the immune system that is difficult to correct.

Increased Plasma Interleukin-7 Level Correlates with Decreased CDl27 and Increased CD132 Extracellular Expression on T Cell Subsets in Patients with HN-1 Infection

BACKGROUND Interleukin (IL)-7 levels are increased in patients with human immunodeficiency virus type 1 (HIV-1)-associated lymphopenia; however, the effects of this on IL-7 receptor (IL-7R) expression, disease progression, and immune reconstitution remain unclear. METHODS Plasma IL-7 levels were measured, by enzyme-linked immunoassay, in patients with primary, chronic, or long-term nonprogressive HIV-1 infection (PHI, CHI, and LTNP, respectively) before and after 40-48 weeks of antiretroviral therapy (ART). Cell-surface expression and intracellular expression of the IL-7R components CD127 and CD132 were measured by flow cytometry. The effects of IL-7 and cycloheximide on IL-7R expression by peripheral blood mononuclear cells were examined in vitro. RESULTS Plasma IL-7 levels were increased in both patients with PHI and those with CHI; administration of ART resulted in normalized plasma IL-7 levels in patients with PHI but not in those with CHI. Plasma IL-7 levels positively correlated with CD4(+) T cell immune reconstitution in patients with PHI. In vitro, exogenous IL-7 rapidly down-regulated cell-surface CD127 expression, but not CD132 expression, whereas subsequent reexpression required active protein synthesis. HIV-1 infection resulted in progressive decreases in the CD127(+)132(-) subset and increases in the CD127(-)132(+) subset of CD4(+) and CD8(+) T cells. Changes in CD4(+) T cell expression of IL-7R components were evident in patients with LTNP who lost viral control, and these changes preceded increases in plasma IL-7 levels. CONCLUSIONS Perturbations in the IL-7/IL-7R system were clearly associated with disease progression but did not reliably predict immune reconstitution.

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.

Proliferation of weakly suppressive regulatory CD4+ T cells is associated with over-active CD4+ T-cell responses in HIV-positive patients with mycobacterial immune restoration disease.

The role of Treg in patients with late‐stage HIV disease, who commence combination antiretroviral therapy (cART) and develop pathogen‐specific immunopathology manifesting as immune restoration disease (IRD) remains unclear. We hypothesised that Treg could be defective in either numbers and/or function and therefore unable to ensure the physiological equilibrium of the immune system in patients with IRD. Phenotypic and functional CD4+ T‐cell subsets of eight late‐stage HIV patients with nadir CD4 count <50 cells/μL, who developed mycobacterial IRD upon commencing cART were compared with six therapy naive HIV+ patients (nadir CD4 count <50 cells/μL), who did not develop an IRD after cART. Mycobacterium‐avium‐specific CD4+ T cells from IRD patients produced high levels of IFN‐γ and IL‐2 compared with controls (p<0.001). Surprisingly, we found a significant expansion of CD127loFoxp3+CD25+ Treg in IRD patients and a higher ratio of Treg to effector/memory subsets (p<0.001). In vitro suppression assays demonstrated reduced functional capacity of suppressor cells and diminished IL‐10 secretion in IRD patients. Plasma levels of IL‐7 were increased in patients and, interestingly, exogenous IL‐7 and other cytokines strongly inhibited Treg suppression. These data suggest that despite substantial Treg expansion in IRD, their ability to induce suppression, and thereby downregulate aberrant immune responses, is compromised.

Increased Natural Killer Cell Activity in Viremic HIV-1 Infection

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-γ and TNF-α than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3−/CD56+/CD94+ and CD3−/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.

Potent Antiretroviral Therapy of Primary Human Immunodeficiency Virus type 1 (HIV-1) Infection: Partial Normalization of T Lymphocyte Subsets and Limited Reduction of HIV-1 DNA Despite Clearance of Plasma Viremia

Antiretroviral therapy commenced during primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) may limit the extent of viral replication and prevent early loss of HIV-specific CD4 lymphocyte function. We studied the effect of current standard therapy (2 nucleoside analogues and a protease inhibitor) in 16 patients with symptomatic PHI. In the 13 patients who completed 1 year of treatment, plasma HIV RNA was <50 copies/mL and median CD4 cell counts were comparable to HIV-uninfected controls, with naive (CD45RA+CD62L+), primed (CD45RO+), and T cell receptor Vbeta subsets all within normal ranges. However, HIV-1 DNA levels in treated and untreated PHI patients were similar. Furthermore, CD8 cell counts remained elevated, including activated (CD38+HLA-DR+), replicating (Ki-67+), and cytotoxic (perforin+CD28-) lymphocytes. In conclusion, early antiretroviral therapy resulted in clearance of viremia and prevented loss of crucial CD4 subsets. The persistence of HIV-1 DNA together with increased CD8 T lymphocyte turnover and activation indicate continued expression of viral antigens.

HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism

HIV‐1 induces apoptosis and leads to CD4+ T‐lymphocyte depletion in humans. It is still unclear whether HIV‐1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV‐1–induced apoptosis, we infected human CD4+ T cells with HIV‐1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro‐caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis‐inducing factor (AIF) from mitochondria was observed in HIV‐infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. HIV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up‐regulation of surface FasL expression, accompanied by a down‐regulation of Fas‐associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.