Dorian McIlroy

Depletion in blood CD11c-positive dendritic cells from HIV-infected patients.

OBJECTIVES To quantify blood dendritic cells from HIV-positive patients and to study the expression of functional molecules, in relation to HIV viral load, CD4 cell counts and antiretroviral treatment. DESIGN AND METHODS Three-colour flow cytometry analysis was used to quantify blood dendritic cells without previous isolation from whole blood and to study the expression of functional molecules (MHC class II, CD11c, CD83, CD86) by dendritic cells from 30 HIV-positive patients, 15 of whom were treated with combined antiretroviral therapy (viral loads from undetectable to 5.4 log copies/ml, CD4 cell counts 1-1895 cells/mm3) and 11 non-infected controls. RESULTS The median proportion of blood dendritic cells from HIV-positive patients was significantly decreased when the plasma viral load was above 200 copies/ml: 0.2% (0.1-1.1, n = 19) compared with 0.4% (0.2-0.8, n = 11) in patients with undetectable viral load whether they were treated or not, and to 0.4% (0.2-1.3, n = 11) in controls (P = 0.02). A major decrease of the CD11c positive dendritic cells was observed in all HIV-positive samples, with only 18% (mean; range: 0.3-80%, median 4.2%) compared with 44% (11-70%, median 42%) of control dendritic cells (P = 0.0006). In contrast, the proportion of dendritic cells expressing CD86, was slightly higher in HIV-positive patients than in controls (P = 0.03). CONCLUSIONS The decreased proportion of blood dendritic cells correlated with virus replication and the lack of dendritic cells expressing CD11c are the first evidence of strong dendritic cell alterations in HIV-positive patients. Although the proportion of blood dendritic cells are in the normal range in treated HIV-positive patients with undetectable viral load, the CD11c alterations persist indicating that antiretroviral therapy might only partly correct the alterations of the circulating dendritic cells.

Protective Effect of Interleukin-4 -589T Polymorphism on Human Immunodeficiency Virus Type 1 Disease Progression: Relationship with Virus Load

The interleukin (IL)-4 -589T allele bears a single nucleotide polymorphism at position -589 upstream from the open-reading frame of the IL-4 gene. To determine the influence of this allele on human immunodeficiency virus (HIV) type 1 disease, disease progression and serum virus load were assessed by IL-4 genotype in 427 white patients with known seroconversion dates who were followed in the French SEROCO cohort between 1988 and 1996. Serum virus load was 0.20 log lower during the 6-24-month plateau phase after seroconversion in patients with IL-4 -589T than in those without this allele (P=.02). Kaplan-Meier analysis survival curves showed a slower progression to clinical AIDS in carriers of IL-4 -589T (P=.04). Adjustment for early serum virus load greatly diminished the strength of this association. These results suggest that IL-4 -589T protects against HIV-1 disease progression by reducing virus load.

A triple-mutated allele of granzyme B incapable of inducing apoptosis

Granzyme B (GzmB) is a serine protease involved in many pathologies, including viral infections, autoimmunity, transplant rejection, and antitumor immunity. To measure the extent of genetic variation in GzmB, we screened the GzmB gene for polymorphisms and defined a frequently represented triple-mutated GzmB allele. In this variant, three amino acids of the mature protein Q48P88Y245 are mutated to R48A88H245. In CD8+ cytotoxic T lymphocytes, GzmB was expressed at similar levels in QPY homozygous, QPY/RAH heterozygous, and RAH homozygous individuals, demonstrating that RAH GzmB is a stable protein. Active RAH GzmB expressed in glioblastoma cell lines displayed proteolytic activity, but in contrast to QPY GzmB, it did not accumulate in the nucleus and was unable to induce Bid cleavage, cytochrome c release, or apoptosis. Molecular modeling showed that the three amino acid substitutions clustered near the C-terminal α-helix of the protein, indicating that this region of the protein may be involved in the intracellular targeting of GzmB. The triple-mutated GzmB allele that we describe appears to be incapable of inducing apoptosis in tumor cell lines, and its presence could, therefore, influence both the prognosis of cancer patients and the success rates of antitumor cellular immunotherapy.

Profiling dendritic cell maturation with dedicated microarrays

Dendritic cell (DC) maturation is the process by which immature DC in the periphery differentiate into fully competent antigen‐presenting cells that initiate the T cell response. However, DC respond to many distinct maturation stimuli, and different types of mature DC induce qualitatively different T cell responses. As DC maturation involves the coordinated regulation of hundreds of genes, comprehensive assessment of DC maturation status would ideally involve monitoring the expression of all of these transcripts. However, whole‐genome microarrays are not well‐suited for routine phenotyping of DC, as the vast majority of genes represented on such chips are not relevant to DC biology, and their cost limits their use for most laboratories. We therefore developed a DC‐dedicated microarray, or “DC Chip”, incorporating probes for 121 genes up‐regulated during DC maturation, 93 genes down‐regulated during maturation, 14 DC‐specific genes, and 90 other genes with known or probable immune functions. These microarrays were used to study the kinetics of DC maturation and the differences in maturation profiles among five healthy donors after stimulation with tumor necrosis factor‐α + polyI:C. Results obtained with the DC Chip were consistent with flow cytometry, enzyme‐linked immunosorbent assay, and real‐time polymerase chain reaction, as well as previously published data. Furthermore, the coordinated regulation of a cluster of genes (indoleamine dioxygenase, kynureninase, kynurenine monoxygenase, tryptophanyl tRNA synthetase, and 3‐hydroxyanthranilate 3,4‐dioxygenase) involved in tryptophan metabolism was observed. These data demonstrate the use of the DC Chip for monitoring the molecular processes involved in the orientation of the immune response by DC.

Despite an impaired response to IL-7, T cells from HIV-positive patients proliferate normally in response to IL-15 and its superagonist, RLI

Objective:In phase I/II trials, IL-7 immunotherapy has been shown to expand CD4+ T cells. However, expression of the IL-7 receptor &agr;-chain, CD127, is reduced on CD4+ T cells from HIV-positive patients, and defects in CD127 signaling have also been reported. To refine and improve cytokine immunotherapy, it is important to identify stimuli that can restore proliferation of CD4+ cells with defective responses to IL-7. Design:Observational study comparing viremic HIV-positive patients with HIV-negative controls. Methods:Peripheral blood mononuclear cells were cultured in the presence of 1 nmol/l IL-2, IL-7, IL-15 or RLI (an IL-15R&agr;/IL-15 fusion protein). Proliferation of different T-cell subsets was assessed by carboxyfluorescein succinimidyl ester fluorescence. Expression of CD127 on CD4+ T-cell subsets was also analyzed. Results:In HIV-positive patients, CD127 expression was correlated with CD4+ T-cell count in the (R2 = 0.36; P < 0.01) and (R2 = 0.45; P < 0.001) populations, whereas CD127 expression on cells was significantly reduced in HIV-positive individuals compared with controls (P = 0.001) independently of CD4+ T-cell count. In patients with high CD4+ T-cell counts, proliferation in response to IL-7 was significantly reduced only in cells (P < 0.05). RLI, and to a lesser extent IL-15, induced strong proliferation of cells from both HIV-positive patients and controls. Neither agent stimulated proliferation of or cells. Conclusion:In HIV-positive patients, cells are deficient in both CD127 expression and proliferation in response to IL-7. RLI and IL-15 specifically induced proliferation of cells, suggesting that they may have a unique potential to complement IL-7 immunotherapy.