Luc Kestens

Cutting edge : Resistance to HIV-1 infection among african female sex workers is associated with inhibitory KIR in the absence of their HLA ligands

NK cells are regulated in part by killer Ig-like receptors (KIR) that interact with HLA molecules on potential target cells. KIR and HLA loci are highly polymorphic and certain KIR/HLA combinations were found to protect against HIV disease progression. We show in this study that KIR/HLA interactions also influence resistance to HIV transmission. HIV-exposed but seronegative female sex workers in Abidjan, Côte d’Ivoire, frequently possessed inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1 and KIR3DL1 homozygosity in the absence of HLA-Bw4. HIV-seropositive female sex workers were characterized by corresponding inhibitory KIR/HLA pairings: KIR2DL3 homozygosity together with HLA-C1 and a trend toward KIR3DL1/HLA-Bw4 homozygosity. Absence of ligands for inhibitory KIR could lower the threshold for NK cell activation. In addition, exposed seronegatives more frequently possessed AB KIR genotypes, which contain more activating KIR. The data support an important role for NK cells and KIR/HLA interactions in antiviral immunity.

Selective increase of activation antigens HLA-DR and CD38 on CD4+CD45RO+ T lymphocytes during HIV-1 infection

Infection with HIV results in a progressive depletion of CD4* T cells and leads to significant in vivo lymphocyte phenotype changes. In this regard, the expression of HLA‐DR and CD38 on CD8* T cells has been shown to increase dramatically with disease progression. We investigated the expression of both aetivation markers on CD4* T cells in HIV‐1‐infected subjeets at different clinical stages of infection and compared the in vivo activation of CD4* T cells with parameters of viral activity and CD8* T cell activation. Eresh peripheral venous blood was obtained from 54 HIVinfeeted subjects and from 28 uninfected healthy controls. Three‐colour immunophenotyping of the CD4’T Cell subset showed that the proportion of CD4* T cells expressing HLA‐DR (10% in HIV negative controls) or CD38 (62%. in HIV‐negative controls) was higher in asymptomatic (P < 0.05 for CD38) and symptomatic (P< 0.001 for HLA‐DR and CD38) HIV‐infected subjects than in controls, whereas the proportion of CD4* T cells expressing CD45RO (54% in controls) remained relatively unchanged. Simultaneous expression of HLA‐DR and CD38 on CD4 * T cells increased from 2‐3% in controls to 11% (P<0.001) in asymptomatic and 22% (P < 0.001) in symptomatic HIV‐infected subjects. This relative increase of CD38 and HLA‐DR expression occurred mainly on CD4* T cells co‐expressing CD45RO. Changes in expression of HLA‐DR and CD38 on CD4 * T cells correlated with similar changes on CD8 * T lymphocytes, with the presence of HIV antigen in the circulation, and with the disease stage of HIV infection.

Increased cytolytic T lymphocyte activity and decreased B7 responsiveness are associated with CD28 down-regulation on CD8+ T cells from HIV-infected subjects.

The CD28 receptor on CD4+ and CD8+ T cells interacts with B7 molecules on antigen‐presenting cells (APC) to generate essential costimulatory signals. The cytolytic potential of CD8+ T cells could be linked to CD28 expression. Since HIV induces dysfunction of both CD4+ and CD8+ T cells, we evaluated CD28 expression and function in both subsets during HIV infection. CD28 expression on CD8+ T cells from HIV+ subjects was strongly reduced in a disease stage‐related fashion. CD28‐CD8+ T cells preferentially expressed CD57 and CD11b, but lacked CD26 and IL‐2Rα. The CD8+ T cells from the patients showed a significantly reduced proliferative response to co‐stimulation with cell‐bound anti‐CD3 and B7. Nevertheless, when stimulated with plate‐fixed anti‐CD3, CD8+ T cells from HIV‐infected subjects proliferated normally, and normal levels of IL‐2Rα nod transferrin‐receptor could be induced on CD28‐CD8+ T cells from the patients. In addition, stimulation with plate‐fixed anti‐CD3 induced proliferative responses in highly purified CD28‐CD8+ T cells from both HIV‐ and HIV+ persons. Furthermore, the increased cytotoxic activity of peripheral blood mononuclear cells (PBMC) from HIV+ subjects, measured in an anti‐CD3 redirected assay, was predominantly exerted by CD28‐CD57+ T cells. CD4+ T cells from the patients showed a slight but significant CD28 down‐regulation and were slightly hyporesponsive to B7 co‐stimulation. Decrease of CD28 on CD8+ T cells from HIV+ subjects is associated with an impaired response to co‐stimulation via B7. CD28‐CD8+ T cells from seropositives, however, are not completely inert, since they contain in vivo activated CTL and they can be additionally activated through a B7‐independent stimulation.

Expression patterns of Fcγ receptors, HLA-DR and selected adhesion molecules on monocytes from normal and HIV-infected individuals

The expression and co‐expression profiles of functionally important monocyte surface markers were compared between control and HIV+ individuals using combined physical gating and dim CD4 expression to delineate the monocytes. The FcγRII (CD32), the MHC class II antigen HLADR and the adhesion molecules CDIIa (LFA‐1α), CD18 and CD54 (ICAM‐1) showed an unimodal distribution. Of these markers, CDI Ia and HLA‐DR were up‐regulated in the HIV+ subjects compared with controls. The expression levels of the adhesion molecules correlated with each other in both patients and controls. The CD11b (CR3‐α), CD14, FcγRI, and FcγRIII markers were bimodally distributed. Compared with controls, monocytes from seropositives contained fewer CDbright+ cells, an equal proportion of FcγIbright+ cells, but twice as many FCγ RIII+ cells. The expression level of FcγRI and CD11b within their brightly positive subset increased as CD4T cells decreased. Both in patients and controls, co‐expression of bright CD11b, CD14 and FCγRI was shown, whereas the FcγRIII+ cells were negative or dim positive for the former triad. We conclude that the expression of two FcγR (I and III), of the adhesion molecules CD11a and CD11b and of HLA‐DR showed particular alterations on monocytes from HIV+ subjects. The relationship of these phenotypic observations with altered cytokine profiles and altered monocyte function is discussed.

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15: the Amplispot assay

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.

Decreased CD40 ligand induction in CD4 T cells and dysregulated IL-12 production during HIV infection

During HIV infection various cytokines are overproduced in early stages, whereas in advanced disease cytokines of the T helper 1 type (e.g. interferon‐gamma (IFN‐γ)) are selectively deficient. During antigenic stimulation, the production of type‐1 cytokines is enhanced by IL‐12, secreted by antigen‐presenting cells (APC) after their interaction with activated CD4 T cells. Two factors are essential in this process: priming APC with IFN‐γ and triggering the CD40 receptor on APC by CD40 ligand (CD40L). In view of the importance of this pathway, we compared its regulation in HIV‐infected and control subjects. After cross‐linking of the T cell receptor (TCR)/CD3 complex, the proportional expression of CD40L was similar on CD4+ T cells from controls and from patients with high circulating CD4 T counts (>u2003500/μl), but CD40L up‐regulation was significantly reduced in patients with more advanced disease. Simultaneous triggering of the costimulatory receptor CD28 on T cells through its natural ligand CD80 partly corrected the CD40L defect in patients with intermediate CD4 T counts (200–500), but not in AIDS patients. Early production of IFN‐γ was preserved in lymphocytes from HIV+ patients. The expression of CD40 on peripheral monocytes from HIV+ subjects was increased in a disease stage‐related fashion. Stimulation of mononuclear cells through cell‐bound CD40L and soluble IFN‐γ induced significantly higher IL‐12 in cultures from patients with >u2003200 circulating CD4 T cells, whereas IL‐12 production was marginally decreased in cultures from patients with <u2003200u2003CD4 T cells, compared with healthy control cultures. In conclusion, our data suggest that impaired CD40L induction on CD4 T cells contributes to deficient type‐1 responses through decreased IL‐12 production in AIDS infection, whereas enhanced CD40‐mediated IL‐12 production in less advanced stages might contribute to increased levels of various cytokines in early disease

Altered receptor expression and decreased sensitivity of T-cells to the stimulatory cytokines IL-2, IL-7 and IL-12 in HIV infection

A dysregulated production of regulatory cytokines has been proposed as a determinant in the progression of HIV infection. The sensitivity of T-cells to these cytokines has, however, not fully been investigated. Therefore, the responses of PBMC and T-cell subsets to the stimulatory cytokines IL-2, IL-7 and IL-12 in HIV-infected patients and HIV-negative controls were compared by examining their effect on the production of secondary cytokines (IFNgamma, IL-4 and IL-10), by simultaneous determination of T-cell activation and apoptosis and by measuring cytokine receptor expression. Production of IFNgamma was decreased in PBMC from the patients after stimulation with several combinations of stimulatory cytokines. IL-10 was only induced upon stimulation with IL-2 and IL-12 and tended to be produced more in patients. Expression of the different cytokine receptor chains showed complex alterations in HIV+ patients as compared to controls. The most pronounced changes were decreased expression of both IL-2Ralpha and IL-7Ralpha chain on CD8+ T-cells and an increase of IL-12Rbeta on both T-cell subsets from the patients. Evaluation of CD25 upregulation and blast formation revealed a deficient response to all three stimulatory cytokines in CD8+ but not in CD4+ T-cells from patients as compared to controls. Both CD4+ and CD8+ T-cells from the patients were less sensitive to the anti-apoptotic effect of IL-7 whereas only CD8+ T-cells were less sensitive to the anti-apoptotic effect of IL-2. The present data show that CD8+ T-cells, and to a lesser extent CD4+ T-cells, become less sensitive to IL-2, IL-7 and IL-12 during HIV infection. The decreased capacity of T-cells to respond to these cytokines could contribute to the HIV-related immune dysfunction.

Deficient T-cell responses in non-responders to hepatitis B vaccination: absence of TH1 cytokine production.

The inability to mount a protective level (> or = 10 IU/l) of hepatitis B surface antigen (HBsAg)-specific antibodies after vaccination is presumably the consequence of a defect in the cellular immune regulation. We compared the in vitro immune responses of peripheral blood mononuclear cells (PBMC) from high, intermediate and non-responders, after stimulation with recombinant HBsAg. The absence of a proliferative response in non-responders was not reversed by removal of CD8+ T cells, indicating that HBsAg-specific CD8+ T-cell-induced suppression was not the underlying cause of non-responsiveness. Non-responders did not produce cytokines after HBsAg stimulation. High responders displayed a typical Th1-like profile since their PBMC produced interleukin-2 (IL-2) and gamma-interferon (IFN gamma) and no detectable IL-4 or IL-5 upon stimulation with HBsAg.

Subset markers of CD8(+) cells and their relation to enhanced cytotoxic T-cell activity during human immunodeficiency virus infection

Using fresh whole blood or isolated lymphocytes, the activity ofin vivo generated cytotoxic T-lymphocytes (CTL) was measured as the OKT3-specific lysis of HL-60 targets, in a cross-sectional study of 53 HIV(+) patients. CTL activity in the entire HIV(+) group was two to three times higher than in HIV(−) controls, with WHO stage 3 (=pre-AIDS) patients showing the highest cytolytic function. The whole-blood CTL assay was validated and its practical and theoretical advantages are discussed. Within the CD8(+) cells, the number and proportion of the CD45RO(+) “memory” subset were significantly increased in HIV(+) subjects. The HLA-DR(+) subset rose most spectacularly in the asymptomatic stage of the infection, while the CD38(+) subset was the only one still significantly rising between the pre-AIDS and the AIDS stage. CTL activity was most closely correlated with T8 cells expressing the CD38 marker. In the context of CTL, CD38 thus seems to reflect activation rather than immaturity. Lymphocytes from HIV(+) subjects with a high OKT3-specific lytic capacity also destroyed normal lymphoblasts to a significant extent, pointing to their possible involvement in an autodestructive process. Our data thus suggest the importance of T8 cytolytic function and/or T8 subtyping in the immunopathogenesis and the prognosis of HIV infection.

Seroepidemiology of HTLV-III antibodies in a remote population of eastern Zaire.

A human retrovirus--human T cell lymphotropic virus-III (HTLV-III)--has recently emerged as the probable cause of acquired immunodeficiency syndrome (AIDS). In May 1984, 250 outpatients at a hospital in a remote area of eastern Zaire were surveyed for AIDS type illnesses and the prevalence of antibodies against HTLV-III determined by an enzyme linked immunosorbent assay using disrupted whole HTLV-III virus as the antigen. No clinical cases of AIDS were diagnosed among these patients. Overall, 31 (12.4%) had clearly positive ratios (greater than or equal to 5.0) and a further 30 (12.0%) had borderline ratios (3.0- less than 5.0). Western blots of serum samples from subjects with antibodies yielded bands consistent with HTLV-III as found in American patients with AIDS and members of groups at risk of AIDS. The prevalence of antibody was highest in childhood (p = 0.02); among adults prevalence rose slightly with age. HTLV-III antibodies were more common among the uneducated (p = 0.006), agricultural workers (p = 0.03), and rural residents (p = 0.006), but the Western blot bands were generally weak in this group. By contrast, one urban resident had strong bands. The relatively high prevalence of antibodies among the rural poor in this area of Zaire suggests that HTLV-III or a closely related, cross reactive virus may be endemic in the region. A different natural history of infection, perhaps in childhood, may also explain the findings.