P. L. J. Gigase

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.

Expression of activation antigens, HLA-DR and CD38, on CD8 lymphocytes during HIV-1 infection

Objective.To study the expression of the activation markers human leukocyte antigen (HLA)-DR and CD38 antigen on CD8+ T-lymphocytes in HIV-infected subjects and HIV-negative controls. Design.Two- and three-colour flow-cytometric analysis. Methods.Fresh peripheral venous blood was obtained from 16 HIV-infected subjects, representing four different stages of HIV disease, and from six HIV-negative controls. Three-colour lymphocyte immunophenotyping was performed using peridinyl chlorophyll-A protein (PerCP)-conjugated anti-CD8 monoclonal antibody (MAb) in combination with anti-HLA-DR (phycoerythrin) and anti-CD38 (fluorescein isothiocyanate) MAb. Results.The relative percentage of the lymphocyte populations thus defined differed between HIV-negative and HIV-positive subjects and between HIV-infected subjects at different clinical stages of disease. Simultaneous expression of HLA-DR and CD38 within the CD8 T-lymphocyte compartment increased from 8% in controls to 49% in asymptomatic HIV-infected subjects (P< 0.005). Symptomatic patients differed from asymptomatic seropositives by a further increase in the HLA-DR + CD38 + CD8 subset. In AIDS patients, the HLA-DR + CD38− CD8 subset decreased (P<0.05) and the HLA-DR- CD38+ CD8 subset increased (P<0.05), compared with the other HIV disease stage patients. Conclusion.There is a stage-associated pattern of HLA-DR and CD38 expression on CD8 T-lymphocytes during HIV infection; specific phenotypic patterns may have functional correlates in the host response to the virus.

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.

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.

Evidence for circulating activated cytotoxic T cells in HIV‐infected subjects before the onset of opportunistic infections

The activity of both cytotoxic T lymphocyte (CTL) and natural killer (NK) cells were measured cross‐sectionally in 43 subjects seropositive for HIV, in 27 HIV‐ blood donors and in 24 HIV persons from the Outpatients Clinic for sexually transmitted diseases. CTL activity was evaluated using the HL‐60 cells coated with OKT3 as the targets and freshly separated peripheral blood lymphocytes as the effectors. In 20 out of 43 HIV+ subjects, CTL activity was significantly enhanced in comparison to the HIV subjects. This lytic activity correlated positively with the percentages of CD3+ HLA‐DR+, of CD8+ CR3‐ and of CD57+CD16‐ lymphocytes, and was greatly reduced after elimination of CD8+ of HLA‐DR+ or of CD57+ cells. The median CTL activity seemed to increase from CDC group II to CDC group IV (Centers for Disease Control classification), but to return back to control levels in those patients with a history of opportunistic infections. NK function in HIV+ subjects was not significantly different from that in the blood donors. In seropositive patients, NK activity correlated positively with the percentages of both CD16+CD57+ and of CD8+ CR3+ cells and was strongly diminished after elimination of CD16 + or of CD57+ cells. There was no significant change in NK function according to the clinical stage. The data show that circulating CD8+HLA‐DR+ CTJ57+ T cells in HIV+ subjects are activated cytotoxic T cells and point to progressive (over) activation of this T cell compartment until the onset of opportunistic infections.

Costimulation of CD4 and CD8 T cells through CD26: The ADA-binding epitope is not essential for complete signaling

It was previously shown that CD26 (DPP IV, EC 3.4.14.5) is a binding site for adenosine deaminase (ADA, EC 3.5.4.4) on T cells and that costimulation by some anti‐CD26 monoclonal antibodies (mAbs) and anti‐CD3 induces CD4+ T cell proliferation. The CD26 epitopes involved in costimulation, the precise sequence of the events preceding proliferation, and the response of CD8+ compared with CD4+ T cells to CD26 were not extensively studied. We therefore compared the effects of the novel TA5.9 anti‐CD26 mAb, recognizing an ADA‐binding epitope, and the clearly distinct anti‐Ta1 reference anti‐CD26 mAb for their costimulatory properties in various T cell subsets. Both purified CD4+ and CD8+ T cells proliferated upon costimulation with anti‐CD3 and either anti‐CD26 mAb, but anti‐TA5.9 mAb induced a more potent response than anti‐Tal. Either anti‐CD26 mAb, together with anti‐CD3, caused a similar sequential up‐regulation of CD69, CD25 (IL‐2Rα), and CD71 (transferrin receptor) expression on CD4+ and CD8+ T cells. The activation markers appeared faster on the CD45R0+ than on the CD45R0− subsets. After costimulation, CD4+ T cell cultures contained significant amounts of the Th1 cytokines EL‐2, interferon‐γ (IFN‐γ), and tumor necrosis factor‐α (TNF‐α). In CD8+ T cell cultures relatively more IFN‐γ and TNF‐α but almost no IL‐2 was measured after triggering of CD3 and CD26. Our data demonstrate that the recognition of the ADA‐binding epitope is not a prerequisite for the costimulatory capacity of anti‐CD26 mAbs. Both CD4+ and CD8+ T cells and their CD45R0−and CD45R0+ subsets are sensitive to various aspects of activation via CD26, but the magnitude and/or kinetics differ according to the anti‐CD26 used and the T cell subset studied.

HIV antigen detection in circulating immune complexes

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.

Immunohistochemical characterization of the mononuclear cells in the brain of the rat with an experimental chronic Trypanosoma brucei gambiense infection.

The distribution of T-cell subsets, B cells, and class II MHC antigens was examined within the CNS of rats chronically infected withTrypanosoma brucei gambiense, using appropriate mouse monoclonal antibodies. The mononuclear infiltrates of the leptomeninges and the perivascular areas (Virchow-Robin spaces) were composed of IgM-producing plasma cells and Mott cells and T-helper/inducer cells. Cells defined phenotypically as suppressor/cytotoxic T cells were rare. Anti-Ia reactive cells were also abundant in these inflammatory lesions and in the white matter, representing Ia-expressing neuroglial cells, B cells, activated T cells, and macrophages. The Ia-positive neuroglial cells, possibly acting as accessory cells, associated with numerous T-helper/inducer cells and cells from the B-cell lineage, suggest that a T-dependent B-cell immune response can be initiated within the CNS of rats with a chronicT. b. gambiense infection.