AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 7, 1990 Mary Ann Liebert, Inc., Publishers
Interferon-7 Marks Activated T Lymphocytes in AIDS Patients
A.
CARUSO,1 R. GONZALES,1 R. STELLINI,2 A. SCALZINI,2 L. PERONI,1 and A.
TURANO1
ABSTRACT
Lymphocytes expressing interferon-7 (IFN-7) on their surface were evaluated in 61 patients, all IV drug abusers, infected with human immunodeficiency virus type 1 (HTV-1), and in 85 healthy subjects (61 of whom were blood donors and 24 HIV-1 seronegative IV drug abusers). Data obtained demonstrated that IFN-7-expressing T lymphocytes, mostly CD8+ cells, were present in HIV-1-infected patients, and that their percentage, always higher in HIV1-infected patients than in healthy subjects (p =s 0.001), increased with progressive stages of HIV-1 infection. At the same time other markers of T-cell activation, namely interleukin-2 receptor (rIL-2), transferrin receptor, and HLA-DR were also found to be positive in some of the HIV-1-infected subjects. The presence in the HIV-1-infected patients of activated CD8+ T cells, which are resistant to HIV-1 infection, may suggest that these cells are able to respond to continuous and progressive viral expression (HIV or/and other viruses) and may be a component of the specific response to HIV-1. (AIDS) is characterized by severe T-cell deficiency and reflected in an increased frequency of opportunistic infections and of Kaposi's A broad spectrum of abnormalities is common in AIDS patients, ranging from a drastic reduction in the number of CD4 lymphocytes, to a depressed proliferative response to mitogens, antigens, alloantigens, and autoantigens. '~5 Although the activation of the cellular immune system seems to contrast with the clinical features of AIDS, the high serum levels of a,-thymosin, acid-labile interferon-a,6 soluble rIL-2,7 and IFN-7,8 as well as the high urinary levels of neopterin9 in AIDS patients are consistent with such activation. In addition, several authors reported a trend of increased proportion of activated T cells in patients with AIDS and AIDS-related complex (ARC) when compared to healthy controls. 10~13 We recently developed an immunofluorescence assay to evaluate the expression of IFN-7 on the surface of circulating lymphocytes, using flow cytometry and an anti-IFN-7 monoclonal antibody (MAb), named IGMB-14, as a specific reagent.I4 We previously found that the expression of IFN-7 on T-lymphocytes marks activated T cells both in vitro14 and in vivo.15 The present study evaluates the number of IFN-7-expressing lymphocytes in blood samples taken from HIV-1-infected individuals, and compares it with other markers defining T-cell subsets and activated T-lymphocytes. Blood was obtained from 61 HIV-1-infected patients (all IV drug abusers; 45 men, 16 women; age range 17-54 years, with a mean ± SD of 29.3 ± 6.7). Infection was established with an enzyme-linked
Acquiredsarcoma.1-4behavior, clinically
immune deficiency syndrome
abnormal B-cell
'Institute of Microbiology, University of Brescia, Brescia. Italy. 2Division of Infectious Diseases. Spedali Civili, Brescia, Italy. 899
CARUSO ET AL.
immunosorbent assay (ELISA) (Wellcozyme anti-HTLV-III; Wellcome Diagnostics, Dartford, England) and confirmed by Western blot analysis.16 Clinical status was defined by the physician according to the Centers for Disease Control (CDC) classification system.I7 Subjects were grouped according to the stage they presented:
patients were in Stage IV-C and yet had no acute opportunistic infection at the time of investigation; 18 in Stage III; the remaining 19 patients were in Stage II. Blood was also obtained from the following two groups of subjects: (a) 61 healthy blood donors, age- and sex-matched with the 61 HIV-1-infected patients, and (b) 24 HIV-1-seronegative healthy IV drug abusers (18 men, 6 women; age range 19-32 years with a 24
were
mean
±SDof23 ±4.6).
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood by Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Sweden), and the cells stained either by direct or by indirect immunofluorescence method using a panel of monoclonal antibodies. These included Leu 4 (CD3; pan T), Leu 3 (CD4; helper/inducer T cells), Leu2(CD8; suppressor/cytotoxicT cells), Leu 11 (CD 16; natural killer cells), Leu 12 (CD19; B cells), anti-rIL-2 (CD25; activated T cells), anti-transferrin-receptor (CD71; activated T cells) and anti-HLA-DR (Simultest Leu 4/anti- HLA-DR; activated T-cells)—all supplied by Becton-Dickinson (Mountainview, CA)—and MAb IGMB-14 (IFN-7; activated T-cells).14 Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG was supplied by Becton-Dickinson. Immunofluorescence assay was performed by incubating 1 x 106 gradient-separated PBMC for 30 min on ice with appropriate amounts of reagent diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) (w/v) and 0.2% NaN3 (assay buffer). The cells were then washed twice with assay buffer and analyzed on the cytofluorimeter FACStar (Becton-Dickinson). Cells treated with unlabeled MAbs were incubated with FITC-goat anti-mouse IgG for 30 min on ice. They were then washed four times and analyzed on the FACStar. For dual-color immunofluorescence, we employed phycoerythrin (PE)-conjugated Leu 4, Leu 3, Leu 2, Leu 11, and Leu 12 all supplied by Becton-Dickinson. Immunofluorescence analysis was performed on the FACStar flow çytometer. Data were analyzed by Mann-Whitney U test to determine significant differences between group means. Linear correlation coefficients were determined between the number of IFN-7-expressing lymphocytes
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Interferon-7 Marks Activated T Lymphocytes in AIDS Patients
A.
CARUSO,1 R. GONZALES,1 R. STELLINI,2 A. SCALZINI,2 L. PERONI,1 and A.
TURANO1
ABSTRACT
Lymphocytes expressing interferon-7 (IFN-7) on their surface were evaluated in 61 patients, all IV drug abusers, infected with human immunodeficiency virus type 1 (HTV-1), and in 85 healthy subjects (61 of whom were blood donors and 24 HIV-1 seronegative IV drug abusers). Data obtained demonstrated that IFN-7-expressing T lymphocytes, mostly CD8+ cells, were present in HIV-1-infected patients, and that their percentage, always higher in HIV1-infected patients than in healthy subjects (p =s 0.001), increased with progressive stages of HIV-1 infection. At the same time other markers of T-cell activation, namely interleukin-2 receptor (rIL-2), transferrin receptor, and HLA-DR were also found to be positive in some of the HIV-1-infected subjects. The presence in the HIV-1-infected patients of activated CD8+ T cells, which are resistant to HIV-1 infection, may suggest that these cells are able to respond to continuous and progressive viral expression (HIV or/and other viruses) and may be a component of the specific response to HIV-1. (AIDS) is characterized by severe T-cell deficiency and reflected in an increased frequency of opportunistic infections and of Kaposi's A broad spectrum of abnormalities is common in AIDS patients, ranging from a drastic reduction in the number of CD4 lymphocytes, to a depressed proliferative response to mitogens, antigens, alloantigens, and autoantigens. '~5 Although the activation of the cellular immune system seems to contrast with the clinical features of AIDS, the high serum levels of a,-thymosin, acid-labile interferon-a,6 soluble rIL-2,7 and IFN-7,8 as well as the high urinary levels of neopterin9 in AIDS patients are consistent with such activation. In addition, several authors reported a trend of increased proportion of activated T cells in patients with AIDS and AIDS-related complex (ARC) when compared to healthy controls. 10~13 We recently developed an immunofluorescence assay to evaluate the expression of IFN-7 on the surface of circulating lymphocytes, using flow cytometry and an anti-IFN-7 monoclonal antibody (MAb), named IGMB-14, as a specific reagent.I4 We previously found that the expression of IFN-7 on T-lymphocytes marks activated T cells both in vitro14 and in vivo.15 The present study evaluates the number of IFN-7-expressing lymphocytes in blood samples taken from HIV-1-infected individuals, and compares it with other markers defining T-cell subsets and activated T-lymphocytes. Blood was obtained from 61 HIV-1-infected patients (all IV drug abusers; 45 men, 16 women; age range 17-54 years, with a mean ± SD of 29.3 ± 6.7). Infection was established with an enzyme-linked
Acquiredsarcoma.1-4behavior, clinically
immune deficiency syndrome
abnormal B-cell
'Institute of Microbiology, University of Brescia, Brescia. Italy. 2Division of Infectious Diseases. Spedali Civili, Brescia, Italy. 899
CARUSO ET AL.
immunosorbent assay (ELISA) (Wellcozyme anti-HTLV-III; Wellcome Diagnostics, Dartford, England) and confirmed by Western blot analysis.16 Clinical status was defined by the physician according to the Centers for Disease Control (CDC) classification system.I7 Subjects were grouped according to the stage they presented:
patients were in Stage IV-C and yet had no acute opportunistic infection at the time of investigation; 18 in Stage III; the remaining 19 patients were in Stage II. Blood was also obtained from the following two groups of subjects: (a) 61 healthy blood donors, age- and sex-matched with the 61 HIV-1-infected patients, and (b) 24 HIV-1-seronegative healthy IV drug abusers (18 men, 6 women; age range 19-32 years with a 24
were
mean
±SDof23 ±4.6).
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood by Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Sweden), and the cells stained either by direct or by indirect immunofluorescence method using a panel of monoclonal antibodies. These included Leu 4 (CD3; pan T), Leu 3 (CD4; helper/inducer T cells), Leu2(CD8; suppressor/cytotoxicT cells), Leu 11 (CD 16; natural killer cells), Leu 12 (CD19; B cells), anti-rIL-2 (CD25; activated T cells), anti-transferrin-receptor (CD71; activated T cells) and anti-HLA-DR (Simultest Leu 4/anti- HLA-DR; activated T-cells)—all supplied by Becton-Dickinson (Mountainview, CA)—and MAb IGMB-14 (IFN-7; activated T-cells).14 Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG was supplied by Becton-Dickinson. Immunofluorescence assay was performed by incubating 1 x 106 gradient-separated PBMC for 30 min on ice with appropriate amounts of reagent diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) (w/v) and 0.2% NaN3 (assay buffer). The cells were then washed twice with assay buffer and analyzed on the cytofluorimeter FACStar (Becton-Dickinson). Cells treated with unlabeled MAbs were incubated with FITC-goat anti-mouse IgG for 30 min on ice. They were then washed four times and analyzed on the FACStar. For dual-color immunofluorescence, we employed phycoerythrin (PE)-conjugated Leu 4, Leu 3, Leu 2, Leu 11, and Leu 12 all supplied by Becton-Dickinson. Immunofluorescence analysis was performed on the FACStar flow çytometer. Data were analyzed by Mann-Whitney U test to determine significant differences between group means. Linear correlation coefficients were determined between the number of IFN-7-expressing lymphocytes
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