Significant Enlargement of a Specific Subset of CD3+CD8+ Peripheral Blood Leukocytes Mediating Cytotoxic T-Lymphocyte Activity D
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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9427-9430, October 1993 Immunology Significant enlargement of a specific subset of CD3+CD8+ peripheral blood leukocytes mediating cytotoxic T-lymphocyte activity during human immunodeficiency virus infection (T lymphocyte subset/monoclonal antibody/AIDS) A. BENSUSSAN*t, C. RABIANt, V. SCHIAVON*, D. BENGOUFAt, G. LECA*, AND L. BOUMSELL* Institut Nationale de la Sant6 et de la Recherche Medicale U93, Association Claude Bernard, and tLaboratoire d'Immunologie et d'Histocompatibilite, H6pital Saint-Louis, 1 Avenue Claude Veliefaux, 75475 Paris cedex 10, France Communicated by Jean Dausset, July 20, 1993 ABSTRACT We have obtained a monoclonal antibody studies have shown that human immunodeficiency virus termed BY55 that defines an 80-kDa cell-surface structure on (HIV) infection significantly changes the phenotype of cir- a subset ofcirculating peripheral blood mononucleocytes. This culating lymphocytes and increases CD8 T lymphocytes (19), structure, which was not detected on most cell lines or activated it was interesting to look for this cytotoxic CD3+CD8+BY55+ lymphocytes, was expressed exclusively on 15-25% of CD2+ circulating cell subset in asymptomatic HIV-positive individ- circulating lymphocytes, including a major subset within the uals with various numbers of CD4 cells. Results indicated CD3- and the T-cell receptor y6+ lymphocytes and a small that peripheral blood CD3+CD8+BY55+ cells significantly percentage of the CD3+CD8+ cells. Moreover, we have shown increased in HIV-positive individuals. that the BY55 molecule delineated the competent killer circu- lating lymphocytes. In the present report, additional two- and MATERIALS AND METHODS three-color immunofluorescence studies of peripheral blood lymphocytes were done to precisely determine BY55 expression mAbs. BY55 mAb was obtained by immunization of within the T-cell population. In normal individuals, peripheral BALB/c mice with the YT2C2 cell line (18). Briefly, spleen blood CD3+CD8+BY55+ cells represented only 5-6% of the cells from immunized mice were fused to the NS1 cell line 5 lymphocytes, and these cells possessed cytolytic activity. Inter- days after the last injection. The initial screening by indirect estingly., we found that the percentage of total BY55+ lympho- immunofluorescence and flow cytometry with a FACStar cytes as well as the percentage of CD3+CD8+BY55+ was microfluorometer (Becton Dickinson) retained all the hybri- significantly increased in peripheral blood lymphocytes of doma-culture supematants reactive with the immunizing human immunodeficiency virus-seropositive individuals. cells but weakly reactive with resting peripheral blood mono- nuclear cells. Cultures containing selected antibodies were cloned twice by limiting dilution. The cloned hybridoma was Use of monoclonal antibodies (mAbs) has allowed the iden- serially passaged by i.p. injection into BALB/c mice primed tification of various cell-membrane glycoproteins that define with pristane. Ascites fluid was collected, ultracentrifuged, phenotypic and functional human lymphocyte subsets (1-3). and affinity-purified, when required. BY55 mAb is an IgM. In particular, it was well established that CD8 and CD4 Other mAbs such as CD3, CD4, CD8, and CD38 were subsets were associated with the major histocompatibility produced locally. These mAbs were used as ascites fluid and, complex (MHC) class I/II restriction ofT-cell recognition (4, when needed, coupled to fluorescein isothiocyanate (FITC) 5). Several cell-surface structures were described as being or biotin. Some antibodies were purchased from Immunotech restricted to a subset of circulating T cells. These included (Marseille, France). dipeptidylpeptidase IV CD26 (6), CD28 (7), CD31 (8), and Isolation of Cell Populations. Human peripheral blood lym- CD38 (9). Except for CD28, the expression of all these phocytes (PBL) were prepared by Ficoll/Hypaque density- molecules increases during cellular activation. Other struc- gradient centrifugation. Cells were washed twice in RPMI tures such as CD25 (10), CD39 (11), CD69 (12), and CD71 (13) 1640 medium (GIBCO) supplemented with 2 mM L-gluta- are characteristic of activated T lymphocytes. In an attempt mine, penicillin/streptomycin (100 units/ml and 100 Mg/ml, to define specific cell subset surface glycoproteins, we char- respectively), and 10% heat-inactivated human serum and acterized various mAbs obtained by repeated immunizations used for immunofluorescence assays. HIV-negative and with functionally defined cell lines (14-16). In particular, we HIV-positive blood samples were obtained from the Labo- had recently reported a mAb, termed BY55 (17), that recog- ratory of Immunology (H6pital Saint-Louis). The HIV- nizes a distinctive 80-kDa structure on the immunizing leu- seropositive population studied was a heterogeneous cohort. kemic natural killer cell line YT2C2 (18). Flow cytometry For isolation of CD8+BY55+ and CD8+BY55- cell popula- analysis revealed that BY55 mAb exclusively stained 15-25% tions, fresh PBL were first depleted in CD4+ cells by passage of the circulating CD2+CD4- lymphocytes of a normal indi- over a column and next were simultaneously labeled with vidual. These cells include a CD2+CD3- competent killer- phycoerythrin (PE)-conjugated CD8 mAb and FITC- cell subset and a minor CD3+ cell subset coexpressing conjugated BY55 mAb. After a final wash, cells were sorted CD8bight molecules. In the present report, we show further into CD8brht+BY55+ (representing 5-7% of the CD4- PBL) antigenic phenotype from studies on 10 normal individuals. and CD8bright+BY55- (representing >60% ofthe CD4- PBL) BY55 mAb stained only 5-6% of circulating CD3+CD8brigh+ fractions using a FACStar microfluorometer (Becton Dick- cells. In addition, we reveal that these cells possess cytolytic Sorted were collected in RPMI 1640 activity, as they mediate CD3-redirected lysis against the Fc inson). populations receptor-bearing tumor cell line K-562. As a number of Abbreviations: mAb, monoclonal antibody; HIV, human immuno- deficiency virus; PBL, peripheral blood lymphocyte(s); CTL, cyto- The publication costs of this article were defrayed in part by page charge toxic T lymphocyte(s); MHC, major histocompatibility complex; PE, payment. This article must therefore be hereby marked "advertisement" whycoerythrin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. TTo whom reprint requests should be addressed. 9427 Downloaded by guest on October 1, 2021 9428 Immunology: Bensussan et al. Proc. Natl. Acad. Sci. USA 90 (1993) medium/10% heat-inactivated human serum, washed twice, ceptor y8. Although no significant amounts of CD4+ circu- and used as effector cells in cytotoxicity assays. Trypan blue lating cells coexpressed BY55 molecule, a major subset of exclusion was performed on the unsorted population and on T-cell receptor y3+ cells were stained by BY55 mAb (17). sorted populations, and the viability was always >90%. Interestingly, we found that 6% of peripheral blood mono- Immunofluorescence Assays. Phenotypic analyses were nuclear cells coexpressed BY55, CD3, and CD8 molecules, done on whole peripheral blood or Ficoll/Hypaque PBL indicating that only a minor subset of the CD3+CD8+ cells gated on lymphocytes. For two-color immunofluorescence coexpressed BY55 molecule. experiments, cells were simultaneously incubated during 30 Cell-Mediated Cytotoxic Activity of BY55+ Circulating min with a PE-conjugated mAb and FITC-labeled BY55 Cells. We had previously shown that BY55+ circulating cells mAb. Red blood cells were lysed by adding 2 ml of cell contained all the competent MHC-unrestricted killer lym- cytometry lysis solution (Becton Dickinson). The overlap in phocytes (17). However, as the BY55+ lymphocytes consist FITC and PE fluorescence emissions was corrected by using of CD3+CD8+BY55+ cells, corresponding to T lymphocytes an electronic compensation network. For three-color immu- expressing T-cell receptor af3, natural killer CD3-CD8-- nofluorescence assays, 2 x 106 cells or 100 ,l of blood was BY55+, and CD3- or CD3+CD8dim+BY55+ cells, we could simultaneously stained with CD3 Tri-Color (Caltag Labora- not distinguish their respective contribution to the cell- tories, South San Francisco, CA), CD8PE, and FITC-BY55 mediated cytotoxic function. In particular, we could not mAb. Analysis was performed on a FACScan microfluorom- determine whether the MHC-restricted CD3+CD8bright+- eter after compensating spectrum overlaps between FITC BY55+ circulating cell subset exhibited killer activity. There- and PE and between PE and Tri-Color (excitation wavelength fore, to study MHC-restricted killer activity of freshly iso- of Tri-Color was 488 nm; emission wavelength was >650 lated CD3+CD8bright+BY55+ and CD3+CD8bright+BY55- cell nm). populations, CD3-redirected cytotoxic assays were done CD3-Induced Redirected Cytotoxicity Assay. The K-562 with these sorted cells. Moreover, to exclude the natural cells were labeled with 100 ,uCi of 51Cr (1 Ci = 37 GBq) for killer activity ofthe CD3-CD8dim+BY55+ cells against K-562 1 hr, washed, and used as target cells, Anti-CD3 mAb (Leu4, target cells, we separated BY55+ and BY55- cells within IgGl) (final concentration, 10 jig/ml) was added to 51Cr- CD8bnght+ gated cells. A representative cell-mediated cyto- labeled targets 15 min before addition of effector cells. toxicity assay done with unsorted and sorted effector cells is Assays at various effector to target cell (E:T) ratios with 5 x presented in Fig. 1. As expected, a weak