A collaborative study was organized to identify monoclonal antibodies (MAbs) that may be broadly and potently neutralizing for a panel of human immunodeficiency virus type 1 (HIV-1) low-passaged adult and pediatric primary isolates in peripheral blood mononuclear cells. Five laboratories evaluated a coded panel of seven human MAbs to HIV-1 subtype B envelope V3, CD4 binding region, gp41, and other conformationally sensitive determinants. Each laboratory measured neutralizing activity of the MAbs against the laboratory isolate HIV(MN) and a panel of 9 subtype B primary isolates. Antibodies were classified as suitable candidates for future clinical studies if they could neutralize at least half of the 9 primary isolates at a concentration of < or = 25 microg/mL for 90% viral inhibition. The study identified three MAbs that met stated performance criteria: IgG1b12, 2G12, and 2F5. These results may provide a rationale for examining the clinical efficacy, either singly or in combination, of the three MAbs.
Since the publication of the "Three-color supplement to the NIAID/DAIDS Guideline for Flow Cytometric Immunophenotyping" in 1996 (1), significant scientific and technological advances in the development and production of reagents, instrumentation, and software have increased the use of multicolor flow cytometry in both research and clinical laboratories. With the increased adoption of three and four-color flow cytometry as the preferred methodology in determining patients' CD4 and CD8 T-cell counts, it has become apparent that a gating strategy that integrates the bright CD45 cells reduces interlaboratory and intralaboratory variability. Traditionally, a lymphocyte gate for immunophenotyping is derived from a bivariate frequency distribution histogram that includes 90°side scatter (SSC) and forward light scatter (FSC) frequency patterns. This type of histogram configuration is called a homogenous gating protocol (2). The advantage of the combination of bright CD45 fluorescence and light scatter, a heterogeneous gating protocol, was first reported in 1993 (3). Over the past few years, it has been determined that this alternative approach provides a more reproducible and accurate lymphocyte gate (4 -6). This heterogeneous method will be referred to as the CD45 gating method.The purpose of this article is to update the 1996 NIAID/ DAIDS Guideline and include the use of the CD45 gating method to minimize measurement variability with multicolor flow cytometry for the enumeration of T-cell subsets. Some information is provided about the advantages of four-color flow cytometry and the use of single-platform bead-based technology for determining absolute and percentage of lymphocytes. The addition of integrated fluorosphere counting provides a single-platform protocol that facilitates the simultaneous determination of both absolute and percentage of lymphocyte subsets. The specifications and recommendations were developed for use in laboratories supporting clinical trials and epidemiolog-
Health and Welfare Canada, Ottawa, Ontario (F.M.); and Division of AIDS, NIAID, NIH, Bethesda, Maryland (D.L., J.K.)'Section nurnberingsystern isdesigned to maintain continuity with the 1993 "NIAID DAIDS Guidelinesfor Flow Cytornetric Immunotyping" (1).
It has become increasingly apparent that B lymphomas provide clonal models for antigen-specific lymphocytes and for the analysis of both positive and negative signalling in B cells. In addition, anti-Ig reagents have been used to mimic antigen/tolerogen in their interaction with B cell receptors at a polyclonal level (1-3). We have been studying the effects of anti-Ig on a group of unique B cell lymphomas as models for either tolerogenic or immunogenic signalling (4-6). One such line, WEHI-231, has been shown (7, 8) to be readily inhibited in its growth by anti-Ig reagents. We confirmed the sensitivity of this line to growth inhibition by anti-u and anti-K reagents, and determined its kinetics, specificity, and site of the block in the cell cycle at the G1/S interface (4). In this report, we have enriched WEHI-231 lymphoma cells at various points in G1 to S and analyzed the effects of anti-# on progression through S phase. Our data suggest that critical events occur early in GI and are uniquely sensitive to modulation by anti-~. These studies now will allow sensitive molecular approaches towards an understanding of B cell signalling, as well as methods to regulate lymphoma growth per se. Materials and MethodsCell Lines and Their Maintenance. WEHI-231 B lymphoma cells arose in a (BALB/c x NZB) F~ mouse after mineral oil induction (9). Clone 28, obtained from Dr. Noel Warner (Becton Dickinson Research Laboratories, Mountain View, CA), has been maintained in our laboratory for several years in DMEM supplemented with 5-10% FCS, 100 U/ml penicillin, 100 #g/ml streptomycin, 2 mM L-glutamine, and 5 x 10 -5 M 2-ME (4, 5). It is grown as a single cell suspension with occasional aggregates, and is split twice weekly. As a standard test fbr anti-# sensitivity, 104 WEHI 231 cells were cultured in a total volume of 0.2 ml with various concentrations of goat anti-/~ or other antisera for 48 h, at which time they were pulsed with 1 ~Ci of tritiated thymidine. 18 h later they were harvested on glass fiber filters and assayed by liquid scintillation spectrometry.
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