Antiserum to a glycoprotein antigen complex of 23,000 and 30,000 dalton subunits (p23,30), isolated and purified from a human lymphoblastoid B cell line, was shown to be highly specific for human bursal-equivalent-processed (B)

It has been shown in the mouse that antigen triggers distinct subpopulations of thymusderived lymphocytes to express specific immunoregulatory activities, and that these activities are mediated in part by intercellular signals involving products of the I-region (Ia antigens) of the major histocompatibility complex. Thus, discrete Ia antigens have been detected on soluble T-cell factors which have either helper (1) or suppressor (2) activities, and are also present on the surface membrane of suppressor T cells (3), and Con A-induced T-cell blasts (4).Although Ia antigens have been generally thought not to be expressed by normal or leukemic T cells in man (5, 6), this view was re-examined when we found that antibody to a human Ialike antigen, p23,30 occasionally reacted with leukemic blasts which also expressed thymusdependent markers, p23,30 is a glycoprotein complex of 23,000 and 30,000 dalton subunits, isolated from the papain-solubilized membrane of a human lymphoblastoid B-cell line (7). A rabbit anti-p23,30 serum binds to peripheral B cells, monocytes, and a subpopulation of null cells, but is unreactive with normal human T cells or thymocytes (6,8). In addition to conforming in molecular weight and normal tissue distribution with murine Ia antigen, the detergent-solubilized form of p23,30, p29,34, reacts with B-cell alloantisera which are specific for determinants coded by the HLA-D locus, or I-region counterpart in man (9).We report here that determinants recognized by anti-p23,30 are expressed on the surface membrane of T cells which are transformed by alloantigen in mixed leukocyte culture (MLC), but are not detectable on T cells which are either freshly purified or maintained without stimulation in culture for 6 days. Moreover, allosensitized T cells were shown to elaborate and to incorporate into their surface membranes a 29,000 and 34,000 dalton, HLA-D-related complex, Materials and MethodsIsolation of Human TLymphocytes. T cells were isolated from peripheral blood mononuclear cells by nylon wool purification followed by formation and density gradient sedimentation of sheep erythrocyte-rosetting cells (10).Sensitization Cultures. T cells were sensitized to aUogeneic mitomycin-C-treated mononuclear cells (10). Of the cells recovered after a 6-day sensitization, greater than 98% were reactive with a heterologous antiserum which was specific for thymus-derived lymphocytes.

Background: We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers. Methods: We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1_141–155 peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1_141–155 epitope chemically conjugated to the Ii-Key peptide. Results: Identification of Bet v 1_141–155 as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1_141–155-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1_141–155 Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1_141–155-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1_141–155 tetramer-positive T cells produce IFN-γ and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10^–5 to 10^–3 CD4+ T cells, outside or within the pollen season, respectively. Conclusions: MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses.

Lymphocytes bear many types of surface molecules, some of which are restricted to subpopulations of lymphocytes and may be necessary for their specialized immune functions. Examples of such antigens include: the Ia antigens; cell surface immunoglobulin; the Fc receptor, complement (C) receptors, and Ly-4 all present predominantly on B lymphocytes and Thy (theta) antigen; and sheep erythrocyte receptor, and the Ly-1, 2, and 3 antigens all present on T cells. H antigens (H-2 or HL-A) and ~-microglobulin are found on all populations on lymphocytes. Because a non-H-2, B-lymphocyte-specific antigen had been serologically defined on the murine lymphoblast line L1210 (reference i and footnote 1) and because a similar human, non-HL-A antigen could be present on the human B-lymphoblast line IM-1, from which the isolation of the HL-A antigen was in progress, attention has been paid to the "contaminant" molecules of the HL-A antigen preparations/ Several cell surface antigens have been identified, one of which (p23,30) is similar to Ia antigens which have been described on mouse B lymphocytes. The purification of that antigen, its distribution on distinct subsets of human lymphocytes, and the properties of heteroantisera to p23,30 in lymphocyte functional assays are described in this and the accompanying paper (3).

No abstract