Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (approximately 2.5 x 10(5) cells per cm2), and the incubation temperature was maintained at 35-37 degrees C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.

Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements.

Isolated normal human peripheral lymphocytes were analyzed for their ability to bind IgE as shown by rosette formation with aldehydefixed ox red cells coated with an IgE myeloma protein (Eo'-IgE) as indicator cells. An average of 4.3% of the cells in the lymphocyte preparations of 12 donors formed Eo'-IgE rosettes. The lymphocyte preparations contained an average of 0.36% basophils which also formed Eo'-IgE' rosettes, suggestiing that about 4% of the lymphocytes bound IgE. The rosette formation was inhibited by IgE myeloma protiens or IgE Fc fragments but not by IgE Fab fragments or Ig of the other classes. On the average, the lymphocytes of the 12 donors contained 70.5% cells forming spontaneous rosettes with sheep erythrocytes (E), 10.6% cells having surface immunoglobulin (SIg), and 15.5% bunding IgG as shown by rosette formation with IgG-coated ox red cells (EoA). Fractionation of the lymphocytes into populations rich in spontaneously E-rosetting cells and cells with SIg indicated that the majority of the lymphocytes forming Eo'-IgE rosettes belonged to the SIg-positive lymphocytes. Analyses of lymphocyte populations lacking cells forming EoA rosettes and of rosetting with mixed indicator cells both demonstrated that over 90% of the lymphocytes forming Eo'-IgE rosettes did not form EoA rosettes and apparently had no Fc receptors for IgG. Pretreatment of the lymphocytes with trypsin in amounts that did not alter the number of EoA-rosetting cells abolished the Eo'-IgE rosette formation. These data indicate that a subpopulation of normal human peripheral lymphocytes, probably belonging to the B-cell type, binds IgE presumably via trypsin-sensitive receptors specific for the Fc fragment of IgE. The surface markers of these lymphocytes resemble those of cultured human lymphoblastoid cells that have recently been shown to bind IgE.

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A subpopulation of normal human peripheral B lymphcytes that bind IgE.

Multimarker classification of acute lymphoblastic leukemia: evidence for further T subgroups and evaluation of their clinical significance.

Human peripheral blood lymphocytes were separated into subpopulations enriched or depleted with respect to B lymphocytes (Ig-bearing cells), T lymphocytes, (cells forming rosettes with sheep erythrocytes: E-RFC) and Fc receptor-bearing lymphocytes (EA-RFC).



From the distributions and recoveries of the various cell types it could be concluded that there was very little overlap between Ig-bearing lymphocytes and EA-RFC. The latter cells partly belonged to “null” (non-T, non-B) cells; it was however demonstrated that 30 % of the EA-RFC were T cells (E-RFC).



The lytic capacity in antibody-dependent lymphocytotoxicity (ADL) was shown to correspond with the proportions of EA-RFC in the various fractions. Non-T cells showed enhanced ADL activity when compared to the unseparated cells. Purified T cell populations also displayed ADL activity. Since the latter could not be due to contaminating non-T cells, this activity was ascribed to Fc receptor-bearing T lymphocytes.

Separation and properties of EA‐rosette‐forming lymphocytes in humans

To characterize the effector cells involved in ADCC in a model system (lysis of 51 Cr-labeled chicken erythrocytes induced by rabbit IgG antibodies), in “spontaneous”, and in tumor-associated cytotoxicity, we prepared subpopulations of peripheral blood lymphocytes from healthy individuals and from cancer patients by fractionation on columns according to three different principles. Columns with anti-immunoglobulin activity but lacking Fc-structures (F(ab′) 2 /anti-F(ab′) 2 columns) removed ∼99% of the SIg + cells (B cells), but only ∼20% of the Fc-receptor-bearing cells, as characterized by rosette formation with IgG-coated bovine erythrocytes (EA + cells), and ∼25% of the complement receptor-bearing cells (EAC + cells). Cells with receptor for sheep erythrocytes (E + cells) were enriched. The K cell activity of this fraction was unchanged. Columns charged with immune complexes (OA/anti-OA columns) which display no anti-immunoglobulin activity retained ∼70% of the EA + cells but only ∼20% of the SIg + cells. About 50% of the EAC + cells and ∼10% of the E + cells were also removed. The K cell activity of this fraction was strongly reduced. IgG/anti-IgG columns very efficiently remove SIg + cells, EA + cells, and EAC + cells whereas E + cells are enriched. Again K cell activity is strongly reduced. The results indicate that B cells with high density SIg are inactive in the K cell assay and that the majority of mature B cells either have Fc-receptors of low avidity or have receptors for immunoglobulin which are qualitatively different from the Fc-receptors of SIg - cells. Furthermore, a significant fraction of the lymphocytes with complement receptors is different from these B cells. It can also be inferred that a significant subpopulation of the cells with receptors for sheep erythrocytes also have Fc-receptors for IgG. The cytotoxicity of these different lymphocyte fractions against a variety of human tumor cells will be reported in the following paper.

Characterization of Cytolytic Effector Cells in Peripheral Blood of Healthy Individuals and Cancer Patients: I. Surface Markers and K Cell Activity After Separation of B Cells and Lymphocytes with Fc-Receptors by Column Fractionation

Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regulary in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered.

The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.

J. W. Chiao

Papers

Studies of the presence of membrane receptors for complement, IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes