A genetic defect in "acquired" agammaglobulinemia.

Phytohemagglutinin (PHA) has been shown to effect biochemical and morphologic changes in the small lymphocytes of normal human peripheral blood.1' 2 The percentage of morphologic transformation of small lymphocytes to blastlike cells on exposure to PHA is reduced in a variety of conditions associated with abnormalities of the lymphoid system, such as Hodgkin's disease, sarcoidosis, and chronic lymphocytic leukemia.3' 4 Recent studies from our laboratory have demonstrated that the in vitro, PHA-induced incorporation of labeled precursors into DNA and RNA by lymphocytes is significantly diminished in cells of adult "acquired" agammaglobulinemic individuals when compared to the incorporation by normal control cells. This difference between agammaglobulinemics and normals is independent of the culture-medium serum supplement (bovine, normal human, or autologous), implicating a cellular abnormality in the againmaglobulinemic patient.5 In an effort to investigate further the possible genetic aspects of this disease, the in vitro response of peripheral blood lymphocytes obtained from the parents of such patients was studied in our assay systems.' An autosomal recessive mode of inheritance can be inferred. Methods.-The study group included 4 adult patients (ages 30-45) with "acquired" agammaglobulinemia whose levels of serum immunoglobulins G, A, and M were less than 10% of normal; a 21-year-old male with repeated infection but normal serum immunoglobulin levels (a second cousin of a known adult "acquired" agammaglobulinemic, see Discussion); 7 parents (ages 55-75) of the patients, whose serum immunoglobulin levels were normal; and 23 normal control individuals (ages 25-65), of both sexes, with normal serum immunoglobulin levels. None of the individuals studied were receiving medications. Peripheral venous blood was collected in heparin (heparin sodium, Upjohn Com-' pany, Kalamazoo, Michigan), and the erythrocytes were separated by gravity sedimentation, in 16 X 125-mm screw-cap tubes, for 2 hr at room temperature. When necessary, blood specimens from agammaglobulinemic patients were gently centrifuged to facilitate sedimentation. The leucocyte-rich plasma supernatant was aspirated and the lymphocyte concentration determined. The leucocyte suspension was then centrifuged, and the pellet was washed in Eagle's minimal essential medium (MEM) Spinner salts solution and resuspended in culture medium at 350,000 lymphocytes/ml. Three-ml aliquots of each cell suspension were dispensed into 16 X 150-mm rubber-stoppered tubes, so that each culture tube contained approximately 1 X 106 lymphocytes. The cells were then cultivated as described previously.5 The culture medium consisted of MEM Spinner medium supplemented with L-glutamine, MEM nonessential amino acids, penicillin (50 units/ml), streptomycin (50 mcg/ ml), and 10% bovine serum. Culture medium ingredients were obtained from Grand Island Biological Company, Grand Island, New York. A single lot of bovine serum was used in all studies. PHA-M (control 485336; Difco Laboratories, Detroit, Michigan) was used as the mitogen. Each vial, dissolved in 5 ml Spinner salts, was diluted 1:10, and 0.1 ml of this dilution was added to each of the PHA-stimulated cultures at the appropriate time. Control cultures received an equivalent volume of diluent. Separate assays were performed to test the incorporation of C14-thymidine (Tdr-2-C14)