Antonio Coutinho

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.

Genetic basis for unresponsiveness to lipopolysaccharide in C57BL/10Cr mice.

The unresponsiveness to LPS detected in C57BL/10Cr mice is inherited as a recessive trait and is determined by an autosomal gene linked to theMup-1 locus on chromosome 4. Since no complementation for LPS responsiveness was observed in F1 hybrid mice between C3H/HeJ and C57BL/10Cr, we conclude that C57BL/10Cr mice carry a defective allele at the sameLps locus, previously identified by the mutation detected in the C3H/HeJ strain.

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100

Addition of 3 × 106 thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.

Continuous Growth of Mitogen-Reactive B Lymphocytes

B‐cell mitogens induce continous growth of murine lymphocytes in suspension cultures. Continuous growth is observed only at low cell densitiess, below 106 cells/ml, in regularly renewed medium containing β‐mercaptoethanol and a growth‐supporting fetal calf serum. Continuous growth of B lymphocytes in culture appears to be favored by conditions under which maturation of Ig‐secreting plaque‐forming cells does not occur. On the other hand, maturation is observed whenever stimulated B cells cease to divide. This maturation leads to IgM secretion followed by secretion of IgG or IgD‐like molecules. Neither continuous growth of B cells nor the ‘switch’ to secretion of IgG or IgD‐like molecules requires the presence of T cells.

Independent Segregation of Two Functional Markers Expressed on the Same B‐Cell Subset in the Mouse: The Mls Determinants and LPS Receptors

Mice of the C3H/Tif strain display a mixed leukocyte reaction (MLR) with all H‐2k strains tarrying any of the known alleles of the Mls locus. In particular, C3H/Tif is incompatible with the related substrain C3H/HeJ, from which it also differs at the locus responsible for the recognition of lipopolysaccharides (LPS) as B‐cell mitogens, and at the Mod‐1 locus. Our genetic analysis indicates that the MLR incompatibility between these strains is not H‐2‐linked and segregates as controlled by a single locus, most probably identical to Mls, for which the C3H/Tif strain expresses a previously unidentified allele, Mlse. Moreover, segregation data show that this locus assorts independently of LPS responsiveness and that neither marker is closely linked to the Mod‐1 locus in linkage group II.

The Vβ8 gene family is preferentially used by naturally activated T cells

Receptors encoded by the Vβ8 gene family detected by the monoclonal antibody F23.1 are expressed among ‘naturally’ activated T cells in normal spleen at frequencies significantly higher than in the total CD4+ and CD8+ cell populations. The positive selection of these clones into ‘natural’ T‐cell activity could be the reason for the high frequencies of cells expressing Vβ8 genes. This phenotype is strain‐dependent.