Irwin Scher

The cba/n mouse strain: an experimental model illustrating the influence of the x-chromosome on immunity.

Publisher Summary Studies of the mouse strain have identified the gene, or closely linked group of genes, responsible for the immune defect, and it has been named “ xid .” This chapter summarizes the known functional immune defects associated with xid and discusses the cellular basis for these defects. The X-chromosomes of mice and men influence the immunological function of these species in a number of interesting ways. In studies of the influence of the murine X-chromosome on serum IgM levels, it was shown that the mean values of serum IgM were higher in female mice than in males of two different mouse strains. However, in contrast to humans with Turners syndrome (XO), who had lower mean serum IgM values than did normal XX females, the mean serum concentrations of IgM in XO mice were similar to that found in normal female mice. An X-linked form of severe combined immunodeficiency disease is described in the chapter. Children with this disease have variable degrees of lymphopenia, with small foci of lymphocytes in their lymph nodes and spleens. It is unclear if these patients have a primary B- or T-lymphocyte or stem cell defect. Spleens from either CBA/N or defective F 1 male mice are considerably smaller than those of normal mice and the number of nucleated cells/spleen reflects this difference. The absence of thymus-independent type 2 (TI-2) responses in immune-defective mice and the apparent B-cell abnormality in this strain is related to an isolated dysfunction in otherwise normal B cells of these mice. It has been detected in studies that the xid defect could impair thymic-dependent (TD) responses by the induction of hapten-specific suppression without altering the development of memory B cells.

Activation of mouse lymphocytes by anti-immunoglobulin: IV. Stimulation with soluble heterologous anti-δ antibodies

Mouse spleen cells were stimulated to proliferate in vitro by soluble affinity-purified heterologous antibodies to mouse delta. Antibodies from goat or rabbit antisera to TEPC 1017, a mouse IgD myeloma protein, were purified on an affinity column of TEPC 1033, a second mouse IgD myeloma protein. Maximum uptake of [3H]thymidine in the range of 60,000 cpm was obtained after 48 hr of culture with anti-delta at concentrations of 50 micrograms/ml. In contrast, the hybridoma 10-4.22 anti-delta was nonmitogenic at similar concentrations. The proliferative response was not impaired upon removal of T cells by treatment with an anti-thymocyte serum (ATS), nor by removal of adherent cells by passage of spleen cells over Sephadex G-10 columns and counter-flow centrifugation. Splenic lymphocytes isolated on the fluorescence activated cell sorter (FACS) with intermediate-to-high amounts of surface IgD (sIgD) were responsive to soluble anti-delta, while IgD-negative cells, or cells with low amounts of sIgD, were unresponsive. Spleen cells from mice less than 4 weeks of age, or from mice carrying the X-linked B cell defect (xid), were unresponsive to anti-delta. These results indicate that anti-delta acts similarly to anti-mu in stimulating a proliferative response by later maturing B cells, which are characterized by a high density of sIgD.

A method for size separation of murine spleen cells using counterflow centrifugation.

A method for the rapid separation of murine spleen cells into subpopulations on the basis of their size has been developed using counterflow centrifugation. Upon separation of normal spleen cells with a mean cell volume of 125.5 +/- 6.0, 5 fractions of cells were obtained with mean cell volumes which ranged from 107.8 +/- 3.2 microns3 in fraction 1 to 152.7 +/- 4.9 microns3 in fraction 5. The cells in these 5 fractions were characterized by analysis on a fluorescence activated cell sorter (FACS) after staining with fluorescein conjugated anti-mu, delta, Ia, or Thy 1.2 antibodies, and by assaying for the presence of non-specific esterase activity. Surface Ig+, Ia+ B lymphocytes and Thy 1.2 T lymphocytes were present in all 5 fractions. However, while these T and B lymphocytes accounted for virtually all of the cells in the first 3 fractions, non-T, non-B cells were found in fractions 4 and 5, and represented 30% of the total population in the 5th fraction. Comparison of the intensity of anti-mu, delta or Ia staining of the B cells in fractions 1-5 revealed differences which suggested that B cell size correlated with different activation states of these cells. Increases in the intensity of the staining of T lymphocytes by anti-theta antibodies were also noted in the various fractions. The capacity of the B and T cells in each fraction to proliferate to B or T cell mitogens, respectively, was proportional to their frequency within the fractions. By contrast, the fractions containing larger cells were enriched in cells which proliferated in vitro in the absence of added mitogen. Furthermore, only fractions containing larger cells had the capacity to stimulate allogeneic T cell proliferation in a mixed lymphocyte reaction. Our data suggest that this technique provides a useful method for separating splenocytes on the basis of cell size. Use of this methodology provides a way to correlate cell size with phenotypic surface markers and functional abilities.

B-Lymphocyte Development and Heterogeneity

CBA/N (CN) mice (originally referred to as CBA/HN) are a distinct subline, derived originally from the CBA/Harwell line at the National Institutes of Health (NIH), Bethesda, Maryland. Mice of the Harwell line, which were heterozygous for the lethal foam cell reticulosis gene (fm), were obtained at the NIH in 1966. These mice were bred and normal homozygous progeny were used to reestablish the inbred strain by brothersister matings. However, because of a breeding crisis at the seventh generation, subsequent mice were derived from the litter of a single pregnant female. At the time of the first publication concerning the immune-defective CN mice, 15–25 generations of brother-sister matings had occurred (Amsbaugh et ai, 1972). CN mice reproduce as well as other inbred mouse lines and exist as a vigorous line that has no difficulty in dealing with the routine laboratory environment.

Genetic Control of Murine Resistance to Salmonella Typhimurium Infection

Mice infected with Salmonella typhimurium develop a disease which is similar in its pathogenesis to typhoid fever (42). This facultative intracellular organism multiplies in the phagocytic cells of the murine reticuloendothelial cell system, and unrestricted bacterial growth results in death of the host. However, mice of various inbred strains differ in response to S. typhimurium infection. Some strains of mice invariably succumb to infection with < 10 organisms, whereas other strains survive challenge doses of ≥ 104 bacteria (21, 24). Webster, Schott and Gowen (9, 34, 39–41) were the first to recognize that this differential susceptibility was genetically regulated, and they subsequently developed salmonella-susceptible and resistant mouse strains (39). Although their pioneering work was performed in the 1930’s, the delineation of the genes involved in resistance to murine typhoid occurred only recently.

CHARACTERIZATION OF A NON-H-2 LINKED GENE CLUSTER CODING FOR THE MURINE B CELL ALLOANTIGENS LYB-2, LYB-4, AND LYB-6

ABSTRACT Lyb-6 is the 45,000 dalton polypeptide, B lymphocyte surface membrane target of an antiserum raised in CBA/N mice against CBA/J spleen cells. The antigen is identified by its mobility (reduced) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation from radioiodinated cells of appropriate mouse strains. The B cell localization of this antigen was verified by the inability of extensive absorptions with cells from nonlymphoid tissues to remove Lyb-6 reactivity from this serum, and by the ability to isolate this antigen from B cell populations but not from B cell-depleted populations. Because the strain distribution of Lyb-6 (further defined as the 6.1 allele) was strikingly similar to the published distributions of Lyb-2.1 and Lyb-4.1 (defined serologically), we performed linkage analyses for these and other markers in (C57BL/6 × DBA/2)F 1 × C57BL/6 back-cross mice. The results indicated conclusively that Lyb-2, −4, and −6 are products of distinct genes which are linked to the Mup-1 locus on chromosome 4. The pattern of recombinations observed establishes the probable gene order as Lyb-2-Lyb-4-Lyb-6-Mup-1, with a recombination frequency of 3–5% between the Lyb-2 and Lyb-6 loci.