Edward A. Boyse

Serological demonstration of H-Y (male) antigen on mouse sperm.

WE have recently overcome certain technical difficulties in applying the cytotoxicity test to mouse spermatozoa and have been able to show directly that H-2 antigens are expressed on these cells1, thus confirming less direct evidence leading to the same conclusion2. We have since applied the cytotoxicity test to sperm with antisera directed against a number of other systems of mouse alloantigens, TL3, θ4, Ly-A5 and Ly-B5, but none of these has given a positive reaction with sperm. This is not surprising because these are all “differentiation antigens”6 expressed primarily on lymphoid cells. Another antigenic system which distinguishes one mouse from another is H-Y. The H-Y antigen is carried by male cells only and is responsible for the rejection of male tissues by females of the same inbred strain7.

Ly-A and Ly-B: two systems of lymphocyte isoantigens in the mouse

Two systems of isoantigens confined to thym ocytes and lymphocytes have been defined in the mouse by cytotoxic isoantisera. The two genetic loci have been designated Ly-A and Ly-B. Typing of 25 mouse strains and sublines indicates that each system comprises only 2 alleles, determining alternative isoantigens Ly-A. 1 or Ly-A. 2, and Ly-B. 1 or Ly-B. 2, respectively. There is no evidence of multiple alleles or that either locus is compound. Tests for genetic linkage were performed by serological typing of mice from segregating generations. None of the four loci H-2 (group IX ), θ,Ly-A and Ly-B is closely linked to any other of the four. Ly antigens are found in high concentration on thymocytes and in lesser amounts on lymphocytes. The small absorptive capacity of bone marrow cell suspensions is probably due to the presence of mature lymphocytes rather than thymocyte-lymphocyte precursors, which according to preliminary evidence lack Ly antigens. Ly-B. 1 is exceptional in that the disparity between thymocytes and lymphocytes in content of antigen is greater than that of the three other antigens. When Ly antiserum is injected into mice of the relevant Ly type, under conditions which give complete absorption of H-2 antibody in vivo, Ly antibody is not extensively absorbed ; this is to be expected from the limited tissue distribution of Ly antigens. Absorption of Ly and θ iosantibodies in vivo is reduced by treatment of the recipients with cortisone or lethal total-body irradiation. The reduction in titre of Ly and θ antibodies in such mice may be no more than that resulting from dilution. Absorption of injected H-2 antibody also may be reduced by treatment of the recipients with cortisone but in this case the effect of cortisone does not approach the virtual abolition of absorption that is seen with Ly and θ antibodies. The thymocytes and lymphocytes of chimeras formed by restoring lethally irradiated mice with allogeneic bone marrow have the Ly-A type, Ly-B type and θ type o f the donor. The content of Ly antigens on cells of different leukaemias varies widely and shows no correlation with presence or absence of TL (thymus-leukaemia) antigen; TL + leukaemias may be Ly — or Ly + and TL — leukaemias may be Ly — or Ly + . Five systems of isoantigens are now demonstrable on mouse thymocytes by means of cytotoxic isoantisera. Of these, TL is an exclusively thymic antigen. Ly-A and Ly-B antigens occur also on lymphocytes. θ occurs on thymocytes, on lymphocytes, and in brain. The fifth antigen, H-2. is still more widely distributed and differs from the other four in being represented less strongly on thymocytes than on lymphocytes.

Asparagine Synthetase Activity of Mouse Leukemias

Various transplanted leukemias and normal tissues of the mouse were tested for asparagine synthetase activity. Leukemias susceptible to suppression by asparaginase have little or no synthetase activity. In contrast, leukemias insensitive to asparaginase exhibit substantial and often very high asparagine synthetase activity. Asparaginase-resistant variants of sensitive leukemias also have considerable synthetase activity. Thus the requirement by certain malignant cells of exogenous asparagine, which entails sensitivity to asparaginase, may be ascribed to lack of asparagine synthetase. Development of asparaginase-resistant variants from asparaginase-sensitive lines is consistently associated with acquisition of asparagine synthetase activity.

Ly antigens as markers for functionally distinct subpopulations of thymus-derived lymphocytes of the mouse.

THYMUS-DERIVED lymphocytes (T cells) have several functions1–3, and in some instances it appears that two sub-populations of T cells interact to amplify their response4,5. But recognition of the diverse functions and interactions of T cells has not been matched by the development of techniques for identifying and separating the different T-cell subpopulations involved. The ability to do so would greatly aid study of the many roles of T cells. We report here experiments with mice that show that antisera to different Ly alloantigens can identify functionally-distinct subpopulations of T cells.

Genetic linkage relationships of loci specifying differentiation alloantigens in the mouse.

Data concerning the linkage relationships of loci determining cell surface differentiation alloantigens in the mouse are reported. The order of the loci theta antigen (&thetas;), dilute (d), and malic enzyme (Mod-1) in genetic linkage group (LG) II is &thetas;-d-Mod-1. Recombination between &thetas; and d is 15.0 \Pm 4.0%. The lymphocyte antigen C locus (Ly-C) and the lymphocyte antigen B locus (Ly-B) are closely linked; no recombinants were observed among 370 backcross segregants. The provisional symbols, Ly-A and Ly-B,C, therefore, provide a useful descriptive notation for the loci of the Ly series so far recognized. Recombination between the lactic dehydrogenase regulatory locus (Ldr-1) and the Ly-B,C locus is 37.5 \Pm 5.4% and between Ly-B,C and the microphthalmia locus (mi), 7.3 \Pm 2.2% in linkage group XI. The tentative order of the three loci is Ly-B,C-mi-Ldr-1. Other (negative) linkage data, involving biochemical, serological, and other markers, are summarized.

Treatment of lymphosarcoma in the dog with L-asparaginase

Three dogs with advanced lymphosarcoma were treated with L‐asparaginase from E. coli. All three responded to therapy by marked regression of lymph nodes and dramatic improvement in general condition without evidence of toxicity. Two dogs returned temporarily to normal health and were clinically free of disease; the other showed a marked partial remission. One of the three dogs has remained in good health for 50 days after treatment was discontinued although the disease now shows signs of recurrence. Early relapse in the other two cases may be attributed to insufficient dosage of L‐asparaginase.

Group-specific viral antigens in the milk and tissues of mice naturally infected with mammary tumor virus or Gross leukemia virus☆

Abstract Group-specific antigens of the mammary tumor virus (MTV-s1) and of Gross (wild-type) murine leukemia virus (MuLV-s1), demonstrable with specific precipitating antisera, are present in high concentration in the milk of naturally infected mice. With two exceptions, MTV-s1 was restricted to mice of strains with a high incidence of mammary tumors; the two exceptions were individual mice of strains free of MTV by foster-nursing and may be explained by the presence of virus of the same antigenic group. MuLV-s1 has a wide distribution among mouse strains. It occurs not only in mice with a high incidence of leukemia, but also in mice of low-incidence strains. This points to the existence of naturally occurring variants with MuLV-s1 group specificity and G (Gross) type specificity, but with little or no leukemogenic activity. An analogous relationship exists between the weakly oncogenic nodule-inducing virus and MTV, which belong to the same antigenic group. In only one mouse strain were MTV-s1 and MuLV-s1 found together in the milk, an observation suggesting that viruses of the two types may to some extent exclude one another. NZB mice are G+, have MuLV-s1 in their milk and tissues, and carry virus morphologically indistinguishable from murine leukemia virus. It is an open question whether this virus is etiologically related to the autoimmune disease of NZB mice.

THE PHAGOCYTOSIS OF TUMOR CELLS IN VITRO.

The phagocytosis of tumor cells by peritoneal macrophages from mice was demonstrated in vitro following opsonization with isoanti-body. Phagocytosis of tumor cells occurred in the presence of isoimmune serum prepared in either H-2 compatible or H-2 incompatible strains. After ingestion, the tumor cells were destroyed by the engulfing macrophages. Normal mouse serum contained nonspecific factors that enhanced phagocytosis in the presence of specific isoimmune serum. Peritoneal macrophages from immunized animals had no inherent, specific ability to phagocytize tumor cells in vitro, apart from that due to the presence of humoral antibody in the medium. Their ability to phagocytize tumor cells was abolished by washing the peritoneal cells, but was restored by the addition of a medium containing isoantibody. Peritoneal macrophages collected from animals that had been given an i.p. injection of starch phagocytized tumor cells in greater numbers than did peritoneal macrophages from normal animals. In contrast to viable tumor cells or cells killed by prolonged storage, tumor cells killed by methanol or heat were phagocytized in the absence of isoantibody.

A Study of Passive Immunization Against a Transplanted G+ Leukemia with Specific Antiserum.∗

Summary Antiserum with high titers of cytotoxic antibody against G-f leukemia cells can be prepared in inbred rats by immunization with histocompatible rat leukemias induced in the same inbred strain by wild-type Gross virus. Under these conditions heteroantibody and isoantibody to normal tissue antigens are not formed. Passive immunization with this antiserum was successful against a G + transplanted mouse leukemia but not against a G— mouse leukemia of the same inbred strain. These results conform to the specificity of the antiserum for G+ cells, as shown by the cytotoxic test in vitro. Complete protection can be achieved in some mice by serum given as late as 3 days after intravenous inoculation of leukemia cells. Protected mice are not immune to rechallenge with minimal numbers of G + leukemia cells after the transferred G antibody has been cleared.

Continued expression of H-Y antigen on male lymphoid cells resident in female (chimaeric) mice.

REJECTION of male skin grafts by female mice of the same inbred strain is due to male-specific (H-Y) antigen1,2 (review3). As the only functions so far definitely traced to the Y chromosome are concerned with sex determination, a question arises as to whether the expression of H-Y antigen on male cells is secondary to development of the male phenotype, or whether a gene (or genes) on the Y chromosome specifies H-Y antigen (either directly or under regulation of an autosomal locus) independently of other sex-related characteristics. The fact that H-Y antigen is found on all male tissues that have been tested, rather than being restricted to sex-related tissues3,4, is perhaps an indication that it is not directly concerned with the attributes of male sexuality. Its appearance on male but not on female cells might instead be a fortuitous consequence of the lack of crossing over between the X and Y chromosomes; in this case, any change in cell surface antigenicity brought about by mutation of genes on the Y chromosome would automatically be transmitted only to males.