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GM2, GD2, and GD3 gangliosides are expressed on the surface of human melanoma cells and represent potential targets for immunological control of melanoma growth by monoclonal antibodies and active immunization. The immunogenicity of GM2 was investigated by analyzing the humoral immune response of melanoma patients to vaccination with cell lines selected for high GM2 expression and with vaccines containing purified GM2. The whole-cell vaccine and vaccines containing purified GM2 and bacillus Calmette-Guérin (BCG) elicited GM2 antibody in a high proportion of patients, particularly in GM2/BCG-vaccinated patients pretreated with cyclophosphamide and given a GM2/BCG booster immunization. Vaccines containing purified GM2 and Salmonella minnesota R595 as the adjuvant were also effective, but only in patients pretreated with cyclophosphamide. GM2 antibodies in vaccinated patients were of the IgM class and were cytotoxic for GM2-positive targets in the presence of human complement.

TL (thymus leukemia) antigens are encoded by genes in the major histocompatibility complex (MHC) of the mouse. Although similar in overall structure to other class I MHC antigens (H-2, Qa), TL expression is regulated in a highly distinctive fashion. In contrast to the broad distribution of H-2 and the intermediate distribution of Qa, TL expression is restricted to cells of T-cell derivation during development in the thymus and is lost when T cells migrate to the periphery. Some mouse strains do not express TL antigens on thymocytes (TL- strains), but leukemias occurring in these mice can have a TL+ phenotype, indicating activation of normally silent TL genes. In transfection studies with H-2 or TL genes in L cells (mouse fibroblasts), H-2 is expressed at high levels, whereas TL is poorly expressed. To identify genetic elements that regulate expression in transfected L cells, chimeric genes were constructed by transposing the 5' and 3' regions of TL and H-2 genes. Antigen expression was not influenced by transposing the cytoplasmic domain and 3' untranslated region. In contrast, interchanging the 5' flanking sequences and exon 1 had a marked influence on antigen expression, with 5' sequences from the H-2 gene increasing TL expression 10- to 50-fold, and 5' sequences from the TL gene markedly decreasing H-2 expression. With both the parental TL gene (p20-TL) and the highly expressed chimeric TL gene (construct 3), levels of TL mRNA and TL antigen correlated with the number of transfected gene copies. However, in cells transfected with equal copy numbers, much higher levels of TL mRNA and TL antigen were found in construct-3 transfectants than in p20-TL transfectants. In addition, there was marked heterogeneity in TL mRNA size in L cells transfected with p20-TL, in contrast to a more homogeneous transcript size in construct-3 transfectants. These results point to regulatory sequences in the 5' flanking region of class I genes that control proper initiation and processing of TL transcripts.

Comparative tryptic peptide analysis was used to probe the structure of the TL products coded for by normally expressed TL alleles (Tlaa and Tlac haplotypes) and the structure of the TL product on ERLD, a TL+ leukemia occurring in a strain that does not normally express TL antigens (Tlab haplotype). In mice that have the Tlaa haplotype, the peptide maps of TL glycoproteins from normal thymocytes and from TL+ leukemias were identical, a finding that is consistent with the indistinguishable serologically defined TL phenotype of these cells. Analysis of the TL product on ERLD leukemia cells (TL phenotype, TL.1,2,4) indicates a single TL glycoprotein species with the TL.4 determinant (restricted to leukemias of Tlab and Tlac haplotypes) coexisting on the same glycoprotein as the Tl.1 determinant (normally expressed by thymocytes of Tlaa haplotype). Comparison of the peptides of the TL product of ERLD and the products of the normally expressed Tlaa and Tlac loci showed a high degree of structural similarity (i.e., 70-80%) of shared peptides. In contrast, the products of the Tlaa, Tlab, and Tlac loci shared relatively few peptides with the products of the K, D, and L loci of the closely linked major histocompatibility complex (15-35%). The TL family, therefore, consists of alleles that are highly homologous. This contrasts with the marked diversity and polymorphism among the alleles of the K, D, and L loci.

Thymocytes from preleukemic AKR mice aged 5-6 mo have an altered pattern of cell surface antigens. The expression of four MuLV-related antigens on the cell surface (GIX, GCSA, gp70, p30) is markedly increased in comparison to 2-mo-old AKR mice and approximates the heightened levels of these antigens found on thymic leukemia cells. H-2 and Thy-1 alloantigens also show characteristic modifications in relation to age and leukemia development. In contrast to the high Thy-1/low H-2 levels on 2-mo-old AKR thymocytes, thymocytes from 6-mo-old mice and thymic leukemia cells frequently show a low Thy-1/high H-2 surface phenotype. As thymocytes from mouse strains with a low incidence of leukemia do not show these changes, they appear to represent a stage in the conversion of normal cells to leukemia cells.

A number of ecotropic and xenotropic murine leukemia viruses were examined for their ability to induce the GIX antigen and Gross cell surface antigen (GCSA) in tissue culture fibroblasts. GIX appears to be a constituent of murine leukemia virus gp70; a molecular characterization of GCSA has not yet been reported. Antigen induction was measured by the ability of productively infected cells to absorb cytotoxic activity from the standard GIX- and GCSA-typing antisera. Cells infected by ecotropic viruses displayed four distinct phenotypes GIX:+/GCSA++, GIX-/GCSA++, GIX++/GCSA+, and GIX-/GSCA+; cells infected by xenotropic viruses were either GIX-/GCSA+ or GIX-/GCSA-. GIX induction appeared to be a type-specific property of some but not all Gross-AKR type ecotropic viruses. Differences in the degree of absorption of the GCSA antiserum by ecotropic virus- and xenotropic virus-infected cells indicated that GCSA may comprise multiple antigenic determinants.

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.

A new cell surface antigenic system of the mouse, designated G(RADA1), is described. The antigen is defined by cytotoxic tests with the A strain X-ray-induced leukemia RADA1 and naturally occurring antibody from random-bred Swiss mice and can be distinguished from all other serologically detected cell surface antigens of the mouse. Absorption tests indicate that G(RADA1) is present in the normal lymphatic tissue and leukemias of mouse strains with high spontaneous leukemia-incidence, e.g., AKR, C58, and C3H/Figge. Low leukemia-incidence strains, e.g., C57BL/6, BALB/c, and A lack G(RADA1) in their normal tissues, but a proportion of leukemias and solid tumors arising in these strains are G(RADA1)+. The relation of G(RADA1) to MuLV is shown by G(RADA1) appearance after MuLV infection of permissive cells in vitro; four of five N-tropic MuLV isolates, one of four B-tropic MuLV, and none of four xenotropic MuLV induce G(RADA1). Two MCF MuLV, thought to represent recombinants between N-ecotropic and xenotropic MuLV, also induce G(RADA1). Serological and biochemical characterization indicates that G(RADA1) is a type-specific determinant of the gp70 component of certain MuLV. The presence of natural antibody to RADA1 in various mouse strains and the emergence of G(RADA1)+ leukemias and solid tumors in mice of G(RADA1)- phenotype suggest widespread occurrence of genetic information coding for this antigen.

For several reasons the G(IX) antigen (1) has a prominent place in current work on murine leukemia virus (MuLV): In the prototype G(IX+) mouse strain 129, the G(IX) trait is mendelian, and is expressed selectively (though not exclusively) on thymocytes. Thus, expression of this cell surface component is under the control of cellular genes and is subject to the controls governing the differentiation of T lymphocytes (2). Although the 129 mouse produces no demonstrable leukemia virus such as that found in the AKR strain, it was soon realized that G(IX) antigen must in some way be related to MuLV, because productive infection with MuLV is frequently associated with appearance of G(IX) antigen on cells that are genotypically G(IX-), most notably on MuLV-infected rat cells, or cells that belong to other differentiation pathways (1). The basis of this connection between G(IX) and MuLV has recently become clear from the demonstration that G(IX) is one of MuLV envelope. Therefore, our working hypothesis is that the presence of G(IX) is one of the antigens present on gp69/71 (3,4), the major glycoprotein component of the MuLV envelope. Therefore, our working hypothesis is that the presence of G(IX) antigen always denotes the presence of gp69/71 (though not all variants of gp69/71 need necessarily carry G(IX)). Study of the circumstances under which G(IX) is expressed on the cell surface is thus potentially a powerful approach to understanding how the expression of C-type viral genomes is controlled. Such studies are greatly facilitated by the availability of mutant and congenic strains of inbred mice which differ from the nonmutant or partner strains only with respect to one or another manifestation of the viral genome. It is for this reason that we record here (Table I) some details of two G(IX) mutant and two G(IX) congenic stocks derived in our colonies at Memorial Sloan-Kettering Cancer Center (MSKCC). In addition, to these four strains, Table I includes data for the three relevant partner strains, and for strain AKR, for comparison. These eight strains all differ from one another with respect to one or more MuLV-related traits.

Three Tla region-specific probes have been generated from the BALB/c genomic cosmid clone C6.3. One probe, pTL1, corresponds to 3' sequences of a thymus leukemia (TL)-encoding gene, whereas pTL2 and pTL3 detect noncoding flanking sequences. The TL specificity of pTL1 was demonstrated by studies of RNA from thymocytes of TL+ and TL- mouse strains and from TL+ and TL- leukemias; presence/absence of pTL1+ transcripts correlated with presence/absence of TL antigens detected serologically. Nine Tla haplotypes were defined by restriction fragment polymorphism with pTL1, and the number of TL genes has been estimated to be greater than or equal to 4 in Tlaa, Tlac and Tlae mice, greater than or equal to 3 in Tlad mice, and greater than or equal to 2 in Tlab and Tlaf mice. A TL-encoding gene (C25.1) from the C57BL/6TL+ leukemia ERLD has been cloned and sequenced, and the exon/intron organization of C25.1 has been deduced from the structure of pTL1+ cDNA clones and from the known organization of H-2 genes. The major structural differences between TL and H-2 genes are in exons coding for the cytoplasmic domain.

We recently reported that thymocytes from 6-month-old preleukemic AKR mice express higher levels of murine leukemia virus (MuLV)-related antigens that thymocytes from 2-month-old mice. We have now found that the level of xenotropic MuLV (defined operationally as MuLV able to infect mink cell cultures) is also markedly increased in thymus of 6-month-old AKR mice and that this increase in virus correlates closely with increased MuLV-antigen expression. There is no increase of MuLV antigen or xenotropic virus in spleen or lymph nodes. Production of ecotropic MuLV remains unchanged with age in thymus, lymph nodes, and spleen. Thymic grafts from 6-month-old AKR mice, but not from 2-month-old mice, induce both amplified MuLV-antigen expression and xenotropic virus production in the thymus of young AKR recipients. Experiments with lethally irradiated AKR mice reconstituted with syngeneic bone marrow cells indicate that age-related changes in the thymus rather than in bone marrow precursor cells are responsible for MuLV-antigen amplification.

The GIX antigen is a feature of the gp70 envelope glycoprotein of murine luekemia virus (MuLV). This GIX-gp70 molecule is found on the thymocytes of some (GIX+) strains of mice, where its expression is controlled by two mendelian genes, Gv-1 and Gv-2. Previous recombination data involving the prototype GIX+ strain 129 indicated that the H-2 (chromosome 17) and Gv-1 loci are linked, at a distance of 36 units from one another. New data indicate that the association of H-2 and GIX phenotypes is an example of quasi-linkage, evidently dependent in this instance on heterozygosity at a locus or loci in the vicinity of H-2. Other previous recombination data, involving the GIX+ strain AKR, had indicated that the Gpd-1 (chromosome 4) and Gv-1 loci are linked at a distance of 19 units from one another. New data from other crosses show that this association of Gpd-1 (glucose-6-phosphate dehydrogenase 1) and GIX phenotypes also constitutes quasi-linkage, evidently due to heterozygosity at the Fv-1 locus. An important theoretical consequence of quasi-linkage in general is that it should enhance the heritability of particular constellations of unlinked genes, and so influence population structure. Our new data are discussed from the viewpoint that MuLV genomes are apparently concerned in quasi-linkage, and therefore by the same arguments may influence the genetic structure of populations. This in turn may strengthen the view that integrated MuLV genomes are not simply intruded into a self-sufficient cellular genome, but are themselves elements of the cellular genome with primary functions, perhaps in reproduction or embryogenesis.

We report here the serologic detection of a cell surface antigen common to cells transformed by the Abelson murine leukemia virus (A-MuLV) and to normal hematopoietic cells from certain strains of mice. Serum from C57BL/6 mice hyperimmunized with syngeneic A-MuLV lymphoma cells was cytotoxic for the immunizing cells; this reaction was used as the serologic test system for recognition of A-MuLV antigens. Absorption analysis using 40 tumors and 21 cell lines revealed that two serologic specificities were detected by this test system: (i) FMR antigen(s) related to the Moloney MuLV helper (the virus from which A-MuLV was originally derived), and (ii) an antigen expressed on all cells transformed by A-MuLV. The A-MuLV-specific antigen was also present on uninfected cells from BALB/c bone marrow, spleen, and fetal liver but not from adult liver, thymus, lymph nodes, or peripheral blood. Abelson antigen was not expressed on bone marrow or spleen cells of 12 other mouse strains. In light of the original isolation of A-MuLV from a BALB/c mouse infected with Moloney virus, it is possible that Abelson antigen is a serologic marker for a gene of BALB/c mice, normally encoding a cell surface molecule, that was incorporated into the Moloney virus genome during the generation of A-MuLV.

Intrathymic injection of SMX-1, a dualtropic murine leukemia virus (MuLV) originally derived from Moloney murine leukemia virus stocks, protects AKR mice from developing MuLV-accelerated leukemia and spontaneous leukemia. Thymuses of SMX-1-injected mice show no change in weight, morphology, or thymocyte size, and quantitative expression of Thy-1 and Lyt-2 differentiation antigens is identical to control mice. The amplified thymic expression of MuLV-related antigens that occurs spontaneously in 6-month-old preleukemic AKR mice or that can be induced in young AKR mice by leukemogenic AKR dualtropic MuLV is prevented by SMX-1. It appears unlikely that the protective effect of SMX-1 is explicable in terms of cross-immunogenicity with transforming MuLV or transformed cells. As SMX-1 persists for long periods after intrathymic injection and does not alter levels of thymic ecotropic MuLV, SMX-1 may interfere with the generation, spread, or leukemogenicity of dualtropic MuLV that form de novo in AKR thymus during the late preleukemic phase. SMX-1 provides a way to probe the events leading to cell transformation in AKR mice.

A thymus-leukemia (TL)-specific probe, pTL1, has been generated from a TL-coding gene of BALB/c mice. Multiple species of TL mRNA were detected in TL+ cells by Northern blot analysis with pTL1, and different Tla haplotypes could be distinguished on the basis of characteristic patterns of TL mRNA. No TL-related message was found in normal or leukemic TL- cells, including thymocytes from Tlab mice. However, TL mRNA could be detected in TL+ leukemias occurring in Tlab mice. A cDNA library has been made from ASL1 (a TL+ leukemia of A mice [Tlaa]), and pTL1+ clones have been sequenced. At least three structurally distinct TL genes are expressed in ASL1. Sequence comparison of TL genes from three Tla haplotypes indicates that TL genes are highly conserved (greater than 90% homology) and are more distantly related to H-2 genes. Several polyadenylation sites have been found in the 3' untranslated region of TL genes, and differential polyadenylation contributes to the size heterogeneity of TL transcripts. The predicted amino acid sequence of TL products indicates that TL and H-2 are similar in domain structure and disulfide bonds, but differ in glycosylation sites and in cytoplasmic domain sequences.

To investigate the genetic regulation of TL expression, 12 transgenic mouse strains on a C3H (TL-nonexpressing) background have been derived: two Tg.Tlaa-3 strains with Tlaa-3 isolated from A-strain TL+ thymocytes, four Tg.T3b strains with T3b from a TL+ leukemia arising in a C57BL/6 (TL-) mouse, three Tg.Con.3 strains with an H-2Kb/T3b chimeric gene (construct 3,5'flanking region and exon 1 of H-2Kb and exons 2-6 of T3b), one Tg.Con.4 strain with a T3b/H-2Kb chimeric gene (construct 4, 5' flanking region and exon 1 of T3b and exons 2-8 of H-2Kb), and two Tg.H-2Kb strains with H-2Kb. Expression of the transgenes was determined by the presence of TL or H-2Kb products or transcripts. Both Tg.Tlaa-3 strains expressed high levels of TL antigen in thymus, indicating that (i) the 9.6-kilobase Tlaa-3 DNA fragment contains sufficient information for correct tissue-specific expression in thymocytes and (ii) TL- thymocytes of C3H provide conditions for the transcriptional activation of Tlaa-3. In contrast, neither the four Tg.T3b strains nor the Tg.Con.4 strain expressed transgenes, indicating that (i) T3b lacks elements necessary for TL expression in normal thymocytes and (ii) the corresponding endogenous TL genes of C3H mice also lack these elements. The pattern of TL expression in two of the three Tg.Con.3 strains was similar to that of H-2Kb expression, indicating that transcription of this H-2Kb/T3b chimeric gene was driven by the regulatory sequences of H-2Kb. The thymuses of mice derived from the Tg.Tlaa-3-1 strain were smaller than C3H thymuses, and the surface phenotype of Tg.Tlaa-3-1 thymocytes resembled thymocyte precursors (TL+L3T4-Lyt-2-Thy-1+H-2+). These mice developed a high incidence of lymphomas with the same thymocyte precursor phenotype. The study of TL transgenic strains should prove useful in defining the role of TL in normal and abnormal T-cell differentiation.

SMX-1 virus delays the appearance of spontaneous thymomas in AKR mice which have been inoculated as young adults by the intrathymic route. Analyses of high-molecular-weight thymus DNAs from SMX-1 virus-inoculated AKR mice indicated the absence of 3' recombinant proviral-cellular DNA junction fragments generated by EcoRI and PvuII digestion. An average of five recombinant proviral fragments were detected in DNAs from spontaneous thymomas that developed in medium-injected control mice. Preleukemic mice that amplify murine leukemia virus-related antigens on their thymocyte surface contained unintegrated proviruses in their thymus DNAs, and 2.3-kilobase EcoRI and 2.1-kilobase PvuII recombinant DNA fragments were detected.

X-irradiation of BALB/c mice in the second month of life induced a high incidence of generalized lymphatic leukemia of T-cell origin, beginning at 7 months of age. Infectious ecotropic murine leukemia virus (B-tropic predominant over N-tropic) was isolable from all tumor extracts but exhibited a wide titer range among individual leukemias. Detection of infectious xenotropic virus usually required extensive amplification on indicator cells. Dual-tropic (mink cell focus-forming) virus has not been found in the leukemias. Expression of ecotropic virus in tail extracts prepared at 6.5 months of age, although greatly enhanced compared with unirradiated controls, was not found to be prognostic of tumor development in individual mice. We conclude that leukemogenesis does not show a simple dependence on infectious murine leukemia virus expression in these mice.

The GIX antigen expressed on the thymocytes of GIX+ mice is a type-specific constituent of glycoprotein gp70, which forms the major envelope component of murine leukemia virus. In the prototype GIX+ mouse strain 129, this glycoprotein is a Mendelian character expressed independently of virus production. In the intact thymocyte plasma membrane, part of this glycoprotein, bearing group-specific (gs) antigen, is inaccessible to antibody. The moiety bearing the type-specific GIX determinant is accessible to GIX antibody, which may be an important factor in determining the consequences of autoimmune responses involving GIX. Previously, all attempts to induce GIX antibody in mice had failed. We now find that the hybrid mouse (B6-GIX+ X 129) spontaneously produces substantial amounts of GIX antibody, presumably of the IgM class appearing as early as 2 mo of age. The specificity of the GIX natural mouse antibody is the same as that recognized by the conventional GIX typing serum produced in rats ("anti-NTD"). As neither parent strain produces appreciable GIX antibody, we surmise that this autoimmune response requires two dominant genes, each parent contributing a high-response allele to the hybrid. These can be envisaged as two immune response loci, controlling different immunocompetent cells which must cooperate to produce GIX antibody. Production of GIX antibody by the hybrids increases progressively with age. This is accompanied by decreased expression of GIX antigen on their thymocytes. We attribute this to antigenic modulation. Antibody to gs antigen of gp70 is also found in autoimmune (B6-GIX+ X 129) hybrids but not in either parent strain. We are investigating evidence of a pathological autoimmune syndrome in these hybrids. The special interest of this syndrome is that it presumably signifies the consequences of autoimmunization to a single C-type virus component, expressed without significant virus production, in a mouse with no evident genetic predisposition to such disease in the absence of that antigen.

Expression of Gix surface antigen on thymocytes is an inherited mendelian train of certain strains of mice. We report here the following new findings: (a) Gix antigen was found free in the serum of Gix+ mouse strains. (b) Expression vs. nonexpression of Gix antigen was invariably correlated with presence or absence of the group-specific antigen of Murine leukemia virus (MuLV) gp69/71 in the serum of mice of inbred and segregating populations. (c) Gix antigen could be removed from normal Gix+ mouse serum by precipitation with antiserum to MuLV gp 69/71. (d) Anti-gp69/71 serum was weakly cytotoxic for Gix+ thymocytes, and partially blocked the cytotoxic activity of Gix antibody for Gix+ thymocytes. (e) Purified AKR virus absorbed Gix activity, and disruption of the virions did not increase their absorbing capacity. These serological data indicate that Gix antigen is a constituent of gp69/71, the glycoprotein which is the major component of the MuLV envelope. On present evidence, Gix antigen is represented in intact virions and is probably accessible to Gix antibody.

Anomalous appearance of TL (thymus-leukemia) antigens is a characteristic feature of radiation-induced leukemias of C57bl/6 mice. We now report that thymocytes of irradiated C57BL/6 mice express TL antigens long before the development of overt leukemia. Thus, TL is a marker for preleukemic changes occurring during radiation leukemogenesis. Low levels of murine leukemia virus (MuLV)-related antigens are also detected on preleukemic thymocytes. Comparative tests on individual mice show no direct correlation between TL and MuLV antigen expression.

In a further genetic study of murine leukemia virus (MuLV) and its components we examined the backcross C57L X (C57L X AKR). This population was selected because strains AKR and C57L are both Fv-1n, and the restriction which the Fu-1b allele imposes on the output of virus was thereby obviated. The segregants were scored for three characters: (a) infectious Gross-AKR-type MuLV (V), in the tail; (b) group-specific antigen indicative of p30 internal viral protein, in spleen; and (c) GIX antigen, now thought to be indicative of gp69/71 viral envelope glycoprotein, on thymocytes. Our conclusions are: (a) It is confirmed that the AKR mouse has two unlinked chromosomal genes, Akv-1 and Akv-2, each of which can independently give rise to the life-long high output of MuLV that is characteristic of AKR mice. (b) Of the eight phenotypes that could possibly be derived from segregation of the three pairs of independent alternative traits, seven were observed, but on progeny testing only three were shown to reflect stably heritable genotypes; these were V+p30+GIX+ and V-p30-GIX- (the parental types) and V-p30+GIX+. A third, newly identified AKR gene, designated Akvp, segregating independently of Akv-1 and Akv-2, also determines expression of p30 and GIX but in this case independently of XC-detectable MuLV. (c) The four remaining observed phenotypes, which did not breed true on progeny testing, involved mostly antigen-negative parents yielding antigen-positive progeny; it is likely that these discrepancies represented suppression of phenotype by a maternal resistance factor.

Normal mouse sera were tested for cytotoxic antibody to surface antigens of cultured monolayer cells infected with AKR-derived ecotropic MuLV, xentropic MuLV, or dualtropic MCF 247 MuLV. Antibody to ecotropic MuLV-infected cells was found in a proportion of C57BL/6, C3Hf/Bi, AKR-Fv-1b, and (C3Hf/Bi X AKR)F1 mice, but not AKR or (AKR X C3Hf/Bi)F1 mice. Antibody to xenotropic MuLV-infected cells was virtually restricted to C57BL/6 mice. Antibody to MCF 247-infected cells was found in all strains tested, including AKR mice. Absorption analysis of (C3Hf/Bi x akr)f1 and AKR-Fv-1b sera with selective reactivity for MCF 247-infected cells showed that these sera recognize distinctive antigens on MCF 247-infected cells that are not present on ecotropic or xenotropic MuLV-infected cells. The transplantable AKR spontaneous leukemia AKSL2 was found to be uniquely sensitive to the cytotoxic action of naturally occurring antibody to MCF 247-related antigens and absorption tests with AKSL2 as the target cell and sera from a single AKR-Fv-1b mouse have permitted the definition of a new MuLV-related cell surface antigen, which has been designated G(AKSL2). Thymocytes from young mice of high leukemia-incidence strains (AKR, C58, and PL) express G(AKSL2), whereas thymocytes from 12 other strains do not. In AKR mice, the antigen is expressed in higher amounts on cells from thymus and bone marrow than on spleen cells. All AKR spontaneous leukemias tested express G(AKSL2), as did three MuLV-induced leukemias arising in G(AKSL2)- strains. Five X-ray-induced leukemias of G(AKSL2)- strains were G(AKSL2)-, as were MuLV+ and MuLV- chemically induced sarcomas. In the limited survey conducted to date, natural antibody to G(AKSL2) has been restricted to strains expressing G(AKSL2) in their normal tissues: AKR, AKR congenic mice AKR-Fv-1b and AKR hybrid mice (C3Hf/Bi x akr)f1 and (C57BL/6 X AKR)F1. In vitro G(AKSL2) induction tests involving MuLV infection of cultured monolayer cells showed that 8 of 12 newly isolated dualtropic MuLV shared the property of G(AKSL2) induction with the prototype MCF MuLV, MCF 247. Of the 12 ecotropic MuLV tested, only the N-tropic MuLV isolated from a leukemia originally induced by Passage A Gross virus induced G(AKSL2). The xenotropic and amphotropic MuLV isolates tested lacked G(AKSL2) inducing activity. Recognition of the g(aksl2) system provides a way to trace the origin and natural history of a class of dualtropic MCF MuLV in the mouse and to determine whether natural antibody to G(AKSL2) plays a role in AKR leukemogenesis.

The gp70 family of glycoproteins is distinguished by the role of these molecules as constituents of C-type viral envelopes and also as Mendelian cellular constituents expressed independently of virus production. The source of G(IX)-gp70 in the serum of 129 strain mice, which are not overt producers of virus, could not be traced to any organ or tissue that is known to be G(IX)-positive by serological tests. Hematopoietic tissues were excluded as source of serum G(IX)-gp70 by tests with reciprocal radiation chimeras made from 129 and 129-G(IX)(-) donors and recipients. Thymus and spleen were excluded because excision of these organs did not affect levels of G(IX)-gp70 in the serum. The serum of young adult 129 males contains roughly four times as much G(IX)-gp70 as adult 129 females and the levels rise in both sexes with increasing age. Castration of 129 males reduced the level of serum G(IX)-gp70 to that of females, and the level was fully restored by testosterone. Thus the epididymis and seminal fluid, though rich in G(IX)-gp70, do not contribute significant amounts of G(IX)-gp70 to the serum. The level of G(IX)-gp70 in the serum of testosterone-treated females, though more than double that of untreated females, did not reach the level of normal males, under the conditions tested. This may signify that G(IX)-gp70 production by males is subject to imprinting by testosterone in early life. Evidently the main source of serum G(IX)-gp70 is a tissue or organ that is common to males and females, is directly or indirectly responsive to testosterone, and has not so far been identified serologically as G(IX)- positive.

The specificity of transplantation immunity and T cell cytotoxicity against leukemias induced by RadLV was examined. Subcutaneous inoculation of two RadLV leukemias induced in BALB/c mice, BALBRVB and BALBRVD, resulted in initial tumor growth in CB6F1 mice, followed by complete tumor regression. Mice that had rejected leukemias BALBRVB or BALBRVD were subsequently challenged with various tumors of BALB/c origin. The growth of all five RadLV leukemias tested, and of one radiation-induced leukemia, was significantly inhibited. Another radiation-induced leukemia, a methylcholanthrene-induced sarcoma, and a leukemia induced by the Moloney leukemia virus, were not inhibited. The results indicate that RadLV leukemias share cell surface antigens that induce transplantation immunity in vivo. Cytotoxic lymphocytes were generated by coculturing spleen cells from mice that had rejected leukemia BALBRVB or BALBRVD with the corresponding leukemia cells. Direct tests and inhibition tests showed that such cytotoxic cells recognized individually specific antigens on leukemias BALBRVB and BALBRVD, distinct from the shared antigens detected in transplantation experiments. The effector cells in cytotoxicity assays were Thy-1+, Lyt-1+,-, Lyt-2+, and Lyt-3+ T cells.

Genes specifying or controlling the expression of GIX (cell surface), GCSA (cell surface), and gs (internal viral) antigens are located in chromosome 4 (linkage group [LG] VIII) of the AKR mouse. All three antigens may exhibit mendelian inheritance, mice being antigen positive or antigen negative, but each may also appear in leukemic cells of mice whose inherited genotype was antigen negative. The GIX-determining gene in LG VIII of AKR mice apparently is equivalent to Gv-1, which determines expression of the same antigen in 129 strain mice, but which in the latter strain is located in LG IX. As the estimated distance of Gv-1 from H-2 in 129 mice is considerable (37 units) further tests are now indicated to assess the possibility of pseudolinkage in this case. The Fv-1 locus, also located in LG VIII, influences the mouse's titer of MuLV, and might thereby be thought to regulate the GIX and gs phenotypes of AKR backcross segregants. But the data indicate a discrete LG VIII locus for GIX, since expression of this antigen is mendelian and independent of infectious virus titer. Since the GIX and GCSA phenotypes of AKR backcross segregants were invariably concordant, these two antigens must be specified or controlled by closely linked genes, and the latter also is presumably independent of virus titer. The question as to what extent expression of gs antigen in the segregants is secondary to virus production is undecided.