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8-Methoxypsoralen (8-MOP) plus long-wavelength UV radiation (UVA, 320-400 nm) have been used to treat various diseases such as cutaneous T-cell lymphoma, systemic scleroderma, rheumatoid arthritis and rejection of heart transplants. However, the immunological mechanism of this treatment remains unknown. In this report, we investigated the effect of 8-MOP/UVA on the modulation of the immunogenicity of a T-cell leukemia cell line (RL male 1 cells). The results demonstrated that the stimulator function of the in vitro 8-MOP/UVA-treated RL male 1 cells was enhanced in both RL male 1-specific allogeneic and syngeneic immune responses. Furthermore, the enhancement of the immunogenicity of the 8-MOP/UVA-treated RL male 1 cells was found to be strongly associated with the increase of intercellular adhesion molecule-1 expression on these 8-MOP/UVA-treated tumor cells. Therefore, our findings suggested that the alteration of the expression of the immune-related cell surface molecules might be an important effect of 8-MOP/UVA treatment on the elevation of the immunogenicity of the 8-MOP/UVA-treated tumor cells.

CD45 antigens are protein tyrosine phosphatases. A possible link was evaluated between expression of CD45 antigens on human myeloid progenitor cells (MPC) (colony-forming unit-granulocyte/macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], and colony-forming unit-granulocyte/erythroid/macrophage/megakaryocyte [CFU-GEMM]) and regulation of MPC by colony-stimulating factors (CSF) (interleukin 3 [IL-3], GM-CSF, G-CSF, M-CSF, and erythropoietin [Epo]), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (MGF; a c-kit ligand). Treatment of cells with antisense oligodeoxynucleotides (oligos) to exons 1 and 2, but not 4, 5, or 6, of the CD45 gene, or with monoclonal anti-CD45, significantly decreased CFU-GM colony formation stimulated with GM-CSF, IL-3, fusion protein, and GM-CSF + MGF, but not with G-CSF or M-CSF. It also decreased GM-CSF, IL-3, fusion protein, and MGF-enhanced Epo-dependent BFU-E and CFU-GEMM colony formation, but had little or no effect on BFU-E or CFU-GEMM colony formation stimulated by Epo alone. Similar results were obtained with unseparated or purified (greater than or equal to one of two cells being a MPC) bone marrow cells. Sorted populations of CD343+ HLA-DR+ marrow cells composed of 90% MPC were used to demonstrate capping of CD45 after crosslinking protocols. Also, a decreased percent of CD45+ cells and CD45 antigen density was noted after treatment of column-separated CD34+ cells with antisense oligos to exon 1 of the CD45 gene. These results demonstrate that CD45 cell surface antigens are linked to stimulation of early human MPC by IL-3, GM-CSF, a GM-CSF/IL-3 fusion protein, and MGF.

The unique feature of the Ly-5 system is that it is a major cell surface glycoprotein, representing up to 10 % of the total cell surface complement, confined to the hematopoietic cells as a family of isoforms generated by alternative splicing of a single Ly-5 gene. The cytoplasmic domain of Ly-5 has protein tyrosine phosphatase activity suggesting that Ly5 is involved in signal transduction. We used Ly-5 anti-sense oligodeoxyribonucleotides (oligo) and Ly-5 monoclonal antibody (mAb) to study the functional role of Ly-5 in the concanavalin A mitogenesis response by spleen cells, as well as in the generation of lymphokine-activated killer cells and the proliferative response by spleen cells induced by recombinant human interleukin-2 (rhIL-2). Our results indicate that the Ly-5 mAb could enhance these activities whereas the anti-sense oligo was inhibitory. These data clearly suggest that Ly-5 is involved in the IL-2 and IL-2 receptor responsive circuit.

Genetically determined body odors that distinguish one mouse from another are termed odortypes. The best known odortypes, highly expressed in urine, are those specified by H-2, the major histocompatibility complex of the mouse, but other odortypes originate from unidentified loci in the rest of the genome, including both sex chromosomes. The definition of H-2 odortypes and evidence that their perception affects reproductive behavior have so far depended on studies with inbred mouse strains whose genetic differences are confined to the H-2 complex of genes. To simulate feral conditions more closely, a freely segregating population was bred from crosses involving four unrelated inbred strains contributing four different H-2 haplotypes. After H-2 typing, this outbred population was divided into four groups of freely segregating mice, comprising the four distinct H-2 genotypes represented, to serve as conventional donors of urine for evaluation in the standard Y-maze system used in the training and testing of mice for H-2 odortype discrimination. With respect to utility in training mice for H-2 odortype discrimination, and to degrees of concordance attained in the Y-maze by trained mice, these urinary H-2 odortype sources from outbred mice were no less effective than urines customarily obtained for those purposes from nonsegregating inbred donors. We conclude that discrimination of H-2 odortypes is not appreciably affected or impaired by the usual concurrent segregation within the genome as a whole.

T18d of BALB/c mice is a member of the Tla category of class I genes of the major histocompatibility complex of the mouse and is highly restricted in expression. Deletion analysis implies that an element essential to T18d expression resides within the region −4 to +54. The homologous region of T3d, a Tla gene which normally is not expressed in BALB/c mice, also has promoter activity. Thus the expressibility of T18d vs T3d is unlikely to be due to sequence differences in this region. A DNA-binding protein, factor VI, was found to bind to the region −33 to +54. DNase I footprinting analysis indicated that the DNA fragment 5'-ACTATAGTTTCACTTTTT-3' (+3 to +20) was protected by factor VI. This region includes the interferon response sequence (IRS). Homologous DNA segments of other class I genes, Ld and Dd, competed for factor VI in DNA-protein binding assay with lower affinity as compared with T18d. In mutation analysis, the 3' portion of the IRS is more important than the 5' portion with respect to binding affinity of factor VI and to transcriptional activity in transfected cells. This result signifies a role of IRS in T18d transcription and suggests that the mechanism of T18d transcription might be unusual.

Previous inferences that Ly-5 glycoprotein isoforms of murine hematopoietic cells are generated by alternative splicing of primary transcripts of a single Ly-5 gene are supported by the present study. A cDNA library was prepared from B cells by extension from primer representing a known T-cell cDNA sequence. Three different Ly-5 clones from this library included sequences missing in T-cell cDNA clones. From the constitution of cDNA clones and of the Ly-5 gene, and from S1 nuclease mapping, it is concluded that at least two exons, provisionally numbered Ex-6(B) and Ex-7(B), in the 5'-proximal region are mainly represented in mRNA of the B-cell lines examined but not of the T-cell lines examined. Also, exons 1 and 2 appear to be used alternatively in different species of B-cell mRNA and probably also in different species of T-cell mRNA.

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tlaa, Tlad, Tlac), intermediate (Tlac, Tlaf), and low (Tlab). (Relative amounts: 1000 : 100 : 1.) Some thymic leukemias arising in (Tlab, Tlac, Tlac) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.

Immunogenetic cell-surface markers that distinguish sets and subsets of lymphocytes, representing different stages and different lines of maturation within the total lymphoid population, have proved to be oI great value. They have helped to elucidate the immunological functions of different lymphocyte sets, and .have led to findings of much interest in other branches of biology. A serious impediment to wider use of such serological techniques is that the antisera required are often difficult to produce. Among the main problems is that, commonly, only a few immunized mice produce antiserum of adequate titer in the cytotoxicity assay, and of these, many must be discarded because they produce too much T-cell autoantibody. Thus, the volumes of satisfactory antiserum available are grossly inadequate. For this and other reasons (discussed below), we have explored the possibility that antiserum production can be improved by adoptive transfer of lymphoid cells from satisfactorily immunized donors to irradiated syngeneic recipients. The following progress report is sufficiently promising to warrant the attention of interested colleagues. The data refer exclusively to the cytotoxicity assay performed according to Boyse and coworkers (1970) with this exception: we no longer use preabsorbed rabbit serum as the source of complement. In our experience, the best and most ample source of complement is a pool of serum from a few among many rabbits whose serum has been rigorously screened (both before and after exsanguination) for low toxicity for mouse thymocytes combined with high lytic efficiency for thymocytes in cytotoxicity assays with an Lyt antiserum which has a high demand for complement. The conditions for testing the use of adoptive immunization in the three antigenic systems named below were: (i) All mice were females. (ii) All donors had a satisfactory record of antibody production, as defined below for each system, and had reached the limit of their usefulness after many immunizations and bleedings; we could not afford to sacrifice younger donors for an untried procedure. (iii) For adoptive immunization, each syngeneic recipient (aged 8-12 weeks) was subjected to 500R total-body irradiation (Gamma Cell/40, Atomic Energy of Canada, Ltd.)

Thymocytes and leukemia cells of some mouse strains yield TL proteins, precipitable by anti-TL antiserum and by anti-TL monoclonal antibodies, that include not only the familiar heavy (H) chain of 45-50 kDa but also products of higher molecular mass. Production of a 53-kDa TL form by Tlad thymocytes was studied in detail. A cross was made between B10.M (Tlad) mice, which produce the 53-kDa TL, and mice of the A strain (Tlaa), which make only the usual H chain. Hemi-expression of apparently unaltered 53-kDa TL was observed in thymocytes of the Tlad/Tlaa heterozygous F1 progeny. Thus, there was no indication of positive or negative trans interaction with respect to production of the 53-kDa TL form associated with Tlad. We conclude that production of 53-kDa TL is governed intrachromosomally. Two-dimensional chymotryptic peptide maps of the TL H chain and the 53-kDa TL of Tlad thymocytes differed only by added features found in the map of the 53-kDa TL. With the exception of Tlaa, all Tla alleles (Tlab-f) yielded TL products of higher molecular weight than the accompanying H chain, although in the case of Tlab this was evident only in TL+ leukemia cells because Tlab thymocytes are TL-. For H-2, representing other class I genes, no products other than the familiar H chain were demonstrable under similar conditions.

We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors.

The antiserum (BALB.I-H-2j x SWR/J)F1 anti-I.29 ascites cells, in reaction with B6 lymph-node cells (LNC) in the cytotoxicity assay, defines an alloantigen system called Lna-1 (lymph-node alloantigen-l) which in normal, untreated mice is expressed on the cells of lymph nodes but not of other lymphoid organs. The T- and B-cell populations of lymph nodes evidently include Lna-1+ subpopulations representing 30-40 percent of the total population The Lna-+ phenotype could be induced on cells of thymus and spleen but not of bone marrow. Congenitally asplenic +/Dh mice have no Lna-1+ cells in their lymph nodes, but their LNC can be induced to express Lna-1; this suggests that the spleen is normally required for the differentiation of Lna-1+ cells from Lna-1- precursors. It is not yet known whether thymus is also required for the expression of Lna-1 in lymph nodes. It remains to be seen whether the existence of the Lna-1+ B-cell subpopulation of lymph nodes depends on Lna-1+ T cells, and whether the Lna-1+ phenotype of B cells may be acquired rather than intrinsic. One hypothesis which is the basis of further study is that there is a T-cell pathway in which noninducible bone-marrow cells become Lna-1-inducible in the thymus, then travel to the spleen, where they are induced to become Lna-1+, after which they reside in lymph nodes.

Type-variants of gp70 (glycoprotein-70), which is the major envelope protein of C-type mouse virus and is also found in plasma membranes, are identified immunogenetically by the antigens Gix and Ec. Cellular expression of Gix+ gp70 does not depend on production of virus, but expression of Ec+ gp70 (formerly X-gp70) has been observed only in AKR and other strains of mice that produce large amounts of virus throughout life. To test the inference that cellular expression of Ec+ gp70 is secondary to production of virus we examined the effect of Fv-1 alleles, which govern the replicability of N-tropic and B-tropic C-type virus, on the expression of Ec+ gp70 on thymocytes. By typing thymocytes of Fv-1-congenic mice for Ec+ gp70 was found that manifestation of the Ec+ gp70 phenotype requires the Fv-1n allele, which is permissive for replication of N-tropic virus produced by AKR and other virus-producing mouse strains. Substitution of the Fv-1b allele for the Fv-1n allele abolishes demonstrable expression of Ec+ gp70 by AKR thymocytes at ages up to 9 mo, the oldest AKR mice tested.

This study was designed to investigate the phenotypes of immune cells concerned in the rejection of chemically induced tumors. Counted numbers of viable dissociated cells of a selected C57BL/6 (B6) tumor were combined with counted numbers of peritoneal cells from highly immunized syngeneic B6 donors and inoculated subcutaneously into untreated syngeneic B6 mice. Progressive tumor growth was prevented by nonadherent immune peritoneal cells in numbers less than one-quarter the number of tumor cells, and the cells responsible belonged mainly or entirely to the nonadherent fraction of the peritoneal population. There was no cross-protection between this tumor and a second B6 tumor induced by the same carcinogen, indicating that immunity in this experimental system, as reported previously, is directed to antigens that individually characterize chemically induced tumors. Elimination of Thy-1(+) cells by Thy-1 antiserum and complement abolished the protective capacity of the immune peritoneal cells, showing that immunity under these conditions is mediated by T cells. Protection was also precluded by elimination of Lyt-1(+) cells or of Lyt-2(+) cells, showing that protection could not be attributed to either the Lyt-23 or the Lyt-1 set of T cells alone. Nor was protection conferred by various combinations of these selected Lyt-1 and Lyt-23 cell sets. Thus, participation of the third well defined Lyt cells set, Lyt-123, which is automatically eliminated when the Lyt-23 and Lyt-1 sets are prepared by selective immune cytolysis is evidently necessary to rejection, in these experimental circumstances. Although this might imply that cytotoxicity for syngeneic tumors is effected by Lyt-123 cells, it is emphasized that this assay system allows time for the differentiation and proliferation of cells sets. Thus, the data are also consistent with a uniform hypothesis that cell-mediated cytotoxicity is an exclusive function of Lyt123:CS cells, and that these, and possible Lyt1:H amplifier cells, are generated in the course of the assay from a specifically primed antecedent Lyt123:ARC cell set.

The technique of cell-surface iodination, followed by immuno-precipitation withPC-1.1 antiserum, was applied to normal spleen cells and to MOPC-70A BALB myeloma cells. The results indicate that the cell-surface component bearing PC-1 alloantigen has a molecular weight of from 105,000 to 110,000 daltons and does not resemble any constituent of plasma membranes or MuLV-type virus so far categorized by similar methods. The PC-1 specificity of the molecule was confirmed by comparison of the spleen cells of two mouse strains with spleen cells of their respective PC-congenic partner strains. MOPC-70A myeloma cells, but not spleen cells, yield a fainter band in the PC-1 position in controls in which antiserum is omitted, but peptide maps show that this similarly placed band has no relation to the PC-1 molecule.

Generation of anti-sheep erythrocyte plaque-forming cells (PFC) is greatly reduced in the presence of monoclonal Ly-5 alloantibody. Although Ly-5 is expressed in one of its molecular forms on T cells and macrophages (M phi ) involved in this response, the only demonstrated action of Ly-5 antibody was on B cells. Evidence from elimination of Lyt-2+ cells, and from the responses of serial proportions of Ly-5.1 and Ly-5.2 cells and of Ly-5 heterozygous cells, signifies that PFC reduction cannot be ascribed to any known mechanism of suppression or to a direct suppressive action of Ly-5 antibody on B cells. A critical distinction of Ly-5 from Lyb-2 is that Ly-5 antibody reduces PFC generation to trinitrophenylated Ficoll, a thymus-independent type 2 antigen requiring T cells and M phi for maximal PFC generation in vitro. A second distinction is that PFC reduction by Ly-5 antibody is strictly tied to the time of operation of M phi factor, whereas PFC reduction by Lyb-2 antibody relates to the time of B cell triggering by antigen. Accordingly, M phi factor competitively and quantitatively inhibits the action of Ly-5 antibody in reducing PFC generation. It is likely that the Ly-5 system is concerned in the reception or handling of M phi message by B cells.

Three variants of the gp70 envelope component of MuLV are now recognizable serologically: GIX-gp70, 0-gp70, and X-gp70. The last of these, X-gp70, has so far been found only in mice or cells producing abundant C-type virus. This distinguishes X-gp70, provisionally, from the GIX-gp70 and 0-gp70 variants, each of which can be expressed on normal thymocytes without accompanying virus production, as exemplified by mouse strains 129 and B6, respectively. The X-gp70 genotype, however, is not limited to strains of mice-producing abundant virus, because X-gp70+ leukemias occur in strains of mice which do not produce a great deal of virus and whose thymocytes and other tissues are X-gp70-; this is analogous to the appearance of GIX+ leukemias in GIX- mouse strains.

The generation of plaque-forming cells (PFC) to T-dependent antigen, but not to T-independent antigen, is reduced in vitro by Lyb-2 antibody. Monoclonal Lyb-2 antibody, added to Mishell-Dutton cultures within the first 2 d, but not later, greatly reduces the generation of alpha-sheep erythrocyte (SRBC) PFC from T-depleted spleen cells whether help is provided in the form of intact T cells or as soluble factors contained in mixed lymphocyte culture (MLC) supernatants. Generation of alpha-SRBC PFC from purified B cells, assisted by soluble factors in MLC and macrophage (P388D.1 cell) supernatants, is similarly reduced by Lyb-2 antibody. The initial 2-d period, during which cultures are diminishingly sensitive to reduction of PFC generation by Lyb-2 antibody, is not affected by the time at which such soluble factors are added. Thus, Lyb-2 cell surface molecules evidently do not function as receptors for these differentiative signals. Reduction of PFC generation by Lyb-2 antibody is antigen dependent in the sense that reduction of the PFC response to one antigen (SRBC) does not affect subsequent generation of PFC to a second antigen (horse erythrocytes) from the same cell population. These findings accord with the view that the Lyb-2 molecule participates in a B cell differentiative phase, probably proliferative, which begins with binding of antigen and precedes the phase in which B cells become fully receptive to signals from T and other cells.

Killer T cells with specificity for major histocompatibility antigens have been shown in mice and rats to display idiotypic receptors allowing the lysis of such cells at the effector phase by anti-idiotypic antibodies and complement. A comparison was made between idiotypes displayed by Lyt-1-2+3+ and Lyt-1+2-3- T blasts, generated in the same mixed leucocyte culture (MLC), across an entire H-2 locus barrier. This was done by absorption of anti-idiotypic antibodies with respective T blasts, followed by estimation of the ability of the absorbed antiserum to inhibit MLC or killer T-cell function. Further, the capacity of Lyt-purified, MLC-generated T blasts to provoke specific unresponsiveness via anti-idiotypic immunity in syngeneic recipients was analyzed. Taken together, the results demonstrate that Lyt-1+2-3- T blasts responsible for the major part of MLC proliferation have distincly different idiotypes from those on the Lyt 1-2+3+ killer T cells. That the idiotypes on the killer T-cell presursors can serve as triggering sites for induction of effector T-cell function was then suggested by experiments with Lyt-1-2+3+-purified, normal T cells as precursor cells in vitro. The fact that autoanti-idiotypic antibodies may circumvent the need for helper Lyt-1+2-3- T cells in the generation of allospecific killer T cells indicates that the former cells may normally function partly via such anti-idiotypic reactions.

These experiments test the hypothesis that cells carrying the Ly1+23- surface phenotype are programmed exclusively for helper and not suppressive activity regardless of external conditions such as the mode or type of antigen stimulation. To this end, we have stimulated purified populations of Ly1 cells with antigen in vitro using conditions devised to induce unselected T cells to express optimal levels of antigen specific T-suppressor activity. We find that after such stimulation, Ly1 cells generate SRBC-specific T-helper activity but not T-suppressive activity. These findings establish that the Ly1.2+,2.2/3.2- surface phenotype is a stable, and probably invariant, marker of T cells that are programmed to express only helper activity and have lost the capacity to directly suppress the antibody response. These findings support the concept that the genetic program for a single differentiated set of cells combines information for cell surface phenotype and function. We also demonstrate that antigen-stimulated Ly1 cells, in addition to inducing B cells to secrete antibody, can induce or activate other sets of resting T cells to develop profound suppressive effects. The surface phenotype of this feedback suppressive T-cell set is shown to be: Ly1+2+3+Qa1+. These findings, taken together, indicate that activation of resting Ly123 cells by immune Ly1 TH cells may represent an important homeostatic immunoregulatory mechanism.

We have described an interaction between two T cells subsets that results in interference with the expression of Ly-1-, 2+ (Ly-2) T cell-mediated suppression. We refer to this novel immunoregulatory activity as contrasuppression. The T cell responsible for the induction of contrasuppression (inducer cell) expresses the phenotype Ly-1-, 2+;I-J+;Qa-1+. This phenotype distinguishes it from the suppressor effector cells which we find to be I-J-2.3. An I-J+ soluble mediator from the contrasuppressor inducer cell acts on another cell (acceptor cell) that expresses the phenotype Ly-1+, 2+; I-J+; Qa-1+. This phenotype distinguishes it from T helper cells. Both the inducer cell (or its biologically active mediator) and its acceptor cell are required for the expression of contrasuppression. Because contrasuppressor cells can block the suppressive activity of cell-free mediators released by Ly-2 suppressor T cells, the mechanism of contrasuppression is either separated from or in addition to the inactivation of suppressor cells themselves. The potential importance of contrasuppressor activity in the regulation of suppressor T cell activity in allowing immunologic memory to be expressed and in permitting microenvironmental immune regulation is discussed.

A single Ly-5 gene is known to generate a variety of transmembrane glycoprotein isoforms that distinguish various cell lineages and stages of differentiation within the hematopoietic developmental compartment of the mouse. Systems homologous to Ly-5 are known in rats and in humans. The complete exon-intron organization of the Ly-5 gene is described in this report. The Ly-5 gene occupies about 120 kilobases of chromosome 1 and comprises 34 exons, of which 32 (Ex-3 to Ex-34) are protein coding. Ex-1, Ex-2, and parts of Ex-3 and Ex-34 are untranslated. In all cDNA clones examined, either Ex-1 or Ex-2 was represented, but not both, implying that Ex-1 and Ex-2 in Ly-5 mRNA may be mutually exclusive. Primer extension and S1 nuclease protection mapping were used to identify initiation (cap) sites for transcription. The finding of putative cap sites for Ex-1 and Ex-2, and of corresponding TATA-like sequences, suggests the presence of two promoters. In both Ex-1+ and Ex-2+ cDNA clones the next exon is Ex-3, which has a translation-initiating codon. The intron between Ex-3 and Ex-4 is unusually long, about 50 kilobases. Evidence is given that Ex-5, like Ex-6 and Ex-7 (studied previously), is another alternative exon that is selectively programmed, alone or together with Ex-6 or Ex-7 or both, to generate actual or potential Ly-5 isoforms by alternative splicing.

Biosynthetic incorporation of 3H-leucine or lactoperoxidase-catalysed radioiodination of cell surface proteins was used to radiolabel antigen-specific molecules produced by murine splenic T cells cultured in vitro with heterologous erythrocytcs. Antigen-binding proteins present in detergent lysates of labelled cells or in culture media in which labelled cells were incubated were isolated by specific absorption to and elution from erythrocytes. The eluted proteins were characterized by gel electrophoresis and column chromatography and were found to be primarily 68,000–72,000 d ‡ polypeptides, although lower molecular species (35,000d, 30,000d) believed to be proteolytic fragments of the larger polypeptides were sometimes detected. Lyl +,2−, Lyl −,2+, and (by inference) Lyl +,2+ cells produced antigen-binding proteins. However, the majority of cell-associated polypeptides were derived from Ly2 + cells, while antigen-binding proteins secreted by the cells were derived primarily from Lyl + cells. Moreover, the production of detectable levels of antigen-binding proteins by Ly2 + cells required interaction with Lyl + cells.

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.

Applying a sensitive colorimetric method for serum lipase activity in murine serum, BALB/c were found to have high levels. By contrast. B6 mice and mice of several other strains studied showed low serum lipase activity. Selecting BALB/c and B6 mice for analysis, we studied the influence of genetic crossing and bone marrow transplantation to investigate mechanisms involved in control of serum lipase activity. Compared to BALB/c, F 1 hybrids of these two strains exhibited decreased serum lipase activity, as did B6 mice, suggesting that serum lipase levels may be genetically determined. High levels of serum lipase behaved as an apparent autosomal recessive trait; an autosomal codominant inheritance could not be ruled out, however. Bone marrow transplantation, using a method based on immunologic elimination of post-thymic cells to prevent graft-vs-host disease, showed that B 6→ BALB/c, B 6→ B 6, and BALB/c→ B 6 had low lipase levels, while BALB/c→ BALB/c had high lipase levels. These findings suggest that transplanted marrow cells can influence the level of serum lipase activity if the marrow comes from a donor bearing an apparent genetically dominant trait, even though bone marrow cells themselves, or their descendents, do not exhibit lipase activity or generate lipase.

Relatively large numbers of nonimmune spleen cells do not protect against the local growth of two lymphomas. However, this heterogeneous population of splenic lymphocytes contains a subset of cells that efficiently protects against in vivo tumor growth. This cell population (cell-surface phenotype Thyl.2(-)Ig(-)Ly5.1(+)) represents less than 5 percent of the spleen cell population and is responsible for in vitro NK-mediated lysis. Although these studies clearly and directly demonstrate that Ly5(+) NK cells selected from a heterogeneous lymphoid population from nonimmune mice can protect syngeneic mice against local in vivo growth of two different types of tumor cells (in contrast to other lymphocyte sets within the spleen), they do not directly bear upon the role of NK cells in immunosurveillance. They do indicate that highly enriched Ig(-)Thyl(-)Ly5(+) cells, which account for virtually all in vitro NK activity, can retard tumor growth in vivo. It is difficult to ascribe all anti-tumor surveillance activity to NK cells, because they probably do not recirculate freely throughout the various organ systems of the body. Perhaps NK ceils may play a role in prevention of neoplastic growth within discrete anatomic compartments where there is rapid differentiation of stem cells to mature progeny (e.g., bone marrow, spleen, and portions of the gastrointestinal tract)and may normally act to regulate the growth and differentiation of non-neoplastic stem cells. Long-term observation of chimeric mice repopulated with bone marrow from congenic or mutant donors expressing very low or very high NK activity may help to answer these questions.

Purified Ly1 cells induce other T-cell sets to exert potent feedback inhibitory activity and this T-T interaction has been shown to play an important role in regulating in vivo immune responses. Approximately 2/3 of Ly1 cells also express the Qa1 surface phenotype (Ly1:Qa1+ cells). The experiments reported here indicate that Ly1:Qal+ cells are responsible for induction of feedback inhibition and that signals from both Ly1:Qal+ cells and Ly1:Qal- cells are required for optimal formation of antibody by B cells.

When B mice are supplied with Ly1 or Ly23 cells they acquire, over the next 6 mo, only the immune functions associated with each of these T-cell subclasses, respectively. The T-cell population of these "B-Ly1" and "B-Ly23" mice mice also remains restricted to the Ly1 and Ly23 subclass phenotypes. Thus the Ly1 and Ly23 populations are derived from two separate lines of differentiation and are not sequential stages of a single differentiative pathway.

Antigen-stimulated Ly1:Qa1+ cells induce a nonimmune set of T-acceptor cells (surface phenotype Ly123+Qa1+) to participate in the generation of specific suppressive activity. The experiments reported here were designed to test the possibility that the interaction between T-inducer and T-acceptor cells might be governed by genes linked to the Ig locus. We find that inducer:acceptor interactions occur only if the inducer and acceptor T-cell sets are obtained from donor that are identical at the Ig locus and are independent of the Ig locus expressed on the B cells used for assay of T-helper activity. In addition, experiments using inducer and acceptor T cells from the congenic recombinant BAB. 14 strain show that T-T interactions are not governed by Ig-CH genes, per se. These data indicate that T-inducer: T-acceptor interactions are governed by Ig-linked genes that may control expression of VH-like structures on T cells, or control expression of as yet unidentified cell-surface molecules.

This report that (1) cells mediating NK activity in different inbred mouse strains selectively express one of two allelic products specified by theLy-5 locus (or a locus tightly linked to it) and (2) this surface structure may directly contribute to NK-mediated cytolysis, since Ly 5 antiserum specifically inhibits NK activity in vitro in the absence of complement.

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.

CELL-MEDIATED CYTOTOXICITY (CMC) against C3H/Umc (C3H) syngeneic mammary tumours measured in vitro, after in vivo immunisation, is mediated by cytotoxic T lymphocytes (CTL) and is directed mostly against cross-reacting antigens related to the mouse mammary tumour virus (MTV)1–3. The long-term CMC assay required for detecting this response against adherent target cells has complex kinetics and at least two superimposing components: (1) an initial early peak of cytotoxicity, detectable at approximately 6 h and accounting for 20–30% of the CMC, and (2) a later peak, detectable after 18–20 h and representing an additional 40–50% of the cytotoxic effect3. Both peaks are mediated by CTL with the Ly23 phenotype (that is, Ly1−Ly2,3+); however, non-cytotoxic T cells of Ly1 and probably Ly123 phenotype (that is, Ly1+Ly2,3− and Ly1+Ly2,3+, see refs 3 and 4 for nomenclature and functional characteristics of these T-cell subsets) are required for the expression of the second cytotoxic peak3. On the other hand, the short-term assays of CTL-mediated CMC show single-hit kinetics5, perhaps comparable to part of the early CMC peak in our system. During our studies of immunity to syngeneic mammary tumours, we observed that when C3H or C3Hf immune CTL were tested against allogeneic MTV-induced mammary tumour targets, a relative degree of H–2 restriction of CMC was observed2, although not of the magnitude detected in other systems using mostly short-term CMC assays6–11. The experiments reported here show that when the kinetics of the CMC response against adherent syngeneic and allogeneic mammary tumour targets were compared, some differences became apparent: whereas the early CMC peak showed absolute H–2 restriction, no restriction was observed in the late CMC peak, although both events were mediated by Ly23 CTL (ref. 3).

A TL+ leukemia of a (B6 X A)F1 hybrid mouse (H-2b/H-2a) was previously subjected to immunoselection against H-2a by passage in (B6 X A.SW)F1 mice (H-2b/H-2s). A variant leukemia line was obtained that serologically lacked not only the H-2a phenotype but also the TL phenotype determined by the linked cis Tlaa allele of strain A. The H-2b phenotype and the TL phenotype of the Tlab allele of the B6 strain, which is expressed only by leukemia cells, were retained by the variant. Southern blotting with an H-2 cDNA probe that identifies restriction fragment polymorphisms distinguishing alleles of the H-2 and Tla regions of the B6 and A strains indicates that both the H-2a and Tlaa alleles are missing from the genome of this H-2a:Tlaa negative variant. Since the variant has two apparently unaltered chromosomes 17, where the H-2:Tla complex is situated, and since the intensity of bands in Southern blotting is suggestive of H-2b homozygosity, it is considered that loss of the H-2a:Tlaa haplotype by the variant was accompanied by duplication of the H-2b:Tlab haplotype. The implied change from heterozygosity to homozygosity that the variant has undergone with respect to H-2:Tla was not paralleled by a similar change at the three other loci tested, since the variant retained heterozygosity for Pep-3 (chromosome 1), Gpi-1 (chromosome 7), and Es-1 (chromosome 8).

Trisomy of chromosome 15 is a common feature in murine T-cell leukemias of chemical, viral and spontaneous origin. It is not present in all T-cell derived lymphomas, however. In order to study whether trisomy 15 was restricted to a specific T-cell subtype, diploid and chromosome 15 trisomic M-MuLV induced leukemias and derived in vitro lines were compared with regard to histology, karyotype, Thy-1,2 and Lyt-1, 2, 3 and 5 antigen expression. Histologically trisomic lymphomas showed a higher mitotic rate, higher peri-cellular reticulum content and less small cell infiltration than diploid tumors. It was not possible to distinguish trisomic from diploid lymphomas in blind tests based on these criterias, however. Trisomic and diploid lymphoma cells were indistinguishable in morphology. Three of nine lymphomas induced by M-MuLV inoculation were trisomic; two of them were thymomas. Transplantation to syngeneic recipients of the opposite sex and study of the sex chromosome marker was used to establish the origin of the enlarged organ. Upon subcutaneous transplantation, trisomic tumors and thymomas grew locally and were of donor cell type whereas splenic lymphomas caused spleen enlargement, as a rule, and were of donor or recipient type. Transplantation of bone marrow from mice infected with M-MuLV as newborns gave rise to lymphomas with the same sex as the donor and established the presence of preleukemic T-cells in the bone marrow of the donor. In vitro lymphoma lines expressed four different patterns of Lyt antigens: (a) 1.2 + , 2.2 + , 3.2 + , 5.1 + (4 lines); (b) 1.2 + , 2.2 − , 3.2 − , 5.1 + (5 lines); (c) 1.2 + , 2.2 + , 3.2 − , 5.1 + (7 lines); (d) individually distinct patterns (3 lines). Each group contained Thy-1.2 − and Thy-1.2 + lines. The trisomic tumors were mostly in group (a), all of them were Thy-1.2 positive. Diploid tumors were randomly distributed largely between groups (b) and (c). Two of three tumors in group (d) were aneuploid (not trisomic for 15). In spite of the differences observed between trisomic and diploid lymphomas, no specific T-cell subset could be assigned to the two groups. This is in line with the finding of trisomy 15 in some B-cell lymphomas suggesting that this anomaly may contribute to the neoplastic change in a variety of lymphocyte subsets, representing different lineages and steps of differentiation.

Two-dimensional chymotryptic peptide maps of Lyt-1.1 and Lyt-1.2 from Lyt-1 congenic thymocytes labeled with 125I in the usual way before lysis did not significantly differ, but there was a characteristic difference between maps of Lyt-1.1 and Lyt-1.2 obtained from 125I-labeled solubilized membrane fragments. We conclude that the Lyt-1 locus of chromosome 19 includes the protein-structural gene for Lyt-1. This conclusion is further supported by evidence with 35S-cysteine-labeled thymocytes: Thus an early 62K intermediate form, and a 60K form from tunicamycin-treated cells devoid of N-linked and O-linked carbohydrates, were precipitated by Lyt-1 alloantibody, which implies that the allo-specificity of Lyt-1 glycoprotein (67K) resides partly or wholly in protein.

An I-J-subregion controlled determinant is expressed on Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit. Ly-1 T cells absorb the I-J antibody reactive with the Ly-1,2 acceptor T cell, suggesting that both inducer and acceptor T cells have the same 1-J determinant. Since less than 10 percent of Ly-1 or Ly-1,2 T cells are killed by anti-I-J plus complement treatment, the I-J determinant demarcates functionally distinct subsets of both the Ly-1 and Ly-1,2 T-cell sets. This I-J determinant is not expressed on a detectable number of Ly-1 helper T cells which induce B lymphocytes to produce anti-sheep red cell antibody in tissue culture.

A notable feature of Ly-5, among immunogenetic systems that identify glycoproteins of the cell surface and define the surface phenotype of cells according to their lineage, is that the Ly-5 locus specifies a range of molecular isoforms that distinguish cells of different stages and branches of hematopoietic development. The composition of the Ly-5 locus is of much interest in regard to how these isoforms are constructed and differentially regulated according to cell lineage. We describe here a cDNA clone, pLy-5-68, that identifies Ly-5. The Ly-5 specificity of the pLy-5-68 clone was first indicated by a restriction fragment length polymorphism (RFLP), which in Southern blotting distinguishes genomic DNA of C57BL/6 (B6) mice (Ly-5a) from that of B6-Ly-5b congeneic mice whose genome is the same as B6 except for the segment of chromosome 1 that bears Ly-5b. For the following reasons it is unlikely that pLy-5-68 represents a gene linked to Ly-5 that was carried over with Ly-5b during serial backcrossing to make the B6-Ly-5b congeneic strain. In all mouse strains tested, the serological Ly-5 allotype (Ly-5.1 vs. Ly-5.2) accorded with the RFLP pattern. Cells of the ST/bJ mouse strain have unique Ly-5 serological reactions and ST/bJ DNA gives a unique (third) RFLP pattern (Ly-5c) with pLy-5-68. All Ly-5+ cell types reacted positively with pLy-5-68 in RNA transfer blotting, and all Ly-5- cell types tested did not. The difference in size of mRNA reactive with pLy-5-68 in cells expressing the 200-kDa Ly-5 isoform as compared with cells expressing the 220-kDa Ly-5 isoform corresponded with the difference in size of the protein components of those isoforms.