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To determine whether the gene that controls the expression of H-Y ("male") antigen on human cells is Y-linked, we have compared the H-Y antigen level in normal males with that in three males with two Y chromosomes. Leukocytes from one XXYY and two XYY males express more H-Y antigen than leukocytes from normal XY males. We conclude that a structural gene or positive regulatory gene for H-Y antigen is on the human Y chromosome. Testing for the H-Y antigen may be of benefit in patients who have signs of masculinization but who lack an identifiable Y chromosome. Positive results for the H-Y antigen would be tentative evidence that the corresponding region of the Y chromosome was present, perhaps as part of a translocation, despite the absence of a typical Y chromosome.

Specific cell-mediated cytotoxicity (CMC) of lymph node cells from immunized C3Hf mice, against syngeneic C3H/Umc mammary tumor cells, assayed in vitro, is effected by T lymphocytes. This CMC response is biphasic, with an early peak attained within 6 hr and a second major peak beginning at about 18 hr. Effector cells of both the early minor and late major phases of the response belong to the Ly23 set. Other T cell sets evidently play no part in the early effector response. But specifically activated Ly1 cells help or amplify the major late-phase response. Nevertheless, the mixture of specifically activated Ly1 and Ly23 sets still does not completely reconstitute the late response, which implies that the Ly123 set is also needed for maximal expression of CMC in this system. These Ly123 cells must come from specifically immunized donors. It appears, therefore, that maximal CMC is achieved by the participation of specific Ly123 cells which in the late phase directly or indirectly give rise to Ly23 killer cells. Thus, although killing of syngeneic mammary tumor cells in the CMC assay is invariably effected by cells of the Ly23 set, specifically activated cells of the Ly1 set, and probably of the Ly123 set also, are participants in the interactions needed to produce a maximal CMC response.

Previous studies of mating preference signified that mice can sense one another's major histocompatibility complex (MHC) types, probably by olfaction. This conclusion has now been substantiated by the use of a Y-maze whose two arms were differentially scented with currents of air conducted through boxes occupied by B6 (H-2b) males and by B6-H-2k congenic males. Four B6 mice, two males and two females, were successfully trained, by water deprivation and reward, to enter the arm scented by B6 or B6-H-2k males. One of the males and one of the females were trained to select the B6-scented arm; the other male and female were trained to select the B6-H-2k-scented arm. Untrained mice showed no MHC discrimination in the maze. The performance of the trained mice in distinguishing between MHC congenic homozygous F2 segregants derived from a cross of B6-H-2k with B6 was as good as their performance in distinguishing the respective inbred strains, thus essentially eliminating alternative and significant additional explanations of MHC-associated sensory discrimination. The data further indicate that chemosensory discrimination of MHC types can be entirely dissociated from sex differences and from the circumstances of mating.

Mice can recognize one another by individually characteristic body scents that reflect their genetic constitution at the extremely polymorphic major histocompatibility complex (MHC) of genes on chromosome 17. Reproductive behavioral manifestations of this sensory communication system include MHC-related mating preferences and neuroendocrine responses that affect preimplantation pregnancy and arise from the MHC-related scent of alien males. We have shown previously that mice can be trained in a Y maze to distinguish the scents of urine of congeneic mice that differ genetically only at the MHC. By means of an automated olfactometer, we now show that rats also can similarly distinguish the urinary scents of MHC congeneic mice. Thus, the mode of individual recognition that depends on scents determined by MHC genes can operate across species barriers.

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.

The cell-mediated cytotoxicity (CMC) of nonadherent cells from the peritoneal cavity (NAPC) of alloimmunized mice can be measured by the [3H]proline microassay. The exhibition of thymus-derived (T) cell antigens on these killer cells was studied by incubating them with the relevant T-cell antisera and complement (C), under optimal conditions for lysis, before performance of the CMC assay. Under these conditions, the following T-cell antigens were demonstrable on the killer population in terms of percent reduction in CMC by the respective antisera: (a) Thy-1.1 (83%) and Thy-1.2 (100%), (b) MSLA (86%), (c) NTA-RA (a T-cell antigen recognized by naturally occurring autoantibody of NZB mice) (62%), (d) Ly1.1 )58%, (e) Ly-2.1 (11%; considered a marginal result) and Ly-2.2 (63%), and (f) Ly-3.2 (77%). The following were not demonstrable: (g) TL, and (h) Ly-1.2. (i) The antigen Ly-3.1 was not studied. Omission of C deprived all T-cell antisera tested of their capacity to suppress CMC, indicating that the cell components recognized by such antisera may perform no direct function in CMC. On the assumption that all Ly+ cells are Thy-1+, it is clear that the T-cell members of the immune NAPC population must be heterogenous. This follows from the fact that the proportions of T cells lysed by different Ly antisera did not correspond with ensuing degree of loss of CMC capacity. The extremes were represented by anti-Ly-1.2 (74% Thy-1+ cells lysed, but no reduction in CMC) and Ly-3.2 (54% Thy-1+ cells lysed, with 77% reduction in CMC). From this initial survey it appears that the C57BL/6 mice killer T-cell population active in CMC in vitro is relatively rich in surface antigens of the Ly-2/Ly-3 category and relatively poor in representation of the Ly-1 surface antigens. It remains to be seen whether this killer cell phenotype, poor in Ly-1 and rich in Ly-2/Ly-3, is characteristic of the mouse generally. From these results it appears that subsets of T cells with different immunological functions may exhibit qualitative or quantitative differences in surface antigens specified by different Ly loci; this will be easier to assess in the future when the results of experiments with the same Ly antisera but dealing with T-cell functions other than CMC become available.

Ly alloantigens coded by two unlinked genetic loci (Ly-1 and Ly-2/Ly-3) are expressed on lymphoid cells undergoing thymus-dependent differentiation. Peripheral Thy-1+ cells from C57BL/6 mice can be divided into three subclasses on the basis of differential expression of Ly-1, Ly-2, and Ly-3; about 50% express all three Ly antigens (Ly -123+), about 33% only Ly-1 (Ly-1+), and about 6-8% Ly-2 and Ly-3 (Ly-23+). Cells of the Ly-123+ subclasses are the first peripheral Thy-1+ cells to appear in ontogeny, and are reduced in the periphery shortly after adult thymectomy. In contrast, Ly-1+ and Ly-23+ subclasses appear later in the peripheral tissues than do Ly-123+ cells, and are resistant to the early effects of adult thymectomymperiheral lymphoid populations depleted of Ly-1+ cells and Ly-123+ cells (and thereby enriched for Ly-23+ cells) were incapable of developing significant helper activity to SRBC but generated substantial levels of cytotoxic activity to allogeneic target cells. The same lymphoid populations, depleted of Ly-23+ cells and Ly-123+ cells (and thereby enriched for Ly-1+ cells), produced substantial helper responses but were unable to generate appreciable levels of killer activity. These experiments imply that commitment of T cells to participate exclusively in either helper or cytotoxic function is a differentiative process that takes place before they encounter antigen, and is accompanied by exclusion of different Ly groups, Lu-23 or Ly-1 respectively, from TL+Ly-123+ T-cell precursors. It is yet to be decided whether the TL-phase by Ly-123+ subclass is a transitional form or a separately differentiated subclass with a discrete immunologic function.

The Q genes, specifying Qa antigens and situated in the extended part of the major histocompatibility complex (MHC) of the mouse, comprise a subgroup of MHC class I genes whose significance and function are still largely unknown. In screening a cDNA library made from the BALB/c inducer T-cell line Cl.Ly1-T1, we isolated 11 clones representing Q8/9, but none representing Q6 or Q7. Confirmatory evidence is given that the Q8/9 gene originated from fusion of the 5' region of the Q8 gene with the 3' region of the Q9 gene at a recombination site or hot spot in the vicinity of intron 4. Contrary to previous impressions that Q8/9 is an inert pseudogene, we find that the Q8/9 gene can be functional and encode a Qa-2, 3 antigen. One variety of the 11 Q8/9 clones isolated lacked exon 5, which encodes the transmembrane domain of class I glycoproteins, and thus may account for secretion of a soluble form of Qa-2, 3 antigen thought to be released by activated T cells.

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.

When a male mouse is presented with two H-2 congenic two female in estrus, his choice of a mate is influenced by their H-2 types. The term "strain preference" is used to describe the general tendency of the male population of one inbred strain to prefer two female of one H-2 type rather than another. The term "consistency of choice" is used to describe the added tendency of particular two males of one inbred strain, in sequential mating trials, to prefer two females of the H-2 type they chose in previous trials. Statistical analysis showed trends in the data that support the following conclusions: (a) The choice is made by the male, not the female. (b) The strain preference of two males may favor two females of dissimilar H-2 type (four of six comparisons), or of similar H-2 type (one of six comparisons). (c) Consistency of choice does not always correspond with strain preference. In one of six comparisons of H-2 genotypes there was no strain preference but pronounced consistency of choice by individual two male. This suggests memory, but fortuitous bias is not excluded. (d) Strain preference of the same male population may favor two male of the same or a different H-2 type, depending on which different H-2 type is offered as the choice alternative to self. These findings conform to a provisional model in which olfactory mating preference is governed by two linked genes in the region of H-2, one for the female signal and one for the male receptor. These mating preferences could in natural populations serve the purpose of increasing the representation of particular H-2 haplotypes or of maintaining heterozygosity of genes in the region of H-2.

Six new monoclonal TL antibodies are described. At least one new TL antigen is defined (TL.7), and at least one more Tla allele, bringing the total number of known Tla alleles to six. Five of the monoclonal antibodies, and probably all six, identify distinct TL antigenic specificities. Four of these antigens conform in strain distribution and expression on leukemia cells to antigens defined by conventional antisera. The data contain a hint that monoclonal TL antibodies like TL.m6 may serve to identify a region of the Tla gene, which determines whether or not prothymocytes will respond to physiological induction by expressing TL, and thus may provide a means to study the regulatory mechanism that determines whether mouse strains are phenotypically TL+ or TL-.

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.

The extent and diversity of T cell differentiation in nude athymic mice are matters of dispute. In this study, we examined the splenic T cell population of pathogen-free and germ-free nu/nu mice, treated or not treated with the pentapeptide analogue of thymopoietin (TP-5), in terms of TL, Qa-1, and Lyt phenotypes. At all ages, 50-60% of nu/nu splenocytes, enriched for T lymphocytes by removal of sIg+ cells, expressed T markers, as compared with greater than 85% in normal mice. At 2 mo of age, all nu/nu splenic T cells expressed the surface phenotype TL+:Thy-1+:Ly-123. This is abnormal in two respects: first, because expression of TL is normally confined to thymocytes; and second, because there was no evidence of the usual diversification into the subsets Ly-1 and Ly-23. From 10 wk of age onwards, diversification into Ly subsets was evident in nu/nu spleen, although the usual predominance of Ly-1 over Ly-123 cells was not attained, and some TL+ cells persisted. Also, the ratio of Qa-1+ to Qa-1- cells rose progressively to as high as 4:1 at 4-6 mo, in contrast to the usual ratio of approximately 1:1, regardless of age. In the spleens of nu/nu mice treated with TP-5 from 5-8 weeks of age and tested 1 wk later, the proportion of T cells was raised, though not to normal levels, the number of TL+ cells was reduced, and there was diversification into Ly sets.

In comparing spleen cells of inbred and congenic mice for spontaneous capacity to lyse cells of the BALB/c leukemia RLmale1 in vitro, we found that the activity of 129 spleen cells was more than double that of 129-Gix- spleen cells. The only known difference between these two strains is that 129-Gix- mice express no known demonstrable gp70 or p30, whereas 129 mice express both these MuLV-related components as mendelian traits not associated with the production of virions. We infer that MuLV-related components at the cell surface are concerned in effector-target interactions leading to cytolysis under the conditions described. Although the congenic strains B6 (Gix-) and B6-Gix+ differ likewise in expression of the type-variant Gix-gp70, both strains express a second type-variant of gp70. The lytic activity of spleen cells of these two strains for RLmale1 cells was equally high, suggesting that involvement in lytic effector-target interactions is common to gp70 molecules in general. When used as targets rather than as effectors 129 spleen cells were more sensitive to lysis than 129-Gix- spleen cells. Pre-exposure to gp70, purified from R-MuLV, rendered splenic effector cells less lytic. Pre-exposure to gp70 also rendered RLmale1 target cells less sensitive to lysis. One explanation of these findings is that both target cells and effector cells express gp70 and also receptors for gp70 and that this is the basis of mutual cellular recognition leading to lysis in the circumstances described.

It is known that mice can distinguish the H-2 haplotypes of other mice by scent and that urine is a potent source of the characteristic odors governed by H-2. It now is shown that genetic disparity confined to the Qa:Tla region of chromosome 17, adjacent to H-2, is accompanied by distinctive urinary scents that can be distinguished by trained mice in a Y maze. This was confirmed by the transfer of training procedure in which mice trained to distinguish the urine of B6 mice from the urine of recombinant congeneic B6-Tlaa mice successfully distinguished the urines of Tlaa and Tlab homozygous F2 segregants of the cross B6 X B6-Tlaa in the absence of reward, including blind trials with coded urine samples. It also is shown that genetic disparity confined to the K end of H-2 is accompanied by distinctive urinary scents recognized in the Y maze. Thus, mice trained to distinguish the urines of B10 from B10.A congeneic mice successfully distinguished various combinations of B10, B10.A, and B10.A (2R, 3R, 5R, and 18R) recombinant congeneic mice representing genetic differences limited to the K end of H-2. It is suggested that the individual scents of mice comprise several odorous components, at least some of which may be incidental to quantitative or qualitative metabolic variations arising from polymorphism of genes such as H-2.

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.

Ly-5 is expressed by cells of the hematopoietic branch of development. Further serological analysis of the Ly-5 system, aided by Ly-5 monoclonal antibodies and by two Ly-5 congenic mouse strains, reveals two new Ly-5 alloantigens, Ly-5.3 and Ly-5.4. The data define three thymocyte phenotypes, Ly-5.1,3, Ly-5.2,4, and Ly-5.2,3, and three corresponding genotypes, Ly-5a, Ly-5b, and Ly-5c, respectively. Ly-5a is by far the most common allele. The Ly-5c allele is found only in the ST/bJ strain, a finding that accords with the presently unique pattern of restriction fragments previously observed in Southern blotting of ST/bJ DNA with an Ly-5 cDNA probe. Present serological and biochemical data favor the interpretation that the compound Ly-5 phenotype of thymocytes is attributable to two separate Ly-5 molecular isoforms that exhibit a discrete difference in protein composition, bear different Ly-5 antigens, and are produced jointly by thymocytes, unlike other Ly-5 isoforms previously shown to distinguish different hematopoietic cell lineages.

The Ly and Ia phenotypes of T lymphocytes involved in different functions were characterized by the use of specific antisera. T cells responsible for delayed-type hypersensitivity (DTH) and for helper functions were found to be Ly-1+,2- in contrast to cytotoxic T cells and T cells responsible for suppression of antibody responses which were Ly-1-,2+. Unlike some primed helper cells, T cells involved in DTH were Ia-. Suppressor cells in the system were Ia+.

Inbred male mice typically prefer to mate with females of a different, non-self H-2 haplotype. To determine whether this natural preference is irrevocable or results from familial imprinting, a test system was used which relied on previous observations that B6 males (H-2b) mate preferentially with congenic B6-H-2k rather than B6 females, and B6-H-2k males with B6 females. This preference was reversed in B6 males fostered by B6-H-2k parents and in B6-H-2k males fostered by B6 parents, preference in these cases favoring the same H-2 type. Thus, H-2 selective mating preference is acquired by imprinting on familial H-2 types.

The Ly-5 system of the mouse defines a set of transmembrane glycoprotein isoforms (T200, B220, etc) that hallmark various lineages and stages of hematopoietic differentiation. These isoforms are the products of a single Ly-5 gene comprising 34 exons, 32 of them (Exs-3-34) protein-coding and three (Exs-5-7) selectively represented in different isoforms (e.g., all three in isoform B220 but none in isoform T200). Probable structural features of Ly-5 glycoproteins, largely inferred from Ly-5 gene composition, are presented and compared with the rat L-CA and human LCA/T200 systems, which are phylogenetic counterparts of Ly-5 as an index of the extent and nature of structural conservation. The outer (N-terminal) region of the Ly-5 T200 isoform comprises three broadly similar domains (Exs-4, 8, 9) with salient features that jointly favor free interaction with the aqueous environment and are shared by the L-CA and human LCA/T200 systems despite an overall interspecies protein sequence similarity in this region of only about 50%. In the larger B220 isoform this region includes epitopes dictated by the selective exons Exs-5, 6, 7, these being more conserved than the shared exons Exs-4, 8, 9 and no doubt sustaining the differential functions of the respective isoforms. Comparison of the genomic sequences of Ex-5 in the Ly-5 and human systems suggests that a shift in splice donor site accounts for an extra 23 amino acids in the human Ex-5-coding domain, which is the only salient structural difference between the mouse Ly-5 and human systems. The inner extracellular region (Exs-10-16) includes subregions of high variability, but again there are shared salient interspecies similarities such as sites and numbers of Cys residues that imply a conserved, tightly-folded conformation, in contrast to the more open conformation predicted for the outer extracellular region. The transmembrane region (Ex-17) is highly conserved, as is the very large cytoplasmic region (Exs-17-34) which may interact with the plasma membrane but probably does not traverse it.

Antisera to purified structural proteins of Rauscher murine leukemia virus, the major envelope glycoprotein, gp69/71, and the major internal protein, p30, were studied by immunofluorescence of viable and fixed virus-infected cells and by virus neutralization. Group-specific and type-specific determinants of gp69/71 were demonstrated by immunofluorescence and virus neutralization tests, indicating that these determinants are located in the cytoplasm and probably on the cell surface as well as on virus envelope. Antisera against p30 showed anti-group and anti-interspecies activities by immunofluorescence with no virus-neutralizing activity. Both antigenic determinants of gp69/71 were sensitive to guanidine-hydrochloride and to a lesser degree to ether treatment, whereas the group-specific determinants of p30 were relatively stable to these treatments.

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.

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.

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, Tlae), intermediate (Tlac, Tlaf), and low (Tlab). (Relative amounts: 1000: 100: 1.) Some thymic leukemias arising in (Tlab, 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.

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.

The Ly-5 system of the mouse is expressed exclusively by hematopoietic cells and comprises a series of glycoprotein isoforms that typify different hematopoietic cell lineages. The 200-kDa isoform of T cells and the 220-kDa isoform of B cells are known to differ in peptide composition. The complete 1152 amino acid sequence of the 200-kDa isoform protein deduced from cDNA sequence appears to comprise a leader sequence of some 30 residues, an external N-terminal domain of 370 residues, a probably single transmembrane domain of 22 residues, and an unusually large cytoplasmic domain of 730 residues. Both the external and cytoplasmic domains include regions of internal homology suggestive of evolution from a smaller ancestral gene. RNA transfer blotting has previously shown that B-cell mRNA for Ly-5 is larger than T-cell mRNA. S1 nuclease protection mapping with Ly-5 cDNA probes suggests that this difference can be ascribed to interpolation of an extra B-cell sequence located at the 5' end of B-cell mRNA, probably immediately following the leader sequence. From restriction mapping of overlapping Ly-5 genomic clones spanning 60 kilobases it is concluded that Ly-5 isoforms are generated by differential processing of transcripts of a single gene, rather than from a family of linked Ly-5 genes.

C57BL/6 and C57BL/10 female mice were grafted with skin from male or female donors incompatible for H-2 and/or non-H-2 antigens. Syngeneic male grafts applied after the rejection of primary allografts or syngeneic male grafts were rejected in accelerated (second set) fashion, whereas male grafts applied after primary female grafts were not. In addition, C57BL/10 female spleen cells, primed in vivo with an allogeneic (BALB/c, CBA, or B10.BR) male graft and challenged in vitro in mixed lymphocyte culture with syngeneic (C57BL/10) male cells, produced cytotoxic cells specific for syngeneic male target cells. We conclude that at least some component of H-Y is detected by female responder cells on allogeneic male cells, and that the second set cell mediated response to H-Y is not necessarily restricted by the H-2 haplotype of the primary sensitizing strain. Moreover, (CBA X B10) F1 females, primed in vivo with male cells of one parental haplotype (B10 or CBA) and challenged in vitro with male cells of the other parental haplotype (CBA or B10), fail to lyse male target cells of either parental haplotype. It therefore seems unlikely that a helper determinant shared between B10 and CBA is sufficient to explain the ability of CBA male cells to prime H-2-restricted T-cell cytotoxic responses by B10 females.

Radiation chimeras were made by restoring lethally irradiated inbred mice with bone marrow cells of F1 hybrid mice of crosses between that inbred strain and an H-2-congenic strain. The urine of these chimeras was tested by the Y maze method, and shown to have acquired a scent indicative of the reconstituting donors' H-2 type. Thus, cells of the hematopoietic system contribute to the H-2-related odorant properties that enable mice to distinguish one another according to their H-2 types.

A polypeptide of 8500 molecular weight is described that induces the differentiation of T (thymus-derived) cell and B (bone-marrow-derived) cell immunocytes in vitro, apparently via beta-adrenergic receptors and adenylate cyclase activation. This polypeptide shows a high degree of evolutionary conservation, exhibiting close structural, functional, and immunological similarity when isolated from such diverse origins as cells of mammals and higher plants. This polypeptide was detected in animal cells, yeast, bacteria, and higher plants, and so may well be a universal constituent of living cells.

Class I products of the major histocompatibility complex (MHC) comprise a heavy chain of about 45 kDa noncovalently linked to a 12-kDa beta 2-microglobulin (beta 2m) light chain encoded on a different chromosome. We find that class I products of some mouse strains include an additional 62-kDa molecule which on the following evidence consists of a heavy chain linked covalently with beta 2m. Production of the 62-kDa protein invariably accorded with the occurrence of cysteine at position 121 of the heavy chain (Kb,Kbm1,Kbm3,Dd, and Ld). Substitution of arginine at position 121 invariably accorded with absence of the 62-kDa protein (Kbm6,Kbm7,Kbm9,Kd, and Db). On the basis of observed production versus nonproduction of the 62-kDa molecule, predictions are made regarding residue 121 in class I products for which this is not yet known; namely, Kk, Ks, and Dk, which produce the 62-kDa molecule, as compared with Kj, Qa-2, and TL, which do not. Reported differences in immunologic reactivity between Kb mutant strains with Arg-121 in place of Cys-121 imply that the occurrence of 62-kDa class I products in mice of Cys-121 genotype has functional consequences.

Lymphocytes from BALB/c or C57B6/6 mice that develop killer activity to alloantigens belong to the numerically small Ly-23 subclass of peripheral T cells, distinguished by selective expression of Ly-23 determinants on their surfaces. The maturation of these cells to killer cells can be amplified by Ly-1+ cells, which do not themselves contribute to the killer cell pool. This amplification was abolished by escluding Ia("Beta")+ cells from the stimulator population during mixed lymphocyte culture (MCL), suggesting that amplification is due to selective recognition of I region antigens by L-1+ cells, a conclusion already drawn from our previous evidence that Ia differences activate Ly-1+ cells but not Ly-23+ cells. These and other experiments indicate that amplification of killer cell production in vitro by Ly-1+ cells is not due to their conversion to Ly-23+ cells during MLC, but to their ability to recognize major histocompatibility complex determinants not recognized by Ly-23+ cells.

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.

The development of congenic mouse strains, strains which differ from their inbred partner strains by only a single selected character, was described; and three a examples of important finding made possible by the use of such strains were given. The first example showed how a gene in a major histocompatibility complex influences susceptibility to spontaneous leukemia. The second example illustrated how a particular constellation of genes in a congenic hybrid may lead to autoimmune disease that does not occur in either parental strain. The third example was concerned with function of major histocompatibility genes as identification markers which enable individual mice to recognize one an other by scent.

Thymectomy of mice aged 6-8 weeks causes a disproportion of splenic T cell sets, the Ly123 set being relatively decreased and the Ly23 set relatively increased (Ly123 decrease: Ly23 increase). A similar disproportion of splenic T cell sets was found to occur spontaneously with advancing age (12-18 months). By PA-SRBC assay, the total number of splenic Lyt+ cells is not appreciably reduced by thymectomy or by aging, but the Thy-1+ cell count falls by about 40% according to both PA-SRBC and cytotoxicity assays. Thus there is an increase in the number of Lyt+ cells expressing sub-threshold amounts of Thy-1. The following observations show that thymopentin (TP-5), a synthetic pentapeptide analogue of thymopoietin, counteracts these changes in thymectomized and aged mice. As reported previously, the capacity of C3H/HeJ female mice to reject C3H/HeJ male skin was raised by thymectomy, or with age, and treatment with TP-5 substantially normalized the rejection response. Here we correlate these findings with changes in the profile of splenic T cell sets. The splenic T cell set profile of thymectomized B6-Tlaa male and female mice was essentially restored by TP-5. The Ly123 decrease: Ly123 increase change caused by thymectomy was not associated with obviously altered proportions of Qa-1+ and Qa-1- subsets. Treatment of aged mice with TP-5 also prevented the onset of changes in splenic T cell sets that occur spontaneously with age. Thus thymectomy and aging give rise to disproportions of splenic T cell sets, and in C3H female mice to a heightened capacity for male skin rejection, both effects being largely abrogated by the TP-5 derivative of thymopoietin.

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.

Molecules specified by the H-2-linked T/t locus in the mouse are expressed on H-2-negative cells such as early embryos, sperm, and teratoma cells. By means of enzymatic radioiodination of cells and immunoprecipitation of lysates with congenic antiserum, one of these molecules, known as F9, has been obtained from sperm and teratoma cells and compared to H-2 isolated from murine splenocytes. Our studies indicate that both H-2 and F9 have identical molecular weights and subunit structure, including the presence of a B2-microglobulin-like subunit. These findings, taken together with previous studies of TL alloantigens, suggest that a number of gene products of the 9th linkage group show structural homology with each other and, in addition, with immunoglobulin. The genes in question may therefore have arisen from a primitive gene concerned with cellular recognition.