Definition of new alloantigens encoded by genes in the Ly-6 complex

Three alloantigens encoded by Ly-6-linked genes are defined by monoclonal antibodies. The Ly-27.2 antigen is defined by antibody 5075-19.1, Ly-28.2 by 5075-3.6, -12.1, -16.10 and by 5095-16.6. The strain distribution pattern of these antibodies is the same and identical with Ly-6.2. However the tissue distribution of these antigens is unique and distinguishes these antigens from the Ly-6.2 antigen or any known antigen encoded by Ly-6-linked genes. Ly-27.2 is present on all thymocytes, T cells, and B cells but is absent from bone marrow cells, whereas Ly-28.2 is absent from most thymocytes and is present on a subpopulation of T cells and B cells but is found on 60–70% of bone marrow cells. No recombination between the Ly-6/Ly-27/Ly-28 loci was found in linkage studies using 41 recombinant inbred strains and 57 backcross mice and indicates very close linkage of these genes. In addition, close linkage to 24 minor histocompatibility genes was excluded using the Bailey HW bilineal congenic mice. The data presented indicate that either the Ly-6 complex is composed of a family of tightly linked genes or the antigens are the products of a single gene that undergoes extensive modification during differentiation.     

T cell recognition of Mlsc,x determinants

Among a large number of cow insulin-specific T cell clones derived from both C57BL/10 and B10.A strains, several were found to react to non-MHC-linked gene products of a number of allogeneic strains. The stimulatory moiety for three of these clones correlates, in part, with expression of Mlsc, as defined by mouse strains C3H/HeJ and A/J. In addition, all three of these clones are stimulated by cells from strain PL/J, which has the poorly defined Mlsx allele. The data strongly suggest that Mlsx may, in fact, be Mlsc or is, at least, highly cross-reactive with Mlsc. Segregation analysis by using (B10.D2 X PL/J)F2 mice demonstrates that the Mlsx gene is genetically independent of the Mlsa linked Ly-9 marker on chromosome 1. Further studies with the use of these Mlsc,x-reactive clones reveal that they also recognize a gene product present in many mouse strains including DBA/2 which were previously phenotyped as Mlsa. However, testing of BxD recombinant inbred lines excludes Mlsa as being the stimulatory moiety. We therefore propose reclassification of the Mls phenotypes of several mouse strains based upon a two-locus model for Mls.     

Monoclonal antibodies to ThB detect close linkage ofLy-6 and a gene regulating ThB expression

We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976).Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene,Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying theThb ^h allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry theThb ^l allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.This work was supported in part by grants from the National Institutes of Health (AI-08917, CA-04681, GM-17367).     

Investigations into the nature of Igh-V region-restricted T cell interactions by using antibodies to antigens on methylcholanthrene-induced sarcomas. I. Analysis of an Igh-V-restricted suppressor-inducer factor

We have previously demonstrated the relationship between antigens on BALB/c methylcholanthrene (MC)-induced fibrosarcomas and T cell regulatory molecules by using a variety of antisera raised to these sarcomas in BALB/c and BALB/c X C57BL/6 (CB6F1) mice. One such pool of antiserum, a CB6F1 anti-CMS 4 (Pool XIV) serum, was used to investigate the nature of the T cell regulatory structures recognized by these antibodies. Pool XIV antiserum was capable of blocking the induction of feedback suppression by Ly-1 TsiF, an SRBC-specific suppressor T cell factor secreted by Ly-1+, 2- I-J+ T cells. Ly-1 TsiF induces suppression by interacting with an Ly-1+,2+ I-J+ T cell target. Successful interaction of Ly-1 TsiF with its target cell requires genetic homology between inducer and target cells at the variable region of the immunoglobulin heavy chain gene complex (Igh-V). The addition of Pool XIV antiserum to primary in vitro anti-SRBC cultures resulted in blocking the ability of Ly-1 TsiF from Igha (BALB/c) and Ighj (CBA/J) mice to induce suppression on syngeneic cells, whereas suppression induced by Ly-1 TsiF in Ighb (B6), Ighc (DBA/2), Ighd (A/J), and Ighe (AKR) mice are unaffected by addition of the Pool XIV antiserum. The ability of Pool XIV antiserum to block Ly-1 TsiF activity is linked to the Igh region, because Pool XIV antiserum can block Ly-1 TsiF from BALB/c (H-2d, Igha) and the Igh congenic B.C9 (H-2b, Igha) while not affecting Ly-1 TsiF activity on B6 (H-2b, Ighb) or its Igh congenic C.B20 (H-2d, Ighb). In CB6F1 animals, Pool XIV antiserum could block the ability of CB6F1 Ly-1 TsiF to suppress BALB/c spleen cells but not B6 spleen cells. Conversely, Pool XIV antiserum could block the ability of BALB/c Ly-1 TsiF to suppress CB6F1 spleen cells, whereas B6 Ly-1 TsiF showed normal suppressive activity in the presence of Pool XIV antiserum. In contrast, Pool XIV was capable of blocking the ability of Ly-1 TsiF from BALB/c into CB6F1 bone marrow chimeras (BMC) to suppress both BALB/c and B6 mice, whereas the activity of Ly-1 TsiF from B6 into CB6F1 BMC on BALB/c or B6 spleen cells was unaffected by the addition of Pool XIV antiserum. We then investigated the molecular nature of the molecule recognized by Pool XIV antiserum on the Ly-1 TsiF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of Ly-6 on activated T and B cells: Possible identity with Ala-1

The antigens Ala-1 and Ly-6 were first thought to be different on the grounds that Ala-1 was present only on activated T and B lymphocytes while Ly-6 was present only on post-thymic T lymphocytes. In this paper, it is shown that Ly-6 is expressed on activated B cells, including PFC and LPS blasts, and that after typing of several recombinant inbred lines,Ala-1 andLy-6 remain genetically inseparable. Based on available data, it is most likely that Ly-6 is in fact Ala-1, although further testing is required to confirm the absence of Ly-6 from nonactivated lymphocytes.Abbreviations used in this paper Con A concavalin A - LPS lipopolysaccharide - SRBC sheep red blood cells - PFC plaque-forming cells - RI recombinant inbred strains - B6 C57BL/6 mice     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

In vivo effects of hyperdiploid Ly-1+ B cells of NZB origin

Cells with increased chromosome number and DNA content have been found in the spleens of old NZB mice. These hyperdiploid cells are of clonal origin and demonstrate discrete IgH chain gene rearrangements by Southern blot analysis. In this report, hyperdiploid cells were analyzed by three-color flow cytometric techniques and found to be Ly-1+ B cells which were dull for Ly-1 and bright for surface IgM. These cells, unlike typical diploid Ly-1+ B cells, were negative for B220/6B2 and surface IgD. Hyperdiploid Ly-1+ B cells were found to be the predominant splenic subpopulation in animals receiving a spleen cell transfer from donors which possessed hyperdiploid Ly-1+ B cells. (NZB x DBA/2)F1 recipients of NZB spleen cells demonstrated a 10- to 1000-fold increase in Ly-1+ B cells in the spleen but showed no increased levels of Ly-1+ B cells in the peritoneum. Nearly all the splenic Ly-1+ B cells were hyperdiploid with the phenotype of the NZB parent. Cytogenetic analysis revealed that all the hyperdiploid cells were NZB donor cells. These findings suggest that the increase in splenic Ly-1+ B cells in the F1 recipients was due to expansion of injected splenic hyperdiploid Ly-1+ B cells of NZB origin. All of the F1 recipients of NZB hyperdiploid Ly-1+ B cells demonstrated a significant decrease in endogenous B cells as well as decreased serum IgM and anti-ssDNA autoantibodies. These studies suggest that hyperdiploid Ly-1+ B cells are different from typical peritoneal Ly-1+ B cells both in the lymphoid organs to which they home and in their proliferative capacity. NZB hyperdiploid Ly-1+ B cells, which may arise as a natural consequence of hyperactive Ly-1+ B cells, may play an immunoregulatory role in the spleen.     

Absence of cross-reactivity between murine Ly-6C and Ly-6G.

BACKGROUND: The Ly-6 family has many members, including Ly-6C and Ly-6G. A previous study suggested that the anti-Ly-6G antibody, RB6-8C5, may react with Ly-6Chi murine bone marrow (BM) cells. This finding has been interpreted as cross-reactivity of RB6-8C5 with the Ly-6C antigen, and has been generalized to many hematopoietic cell types, using the terminology Ly-6G/C. The present study was undertaken to determine whether anti-Ly-6G antibodies truly cross-react with the Ly-6C antigen on multiple hematopoietic cell types. METHODS: Splenocytes, thymocytes, and BM cells obtained from Ly-6.1 and Ly-6.2 strains of mice were stained with a variety of antibodies to Ly-6C and Ly-6G. Flow cytometric analysis was performed on these populations. RESULTS: Evaluation of anti-Ly-6C and anti-Ly-6G staining showed only Ly-6C expression and no Ly-6G expression on subsets of splenic T and B cells and thymocytes from Ly-6.1 and Ly-6.2 mice. Bone marrow cells were identified that express both Ly-6G and Ly-6C; no Ly-6G+Ly-6C- populations were seen. CONCLUSIONS: Multiple Ly-6C+ hematopoietic cell populations were identified that do not stain with anti-Ly-6G antibodies. This calls into question the use of the Ly-6G/C nomenclature and suggests that epitopes recognized by anti-Ly-6G antibodies should simply be designated Ly-6G.     

Ly-1 B helper cells in autoimmune "viable motheaten" mice

Previous work has demonstrated that Ly-1 B cells from normal C57BL/6J mice help the response of B cell subsets to the 4-hydroxy-3-nitrophenylacetyl hapten (NP). This regulatory cell population, called BH, preferentially helps the expression of plaque-forming B cells which express a predominant set of serologically related determinants collectively known as the NPb idiotype family. The specificity of BH cell activity in the NP system is a reflection of NPb idiotype-specific BH cell surface receptors. Thus, BH cells recognize autologous (i.e., idiotype) antigens. Given these observations and previous associations of increased Ly-1 B cell frequency in autoimmune mice, it was hypothesized that autoreactive Ly-1 BH cells may be present in high frequencies and in an activated state in autoimmune mice. To test this hypothesis the immunologic activity of BH cells in autoimmune viable motheaten (mev/mev) mice was studied. It was determined that splenic BH cells are approximately 10 times more frequent in viable motheaten than normal mice. The fact that BH cells from viable motheaten mice are activated was suggested by the presence of NPb idiotype-specific BH replacing helper activity in sera or B cell supernatants from these autoimmune mice. The soluble helper activities constitutively produced in mev/mev splenic B cell cultures and detected in mev/mev serum were resolved into two moieties, an NPb idiotype-specific immunoglobulin and a nonimmunoglobulin lymphokine(s) fraction. Purified mev/mev B cell-derived B cell maturation factor could substitute for the lymphokine moiety in the NPb idiotype helper cell assay. These results suggest that at least two signals, anti-idiotype immunoglobulin and a late-acting B cell maturation factor, are required for BH-dependent helper activity. The relationships of these results to current concepts of B cell activation mechanisms and the possible association of Ly-1 BH cells with autoimmunity are discussed.     

Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1--Gdc-1 region to mouse chromosome 15

Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results of preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships. A cloned genomic DNA fragment derived from a retroviral site has been used to define a novel locus, Pol-5, that is tightly linked to both H-30 and Ril-1 as shown by analysis of the B6.C-H-30 ^c congenic mouse strain. Following the segregation of the Pol-5 mouse-specific DNA fragment in a series of somatic cell hybrids carrying various combinations of mouse chromosomes on a rat or Chinese hamster background mapped Pol-5 to mouse chromosome 15. During the course of these studies, restriction fragment length polymorphisms were defined associated with several loci, including Pol-5, Ly-6, Sis, Ins-3, Krt-1, Int-1, and Gdc-1. Three of these loci, Sis, Int-1, and Gdc-1, have been previously mapped to chromosome 15 by others using somatic cell hybrids or isoenzyme analyses. Following the inheritance of these eight loci in recombinant inbred strains of mice allowed the definition of a linkage group on the chromosome with the order Ly-6-Ril-1--Sis--H-30--Pol-5--Ins-3--Krt-1--Int-1--Gdc-1. Analyses of alleles inherited as passengers in B6.C-H-30 ^c, C3H.B-Ly-6 ^b, and C57BL/6By-Eh/+ congenic mouse strains and in situ hybridization experiments support the above gene order and indicate further that the cluster is located on distal chromosome 15, with Ly-6 and Sis near Eh.Abbreviations A agouti - Abl cellular homolog of the Abelson leukemia virus oncogene - Ada adenosine deaminase - Ak-1 adenylate kinase-1 - AXB A/J × C57BL/6J recombinant inbred strain - B2m beta-2 microglobulin - BXA C57BL/6J × A/J recombinant inbred strain - BXD C57BL/6J × DBA/2J recombinant inbred strain - BXH C57BL/6J × C3H/HeJ recombinant inbred strain - CXB BALB/cBy × C57BL/6By recombinant inbred strain - DNA deoxyribonucleic acid - Eh hairy ears - Fpgs folypolyglutamyl synthetase - FXI fractionated x-irradiation - Gdc-1 glycerol phosphate dehydrogenase-1 - Il2r IL-2 receptor - Ins-3 a novel insulinlike gene - Int-1 mammary tumor integration site-1 - Itp inosine triphosphatase - Krt-1 the locus designated here includes a cluster of at least three keratin genes - LTR long terminal repeat - Ly lymphocyte - Lv-6 lymphocyte antigen-6 - Ly-11 lymphocyte antigen-11 - MIH minor histocompatibility - Myc cellular homolog of the Abelson leukemia virus oncogene; pa, pallid; - Pol-5 locus encoding retroviral polymerase-5 - RFLP restriction fragment length polymorphism - RI recombinant inbred mouse strains - Ril-1 radiation-induced leukemia susceptibility-1 locus - SDP strain distribution pattern - Sis cellular homolog of the simian sarcoma virus oncogene - SFFV spleen focus-forming virus - Tpi-1 triosephosphate isomerase-1 - Ve velvet     

Characterization and differentiation of CD4-, CD8- thymocytes sorted with the Ly-24 marker

Heterogeneity within the CD4-, CD8- thymocyte population was explored by flow microfluorometry on thymocytes from 6-wk-old female C57BL/6 mice. Double negative cells were obtained by twice killing thymocytes with anti-CD4 and anti-CD8 antibodies. The resultant population lacked CD4, CD8, and Ig on cell surfaces; it contained cells bearing Ly-24 (27%), Ly-6C (6%), and Ly-5 (B220) detected by 6B2 (6%). These markers are the same as those characteristic of lpr/lpr T cells; they also are found on bone marrow cells. In order to investigate the maturational pathway of CD4-, CD8- thymocytes, such cells were cultured in vitro for 6 days with phorbol myristate acetate + calcium ionophore. There was a marked increase in cells bearing Ly-24 with time; by 6 days essentially all bore Ly-24. A lesser increase (to 15%) in 6B2 + Thy-1 positive cells was observed. Small numbers of cells bearing CD4 and/or CD8 also were found after 6 days in vitro. In additional studies, CD4-, CD8- cells were first sorted with respect to Ly-24 and then cultured with phorbol myristate acetate + calcium ionophore. Ly-24+ cells proliferated vigorously and formed clusters whereas Ly-24- cells did not. The former gave rise to large numbers of CD4+, CD8+ cells; the latter exhibited little differentiation. These studies demonstrate substantial heterogeneity within the CD4-, CD8- thymocyte population. Use of the markers Ly-24, Ly6C, and 6B2 allows a subdivision of such progenitor thymocytes. Different stages of maturation as well as possible lineages of cells may be investigated by combining such hemopoietic cell surface markers with in vitro culture.     

Evidence for the existence of distinct heterogeneity among the peripheral CD4-CD8- T cells from MRL-lpr/lpr mice based on the expression of the J11d marker, activation requirements, and functional properties

Autoimmune-susceptible, MRL-lpr/lpr (lpr) mice develop a profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in peripheral lymphoid organs. The source and the mechanism of this abnormal accumulation of cells is still unknown. Recently, we reported that a significant number (approximately 35%) of the CD4-CD8- cells expressed J11d, a marker expressed by immature thymocytes but not by mature functional peripheral T cells. In the present study, we investigated the phenotype, growth requirements, and functional properties of purified J11d+ and J11d- subpopulations. Using the mAb, F23.1, which recognizes a TCR determinant encoded by the V beta 8 gene family, it was observed that approximately 30% of the J11d+ and J11d- DN cells expressed this determinant. Further studies on the thymus revealed that J11d+ DN cells from lpr thymus also contained F23.1+ cells (approximately 25%), whereas, similar cells from normal MRL(-)+/+mice were all F23.1-, consistent with earlier reports in other normal strains. Further phenotypic studies revealed that the peripheral J11d+ and J11d- cells from lpr mice were similar in expressing CD3, Ly-5 (B220), and Ly-24 (Pgp-1) determinants. When stimulated with phorbol myristic acetate (PMA) and recombinant IL-2 (rIL-2), only J11d- cells but not J11d+ cells responded by proliferation. However, in the presence of calcium ionophore (A23187) and PMA, both J11d+ and J11d- subpopulations proliferated by producing and responding to endogenous IL-2 but not IL-4. The lymph node T cells from 1-month-old MRL-lpr/lpr mice responded strongly when stimulated with PMA + rIL-4 or PMA + rIL-6. In contrast both J11d+ and J11d- subpopulations failed to respond when similarly stimulated. The J11d+ but not J11d- cells demonstrated spontaneous cytotoxic activity against the NK-sensitive YAC-1 tumor targets. The J11d- cells did not exhibit cytotoxic potential in spite of culture with PMA + rIL-2. Even after repeated culture in vitro with PMA + A23187 or PMA + rIL-2, both J11d+ and J11d- subpopulations failed to express the mature phenotype bearing CD4 and/or CD8 antigens. The present study demonstrates the expansion of unique J11d+, alpha beta-TCR+, DN T cells with cytotoxic potential in lpr mice and further suggests the existence of phenotypic and functional heterogeneity among the abnormal lpr DN cells.     

Analysis of Ly-6.2-bearing murine lymphocyte subpopulations in relation to the T-lymphocyte markers,Thy-1, Lyt-1, and Lyt-2

Using immunofluorescence with a monoclonal anti-Ly-6.2 antibody and FACS analysis we have confirmed that the Ly-6.2 antigen is present on approximately 70% of mature T cells and B cells but on few immature lymphocytes. There is a wide range of antigen density among the Ly-6.2⁺ populations, with the mean density higher on T cells than B cells. Following Con A activation of splenocytes there was a sixfold increase in Ly-6.2 antigen density though approximately 20% of the activated lymphocytes were Ly-6.2^?. The increase in Ly-6.2 density was specific since similar density increases did not occur for the closely linked antigens ThB and H $\text{9}\text{25}$. By panning a predominantly T-cell population for Lyt-2-bearing cells, it was found that Lyt-2⁺ lymphocytes were either negative or dully staining for Ly-6.2. However, activated cells bearing the Lyt-2 antigen were all Ly-6.2 positive. Double-staining experiments showed that T cells which had high Ly-6.2 antigen densities also had high Thy-1 antigen densities. Corticosteroid-resistant thymocytes were highly enriched for Ly-6.2-bearing cells compared to untreated thymocytes and had staining profiles for Ly-6.2 which were similar to peripheral T cells, supporting the idea that steroid treatment selects for a phenotypically mature thymic population.     

Evidence for close linkage of a mouse light chain marker with the Ly-2,3 locus.

Light chains associated with normal serum immunoglobulin can be resolved into a finite number of discrete focusing bands by isoelectric focusing. Four distinct light chain patterns can be distinguished among the inbred mouse strains. In the present studies inheritance of the characteristic light chain patterns has been studied in the AKXL recombinant inbred lines (derived from C57L/J and AKR/J parental lines) and in the inbred Ly-2a,3a congenic line B6.PL-Ly-2aLy-3a/Cy as well as in individual backcross animals of an incipient Ly-2a,3a congenic strain. Virtually complete concordance was observed for the expression of light chains characteristic of phenotype B (AKR-J-like) and the expression of the Ly-2a,3a allele. This observation indicates that a locus controlling light chain structure and/or expression is closely linked (less than 2.6 map units) to the Ly-2,3 locus on mouse Chromosome 6. The locus controlling normal light chain IF-patterns has been designated Ef1.     

Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6 a and Ly-6 b haplotypes

We have studied the cellular basis for differential expression of the Ly-6A/E alloantigen on T cells obtained from mice of the Ly-6 ^a (10–20% Ly-6A/E ⁺) and Ly-6 ^b (50–60% Ly-6A/E ⁺) haplotypes. During T-cell ontogeny only a small fraction (< 12 %) of thymocytes expressed Ly-6A/E. By 4 weeks of age adult levels of Ly-6A/E bearing lymphocytes were seen in peripheral lymphoid tissue. Immunohistochemical studies of the thymus revealed that Ly-6A/E⁺ cells were located predominantly in the medulla with small clusters of Ly-6A/E⁺ cells throughout the cortex. Consistent with this result, phenotypic studies showed that in the adult thymus the majority of Ly-6A/E expression was on mature CD4⁺ CD8^– and CD4^– CD8⁺ cortisone-resistant and precursor CD4^– CD8^– thymocytes. However, a much higher percentage of CD4⁺ CD8^– and CD4^– CD8^– thymocytes as well as CD4⁺ CD8^– peripheral T cells expressed Ly-6A/E from Ly-6 ^b mice. Furthermore, although gamma interferon induced increased Ly-6A/E expression in certain thymocyte and T-cell subsets, this induction functioned preferentially for cells obtained from Ly-6 ^b mice. Studies using F₁ hybrid mice (Ly-6 ^a × Ly-6 ^b) indicated that the basal level of Ly-6A/E expression on these subsets appeared to be under codominant genetic control, whereas gamma interferon-induced regulation of Ly-6A/E expression appeared to be under dominant genetic control. Collectively, these results suggest that the expression of Ly-6A/E on a particular T-cell subset is established in the thymus and is a stable characteristic of each haplotype. In addition, the low levels of Ly-6A/E expression for the Ly-6 ^a haplotype appear to be partially due to the inability of the majority of resting CD4⁺ T cells to express Ly-6A/E and to the relatively poor induction of this protein by gamma interferon.     

IgE formation and Fc receptor-positive lymphocytes in normal, immuno-deficient, and auto-immune mice infected with Nippostrongylus brasiliensis

The IgE serum levels and IgE FcR-positive lymphocytes (Fc epsilon R) in the spleen and mesenteric lymph nodes (MLN) of normal and immunologically mutant strains of mice were determined before and 14 days after infection with Nippostrongylus brasiliensis (Nbr) parasites. By IgE rosetting of cells immunofluorescently stained for sIg. Thy-1.2, Lyt-2, and L3T4, only sIg+ IgE rosetting lymphocytes were detected in both normal and Nbr-infected mice. IgE high responder mice had the same percentage of Fc epsilon R+ spleen and MLN lymphocytes as low responder mice. After Nbr infection, the percentages of splenic and MLN Fc epsilon R+ cells increased in parallel to a similar increase of sIg+ B cells. Athymic C57BL/6J-nu mice had 62% Fc epsilon R+ spleen and 85% Fc epsilon R+ MLN cells before and after Nbr infection, but IgE serum levels were less than 5 ng IgE/ml. C57BL/6J mice with the viable moth-eaten mutation mev which have almost exclusively Ly-1+ B cells, had less than 1% Fc epsilon R+ lymphocytes and formed only small amounts of IgE. C57BL/6J mice with the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations had low numbers of Fc epsilon R+ cells but formed 15 to 30 times more IgE after Nbr infection than control C57BL/6J mice. The IgE response of mice with the beige mutation (bg) did not differ from control mice. Mice with the xid mutation had few Fc epsilon R+ and sIg+ cells but showed high IgE responses. These data demonstrate that Fc epsilon R are typical cell surface markers for approximately 90% of murine Ly-1-, sIg+ B cells and that the number of Fc epsilon R+ cells does not correlate with the capacity of the mice to form IgE. The IgE response to Nbr infection is normal in mice homozygous for the bg mutation, elevated in mice homozygous for the xid, lpr, and gld mutations, and decreased in mice homozygous for the mev and nu mutations.     

Chromosomal location of the Ly-49 (A1, YE1/48) multigene family. Genetic association with the NK 1.1 antigen

The Ly-49 (A1, YE1/48) Ag is a disulfide-linked dimer with 44-kDa subunits, and is expressed on the cell surface of rare T cell tumors of C57BL/6 origin. Although this Ag is undetectable by flow microfluorimetry analysis, normal cells have been shown to express the A1 Ag by immunoprecipitation experiments performed on surface radioiodinated spleen and thymus cells. We (J. Immunol. 143:1379, 1989) and others (J. Immunol. 142:1727, 1989) have recently isolated cDNA encoding the Ly-49 Ag. Southern blots with an Ly-49 cDNA probe revealed multiple bands, consistent with cross-hybridization to other members of a multigene family, and significant RFLP between the C57BL/6 and BALB/c strains. When the RFLP patterns displayed by other common laboratory strains as well as informative recombinant inbred strains were examined, the Ly-49 gene family displayed five RFLP patterns and the entire family was found to reside on a contiguous stretch of the distal portion of mouse chromosome 6, the same region to which the NK1.1 Ag has been mapped. Although tissue distribution studies and transfection analysis ruled out the possibility that Ly-49 was identical to NK1.1 Ag, approximately 20% of NK1.1 cells isolated from normal spleen coexpressed Ly-49 and all Ly-49+ cells were CD3-. Although spleen cells cultured in high doses of rIL-2 demonstrated similar coexpression of NK1.1 and Ly-49, approximately 10% of CD3+ cells coexpressed Ly-49. The chromosomal mapping data and the expression of the Ly-49 and NK1.1 Ag suggest that the NK1.1 Ag may be a member of the Ly-49 multigene family.     

Activation induced differential regulation of stem cell antigen-1 (Ly-6A/E) expression in murine B cells

Expression of stem cell antigen-1 (Ly-6A/E) is developmentally regulated in murine B cells. However, little is known about its modulation during B cell activation. We report here the differential regulation of Ly-6A/E expression in response to diverse activation signals in mature B cells. Stimulation of resting B cells through the antigen receptor (BCR) inhibited, Ly-6A/E surface expression in dose dependent manner. Activation induced downregulation of Ly-6A/E is specific to BCR mediated signaling events as stimulation of B cells with anti-CD40, lipopolysaccharide or interferon-γ induced upregulation of Ly-6A/E surface expression. The activation induced differential modulation of Ly-6A/E expression is mediated at the mRNA levels. A role for BCR signaling in inhibition of Ly-6A/E expression was further confirmed using STAT-1^−/− B cells, which expressed constitutive, but not inducible Ly-6A/E. The BCR induced inhibition of Ly-6A/E RNA and surface expression was mimicked by ionomycin, but not phorbol myristate acetate, indicating a role for calcium but not protein kinase C dependent signaling events. Inhibition of calcineurin reversed the BCR or ionomycin inhibited Ly-6A/E expression. Interestingly, in vitro differentiation analysis of Ly-6A/E⁺ and Ly-6A/E⁻ splenic B cells revealed the Ly-6A/E⁺ cells to be the major source of antibody production, suggesting a potential role for Ly-6A/E in B cell differentiation. These studies provide the first evidence for activation induced differential modulation and differentiation of Ly-6A/E⁺ B cells.     

T cell antigen receptor expression by subsets of Ly-2-L3T4- (CD8-CD4-) thymocytes

The V beta 8-specific mAb F23.1 and KJ16 were used as fluorescent stains to test for TCR expression on the surface of subpopulations of early, CD4-CD8- (L3T4-Ly-2-) thymocytes from adult CBA mice. A surprisingly high proportion (27%) of Ly-2-L3T4- thymocytes were strongly F23.1 and KJ16 positive. No positive cells were detected among Ly-2-L3T4- thymocytes from V beta 8-negative SJL mice. In contrast to the adult thymus, Ly-2-L3T4- cells from embryonic CBA thymus lacked F23.1-positive cells. Subsets of adult CBA Ly-2-L3T4- thymocytes were separated to determine which expressed V beta 8. The major subset, Ly-1 low B2A2-M1/69+Thy-1+Pgp-1-, representing a phenotype similar to embryonic Ly-2-L3T4- thymocytes and the phenotype commonly isolated from adult thymocytes as Ly-1 "dull," lacked cells strongly positive for F23.1. In contrast, a series of subsets of adult CBA Ly-2-L3T4- thymocytes which were B2A2-M1/69- and Pgp-1+ all included strongly F23.1-positive cells. A minor subset, negative for most markers except Pgp-1 and presumed on the basis of this phenotype and some reconstitution studies to include the earliest intrathymic precursors, contained 28% F23.1-positive cells. However, no F.23.1-positive cells were detected in equivalent "prethymic" populations from bone marrow or from athymic mouse spleen. The subsets of Ly-2-L3T4- thymocytes which were Ly-1 high, B2A2-M1/69-, and Pgp-1+ all contained about 70% F23.1-positive cells, indicating a V beta 8 usage much higher than the mature T cell average. These results indicate that a series of distinct developmental events have occurred within these CD4-CD8- thymocytes previously considered as a single group of early precursor cells, and that some aspects of repertoire selection may be occurring amongst thymocytes which lack CD4 or CD8.