Identity of murine lymphocyte alloantigens DAG,ALA-1, Ly-8, and Ly-6?

The DAG, Ala-1, Ly-8 and Ly-6 lymphocyte membrane alloantigens, identified by immune sera raised in different strain combinations, were originally thought to be distinct specificities. With the use of a congenic strain, BALB-DAG, we have now further evidence for antigenic identity based on serological and genetic criteria.     

Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2.

On the basis of the pattern of lymphokines they secrete, murine T helper clones can be divided into two subsets, TH1 and TH2. This concept of two different T helper effector cells helps to explain the diversity of immune reactions occurring in different parts of the body. The in vivo localization of T helper subtypes is of great interest, but up to now no biochemical or surface markers were available to distinguish between them. We analyzed the glycolipids from altogether 12 murine TH1 and TH2 cell lines or clones. A comparison of the gangliosides by thin-layer chromatography showed differences between the TH1 and TH2 cells. Binding studies with specific antibodies to asialo backbone structures after degradation by neuraminidases showed that the main gangliosides from these lymphocytes shared a common GgOse4 backbone and thus differed only in their degree or position of sialylation. Two disialogangliosides appeared to be characteristic. They were isolated from the D10.G4.1 TH2 cell clone and identified by fast atom bombardment mass spectrometry as IVNeuAc,IINeuAc-GgOse4Cer (GD1a) and IVNeuAc,IIINeuAc-GgOse4Cer (GD1 alpha), respectively. GD1a was characteristically only detected in TH2 cells, whereas GD1 alpha was preferably, but not exclusively, expressed by TH1 lymphocytes. Although GD1a was also found in the lung, heart, kidney, and spleen, its expression within the murine immune cells under investigation was unique to TH2 lymphocytes. Scarcely any GD1a was found in thymocytes, B cells, or CD8 positive (cytolytic) T cells, but significant expression was seen in CD4 positive (helper) T cells which include the TH2 subpopulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in the relative numbers of Thy-1- and Lyt-2-bearing peripheral blood lymphocytes in mice: a longitudinal approach

Longitudinal and cross-sectional analyses of Lyt-2+ and Thy-1+ cell populations in the peripheral blood of aging male CBA and C57BL mice revealed that the relative number of Thy-1+, Lyt-2+ cells in the peripheral blood of both mouse strains remained relatively constant during the entire lifespan. The proportion of Thy-1+, Lyt-2- cells decreases with age, which indicates that major changes in the T-cell compartment with age must be attributed to the Lyt-2- helper compartment. For individual CBA mice, a direct relation between the relative number of Thy-1+, Lyt-2- cells at a certain age and the time remaining to live is demonstrated. The changes in the proportion of Lyt-2+ of total Thy-1+ cells in CBA mice show a regular pattern of slow increase with age followed by a rapid increase phase preceding the death of the animal. In C57BL mice, the development of the proportion of Lyt-2+ T cells with age showed various patterns. Rapid changes both positive and negative in these mice seem to be indicative of approaching death. The predictive value of Lyt-2+/Thy-1+ ratios at a given age for the remaining lifespan of individual mice is discussed.     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to the Lyt-1 locus

Spleen cells from a (BALB/c x C57BL/6)F1 mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called "Ly-10.1". This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.     

A new murine lymphocyte alloantigen,Ly-21.2, mapping to the seventh chromosome

Using a monoclonal antibody raised by fusing spleen cells from A/J mice, immunized with B10.A splenocytes and lymph-node cells, with a BALB/c myeloma, we have described a new surface alloantigen, Ly-21.2. Ly-21.2 is present in varying amounts in all lymphoid tissues, is not detectable in the brain, kidney, lung or erythrocytes, and is found in only trace amounts in the liver. Strain distribution studies showed that Ly-21.2 is present in all strains examined, including B10, except the A strain and segregation analysis of (A/J × 1310) F₂ mice showed that Ly-21.2 expression (1) is encoded by one gene and (2) is linked to albinism on chromosome 7. Studies performed on mice developing T-cell leukemia showed that, regardless of the etiologic agent, Ly-21.2 expression increases dramatically in mice with overt leukemia. In addition, preliminary studies suggest that expression of Ly-21.2 is linked to increased susceptibility of mice to Friend-virus-induced erythroleukemia.Abbreviations used in this paper B10 C57BL/10 - CPM counts per minute - FFU focus-forming units - FV Friend virus - i.v. intravenous - NMS normal mouse serum - PBS phosphate-buffered saline - RAMIG rabbit anti-mouse IgG - RIA radioimmunoassay     

Thy-1-negative and Ly-1-negative variants of T cells produce interleukin 2 in response to mitogens

IL 2 production by T cell variants, which lack the Thy-1 or Ly-1 surface glycoproteins, was studied. Cross-linking of the Thy-1 molecule resulted in IL 2 production by the EL4 thymoma and by a T cell hybridoma, suggesting that Thy-1 may play a role in T lymphocyte triggering. To further study the functional role of this molecule, Thy-1-negative variants were selected and analyzed for IL 2 production in response to phorbol-12-myristate-13-acetate (PMA) or to Con A. It was demonstrated that in spite of their failure to express Thy-1, the Thy-1-negative clones were capable of IL 2 production. These results indicated that although Thy-1 cross-linking triggers cell activation, a signal provided by Thy-1 is not indispensable for cell activation by mitogens. The T cell tumor line LBRM331A5 responds synergistically to IL 1 and PHA by releasing IL 2. It was demonstrated that anti Ly-1 monoclonal antibodies and PHA co-stimulated LBRM331A5 cells, as did IL 1 plus PHA. Thus, anti Ly-1 antibodies mimic the effect of IL 1, suggesting a role for Ly-1 antigen in T cell activation, perhaps by serving as an IL 1 receptor or as an associated molecule. To further study the functional role of Ly-1 and its relation to IL 1 receptor, Ly-1-negative variants of the LBRM331A5 cell line were selected and analyzed for IL 2 production in response to PHA plus IL 1. It was demonstrated that the Ly-1-negative clones were capable of IL 2 production as efficiently as Ly-1-positive clones. These results indicate that the Ly-1 and IL 1 receptor are distinct molecules, which are involved in different activation pathways.     

Biochemical characterization of the murine activated lymphocyte alloantigen Ly-6E.1 controlled by the Ly-6 locus

The Ly-6.1 alloantigen defined by monoclonal antibody SK70.94 and expressed predominantly on activated lymphocytes has been immunoprecipitated from lysates of cells biosynthetically radiolabeled with 35S-methionine. On SDS-polyacrylamide gel electrophoresis the reduced antigen appeared as a doublet of 17 and 18 kD. The nonreduced polypeptides had higher mobilities indicating intrachain but not interchain disulfide linkages. The nonreduced form was detectable by immunoperoxidase stain on blots after SDS-PAGE. This showed both polypeptide species contained the antigenic epitope. Labeling in the presence of tunicamycin did not change the apparent m.w. suggesting the absence of N-linked carbohydrate. Pulse-chase data are inconsistent with a strict precursor-product relationship between the two polypeptides and give a half-life for the antigen in 2-day Con A blast cells of about 4 1/2 hr. A highly purified preparation of this antigen displayed a similar electrophoretic pattern and, in the presence of deoxycholate, eluted from a Sephacryl S-200 gel filtration column with a partition coefficient equivalent to that of a 31-kD soluble globular protein. Because of the associated detergent and probable deviation from globularity, this is an over-estimate of the size of the native molecule, and is more consistent with the native molecule being a single polypeptide chain rather than a dimer. Isoelectric focusing of this material showed microheterogeneity with at least five bands between pI 4 and 5. Another monoclonal antibody, HD-42, which had been isolated on the basis of its specificity for a fibrosarcoma antigen subsequently found to be Ly-6 related, precipitated the same polypeptides. Furthermore, no obvious difference was evident between precipitates from Con A-activated lymphocytes and Meth A fibrosarcoma cells.     

N-terminal and cDNA characterization of murine lymphocyte antigen Ly-6C.2

The Ly-6C.2 molecule was purified from K36 tumor cells by affinity chromatography and gel filtration. The electrophoretically homogeneous preparation, with m.w. 15,000, was tested with a panel of antibodies that confirmed the presence of the LY-6C.2 epitope. An N-terminal sequence of 39 amino acids was obtained showing 59% homology with the corresponding portion of the Ly-6A.2 polypeptide. Based on the least homologous (29%) 14 amino acid segment, an oligonucleotide probe was constructed, and Ly-6C.2 cDNA was cloned from a BW5147 cDNA library. A 794-base pair cDNA containing the entire coding region had 82% homology with Ly-6A.2 cDNA. The encoded polypeptide sequence of 131 amino acids containing a perfect correlation with the N-terminal sequence data was 63% homologous with that of Ly-6A.2. The greatest homology was in the leader, first 16 N-terminal and last 39 C-terminal amino acids. The latter are likely to be important in determining the attachment of glycophosphatidylinositol. Despite results indicating fewer disulfide constraints in the Ly-6C molecule, the predicted sequence contains 10 cysteine residues nearly perfectly matched with those predicted in Ly-6A.     

Immunochemical characterization of the Ly-8.2 murine lymphocyte alloantigen: possible relationship to actin.

An immunochemical analysis of the Ly-8.2 antigen was performed. Reactivity of B10-radiolabeled T and B cell lysates with a C3H anti-AKR serum immunoprecipitates a major 43,000 m.w. protein detectable by one-dimensional gel electrophoresis. The protein can be radioiodinated, however, it does not appear to blind to lentil lectin. Two-dimensional gel electro phoresis and peptide-mapping studies indicate that this protein is structurally very similar to lymphocyte actin. The possible molecular relationship between Ly-8.2, other lymphocyte membrane antigens, and actin is discussed.     

Analysis of subpopulations of glass-adherent mouse skin cells controlling resistance/susceptibility to infection with Leishmania tropica,and correlation with the development of independent proliferative signals to Lyt-1+/Lyt-2+ T lymphocytes

A comparison has been made between the course of Leishmania tropica infection of BALB/ c, CBA, and (BALB/c × CBA)F₁ mice in vivo and the growth of the parasite in isolated adherent skin cells in vitro. The susceptible phenotype of the BALB/c mouse was reflected in an innate susceptibility of a discrete subpopulation of adherent skin cells to permit extensive and prolonged growth and replication of the parasite in tissue culture. When cells infected in culture were used to stimulate proliferation of immune lymphocytes from “cured” mice, the skin cells of susceptible BALB/c mice were deficient in their ability to induce proliferation of lymphocytes of BALB/c, CBA, or BCF₁ origin (all immunized in the appropriate bone marrow reconstituted irradiated BCF₁ hosts). In contrast, these skin cells were able to induce proliferation of immune lymphocytes if the L. tropica antigen source used was a soluble excreted extract (EF), rather than that produced by a live parasite infection. Stimulation of naive lymphocytes using an infected adherent skin cell population from BALB/c mice was found to produce a cell population(s) (Thy-1.2⁺, Lyt-2⁺ and including some Lyt-1⁺ cells) able to inhibit subsequent sensitization of normal BCF₁ lymph node cells by L. tropica antigens. The susceptibility of the BALB/c mouse in vivo thus may be attributable to the early contact of T-lymphocyte subsets in BALB/c mice with the high-antigen load maintained in this discrete skin cell population. These particular skin cells were also found to express low levels of Ia antigens.     

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.     

Antigen-presenting cells for Lyt-2+ cells. I. Stimulation of unprimed Lyt-2+ cells by H-2 different Thy-1-Ia- cells prepared from spleen and bone marrow

In agreement with previous studies on Ia- tumor cells, evidence is presented that primary MLR of purified Lyt-2+ T cells to class I alloantigens can be elicited by a minor population of Thy 1- Ia- cells present in normal spleen, bone marrow, and day-13 fetal liver; these cells are non-stimulatory for L3T4+ T cells. The data strengthen the view that primary responses of Lyt-2+ cells do not require the presence of Ia+ cells.     

Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st(-/-)) by embryonic stem cell technology. C6st(-/-) mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st(-/-) mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st(-/-) mice. Further analysis revealed that the number of CD62L(+)CD44(low) T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st(-/-) mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.     

Characterization of the interaction between galectin-1 and lymphocyte glycoproteins CD45 and Thy-1

Galectin-1 is a sugar binding protein specific for beta-galactosides and not requiring metal ions for binding activity. It exists as a soluble protein which forms a noncovalent homodimer and is expressed with a broad tissue distribution. Recently, galectin-1 has been shown to play a possible role in the immune system mediating apoptosis of activated T cells with indirect evidence suggesting that galectin-1 interacts with the heavily glycosylated, transmembrane, protein phosphotyrosine phosphatase CD45. The interaction of galectin-1 with purified lymphocyte cell surface proteins was studied using surface plasmon resonance in a BIAcoretrade mark. Galectin-1 was shown to bind CD45 and Thy-1 in a carbohydrate-dependent manner. Several galectin-1 molecules could bind each CD45 molecule. The dissociation constant of dimeric galectin-1 binding to CD45 was measured at approximately 5 microM, indicating the concentration at which cross-linking of cell surface glycoproteins by galectin-1 would occur. A possible role for galectin-1 in the organization of cell surface glycoproteins is discussed.     

Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Expansion of NK cells with reduction of their inhibitory Ly-49A, Ly-49C, and Ly-49G2 receptor-expressing subsets in a murine helminth infection: contribution to parasite control

Natural killer cell-associated direct cytotoxicity and cytokine production are crucial mechanisms for early innate host resistance against viruses, bacteria, or protozoa. The engagement of inhibitory NK cell receptors can influence host responses to viruses. However, these receptors have not been investigated to date in parasitic infections, and little is known about the role of NK cells in the defense against helminths. Therefore, we have correlated the frequencies of cells expressing the pan-NK marker DX5 and subsets bearing inhibitory Ly-49 receptors with worm survival and cytokine production during infection with Litomosoides sigmodontis in BALB/c mice (H2(d)), the only fully permissive model of filariasis. A marked influx of DX5(+)/CD3(-) NK cells and DX5(+)/CD3(+) T cells into the pleural cavity, where the parasites were located, was observed. The frequency of pleural NK cells expressing the H2(d)-reactive inhibitory receptors Ly-49A, Ly-49C, or Ly-49G2 declined most strongly compared with spleen and blood. In the peripheral blood, longitudinal analysis revealed an early and stable reduction of Ly-49C(+) and Ly-49G2(+) NK cells, a subsequent significant increase of the entire NK cell and DX5(+)/CD3(+) T cell populations, and a reduction in the Ly-49A(+) subset. The in vivo depletion of NK cells strongly enhanced the worm load and influenced IL-4 and IL-5 plasma levels. These data demonstrate a new role for NK cells in the host defense against filariae and, for the first time, alterations of Ly-49 receptor-expressing NK cell subsets in a parasitic infection.