The Ly-10 antigen is a marker of mouse-activated T lymphocytes

Ly-10.1 is a lymphocyte surface antigen controlled by a gene linked to the Ly-1.1 locus and expressed on activated T helper, T suppressor (Ts), and cytotoxic T lymphocytes (CTL). In this report, we describe the following:

Activation of T lymphocytes through the Ly-6 pathway is defective in A strain mice

The Ly-6 alloantigens have been shown to participate in the process of T cell activation based on the ability of anti-Ly-6 mAb to induce IL-2 production and proliferation of T lymphocytes. In the present investigation we have demonstrated that peripheral T lymphocytes from A strain mice exhibited abnormally low proliferative responses after stimulation through Ly-6A/E and Ly-6C molecules when compared to responses of T cells from numerous other mouse strains. The abnormal activation of the Ly-6 pathway of A strain T cells was not due to ineffective FcR cross-linking of the anti-Ly-6 mAb, to inappropriate cellular expression of the Ly-6A/E alloantigen in A strain T cells, or to an active suppressive phenomenon. T lymphocytes from A strain mice proliferated normally when the cells were activated by mAb to Thy-1 or the CD3/TCR complex suggesting that A strain mice did not exhibit a generalized T cell activation defect. Cell separation studies of T cells and accessory cells demonstrated that this defect was quantitative, rather than qualitative, and that it was complex, residing at both the T cell and accessory cell levels. These results suggest that activation of T lymphocytes via the Ly-6 molecule may involve a signaling pathway and/or cell-cell interactions distinct from those required for optimal activation via CD3/TCR.     

The distribution of the Qa-2 alloantigen on functional T lymphocytes

The expression of Qa-2 on functional lymphocytes was investigated in vitro and in vivo by using a monoclonal anti-Qa-2 antibody. In vitro treatment of T cells with antibody and complement demonstrated that T cells mediating help or delayed-type hypersensitivity for anti-SRBC responses were Qa-2+. In addition, cytotoxic T cells and either their precursors or cells involved in their generation were Qa-2+, as were anti-HGG suppressor T cells. Panning techniques were also used to show that secondary suppressor T cells were Qa-2+ and that there may be heterogeneity in suppressor T cells defined by Qa-2 expression. In vivo treatment of mice with anti-Qa-2 resulted in decrease in immune responsiveness seen by i) prolongation of skin grafts with either H-2D or I-A differences, ii) suppression of delayed-type hypersensitivity, and iii) inhibition of T cell-mediated suppression. Finally, IgG, but not IgM, anti-body-forming cells were Qa-2+.     

Distribution of the alloantigen Ly-4.2 on murine B cells

The Ly-4.2 alloantigenic specificity has been described as a possible marker for B cells in the mouse, as, on testing by the lymphocytotoxicity method, there was a restricted distribution of this specificity on lymphocytes. Using the highly sensitive method of immunoelectron microscopy with a hybrid antibody, it was found that only 5.5% of cells in the thymus carried the Ly-4.2 specificity, compared with 55.7% in the spleen and 20.9% in lymph nodes. This distribution suggests that Ly-4.2 is present on B cells, and confirms other functional and population studies. However, by immunoelectron microscopy Ly-4.2 was also detected on a few macrophages in the thymus, spleen, and lymph nodes. As with other alloantigens, the location of Ly-4.2 was found in restricted areas of various sizes on the cells bearing this specificity.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

Variations in the expression of theta-C3H alloantigen on thymic and peripheral lymphocytes of newborn and young adult mice

The relative frequency of lymphocytes of mice showing varying degrees of surface θ-positivity (circumferential fluorescence) was recorded. Thymocytes were nearly 100% θ-positive. The relative proportions of θ-positive cells in Peyer's patches and lymph nodes of newborn mice varied in an almost identical fashion as a function of age. At 4 weeks of age and beyond, the relative numbers of θ-positive cells in Peyer's patches were consistently lower than in lymph nodes. As opposed to the predominance of thymocytes with complete rings, peripheralized thymic (T) lymphocytes showed a broad, age- and organ-dependent range of surface θ-positivity. These results suggest that surface θ may be lost rather rapidly upon emigration of lymphocytes from the thymus and/or that many θ-positive T cells with complete rings disappear within a short time. Variations in the relative proportion of complete rings on mesenteric lymph node cells on Days 1 and 4, were tentatively related to antigen-induced changes in the magnitude of thymocyte emigration.The pattern of surface θ-antigen of a given thymocyte or T cell with its size and DNA synthetic activity was compared. The findings suggest that incomplete ring fluorescence may especially be observed on proliferating lymphoblasts in the outer thymic cortex on their way to acquire the full complement of θ-antigen, and on medullary thymic lymphocytes or T cells having reentered mitotic activity, in response to antigenic and/or other microenvironmental stimuli. Our study yielded data consistent with the hypothesis that the progressive loss of surface θ-antigen does not represent a fully autonomous time-dependent process. Moreover, it is not clear if continued loss of θ-antigen by T cells below a certain threshold would render these cells undetectable by anti-θ sera.     

Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.     

Linkage mapping of genes controlling endosperm storage proteins in wheat

Summary A translocation mapping procedure was used to map gene-centromere distances for the genes controlling endosperm proteins on the short arm of each of the chromosomes 1A, 1B and 1D in wheat. The genes controlling triplet proteins (tentatively designated Tri-1) were found to be closely linked to the centromere on chromosome arms 1AS and 1DS and loosely linked to the gliadin genes (Gli-1) on the same arms. The Gli-1 genes segregated independently or were very loosely linked to their respective centromeres. The Gli-B1-centromere map distance on 1BS was also estimated using conventional telocentric mapping and the result was similar to that obtained with the translocation mapping. A simple two-step one-dimensional electrophoretic procedure is described which allows the low-molecular-weight (LMW) glutenin subunits to be separated from the gliadin bands, thus facilitating the genetic analysis of these LMW subunits. No recombination was observed between the genes (designated Glu-3) controlling some major LMW glutenin subunits and those controlling gliadins on chromosome arms 1AS and 1DS. However, in a separate experiment, the genes controlling LMW glutenin subunits on 1BS (Glu-B3) showed a low frequency of recombination with the gliadin genes.Portion of the Ph.D. thesis submitted by the senior author     

Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.     

Characterization of the two heavy chains of the T3 complex on the surface of human T lymphocytes

The T3 complex has been defined by a group of monoclonal antibodies which react with all human peripheral blood T lymphocytes and a subpopulation of thymocytes. This membrane structure includes glycoproteins of 44 (alpha), 37 (beta), 25 (gamma), and 20 kDa (delta) as well as a nonglycosylated polypeptide of 20 kDa (epsilon). The characterization of the alpha and beta chains has been of particular interest because they may constitute the T cell receptor for antigen. Here we show that the T3 complex prepared by immunoprecipitation from T lymphocytes of a leukemic patient (Sezary syndrome) displays an unusually strong association of the alpha and beta chains with the 20/25-kDa T3 proteins. The alpha and beta chains (48 and 44 kDa) were co-precipitated by anti-20-kDa T3 monoclonal antibodies as a disulfide-linked 90-kDa heterodimer. A minor 220-kDa multimer composed of proteins similar to the alpha and beta chains was also present in these immunoprecipitates. This multimer could be independently precipitated with a new monoclonal antibody WT-31, which detects the larger polypeptide chains of the T3 complex on all human T lymphocytes. After removal of N-linked oligosaccharides, both the alpha and beta chain were found to have 33-kDa peptide backbones with distinct isoelectric points. Using a monoclonal reagent T40/25, a 90-kDa heterodimer, consisting of 40- and 49-kDa chains with peptide backbones of 34 kDa was found to be T3-associated on the T leukemic cell line HPB-ALL. When the alpha and beta chains from the Sezary patient were compared with the corresponding chains from HPB-ALL by peptide mapping, large differences were observed. Taken together, the data presented here provide strong evidence that the T cell receptor for antigen is part of the T3 complex on the surface of human T lymphocytes.     

On estimating the linkage of marker genes to viability genes controlling inbreeding depression

Statistical properties and extensions of Hedrick and Muona's method for mapping viability alleles causing inbreeding depression are discussed in this paper. Their method uses the segregation ratios among selfed progeny of a marker-locus heterozygote to estimate the viability reduction, s, of an allele and its recombination fraction, c, with the marker. Explicit estimators are derived for c and s, including expressions for their variances. The degree of estimation bias is examined for cases when (1) the viability allele is partially recessive and (2) the marker locus is linked to two viability loci. If linkage or viability reduction is moderate, very large sample sizes are required to obtain reliable estimates of c and s, in part because these estimates show a statistical correlation close to unity. Power is further reduced because alleles causing viability reduction often occur at low frequency at specific loci in a population. To increase power, we present a statistical model for the joint analysis of several selfed progeny arrays selected at random from a population. Assuming a fixed total number of progeny, we determine the optimal number of progeny arrays versus number of progeny per array under this model. We also examine the increase of information provided by a second, flanking marker. Two flanking markers provide vastly superior estimation properties, reducing sample sizes by approximately 95% from those required by a single marker.     

Role of cholesterol in the capping of surface immunoglobulin receptors on murine lymphocytes

Previously, we have shown that the capping of surface immunoglobulins on murine lymphocytes can be affected by modulating the lipid environment of the surface membrane with free fatty acids. In the present study, murine lymphocytes were depleted of cholesterol by incubation with phospholipid vesicles. As the cellular cholesterol:phospholipid ratio decreased, the capping of the surface immunoglobulin was seen to decrease. This inhibition of capping could not be reversed by calcium and is not accompanied by changes in either the cytoskeletal element alpha-actinin or cellular ATP levels. Incubation of the cholesterol-depleted cells with cholesterol-containing phospholipid vesicles raised both the cholesterol:phospholipid ratio and capping levels to values close to those of untreated control cells. Remarkably, stearic acid, a saturated fatty acid, could also restore the capping levels in the cholesterol-depleted cells. On the basis of the present data and measurements of the fluorescence polarization of the probe diphenyl hexatriene, we propose a model in which the protein(s) involved in capping is located in a gel-like lipid domain, and that removal of cholesterol makes this domain less gel-like and inhibits capping. Restoration of the gel-like nature of this domain by the addition of either cholesterol or stearic acid enables the protein(s) to function normally.