A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor

The Lyb-2 system of the mouse is involved in regulation of a proliferative step in the differentiation of B cells responding to T-dependent antigen. The present study concerns the role of Lyb-2 in an early phase of B cell activation with respect to B cell receptor functions for activation factors. It is shown that interaction of monoclonal anti (alpha)-Lyb-2 antibody with Lyb-2 on the B cell surface induces B cell proliferation by synergistic action with B cell growth factor II-containing factor or interleukin 1. In contrast, alpha-Lyb-2 antibody could not synergize with the Con A-induced culture supernatant of T cell hybridoma FS6-14.13 (FS6) containing B cell stimulatory factor-1 (BSF-1; formerly called BCGF I), and the effect of combining the two was only additive on B cell proliferation. Absorption studies showed that BSF-1 in FS6 could be absorbed by unstimulated B cells, about 95% of which were at Go phase of the cell cycle, but not by thymocytes, and more importantly that alpha-Lyb-2 antibody blocked the absorption in an Lyb-2-specific manner, possibly by competing with BSF-1. It is thus likely that alpha-Lyb-2 antibody may interact with a BSF-1 receptor on B cells or a molecule closely associated with it. Interestingly, alpha-Lyb-2 antibody mimicked the action of BSF-1 in a costimulator assay with affinity-purified goat alpha-mouse IgM antibody, but could not replace all the activities ascribed to BSF-1. Possible mechanisms involved are discussed.     

Biochemical similarity of Ly-19, Ly-32, and Lyb-2 alloantigens encoded in the gene cluster on mouse chromosome 4

Mouse lymphocyte alloantigens Ly-19 and Ly-32 are controlled by the genes tightly linked to the Lyb-2 locus on chromosome 4. Despite the similarity in mouse strain distribution patterns, Ly-19 and Ly-32 antigens which have been detected on both B- and T-cell lineages are distinct from Lyb-2 antigen whose expression is restricted to the B cells. In this report, the close linkage of these three loci was confirmed by the typings of three sets of recombinant inbred mice including BXD, CXS, and OXA. Furthermore, the biochemical characterization of these Lyb-2-linked proteins, i. e., Ly-19, Ly-32, and Lyb-2, demonstrated their similarities on a molecular level. Two polypeptides of 45 000 and 95 000 were the components of these three alloantigens. Furthermore, sequential immunoprecipitation experiments indicated that the three alloantigenic determinants were located on the same molecular components. These findings may provide insight into the complexities and functional roles of Lyb-2 gene-cluster products.     

Inhibition of the mixed lymphocyte culture response with anti-Lyb-4.1 serum 1,2,.

C57BL/Ks anti-L1210 serum, which recognized a non-H-2-linked B cell alloantigen, designated Lyb-4.1, specifically blocked the mixed lymphocyte culture (MLC) response to allogeneic cells that expressed the Lyb-4.1 determinant. Anti-Lyb-4,1 serum blocked the MLC response across H-2 and MLC disparities. To test that this effect was not the result of a toxic or nonspecific cell-coating action, the response of parental cells to F1 lymphocytes was studied in combinations in which only one parent expressed the recognized allele. MLC stimulation was blocked only when the responding parental cell recognized on the F1 cell H-2 or MLs disparities which were derived from the parent which possessed the Lyb-4.1 antigen. Several DBA/2 tumors were characterized by cytotoxic and quantitation absorption assays for the presence of the B cell antigen. The presence of the antigen correlated with the ability of a limited number of tumors to stimulate the MLC response of H-2d identical BALB/c lymphocytes. An increased representation of the B cell alloantigen was found on the transformed B lymphoblast cell line in comparison to splenic B lymphocytes.     

Ly-m19: The Lyb-2 region of mouse chromosome 4 controls a new surface alloantigen

Spleen cells from an SJL mouse immunized with 70'/3 cells, an established pre-B cell line, were fused with cells of the nonsecretor myeloma line NS.1. One established hybridoma cell line (clone K10.6) continuously secreted antibody that recognized a new antigenic specificity tentatively named Ly-m19. This newly found antigen is detectable on both T and B cells. Cytotoxicity assays reveal that 75 percent of the spleen and lymph-node cells, 35 percent of bone-marrow cells, and 15 percent of thymus cells reacted with antibody of clone K10.6. Strains expressing the specificity Ly-m19.1 are characterized by negative reactions and include the strains AKR, CE/J, RF/J, GR/A, SJL, P/J, BDP/J, and LG/J. All other strains so far tested are Ly-m19.2. This strain distribution pattern distinguishes Ly-m19 from any known murine lymphocyte alloantigen, but it parallels the Lyb-2 ^c haplotype. Linkage test of a set of AKXL recombinant inbred strains revealed close linkage of Ly-m19 and Lyb-2 loci on mouse chromosome 4.Abbreviations used in this paper LPS lipopolysaccharide - B6 C57BL/6 - Con-A concanavalin A - MLC mixed-lymphocyte culture The prefix m (monoclonal) is used following a suggestion by Klein and co-workers (1979).     

Mapping of theLyb-4 gene to different chromosomes in DBA/2J and C3H/HeJ mice

Linkage has been established between the Lyb-4 alloantigen locus and the chromosome 4 markersLyb- 2 andMup- 1 using recombinant inbred (RI) strains. Only 2 of 24 BXD RI strains possess recombinant genotypes with respect to the B cell alloantigen lociLyb- 4 andLyb- 2, for an estimated recombination frequency of 0.024 ±0.019. One additional BXD RI strain was a recombinant with respect toLyb- 4 andMup- 1 (major urinary protein locus) for an estimated recombination frequency of 0.039 ± 0.026. These linkages were confirmed and further quantitated in a (C57BL/6J × DBA/2J)F₁ × C57BL/6J backcross population, in which the recombination frequency betweenLyb- 4 andMup- 1 was 0.049 ± 0.019. No recombination between the expression of Lyb-4.1 antigen and the ability of anti-Lyb-4.1 serum to suppress MLC reactivity was found, indicating that the genes controlling the antigenic determinant which is recognized with cytotoxic antibodies in anti-Lyb-4.1 serum is the same as, or is very closely linked to, the gene which is responsible for augmentation of the MLC response. In contrast, no linkage was observed between the gene controlling the Lyb-4.1 determinant andMup- 1 in RI strain and backcross mice derived from the cross of C3H/HeJ and C57BL/6J. Again, there was complete concordance between the serologically recognized determinant and the ability of anti-Lyb-4.1 serum to suppress the MLC response. Absorption of anti-Lyb-4.1 serum with C3H/HeJ, DBA/2J, and C57BL/6J lymphocytes, followed by the cytotoxic assay of the absorbed sera on lymphocytes of each of these three strains showed that serologically the Lyb-4.1 antigenic determinant on DBA/2 mice was indistinguishable from that on C3H/HeJ mice. Thus, both traits appear to be under the control of single genes in both DBA/2J and C3H/HeJ, but the C3H/HeJ gene appears to be nonallelic and unlinked to the DBA/2J gene.Abbreviations used in this paper LAD lymphocyte activating determinants - LPS lipopolysaccharide - MLC mixed lymphocyte culture - RI recombinant inbred     

Functional heterogeneity of the Lyb-5- B cell subpopulation: mutant xid B cells and normal Lyb-5- B cells differ in their responsiveness to phenol-extracted lipopolysaccharide

In the present study, responses stimulated by phenol-extracted lipopolysaccharide (LPS(phenol)) and butanol-extracted LPS (LPS(butanol)) were used to assess the possibility that xid B cells might not be identical to the Lyb-5- B cells present in normal mice. It was found that xid B cells responded well only to LPS(butanol) whereas normal B cells responded well to both LPS(butanol) and LPS(phenol). Thus, LPS(butanol) appeared to be a TI-1 antigen and LPS(phenol) appeared to be a TI-2 antigen. In contrast to classical TI-2 responses, however, responses stimulated by LPS(phenol) did not exhibit a stringent requirement for accessory cells. Furthermore, if LPS(phenol) were a classical TI-2 antigen, it should only activate Lyb-5+ B cells. To determine if the responsiveness of normal B cells to LPS(phenol) were due, at least in part, to the stimulation of normal Lyb-5- B cells, the responsiveness of normal neonatal B cells and normal adult B cells that had been pretreated with anti-Lyb-5.1 + C was assessed. It was found that both normal neonatal B cells and normal adult Lyb-5- B cells did respond well to LPS(phenol). Thus, even though LPS(phenol) does not stimulate xid B cells, these data demonstrate that LPS(phenol) is different from other TI-2 antigens. More importantly, these data also demonstrate that xid B cells and normal Lyb-5- B cells are not identical. It is hypothesized that the normal Lyb-5- B cell subpopulation is heterogeneous, consisting of an Lyb-5(1)- and an Lyb-5(2)-B cell subset with the xid mutation blocking the differentiation of Lyb-5(1)-B cells into Lyb-5(2)-B cells.     

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.     

Mouse Ly-31.1 is an alloantigenic determinant of alkaline phosphatase predominantly expressed in the kidney and bone

The mouse lymphocyte surface alloantigen, Ly-31, defined by monoclonal antibody N1.10 (IgG2b, k) and controlled by a gene locus closely linked to theAkp-2 locus on chromosome 4, was biochemically investigated. By employing a quantitative immunoassay system, it was found that the Ly-31.1-specific antibody detected an allotypic determinant of mouse alkaline phosphatase. Ly-31.1, i. e., mouse alkaline phosphatase, was expressed predominantly in kidney and bone and was also detected in placenta, lung, and testis. Concerning tumor cell lines, they varied in the amount of antigen present, with both T and B lymphoid lineages selectively possessing the antigen. In normal lymphoid tissues, lesser amounts of antigen were detected. The binding of mouse alkaline phosphatase to Ly-31.1-specific monoclonal antibodies was specific in nature. The Ly-31.1 antigen was immunoprecipitated from the lysates of surface-radiolabeled YAC-1 moloney leukemia cells, and appeared as a single band of about 78 000 under both reduced and nonreduced conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, treatment of tumor cell lines with phosphatidylinositol-specific-phospholipase C resulted in the removal of Ly-31 antigen from the cell surface. These results suggest that a gene cluster containing theLy-31 andAkp-2 loci which control the alkaline phosphatase is formed on mouse chromosome 4. The Ly-31 antigen is the first enzyme demonstrated to be a lymphocyte surface alloantigen.     

Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails

In order to target specifically double-Ag-positive cells in vivo, we synthesized chemically two mAb conjugates with specificities for both an allelic murine B cell-surface Ag and for a synthetic hapten. One conjugate was designed for its specificities for I-Ek and for N-epsilon-(2,4-DNP)-amino-caproate, and the other one for its reactivity to Lyb-8.2 and to indium-diethylenetriamine pentaacetate. A radiolabeled tracer, containing both the N-epsilon-(2,4-DNP)-amino-caproate and the indium-diethylenetriamine pentaacetate haptens, was obtained by reacting diethylenetriamine pentaacetic acid dianhydride with mono-[N-epsilon-(2,4-DNP)-amino-caproyl]-tyrosyl-lysine and labeling with indium-111. Mice from various strains (CBA/N: I-Ek+, Lyb-8.2+; AKR/N: I-Ek+, Lyb-8.2-; BALB/c: I-Ek-, Lyb-8.2+; and DBA/2: I-Ek-, Lyb-8.2-) were given simultaneous i.v. injections of microgram amounts of less than anti-[N-epsilon-(2,4-DNP)-amino-caproate], anti-I-Ek greater than and of [anti-(indium-diethylene-triaminepentaacetate), anti-Lyb-8.2] antibody conjugates and picomole amounts of the tracer. As expected, specific uptake of the tracer by the spleen was observed in strains where spleen cells expressed at least one Ag (CBA/N, AKR/N, and BALB/c). Furthermore, spleen cells from the double-Ag-positive mouse strain (CBA/N), when compared with spleen cells from single-positive mouse strains, exhibited a significantly higher uptake of the bivalent hapten. This specificity for double-Ag-positive cells, it is suggested, occurs through the formation of stable complexes between both cell-surface Ag, both conjugates, and the asymmetric bivalent hapten. The use of such asymmetric bivalent haptens, together with matched (anti-hapten, anti-cell) antibody conjugates, is proposed as a general method for increasing the in vivo specificity of immunoimaging and radioimmunotherapy.     

T lymphocyte-dependent B lymphocyte proliferative response to antigen. II. Induction of polyclonal B lymphocyte activation of normal B cells and of Lyb-5- B cells from xid mice via recognition of self-IA antigens

We extended our investigations into the genetic requirements and antigen dependence for the induction of polyclonal B lymphocyte proliferation by primed T lymphocytes. By using recombinant inbred mouse strains and antigen-specific T lymphocyte clones that lack alloreactivity, the genetic requirement was mapped to the IA subregion of the MHC. Furthermore, approaches that prevented or limited the accessibility of antigens to the B lymphocyte surface demonstrated that antigen binding onto the B lymphocyte surface was probably not necessary for induction of B lymphocyte proliferation. These experiments suggest strongly that T lymphocyte recognition of B lymphocyte Ia molecules in the absence of sIg cross-linking or in the absence of antigen bound nonspecifically to B lymphocytes can cause cellular activation. Similar T lymphocyte-dependent B lymphocyte activation was seen when Lyb-5- cells from CBA/N mice with the xid defect were cultured. Increases in the number of cells secreting immunoglobulins could be detected in the proliferating B lymphocyte cultures, suggesting that the culture conditions had fulfilled the requirements for B lymphocyte differentiation into antibody-producing cells. Although anti-Ig did not interfere with the B lymphocyte proliferative responses, it did diminish the number of cells secreting immunoglobulins. The implications of these experiments in extending our understanding of the activation pathway of Lyb-5- and Lyb-5+ B lymphocytes are discussed.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.     

Contribution of Lyb 5+ and Lyb 5- B cells to the primary and secondary phosphocholine-specific antibody response

Due to a mutation on their X-chromosome, CBA/N mice lack the Lyb-5+ subset of B cells. The loss of this B cell subset results in a profound alteration in the immune response of these mice to the hapten phosphocholine (PC). Thus, when these mice are immunized with high doses of PC-KLH (200 micrograms) in CFA, they: 1) fail to produce IgM anti-PC antibodies; 2) produce little or no anti-PC antibody bearing the normally predominant T15-idiotype; and 3) produce IgG anti-PC antibodies only late in the primary response. In order to more fully delineate this defect in responsiveness to PC, the splenic focus assay was used to analyze Lyb-5- B cell precursors from both normal and immune defective mice. Lyb-5- cells were obtained from normal (CBA/N x DBA/2)F1 (CD) female spleens by treatment with anti-Lyb-5 serum and complement. These normal Lyb-5- cells and Lyb-5- cells from immune defective CD male mice were stimulated in vitro with either PC-Hy or TNP-Hy in the presence of Hy-primed T helper cells. The results demonstrate that primary Lyb-5- PC-specific B cells fail to respond in the splenic focus assay, while secondary Lyb-5- PC-specific precursors respond normally, and that both primary and secondary Lyb-5- TNP-specific precursors respond in the splenic focus assay. These data suggest that Lyb-5- PC-specific precursors must differentiate into memory cells before they can be activated to secrete antibody, and they also indicate that the Lyb-5- B cell subset may be composed of two subsets with different activation requirements.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to synergize with anti-mu in induction of B-cell proliferation. The relation between Lyb-2 molecules and BSF-1 receptors was tested in assays that measure binding of anti-Lyb-2 or BSF-1 in B cells and were found not to compete with each other. It appears that the two B-cell agonists anti-Lyb-2 and BSF-1 may exert their effects on B cells through different cell surface moieties as well as different intracellular pathways.     

A new alloantigen expressed selectively on B cells: the Lyb-2 system

The new alloantigenic system of the mouse -Lyb-2-is described, and the strain distribution of the phenotype Lyb-2.1 recorded. So far no antiserum to the alternative Lyb-2.2 or other phenotypes is available. On present evidence, the cell-surface component represented by Lyb-2.1 antigen is expressed only on cells of the B-lymphocyte lineage. Cytotoxicity titers of Lyb-2.1 antisera may exceed 1:640 against spleen cells, with maximal lysis usually around 1:80. In two Lyb-2.1⁺ mouse sublines — CBA/J and C3Hf/Bi — the proportion of spleen cells lysed is lower than in other Lyb-2.1⁺ strains. The position of theLyb-2 locus has not been found, but close linkage with several loci has been excluded.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Genetic control of B- and T-Lymphocyte abnormalities of NZB mice in crosses with B10.D2 mice

Parental NZB and B10.D2, F₁ and F₁ × B10.D2 mice were studied to determine the genetic control of (1) altered B-cell IgD expression, (2) plasma cell frequency, (3) IgM secretion per plasma cell, (4) primary in vitro cytotoxic T-cell responses to H-2-compatible cells, (5) production of thymocyte-binding antibodies, and (6) production of red-cell-specific antibodies. The results demonstrate that, in this cross, IgD abnormalities and production of red-cell-specific antibodies were recessive traits. There was a common genetic influence on plasma cell frequency, IgM secretion per plasma cell and production of thymocyte-binding antibodies which was distinct from the genes governing the ability to generate a cytotoxic T lymphocyte response to H-2-compatible cells.Abbreviations used in this paper CTL cytotoxic T lymphocyte - F1 anti-Fab fluorescein-labeled antimouse Fab - FMF flow microfluorometry - Ig immunoglobulin - IgM/PC IgM secretion per PC - PC plasma cell - sIg surface immunoglobulin - TBA thymocyte-binding antibody     

Lyb-5+ B cells can be activated by major histocompatibility complex-restricted as well as unrestricted activation pathways

It has previously been demonstrated that B cells can be activated through two distinct T helper (Th) cell-dependent pathways, one requiring both carrier-hapten linkage and MHC-restricted T-B interaction and the other requiring neither. In addition, it has been shown that different B cell subpopulations exist and that these subpopulations differ in their activation requirements. Previous studies demonstrated that resting B cells containing an Lyb-5+ subpopulation were activated by MHC-unrestricted T cell signals, whereas resting Lyb-5- B cells were activated only through MHC-restricted T-B interaction. It was suggested that this difference resulted from the ability of Lyb-5+ but not Lyb-5-B cells to respond to soluble MHC-unrestricted Th signals. Because Lyb-5+ B cells were responsive in these previous experiments to MHC-unrestricted Th signals, it could not be determined whether Lyb-5+ B cells were also responsive to MHC-restricted Th signals. Consequently, the present study was undertaken to directly address the question of whether Lyb-5+ B cells can be activated under appropriate conditions by MHC-restricted as well as unrestricted T cell-B cell interactions. It was found that unprimed normal B cells (containing Lyb-5+ and Lyb-5-B cells) but not unprimed xid-defective populations (Lyb-5- only) can be activated by cloned KLH-specific and MHC-restricted Th cells in response to either high or low concentrations of TNP-KLH. The IgM response of Lyb-5+-containing B cells to a high concentration of antigen (10 micrograms/ml) was MHC unrestricted, whereas the IgM response of unprimed Lyb-5+ B cells to a low concentration of antigen (0.001 micrograms/ml) was MHC restricted. Thus, unprimed Lyb-5+ B cells can be activated through both MHC-restricted and unrestricted pathways. It was further demonstrated that the activation requirements of Lyb-5+ and Lyb-5- B cells differed even for MHC-restricted B cell activation.     

A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.