B6 Spleen Cells Research Articles

Drug-induced in vitro tolerance to allogeneic antigens. II. Further analysis of in vitro-tolerized spleen cells in a fully allogeneic murine combination.

C3H/HeSlc (C3H, H-2k) spleen cells were made tolerant in vitro to C57BL/6CrSlc (B6, H-2b) at the cell-mediated cytotoxicity (CMC) level by in vitro stimulation for 48 hr with mitomycin C (MMC)-treated B6 spleen cells, and treatment with 5 micrograms/ml of 5-fluorouracil for a further 9 hr. These cells were given intraperitoneally to neonate (C3HxB6) F1 mice to examine whether these tolerized spleen cells would cause lethal graft-versus-host disease (GVHD). Despite the lack of CMC, the tolerized C3H spleen cells caused lethal GVHD in most of the neonate F1 mice. Evaluating from various immune parameters, it was evident that T cell populations responsible for IL-2 production, cytostasis, and delayed footpad reaction (DFR) were retained intact after in vitro tolerance induction, probably because of their less-proliferative characteristics in response to fully allogeneic antigen stimulation, and were considered to be responsible for lethal GVHD. Contribution of natural killer (NK) cells to lethal GVHD was not ruled out.

Augmentation of Killer Activity of Murine Spleen Cells against Allogeneic and Syngeneic Tumor Cells by Inoculation of Alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.

Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens.

Abstract Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.

Drug-induced tolerance to allografts in mice. XII. The relationships between tolerance, chimerism, and graft-versus-host disease.

When AKR/J Sea (AKR, H-2k) mice were primed i.v. with 1 X 10(8) viable spleen cells from naive C3H/He Slc (C3H, H-2k) mice and treated i.p. with 200 mg/kg cyclophosphamide (CP) 2 days later, a minimal degree of mixed chimerism associated with tolerance to C3H skin was established without graft-versus-host disease (GVHD) and maintained for at least one month. When AKR mice were primed i.v. with 1 X 10(8) viable spleen cells from C3H mice preimmunized i.v. 7 days earlier with 5 X 10(7) viable AKR spleen cells, and treated with 200 mg/kg CP, chimerism became exclusive, but lethal GVHD occurred in the AKR mice. Moreover, most of normal AKR mice primed with the preimmunized C3H spleen cells without CP died of GVHD. In contrast, in a major histocompatibility complex (MHC)-incompatible combination of AKR (H-2k)-C57BL/6 Cr Slc (B6, H-2b), mixed chimerism, tolerance to skin allografts, and GVHD were not observed, whether or not the mice had been treated with naive or preimmunized B6 spleen cells with or without CP.

Specific and nonspecific resistance to local graft-versus-host reaction in F1 hybrids pretreated intravenously with parent-strain spleen cells. I. Two distinct mechanisms.

B6D2F1 hybrid mice pretreated i.v. with 5 X 10(7) spleen cells from B6 donors seven days earlier (B6-pretreated B6D2F1 hybrids) develop resistance to local GVHR induced by the subcutaneous injection of spleen cells of either B6 (GVHR-B6) or D2 (GVHR-D2) origin. This resistance has specific and a nonspecific components that concern the GVHR-B6 and the GVHR-D2, respectively. The two types of resistance to GVHR are neither induced under the same conditions nor mediated by the same mechanism. Specific resistance to GVHR is observed in B6D2F1 hybrids pretreated with unseparated, anti-Lyt-1.2+C' treated or 1000 rads-irradiated B6 cells, but not in B6D2F1 hybrids pretreated with anti-Thy-1.2+C' or anti Lyt-2.2+C'-treated B6 cells. In contrast, nonspecific resistance to GVHR is induced only by pretreatment with unseparated B6 cells. Treatment of B6 cells with anti-Thy-1.2, anti-Lyt-1.2, or anti-Lyt-2.2 moAb plus C', or their irradiation at 1000 rads completely abolishes their capacity to induce the nonspecific resistance to GVHR. Moreover, specific resistance to GVHR can be transferred to normal B6D2F1 mice by injection of nylon-adherent, anti-Thy-1.2+C'-treated or 2000-rads-irradiated, but not unseparated or nylon-nonadherent, B6-pretreated B6D2F1 spleen cells. Treatment of nylon-adherent B6-pretreated B6D2F1 cells with anti H-2d antiserum plus C' does not affect their capacity to transfer specific resistance to GVHR. Nonspecific resistance to GVHR can be transferred by unseparated, anti-Lyt-1.1+C' or anti Lyt-2.1+C'-treated, but not by anti-Thy-1.2+C' anti-Lyt-1.2+C', anti-Lyt-2.2+C'-treated or 2000-rads-irradiated B6-pretreated B6D2F1 spleen cells. Both types of resistance are observed in B6D2F1 hybrids pretreated with more than 2.5 X 10(7) B6 spleen cells.

Drug-induced tolerance to allografts in mice. X. Augmentation of split tolerance in murine combinations disparate at both h-2 and non-h-2 antigens by the use of spleen cells from donors preimmunized with recipient antigens

In a fully allogeneic murine combination of C3H/HeSlc (OH) (H-2 k) and C57BL/6CrSlc (B6) (H-2 b), C3H mice were primed i.v. with 1 × 10 8 spleen cells from B6 mice preimmunized i.v. with 5 × 107 C3H spleen cells and then were given i.p. 200 mg/kg cyclophosphamide (CP) 2 days later (Im-B6-Sc plus CP group). The tolerant state in those recipient mice was compared with that in mice made tolerant conventionally with 1 × 10 8 naive B6 spleen cells plus 200 mg/ kg CP (naive-B6-Sc plus CP group). B6 skin was rejected in an almost normal fashion in both the naive-B6-Sc plus CP group and the Im-B6-Sc plus CP group. However, EL4 tumor allografts (B6 origin) inoculated after complete rejection of B6 skin grafts were specifically accepted in both groups. Moreover, the tumor growth in the Im-B6-Sc plus CP group was faster than that in the naive-B6-Sc plus CP group. Mixed lymphocyte reaction, cytotoxic T lymphocyte activity, antibody production against the tolerogen were depressed more profoundly in the Im-B6-Sc plus CP group than in the naive-B6-Sc plus CP group. These observations were consistent with the results from tumor allografting. The other immunological parameters examined in the present study, including helper T cell activity and delayed footpad reaction, were retained in the Im-B6-Sc plus CP group at the same levels as in the naiveB6-Sc plus CP group. These observations were consistent with the results from skin allografting. In conclusion, tumor allograft tolerance was made more profound by the use of spleen cells from donors preimmunized with recipient antigens as the tolerogen than by the use of naive spleen cells. However, skin allograft tolerance was not achieved at all by these same treatments. The contribution of graft-versus-host disease to this phenomenon was excluded by the chimeric analysis in AKR/JSea (H-2k) mice given the preimmunized (with AKR antigens) B6 spleen cells plus CP. These results strongly support the existence of a less proliferative lymphocyte population which does not evoke cell divisions to mature even after the strong stimulation with the preimmunized spleen cells and is resistant to tolerance induction.

Specificity repertoire of splenic Lyt-2 +/F23 + cytotoxic lymphocyte precursors from B6 mice

As revealed by flow cytometric analysis, about 30% of nylon wool nonadherent Lyt-2 + B6 spleen cells were F23 +, i.e., were stained with the monoclonal antibody F23.1 directed against an allotypic T-cell receptor determinant. The specificity repertoire of splenic Lyt-2 +/F23 + cytotoxic lymphocyte precursors (CLP) from B6 mice was investigated in a limiting dilution (LD) system designed to support clonal expansion in vitro of a representative fraction of this T-cell subset: in highly purified Lyt-2 + responder cells cocultured with mitomycin-treated F23 hybridoma cells in the presence of (recombinant) interleukin 2 under LD conditions, one out of three Lyt-2 +/F23 + CLP gave rise to a functional cytotoxic T lymphocyte (CTL) clone. The split-well analysis of individual CTL populations demonstrated a clear-cut segregation of the lytic reactivities toward different allogeneic Con A blast targets. A large fraction of B6-derived CTL clones (3–10%) specifically lysed fully H-2 allogeneic (H-2 k, H-2 d), or H-2K mutant (bm1) targets. Self-reactive and allorestricted lytic patterns were not found.

Graft-vs-host reaction limited to a class II MHC difference results in a selective deficiency in L3T4+ but not in Lyt-2+ T helper cell function.

Hybrid mice of the (B6 X bm12)F1 combination were inoculated i.v. with parental B6 spleen cells to induce a class II graft-vs-host disease (GVH). Such mice failed to generate in vitro cytotoxic T lymphocyte (CTL) responses that were dependent upon L3T4+ T helper cell (Th) function (e.g., anti-B6-TNP) but were capable of generating in vitro CTL responses that could be mediated by Lyt-2+ Th cells (anti-allo class I). When Th function was assayed directly by interleukin 2 (IL 2) secretion, class II GVH animals were found to be deficient in L3T4+ but not Lyt-2+ IL 2-secreting Th cells. This selective deficiency in L3T4+ Th function correlates with a selective decrease in class II GVH mice of host-derived derived L3T4+ T cells. In addition, it was found that the spleens of class II GVH mice contained cells capable of selectively suppressing L3T4+ Th function. In contrast, mice in which a class I + II GVH occurred were depleted of both L3T4+ and Lyt-2+ Th function as assessed by IL 2 production. The findings that class II GVH selectively depletes L3T4+ T cells and T cell functions are discussed with respect to the immune function of distinct T cell subsets in normal and diseased states.

Anti-receptor anti-MHC cytotoxic T lymphocytes: their role in the resistance to graft vs host reaction.

Specific resistance to local graft vs host reaction (GvHR) observed in F1 hybrids pretreated s.c., i.p., or i.v. with parent strain spleen cells has been explained as being due to cytotoxic cells induced in these pretreated F1 hybrids and directed against cells bearing receptors that recognize the major histocompatibility complex (MHC) alloantigens of the opposite parent strain (anti-receptor anti-MHC cytotoxic T lymphocytes; CTL). These anti-receptor anti-MHC CTL, however, have never been detected directly in popliteal lymph nodes (PLN), which develop a specific resistance to GvHR. In this paper, we describe the detection of anti-receptor anti-D2 cytotoxic activity in both right and left PLN of B6D2F1 hybrids injected s.c. in the right footpad only with B6 spleen cells. This cytotoxic activity appears 4 days after the injection of B6 cells and diminishes by day 7. It is mediated by a Thy-1+, L3T4-, Lyt-2+ cell of B6D2F1 origin and induced by the injection of either Thy-1+L3T4+Lyt-2- or Thy-1+L3T4-Lyt-2+ B6 spleen cells. The anti-receptor anti-D2 CTL activity is not observed in PLN of B6D2F1 hybrids pretreated i.p. or i.v. with B6 spleen cells. Our results demonstrate that anti-receptor anti-MHC CTL can fully explain the specific resistance to GvHR induced by the s.c. pretreatment of F1 hybrids with parent strain spleen cells. Their role, however, in the resistance to GvHR observed in F1 hybrids i.v. or i.p. pretreated is far from being entirely proven.

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin.

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.

Specific suppression of the in vitro parent anti-hybrid reaction : II. Parameters influencing suppressor cell induction in the F1 hybrid

In the accompanying paper (K. Kosmatopoulos et al. Cell. Immunol. 104, 319–334, 1987) we have reported that the spleens of B6D2F1 hybrids pretreated with B6 spleen cells 7 days earlier contain a cell which specifically suppresses the in vitro proliferative and cytotoxic B6 anti-B6D2F1 responses. The results we present here concern the in vivo conditions under which this suppressor cell can be induced. Suppressor cell activity appears early after the injection of B6 spleen cells (day +1), increases on Day 7, and disappears by Day 30; it is always detectable after the injection of 5 × 10 7 B6 spleen cells and never after the injection of 1.25 × 10 7 cells, the intermediate dose of 2.5 × 10 7 cells being followed by variable results. This variability is attributable to the age of B6 donor and B6D2F1 recipient mice, and suppression is never observed when 2.5 × 10 7 spleen cells from 6-week-old B6 mice are injected into 6-week-old B6D2F1 hybrids. The suppressor cell is induced by the injection of B6 spleen cells of the Thy-1 + Ly-1 −2 + phenotype, even if they are irradiated at 1000 R just before their injection. Lymph node cells from B6 mice induce the suppressor cell, whereas thymocytes do not. Irradiation of B6D2F1 hybrids at 600 or 950 R does not prevent the induction of suppressor cell, nor does thymectomy. Moreover, in the thymectomized or 600 R-irradiated B6D2F1 animals suppression can be induced even by the injection of only 1.25 × 10 7 B6 spleen cells. This phenomenon of specific suppression is not limited to the B6-B6D2F1 genetic combination since it has been observed in all parent-hybrid combinations tested to date.

Specific suppression of the in vitro parent anti-hybrid reaction : I. Characterization of a suppressor cell induced in hybrids by pretreatment with parent-strain spleen cells

Spleen cells from B6D2F1 hybrid mice pretreated with 5 × 10 7 B6 spleen cells iv 7 days earlier (B6-pretreated B6D2F1) exhibit a reduced capacity to stimulate the in vitro proliferative and anti-D2 CTL responses of B6 spleen cells. This inability of B6-pretreated B6D2F1 spleen cells to stimulate B6 spleen cells efficiently is due neither to the absence of stimulating cells bearing the D2 alloantigens nor to the destruction of B6 responding cells, but to the presence in the B6-pretreated B6D2F1 cell population of a suppressor mechanism, since the addition of B6-pretreated B6D2F1 spleen cells to a culture of normal B6 responding and irradiated B6D2F1-stimulating spleen cells can suppress the B6 anti-B6D2F1 response. This suppression is mediated by a nylon adherent, Thy-1-negative cell of parent-strain origin which is radioresistant at 2000 R. This suppressor cell is not induced by the injection to B6D2F1 hybrids of spleen cells from the other parent strain (D2) or an allogeneic strain (C3H). It does not suppress either the response of the other parent (D2) or an allogeneic strain (C3H) to B6D2F1 antigens, or the response of B6 cells to an allogeneic strain (C3H).

Cytotoxic T lymphocyte recognition of a xenogeneic major histocompatibility complex antigen expressed in transgenic mice

Introduction of a porcine major histocompatability complex (MHC) class I gene (PD1) into the genome of a C57BL/10 (B10) mouse has been shown to lead to cell surface expression of the porcine MHC antigen, SLAPD1 in a transgenic mouse. The PD1 product expressed on spleen cells from the transgenic mice stimulated B10 spleen cells in a mixed lymphocyte culture to generate PD1-specific cytotoxic T lymphocytes (CTL). The CTL were PD1 specific since they lysed transgenic splenic blast cells and PD1-transfected L cells, but not B10 blasts or control L cells. The CTL were L3T4-, Lyt-2+ and their activity was partially inhibited by either anti-Lyt-2 antibody or by anti-swine MHC alloantibodies. The repertoire of responding B10 anti-transgenic CTL was assessed by examining their cross-reactivity on a series of murine allogeneic targets. The B10 anti-transgenic CTL showed some cross-reactivity on conventional allogeneic targets, but reacted strongly on a series of mutant H-2Kbm blast cells. In addition, B10 anti-B6.cH-2bm6 CTL cross-reacted extensively on the transgenic target cells. These results demonstrated that normal B10 CTL possess a repertoire specific for the products of the xenogeneic class I gene PD1, that this repertoire is cross-reactive with the conventional alloreactive CTL repertoire, and that there exists an unanticipated relationship between PD1-specific CTL and CTL specific for Kb mutant determinants.

Clonal analysis of murine graft-vs-host disease. II. Leukokines that stimulate fibroblast proliferation and collagen synthesis in graft-vs. host disease.

T lymphocyte clones were established from mice with acute and chronic graft-vs-host disease (GVHD) [C57B6/6 (B6) mice transplanted i.v. with LP/J spleen cells] and immune mice (LP/J mice immunized intraperitoneally with B6 spleen cells). To determine the role of leukokines in the increased collagen production that characterizes chronic GVHD, the supernatants of in vitro stimulated clones were assayed for their effect on 1) B6 embryonic fibroblast proliferation, 2) total fibroblast collagen secretion, and 3) collagen secretion per fibroblast. Four of nine chronic GVHD (CG) clones stimulated both total collagen secretion and collagen secretion per fibroblast, but none stimulated fibroblast proliferation. Six of 10 immune (I) clones stimulated fibroblast proliferation, and none stimulated collagen secretion per fibroblast. Acute GVHD (G) clones were heterogeneous; 2/10 G clones stimulated fibroblast proliferation, 5/10 stimulated total collagen secretion, and 4/10 stimulated collagen secretion per fibroblast. I and CG clones may act synergistically in vivo. The presence of CG-like clones in mice with acute GVHD suggest that the immunologic events that culminate in chronic GVHD are initiated early after transplantation.

Effect of thyroxine on skeletal muscles of dystrophic mice

The number of M-cholinergic receptors on spleen B-lymphocytes of CBA mice was determined using radioactive blocker 3H-Quinuclidinil benzilate. 3 and 4 days after the animals' immunization with ovalbumin the number of M-cholinergic receptors somewhat increased. Specific antigen attenuated M-cholinergic receptor expression on B-lymphocytes, most pronounced on day 4 after immunization, without affecting the receptor expression in control animals. Possible steric interaction between antigen-binding immunoglobulins and B-lymphocyte M-cholinergic receptors during immune response is suggested.

Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response

As an alternative to the measurement of radiolabeled thymidine incorporated into DNA, a method is presented in which thymidine has been replaced by its analogue, 5-bromo-2-deoxyuridine (BUdR). BUdR incorporated into DNA (BUdR-DNA) is measured by a sandwich-type enzyme immunoassay using a monoclonal anti-BUdR antibody. This method allows the quantitation of 4 ng of BUdR-DNA. Comparative experiments with myeloma cells and LPS stimulated spleen B-cells have shown that this technique is at least as sensitive as the traditiona; counting of [ 3H]thymidine.

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)

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity

The selective toxicity of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) for the thymus, consisting primarily of immature T-cells, led us to search for an analogous selective toxicity for the immature B-lymphocytes in the bone marrow. In the dose-response study C57B1/6 male mice were injected with either vehicle alone (corn oil), 30, 60, or 120 micrograms/kg of TCDD i.p. The mice were killed by cervical dislocation 7 days later. In the time-response study, mice were injected with either saline or 120 micrograms/kg i.p. TCDD, 3, 7, 14, or 21 days before killing. In both studies, the following were analyzed: change in body weight, thymus weight, spleen and bone marrow cellularity, and spleen and marrow B-lymphocyte function, measured using the in vitro B-lymphocyte colony forming unit in culture assay, with the mitogen lipopolysaccharide (LPS) from Salmonella typhosa, and the in vitro plaque forming cell assay, with the thymus independent antigen, TNP-LPS. In the dose-response study there was a reduction in thymic weight, spleen B-cell functional response (per spleen), and bone marrow B-cell functional response to 14%, 35-54%, and 20-32% of control, respectively, at a dosage of 120 micrograms/kg. In the time-response study, thymic weight and bone marrow B-cell functional response (per femur) were reduced to 6% and 18% of control, respectively, at day 21. The results indicate that TCDD was selectively more toxic to the immature B-cells in the bone marrow than the more mature B-cells in the spleen. This immunotoxicity was dose-dependent.

Endocytosis of the membrane immunoglobulins of mouse spleen B-cells: a quantitative study of its rate, amount and sensitivity to physiological, physical and cross-linking agents.

A quantitative analysis by flow cytometry of the rate and extent of endocytosis of ligands bound to the membrane immunoglobulins of mouse B-splenocytes is reported. The temperature dependence and the response to inhibitors of oxidative metabolism are described. Inhibitors of the cytoskeleton (cytochalasin B, vinblastine and colchicine) and of calmodulin (trifluoperazine) do not interfere with endocytosis at non-lethal doses. Similarly fetal calf serum does not modify the rate and extent of the process. Endocytosis occurs in a similar time range, but to a lesser extent, when the ligand is monovalent than when it cross-links the membrane immunoglobulins. Finally, it is shown that, within minutes after its internalization, the divalent ligand is found in an acidic environment, while the monovalent ligand is not. These results, in agreement with the model of receptor recycling, suggest that the divalent ligand-receptor complex is indeed internalized and captured in an acidic environment while the monovalent ligand-receptor complex is internalized and probably rapidly recycled back to the cell surface.

Inhibition of capping of antigen-antibody complexes on the surface of normal mouse B-lymphocytes by hyperthermia

1. 1.|Mouse spleen B-lymphocytes were tested for the ability to cap plasma membrane antigen-antibody complexes during of following in vitro hyperthermia. After a 30-min heat pretreatment at six different temperatures (37–42°C) the percentage of capping gradually dropped from more than 90% at 37°C to less than 10% at 42°C. 2. 2.|While less than 2h were required for cells to recover the ability to cap surface Ig, no such recovery was observed for cells treated at 43°C.