Lymph Node Cells Of Mice Research Articles

Induction of Th2 cytokines and control of collagen-induced arthritis by nondepleting anti-CD4 Abs.

CD4+ T cells play a key role in the development of cell-mediated autoimmune diseases, and their modulation has been used to prevent autoimmune diseases in animal models. The effect of the nondepleting anti-CD4 mAb (KT6) was investigated in collagen-induced arthritis (CIA) in DBA/1 mice and in adoptive transfer of CIA into SCID mice. KT6 (200 microg/dose/mouse) was administered systematically from the day of collagen type II (CII) immunization and continued by alternate-day injection until day 11. Only 20% of KT6-treated mice developed arthritis compared with 100% of the isotype control treated mice (p < 0.001). KT6 treatment in a similar regimen also abrogated the adoptive transfer of CIA into SCID mice. Serum levels of IgG2a anti-CII Abs in KT6-treated DBA/1 mice were significantly reduced (p < 0.001), while IgG1 anti-CII were not significantly changed (p > 0.05). Lymph node cells of mice treated in vivo with KT6 had a reduced production of IFN-gamma but increased IL-4 upon in vitro challenge with CII. Furthermore, KT6 could also reverse the profile of cytokine release of in vivo primed and pathogenic CII-specific T cells. These results demonstrate that in vivo modulation with nondepleting anti-CD4 Abs prevents CIA, likely by altering the functional profile of Th1 T cells to Th2. Furthermore, we demonstrate that this treatment can not only prevent, but more importantly also control pathogenic T cells by switching their cytokine production from a Th1 to a Th2-like profile. Our results thus provide compelling evidence of how treatment with nondepleting anti-CD4 may control an autoimmune process. They also indicate that this approach not only prevents, but also could control ongoing autoimmune diseases.

Antigen-specific B cells are required for the secondary response of T cells but not for their priming.

We studied the potential role of B cells in T cell responses using severe-combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B-17 mice (TG mice). These mice developed both CD4+ and CD8+ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed-type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)-2, IL-6 and interferon (IFN)-gamma and the expression of IL-4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B-17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen-primed B cells, but not by naive B cells, lipopolysaccharide-activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen-primed B cells could induce T cell responses without professional antigen-presenting cells (APC). T and B cells were purified from OVA-immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN-gamma in the absence of non-B APC. When anti-CD80 or anti-CD86 was added in the assay, proliferation and IFN-gamma production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.

Hair Cycle-Dependent Changes in Skin Immune Functions: Anagen-Associated Depression of Sensitization for Contact Hypersensitivity in Mice

To assess whether hair follicle cycling influences skin immunity, we examined the association between highly synchronized hair follicle cycling and experimental contact hypersensitivity in C57BL/6 mice. Hair cycle synchronization was performed by depilation of hair shafts on the back with telogen skin. Mice were sensitized on the lower back skin with picryl chloride between 0 and 25 d, after anagen induction by depilation, and challenged on the earlobes with picryl chloride 5 d later. The magnitude of contact hypersensitivity was significantly decreased in mice sensitized on day 1, was minimal on day 3 (early anagen), and slowly increased thereafter, reaching level comparable to day 0 on day 25 (telogen). The significantly depressed contact hypersensitivity response in anagen skin was confirmed in mice with spontaneously developed follicles. Lymph node cells taken from mice sensitized with picryl chloride on days 0, 1, and 3 after depilation were cultured in vitro in the presence of syngeneic, haptenized, Langerhans cell-enriched epidermal cells. Marked proliferative responses of lymph node cells to haptenized cells were found in mice not only of day 0, but also of days 1 and 3, suggesting that immune T cells exist even lymph node cells of the low-responsive mice. Flow cytometric analyses demonstrated that the number of intraepidermal Langerhans cells and their functions, including the expression of major histocompatibility complex class II, CD54, and CD86, and mixed epidermal cell lymphocyte reactions, were not changed in skin on days 0,1, and 3. These findings demonstrated that contact hypersensitivity is induced most effectively via skin with telogen hair follicles and that the depressed response in early anagen skin is not simply due to failure in Langerhans cell function or sensitization of T cells.

Identification of a mouse protein homologous to the human CD6 T cell surface protein and sequence of the corresponding cDNA.

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.

Anti-lymphoproliferative activity of brown adipose tissue of hibernating ground squirrels is mainly caused by AMP

A fraction with mol wt < 1 kDa was obtained from the brown fat of hibernating ground squirrels ( Citellus undulatus) by means of delipidization, acid extraction, ultracentrifugation and ultrafiltration. This fraction suppressed the proliferation of mouse lymph node cells under standard mitogenic stimuli for T lymphocytes. In contrast, the fraction with mol wt < 1 kDa obtained from the brown fat of active ground squirrels in spring did not display such activity. Further HPLC purification of the biologically active fraction and chemical and structural analysis of its most potent antilymphoproliferative component revealed that this is adenosine 5′-monophosphate (AMP). These data lend support to the notion that in hibernating mammals AMP originating, at least partly, from the brown fat down-regulates the seasonally-dependent proliferation of the thymus.

The immune response to cytochrome c in BALB/c mice is delayed due to inability of their non-specific antigen-presenting cells to provide its immunodominant epitope

The antibody response to horse cytochrome c (cyt. c) in BALB/c mice developed slowly and a substantial production of IgG antibodies was observed only 26–30 days after immunization. Lymph node cells (LNC) of unimmunized mice proliferated weakly in response to both native cyt. c and its synthetic peptides. On day 8 after immunization, LNC could not be stimulated with native cyt. c and peptide 92–104. However, they did proliferate in response to cyt. c peptides 1–6, 1–13, 2–13, 14–22, 46–56, 57–77, 61–77 and 61–69 which are closely related in horse and mouse cyt. c. On day 26, both native cyt. c and the peptides, including 92–104, were equally active in stimulating LNC proliferation. Both plastic-adherent and cyt. c-specific cells panned from day 8 cells enhanced the response of unprimed cells to native cyt. c. Elimination of B cells demonstrated that primary recognition of cyt. c was mediated, at least partly, by non-specific antigen-presenting cells (APC) while later B cells of additional specificities were involved. It is concluded that immunization with horse cyt. c initiated an autoimmune response resulting in T-dependent anergy. Peptide determinants processed by non-specific APC stimulated corresponding autoreactive T cells. Specific B cells which appeared as a result of the response maturation processed successfully the immunodominant epitope and finally mediated proliferative and antibody responses.

Comparison of the T helper cell response induced by respiratory syncytial virus and its fusion protein in BALB/c mice

Specific proliferative T-cell responses were induced in the lymph node cells (LNC) of mice immunised with a sucrose density gradient purified preparation of respiratory syncytial (RS) virus or an immunoaffinity purified preparation of the F glycoprotein. Inhibition studies and flow cytometric analysis showed that the responding cell population were CD4 + T cells. The cytokines produced by virus-specific and F-specific cells were assessed using the CTLL cell line. Peak quantities of cytokine were consistently detected in the supernatants of stimulated cultures 24 h prior to maximum proliferation. The proportion of IL-2 released was determined by blocking IL-2 activity with an anti-IL-2 monoclonal antibody. In cultures of RS virus primed LNC challenged with whole virus there was a switch of cytokine production from 70% IL-2 at day 3 to 80% IL-4 by 6 days of culture. In contrast, LNC cultures from mice immunised with F protein secreted 75–100% IL-2 throughout the culture period. These data suggest that after 6 days of challenge with viral antigen, the RS virus-primed LNC response consists of T helper cells which are predominantly of the Th2 subset, secreting IL-4, whilst F protein-primed LNC secrete large quantities of IL-2 and can therefore be classified as predominantly of the Th1 subset.

Concanavalin A—induced T-cell—Mediated hepatic injury in mice: The role of tumor necrosis factor

Concanavalin A activates T lymphocytes in vitro and causes T-cell-dependent hepatic injury in mice. T lymphocytes were previously identified as effector cells of concanavalin A—induced liver injury. Here we report that hepatic injury is characterized by apoptotic cell death. On concanavalin A challenge, the cytokines tumor necrosis factor-α (TNFα), interleukin-2, granulocyte macrophage-colony stimulating factor, and interferon-γ were detectable in the circulation of the mice. Pretreatment of mice with anti-mouse TNF-α antiserum protected them from concanavalin A-induced liver injury. Nude mice failed to release TNF-α or interleukin-2 after concanavalin A challenge and were protected from liver injury. Lymph node cell transfer from responder mice to resistant nude mice resulted in susceptibility of the latter towards concanavalin A, i.e., to induction of cytokine release and hepatotoxicity. These experiments suggest that immunocompetent T cells play a pivotal role in concanavalin A—stimulated TNF-α release in vivo. After intravenous administration of fluorescein isothiocyanate-labeled concanavalin A to mice, the most fluorescence was found within the liver. In vitro, concanavalin A stimulation of separate cultures of mouse lymph node cells or nonparenchymal liver cells induced the release of minute amounts of TNF, whereas stimulation of cocultures of these cells resulted in production of substantial amounts of TNF-α. These findings may explain the hepatotropic effect of concanavalin A. In conclusion, T-cell-dependent concanavalin A-induced apoptotic liver injury in mice is related to immunological and cytokinemediated disorders and possibly to autoreactive hepatic processes.

Concanavalin A—induced T-cell—mediated hepatic injury in mice: The role of tumor necrosis factor

Concanavalin A activates T lymphocytes in vitro and causes T-cell-dependent hepatic injury in mice. T lymphocytes were previously identified as effector cells of concanavalin A-induced liver injury. Here we report that hepatic injury is characterized by apoptotic cell death. On concanavalin A challenge, the cytokines tumor necrosis factor-α (TNF α), interleukin-2, granulocyte macrophage-colony stimulating factor, and interferon-γ were detectable in the circulation of the mice. Pretreatment of mice with anti-mouse TNF-α antiserum protected them from concanavalin A-induced liver injury. Nude mice failed to release TNF-α or interleukin-2 after concanavalin A challenge and were protected from liver injury. Lymph node cell transfer from responder mice to resistant nude mice resulted in susceptibility of the latter towards concanavalin A, i.e., to induction of cytokine release and hepatotoxicity. These experiments suggest that immunocompetent T cells play a pivotal role in concanavalin A-stimulated TNF-α release in vivo. After intravenous administration of fluorescein isothiocyanate-labeled concanavalin A to mice, the most fluorescence was found within the liver. In vitro, concanavalin A stimulation of separate cultures of mouse lymph node cells or nonparenchymal liver cells induced the release of minute amounts of TNF, whereas stimulation of cocultures of these cells resulted in production of substantial amounts of TNF-α. These findings may explain the hepatotropic effect of concanavalin A. In conclusion, T-cell-dependent concanavalin A-induced apoptotic liver injury in mice is related to immunological and cytokinemediated disorders and possibly to autoreactive hepatic processes. (Hepatology 1995;21:190-198).

IL-4 production in mediastinal lymph node cells in mice intratracheally instilled with diesel exhaust particulates and antigen

To clarify the relationship between air pollutants and IgE antibody production, interleukin 4 (IL-4) production was investigated in BALB/c mice intratracheally injected with diesel exhaust particulates (DEP) mixed with antigen (Ovalbumin (OA) or Japanese Ceder Pollen (JCP)). BALB/c mice were injected with DEP plus OA or OA alone three times with a 3-week interval. After the last instillation, proliferative response and lymphokine-producing activity of mediastinal lymph node cells (LNC) were examined in vitro. Proliferative response to OA in mediastinal LNC from mice injected with DEP plus OA was enhanced 4–17 times of that from control mice. IL-4-producing activity by OA stimulation also enhanced in mediastinal LNC from mice injected with DEP plus OA. A significantly larger amount of anti-OA IgE antibody was detected in sera from DEP- and OA-injected mice compared with those from control mice. The levels of IL-4, estimated by JCP antigen in mediastinal LNC, from mice injected with DEP plus JCP were two-fold higher than those from mice injected with JCP alone. These results suggest that intratracheal instillation of DEP affects antigen-specific IgE antibody responses via local T-cell activation, especially enhanced IL-4 production.

Modulation of T‐cell responsiveness during Trypanosoma cruzi infection: analysis in different lymphoid compartments

Spleen and lymph node cells of Trypanosoma cruzi-infected mice were studied for mitogen-induced responsiveness in terms of proliferation and lymphokine production (IL-2, IFN-gamma). Splenocyte (SP) as well as lymph node cell (LN) proliferation and IL-2 production were depressed during the acute phase of the infection. Proliferative capacity of LN cells recovered completely and that of SP partially during the chronic phase. In contrast to these suppressive effects, the mitogen-induced IFN-gamma response was enhanced. In vitro co-incubation of normal SP or LN cells with trypomastigotes resulted in a reduced mitogen-induced cell proliferation and IL-2 secretion, similar to those seen with cells taken from infected mice. In contrast, trypomastigotes exerted a stimulatory activity on the mitogen-induced IFN-gamma response of both SP and LN cells. Addition of lymph node cells from T. cruzi-infected mice (LN-I) to lymph node cells of control mice (LN-C) suppressed strongly the mitogen-induced responsiveness of such cocultures. A marginal level of suppression was recorded in cocultures of spleen cells from infected mice (SP-I) and control spleen cells (SP-C). The potent suppressive cells within LN-I populations were identified as macrophage-like and such cells were absent in SP-C and peritoneal exudate cells from T. cruzi infected animals.

Split tolerance between spleen and lymph node cells in severe combined immunodeficiency mice grafted with AKR fetal liver cells.

Severe combined immunodeficient (SCID) mice defective in stem cells for T and B cells appear to be an ideal host for construction of chimeric mice. When bone marrow cells are used as a source of stem cells, however, host SCID mice do not always show sufficient reconstitution. In this study, fetal liver cells from AKR embryos were transplanted into SCID mice without prior irradiation. This treatment induced full reconstitution of lymphopoiesis as evaluated by flow cytometry analysis and serum Ig production 2 months after transplantation. Thus, fetal liver cells seem to be a better source for reconstitution of SCID mice than bone marrow cells. Lymph node (LN) cells of these mice (FLT mice) had no proliferative or cytotoxic activities against either host-type (C.B-17) or donor-type (AKR) spleen cells. However, spleen cells from FLT mice exhibited marked proliferative and cytotoxic activities against C.B-17 cells, with no activities against AKR cells. Split tolerance against C.B-17 cells in spleen and LN cells was not a transient phenomenon, since similar results were obtained from a cytotoxic T lymphocyte assay 4 months later. In spite of the strong host reactivity in vitro, aberration of clonal deletion or development of a graft-versus-host disease was not seen in FLT mice. As IL-2 induced the host reactivity of LN cells in a mixed lymphocyte reaction, potentially host-reactive T cells were present in LN but were rendered anergic. Tolerance in FLT mice seems to be regulated by a peripheral mechanism. We supposed that the split tolerance in FLT mice was induced by the different antigenicity between the spleen and LN.

Trypanosoma cruzi: Antibody Production and T Cell Response Induced by Stage-Specific Surface Glycoproteins Purified from Metacyclic Trypomastigotes

The main surface glycoproteins of metacyclic trypomastigotes of Trypanosoma cruzi, gp90, gp82, and gp35/50, were purified and the immune response elicited by these antigens was analyzed. Balb/c mice immunized with antibody-affinity-purified gp82, plus alum as adjuvant, produced antibodies that recognized both the gp82 and the heterologous gp90 and gp35/50. On the other hand, antisera to gp90 reacted only with the homologous antigen, either by immunoprecipitation or by immunoblotting. Neither sera reacted with unrelated proteins in ELISA. Both antisera lysed 90-100% metacyclic forms in a complement-mediated reaction, a property associated with protection. However, in contrast to gp90, previously shown to induce protective immunity against acute T. cruzi infection, gp82 was not immunoprotective. Lymph node (LN) cells of mice primed with gp82 or gp90, which display 40% amino acid sequence identity at the carboxy terminal domain, were strongly stimulated in vitro by either one of these antigens. Proliferation, inhibitable by anti-CD4 but not by anti-CD8 antibodies, was T. cruzi-specific, no activation being observed with irrelevant antigens. LN cells of mice immunized with unrelated proteins did not proliferate in vitro in the presence of gp82 or gp90. The 35/50-kDa glycoconjugate, which was phenol-extracted, did not elicit any detectable antibody or T cell response.

Contrasting effect of delta-9-tetrahydrocannabinol on IL-2 activity in spleen and lymph node cells of mice of different ages

The purpose of this study was to investigate the immunomodulatory effect of delta-9-tetrahydrocannabinol (THC) on spleen and lymph node cells of adult and young mice. When plant mitogen (Con A or PHA) stimulated cells were treated with varying doses of THC, the proliferation of spleen and lymph node cells of adult and young mice was suppressed. This suppression was found to be compatible with a decrease in number of Tac positive cells as well as a down-regulation of IL-2 activity. In contrast, when these cells were stimulated with anti-CD3 antibody in combination with THC, the pattern of the immunomodulation as well as the absolute effects of the drug differed with the age of the mice as well as the organ of origin of the cells. In this regard, anti-CD3 antibody stimulation in combination with low doses of THC stimulated proliferation of splenocytes obtained from adult mice. The stimulation was found to reflect an increase in the number of both suppressor/cytotoxic T cells (Ly2 cells) as well as Tac positive cells. IL-2 activity was found to be enhanced under these conditions. This upregulation of responsiveness was not seen in either lymph node cells of adult or young mice or in spleen cells of young mice. The THC modulating activity is directly related to its effect on both IL-2 activity and generation of Tac positive cells in spleen and lymph node cells of both adult and young mice. It appears, therefore, that not only age, but also lymphoid organ system differences are important factors in terms of considering the effects of drugs abuse.

The role of the 16/6 idiotype network in the induction and manifestations of systemic lupus erythematosus.

Experimental systemic lupus erythematosus (SLE) has been induced in mice by immunization with either a human anti-DNA mAb bearing a common idiotype (Id) designated 16/6 Id (antibody 1, Ab1) or with a murine anti-16/6 Id mAb (Ab2). In the present study a murine mAb (5G12-4, Ab3) that bears the 16/6 Id and binds to DNA was produced and was found to bind rabbit anti-16/6 Id sera and murine anti-16/6 Id mAb similarly to the human mAb 16/6 Id (Ab1). Moreover, mAb 5G12-4 was shown to share T cell epitopes with the human 16/6 Id mAb, since lymph node cells of mice immunized with the mAb 5G12-4 proliferated significantly to the human 16/6 mAb and vice versa. Following immunization of mice with the murine mAb bearing the 16/6 Id, antibodies to dsDNA, ssDNA, 16/6 Id, anti-16/6 Id, and to HeLa nuclear extract proteins were detected, similarly to those observed previously upon immunization with Ab1 or Ab2. Six months following the immunization, the mice exhibited leukopenia, increased erythrocyte sedimentation rates, and proteinuria. Examination of the kidneys of the mice disclosed immune complex deposits, thickening of the Bowman's capsule and glomerular necrosis. These results show the importance of the 16/6 Id network in the induction and progression of SLE in mice.

The Effect of Cyclophosphamide Treatment on Lymphocyte Subsets in the Nonobese Diabetic Mouse: A Comparison of Various Lymphoid organs

The nonobese diabetic (NOD) mouse is a model for human Type 1 diabetes mellitus. Pancreatic beta-cell destruction in NOD mice is mediated by an autoimmune process which can be accelerated by cyclophosphamide (CP). We studied the phenotype of lymphocytes from central, peripheral and regional lymphoid tissues in prediabetic NOD and C3H mice before and after a single large dose of CP. All lymphoid organs showed a greatly diminished cell number and most alterations appeared early after CP and were transient, but an aggressive insulitis was not seen in NOD mice until 14 d after injection. The pancreatic islets in C3H mice remained intact and were not infiltrated. NOD female mice, which are most prone to spontaneous and CP-induced diabetes, exhibited the most unusual lymphoid kinetics after treatment with CP. Their thymus and spleen showed the least relative drop in total cell number and the most rapid rate of recovery. The thymus of these mice was also found to have an increased proportion of CD3+ thymocytes while CD4/CD8 double positive thymocytes decreased 7 d after CP. At 14 d after CP the number of IL-2R+ thymocytes had surpassed that of normal levels. The most dramatic observation was the rapid recovery and overshoot in the number of pancreatic lymph node cells of female NOD mice which coincided with aggressive insulitis.

Lymphokine profiles in contact sensitivity induced by dinitrofluorobenzene and tolerance induced by dinitrothiocyanobenzene

We determined the lymphokines involved in the immune response to epicutaneously applied dinitrofluorobenzene (DNFB), a sensitizer, and dinitrothiocyanobenzene (DNTB), a tolerogen. Hapten-dependent T-cell proliferation and production of interleukin-2, interleukin-3 and interleukin-4 by lymph node cells (LNC) in mice painted with these cross-reactive haptens were measured by specific lymphokine assays. Proliferation of LNC in tolerized animals was lower than in sensitized mice. LNC from DNTB-treated mice produced lower amounts of interleukin-2, interleukin-3 and interleukin-4 than cells from DNFB-painted mice. These results may explain hapten-specific tolerance induced by DNTB which results in deficient production of both type 1 T-helper cell (Th1) and type 2 T-helper cell (Th2) lymphokines in response to hapten re-exposure. Deficient interleukin-4 production by cells from tolerized mice was corrected by the addition of exogenous interleukin-2. The suppressor function of adoptively transferred T cells from animals tolerized with dinitrothiocyanobenzene may be related to a shift in the balance of Th1 and Th2 lymphokines in favour of the latter, since recipient T cells might provide the source of interleukin-2 that induces interleukin-4 production by donor T cells.

Alloreactivity against IE-encoded antigens: Evidence of the discrepancy between graft rejection and reactivity of IE-reactive T cells

Participation of IE antigens (Ag) in immune response as the transplantation Ag was examined. IE − B10.A(4R)(4R; K k, IA k, IE −, D b) mice could not reject skin graft from IE Ag alone-disparate B10.A(2R) (2R; K k, IA k, IE k, D b) mice despite intravenous (iv) injection of 2R spleen cells (SC) before or after skin grafting, indicating that graft rejection could not be caused across IE Agbarrier alone. Furthermore, 4R SC could not induce lethal graft-versus-host disease (GVHD) in supralethally (950 rad) irradiated 2R mice. On the other hand, infiltration of lymphoid cells was observed at the site of transplanted 2R skin in 4R mice. SC of 4R mice unprimed or primed with 2R skin or 2R SC showed the capability to proliferate in vitro in response to 2R Ag. In immunofluorescence analysis of lymph node cells (LNC) of 4R mice injected iv with 2R SC 7 days earlier, IE-reactive CD4 +Vβ11 + T cells did not change in number, but slightly increased the expression of interleukin-2 receptor (IL-2R). In 2R mice irradiated with 670 rad and injected iv with 4R SC 7 days earlier, 4R-derived CD4+Vβ11 + and CD8+Vβ11 + T cells proliferated, changed to blastoid form, and showed a markedly increased expression of IL-2R. To further investigate the influence of IE alloantigens on transplantation immunity, IL-2 production and anti-class I CTL activity were assayed. The 4R SC capable of recognizing IE k and D k Ag of B10.BR (K k, IA k, IE k, D k) generated levels of both IL-2 and CTL activities higher than those of 2R SC capable of recognizing D k Ag alone. These results strongly suggest that IE alloantigens indirectly act as the transplantation Ag by the stimulation of IE-reactive CD4 + helper T cells resulting in the differentiation of Class I-restricted CD8 + T cells.

Inhibition of mouse lymphocyte proliferative response by glycosphingolipids from Leishmania (L.) amazonensis

The effect of a purified preparation of glycosphingolipids (GSLs) extracted from Leishmania (L.) amazonensis amastigotes on murine lymphocytes was investigated. GSLs inhibited both concanavalin A (Con A)- or lipopolisaccharide-induced [ 3H]thymidine uptake by normal and immunized BALB/c mouse lymph node cells. The effect of total GSLs was dose dependent. Total GSLs also suppressed the two-way mixed lymphocyte reaction of BALB/c × C57BL/6 cells and the antigen-specific response of immunized mouse cells. Six pure bands as well as a pool containing a mixture of GSLs with five to seven sugar residues separated by a combination of HPLC and preparative HPTLC were tested and shown to be inhibitors of Con A stimulation. These results suggest that parasite glycosphingolipids may play an immunologically relevant role in leishmaniasis.

Mast cells in female mouse lymph nodes

This study was designed to examine the possible role of sex steroid hormones on mast cells localized in uterus draining lymph nodes (UDLN) in mice. Young virgin estrous animals had more mast cells than diestrous animals in both the UDLN and popliteal lymph nodes (PLN). In retired breeders there were no differences in mast cell numbers of estrous and diestrous animals. There were no differences in mast cell numbers among weanling and older animals in diestrous in the UDLN but, in the PLN, mature animals in either diestrous or estrous had more mast cells than the PLN of weanlings. In mature animals, ovariectomy did not alter mast cell number in either node. However, ovariectomy of weanlings increased mast cell numbers in the PLN but not in the UDLN. These results suggest that the UDLN behaves as a nonclassical target organ for the endocrine system, with mast cell number variations related to gonadal steroid levels.