Rabbit Anti-mouse Thymocyte Serum Research Articles

The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen.

The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.

Specific unresponsiveness to skin allografts in burns

We have examined the potential to provide long-term or even permanent wound coverage in a mouse model of a 30% total body surface area burn using skin allografts. Treatment of the recipient mouse with rabbit anti-mouse thymocyte serum (ATS) followed by donor bone marrow infusion induces a state of specific unresponsiveness to the skin allograft without the need for chronic immunosuppression. Specifically, a B6AF 1 mouse receives a burn on Day −2 relative to grafting, ATS on Day −1, and Day +2, a skin allograft from a C3H/He mouse on Day 0, and infusion of C3H/He donor bone marrow on Day +6. We studied three groups of burned mice: Group I, allograft control ( n = 5); Group II, allograft plus ATS ( n = 12); and Group III, allograft plus ATS and bone marrow infusion ( n = 15). Mean graft survival was compared using a one-way analysis of variance and a Student-Newman-Keuls post hoc test. There was no statistical difference in animal mortality among any of the three groups, and there was no evidence of infectious morbidity. Mean skin allograft survival was as follows: Group I, 9 days; Group II, 29 days; and Group III, 66 days ( P < 0.05 vs Group I and II). Nine animals in Group III had intact hair bearing grafts at 90 days when the study was terminated. This study suggests the potential use of induced specific unresponsiveness to skin allografts for wound coverage in thermal injury without use of chronic immunosuppression. In our animal study this was accomplished without increased mortality or apparent infectious morbidity.

Variation in susceptibility of Balb/c mice to coxsackievirus group B type 3-induced myocarditis with age

The severity of cardiac lesions in coxsackievirus group B, type 3 (CVB3)-infected Balb/c mice depends upon both the age and the sex of the animal at the time of viral inoculation. Suckling animals (1–3 weeks old) of either sex develop few cardiac lesions. Thereafter, males rapidly demonstrate increasing disease susceptibility peaking at 16–18 weeks old and then decreasing susceptibility from 20 to 40 weeks of age. Female susceptibility increases much more gradually and myocarditis in this sex never reaches maximal levels as seen in males. Increased susceptibility correlates with virus concentrations in the heart and anti-CVB3 titers in the serum. Cardiac injury is dependent on functional T lymphocytes since treatment of the animals with rabbit antimouse thymocyte serum abrogates inflammation and myocyte necrosis. Sex-associated steroid hormones influence both virus concentrations and immune responses in mice and are probably responsible for variations in disease susceptibility throughout the animal's life.

Suppression of overt diabetes in NOD mice by anti-thymocyte serum or anti-Thy 1, 2 antibody.

Effects of anti-thymocyte serum (ATS) and anti-Thy 1, 2 monoclonal antibody on the spontaneously occurring diabetes in NOD mice were examined. Spontaneous diabetes in female mice was markedly suppressed by intravenous injection of rabbit anti-mouse thymocyte serum diluted to 1:4 on three consecutive days during the time period from 70 to 100 days after birth; the cumulative incidence of overt diabetes upto 195 days of age was greatly reduced and the onset of diabetes was delayed. Similar effect was observed with anti-Thy 1, 2 antibody treatment. These findings suggest that T lymphocytes play a role in the production of spontaneous diabetes in this mouse strain.

Differential Tumor Immunogenicity of DBA/2 Mouse Lymphoma L1210 and Its Sublines. III. Control of Host Resistance to Drug-Resistant L1210 Sublines by &lt;italic&gt;H&lt;/italic&gt;-&lt;italic&gt;2&lt;/italic&gt;-Linked and Non-&lt;italic&gt;H&lt;/italic&gt;-&lt;italic&gt;2&lt;/italic&gt;-Linked Genes&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

The effects of the host's genetic composition on the host's resistance to the DBA/2 L1210 lymphoma and three drug-resistant L1210 sublines (which show an increased expression of tumor-associated antigens) were investigated. Histocompatible F1 mice between DBA/2 and other mouse strains were given injections ip of graded numbers of these tumor cells, and a marked difference in the resistance of the host to tumor growth was demonstrated. The resistant hybrid mice showed markedly higher resistance to the L1210 sublines than to the L1210 parent lymphoma cells. Experiments with F1 mice between DBA/2 (H-2d) and certain congeneic intra-H-2 recombinant strains showed that the H-2b/d heterozygosity at the H-2K+l-A regions and additional non-H-2 genetic factor(s) confer the resistance to the F1 mice. The resistance was abolished by treating host animals with injections of silica, an antimacrophage agent, or rabbit anti-mouse thymocyte serum. These results suggest that the resistance is immunologic and that the genetically controlled host resistance may be directed to surface changes related to the increased expression of tumor-associated antigens.

Alterations in coxsackievirus B4 heart muscle disease in ICR Swiss mice by anti-thymocyte serum.

Coxsackievirus B4 (CB4) infection in infant ICR Swiss mice induces synchronous peaks in both virus titres and pathologic changes in the heart. Among surviving mice, transmural necrosis is followed by fibrosis and ventricular aneurysm. Rabbit antimouse thymocyte serum (ATS) was given before CB4 infection to determine the effects of thymus-dependent functions upon the course of disease. The mortality in ATS-treated mice was 75.9% (65 of 83 mice died) compared to 21.3% (16 of 75 mice died) in normal rabbit serum-treated controls. Pathologic studies showed that ATS-treated mice had more extensive myofibre necrosis and subsequent mineralization, but during the first 10 days of infection, leukocytic infiltration was decreased. Splenic follicles were not present until the 17th day after infection in this treated group. Serum CB4-neutralizing antibodies were similar in mice from the group treated with ATS and normal rabbit serum. These findings indicate that ATS-suppressible functions contribute importantly to virus elimination, perhaps by an increase in macrophage phagocytosis of CB4.

T lymphocytes that contribute to the immunoregulation of granuloma formation in chronic murine schistosomiasis.

Immunoregulation of Schistosoma mansoni egg-induced granuloma formation in the mouse occurs as chronic infection is being established. Three different modes of manipulation that are known to affect immuno-regulatory T lymphocytes were applied to this model. Adult thymectomy (AdTx) of CBA/J mice, 6 wk after infection, did not affect the size of their granulomas at 8 or 12 wk after infection. However, by 15 wk after infection, sham-treated mice exhibited continued development of modulation of their granulomas, whereas AdTx mice did not. Hemagglutinating antibody titers against soluble egg antigen preparation were marginally elevated in the AdTx group. Adoptive transfer of the suppressor effect of chronic spleen cells was ablated by AdTx. In vivo administration of 1 ml of rabbit anti-mouse thymocyte serum (RAMTS) over a 3-day period to chronically infected mice resulted in a minimal increase in granuloma formation. This treatment was seen, however, to be efficacious in eliminating the suppression normally associated with adoptive transfer of chronic spleen cells. Hydrocortisone acetate (HyC) treatment of chronically infected mice (5.0 to 7.5 mg per mouse over 3 to 5 days) significantly decreased the size of their granulomas. This diminution may be due to the eosinophil-depleting activity of HyC. Adoptive transfer studies demonstrated that the granuloma suppressor mechanism was HyC-sensitive. A continuing supply of newly developed T lymphocytes appears to be required for the establishment and maintenance of a substantial portion of this immunoregulatory process in chronic infection.

Alterations in expression of murine thymocyte proteins and glycoproteins during fetal thymus development

To describe the biochemical alterations that accompany T cell differentiation and proliferation in the mouse thymus, proteins and Con A-binding glycoproteins of adult thymocytes and of fetal thymocytes on days 14–19 of gestation were compared. Cells were labelled with 125I and lactoperoxidase, NP-40 extracts were prepared, and both whole extracts and immunoprecipitates obtained with rabbit anti-mouse thymocyte serum and Staphylococcus aureus, Cowan I strain, were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions. In addition, NP-40 extracts of unlabelled fetal and adult thymocytes were subjected to SDS-PAGE, and the gels reacted with 125I-labelled Con A before drying and autoradiography. Results obtained suggest that: (1) T200-like components of early fetal thymocytes have a higher apparent mol. wt(200 + K) than those of late fetal and adult cells; (2) the quantity of the T25 component which bears the Thy-1 alloantigen increases from very low early in gestation to adult levels by day 17–19; (3) adult thymocytes express a Con A-unreactive surface component of apparent mol. wt 12,000 which is present in much lower amounts on fetal thymocytes; (4) fetal thymocytes on all days of gestation express a Con A-unreactive surface component of apparent mol. wt 10,000 which is barely (if at all) detectable on adult thymocytes.

Studies on the mechanism of action of rabbit anti-mouse thymocyte serum. I. Induction of suppressor cells.

Treatment of B6AF1 (C57BL/6 x A/Jax) mice with either rabbit anti-mouse thymocyte serum (ATS) or with both ATS and sheep red blood cells (SRBCs) resulted in the generation of splenic suppressor cells within 5 days. Suppressor activity was determined by injecting secondary syngeneic recipients with both donor cells and SRBCs. Five days later the SRBCs hemolytic effect of their spleen cells was determined. Suppressor cells were found in either whole spleen or enriched T cell suspensions from mice treated with ATS and SRBCs and in only enriched T cell suspensions from mice treated with ATS alone. These results support the possibility that ATS induces a population of suppressor cells and that these cells may be responsible in part for the immune suppression noted after treatment with ATS.

Mitogenic activity of water-soluble adjuvant obtained from Bacterionema matruchotii.

A butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii, a gram-positive oral bacterium, is a potent B-cell mitogen for murine lymphocytes in vitro. [(3)H]thymidine uptake of cultured spleen cells of BALB/c mice and nude athymic mice was greatly enhanced by the presence of Bu-WSA. Spleen cells which had been treated previously with rabbit anti-mouse thymocyte serum and guinea pig complement responded to Bu-WSA, whereas thymocytes and nylon wool column-filtered spleen cells were unresponsive to it. It was necessary for the adjuvant to be in the culture for at least 24 h in order to obtain significant lymphocyte activation. As macrophage-depleted spleen cells still responded to Bu-WSA, the proliferative response of lymphocytes seems to be independent of the presence of macrophages. The degree of response of spleen cells stimulated by a mixture of Bu-WSA and lipopolysaccharide (LPS) was very close to the sum of the responses stimulated by Bu-WSA and LPS individually. Spleen cells of LPS-injected mice, which were refractory to LPS, responded to Bu-WSA. On the other hand, the spleen cells of Bu-WSA-injected mice responded to LPS but not to Bu-WSA. Furthermore, the cells obtained from the spleens of mice which had been heavily cobalt 60 irradiated and bone marrow reconstituted 3 weeks in advance responded to LPS but not to Bu-WSA. Therefore, it appears that the cells responding to Bu-WSA were not identical to those responding to LPS.

Factors affecting the acquisition of resistance againstSchistosoma mansoniin the mouse. IV. The inability of T-cell-deprived mice to resist re-infection, and otherin vivostudies on the mechanisms of resistance

SUMMARYCBA mice deprived of their T-cells by means of thymectomy and administration of rabbit anti-mouse thymocyte serum before a primary infection ofSchistosoma mansoni, failed to resist re-infection to the same extent as immunologically intact controls. However, little difference was made to the degree of resistance to re-infection acquired by thymectomizedS. mansoni-infected mice when the anti-thymocyte serum was administered after the primary infection and just before the challenge. Hydrocortisone acetate, when administered to primarily infected mice just before reinfection appeared to reduce the degree of acquired resistance, but the drug also had a schistosomicidal effect on the challenge-derived organisms. An attempt to specifically suppress the acquisition of resistance againstS. mansoniby administering serum from homologously infected, resistant donors before and during the course of a primary infection failed, and partial depletion of complement activity with cobra venom factor that was administered just before challenge also failed to affect resistance. Serum obtained from mice with chronicS. mansoniinfections and injected intradermally at the site of subsequent administration of a homologous percutaneous challenge infection failed to markedly inhibit maturation of the challenge in the recipient. Recipients of 3 ml of serum from heavily infected donors were also not significantly more resistant to challenge than untreated controls. However, isolated skin from heavily infected mice allowed fewer cercariae to penetrate and emerge transformed as schistosomula than did isolated skin from uninfected controls.

Nonuniform distribution of concanavalin-A receptors and surface antigens on uropod-forming thymocytes.

Uropods can form spontaneously in a variable fraction of mouse thymocytes incubated for 30--60 min in vitro at temperatures between about 8 degrees and 37 degrees C. The majority of the cells with a typical uropod are medium and large thymocytes. The "normal" distribution of concanavalin-A receptors and antigens recognized by a rabbit anti-mouse thymocyte serum was studied on these cells by electron microscopy using ferritin-conjugated lectin or antibodies. The cells were fixed with glutaraldehyde or formaldehyde before labeling. The distribution was essentially uniform on spherical cells. On the contrary, on cells which had formed a uropod the labeled receptors and antigens appeared to be preferentially concentrated around the nucleus, and depleted over the uropod, and especially over the constriction at the base of the uropod. Uropod formation and inhomogeneous distribution were inhibited or reversed by cytochalasin B, but not by vinblastine or colchicine. When the same ligands were applied to unfixed cells, the labeled and cross-linked components capped normally towards the cytoplasmic pole of the cell. These observations are described in relation to the ability of receptors and antigens to interact with an intracellular mechanical structure, and to the mechanism of capping.

Contrasting effects of immunosuppression on Theiler's virus infection in mice

In the present study, cyclophosphamide and rabbit anti-mouse thymocyte serum were used to immunosuppress SJL/J mice infected with Theiler's mouse encephalomyelitis virus (TMEV) in order to delineate the potential mechanism(s) of virus-induced cellular injury in this infection. Whereas both immunosuppressive agents produced a significant increase in mortality, this treatment had differing effects on the pathological involvement of gray and white-matter structures in the central nervous system. The central nervous system of immunosuppressed TMEV-infected mice had increased microglial cell proliferation and neuronal necrosis, longer maintenance of high virus levels and spread of virus antigen to involve the neocortex and hippocampal complex. These observations indicate that TMEV causes a cytolotic infection of neurons and possibly other cells in gray matter. In contrast, immunosuppression produced a dramatic reduction in mononuclear inflammatory cells in the leptomeninges and spinal cord white matter of infected mice and prevented demyelination. Further, virus antigen was not detected in the leptomeninges and white matter of immunosuppressed and infected mice. These findings suggest that demyelination of TMEV infection is immune mediated.

Studies on production of biologically active substance which inhibits the intracellular multiplication of Toxoplasma within mouse macrophages

In vitro assessments were carried out to study some biological aspects of immune lymphocytes producing a new lymphokine, called by the authors Toxoplasma growth inhibitory factor (Toxo-GIF), which inhibits the intracellular multiplication of Toxoplasma gondii within nonimmune mouse macrophages. Concanavalin A and phytohemagglutinin-P were found to induce vigorous production of Toxo-GIF, whereas bacterial lipopolysaccharide did not. In vitro treatment of splenic lymphocytes with rabbit anti-mouse thymocyte serum plus complement abolished almost completely their ability to produce Toxo-GIF. Treatment of splenic lymphocytes with inhibitors of protein synthesis, cycloheximide or puromycin resulted in a remarkable reduction of the ability of sensitized lymphocytes to produce this lymphokine. Thus the production of Toxo-GIF seems to be dependent on the cellular metabolic events of sensitized T-lymphocytes. The significant activity of Toxo-GIF was demonstrable even in the supernate of lymphocyte cultures incubated in serum-free medium and was also evident after immune lymphocytes and homologous antigen were incubated for the relatively short period of 10 h.

Suppression of delayed type hypersensitivity induction to sheep red blood cells by concanavalin A in mice.

An examination was carried out on the in vivo effect of concanavalin A (Con A) on the induction of delayed-type hypersensitivity (DTH) to heterologous erythrocytes in mice. DTH induction was suppressed by an intravenous injection of 100 microng of Con A 1 day before, or simultaneously with immunization. This suppression was mediated by Con-A-activated spleen cells, and also by their cell-free extracts. However, this suppressive effect was abrogated by absorption of cell-free extracts with immunoadsorbent composed of rabbit anti-mouse thymocyte serum (ATS), indicating that suppressive factors for DTH induction make up one of the components extractable from Con-A-activated T cell population.

Eosinophils and Immune Mechanisms

Abstract The lymphoid cell population responsible for production of eosinophil stimulation promoter (ESP), a lymphokine which increases migration of eosinophils, was investigated in murine Schistosoma mansoni infection. Con A challenge induced ESP production, whereas LPS did not. Prior treatment with anti-θC3H alloantiserum plus complement in vitro eliminated ESP production; in vivo treatment with rabbit anti-mouse thymocyte serum consistently reduced ESP production by splenic lymphoid cells, but affected lymph node cell ESP production only after exceptionally large doses. Thymocytes did not produce significant amounts of ESP; nor did lymphoid cells from congenitally athymic mice. Depletion of B lymphocytes and macrophages by nylon fiber adherence eliminated antigen-induced ESP production; this was partially restored by addition of non-immune, 72-hr peritoneal exudate cells. Con A-induced ESP production was not affected by nylon fiber treatment. These results demonstrate that ESP is produced by an ATS-sensitive, peripheralized T lymphocyte population, and suggest a macrophage requirement for antigen-induced production of this lymphokine.

Functional Heterogeneity Among the T-Derived Lymphocytes of the Mouse

Abstract A number of T cell functions were examined in mice after treatment with rabbit anti-mouse thymocyte serum (ATS). Two populations of T cells were distinguished; the first, sensitive, and a second, resistant, to elimination by ATS. The helper cell involved in the primary humoral response to sheep red blood cells (SRBC) was found within the sensitive population, whereas the T-cells required for the priming of mice with SRBC for helper activity or delayed hypersensitivity were found within both populations. Previous work and experiments utilizing both adult thymectomy and ATS treatment indicated that the ATS-sensitive population is long-lived, whereas the ATS-resistant population is short-lived.

An in vivo test for the immunocompetence of human lymphocytes.

After the injection of mice with human lymphocytes suspended i.d. in Medium 199 a local area of induration was obtained. This was maximal at 48 hr and correlated to the number of lymphocytes injected. Evidence that this normal lymphocyte transfer reaction was graft-versus-host in character was the finding that the reaction size was independent of the administration of rabbit antimouse thymocyte serum to the recipient mice. However, the reaction could be suppressed by administration of rabbit antihuman lymphocyte serum. Lymphocytes from 7 of 10 patients with advanced carcinoma of the urinary bladder, 4 patients with advanced carcinoma of the breast, and 1 case each of carcinoma of the prostate, carcinoma of the uterus, and malignant melanoma showed diminished normal lymphocyte transfer reactivity. This was assessed both in relation to patients of similar age with debilitating nonneoplastic conditions and to patients in a surgical ward whose general condition was good.

Trypanosoma cruzi: Effects of anti-thymocyte serum in mice and neonatal thymectomy in rats

CF 1 mice were given eight injections of normal rabbit serum (NRS), Hanks' balanced salt solution (HBSS), or rabbit anti-mouse thymocyte serum (ATS) beginning 3 days prior to and at 3-day intervals subsequent to intraperitoneal (ip) inoculation with 5 × 10 4 trypomastigotes of a Brazil strain of Trypanosoma cruzi. Markedly enhanced parasitemia, increased numbers of tissue stages (amastigotes), and higher mortality occurred in ATS-treated mice as compared to NRS- or HBSS-treated controls. Administration of three injections of ATS at 3-day intervals during the latter stages of acute Chagas' disease, i.e., when numbers of parasites were declining, resulted in a transitory relapse (increase in numbers) of blood and tissue parasites. No relapse occurred in mice when ATS was administered at 3-day intervals over a period of 15 days during the subacute stage of the disease, i.e., after parasites had disappeared from the blood. Parasitemia and mortality were enhanced in neonatally thymectomized rats when compared to that observed in sham-operated and unoperated control rats following ip injection of 2 × 10 5 trypomastigotes of T. cruzi. Serum obtained from thymectomized and control rats 5 weeks after inoculation with T. cruzi at a time when the blood of all animals had become microscopically negative for parasites were equally protective in passive transfer experiments, while serum from uninfected controls gave no protection. Gamma globulin levels significantly increased in thymectomized as well as intact rats by the third to fourth week of infection with T. cruzi, reached maximum concentrations in 5–6 wk, and remained elevated significantly at the twelfth week post infection as compared with uninfected controls. No significant changes occurred in total serum proteins or α and β fractions of any group, infected or uninfected. Total circulating leukocytes, especially lymphocytes, were diminished in mice and rats subjected to treatment with ATS or neonatal thymectomy. These data clearly indicate that neonatal thymectomy of rats and ATS treatment of mice suppress the acquired immune response to T. cruzi. Further, passive transfer experiments in rats confirm the protective role of circulating antibody in acquired immunity to Chagas' disease.

Reaginic antibody formation in mice injected with rabbit anti-mouse thymocyte serum.

A new model system for inducing reaginic antibody formation in mice is presented, namely a single injection of rabbit anti-mouse thymocyte serum (RAMTS). Using this model we have shown that X-irradiation (450 rad) administered to CBA mice 24 h after a single injection of RAMTS or IgG- (RAMTS) results in a sustained moderately high level of reagins against rabbit serum proteins with a concomitant barely detectable level (titer 1:5) of γ&lt;sub&gt;1&lt;/sub&gt;-antibodies. Splenectomy and thymectomy had no demonstrable effect on reaginic antibody synthesis.