Requirement for a bacterial flora before mice generate cells capable of mediating the delayed hypersensitivity reaction to sheep red blood cells.

A delayed-type hypersensitivity (DTH) reaction can be elicited by an injection of 10(8) sheep red blood cells (SRBC) into a rear footpad of conventional (CV) mice previously immunized with small doses of SRBC. In contrast, immunization of germ-free (GF) mice with the same doses of SRBC produced no DTH when immunization was by the intravenous (i.v.) route, and only weak reactions when immunization was by the subcutaneous (footpad) route. Varying the immunizing dose of SRBC, or the time at which DTH was elicited, did not produce a state of DTH responsiveness in i.v. immunized GF mice. However, the transfer of lymphocytes from CV mice, immunized 4 to 5 days previously with SRBC, into GF mice, conferred on GF mice the capacity to express DTH. Although DTH was not readily demonstrable in GF mice immunized with SRBC, they nevertheless produced normal levels of hemagglutinating antibody to SRBC. Finally, it was shown that GF mice could generate a normal DTH response to SRBC if they were first monoassociated with a Gram-negative bacterial flora.     

Ascaris suum: Immunosuppression in mice during acute infection

Mice inoculated by stomach intubation with 10,000 embryonated Ascaris suum eggs, 4, 11, or 21 days before an intraperitoneal (ip) immunization with 2 × 10⁸ sheep erythrocytes (SRBC) had reduced numbers of direct (IgM) splenic hemolytic plaques measured at 4 days after immunization and only a marginal reduction in indirect plaques (IgG) measured at 9 days after immunization. Lower dosages of Ascaris eggs or simultaneous inoculation of Ascaris eggs and SRBC did not suppress antibody responses to SRBC. No reduction in a secondary antibody response to SRBC injected 4 days after Ascaris inoculation was observed. IgM and IgG hemagglutinin titers, as distinguished by 2-mercaptoethanol sensitivity, were suppressed in mice injected ip with 10⁸ SRBC 10 days following inoculation of 10,000 Ascaris eggs, but titers in both Ig classes were similar in infected and control mice injected with 2 × 10⁹ SRBC. At Day 20, antibody titers following ip injection of 1.0 or 100 μg of ovalbumin in alum were reduced in mice infected with 10,000 Ascaris eggs 4 days before antigen injection.Contact hypersensitivity to oxazalone was not altered in mice sensitized at 5 or 14 days after inoculation of 10,000 Ascaris eggs. The delayed hypersensitivity response, measured by footpad swelling, to an optimum intravenous sensitizing dosage of SRBC was inhibited in mice sensitized 10 days after Ascaris infection, but not inhibited in mice sensitized at 21 or 32 days after infection. In contrast, the delayed hypersensitivity response to subcutaneous sensitization with SRBC 10 days after Ascaris infection was not altered.     

Suppressive effect of nonviable Mycobacterium paratuberculosis on the delayed-type hypersensitivity reaction to sheep erythrocytes in mice.

The effect of nonviable Mycobacterium paratuberculosis on the delayed-type hypersensitivity reaction to sheep erythrocytes (SRBC) in mice was evaluated by means of delayed-type footpad swelling. Intraperitoneal (i.p.) injection with nonviable M. paratuberculosis into mice from 28 days before to 1 day after immunization with SRBC resulted in a significant suppression of foot-pad swelling to SRBC. The suppressive effect could be transferred by i.p. injection of spleen cells or peritoneal exudate cells from mice which had been pre-treated with nonviable M. paratuberculosis into non-treated recipient mice. The suppressive effect of spleen cells was retained even after passing them through a nylon wool column. The suppressive effect of spleen cells was abolished by treatment with anti-Thy 1.2 monoclonal antibody plus complement or anti-Lyt 2.2 monoclonal antibody plus complement. However, treatment of spleen cells with anti-mouse gamma globulin antiserum plus complement or anti-Lyt 1.2 monoclonal antibody plus complement did not affect the suppressive effect of spleen cells. The suppression of footpad swelling to SRBC induced by pre-treatment with nonviable M. paratuberculosis could be reversed by i.p. administration of cyclophosphamide. Serum antibody response to SRBC in mice was not affected by pre-treatment with nonviable M. paratuberculosis. These findings indicate that T cells appear to be involved in the suppression of delayed-type hypersensitivity reaction to SRBC in mice by pre-treatment with nonviable M. paratuberculosis.     

Genetic restrictions of transfer of delayed-type hypersensitivity to sheep erythrocytes: local versus systemic transfer.

Regulation of the transfer of delayed-type hypersensitivity (DTH) reactions to SRBC was studied using two assays. In the systemic transfer, SRBC immune cells were transferred intravenously and the recipient challenged by injecting antigen into the footpad. In the local transfer assay, SRBC immune cells were mixed with antigen before transfer into the footpad of the recipient. These studies utilized B10.D2 and B10.BR mice which are congenic strains differing only at H-2 region. DTH reactions can be transferred across H-2 barriers using a local transfer assay. When the immune cells were transferred intravenously or depleted of adherent cells prior to local transfer, DTH reactions cannot be transferred to an H-2 congenic recipient. Spleen cells from naive mice syngeneic to the intravenously transferred cells supply the necessary accessory cell when mixed with the antigen prior to injection into the footpad. This accessory cell may be a macrophage.     

Effects of Cholera Toxin on Delayed-Type Hypersensitivity to Sheep Red Blood Cells Inoculated Intranasally into Mice

The effects of cholera toxin (CT) on delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) were studied in mice sensitized by intranasal administration of SRBC. CT (1 μg/mouse), given intranasally together with SRBC (2 × 10⁷/mouse), induced a maximally enhanced DTH response, which reached its peak around 7 days after sensitization, and also induced an accelerated DTH response upon a second administration of SRBC 28 days later. The ability of CT to enhance the DTH to SRBC was lost, either when CT was administered via the intraperitoneal or subcutaneous route, or when CT was introduced into the nasal site from which a large proportion of the SRBC was discharged 2 days after SRBC administration. These results indicate that the cells that are located in the nasal site and participate in the earlier events of DTH response were most affected by CT. The following effects of CT on the earlier events, which occur within 24 hr after the intranasal administration of both CT and SRBC, appeared to be involved in the mechanisms by which CT enhances DTH to SRBC: (i) facilitation of the penetration of the antigen into the nasal tissue; (ii) reinforcement of the migration of immunocompetent cells from the blood to the nasal tissues; (iii) promotion of the ability of Ia-positive macrophages to present the antigenic determinants to T cells; (iv) facilitation of the differentiation of primed T cells to DTH-effector T cells.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

The chicken's femoral-lymph nodules: T and B cells and the immune response.

Femoral lymph nodules (FLN), which are barely perceptible in normal birds, after a footpad injection of sheep red blood cells (SRBC) may either significantly enlarge (responder) or remain reduced in size (nonresponder). There were approximately 38% T cells and 53% B cells in the FLN of responder chickens. Significantly more plaque-forming cells (PFC) developed in the FLN than in the spleen after a footpad injection of SRBC. Total antibody, mercaptoethanol- (ME) resistant, and ME-sensitive fractions were significantly higher in birds given i.v. than in those given footpad injections. There were no differences in PFC and agglutinin titers between FLN-responders and nonresponders. The number of PFC in FLN exceeded the number of splenic PFC previously reported. The high PFC response of the FLN may reflect the large percentage of B cells in this lymphomyeloid tissue or the presence of antigen-experienced B cells in the FLN. Although FLN may influence a systemic immune response its major role appears to be restricted to a local response.     

Immunological reactivity of the lung: VII. Effect of corticosteroids and cyclophosphamide on the Fc receptor function of alveolar macrophages

The effects of various in vitro and in vivo regimens of either corticosteroid or cyclophosphamide administration on guinea pig alveolar macrophages were studied. Corticosteroid- and cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the capacity of alveolar macrophages to attach to and/or ingest antibody-coated sheep red blood cells (SRBC). In vitro hydrocortisone (up to 20 μg/ml) had no effect on either the binding or ingestion of antibody-coated SRBC. Two separate regimens of in vivo corticosteroids were given: a single dose of iv hydrocortisone (100 mg/kg), which is a short-acting soluble preparation, and sc doses of cortisone acetate (100 mg/kg for 7 days), which is a depot preparation resulting in sustained levels of plasma cortisol of the magnitude of that found for a brief period of time following iv injection of hydrocortisone. Both regimens resulted in similar degrees of peripheral blood lymphocytopenia and monocytopenia 4 and 24 hr, respectively, following injection. The regimen of hydrocortisone has previously been reported to have no effect on alveolar macrophage cytotoxic effector function in antibody-dependent cellular cytotoxicity (ADCC), whereas the cortisone acetate regimen markedly suppressed ADCC. In the present study, hydrocortisone had no effect on either the binding or ingestion of antibody-coated SRBC by alveolar macrophages. In contrast, cortisone acetate caused a marked decrease in both the binding and ingestion of antibody-coated SRBC. This suppressive effect was maximal at suboptimal concentrations of antibody on the SRBC and could be overcome by increasing the concentrations of anti-SRBC antibody. Alveolar macrophages from animals treated with daily cyclophosphamide (a regimen which suppresses ADCC) were capable of binding and ingesting antibody-coated SRBC normally. Thus, prolonged exposure to corticosteroids in vivo causes an alteration in membrane Fc receptor function of alveolar macrophages, which can explain this impaired ability to kill target cells. Since cyclophosphamide therapy did not interfere with the binding and ingestion of antibody-coated target cells, it is concluded that the impairment in killing of target cells by alveolar macrophages is not directly related to an alteration of Fc receptor function but to a defect in the actual killing process.     

Gallium arsenide exposure impairs processing of particulate antigen by macrophages: modification of the antigen reverses the functional defect

Gallium arsenide (GaAs), a semiconductor used in the electronics industry, causes systemic immunosuppression in animals. The chemical's impact on macrophages to process the particulate antigen, sheep red blood cells (SRBC), for a T cell response in culture was examined after in vivo exposure of mice. GaAs-exposed splenic macrophages were defective in activating SRBC-primed lymph node T cells that could not be attributed to impaired phagocytosis. Modified forms of SRBC were generated to examine the compromised function of GaAs-exposed macrophages. SRBC were fixed to maintain their particulate nature and subsequently delipidated with detergent. Delipidation of intact SRBC was insufficient to restore normal antigen processing in GaAs-exposed macrophages. However, chemically exposed cells efficiently processed soluble sheep proteins. These findings suggest that the problem may lie in the release of sequestered sheep protein antigens, which then could be effectively cleaved to peptides. Furthermore, opsonization of SRBC with IgG compensated for the macrophage processing defect. The influence of signal transduction and phagocytosis via Fcgamma receptors on improved antigen processing could be dissociated. Immobilized anti-Fcgamma receptor antibody activated macrophages to secrete a chemokine, but did not enhance processing of unmodified SRBC by GaAs-exposed macrophages. Restoration of normal processing of particulate SRBC by chemically exposed macrophages involved phagocytosis through Fcgamma receptors. Hence, initial immune responses may be very sensitive to GaAs exposure, and the chemical's immunosuppression may be averted by opsonized particulate antigens.     

Macrophage infiltration in tumors and tumor-surrounding tissue: influence of serotonin and sensitized lymphocytes

Summary In a delayed-type hypersensitivity reaction serotonin released from mast cells plays an important role in the induction of a cellular infiltrate at the site of antigen challenge. In analogy, we have studied whether it is possible to enhance the number of intratumoral macrophages by injecting serotonin into a s.c. SL2 lymphosarcoma. The vessels in the tissue surrounding the tumor responded well to serotonin, as there was an influx of i.v. injected ⁵¹Cr-labeled sensitized spleen cells in this tissue during the first 4 h after intratumoral injection of serotonin. At 24 h after serotonin injection there was an influx of macrophages into this tumor-surrounding tissue. No influx of cells was detected in the tumor itself during the first hours after injection of serotonin. In the tumor, similar phenomena occurred as in the surrounding tissue, but with a delay of about 24 h. This suggests that lymphocytes leave the blood circulation in the tumor-surrounding tissue and migrate to the tumor. The influx of macrophages into the tumor after intratumoral injection of serotonin is probably due to an immunological reaction as the lymphocyte influx preceeds the macrophage influx into tumors. In addition, transfer of sensitized lymphocytes, as well as lymphocytes from a tumor-bearing host caused an enhanced influx of macrophages into the tumor. To test the specificity and serotonin dependency of the phenomenon of infiltrating cells in tumors we have used a footpad swelling assay in which the serotonin dependency and the antigen specificity of the response against syngeneic tumor cells was shown. The following picture emerged: an intratumoral serotonin injection enables lymphocytes to leave blood vessels in the tumor-surrounding tissue. These lymphocytes with specificity for tumor antigens migrate to the tumor. After contact with the antigenic tumor cells, these lymphocytes secrete chemoattractive factors for monocytes/macrophages. Also these monocytes/macrophages leave the circulation in the tumor-surrounding tissue. Subsequently the macrophages invade the tumor. We conclude that the number of intratumoral macrophages can be enhanced by serotonin.     

Serologic and immunochemical characterization of HLA-A9 xenoantisera

There was a pronounced quantitative difference between the helper activities of B6C3F1 splenic T cells sensitized with unmodified vs modified antigens of SRBC. Modified SRBC induced the greater helper activity which was measured by the magnitude of an anti-TNP response (IgM and IgG) elicited in vivo by virgin B lymphocytes. Antigen modification was produced by conjugating SRBC with the hapten or simply by incubating SRBC in cacodylate buffer. There were restrictions with respect to both erythrocyte species and mouse strains for this differential priming to occur. The relatively poor performance of SRBC-primed T lymphocytes was apparently not due to suppressor T cells, but rather to activation of only one of two identified T cell subpopulations required for full helper activity. Unmodified SRBC activated a subpopulation of "helper" cells characterized as sensitive to elimination by ATS and long-lived after ATx, but failed to activate in B6C3F1 mice a second subpopulation of "amplifier" cells resistant to elimination by ATS and short-lived after ATx. In contrast, modified SRBC activated both helper and amplifier cells. Under appropriate conditions these subsets of T cells were strongly synergistic in promoting antihapten antibody formation especially of the IgG class. The involvement of two distinct types of T lymphocytes in the positive regulation of antibody responses raises interesting and novel questions concerning the sequence of events in the triggering of B cells and the subsequent development of the response.     

Effects of injection routes of growing tumors PSK or OK-432 on antiproliferative activity of mouse serum

Summary The present study examined the effects of various treatments on the antiproliferative activity of mouse serum. Its activity was estimated against the growth of EL4 tumor cells and L929 cells and against splenic blastogenesis in culture. The activity varied among mouse strains tested and among individuals in any strain. However, normal outbred NIH Swiss mice showed the highest activity among the strains and the least variation among individuals. The activity of serum from NIH Swiss mice constantly decreased 7 or 14 days after an injection of 10⁶ Ehrlich or sarcoma 180 tumor cells subcutaneously in the right-hind footpad, intradermally in the right side of the chest or into the palm. Other routes, such as intraperitoneal, intravenous in the tail vein, subcutaneous in the right side of the chest and intramuscular in the left thigh, however, hardly affected the activity. The activity also decreased 7 days after an injection into the footpad of a biological response modifier such as PSK or OK-432. The antiproliferative activity of mouse serum seems to depend on macrophages but not natural killer-cell activity, because treatment with silica but not anti-(asialo-GM1) antibody totally reduced the activity. The active fraction was heat-stable (100° C, 30 min) and its molecular mass was 127–140 kDa.     

IL-1 gene expression in lymphoid tissues

We examined the expression of IL-1 mRNA in vivo by in situ hybridization. RNA probes for murine IL-1 alpha and IL-1 beta were used to detect IL-1 mRNA in frozen sections of spleen, lymph node, and thymus of mice injected with Salmonella typhi LPS or SRBC. No IL-1 was detected in lymphoid tissues from un-injected mice. This lack of expression correlated with the absence of IL-1 biologic activity. However, after LPS injection, IL-1 alpha and beta mRNA expression was found in macrophages of the red pulp and marginal zone of the spleen. The periarteriolar lymphoid sheath contained cells that only expressed IL-1 beta mRNA. These cells were not lymphocytes and did not stain with the macrophage marker F4/80. A similar cellular response was found after SRBC injection. Scattered macrophages in lymph nodes and thymus were positive, but only after LPS or SRBC injection. The spleens of mice injected with LPS had megakaryocytes containing IL-1 mRNA.     

A CYTOLOGICAL STUDY OF ARTIFICIAL PARTHENOGENESIS IN THE SEA URCHIN ARBACIA PUNCTULATA

Eggs of the sea urchin Arbacia punctulata were artificially activated with hypertonic seawater. The artificially activated eggs undergo the cortical reaction which is not distinguished by a wavelike progression as in the case of inseminated eggs. The cortical granules are released at random loci at the surface of the egg and result in spaces separated by large cytoplasmic projections. Unreacted cortical granules and ribosomes are found within the matrix comprising the large cytoplasmic projections. No "fertilization cone" is formed. The subsequent release of additional cortical granules results in the formation of a continuous perivitelline space, 15 min following activation. 85 min postactivation, an organization of annulate lamellae, endoplasmic reticulum of the smooth variety, and microtubules around a centriole is observed prior to nuclear division. Before the breakdown of the nuclear envelope a streak stage is formed. The streak is composed of a central core of annulate lamellae and is encompassed by endoplasmic reticulum and vesicular components. Condensation of chromatin is followed by the establishment of the mitotic apparatus. Centrioles were not found in the mature egg; however, they are present after activation prior to the first nuclear division, in the four-cell embryo, multicellular embryo, and at blastula. Artificially activated eggs have been observed to develop to the pluteus stage in more than 50% of the eggs treated.     

Peritoneal macrophages introduced into mouse foot pads enter the germinal center of regional lymph nodes nonspecifically.

Male mice were injected into their foot pads with sheep erythrocytes (SRBC) to form lymph follicles in the germinal centers in the popliteal lymph nodes. 4 weeks later, peritoneal macrophages labeled with carbon from syngeneic donors sensitized with SRBC or typhoid-paratyphoid bacilli (TAB) were separately injected into the foot pads as well. The popliteal lymph nodes were histologically examined at 6 h to 5 days after injection. Labeled macrophages appeared in the marginal sinus, migrated straight across the cortex from the marginal sinus to the lymph follicles and then entered the germinal centers. There was no difference in the mode of appearance, migration and localization of labeled macrophages in the regional lymph nodes between the mice given labeled macrophages from SRBC-sensitized donors and those given macrophages from TAB-sensitized donors. The entrance of lymph macrophages into the germinal centers of the regional lymph nodes would be immunologically nonspecific. After the injection of Pelikan ink into the foot pads, the macrophages which have taken up carbon in the peripheral tissue reached the regional lymph nodes via the afferent lymphatics and then entered the germinal centers, mainly through the medullary pole of the lymph follicles, after migrating along their immediate exterior from their marginal sinus to their medullary pole.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHIC STUDIES OF THE CAROTID BODY FOLLOWING INJECTIONS OF LABELED BIOGENIC AMINE PRECURSORS

Adult Syrian hamsters were given a subcutaneous injection of reserpine 3 days before an intraperitoneal injection of ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan and the carotid bodies were subsequently prepared for electron microscopic radioautography. Other Syrian hamsters were given a subcutaneous injection of reserpine and the carotid bodies were subjected to a sensitive cytochemical test for the detection of unsubstituted amines. These studies were made to determine whether the labeled amine precursors were incorporated into the cells and to see whether the parenchymal cells were affected by reserpine treatment. Material from hamsters treated first with reserpine and subsequently injected with ³H-3,4 dihydroxyphenylalanine or ³H-5, hydroxytryptophan exhibited reduced grains of silver over the cells which were associated mainly with the dense cores of the cytoplasmic granules. These studies offer evidence that the granules of the carotid body incorporate catecholamine and indolamine precursors. Material from hamsters incubated for the presence of unsubstituted amines gave a positive reaction (opaque cytoplasmic granules) for catecholamines but not for indolamines. The latter substances may not be present in quantities sufficient to register a positive reaction in the cytochemical test. The opaque granules, indicative of the presence of catecholamines, decreased in density after reserpine treatment. 5 days after one reserpine injection the granules had regained opacity and were comparable to those seen in the control cells.     

Opsonic effect of antiserum and complement on phagocytosis by macrophages from the peritoneal cavity of the Japanese eel, Anguilla japonica

Macrophage exudates in the Japanese eel, Anguillu japonica, were induced by intraperitoneal injection with a mixture of Edwnrdsiella bacterin, proteose peptone and liquid paraffin, and the opsonic effect of antiserum and complement on the phagocytic activity of the macrophages was studied.
The macrophages contained a round nucleus with a nucleolus, and an agranular cytoplasm with projecting processes. These cells showed phagocytosis of sheep red blood cells (SRBC). An opsonic effect of antiserum was recognized in terms of a significant increase of macrophage phagocytic activity against SRBC treated with antiserum relative to that against SRBC treated with inactivated normal serum. Complement, however, did not enhance the phagocytic activity of macrophages.     

Murine T-cell preparations: Radiosensitivity of helper activity

Sheep red blood cell (SRBC)-primed T-cells obtained by two different methods had differential helper activity kinetics and sensitivities to radiation. The helper activities of primed T-cells (pT) isolated from SRBC-immunized mice by glass wool-nylon fiber column purification and SRBC-activated T-cells (ATC) prepared by injection of thymocytes and SRBC into lethally irradiated mice were assessed in Day 5 in vitro primary immune responses to SRBC, TNP-SRBC, or TNP-KLH. The pT had more helper activity than the ATC, but pT activity was not manifested as long as ATC activity. The helper activity of 2 × 10⁶ pT was radiosensitive over an extended period after SRBC-activation; therefore, pT radiosensitivity was not temporally related to the time of activation. The helper activities of ATC and 4 × 10⁶ pT were relatively radioresistant, and 500 R enhanced ATC activity. Removal of macrophages from the ATC did not substantially effect their activities before or after irradiation. Addition of macrophages to the pT before or after irradiation enhanced their activity but not back to the level of non-irradiated (O R) pT. The helper activities of the pT and ATC resided in an anti-Thy-1 sensitive population. In fact, the 1000 R-radioresistant ATC were more sensitive to anti-Thy-1 plus complement treatment than non-irradiated ATC. The pT appeared to contain T₁ and T₂ cells, but the ATC possessed the activities mainly of T₁ cells. The evidence suggests that specific help which was the major type provided by pT cells (2 × 10⁶) was radiosensitive, but nonspecific help as induced by Con A and provided by the majority of the ATC was radioresistant.     

Immunopotentiator separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi)

Summary The separation and properties of a new immunopotentiator, Benincasa cerifera mitogen (BCM) fraction, were investigated. BCM fraction was separated from hot water extract of the seed of Benincasa cerifera Savi (Tohgashi) by gel filtration using Sepharose 4B. BCM fraction is a heteropolymer consisting of uronic acid, neutral sugars, protein, and phosphorus. The proliferation and differentiation of murine B cells were markedly stimulated by BCM fraction. The in vitro development of peritoneal macrophages into antitumor macrophages was also activated by the addition of BCM fraction to cultures. BCM fraction augmented the IgM and IgG antibody responses against sheep erythrocytes (SRBC) and the induction of delayed-type footpad reaction against SRBC. The antitumor activity of BCM fraction was observed in terms of prolongation of the survival period of mice bearing Meth A fibrosarcoma. After hydrolysis with 1% acetic acid at 100° C for 4 h, marked mitogenic activity was found in a precipitate composed of 29% neutral sugars, 50% uronic acid, 1% protein, and 0.1% phosphorus. The precipitate did not contain detectable amino sugar. The possibility that the biological activities of BCM fraction may be due to contamination by bacterial lipopoly-saccharide was ruled out on the basis of the results of chemical analysis and of marked mitogenicity noted in C3H/HeJ spleen cell cultures. Abbreviations used: BCM, Benincasa cerifera mitogen; SRBC, sheep erythrocytes; PFC, plaque-forming cells; TNP-HRBC, trinitrophenylated horse erythrocytes; PBA, polyclonal B-cell activation; SI, stimulation index     

Studies on primed precursors of effector T cells involved in delayed-type hypersensitivity to sheep red blood cells in mice

The nature of primed precursor T cells (primed pre-TD), capable of differentiating into effector T cells (TD) that mediate delayed-type hypersensitivity (DTH), was investigated in B10 mice which were primed by intravenous (iv) injection of various doses of sheep red blood cells (SRBC). The presence of primed pre-TD was detected by the ability of T cells in the spleens from primed mice, which were treated in vitro with pertussis toxin and then transferred into naive recipient mice, to generate DTH in the recipient mice 14 days after transfer. The primed pre-TD were induced antigen specifically 1 day after mice were primed by iv injection of a suboptimal (10(3)), an optimal (10(5)), or supraoptimal (10(9)) dose of SRBC. They were replaced by TD 4 days after priming in optimally sensitized mice, while they were maintained without generating TD for at least 5 weeks after priming in mice primed with either a suboptimal or a supraoptimal dose of SRBC. They were L3T4-positive and dense cells, fractionated in the high-density layers on a discontinuous Percoll density gradient, and capable of transforming into less dense TD, fractionated in the low-density layers. These results indicate that primed pre-TD, which are induced by an antigen signal and then can be activated by a nonspecific stimulus, are present not only in responsive mice but also in unresponsive mice, suggesting that either the generation of TD from primed pre-TD or primed pre-TD alone is the decisive factor for either responsiveness or unresponsiveness.