Antigen-specific lysis in vitro of lymph node cells of intact mice injected with RNA from viable lymph node cells of immunized animals]

Supernatant fluid obtained after centrifugation of the suspension of viable lymph node cells of immunized animals proved to induce in vivo in the lymph node cells of intact mice sensitivity to lysis with a specific antigen in vitro. This property was possessed after chromatography of the supernatant fluid on Sephadex G-200 by the 3rd fraction (MW about 30000 dalton). DNA-ase, trypsin or deproteinization failed to influence whereas RNA-ase inactivated this fraction in respect to the inducing properties.     

Immunochemical characteristics of a factor causing antigen-specific lysis of thymus-dependent lymphocytes]

A factor capable of lysing in vitro, in the presence of a specific antigen, the cells of the lymph nodes and the thymus of intact mice was revealed in the supernatant obtained after the centrifugation of a suspension of viable cells of the lymph nodes and the thymus of the immunized mice. It was found by immunochemical methods that this factor had a mol. wt. of about 30000 dalton and an electrophoretic mobility in polyacrylamide gel exceeding that of mouse blood serum albumin. Besides, it was revealed by the precipitation reaction in agar that it was not an immunoglobulin or its chains.     

Factor producing lysis of thymus-derived cells in vitro by specific antigen.

Lymph node cells of immunized mice were lysed by specific antigen in vitro. These cells were not lysed by antigen after washing them four times with Hanks' solution. The factor lysing lymph node cells and thymus cells of normal mice in the presence of the specific antigen was found in the supernatant of the lymph node cell suspension of immunized mice. Bone marrow cells were also lysed by the factor and specific antigen only after preincubating the cells in extracts of thymus or lymph nodes of normal mice. When active supernatants were fractionated by gel filtration on Sephadex G-200, the active material eluted with molecules of approximately 20,000–45,000 MW. This factor was homogeneous on electrophoreses in polyacrylamide gels and detected in the prealbumin zone.     

Immunostimulating and protective effects of terrilytin preparations in staphylococcal infection

In mice infected with staphylococci there was observed less pronounced development of the immune response to sheep red blood cells (SRBC) than in intact animals subjected only to immunization. Administration of free terrilytin to the infected mice increased the immune response development induced by SRBC while the use of immobilized terrilytin normalized it. The supernatant liquid of the spleen cells (SLSC) from the mice treated with the free of immobilized terrilytins stimulated development of the immune response to SRBC in the infected animals and inhibited the function of the suppressor cells in the spleen. The immunomodulating and protective effects of the SLSC fractions isolated with column chromatography on Sephadex G-150 were investigated. Substances of the low molecular fraction of the SLSC proteins (molecular weight of 10-15 kD) from the mice treated with the terrilytins showed immunostimulating and protective properties. The factors inducing both the activity types included peptides and the ribonucleotide component playing a significant role in realization of the immunostimulating and protective effects of the free and immobilized proteases.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Studies on macrophage-activating factor (MAF) in antitumor immune responses. I. Tumor-specific Lyt-1+2- T cells are required for producing MAF able to generate cytolytic as well as cytostatic macrophages

In the present study we investigated some of the cellular mechanisms for the generation of macrophage-activating factor(s) (MAF) in immune responses to tumor antigens. C3H/HeN mice were immunized to syngeneic MH134 hepatoma or MCH-1-A1 fibrosarcoma by intradermal inoculation of viable tumor cells, followed by the surgical resection of the tumor. Spleen and lymph node cells from these tumor-immune mice were stimulated in vitro with the corresponding tumor cells, and supernatant from such a culture was tested for an ability to activate macrophages to exert their cytostatic and cytolytic activities as detected on tumor cells unrelated to immunizing tumors. Peritoneal adherent cells as a macrophage source, which were preincubated with supernatant from co-culture of tumor-unimmunized normal spleen and lymph node cells plus tumor cells, failed to exhibit any significant antitumor effect on unrelated X5563 tumor cells, whereas the addition of supernatant from cultures containing immune lymphocytes to adherent cells resulted in appreciably potent cytostatic and cytolytic effects on X5563 tumor cells, indicating the generation of MAF in culture supernatant. The activation of tumor-immune spleen and lymph node cells for MAF generation was tumor-specific, because anti-MH134- and anti-MCH-1-A1-immune lymphocytes produced MAF by the stimulation with the respective but not with the other alternative tumor cells. Such MAF production was abolished by treatment of tumor-immune spleen and lymph node cells with anti-Thy-1.2 or anti-Lyt-1.1 but not with anti-Lyt-2.1 antibody plus complement before culturing. These results indicate that the tumor-specific Lyt-1+2- T cell subset has a crucial role in generating MAF by which an adherent cell population as a source of macrophages acquires the potential for inducing a cytolytic as well as a cytostatic effect on tumor cells.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

Cryptosporidium parvum infection in mice: inability of lymphoid cells or culture supernatants to transfer protection from resistant adults to susceptible infants

The ability of murine lymphoid cells or culture supernatant fractions to transfer protection against Cryptosporidium parvum was examined. Spleen or mesenteric lymph node (MLN) cells were taken from adult mice resistant to C. parvum and given either directly or following in vitro culture to infant mice. Neither spleen or MLN cells, nor cells or supernatant fractions from in vitro cultures transferred protection from resistant adult donors to susceptible infant recipients. These results may be due to limitations in the present model. Alternatively, the resistance of infant mice to C. parvum may not be immunologically mediated.     

Alterations in the Immunogenic Properties of Sheep Erythrocytes by Sonic Disruption

A solubilized sheep red blood cell (SRBC) antigen (supernatant fraction obtained by centrifuging 10^7-2 × 10⁸ sonicated SRBC at 6 × 10⁴ g for 30 min [Sup-SRBC]), whose ability to inhibit anti-SRBC plaque formation was 70% of that of the original sonicated SRBC, was unable to elicit a detectable antibody response in either unprimed or SRBC-primed mice. However, Sup-SRBC as well as intact SRBC antigens generated memory for the secondary response, which was transferable to irradiated syngeneic recipients by injection of immune spleen cells. The memory generated by Sup-SRBC involved helper memory for anti-trinitrophenyl group (TNP) response to challenge with TNP-conjugated SRBC. Increase in the helper T cell memory in the spleens of Sup-SRBC-primed mice was also demonstrated by an in vitro culture experiment and by an adoptive cell transfer experiment. In contrast, no detectable B cell memory was generated by Sup-SRBC. Repeated stimulation with Sup-SRBC never induced significant antibody response but reduced the level of memory. A single injection of a low dose (10⁶) of SRBC also failed to induce a definite primary antibody response generating memory for the secondary response. However, repeated stimulation with this dose of SRBC induced a high antibody response and generated good memory. From these results it is suggested that the intact structure of SRBC is required for the activation of B cells, but is not necessary for the stimulation of T cells.     

Preparative Electrophoretic Separation of Antibody Forming Cells

A procedure is described for preparative electrophoretic separation of lymphoid cells. The separations were performed with a free flow electrophoretic cell separator model VAP IV (Desaga, Heidelberg, Bender & Hobein, Munich, Brinkmann Instruments, Westbury, N. Y.). Rats were immunized with sheep erythrocytes (SRBC), lymph node cells electrophoretically separated at different times after immunization and the fractions obtained subsequently cultured in diffusion chambers. The antibody forming cells and the morphological composition of the fractions was determined after separation and after culture. Lymph node cells could be separated into 16 fractions. Within this heterogeneous distribution profile two narrow distributions of antibody forming cells of the same specificity but of different stages of development could be detected. The distribution of lower electrophoretic mobility contained the primed lymphocytes and blast cells, the faster distribution contained the differentiated plasma cells. It was found that a homogeneous cell population is rather homogeneous in its electrophoretic mobility. This is an indication that the electrophoretic mobility would be a useful parameter enabling separation of functionally defined cell populations.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Cytotoxic properties of the splenocytes of C3HA mice following treatment with exogenous RNA

The influence of fractions of exogenous RNA, isolated from spleens of C3HA mice and of rats, both intact (control--cRNA) and immunized with homogenate of normal syngenic, allogenic and xenogenic tissues (immune--immRNA), on the cytotoxic properties of splenocytes of C3HA intact mice was compared in the in vitro cytotoxic experiments. The splenocytes treated with different RNA fractions were used as effector-cells. In vitro cultivated MGXXIIa cells of strain specific C3HA mice hepatoma, and K562 cells of human erythroleukemia, both labeled with 3H-uridine, served as target cells. Thus, it is only the cytoplasmic fraction of immRNA isolated from the spleens of rats immunized with tissue antigens of C3HA mice that induced a more pronounced stimulation of cytotoxic activity of splenocytes.     

Specific immunosuppression by passive antibody. V. Participation of macrophages in reversal of suppression by peritoneal exudate cells from immune animals

Thioglycollate-stimulated peritoneal exudate cells (PEC), harvested from mice immunized against sheep erythrocytes (SRBC) and transferred to normal syngeneic recipients, reverse the immunosuppression caused by passively administered anti-SRBC antibody. Macrophages purified from PEC on BSA gradients did not reverse immunosuppression; neither did suspensions of cells from mesenteric lymph nodes of immune mice. Mixtures of the purified macrophages and lymph node cells were fully capable of reversing immunosuppression. Thus, two types of cell, one a macrophage and one a lymphocyte, are required. Both must be compatible with the recipient mice at the H-2 complex. However, only the macrophages must necessarily be obtained from an immune donor. When “immune” macrophages were preincubated in vitro with “normal” lymph node cells before transfer to antibody-treated syngeneic recipients, a significant reversal of the immunosuppressive effect occurred. The ability of whole PEC or spleen cells to reverse the immunosuppressive effect of passive antibody is acquired rapidly after injection of a single low dose of antigen. Development of this ability precedes the appearance, in the circulation, of immunosuppressive antibody.     

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).     

Study of the phenomenon of specific suppression of the immune response in an adoptive transfer system]

It was revealed that the administration of the spleen cells (SC) of syngeneic animals immunized with a high dose of sheep red blood cells (SRBC) to intact mice led to a marked specific suppression of the recipients' immune response. The donors' SC obtained on the 14th day after the intraperitoneal injection of SRBC had the greatest suppressive activity. The SC of intact animals and mice given rat erythrocytes preliminarily failed to influence the immune response of the intact recipients in their SRBC immunization. Treatment of immune SC with the anti-T-serum (ATS) or the anti-B-globulin (ABG) and the complement considerably decreased or completely eliminated the suppressive activity. Administration of a mixture of two immune SC suspensions one of which was ATS- and another ABC-treated did not produce any suppression of the immune response in the intact recipients. It is supposed that the suppressor cells in the given model were T-lymphocytes expressing the antigens, common of cross-reacting with the B-cells.     

Analysis of certain mechanisms of antigen-specific suppression of the immune response

CBA mice were immunized with sheep red blood cells (SRBC) to obtain immune spleen cells (ISc) which were used to suppressor cells. Administration of ISC to intact syngeneic recipients on the immunization day led to a more powerful suppression of the immune response as compared to that seen one day after antigen injection. Four days after immunization the animals' immune response was not liable to be suppressed. ISC extract possessed similar effects with respect to the immune response of normal spleen cells which were transplanted to the cyclophosphamide-treated recipients. The immune response of spleen cells from mice immunized with SRBC in a dose of 10(6) was less liable to be suppressed. Hyperimmune spleen cells from donors immunized with SRC in a dose of 10(9) were insensitive to ISC or to the extract. Experiments with the use of adoptive transfer of a mixture of immune and intact T- and B-cells have disclosed that B-cells from hyperimmune donors were resistant to suppression. Therefore, B-lymphocytes are the most probable target cells exposed to T-suppressors in the given system. The mechanism is discussed of the selective effect of T-suppressors on B-cells in the course of the immune response development during immunization with high doses of antigen.     

Plasticity of immune responses suppressing parasitemia during acute Plasmodium chabaudi malaria.

gammadelta T cells have a crucial role in cell-mediated immunity (CMI) against P. chabaudi malaria, but delta-chain knockout (KO) (deltao/o) mice and mice depleted of gammadelta T cells with mAb cure this infection. To address the question of why mice deficient in gammadelta T cells resolve P. chabaudi infections, we immunized deltao/o mice by infection with viable blood-stage parasites. Sera from infection-immunized mice were tested for their ability to protect JHo/o, deltao/o double KO mice passively against P. chabaudi challenge infection. The onset of parasitemia was significantly delayed in mice receiving immune sera, compared with saline or uninfected serum controls. Immune sera were then fractionated into Ig-rich and Ig-depleted fractions by HPLC on a protein G column. Double KO mice were passively immunized with either fraction and challenged with P. chabaudi. The onset of parasitemia was significantly delayed in recipients of the Ig-rich fraction compared with recipients of the Ig-poor fraction of immune sera. We conclude that deltao/o mice, which are unable to activate CMI against the parasite, suppress P. chabaudi infection by a redundant Ab-mediated process.     

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.     

Hymenolepis nana: passive transfer of mouse immunity by T-cell subset of phenotype Lyt-1

Mesenteric lymph node cells obtained from donor mice (BALB/c strain) actively immunized by oral inoculation with Hymenolepis nana eggs were syngeneically transferred by intravenous injection into athymic nude mice previously uninfected. The adoptively immunized recipients were then challenged with 1000 H. nana eggs 2 days after cell transfer. The degree of immunity transferred was assessed by examining cysticercoids developed in the intestinal villi of the recipients on Day 4 of challenge infection. The criterion for success in cell transfer of immunity was the complete rejection of cysticercoids as was generally expected in mice infected previously. The transfer of 1.5 X 10(8) immune mesenteric lymph node cells obtained from donors immunized 4 days before cell collection resulted invariably in the complete rejection of cysticercoids, though not less than this cell dosage. The immunity was passively transferable to recipients by T cells, especially by T-cell subset of phenotype Lyt-1 but not those of phenotype Lyt-2.3 and Lyt-1.2.3. However, 1.5 X 10(8) immune mesenteric lymph node cells obtained from donors immunized 21 days before cell transfer and 1.5 X 10(8) immune spleen cells obtained from donors immunized 4 days before cell transfer had little or no effect on the rejection of cysticercoids.