Passive transfer of cell-mediated immunity in xenogeneic animals

Cell-mediated immunity (delayed hypersensitivity) to 1-chloro-2,4-dinitrobenzene (DNCB) was passively transferred from sensitized guinea pigs to mice. Transfer was accomplished by subcutaneous injection of peritoneal exudate cells of sensitized guinea pigs.     

So-called antireceptor sera

Experiments in delayed type hypersensitivity transfer were carried out with the aim of studying the ability of rabbit antisera against peritoneal exudate cells of rats sensitized with bovine gamma globulin or rabbit kidney tissue antigen to block peritoneal exudate cells of guinea pigs. In the serological test the antisera prepared against the cells of sensitized rats and tentatively named "receptor antisera", reacted not only with the cells of these rats, respectively, but also with guinea pig cells. In hypersensitivity transfer experiments in guinea pigs receptor antisera showed a blocking effect on the transferred cells, making them incapable of transferring hypersensitivity, i. e. rabbit antisera against rat peritoneal exudate cells reacted with guinea pig cells. This interaction was specific: the blocking effect was manifested only when guinea pigs whose cells were used in the transfer were sensitized with the same antigen as the rats against whose cells the receptor antisera had been prepared. The control antisera, taken for the treatment of the transferred cells in the same doses as the receptor antisera, had no blocking effect on the cells.     

Specific depression of delayed hypersensitivity to purified proteins, with relation to production of circulating antibody

In guinea pigs sensitized in the footpads with a purified protein, such as hen egg albumin or diphtheria toxoid, in incomplete Freund's adjuvant, delayed hypersensitivity precedes the appearance of circulating antibody. This expression of delayed hypersensitivity by skin-test declines sharply the day before circulating antibody is detected. Adoptive transfer of spleen and peritoneal exudate cells from guinea pigs showing this decline suppresses the expression of delayed hypersensitivity in already sensitized recipients. This suppression of delayed hypersensitivity is immunologically specific. The intensity of this suppression does not correlate directly with the dose of sensitizing antigen, nor does it depend directly on the amount of circulating antibody.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Macrophage migration-inhibition in desensitized guinea pigs

The migration of peritoneal exudate cells obtained from guinea pigs with delayed skin reactivity to egg albumin (EA) and diphtheria toxoid (DT) was inhibited in the presence of antigen. A dose of 2 mg of EA given intravenously 8 days after sensitization specifically abolished the migration inhibition tested 5 weeks later. When the challenge was given into a foot pad 6 weeks after sensitization the migration inhibition was partially suppressed 3 to 28 days later.Repeated skin testing did not affect the migration results of the challenged or unchallenged guinea pigs.The demonstration in vitro of desensitization argues that the mechanism is either a reduced number or a reduced responsiveness of the specific effector cells of delayed hypersensitivity, or an inhibitory effect of cells stimulated by the specific antigen. If a humoral inhibitory factor is involved, it is either tightly bound by the cells or produced during the migration assay.     

RNA extracts of lymphoid cells sensitized to DNP-oligolysines convert nonresponder lymphoid cells to responder cells which release migration inhibition factor

Strain 13 nonresponder peritoneal exudate cells were converted to responder status to α or ?,DNP-oligolysines after incubation of the cells with RNA extracts prepared from responder guinea pigs skin test sensitive to these synthetic antigens. The conversion of nonresponder strain 13 cells was assessed by the direct cell migration inhibition correlate of delayed hypersensitivity. Nonresponder cells were not converted by RNA extracts prepared from unimmunized responder guinea pigs or from non-responder strain 13 guinea pigs previously injected with DNP-oligolysines. Thus, it seems possible to correct immunological unresponsiveness in vitro in spite of a specific genetically determined deficiency of the immune response related to the Ir gene.     

Delayed Hypersensitivity in Relation to Suppression of Growth of Listeria monocytogenes by Guinea Pig Macrophages

Prototypes of delayed hypersensitivity (tuberculin allergy, graft rejection immunity, and contact dermatitis) were established in guinea pigs. The macrophages from peritoneal exudates of such animals were examined for their capacities to suppress the growth of Listeria monocytogenes in vitro. Only the macrophages from animals sensitized to BCG clearly exhibited this property.     

Dermal and intravascular Langerhans cells at sites of passively induced allergic contact sensitivity.

In order to further define the role of Langerhans cells in contact allergic reactions, passive transfer studies were done in guinea pigs using 2,4-dinitro-1-chlorobenzene (DNCB)-sensitive donor cells. Langerhans cells were found in the lumen of dermal vessels resembling lymphatics at 2, 3, 15, and 48 hr after DNCB challenge. In contrast to the previously reported findings in actively sensitized guinea pigs, the changes involving Langerhans cells in passively sensitized guinea pigs were mainly noted in the dermis. These consisted of increased numbers of Langerhans cells and of mononuclear cells apposed to Langerhans cells 3 or more hours after challenge with DNCB. The increased Langerhans cell population in the dermis and the presence of Langerhans cells in dermal vessels in specifically challenged sites in adoptive immune reactions furnishes further support for a significant role of Langerhans cells in the interaction between antigen and sensitized cells.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.     

Killing effect of normal peritoneal exudate cells in guinea pigs on microfilariae of Dirofilaria immitis evoked by passive transfer of immune humoral factor.

Parasiticidal activity of normal peritoneal exudate cells on microfilariae of Dirofilaria immitis in diffusion chambers implanted into normal guinea pigs was evoked by intraperitoneal passive transfer of anti-D. immitis serum. The immune serum was fractionated into supernatant and sediment by ammonium sulfate precipitation. The parasiticidal effect was reproduced with the supernatant and, to a less extent, with the sediment. Anti-D. immitis serum from the animals sensitized 5 days before was also effective in this respect. From these results it can be concluded that the factor responsible for the phenomenon is some other factor(s) than the antibody. In addition, the in vitro cytotoxicity test demonstrated an enhanced Mf-killing activity of sensitized peritoneal exudate cells by adding with anti-D. immitis serum.     

Suppression of the intradermal hypersensitivity reaction of the delayed type by a serum fraction from patients containing leukocyte migration inhibition factor]

The blood serum of patients with active tuberculosis of the lungs and chronic pneumonia inhibited migration of donor's leukocytes and macrophages of the peritonal exudate of guinea pigs when compared with migration of similar cells in the medium with the serum of cattle or donors. After chromatography these sera were fractionated on the columns with Sephadex G-100. Fractions containing the leukocyte migration inhibition factor (LMIF) suppressed, up to complete abolition, the intradermal reaction to tuberculin in man and guinea pigs sensitized with BCG. The LMIF is supposed to act in the regulation of delayed hypersensitivity reaction.     

Effect of Corynebacterium parvum on the immune response in guinea pigs. II. Passive transfer of enhanced anamnestic response and delayed hypersensitivity observed after treatment with Corynebacterium parvum]

After intradermal immunization with a mixture of Corynebacterium parvum (C. parvum) and ovalbumin guinea pigs show a markedly increased anamnestic response to an intradermal booster of ovalbumin as compared to controls treated with ovalbumin only. At the same time a reaction of delayed type hypersensitivity is observed in the treated animals, but not in controls. The enhanced anamnestic response as well as the posivitive skin reaction were transferred to strain 2 histocompatible guinea pigs by peripheral blood leukocytes as well as by peritoneal exudate cells. Passive transfer was not obtained after prior irradiation of donor animals.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Experimental allergic orchitis and aspermatogenesis. VI. Transfer of allergic orchitis with immune cells.

Typical experimental allergic orchitis (EAO) and aspermatogenesis were successfully transferred to strain 13 guinea pigs with peritoneal exudate and lymph node cells from male and female donor guinea pigs (lacking detectable antibody) previously sensitized with 9 mug of highly purified GP1 glucoprotein isolated from the sperm acrosome. Attempts to transfer the disease with circulating antibody from hyperimmunized animals were not successful. These studies support a cell-mediated basis for the immunopathologic events in EAO.     

A comparative study of RNA fractions mediating delayed sensitivity to a chemically defined antigen in vitro.

Hot-cold phenol extracts of RNA prepared from guinea pigs sensitized to mono (p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) contains limited but distinct fractions able to transfer ARSNAT or KLH sensitivity to guinea pig peritoneal exudate cells in vitro. Each of these fractions have been compared by oligo(dT) affinity chromatography and sucrose density gradient analysis. One RNA fraction initially obtained from a sucrose density gradient (designated as B fraction) possessed two separate peaks and contained polyadenylic acid sequences as evidenced by its ability to bind to an oligo (dT) column. Another fraction (Fraction II) initially isolated by oligo (dT) affinity chromatography possessed two peaks after sucrose density gradient analysis, contained poly-A sequences, and had an S-value range approximating the B fraction. RNA fractions prepared from the liver or skeletal muscle of sensitized guinea pigs fails to transfer ARSNAT sensitivity and all fractions are completely inactivated by bovine pancreatic RNase. The results suggest that portions of density gradient prepared B fraction and Fraction II binding to oligo (dT) cellulose may represent the same and/or similar moieties of immunobiologically active RNA.     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

Atomic spectroscopic evidence for the absence of a low-molecular-weight (486) antigen in RNA extracts shown to transfer delayed-type hypersensitivity in vitro

Ribonucleic acid-rich extracts obtained from the spleens or lymph nodes of guinea pigs skin test sensitive to mono-(p-azobenzenearsonate)-N-chloracetyl-l-tyrosine (ARS-NAT) (MW 486) were able to convert “nonsensitive” peritoneal exudate cells (PEC) to a state of specific immunologic sensitivity, as assessed by the cell-migration-inhibition correlate of delayed hypersensitivity. Specific inhibition of migration of RNA-treated PEC by ARS-NAT antigen was observed while no inhibition of migration occurred with RNA alone or by incubation with unrelated antigens. The RNA used to transfer sensitivity was assessed for arsenic (As) content as a chemical marker for the ARS-NAT antigen utilizing two methods: a Gutzeit As assay, and atomic absorption spectroscopy (AAS). Preliminary chemical analysis utilizing the Gutzeit assay, which detects as little as 1 μg As, failed to detect As in 3200–4800 μg of RNA or in cell suspensions from the spleen, lymph nodes, and liver of immunized guinea pigs. Further attempts to detect As utilizing AAS, where the limit of As sensitivity was 0.1 ng, failed to detect As in 250 μg to 10 mg of “ARS-NAT-sensitive” RNA, suggesting that, if As is associated with the RNA-rich extracts, it could be present in an amount of no more than 5 pg in 500 μg of RNA; this corresponds to less than 0.0000065% ARS-NAT antigen. These results suggest an informational role for the RNA extracts in our delayed hypersensitivity system, paralleling similar evidence for the action of RNA extracts in antibody systems.     

The role of B lymphocytes in cell-mediated immunity II. Delayed hypersensitivity induced by dinitrophenyl-ficoll in dinitrophenyl-keyhole limpet hemocyanin-immunized guinea pigs.

Dinitrophenyl (DNP)-Ficoll will elicit typical delayed hypersensitivity skin reactions in guinea pigs immunized with DNP-keyhole limpet hemocyanin (KLH). We observed that lymph node cells (LNC) from these animals produced the lymphokine, monocyte chemotactic factor (MNL CTX) when stimulated by DNP-Ficoll in vitro. This response was antigen and hapten specific since LNC from nonimmune guinea pigs or those immunized with nonDNP containing antigens were not stimulated by DNP-Ficoll. Lymph node cells were fractionated into T- and B-cell-enriched populations to determine the nature of the DNP-Ficoll-responsive cell. Only the B-lymphocyte-enriched population produced MNL CTX in response to DNP-Ficoll. The purity of the B-cell population was demonstrated by its failure to respond to PHA and by the fact that B cells derived from DNP-although they could no longer respond without T-cell help to the T-dependent antigen, DNP-OVA. These findings suggest that the hapten-specific response of guinea pigs to DNP-Ficoll may be a form of B-cell-mediated delayed hypersensitivity.     

Activity and Properties of Macrophage Migration Inhibitory Factor produced by Mixed Lymphocyte Cultures

THE macrophage migration test is an in vitro demonstration of delayed hypersensitivity. Supernatant fluids of sensitive lymphocytes cultured for 24 h in the presence of specific antigen contain migration inhibitory factor (MIF) that arrests the migration of macrophages of unsensitized animals in vitro¹,². In vivo, it induces delayed skin reactions³. The use of the macrophage migration test, based on differences of transplantation antigens in donor and recipient, to show histocompatibility has been suggested⁴. The test was also recommended as an indicator of immunological reactivity after organ transplantation, to demonstrate impending rejection⁵. It can demonstrate homograft sensitivity, for migration of peritoneal exudate cells (containing lymphocytes and macrophages) of CBA mice previously sensitized by grafts from A/Jax donors was inhibited when they were mixed with peritoneal exudate cells of the donor strain. However, histocompatibility was not demonstrated, for mixtures of peritoneal exudate cells of ungrafted CBA mice and A/Jax mice migrated regularly during the 24 h test⁶.     

Cell-mediated immunity to Dirofilaria immitis.

Cell-mediated immunity to Dirofilaria immitis (DI) in guinea pigs was confirmed by the migration inhibition test (MIT), the blast transformation test (BTT), the delayed skin reaction, and the skin reaction by passive transfer with sensitized peritoneal exudate (PE) cells. All migration inhibition (MI) positive cases were always associated with positive skin reactions and two cases showed positive skin reactions without MI. The cellular antibody confirmed by MIT first appeared on the 4th day after single sensitization, but DNA synthesis in splenic lymphocytes had already started on the 3rd day in the absence of delayed skin reaction and MI. Then, the role of this cellular antibody in the immune mechanism against DI infection was investigated by the in vitro and in vivo cytotoxicity test using microfilariae (Mf) of this species as a target. The cytotoxic activity significantly increased in the sensitized splenic and PE cells, and in vivo normal PE cells implanted into sensitized animals.