Hyperimmune Animals Research Articles

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)’2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)’2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.

BackgroundAlpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species.MethodsThe α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (–CONH2) and free α-carboxylic acid (−COO−) isovariant of the valine residue.ResultsP18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb.ConclusionsOur results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

Passive Transfer of Virus-Specific Antibodies Confers Protection against Reproductive Failure Induced by a Virulent Strain of Porcine Reproductive and Respiratory Syndrome Virus and Establishes Sterilizing Immunity

Immune mechanisms mediating protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) are not well understood. The PRRSV-specific humoral immune response has been dismissed as being ineffective and perhaps deleterious for the host. The function of PRRSV antibodies in protective immunity against infection with a highly abortifacient strain of this virus was examined by passive transfer experiments in pregnant swine. All of a group of pregnant gilts ( n = 6) that received PRRSV immunoglobulin (Ig) from PRRSV-convalescent, hyperimmune animals were fully protected from reproductive failure as judged by 95% viability of offspring at weaning (15 days of age). On the other hand, the totality of animals in a matched control group ( n = 6) receiving anti-pseudorabies virus (PRV) Ig exhibited marked reproductive failure with 4% survival at weaning. Besides protecting the pregnant females from clinical reproductive disease, the passive transfer of PRRSV Ig prevented the challenge virus from infecting the dams and precluded its vertical transmission, as evidenced by the complete absence of infectious PRRSV from the tissues of the dams and lack of infection in their offspring. In summary, these results indicate that PRRSV-Igs are capable of conferring protective immunity against PRRSV and furthermore that these Igs can provide sterilizing immunity in vivo.

Durable elimination of high affinity, T cell-dependent antibodies by low molecular weight antigen arrays in vivo.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Diversity of T cell receptor-alpha chain transcripts from hyperimmune alloreactive T cells.

Alloreactive T cells represent a relatively large fraction of the T cell population compared with the fraction of T cells that are specific for other foreign Ag. Recent findings indicate that most alloreactive T cells recognize endogenous peptides in association with a non-self MHC product. In light of these observations, it is perhaps not surprising that previous studies of the size of the TCR-alpha beta repertoire among alloreactive cells showed that they express many different V alpha and V beta genes. To further access the extent of diversity among alloreactive cells, we examined V alpha J alpha combinatorial diversity in polyclonal populations of a BALB/c anti-BALB.B mixed lymphocyte response. A long term culture from naive mice contained a diverse repertoire of V alpha J alpha combinations that was similar to the diversity present among unstimulated splenic T cells. In contrast, long term cultures from hyperimmunized animals contained "dominant" clones of T cells that expressed a restricted repertoire of V alpha J alpha combinations. Examination of the nucleotide sequences of these alpha-chains suggested that there was selective expansion of T cells with identical alpha-chains. In addition, T cells that express the same V alpha J alpha combination but different junctions were also identified. Consistent with previous results, the isolates from hyperimmune animals did not contain somatic mutations in the CDR3 region of the alpha-chains. Nevertheless, the results suggest that T cells may be subject to in vivo selection and clonal expansion analogous to the process of affinity maturation of Ig.

A species-specific enzyme-linked immunosorbent assay for Nosema furnacalis (Microspora: Nosematidae)

A serological assay has been developed that specifically reacts with Nosema furnacalis proteins. N. furnacalis spores were harvested from laboratory-reared Ostrinia nubilalis and purified by centrifugation through a Percoll gradient. Spores were germinated and soluble proteins were used to immunize rabbits and mice. Antibodies from hyperimmune animals were purified and used to develop an indirect enzyme-linked immunosorbent assay (ELISA). The assay detected N. furnacalis proteins in infected O. nubilalis larvae and pupae, in Helicoverpa zea larvae and pupae, and in H. zea cell culture. Latex beads sensitized with antibody did not agglutinate spores of N. furnacalis or N. pyrausta, indicating that the antigens are probably internal sporoplasm proteins rather than soluble surface proteins. No cross reaction was observed when the assay was tested against five species of Nosema: N. pyrausta in O. nubilalis; N. apis in Apis mellifera; N. locustae in Melanoplus femurrubrum, M. bivitattis, and M. lakinus; N. bombycis in Bombyx mori; and an undescribed species from Malacosoma americanum. Three species of Vairimorpha were also tested, all showing no cross reaction: V. necatrix, another pathogen of O. nubilalis, and two undescribed Vairimorpha spp. in Hyphantria cunea and Alabama argillacea. Western blot analysis confirmed specificity of the ELISA, distinguishing among N. furnacalis infected, N. pyrausta infected, and unifected O. nubilalis.

Characterization of a family of monoclonal antibodies which bind Schistosoma japonicum egg antigens and express an interstrain cross‐reactive idiotype

A family of monoclonal antibodies (MoAb) was derived from the somatic cell fusion of P3NS1 myeloma cells and lymphoid cells from naturally infected mice of hyperimmunized mice. C57BL/6 mice were naturally infected with Schistosoma japonicum, and BALB/c mice were hyperimmunized with preparations of S. japonicum soluble egg antigens (SEA). The MoAbs which reflected the immune repertoire of naturally infected animals versus hyperimmune animals were characterized with regard to antibody isotype, antigen binding specificity, in-vitro immunosuppression of antigen-induced cell-mediated immune responses and the expression of SJ-CRIM, a major cross-reactive idiotype which appears on polyclonal anti-SEA antibodies generated during murine S. japonicum infection. The data indicate that for MoAbs of the IgG isotype which bound SEA by ELISA, the most immunosuppressive anti-SEA MoAbs identified expressed SJ-CRIM and were derived from naturally infected mice. All anti-SEA MoAbs expressing SJ-CRIM showed an identical banding pattern on immunoblot analysis which was abrogated by weak periodate treatment. The generation of expression of SJ-CRIM on MoAbs using differing methodologies across an allotype barrier indicates that the expression of SJ-CRIM is encoded by a germline gene. These data indicate an association between expression of this germline interstrain cross-reactive idiotype and immunosuppressive capacity. In addition, the immunoregulatory network which develops during immune S. japonicum infection is initiated by a carbohydrate epitope(s) found on various SEA. These data have profound implications in the use of the cross-reactive idiotype as a serodiagnostic tool in schistosomiasis.

Monoclonal antibodies against α-bungarotoxin

Hybridomas secreting monoclonal antibodies against ?-bungarotoxin were produced from the fusions of mice lymphocytes from hyperimmune animals with two mice myeloma cell lines ((NSI/1 or Sp2/0). Several anti-?-bungarotoxin monoclonal antibodies were derived and characterized. One of these (spB57) belonged to the IgG(1) subclass and bound potently to ?-bungarotoxin in a radioimmunoassay. This effect was specific to the anti-?-bungarotoxin antibody; a control series of antibodies (against tyrosine hydroxylase, enkephalin, neurofilament and the nerve growth factor receptor) did not bind radiolabelled toxin. Furthermore, the anti-?-bungarotoxin antibody did not interact with other radiolabelled receptor ligands. Using autoradiographic techniques, spB57 was shown to block the binding of [(125)I]?-bungarotoxin to brain sections. Similarly, spB57 blocked radiolabelled toxin binding to brain membranes; again this was an effect specific to the anti-?-bungarotoxin antibody. The decrease in [(125)I]?-bungarotoxin binding suggested that spB57 specifically bound the toxin molecule such that it could no longer interact with its receptor. Since the ?-BGT site has the characteristics of a nicotinic receptor, the effect of the antibody was also tested on the inhibition of [(125)I]?-bungarotoxin binding by cholinergic ligands. SpB57 partially reversed the inhibition of ?-toxin binding observed with nicotinic agonists and d-tubocurarine, but not with other nicotinic antagonists nor with muscarinic receptor ligands. These effects appeared to be specific for spB57, as they occurred to a much lesser extent with two other anti-?-BGT mAbs, nsB8 and spB28. These results suggest that an antibody against the ?-toxin can affect the interaction of nicotinic receptor ligands at the ?-BGT site.

Anti-idiotypic antibodies against UV-induced tumor-specific CTL clones. Preparation in syngeneic combination.

In this study, we first established several CTL clones of (BALB/c x C57BL/6)F1 origin that were specific for either syngeneic UV female 1 or UV male 1 fibrosarcoma cell lines. All the CTL clones had Thy-1+ Lyt-2+ L3T4- phenotypes and showed Kd restriction when lysing the corresponding target cells. Sera obtained from syngeneic animals immunized with three CTL clones, 10B-5 for UV female 1, and CTL9 and CTL10 for UV male 1, showed specific inhibition of target cell lysis with the corresponding CTL clones. The inhibitory activities were found in sera of the majority of immunized animals. Because the inhibitory activity resides in protein A-binding fraction, mAb were produced by hybridizing spleen cells of hyperimmune animals. N1-56 was thus obtained from a mouse immunized with 10B-5 CTL clone reactive with UV female 1. N1-56 was clonotype specific, reacting with 10B-5 but not with other CTL lines or leukemia cell lines. No N1-56+ cells were detectable in thymocytes, lymph node cells, or spleen cells of either naive or UV female 1-immune CB6F1 mice. Immunoprecipitation showed that N1-56 reacts with 90,000 Mr molecules on 10B-5 CTL clone under nonreducing conditions and 45,000 Mr molecules under reducing conditions, indicating its reactivities with idiotypic determinants of TCR on the CTL clone. N1-56 inhibited lytic activity of 10B-5, but neither N1-56 nor alpha-10B-5 hyperimmune serum inhibited that of alpha-UV female 1 mixed lymphocyte tumor cell culture cells. N1-56 induced proliferation of 10B-5 without addition of Ag.

Activation of memory and virgin B cell clones in hyperimmune animals.

To study the long-term memory response, BALB/c mice were allowed to rest for over a year after a secondary immunization with the hapten 2-phenyl-oxazol-5-one (phOx). For the tertiary immunization two different protocols were used. In one protocol mice were injected i.v. and 3 days later spleen cells were fused to a nonproducing hybridoma line. PhOx-specific hybridomas were established and the sequence of the heavy and light chain mRNA was determined. This tertiary response resembled the diversity pattern of the secondary response with a further increase both in somatic mutations and in the average dissociation constant. The high number of somatic mutations demonstrates the persistence of memory B cell clones over a long time period. In the second protocol mice were boosted with an i.p. injection of alumprecipitated antigen phOx and 7 or 14 days later spleen cells were fused. Sequence analysis of heavy and light chain mRNA showed that these tertiary response antibody molecules had surprisingly few somatic mutations, indicating an activation of virgin B cell clones in these hyperimmunized animals. The maturation of these newly stimulated B cell clones seems to follow somewhat similar rules to those found for the primary response. It appears therefore that the two immunization protocols reflect the response of memory and virgin B cells, respectively.

Immunological features of consecutive intramammary infections with Streptococcus agalactiae in vaccinated and non-vaccinated heifers

The antibody response of the bovine udder to experimental Streptococcus agalactiae mastitis was measured using an enzyme-linked immunosorbent assay (ELISA). Ten first-lactation heifers, four of which were rendered systemically hyperimmune to Str. agalactiae, were infected, via the teat canal, in the right front quarter on two or three consecutive occasions at 23 and 30-day intervals. Initially there was no specific antibody in the udder of the non-vaccinated heifers while the hyperimmune animals had evidence of specific whey antibody. During each infection the humoral response was essentially biphasic. In the non-vaccinated heifers the first phase coincided with the acute clinical reaction and specific antibody only appeared in the infected quarter. With the second phase, antibody appeared in both the infected and control quarters. By the third consecutive infection these changes were more marked and occurred more quickly and were very similar to those seen with the hyperimmune animals during their first infection. The results demonstrate that repeated intramammary infection with Str. agalactiae can produce high levels of serum and whey antibodies. However, the response in the udder was of short duration.

Are VH framework antigenic determinants expressed on both rabbit B and T lymphocytes?

AbstractThe extent to which VH framework determinants are represented on T cell receptors is unresolved and controversial. We have studied this question in the rabbit using antia (VH framework) allotype antisera and a goat anti‐rabbit VH antiserum. Flow micro‐ fluorometric analysis of spleen lymphocytes from hyperimmune animals (Freund's adjuvant plus picryl chloride) demonstrated closely similar frequencies of cells stained with the anti‐VH framework antisera compared to anti‐light chain antisera. To search for potentially hidden molecules, additional studies were performed with radioiodinated, detergent‐solubilized plasma membranes derived from immune spleen and lymph node cells. The membranes were analyzed by standard immunoprecipitation techniques. Absorption with anti‐Ig constant region immunoabsorbents removed all radioactivity precipitable by specific anti‐VH framework antisera compared to controls, whereas after absorption with control (normal goat globulin) immunosorbents, VH‐bearing surface IgM and putative surface IgD (presumably of B cell origin) were readily demonstrated by immunoprecipitation. Thus, no evidence for a population of cells or a class of molecule(s) bearing VH in the absence of light or heavy chain constant region determinants was obtained. Although these studies do not support the concept of similar VH framework determinants on T and B cell receptors, they do not exclude a much more limited sharing of determinants or a very minor T cell subpopulation which expresses VH framework that would not be detectable by the methods used in these studies.

Regulation of immune response to tumor antigen: interference with syngeneic tumor immunity by anti-IA alloantisera.

We present evidence for a role of I-A subregion-encoded determinants in syngeneic tumor immunity. In animals rendered immune to the S1509a fibrosarcoma, daily treatment with microliter quantities of antisera directed against Kk and I-Ak determinants expressed on lymphoid cells of host origin decreased the capacity for immune tumor rejection. Absorption studies revealed that anti-I-Ak antibody activity alone was sufficient for the manifestation of this effect. Furthermore, experiments utilizing F1 hybrids showed that an antiserum that was genetically unable to interact with H-2 determinants expressed on the tumor was equally effective in inhibiting tumor immunity. Suggestive evidence that the activity of this antiserum is related to interference with the generation of effector T cell function was provided by the observation that hyperimmune animals pretreated with an anti-Kk,I-Ak antiserum were no longer capable of adoptively transferring tumor immunity to naive recipients. Thus, it is possible to regulate the secondary immune response to tumor antigens by using antisera with specificity for I-A determinants expressed on cells or possibly on factors of the host lymphoid system.

The distribution and fate of blood-borne 125IUdR-labelled tumour cells in immune syngeneic rats.

Radiolabelled sarcoma cells injected into the tail veins of normal rats were held up almost exclusively in the lung, and were not observed to pass through into the systemic circulation. Intramuscularly injected tumour cells were retained at the site of injection. Radioactivity was lost from both sites though more rapidly from the lung than from muscular tissue and was probably the result of tumour-cell death. Alveolar macrophages did not take part in the destruction of tumour cells in the lung. There was an increased rate of radiolabel loss from the lungs of hyperimmune, post-excision and tumour-bearing rats, as compared with normal rats. The destruction was immunologically specific; it was detected earlier, was more comprehensive in the hyperimmune and post-excision animals than in tumour-bearing animals, and correlated with the ability of the hyperimmune and post-excision animals to reject larger numbers of intravenous unlabelled tumour cells, than the tumour-bearing rats.

Cuticular reactivity of early larval stages of Ascaris suum: Binding of fractions of immune serum to the surface of infective and parasitic stage larvae as detected by the mixed antiglobulin test

Infective stage and early parasitic larvae of Ascaris suum were evaluated for surface reactivity with serum from uninfected and hyperimmune guinea-pigs. Cuticular binding of serum components was assessed by the mixed antiglobulin test. Ensheathed infective larvae bound both normal serum proteins and 7S protein from immune serum over the entire sheath surface. Parasitic larvae recovered at 16 and 25 h post infection (h.p.i.) were poorly reactive, and binding occurred only to sites on the head and tail regions. Larvae recovered at 48 and 72 h.p.i. were highly reactive over the entire cuticle. Host serum protein was not detectable on the surface of parasitic larvae when harvested from guineapigs after a primary infection until the larvae had been in the host for 72 h. However larvae recovered from hyperimmune animals had host serum protein detectable on the cuticle as early as 16 h.p.i.

Acute Destruction of Rat Skin Grafts by Alloantisera

Rat skin transplanted to mice or to allogeneic rats can be acutely and severely damaged by injecting the graft recipients with alloimmune serum of appropriate specificity. This susceptibility of rat skin to alloantisera has been obscured in previous studies by the fact that the extent of serum-mediated damage that occurs is decisively influenced by the interval of time between placement of the grafts and the injection of antiserum. Grafts are resistant to damage during the 1st week after transplantation; they rapidly develop sensitivity during the 2nd week, reaching a peak at 14 to 16 days; and during the next few weeks the sensitivity is lost. Grafts surviving beyond 35 to 40 days become totally resistant to antisera. LBNF1 GRAFTS Are more resistant than Le or BN grafts to BN anti-Le serum or Le anti-BN serum, respectively. However, combinations of the two sera can act synergistically to destroy LBN skin that has been grafted to immunosuppressed mice. This form of serum-mediated tissue damage is thought not to play a significant role in skin graft rejection in the usual circumstances of transplantation since cell mediated immunity is well developed before the acquisition by the grafts of sensitivity to humoral agents. Furthermore, antisera from animals that have rejected a single graft have little or no destructive potential, and hyperimmune animals reject skin very shortly after grafting, often before the grafts have become vascularized and hence before they are sensitive to antiserum.

Cytotoxic Reactions of Murine Lymphoid Cells Studied With a Tritiated Proline Microcytotoxicity Test2

A lymphocyte cytotoxicity (CTX) test with 3-H-proline-prelabeled target cells was used to detect the immune response of murine lymphoid cells to H-2 and tumor antigens. The specificity of the reaction was determined by simultaneous tests on unrelated target cells and, for reactions directed against H-2 antigens, by blocking experiments with alloantiserum directed against the H-2 antigens of the target cells. After a single intraperitoneal (ip) injection of allogeneic spleen cells, CTX of unfractionated peritoneal cellswas strong, with a sharp peak on day 5. Repeated ip immunization markedly increased the CTX of unfractionated peritoneal cells. The reaction was strongest when the test was done at 37 degrees C. Sometimes CTX should be detected after as little as 6 hours' incubation. CTX depended primarily on the absolute number of effector or target cells per area rather than on the ratio of effector to target cells. Both nonadherent and adherent peritoneal cells destroyed target cells specifically. The CTX of nonadherent peritoneal cells was increased by 2-mercaptoethanol. The CTX reaction depended on effector cells bearing Tyl-1. Destruction of "innocent bystander" target cells was seen with one of four combinations of unfractionated and nonadherent peritoneal cells from hyperimmune animals.

Homocytotropic Autoantibodies in Rabbits Subjected to Cryosurgery

Abstract Homocytotropic antibodies can be induced in rabbits by parasite infection (1, 2) or by immunization with heterologous antigens (3, 4). The presence of such antibodies in primary responder and hyperimmune animals was recently demonstrated (5, 6). This study reports the induction of homocytotropic autoantibodies in male rabbits subjected to injury of the accessory glands by in vivo freezing. It is known that such treatment is able to evoke a specific hemagglutinating autoantibody response of the 19S and 7S type (7, 8). Materials and Methods. Thirty-five adult rabbits, cross-bred from various generations of New Zealand and Flemish Giant were used. Rabbits were subjected to one or several freezings of the accessory glands following a procedure previously described (8). Bleedings were performed approximately at 3, 7, 10, 15, 20, and 25 days after each cryogenic treatment. Semen was obtained from normal animals by means of the artificial vagina (9).

Association of Synthetic Polypeptides with Ribonucleic Acid of Immunocompetent Tissues

Abstract The association of 125I-labeled synthetic polypeptides with ribonucleic acid (RNA) of rabbit peritoneal exudate (p.e.) cells in in vitro systems has been shown to be a cell-mediated reaction, related to the charge, state of aggregation and optical configuration of the polymer. Although slightly higher levels of several D-amino acid polymers were associated with p.e. cells in in vitro systems, the concentrations of p.e. cell RNA-associated 125I-polypeptides of the L-configuration were significantly greater than the D-form. Intraperitoneal administration of the L or D configuration of the polymer—(Glu60Ala30Tyr10)n—(GAT) to oil-stimulated rabbits resulted in p.e. cell uptake and RNA-association of both isomeric polypeptide forms. Whereas the levels of cellular and RNA-associated L-polypeptide declined rapidly over a 24-hr period, the respective concentrations of the D-polymer significantly increased during this period. Similar observations were noted following i.v. administration of D- or L-GAT to normal or hyperimmune animals. Levels of 125I L-GAT declined rapidly over a 24-hr period in kidney, bone marrow, liver, spleen and lymph node. Tissue and RNA-associated levels of 125I D-GAT remained fairly constant for 7 days after administration. There was essentially no correlation between in vitro and in vivo studies with respect to formation of RNA-antigen complexes. The significance of tissue distribution and polypeptide metabolism in the intact animal, its role in the formation of RNA-antigen complexes and induction of the immune response appear to preclude any oversimplification in attempting to correlate data from a unicom-partmental in vitro cell system with the dynamic multicompartmental in vivo systems.

Qualitative and Quantitative Studies of Cytotoxic Immune Cells

Abstract A quantitative assay of in vitro cell-mediated cytotoxicity has been used to detect the appearance of cytotoxic cells in the spleens of immunized BALB/c mice. Biometric techniques which utilize dose response curves suggested quantitative and qualitative changes in immune activities of different lymphoid populations. The immune responses to intraperitoneal injections of allogeneic tumor cells were studied as functions of both time and immunizing cell dose. Spleen cell-mediated cytotoxicity was detectable 3 days after immunization. The response was maximal after about 10 days and then fell gradually. The highest response to a single intraperitoneal exposure occurred after injection of 107 EL-4 cells. Higher doses of cells resulted in lower responses. The response to a second immunizing dose of EL-4 cells was quicker and reached a peak at about 7 days. In addition to changes in total immune activity, a qualitative change occurred in cell-mediated immunity during the course of primary and secondary responses. With an increase in total immune activity, cytotoxic activity of the average immune cell appeared to increase. The time course and immunizing dose dependence of cytotoxic antibody levels and cell-mediated responses were similar after primary immunization. Hyperimmunization resulted in increasing cytotoxic antibody titers but decreasing cell-mediated activity. Cellular immunity in the hyperimmune animals appeared to have been stimulated but effectively suppressed. This suppression was reversible by transfer of the hyperimmune spleen cells with antigen to sublethally irradiated hosts.