Guinea pig T-cell in vitro proliferative responses to tyrosine-azobenzenearsonate-pulsed and azobenzenearsonate-coupled macrophages have different specificities

The cell interactions involved in azobenzenearsonate-N-acetyl-tyrosine (ABA-tyr)-induced delayed hypersensitivity in the guinea pig were studied by in vitro blastogenesis. The ABA-sensitive lymphocyte was demonstrated to be a T lymphocyte and its presence in peritoneal exudate cells was shown to be much higher than spleen or lymph node populations. The secondary response of ABA-sensitized lymphocytes to ABA-tyr in culture is dependent on the presence of an accessory cell, with both splenic and peritoneal macrophages being equally effective. ABA coupled directly to macrophages as an immunogen induced strong responses to itself and not to ABA-tyr-pulsed macrophages or ABA-tyr in solution. The reverse was true in animals, immunized with ABA-tyr. ABA conjugated to thymocytes, L₂C leukemia cells, and guinea pig erythrocytes however, did not elicit significant responses. The results obtained in animals immunized with ABA- or ABA-tyr-modified cells was similar whether or not CFA was used. The difference in specificity shown between ABA-coupled and ABA-tyr-pulsed macrophages favors a single receptor hypothesis for T-cell recognition.     

Natural regulatory cells in murine bone marrow: inhibition of in vitro proliferative and cytotoxic responses to alloantigens

A lymphocyte-enriched fraction of murine bone marrow (BML), obtained by sucrose density fractionation, contains natural regulatory cells that can profoundly suppress the proliferative and cytotoxic response of syngeneic lymph node cells to irradiated alloantigens in a mixed lymphocyte culture (MLC). A close correlation exists between the inhibition of alloantigen-induced proliferation and the generation of cytotoxic effectors. The suppression of proliferation is dependent on the dose of BML added to the cultures but is not due to cell crowding, since red blood cells, thymocytes, and irradiated splenocytes, all syngeneic to the lymph node responders, do not inhibit proliferation to the degree observed with BML. The addition of BML to cultures does not cause the maximum proliferative response to change from the usual day 5 peak, indicating that there is no change in culture kinetics. The release of nonradioactive thymidine by BML cannot explain the suppression. The target of suppression is maximally affected during the first 24 hr of culture, since adding BML to MLC later than this resulted in negligible inhibition of proliferation. Thus, the natural regulatory cell-mediated suppression reflects inhibition of "early" events in the proliferative response to alloantigens.     

Characterization of homologous and heterologous rotavirus-specific T-cell responses in infant and adult mice

During primary rotavirus (RV) infection, CD8+ T cells play an important role in viral clearance as well as providing partial protection against reinfection. CD4+ T cells are essential for maximal development of RV-specific intestinal immunoglobulin A. In this study, we took advantage of the cytokine flow cytometry technique to obtain a detailed map of H-2b- and H-2d-restricted CD8+ and CD4+ T-cell epitopes from the RV proteins VP6 and VP7. Three new CD8+ T-cell epitopes (H-2d and H-2b restricted) and one new CD4+ T-cell epitope (H-2d and H-2b restricted) were identified. Using these newly identified targets, we characterized the development and specificity of cellular immune responses in C57BL/6 and BALB/c mice during acute infection of infants and adults. We found that both the CD4+ and CD8+ responses peaked on days 5 to 7 after infection and then declined rapidly. Interestingly, both the response kinetics and tissue distributions were different when epitopes on VP6 and VP7 were compared. VP6 elicited a response which predominated in the intestine, while the response to VP7 was more systemic. Additionally, the T-cell responses elicited after homologous versus heterologous infection differed substantially. We found that during homologous infection, there was a greater response toward VP6 than that toward VP7, especially in the intestine, while after heterologous infection, this was not the case. Finally, in suckling mice, we found two peaks in the CD8 response on days 7 and 14 postinfection, which differed from the single peak found in adults and likely mimics the biphasic pattern of rotavirus shedding in infant mice.     

Virus-specific proliferative T-cell responses: Parameters and specificity

The genetic requirements for inducing virus-specific T-cell proliferation were investigated by taking spleen cells from animals primed with vaccinia virus in vivo, then culturing the cells in vitro with vaccinia virus-infected syngeneic peritoneal macrophages, and finally restimulating these cells a second time in vitro with vaccinia virus-infected macrophages from several strains of mice. Under these conditions, T cells proliferated in the tertiary response to virus-specific stimulation, whereas background proliferation caused by allogeneic differences between stimulator and responder cells was minimal. Compatibility between T cells and infected stimulator cells at the K or I regions alone or at I-A or I-A + I-B regions of the major histocompatibility complex (MHC) produced strong proliferative responses, whereas compatibility at D alone often resulted in somewhat weaker responses. However, these responses were rarely as great as in combinations of completely syngeneic stimulator and responder cells. Homology between responding and virus-infected stimulating cells in more than one of the H-2K, D, or I regions resulted in an additive, but not potentiating, effect. Genes coded outside the H-2 region did not seem to play a role in this system. In some rare cases, a weak response occurred across allogeneic barriers, but in general, virus-specific T-cell proliferation was strongly H-2 restricted.     

A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses

A 45-kDa human T cell surface glycoprotein which is tightly bound in the membrane of the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidase/galactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35-kDa and 27-kDa peptide fragments of the surface-iodinated 45-kDa protein are common to the membrane-bound and the released forms, but a third 18-kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane-bound and the released molecule are 30 +/- 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti-(T cell membrane protein) antiserum precipitates the 45-kDa protein. A monoclonal anti-(45-kDa protein) antibody precipitates the membrane-bound 45-kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti-(45-kDa protein) antibody specifically enhances the cell proliferative responses in phytohemagglutinin-treated and mixed lymphocyte cultures. These observations suggest that the 45-kDa protein has a specific receptor function in the regulation of cell proliferative responses.     

Serological and biological cross-reactivity of class II antigens between mice and humans in antigen-specific T-cell proliferative responses

Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from B10.S(9R) which possess I-As and I-Ek molecules serologically cross-reacting with human Class II antigens. On the contrary, B10.S(9R) T cells primed to PPD were stimulated by PPD-pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.     

Distinct T-cell proliferative responses to 13762A rat mammary adenocarcinoma and derived clones

We examined the in vitro responses of immune lymphocytes to the tumor antigens of the syngeneic rat mammary adenocarcinoma 13762A. This tumor readily metastasizes to lymph node and lungs and is poorly immunogenic. Rats were immunized with a highly immunogenic clone (18A) which was isolated as a spontaneous variant from the parental 13762A tumor. Clone 18A grew progressively in irradiated rats but regressed completely in normal rats. Animals immune to 18A tumor were also immune to parental 13762A. Lymphocytes obtained from the spleen and peritoneum of immune rats were tested for specific proliferation to parental 13762A tumor and clone 18A to determine whether similar cross-reactivity to these tumors occurred in vitro. We found an anatomical difference in localization of immune lymphocytes which reacted to the two tumor cell lines. Immune peritoneal exudate cells (PEC) responded strongly to clone 18A but poorly to 13762A, while immune spleen cells from the same animals responded predominantly to 13762A tumor. After 7 days culture, PEC proliferating in response to clone 18A contained 84-95% W3/25+ T-helper cells, and only 5-8% OX8+ cytotoxic/suppressor cells, while analogous cultures of spleen cells responding to parental 13762A tumor consisted of 60-80% W3/25+ cells and 20-23% OX8+ cells. Immune spleen cell cultures stimulated with 13762A tumor generated cytotoxic lymphocytes which specifically lysed both parental 13762A and clone 18A cells. We conclude that despite cross-reactivity in vivo and in vitro, antigens present on 13762A and 18A tumor cells stimulated different subsets of immune T cells.     

Immunopotentiating effects of amphotericin B. II. Enhanced in vitro proliferative responses of murine lymphocytes.

Enhanced in vitro proliferative responses to DNBSO3 were seen in lymph node cells and spleen cells after in vivo sensitization of mice with DNFB plus AmB compared with mice primed with DNFB alone. The T cell proliferation in the nylon column nonadherent fraction for both groups was highly similar, and the enhanced lymph node cell proliferation with AmB was demonstrated to be in the nylon adherent population consisting of both T and B cells. These and earlier studies of immunopotentiation by AmB are consistent with a mechanism that depends on selective interaction of the polyene with a subset of T cells and a resultant impairment of the normally induced suppressor regulation that limits the magnitude and duration of immune responses.     

Cytotoxic and proliferative lymphocyte responses to allogeneic and xenogeneic antigens in vitro.

In vitro lymphoproliferative responses to foreign histocompatibility antigens are phylogenetically restricted. Responses occur most readily to allogeneic or closely related xenogeneic leucocytes, but not to unrelated xenogeneic cells. Specific cytotoxic T cell responses to foreign histocompatibility antigens show the same phylogenetic restriction. This lack of xenoreactivity is not due to a lack of precursor cells for the xenoantigens; guinea-pig lymphocytes, although normally unresponsive to mouse antigens, have a similar precursor frequency for these antigens as do lymphocytes of allogeneic mouse strains. Specific cytotoxic responses of guinea-pig lymphocytes to mouse antigens can be generated if a factor released from con A stimulated guinea-pig spleen cells is added to the culture medium. The factor produced by con A-activated spleen cells (CS) is also phylogenetically restricted in its action; CS must be obtained from animals homologous with the donor of the responding lymphocytes.     

Analysis of L-NAME-dependent and -resistant responses to acetylcholine in the rat

The mechanism by which acetylcholine (ACh) decreases systemic arterial pressure and hindlimb vascular resistance was investigated in the anesthetized rat. ACh injections caused dose-dependent decreases in systemic arterial pressure and hindlimb vascular resistance. N(omega)-nitro-L-arginine methyl ester (L-NAME) had little effect on the magnitude of depressor and vasodilator responses but decreased response duration when baseline parameters were corrected by a nitric oxide (NO) donor infusion. The decrease in the duration of the ACh depressor response was prevented by the administration of excess L-arginine. The L-NAME-resistant component of the depressor response to ACh was attenuated by ebselen, a glutathione peroxidase mimic. The calcium-activated potassium (K(Ca)) antagonists charybdotoxin (ChTX) and apamin decreased the magnitude but not the duration of the hindlimb vasodilator response to ACh. The combination of L-NAME, ChTX, and apamin reduced the magnitude and duration of the vasodilator response to ACh but not to sodium nitroprusside. Vasodepressor and hindlimb vasodilator responses to ACh were not modified by cytochrome P-450 and cyclooxygenase pathway inhibitors. These results suggest that the hindlimb vasodilator response to ACh has an initial L-NAME-resistant component mediated by the activation of K(Ca) channels and a sustained L-NAME-dependent component. The results with ebselen suggest that the L-NAME-resistant component of the depressor response involves a peroxide-sensitive mechanism. The present study suggests that vasodilator responses to ACh are not mediated by cytochrome P-450 products, since miconazole and 1-aminobentriazole alone or in combination did not affect either component of the response. The present data suggest that the hindlimb vasodilator response to ACh in the rat is mediated by two mechanisms with an initial ChTX- and apamin-sensitive, L-NAME-resistant phase not mediated by cytochrome P-450 products and a secondary sustained phase mediated by NO.     

Induction of B- and T-cell responses to cruzipain in the murine model of Trypanosoma cruzi infection

Trypanosoma cruzi, the causative agent of Chagas' disease, is an important cause of heart disease in Latin America. The parasite is transmitted mucosally, with both intra- and extracellular life stages in the human host. Cruzipain, the major cysteinyl proteinase of T. cruzi, has been shown to be antigenic in both humans and mice during infection with the parasite. We extend these observations, showing here that multiple murine immune subsets of potential importance for vaccine-induced protection can be induced by cruzipain. Cruzipain-specific serum IgG responses were induced during chronic infection with T. cruzi. In addition, T. cruzi mucosal infection stimulated the development of cruzipain-specific secretory IgA detectable in fecal extracts from infected mice. Cruzipain-specific type 1 cytokine responses characterized by the production of IFN-gamma but not IL-4 were also detectable during murine infection. Furthermore, immunization of mice with a DNA vaccine encoding cruzipain was shown to stimulate cytotoxic T lymphocyte (CTL) responses capable of recognizing and lysing T. cruzi-infected cells. The induction of serum antibody, mucosal IgA, Th1 cytokine and CTL responses by cruzipain in mice supports the use of this parasite protein for further efforts in T. cruzi vaccine development.     

Ontogeny of proliferative and cytotoxic responses to interleukin 2 and concanavalin A in murine fetal thymus

The ontogeny of proliferative and cytotoxic responses to concanavalin A (Con A) and interleukin 2 (IL 2) in C57BL/6J (B6) fetal thymus (FT) was investigated. Embryonic thymocytes were either taken from embryos at different times of gestation or from 14 day B6 FT that were maintained as organ cultures for various times. It was found that the B6 FT could proliferate to Con A and EL4 SN (an IL 2 containing culture supernatant) in a synergistic fashion. This synergy between Con A and EL4 SN was first observed at the 16th to 17th day of gestation. A similar differentiation process took place in 14-day FT that had been maintained as organ cultures; the synergy between Con A and EL4 SN was first observed after 3 days in organ culture. This synergy increased with increasing time of organ culture, and was most evident after 10 days. The synergy between Con A and EL4 SN was also observed when the EL4 SN was replaced with IL 2 which had been purified from crude EL4 SN to apparent homogeneity. B6 FT could also form cytotoxic T lymphocytes (CTL) on stimulation with Con A and EL4 SN. Con A-activated CTL (polyspecific) were detected by including phytohemagglutinin in the assay medium. CTL response was first detected in the 17-day fetal thymus by using this assay. In organ cultures, CTL responses were first detected after 4 days in organ culture, and reached peak levels after 12 to 14 days. The CTL precursor (CTL-P) frequencies in the B6 FT after 2, 5, 10, and 14 days in organ culture were less than 1/10,000, 1/2232, 1/297, and 1/70, respectively; the corresponding CTL-P frequency in adult thymus was 1/60. After 6 days in organ culture, B6 FT could also form CTL in response to Con A and pure IL 2. This finding suggests that the ability to synthesize other differentiation factors that are required for CTL responses is acquired at an early time of thymic differentiation.     

In vitro tapeworm extract-induced proliferative responses of gut-associated lymphoid cells from Hymenolepis diminuta infected mice

The development of lymphoid cells reactive to tapeworm-associated antigens during the course of Hymenolepis diminuta rejection from mice was studied using an in vitro tapeworm extract (TWE)-induced cell proliferation culture system. Mice infected with three cysticercoids on day 0 developed three adult worms by day 7 but worms were rejected by day 21 post-infection. Concomitant with worm rejection was the development of TWE-sensitized lymphoid cells which responded by proliferation when stimulated in vitro with TWE. Sensitized cells were detected in gut-associated mesenteric lymph nodes but were not detected in spleen, axillary lymph nodes, or Peyer's patches of infected mice, or in lymphoid organs of non-infected mice. These studies suggest that rejection of H. diminuta from mice is associated with the activities of gut-associated, tapeworm antigen-sensitized immune cells localized in the mesenteric lymph nodes.     

Characterization of primate antibody responses to administered murine monoclonal immunoglobulin

Murine monoclonal antibodies (MAb) are currently being assessed for their utility as tools in cancer management. Anti-murine immunoglobulin responses have been observed in many patients receiving monoclonal antibody treatment. In this study, we evaluated the response of primates to the administration of a monoclonal antibody. MAb B6.2, an antibody generated against a human breast tumor metastasis, was used as a prototype MAb. Baboons were inoculated with MAb B6.2 whole IgG, Fab', or F(ab')2 fragments. Blood samples were drawn at periodic intervals post-inoculation and the sera collected. Anti-murine immunoglobulin responses were detected using a solid-phase radioimmunoassay. The specificity of the antibody response was analyzed to determine if the response was directed against the species of origin of the MAb (species specificity), against the class of the MAb (isotype specificity), or against the hypervariable region of the MAb (idiotype specificity). We found that primates develop a humoral immune response against all three forms of the monoclonal antibody [IgG, Fab', and F(ab')2]. Furthermore, this antibody response demonstrated a high degree of specificity for the antigen binding site suggesting an idiotypic specificity. Using a competitive radioimmunoassay, the antibody response was found to interfere with antigen binding of MAb B6.2. These studies suggest that monoclonal antibody treatment can generate an anti-idiotypic response which may alter the efficacy of this mode of treatment.     

Regulation of the human allogeneic proliferative response in vitro

It has previously been shown that presensitized cells in culture medium release suppressor factors (SF) which can inhibit a primary mixed leukocyte reaction (MLA I). This occurs when the presensitized cells are resensitized with an HLA-DR-specific cell, which can be either the primary stimulator or any other DR-identical allogeneic cell. The autologous responders (SF producer cells) and certain allogeneic cells are suppressed, which suggests that restriction takes place. In this paper the effect of preincubation of responder or stimulator cells in SF has been studied: (1) When unprimed responders are preincubated with the suppressor supernates (SF) and tested in MLR I against several stimulators, the cells of the autologous SF producer and certain other allogeneic cells are always inhibited as already observed when SF was added directly to a mixed lymphocyte culture. (2) When the same stimulators are preincubated with the same SF and used as stimulators with the same responders (not preincubated) then inhibition is observed without restriction. This difference in behavior suggests the existence of at least two factors, one acting directly but only on some responders (restricted factor) and the other acting through stimulators on all responders. (3) Filtration of unprimed responders through glass wool (before SF preincubation and coculture with stimulators in MLR I) produces nonadherent T cells which are suppressed more after preincubation with SF than the same cells unfiltrated. This could be due to the existence of a subset of acceptor cells. (4) None of these factors has immunoglobulin characteristics. Their molecular weights are between 40 000 and 70 000 daltons.Abbreviations used in this paper SF suppressor factor - CF control factor - MLR I primary mixed lymphocyte reaction - MHC major histocompatibility complex - MLR II secondary mixed lymphocyte reaction - CML cell-mediated cytotoxicity assay - PHA phytohemagglutinin - ⁵¹Cr chromium 51     

Role of germination in murine airway CD8+ T-cell responses to Aspergillus conidia

Pulmonary exposure to Aspergillus fumigatus has been associated with morbidity and mortality, particularly in immunocompromised individuals. A. fumigatus conidia produce β-glucan, proteases, and other immunostimulatory factors upon germination. Murine models have shown that the ability of A. fumigatus to germinate at physiological temperature may be an important factor that facilitates invasive disease. We observed a significant increase in IFN-γ-producing CD8(+) T cells in bronchoalveolar lavage fluid (BALF) of immunocompetent mice that repeatedly aspirated A. fumigatus conidia in contrast to mice challenged with A. versicolor, a species that is not typically associated with invasive, disseminated disease. Analysis of tissue sections indicated the presence of germinating spores in the lungs of mice challenged with A. fumigatus, but not A. versicolor. Airway IFN-γ(+)CD8(+) T-cells were decreased and lung germination was eliminated in mice that aspirated A. fumigatus conidia that were formaldehyde-fixed or heat-inactivated. Furthermore, A. fumigatus particles exhibited greater persistence in the lungs of recipient mice when compared to non-viable A. fumigatus or A. versicolor, and this correlated with increased maintenance of airway memory-phenotype CD8(+) T cells. Therefore, murine airway CD8(+) T cell-responses to aspiration of Aspergillus conidia may be mediated in part by the ability of conidia to germinate in the host lung tissue. These results provide further evidence of induction of immune responses to fungi based on their ability to invade host tissue.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

Polyoma virus-like particles elicit polarized cytokine responses in APCs from tumor-susceptible and -resistant mice

PERA/Ei (PE) mice are highly susceptible to tumor induction by polyoma virus, whereas C57BR/cdj (BR) mice are highly resistant. PE mice respond to viral infection with a type 2 (IL-10) and BR mice with a type 1 (IL-12) cytokine response, underlining the importance of a sustained T cell response for effective antitumor immunity. PE and BR mice showed comparable Ab responses to the virus, indicating that a Th1 response is fully compatible with strong humoral immunity. Tumor susceptibility is dominant, and a type 2 response prevails in F1 mice derived from these strains. In this study, we show that the different cytokine responses of virus-infected hosts are recapitulated in vitro by exposure of APCs from uninfected PE, BR, and F1 animals to the virus. Importantly, virus-like particles formed from recombinant VP1, the major viral capsid protein, elicited the same host-specific cytokine responses as infectious virus. Assembly of VP1 pentamers into capsid shells is required because unassembled VP1 pentamers were ineffective. Binding of virus-like particles to sialic acid is required because pretreatment of APCs with neuraminidase prevented the response. Expression of TLR2 and TLR4 differed among different subpopulations of APCs and also between resistant and susceptible mice. Evidence is presented indicating that these TLRs play a role in mediating the host-specific cytokine responses to the virus.