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The first paper in this series reported that it was possible to reconstitute the response of low concentrations of mouse spleen lymphocytes to concanavalin A (Con A) by adding small numbers of peritoneal exudate cells (PEC) to the cultures. In this paper it is shown that the role of the PEC in this system can be partially replaced by conditioned medium (CM) prepared from PEC cultures or completely replaced by CM taken from lymphocytes cultured at optimal concentration. These CM were inactive unless fresh Con A was added to the assay cultures. Activity was present in CM which was incubated with lymphocytes or PEC for the shortest possible time but maximal activity was found after 24 hr of incubation. Activity was also found in CM prepared in the absence of Con A. Only in the case of lymphocytes cultured at optimal concentration for 24 hr was there substantially more activity in the CM thus prepared if Con A was present. PEC preparations depleted of T lymphocytes produced as much activity in CM as the untreated control. CM produced by PEC was less sensitive to heat treatment or to freezing and thawing than that produced by lymphocyte cultures. [ABSTRACT FROM AUTHOR]

One of the T suppressor circuits induced by picrylsulphonic acid includes the T suppressor cell (Ts-eff) which acts at the efferent stage of the contact sensitivity reaction and produces antigen-specific T suppressor factor (TsF). This factor does not act directly but arms a T acceptor cell (Tacc). This Tacc liberates a non-specific inhibitor when it is armed with TsF and then exposed to picrylated cells sharing the I-J genotype of the source of the TsF. This paper investigates the role of I-J region gene products in this T suppressor circuit. Two approaches were used. Syngeneic CBA (H-2k) lymphocytes were separated into I-J+ and I-J- cells by treatment with anti-I-Jk serum followed by panning on anti-immunoglobulin plates. The cells were then picrylated and used as a source of antigen. Alternatively, B10.A congeneic mice syngeneic (SR) or allogeneic (3R) with CBA at the I-J locus were picrylated and used similarly. The main findings were as follows. (i) The intravenous injection of picrylated I-J+ spleen cells but not a similar number of I-J- cells induced Ts-eff which blocked the transfer of contact sensitivity. Picrylated unseparated cells syngeneic, but not allogeneic, at the I-J locus were also effective. (ii) It is known that the lymphocytes of mice injected with picrylsulphonic acid and then re-exposed to antigen by painting with picryl chloride liberate TsF in vitro. The re-exposure to antigen can be replaced by the intravenous injection of picrylated I-J+ cells or by cells syngeneic at the I-J locus the day before harvesting the spleen cells. (iii) The release of non-specific inhibitor by Tacc armed with TsF requires exposure to picrylated I-J+ cells or cells syngeneic at the I-J locus. The requirement for antigen on a cell bearing syngeneic I-J suggests that antigen together with I-J is an activation signal in this T-cell circuit. The simplest explanation is that the receptor of the pristine Ts and of the mature Ts-eff is similar to T suppressor factor. [ABSTRACT FROM AUTHOR]

This paper examines helper T-cell responses and antibody titres and isotypes following immunization with a peptide antigen in association with three different adjuvants. B10.A mice were primed with pigeon cytochrome c fragment 81-104 in association with the adjuvants complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum. Strong antibody responses, dominated by IgG1, were observed upon priming with CFA and IFA. In contrast, priming with alum induced a weak antibody response with little or no detectable antigen-specific IgG1. These differences did not correlate with differences in T-cell priming, as immunization with peptide in association with all three adjuvants induced comparable T-cell proliferative responses and frequencies of antigen-specific cells. In addition, no significant differences in iiiterleukin-2 (IL-2), interferon-gamma (IFN-γ) and IL-4 production could be found, suggesting that the adjuvants did not differentially affect Th1 and Th2 cells. [ABSTRACT FROM AUTHOR]

We studied the tolerization of neonatal thymocytes (NT), neonatal splenocytes (NS) and adult thymocytes (AT), transferred to syngeneic nude ( nu/nu) hosts previously injected with semi-allogeneic splenocytes, without any supportive immunosuppressive treatment. This protocol allows the study of peripheral tolerance in the absence of the thymus. BALB/c neonatal T cells and ATs were able to expand in syngeneic BALB/c nu/nu mice and functionally reconstituted an allogeneic response, rejecting (BALB/c × B6.Ba) F1 splenocytes transferred 3–4 weeks after injection of BALB/c cells. However, if (BALB/c × B6.Ba) F1 cells were injected into BALB/c nude hosts 30 days before transfer of NT, NS or AT cells, the F1 population was preserved and specific tolerance to B6 allografts was established. Furthermore, transfer to lymphopenic F1 nu/nu showed that tolerance could be established only for neonatal populations, showing that unique properties of neonatal T cells allow their tolerization in both lymphopenic and non-lymphopenic conditions, in the absence of suppressive immunotherapy. These results bring empirical support to the possibility of T-cell engraftment in immunodeficient patients showing partial identity with donor major histocompatibility complex (MHC) genes; the manipulation of immunological maturity of donor T cells may be the key for successful reconstitution of immunocompetence without induction of graft-versus-host disease. [ABSTRACT FROM AUTHOR]

Memory T-cell responses are faster and more robust than those of their naïve counterparts. The mechanisms by which memory T cells respond better to subsequent antigenic exposure remain unresolved. A portion of the more rapid response is undoubtedly the result of the increased frequency of antigen-specific cells. In addition, there are also differences in the cells themselves with respect to their requirements for costimulation and the apparent avidity of the T cells. We used major histocompatibility complex (MHC) class I tetramers to stimulate T cells to focus on the interaction of T-cell receptor (TCR)/MHC and CD8 in the absence of other molecules that are present on cell surfaces and so contribute to the activation of T cells by undefined mechanisms. Mutated MHC class I tetramers that are unable to engage CD8 were used to investigate the role of CD8 engagement in memory cell activation. Either wild-type tetramers or tetramers carrying the mutation were used to stimulate both memory and naïve TCR transgenic T cellsin vitro. Surprisingly, like naïve cells, memory CD8+ T cells required CD8 engagement for calcium mobilization and optimum proliferation. In contrast, the requirements for cytokine production differed. Unlike naive cells, memory cells were able to produce cytokine in the absence of CD8 engagement. This suggests both a CD8-dependent pathway for early events and a CD8-independent pathway for cytokine production in memory cells. [ABSTRACT FROM AUTHOR]

The role of suppressor cells in control of persistent infections may be of profound importance. Whether a positive immune response or suppression of immunity is generated at the time of initial exposure to pathogens causing such infections may in part be due to the nature of the initial antigen presentation to the specific cells of the immune system. We have shown that in vivo modulation of epidermal APCs by an environmentally encountered stimulus (UV-B light exposure) and subsequent transfer of these APCs together with live HSV-1 to naive syngeneic recipients is sufficient to generate suppression of DH rather than DH to HSV-1. This suppression is T-cell mediated and specific for HSV-l. The phenotype of the Ts cell induced by epidermal cell transfer is Thyl+ L3T4+ Ly2-. [ABSTRACT FROM AUTHOR]

The present study indicates that two Mab specific to Fc∊R (MabER) on human B lymphocytes also react with Fc∊R on macrophage and T-cell lines. More importantly, it is also shown that MabER cross-react with IgE-BFs derived from B, T and macrophage cell lines. This conclusion is supported by the following observations: (i) the binding of MabER to Fc∊R-bearing cells is blocked by the CSN of T, B and macrophage Fc∊R-bearing cell lines, known to contain IgE-BFs as shown by their inhibition of rosette formation between IgE-coated erythrocytes and Fc∊R-bearing cells; (ii) the material purified from the CSN of each Fc∊R( +) cell lines by affinity chromatography on MabER-Affi-gel blocks the rosetting of U937 cells with IgE but not with IgG-coated erythrocytes; (iii) the same affinity-purified material inhibits the binding of 125I-IgE to a selected anti-IgE Mab (Mab 75); and (iv) the CSN of Fc∊R( +) cells but not of Fc∊R(-) cells reacts in a solid-phase sandwich radioimmunoassay with two MabER (135-176). and their reactivity is significantly retained on IgE-Affi-gel from which it may be recovered by glycine elution. This RIA is not influenced by any class of Ig, including IgE, employed at a final concentration of 100 μg/ml. Human serum also reacts in the RIA, and parellel dilution curves are obtained with different CSN and human sera. The RIA proved to be a reproducible and sensitive method to quantify human IgE-BFs. The expression of the same antigenic determinants on Fc∊R from T, B and macrophages as well as on the IgE-BFs secreted by these cells indicates structural homology between IgE receptors and IgE-FBs and suggests that they are encoded by the same gene. [ABSTRACT FROM AUTHOR]

The induction of oral tolerance after ingestion of antigen has been reported in several animal models. The precise mechanisms responsible for this unresponsiveness are not well understood. As some investigations have suggested a key role of Peyer's patches suppressor T cells, an animal model was developed in which the PP were surgically removed. Using this model, the influence of the PP on the induction of oral tolerance against SRBC was investigated. In order to induce tolerance, the rats were fed SRBC on four consecutive days. On Day 5 they were i.p. challenged by injection of SRBC, and 5 days later the number of immunoglobulin-secreting cells against SRBC was determined within the spleen. Using this protocol, the oral tolerance induction could be shown very clearly in control animals as well as in rats without PP. Therefore, tolerance induction is possible in the absence of PP-T cells. Other mechanisms must be responsible for the tolerance induction in this model. [ABSTRACT FROM AUTHOR]

The response of primed T cells to keyhole limpet haemocyanin (KLH) was used to compare the characteristics of antigen presentation by lymphoid dendritic cells, splenic and peritoneal macrophages. In a similar manner to macrophages, purified dendritic cells could be pulsed with antigen and subsequently fixed by brief glutaraldehyde fixation and still retain antigen presenting activity. Also, as previously reported for macrophages, presentation could be inhibited by chloroquine. These functional experiments suggested that the pathway of antigen presentation in dendritic cells and macrophages was similar or identical. However, biochemical studies, using radiolabelled antigen, showed that dendritic cells do not significantly degrade large proteins such as KLH to TCA-soluble form, but partially hydrolyse them to smaller peptide fragments. The significance of these results in terms of a model of the cellular pathways of antigen presentation is discussed. [ABSTRACT FROM AUTHOR]

The stimulating potential of freshly irradiated spleen cells in the generation of primary allogeneic cytolytic T lymphocytes (CTLs) is abrogated with as low as 0003% glutaraldehyde concentration, while their antigenicity begins to decrease only with 01%. We show here that the very high stimulating potential of murine radioresistant spleen cells (RSCs) in the primary allo-CTL generation is thirty times more resistant to glutaraldehyde than the one of total spleen cells, while the resistance of their antigenicity is similar. Analysis of the components of the resistance of RSC immunogenicity during MLC showed that IL- I activity produced by RSCs also decreases after 01% glutaraldehyde, and withstands cyclosporin A inhibition to a higher degree than fresh spleen cells. [ABSTRACT FROM AUTHOR]

Cytotoxic T cells that arise spontaneously in cultures (SCTL) appear to be distinct from natural killer (NK) cells. The specificity of these two types of cytotoxic cell populations has been compared by direct cytotoxicity and cold target inhibition tests. The SCTL population consists of an array of cytotoxic cells each of which is specific for a series of target cells. The NK cells had a more limited range of target selectivity although at least two types of NK effector cells were detected on the basis of specificity measurements. [ABSTRACT FROM AUTHOR]

A large number of cells which can replicate vesicular stomatitis virus (VSV) were generated in pokeweed mitogen (PWM)-stimulated cultures of normal mouse spleen cells. The optimal dose of PWM for the generation of VSV-replicating cells was within the range 25 and 50 µg/ml, which had previously been shown to be optimal for the induction of suppressor cells. The VSV-replicating cells in this system were shown to be Thy 1+ and Lyt 1+2-. These cells, however, were unlikely to be helper T cells, but may be a subset of T cells involved in suppression. Thus, inoculation of VSV into a PWM-stimulated culture of spleen cells resulted in marked augmentation of polyclonal immunoglobulin production. Further, it was shown that the suppressive activity of T cells which had been precultured with a high dose of PWM was abolished by the infection with this virus. [ABSTRACT FROM AUTHOR]

The autologous mixed lymphocyte reaction (MLR) was lower in newborn infants and healthy subjects over 65 years of age than in adults between the ages of 20 and 32. In contrast, the allogeneic MLR, although impaired in newborn infants, was normal in elderly subjects. The degree of impairment of the autologous MLR in elderly subjects was correlated with the impairment, in the response of lymphocytes from elderly subjects to phytohaemagglutinin (PHA) and Staphylococcus aureus protein A (SPA). The percentage of autorosetting T cells and oft cells with the OKT4 phenotype was increased in elderly subjects. These findings are paradoxical as autoreactive T cells in young adults have been reported to be drawn from these two T-cell subpopulations. [ABSTRACT FROM AUTHOR]

The properties of mitomycin-treated and untreated spleen cells from allopregnant mice (bearing allogeneic foetuses) have been compared for suppressive activity of mixed lymphocyte reaction (MLR) of maternal strain spleen cells responding to paternal (or genetically related) strain cells. Mitomycin-treated cells in 1% normal mouse serum suppress an MLR if added at its onset while they cannot alter an already set up (day 2) MLR. Conversely untreated spleen cells are unable to affect an MLR if added at its onset while they suppress it when added on day 2 of an ongoing MLR. The two systems also differ in their m vitro retriggering requirements at the target cell (stimulator) level. Whereas the untreated, late acting population, in a k/d or k/a system, requires the contact with molecules coded by ‘S-G’ (or IC + S G) subregion, also present in the father's strain, the mitomycin-treated, early acting population, tested on similar panel does first appear non-specific. Suggestive evidence, however, is given that this apparent non-specificity may in fact veil clones of cells with specific recognition of ‘private’ Ia determinants. It is suggested that allopregnant mice elaborate two T suppressor cells, acting at different steps of the MLR. The usefulness of the first set (mitomycin-resistant suppression) for exploring the Ia chart is pointed out. [ABSTRACT FROM AUTHOR]

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are: SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells arc less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system. [ABSTRACT FROM AUTHOR]

The complement-mediated lysis of human lymphocytes by three monoclonal anti-human T-cell antibodies OKT3.PAN, OKT4.IND and OKT8.SUP was studied. The percentages of Ficoll-Hypaque-isolated mononuclear cells lysed by these antibodies were respectively: 65% for OKT3.PAN, 39% for OKT4.IND and 20% for OKT8.SUP. Optimal lymphocytotoxic reactions were noticed when unabsorbed rabbit serum was used as the source of complement (C). Addition of heat-inactivated human, mouse and newborn calf sera but not of foetal calf serum inhibited the lyric activity of the antibodies. Treatment of peri- pheral mononuclear blood cells with OKT3.PAN and C abrogated their mitotic response to PHA and Con-A. Sheep erythrocyte rosetting lymphocytes (E+ cells) treated with OKT4.IND or OKT8.SUP and C exhibited no marked changes in responsiveness to PHA, Con-A or allogeneic non-T cells.However, only E+ cells enriched with OKT4.IND-reactive cells responded to purified protein derivative, proliferated in the autologous mixed lymphocyte reaction and were highly sensitive to hydrocortisone suppression when stimulated by PHA. Our data indicate that these monoclonal antibodies can be regarded as invaluable tools for enumeration, characterization and functional assessment of human T cells and their subclasses. [ABSTRACT FROM AUTHOR]

Human autologous-rosette-forming T cells (Tar cells) have many of the characteristics of post- thymic precursor cells. Thus, they bind to sheep erythrocytes but have neither receptors for the Fc portion of IgG nor for that of IgM. They include a subpopulation that binds peanut agglutinin which suggests that they are immature and, as opposed to T cells with either receptors for the FC portion of IgM (Tμ) or of IgG (Tγ), Tar cells adhere to nylon wool, another possible indicator of immaturity, as is their extreme sensitivity to hydrocortisone both in vitro and in vivo. There are more Tar cells in cord blood than in the peripheral blood of young adults and there are more Tar cells in the peripheral blood of young adults than in the peripheral blood of elderly subjects. By co-culturing Tμ and B cells, or Tμ, or Tar and B cells in the presence of pokeweed mitogen (PWM) we were able to determine that these cells cause feedback inhibition, a function considered characteristic of post-thymic precursors. In co-cultures in which we placed mononuclear cells (MNC) or MNC plus Tar cells, or MNC depleted of Tar cells or MNC depleted of Tar cells plus Tar cells stimulated with PWM, we determined that Tar cells play a role in the generation of suppression thereby confirming that human Tar cells are precursor cells. We also found that Tar cells proliferated and generated Tγ and Tμ cells both spontaneously and in greater numbers, under the effect of serum thymic factor. [ABSTRACT FROM AUTHOR]

A non-specific suppressor factor, owl monkey suppressor factor, OMSF, from leukaemic owl monkey peripheral T lymphocytes has been isolated and characterized. OMSF is a glycoprotein of a molecular weight of 66,000 which can be produced from purified owl monkey enhancing factor, OMEF, by both Tris buffer dialysis and by repeated freezing and thawing. SDS gel electrophoresis demonstrates that OMSF is composed of 39,000 mol. wt subunits which co-migrate with the OMEF molecule. This suppressor acts at less than nanogram levels on day 0 of the in vitro immune response to suppress the number of plaque-forming cells produced to sheep red blood cells in a marine assay system and has little effect when added on day 2 to this system. OMSF is bound by antibodies raised specifically against OMEF and inhibits the binding of OMEF to this antiserum in a competitive radioimmunoassay. These results suggest that both enhancement and suppression of the immune response can be mediated by the same molecule under different circumstances. [ABSTRACT FROM AUTHOR]

A non-specific immunoenhancing factor from leukaemia owl monkey peripheral T lymphocytes has been purified and partially characterized. Owl monkey enhancing factor, OMEF, is a glycoprotein with a molecular weight of 39,000 and an isoelectric point of pH 6·5. It acts at nanogram levels on day 2 of the in vitro immune response to enhance the number of plaque-forming cells produced to sheep red blood cells in a routine assay system but has no effect when added on day 0 to this system. Antibodies to the purified factor have been produced and binding studies show that OMEF cross-reacts with other anti- sera raised to an enhancing factor produced by a mixture of human allogeneic lymphocytes but shows no cross-reactivity to a murine enhancing factor. [ABSTRACT FROM AUTHOR]

Nude and normal CBA mice have been used in adoptive transfer experiments to analyse the development of immunological memory. The development of B-cell memory to xenogeneic erythrocyte antigens is to a very large degree dependent on the presence of T cells, wit h IgG memory being somewhat more dependent than IgM memory. In this system, the expression of B memory, that is the transformation of memory cells to antibody-secreting cells under the inductive influence of antigen, is largely dependent on the presence of T cells. Primed (educated) T cells can have an antigen-specific potentiating effect on unprimed B cells in the presence of antigen. [ABSTRACT FROM AUTHOR]

Electron micrographs of material fixed during the first 10 mm of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of Contact with specific antigen, and are involved in the Cytotoxic mechanism is discussed. [ABSTRACT FROM AUTHOR]

It was found that spleen cells from mice injected i.v. with large doses of BCG respond to PHA stimulation less intensely than do normal spleen cells. We were able to show that nylon wool column purified BCG treated T cells also had a low PHA reactivity. Unfractionated spleen cells, adherent cells or T-enriched populations from BCG treated mice, when added to normal T cells lowered their PHA reactivity. When the same BCG treated cell populations were added to tumor cells in vitro, they inhibited their growth. [ABSTRACT FROM AUTHOR]

It was found that spleen cells from mice injected i.v. with large doses of BCG respond to PHA stimulation less intensely than do normal spleen cells. We were able to show that nylon wool column purified BCG treated T cells also had a low PHA reactivity. Unfractionated spleen cells, adherent cells or T-enriched populations from BCG treated mice, when added to normal T cells lowered their PHA reactivity. When the same BCG treated cell populations were added to tumor cells in vitro, they inhibited their growth. [ABSTRACT FROM AUTHOR]

Cytosolic-free calcium changes in cultured rat oligodendrocytes attacked with routine T-cell perforin were measured using digital image processing of fura-2-loaded cells. Permeability to the membrane impermeant dye, propidium iodide, and the ability to retain fura-2 were also assessed. Oligodendrocyte response to perforin attack was heterogeneous, ranging from small, transient increases in cytosolic calcium to rapid cell death. Changes in cytosolic calcium occurred in all cells and about half of the cells also became permeable to propidium iodide. Only a minority of these cells proceeded to lysis, as evidenced by loss of fura-2. The cytosolic-free calcium rises visualized here in single cells could initiate non-lethal effects, thereby disturbing oligodendrocyte function and synthesis and maintenance of myelin. [ABSTRACT FROM AUTHOR]

Using an in vitro primary culture system which we had previously established, the induction phase of Listeria monocytogenes-specific effector cells was analysed with respect to their abilities to produce effector lymphokines, macrophage chemotactic factor (MCF) and macrophage-activating factor (MAF). Listeria-specific effector cells generated after in vitro culture of normal spleen cells with viable L. monocytogenes for 5 days conveyed L3T4+, Lyt-2 , Thy-1+ surface antigens and produced MCF and MAF in response to the secondary stimulation with heat-killed L. monocytogenes. The cells required for the induction of Listeria-specific effector cells, which produce effector lymphokines, MCF and MAF, were L3T4+, Lyt-2-, Thy-1+ cells. The kinetic analysis revealed that the ability of these effector cells to produce MCF was generated earlier than that to produce MAF. Furthermore, using passive transfer of cells, the effector cells producing only MCF, which were generated early in culture, conferred delayed-type hypersensitivity (DTH) alone, but MCF- and MAF-producing effector cells generated late in culture conferred sufficient levels of DTH and acquired cellular resistance (ACR). These results indicate a dissociated production of MCF and MAF by L. rnonocytogenes-specific T cells generated in the primary in vitro culture system. [ABSTRACT FROM AUTHOR]