Search

A palmitate-conjugate derivative of ovalbumin which can be inserted into the membrane of B cells has been prepared. The ability of these cells to act as antigen-presenting cells for specific T lymphocytes obtained from immunized mice was tested. It was found that the conjugates were more efficiently processed and presented than the naive form of the antigen. Palmitate-conjugated antibodies specific to ovalbumin were also inserted into the cell membrane of normal B lymphocytes. These cells were pulsed with the antigen and tested as antigen-presenting cells for T cells obtained from immunized mice. The antibody-decorated B cells presented ovalbumin more efficiently than non-decorated controls. Whether antibody-decorated, antigen-pulsed B cells could prime T cells in vivo was investigated. Some priming activity was found.

We have investigated the ability of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, and interferon-gamma (IFN-gamma) to induce motility in murine, splenic B lymphocytes. Two parameters of cellular locomotion were studied, namely acquisition of motile morphology (polarization) and in vitro migration through polycarbonate filters. Of the tested cytokines, only IL-4 gave a strong motile response among B cells. At the optimal concentration of 3 ng/ml, IL-4 induced polarization in 10-20% of the B cells within 30 min and in up to 40% after overnight exposure. The same amount IL-4 present in a microchemotaxis chamber stimulated migration through polycarbonate filters after overnight incubation in up to 15% of the input B cells. IFN-gamma could also induce some degree of polarization and migration, but only after 19 hr of exposure and to a much lesser extent than IL-4.

Lymphokine production, analysed at the single cell level, was compared in resting and primed T-cell populations. Cells were preactivated in vitro by repeated mitogen stimulations, or isolated as large, low density cells naturally activated in vivo, from normal spleens of unimmunized animals. A similar qualitative shift in the pattern of lymphokines synthesized after restimulation was found as a result of in vivo and in vitro preactivation of cells. Repeated stimulations in vitro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of IL-2 during the first and second culture, to a dominance of IL-4 and IL-5 in the later stimulations. In vivo activation lead to a similar separation of lymphokine production as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5. IFN-gamma was produced by both small and large cells. Preactivation in vitro lead to a more rapid appearance of lymphokines during restimulation. In contrast, the in vivo naturally activated cells responded with a slow onset of lymphokine production when stimulated in vitro.

In this report we investigate if IL 4 can work as a chemoattractant factor by inducing locomotion in B cells. We found that murine recombinant IL 4 (rIL 4) induced motile morphology and migration through polycarbonate micropore filters of murine, splenic B cells at an optimal concentration of 3 ng/ml. Kinetic studies revealed optimal migration at 8-16 h, although a significant response could be detected already after 1 h. Flow cytometric studies confirmed that the migrated cells were indeed B cells. We also compared the activity of small, dense B cells and large, low-density B cells, based on Percoll gradient separation. We found no difference in IL 4-induced motility among the two groups. Furthermore, we looked at B cells activated in vitro by preculture in lipopolysaccharide (LPS) or IL 4. Our data indicate that both LPS and IL 4 can increase the general capacity for motility in B cells after preculture for 24 h. T and B cell collaboration requires close cell-cell contacts in order for T cell help to be administered to the B cell. One way of enhancing such cell contacts could be through directional cell migration induced by helper factors (chemotaxis). We suggest that IL 4 can play a role as a chemoattractant factor that enhances cell contacts between T helper cells and B cells.

A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency and antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of kappa light chain. A major anti-dextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response.

We have studied the specificity of the products of a "T-cell" hybridoma, Th 1, a fusion product of AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized mice, which has been shown to affect the anti-Dx but also the anti-SRC response from culture supernatants and ascitic fluids. The anti-Dx-affecting material was separated from unspecific effector molecules by Sephadex affinity chromatography combined with HPLC DEAE chromatography and gel filtration. The activities of fractions were tested for their effects on anti-Dx, anti-SRC, and anti-SSS-III IgM responses. We show that the anti-Dx response-affecting material binds to Sephadex. Its Ig contamination can be reduced by two DEAE chromatographies at pH 6 and 8.1. At pH 8.1 it starts eluting with 0.13 M NaCl, but is still contaminated with materials that affect the anti-SRC and to a smaller extent the anti-SSS-III response. On gel filtration it localizes in the area of 100-40 kDa. The effects of the active material on anti-Dx IgM varied from suppression to enhancement. The details of that effect are largely unknown but three other findings further confirm the Dx specificity of Th 1 products. The growth of Th 1 in mice induces the production of anti-Dx IgA, detectable in their sera with ELISA. The priming of mice with Th 1 products affects the magnitudes of anti-Dx IgM PFC responses to the subsequent immunization with Dx with or without the product. The binding to Dx of material from in vivo active fractions can be verified in the ELISA with an antiserum produced against Th 1.

Concanavalin A induced polyclonal antibody synthesis in normal spleen cells in vitro. Optimal responses were obtained by Con A concentrations lower than those optimal for induction of DNA synthesis. T cells, but not macrophages, were necessary for the effect. Spleen cells from nude mice were not activated, whereas cells from the LPS non-responder stain C3H/HeJ were activated to polyclonal antibody synthesis by Con A. Supernatants from Con A activated spleen cells could by themselves induce polyclonal antibody synthesis in untreated spleen cell cultures, even when Con A had been removed by absorption with Sephadex G-50 and when alpha-methyl-mannoside was present in the secondary cultures. T cells produced the active supernatants, which were competent to induce polyclonal antibody synthesis, but not DNA synthesis, in both H-2-incompatible and compatible strains. When the supernants were absorbed with erythrocyte antigens, they specifically induced an enhanced response, in secondary cultures, to the antigen used for absorption. Possible mechanisms of this specific effect are discussed.

We have compared previous studies of the immune response to dextran (Dx) B512 in thymus-deficient nu/nu and thymectomized, lethally irradiated, and bone marrow-reconstituted mice with those obtained with cyclosporin A (CyA) as a T-cell-inhibiting drug. Our data show that only immune responses to TD forms of Dx B512 are susceptible to suppression by CyA, whereas anti-alpha 1-6 and anti-DNP antibodies induced by high molecular weight dextran and DNP-native Dx, respectively, were not inhibited. Similar results were obtained when polyclonal responses were studied. The polyclonal response induced by another dextran preparation, the polyanion dextran sulphate (DxS), was inhibited by CyA to the same extent as polyclonal T-cell activation by T-cell mitogens.

Thymus lymphocytes activated in vitro by a T cell mitogen (concanavalin A (Con A)) did not form Fc rosettes nor were they cytotoxic against untreated or antiserum-coated target cells. Con A activated spleen cells were cytotoxic against target cells, but they did not display an increased cytotoxic effect against antibody-coated YAC targets, when compared to normal spleen cells. Con A-activated spleen cells as well as cells activated by a mixed lymphocyte culture did not form Fc rosettes. Thus, activated cytotoxic T cells did not form Fc rosettes nor did they cause cytotoxicity on antibody-coated target cells. Antibody coating of the target cells did not suppress expression of cytotoxicity of activated T cells.

Murine spleen cells were activated with concanavalin A (Con A), pokeweed mitogen (PWM), or phorbol myristate acetate (PMA) and the calcium ionophore A23187. Cells producing gamma interferon (IFN-gamma) or interleukin 4 (IL-4) could be detected by lymphokine-specific monoclonal antibodies and indirect immunofluorescence. The frequency and kinetics of the lymphokine-producing cells were examined and were approximately the same after stimulation with Con A or PMA and A23187. Thirty hours after activation, 3-9% of the cells produced IFN-gamma. There were few IL-4-producing cells, and the maximal frequency was 1 out of 400 spleen cells 48 h after activation. When the cells were activated with PWM, the frequency of IFN-gamma-producing cells was still high 72 h after culture. The majority of the IFN-gamma-producing cells were CD8+ and expressed receptors for IL-2.

We obtained the following results, using carrageenans (CGNs) of three types (kappa, lambda and iota), macrophage-toxic agents. 1. CGNs were toxic to human monocytes, cytotoxicity varying from 40% to 60%, as judged by trypan blue dye exclusion test. 2. CGNs were proved to be newly found mitogens for human T cells, whereas CGNs are polyclonal B-cell activators in mice and induced DNA synthesis that peaked at days 6 or 7. In addition, iota CGN induced an early peak of 3H-thymidine uptake at day 1, which found out to be cytoplasmic uptake of 3H-thymidine. 3. Only lambda and iota CGN induced slightly higher DNA synthesis in purified B cells, but both failed to induce polyclonal antibody synthesis in B cells. 4. In spite of the fact the CGNs are macrophage-toxic agents, CGNs could induce Il-1 production by the surviving monocytes. Thus CGN-induced human T-cell activation requires a relatively smaller number of monocytes. 5. The mitogenic responses of T cells induced by kappa, lambda and iota CGN were significantly inhibited by cyclosporin A (CyA) treatment (250 ng/ml) and Con A and PHA responses were also inhibited by CyA addition. It was concluded that CGNs were newly found human T-cell mitogens, which is in sharp contrast with polyclonal B-cell activators in mice, and the mechanism of T-cell activation induced by CGNs was the same as that by Con A or PHA, though there may be another possibility of inhibition mechanism by CyA.

The process of activation of T lymphocytes by ionophoric substances was analysed according to current concepts of T-lymphocyte activation. Proliferation induced by the ionophore A23-187 was found to be strictly dependent on the exogenous availability of growth factors. Lack of production of growth factors in spleen cell cultures containing the ionophore A23-187 explained the failure to directly stimulate mouse T-lymphocyte proliferation. It was concluded that the main action of A23-187 is the induction of expression of growth receptors, and that the increase in Ca++ uptake is, in itself, not sufficient to induce mitotic events.

The results reported here suggest that there exist two T-cell-mitogenic responsive populations in the spleen--one that is responsive to concanavalin A (Con A) and a second one that is responsive to Con A and phytohemagglutinin (PHA). Using an in vitro restimulation system, we have found that a primary in vivo activation with Con A effectively suppresses the subsequent restimulation response to PHA. However, in the reverse experiment, a primary in vivo PHA activation had little effect on the ability of cells to be restimulated with Con A. The suppression seen was due to a refractory recovery period displayed by all cells after previous activation and not to a nonspecific steric hindrance of mitogenic activation receptors due to the lectin remaining on the cell surfaces.

Immunization with horse erythrocytes (HRBC) and a thymus-independent (TI) antigen of the TI-2 category, such as a native dextran, dinitrophenyl-dextran (DNP-dextran), or DNP-Ficoll, resulted in a three- to four-fold enhancement of the plaque-forming cell response to the TI antigen. The effect was found to be T-cell associated, since cyclosporin A abrogated enhancement. The T cells of T-cell factor involved were elicited early in the response to HRBC and acted on the initial stages of the TI response. The HRBC-induced potentiation affected only B cells activated by the TI antigen; it could not induce a TI response when TI-2 antigen was given under nonresponsive conditions; and it had only a slight effect on the response to a thymus-dependent (TD) antigen. A soluble TD antigen, bovine serum albumin-coupled dextran (BSA-dextran), did not have potentiating ability equivalent to that of the particulate HRBC. It was concluded that non-specific T-cell help can influence TI-2 responses when they are present simultaneously.

When CBA X BALB/c mice are immunized with Th 1 hybrid prepared by fusing spleen cells from Dx-immunized mice with the AKR thymoma BW 5147, which secretes material that affects the anti-Dx response in CBA mice, antibodies are produced that in the ELISA are shown to bind to the material secreted by Th 1 but not to the material secreted by parent BW 5147. A fraction of the Th 1 secreted material that starts eluting on DEAE chromatography in 20 nM Tris with 0.13 M NaCl is shown to bind to Dx but not SRC or uncoated ELISA plates. This binding, like that of anti-Dx Ig, can be inhibited with 1-10 micrograms/ml concentrations of free Dx. However, the affinity of the product of Th 1 for Dx is apparently lower than that of anti-Dx Ig, since high concentrations of anti-Dx interfere with the binding of Th 1 products to Dx. On gel permeation chromatography, a similar Dx binding material elutes in two molecular weight areas, a high area of the molecular weight of IgG and less and a low area of the molecular weight of 43,000 and above, and both are found in samples of Th 1 ascites and hyperimmune CBA anti-Dx serum, but not (or less so) in normal CBA serum and not in the hyperimmune nude C57B1 anti-Dx serum. The Th 1-derived material that affected the anti-Dx response in previously shown experiments had similar elution characteristics. Since we also show that these fractions need not to contain Ig and that anti-Th 1 minimally cross-reacts with anti-Dx IgM or IgG, it is likely that that fraction of Th 1-secreted material represents a Dx-binding, non-Ig, anti-Dx response-regulating factor.

It is still not known how T cells are activated, which T-cell surface structures transmit activation signals, and if antigen-presenting cells possess activation structures for T cells. We have studied whether the T-cell receptor (TcR) must be engaged for T-cell activation to occur. By using membrane-incorporated monoclonal antibodies, we artificially forced T cells to bind to antigen-presenting cells in a mixed lymphocyte reaction system and thereby bypassed the need for TcR engagement and also made it possible for any surface molecule on antigen-presenting cells to deliver a stimulatory signal to the T cells. Theoretically, T cells would become polyclonally activated by this procedure. However, we found that they did not, even though they were intimately bound to the antigen-presenting cell, thus demonstrating that the TcR must participate in antigen/MHC binding in order for the T cells to become activated. This study does not exclude the possibility that antigen-presenting cells possess structures that can activate T cells.

Supernatants from incubated normal mouse spleen cells suppressed the DNA synthetic response induced by polyclonal B- and T-cell activators and the primary immune response to sheep erythrocytes in normal spleen cells in vitro. The inhibitory effects of the supernatants were found to be dependent on the culture system used. The main cell population producing or releasing the inhibitory factors was nonadherent spleen cells, but macrophages and bone marrow cells could also give supernatants with inhibitory effects. Thymocytes did not release any inhibitors under the same conditions. Separation of the supernatants showed that there were at least two factors with inhibitory effects on proliferation in lymphocytes. One of these factors had a molecular weight of less than 10,000 (not degradable by protease) and suppressed activation by all tested polyclonal B- and T-cell activators. Another factor (a protein with a molecular weight of more than 30,000) inhibited activation by some polyclonal B-cell activators (lipopolysaccharide, type III pneumococcal polysaccharide) whereas the proliferation induced by dextran sulfate was less or not at all affected under the same culture conditions. Trivial mechanisms for inhibition by supernatant factors, such as medium depletion, general toxicity, and accumulation of free thymidine in the medium, were excluded.

Concanavalin A (Con-A)-induced suppressor T cells were found to respond to T cell growth factor (TCGF) by proliferation. TCGF abrogated the suppressor activity exerted by these cells on phytohemagglutinin (PHA)- and alloantigen- induced lymphocyte proliferation and on pokeweed mitogen (PWM)-driven immunoglobulin secretion. The Con-A-activated suppressor T cells absorbed the TCGF activity, preincubation of these active suppressor cells with TCGF abolished their suppressor activity and addition of increasing numbers of Con-A-activated T cells reverted the abrogator,/ effect of TCGF. Altogether, these findings suggest that Con-A-induced suppressor T cells exert their function by decreasing the available levels of TCGF. Cyclosporin-A (CYA), which is known to inhibit the expression of receptors for TCGF on T cells, also inhibited the suppressor activity as determined in both indicator systems, namely PHA- or alloantigen-induced DNA synthesis and PWM-induced immunoglobulin synthesis. CYA made Con-A-treated T cells unresponsive to TCGF and unable to absorb the growth factor, supporting the notion that CYA inhibits the expression of TCGF receptors on T cells, a mechanism by which this drug seems to abrogate Con-A-induced suppressor T cell function.

Mononuclear cells from human blood were stimulated to tumor necrosis factor alpha (TNF alpha) or beta (TNF beta) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNF alpha- or TNF beta-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNF alpha or -beta in the Golgi system. TNF alpha-producing cells appeared 2-3 h after activation of the cultures and increased up to 6 h. The majority of these early TNF alpha-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNF beta production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNF beta-producing cells was much larger than that of TNF alpha-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNF beta production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNF beta at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.

The intracellular accumulation of five different lymphokines in individual cells could be identified by lymphokine-specific antibodies and an indirect immunofluorescence technique with UV microscopy. When peripheral blood mononuclear cells from one healthy donor were activated in vitro by the T cell mitogen anti-CD3 monoclonal antibody, the maximal number of cells producing interleukin (IL)2, IL 6 or tumor necrosis factor (TNF)-alpha occurred 6 h after initiation of the cultures, while peak numbers of interferon-gamma, TNF-beta and a second wave of TNF-alpha-synthesizing cells were noted approximately 20 h later. By performing two-color immunofluorescence studies we observed a variegated production pattern with cells making no, one or several lymphokines simultaneously. All five cytokines accumulated in the Golgi organelle resulting in a very characteristic morphology of the staining with or without additional cytoplasmic immunofluorescence.

Antisera directed against the heavy, the light, or reactive against the complex of both chains of HLA-DR antigens strongly inhibited proliferation of T cells induced by TNP- or FITC-labeled autologous cells when added at initiation of the cultures, but not 72 h later. T cells from cultures treated with the anti-DR sera were unresponsive to interleukin-2 (IL-2). Nonetheless, the anti-DR sera did not inhibit proliferation of T cells that had already acquired sensitivity to IL-2. The DR antibodies abrogated the synthesis of IL-2 induced by both TNP-and FITC-conjugated autologous cells. Treatment of TNP-and FITC-labeled autologous cell cultures with the four different types of anti-DR sera significantly inhibited the induction of cytotoxic T cells. However, DR antibodies added at the effector phase of cytotoxicity assays did not inhibit the cytotoxic activity. Effector T cells from cultures treated with the anti-DR sera were unresponsive to IL-2 and addition of IL-2 to these cultures did not restore the cytotoxic activity. In contrast, effector T cells from cultures performed in the absence of the anti-DR sera proliferated to Il-2 stimulation and addition of IL-2 to these cultures significantly increased the generation of killer cells specific for hapten-labeled self structures. From these results we concluded the following: (1) Both the heavy and the light chains of Dr antigens participate actively in the activation of T cells by rendering resting T cells sensitive to IL-2 and by inducing production of the growth factor in TNP-and FITC-conjugated autologous cell cultures. (2) The heavy and light chains of the DR antigens play an essential role in the induction of cytotoxic T cells specific for hapten-labeled self structures, most likely by enabling cytotoxic T cells to respond to Il-2 and by inducing the IL-2 producer T cells to synthesize the growth factor.

Tolerance to the alpha1--6 epitope of native dextran B512 was found to be very stable and could not be broken by the injection of dextran conjugated to several substances, such as protein A, keyhole limpet haemocyanin, edistin, concanvalin A or Staphylococcus bacteria, strain Cowan. However, when tolerant mice were injected with dextranase, all the above conjugates induced a strong anti-alpha1--6 immune response. In contrast, native dextran itself never induced a response in tolerant, dextranase-treated mice. It was concluded that tolerance only affects the specific B-cell subpopulation that can respond to the polyclonal B-cell-activating (PBA) property of dextran, whereas other specific B cells having PBA receptors for, e.g., signals delivered by collaborating T cells remain in a resting state. These B cells can respond in a specific immune response against the tolerogen after removal of the antigen, which blocks the Ig receptors and therefore prevents them from passively focusing the antigen. Thus, immunological tolerance is not caused by clonal elimination of the antigen-specific clone, but only affects a small subfraction of cells with Ig receptors against the tolerogen.

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, is a polyclonal B cell activator in the thymus-independent category, and both RU 41.740 and its fraction F1 induce the production of interleukin 1 (IL 1). RU 41.740 alone appears to have no direct effect on T-enriched cells. However, we show here that when given in conjunction with concanavalin A (Con A), RU 41.740 enabled nylon wool-passed T cells, which had been accessory cell depleted and lost responsiveness to Con A, to proliferate in response to Con A. Analysis of this observation indicated that RU 41.740 probably acted via a residual accessory cell population and that contact of this cell with the T cell was necessary for Con A activation of the T cell. Because both lectin/antigen and a source of IL 1 are required to stimulate accessory cell-depleted T cells, the mechanism of action of RU 41.740 in this system may be by induction of IL 1 from residual accessory cells in the nylon wool-passed, T-enriched cell population. However, two other agents that stimulate IL 1 production, lipopolysaccharide (LPS) and F1, do not enable T-enriched cells to respond to Con A in this system.

We have found that some hybrids produced by fusing AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized CBA mice produce factors that affect the primary CBA IgM PFC response. By the use of HPLC-DEAE chromatography, sometimes combined with affinity chromatography, we have purified from hyperimmune anti-Dx serum, from ascites-growing T hybrids, and from normal CBA serum a family of factors that specifically enhance the anti-Dx IgM response. They show specificity for either Dx or anti-Dx, they may contain an Iak determinant, and they are genetically restricted in their function. The factors from all three sources are found in the same fractions after separation and move in fractions without Ig contamination. The affinity-purified factor was eluted at an NaCl concentration of 0.15-0.20 M. On the basis of these characteristics we believe that we have separated a class of T cell-derived helper factors from serum. The concentration of the factor in serum increases after hyperimmunization with Dx. As a specificity control we show that the same sera contain a natural anti-SRBC IgM response-enhancing factor. It co-separates with the anti-Dx-enhancing factor, but they can be distinguished by their preferential binding to their own antigens.

High-dose unresponsiveness to concanavalin A affected T and B lymphocytes equally, although only T cells were activated by Con A. Thus, activation and inactivation signals are totally unrelated, the latter also lacking target cell specificity. The off-signal caused cell death and is probably biologically unimportant.

The addition of a small proportion (10%) of in vivo concanavalin-A (Con-A)-activated spleen cells to normal spleen cell cultures suppressed the primary immune response to sheep erythrocytes (SRBC) but had no effect on the thymus-independent primary immune response to 3,5-dinitro-4-hydroxy-phenacetyl-conjugated lipopolysaccharide. When Con-A-activated cells were added after 24 h, there was no suppression of the anti-SRBC response but rather an enhanced response when few cells were admixed. Con-A-activated cells did not influence activation of normal cells by polyclonal T- and B-cell activators. It is concluded that Con-A-induced suppressor cells do not act on B cells but rather on helper cells (T cells or macrophages) at a very early stage of the immune response to thymus-dependent antigens.

We have shown that cytotoxic T cells can be polyclonally activated by a short exposure to complexes of polyadenylic:polyuridylic acid (poly A:U). Activation is optimal at a dose of 100 microgram/ml poly A:U and occurs during a 2 h incubation period in the absence of antigen. Killing of allogeneic, but not syngeneic, target cells can be observed after 12 h in culture and peaks after 21-24 h in the absence of any nonspecific binding ligand. The observed cytotoxicity is mediated by T lymphocytes and dose not require accessory macrophages or DNA synthesis for the activation or expression of effector functions. These results suggest that few requirements exist for the activation of cytotoxic T cells.

The selective T cell mitogen Con A was found to induce cytotoxic effectorlymphocytes after in vivo or in vitro treatment. The effector cells exhibited immunologic specificity when tested against tumor cells, killing targets only across H-2 histocompatibility barriers. Thus, Con A caused a polyclonal T cell activation, resulting in the appearance of effector cells capable of recognizing all H-2 antigens, except those coded for by the major histocompatibility loci of the lymphocyte donor. These experiments demonstrate that Con A is capable of activating pre-existing T cells to reveal their genetically determined immunological specificity, and furthermore suggest that there is a deletion of T cell clones with specificity for self. The activated effector cells were characterized as T blasts being relatively insensitive to treatment with anti-O serum plus complement. The precursors of the Con A-activated effector cells were shown to reside primarily in the spleen, to be radio-resistant up to 400 R, and to be short-lived after adult thymectomy. Thus, the population of T cells capable of activation to cytotoxic effector lymphocytes by Con A has similar physical characteristics to the splenic subset(s) of T cells mediating both GVH and cytotoxicity responses upon alloimmunization.

CBA and C57BL mice both possess a VHdex gene coding for antibodies against the alpha 1-6 epitope of dextran B512. After immunization with dextran, CBA mice produce IgM plaque-forming cells (PFC) only, which show regular cyclical fluctuations. The second PFC peak disappeared after injection of dextranase, indicating that it is antigen-dependent. The anti-dextran response in C57BL mice is characterized by only one IgM peak, followed 1 day later by an IgG peak that may exceed the IgM response by a factor of 10. The IgG anti-dextran response in C57BL mice was thymus-independent. CBA mice gave an IgG response to the hapten fluorescein isothiocyanate (FITC) after immunization with FITC-dextran, indicating that dextran can function as a carrier for an IgG response in this strain. Attempts to induce IgG PFC against dextran by immunizing CBA mice with thymus-dependent dextran-protein conjugates consistently failed, althouth the conjugates induced IgG fc in C57BL mice. Spleen cells from CBA mice failed to produce IgG antibodies against dextran after transfer into lethally irradiated C57BL mice, whereas the C57BL spleen cells produced IgG PFC after transfer into CBA mice. The lack of IgG synthesis against the alpha 1-6 epitope of dextran in CBA mice appears to be regulated exclusively at the B cell level and is restricted specifically to the VHdex gene product.