Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. III. Amplification of a generation of tumor-specific killer T-lymphocyte activities by suppressor T-cell-depleted hapten-reactive T lymphocytes.

2,4.6-trinitrophenyl (TNP)-reactive T-cell activities were raised in mice by immunization with TNP-isologous mouse gamma globulin. After establishing that TNP-reactive T lymphocytes can serve as amplifier cells for induction of killer T lymphocytes in allogeneic system, we explored the possibility of this hapten-reactive T-cell system to amplify tumor-specific killer T-lymphocyte activity in the syngeneic system. We utlized relatively weak immunogenic syngeneic plasmacytoma X5563 in C3H/He mice. Analysis of the TNP-reactive T-cell activities revealed that such T lymphocytes express the biological functions of both major subtypes of regulatory T cells, namely suppressors and helpers, and that TNP-reactive suppressor and helper T lymphocytes, respectively, differ in their relative susceptibility to specific inactivation by TNP conjugates of the nonimmunogenic D-amino acid copolymer, D-glutamic acid, and D-lysine (D-GL). By taking advantage of the relative susceptibility-difference to TNP-D-GL, selective inactivation of TNP-reactive suppressor T cells was induced by appropriate treatment with TNP-D-GL, and the generation of TNP-reactive helper T-cell activity was amplified. The supplement of augmented TNP-reactive helper T-cell activity to the system at the immunization with syngeneic X5563 with TNP-haptenation, resulted in a striking augmentation of induction of tumor-specific killer T-lymphocyte activity, and a considerable number of hosts survived after the challenge with lethal dose of viable tumor cells. Thus, appropriate manipulations designed to induce potent hapten-reactive helper T-lymphocytes provided the potential for a very effective mode of immunoprophylaxis against tumor.

Antigen presentation by resting B cells. Radiosensitivity of the antigen-presentation function and two distinct pathways of T cell activation.

In this report we have examined the ability of small resting B cells to act as antigen-presenting cells (APC) to antigen-specific MHC-restricted T cells as assessed by either T cell proliferation or T cell-dependent B cell stimulation. We found that 10 of 14 in vitro antigen-specific MHC-restricted T cell clones and lines and three of four T cell hybridomas could be induced to either proliferate or secrete IL-2 in the presence of lightly irradiated (1,000 rads) purified B cells and the appropriate foreign antigen. All T cell lines and hybridomas were stimulated to proliferate or make IL-2 by macrophage- and dendritic cell-enriched populations and all T cells tested except one hybridoma caused B cell activation when stimulated with B cells as APC. Furthermore, lightly irradiated, highly purified syngeneic B cells were as potent a source of APC for inducing B cell activation as were low density dendritic and macrophage-enriched cells. Lymph node T cells freshly taken from antigen-primed animals were also found to proliferate when cultured with purified B cells and the appropriate antigen. Thus, small resting B cells can function as APC to a variety of T cells. This APC function was easily measured when the cells were irradiated with 1,000 rads, but was greatly diminished or absent when they were irradiated with 3,300 rads. Thus, the failure of some other laboratories to observe this phenomenon may be the result of the relative radiosensitivity of the antigen-presenting function of the B cells. In addition, this radiosensitivity allowed us to easily distinguish B cell antigen presentation from presentation by the dendritic cell and macrophage, as the latter was resistant to 3,300 rads. Finally, one T cell clone that failed to proliferate when B cells were used as APC was able to recruit allogeneic B cells to proliferate in the presence of syngeneic B cells and the appropriate antigen. This result suggests that there are at least two distinct pathways of activation in T cells, one that leads to T cell proliferation and one that leads to the secretion of B cell recruitment factor(s).

Antigen-induced human T cell help. Precursor frequency, radiation sensitivity, and allogeneic effects.

We have recently noted marked differences between the in vitro responses of human B lymphocytes to stimulation with soluble antigens vs. stimulation with mitogens. In the present study, these differences were analyzed in terms of the precursor frequencies for the T cells and B cells involved and in terms of the radiation sensitivity of the T cells providing help in the two systems. Marked differences were found between antigen-induced and mitogen-induced systems with regard to T cell precursor frequencies and radiation sensitivity. In contrast, the precursor frequencies for the B cells involved in the two systems were approximately the same. In addition, having developed a system for the study of human antigen-specific B cell responses, we were interested in delineating the nature of the allogeneic effects that might be operative in this system. Marked allogeneic effects, both positive and negative, were noted in this system and will need to be taken into account in any studies that try to address the question of the genetic restriction, if any, that exists in human antigen-specific T cell-B cell collaboration. Appreciation of the marked differences between the antigen-specific and mitogen-induced activation and immunoregulation of human B cell responses will be of importance in understanding the relationship between specificity and nonspecificity of antibody production in normal and disease states.

Antigen-presenting cells in human decidual tissue.

The responses of peripheral blood human T lymphocytes supported by decidual antigen-presenting cells (DAPCs) to a variety of immunogenic stimuli were studied and compared to those of T cells supported by peripheral blood antigen-presenting cells (PAPCs). Antigen-presenting cells were isolated from early normal decidual tissue or peripheral blood by elution with ethylenediamine tetraacetic acid of cells that after Ficoll-Paque separation bear receptors for all have bound to fibronectin. DAPCs pulsed with soluble or particulate antigens induced proliferation of T cells with an efficiency equivalent to PAPCs. Decidual tissue APCs also showed the ability to stimulate auto- and alloreactivity. Treatment with anti-human lymphocyte antigen (HLA) class II antibody and ultraviolet radiation resulted in substantial inhibition of the accessory cell function of DAPCs as well as of PAPCs. Bromodeoxyuridine and light treatment of alloreactive T cells generated in vitro was used to demonstrate that DAPCs primed with a synthetic polypeptide antigen (T,G)-A-L can stimulate only HLA class II-compatible T lymphocytes.

Immunity to coccidiosis: T-cell control of infection with Eimeria vermiformis in mice does not require co-operation with inflammatory cells.

The necessity for co-operation between lymphocytes and myeloid-derived inflammatory cells in the mediation of anti-coccidial immunity was investigated using mice infected with Eimeria vermiformis. Reciprocal exchange of immune lymphocytes between H-2 compatible strains of contrasting susceptibility to infection (resistant BALB/B and susceptible C57BL/10) resulted in successful transfer of immunity in both homologous and heterologous exchanges. Recipients of immune cells, whatever their original response phenotype, expressed a high degree of immunity to infection, indicating that the differential susceptibility of the strains is a property of their lymphoid cells and is not attributable to their capacity to mount inflammatory responses. This conclusion was confirmed by the successful adoptive transfer of immunity into NIH mice previously exposed to 600 rad X-irradiation; at this level of irradiation inflammatory responsiveness is severely depressed. Additional confirmation that strain-response phenotype is lymphocyte dependent and that immune lymphocytes can mediate their effects against E. vermiformis without the intervention of inflammatory cells was obtained from studies on the mucosal mast cell response to infection. No correlation existed between the development of intestinal mastocytosis, an index of T-cell-mediated inflammatory responsiveness, and the expression of resistance to E. vermiformis in BALB/c (resistant), C57BL/10 (susceptible) and NIH (susceptible) mice.

Radiosensitivity of synthesis of various antibodies.

It has been established by statistical analysis of data from the literature that a three-cell cooperation system of immunocompetent cells is damaged by radiation 2.1 times more seriously than a two-cell system. The synthesis of antibodies to molecular T-dependent antigens (proteins) is 1.4 times more radiosensitive than the synthesis of antibodies to corpuscular T-dependent antigens (erythrocytes).

Histocompatibility requirements for T cell help in specific in vitro antibody responses to influenza virus by human blood lymphocytes.

Specific antibody responses to influenza virus were obtained in vitro from human blood mononuclear cells (PBM). The response was T cell-dependent, as shown by separation of PBM into E rosette-positive (E+) and -negative (E-) populations. The histocompatibility requirements for T-B cells interactions in this response were analyzed by recombining E- and E+ fractions from donors with varying degrees of HLA compatibility. No antibody formation was obtained from any allogeneic combination except for the special case of HLA identical siblings. As these experiments included combinations with shared or identical HLA-DR specificities, it was unlikely that genetic restriction alone could account for the failure of T-B cell collaboration. Evidence that suppression was responsible for the lack of antibody formation was obtained from experiments in which allogeneic E+ cells profoundly depressed specific antibody responses of intact PBM. In contrast, no such suppression was seen in pokeweed mitogen-driven polyclonal Ig synthesis for which there are no major histocompatibility complex requirements for T cell help. The suppressor activity of allogeneic E+ cells was found to be radiation-sensitive. By irradiating E+ cells, it was, therefore, possible to test for T cell help across an HLA barrier without unwanted suppressor effects. Under these conditions, (irradiated) E+ cells were able to collaborate with allogeneic E- cells even with no HLA alleles in common. This was true even when autologous monocytes were depleted from the helper E+ population. Supernatants collected from antigen-driven cultures of allogeneic E- and E+ cells were able to replace helper T cells in the specific antibody response to influenza virus. The apparent lack of genetic restriction in these responses might, therefore, be explained by the production of a nonrestricted helper factor.

Cooperation of murine F T and parental B lymphocytes in rejection of a xenogeneic tumour: adaptive differentiation of B lymphocytes?

The primary humoral immune response to rat Yoshida ascites sarcoma (YAS) grown in mice was used to study thymus-dependent (T) and bone marrow-derived (B) lymphocyte cooperation. It was shown that B6D2F1 T lymphocytes that do not cooperate with parental B lymphocytes enabled parental B lymphocytes from B6 leads to B6D2F1 radiation chimaeras to reject the tumour. However, when the bone marrow cells from B6 leads to B6D2F1 chimaeras were used to reconstitute parental B6 mice, these B6 leads to B6D2F1 leads to B6 mice lost their tolerance to D2 transplantation antigens, and their B lymphocytes were not able to cooperate with B6D2F1 T lymphocytes. In our search for the reasons for the failure of F1 T cells to be effective in parental and P leads to F1 leads to P TIR mice, rejection of F1 T cells was excluded because : (i) immune reactivity in TIR mice was found to be either absent or minimal; (ii) parental TIR mice did not produce any detectable cytotoxic antibodies after an intravenous injection of F1 splenic T cells; and (iii) YAS rejection was not induced at very high doses of F1 T cells. However, in a cell transfer system we were able to demonstrate that injection of spleen cells from parental TIR mice could thwart the successful cooperation of transferred F1 T cells with host B cells. Collectively, these data suggest that the changes of the collaborative potential of parental B cells as achieved in the F1 environment could be ascribed to 'adaptive' differentiation of B lymphocytes. It appears that the differentiation process that has rendered nonsyngeneic chimaeric cells able to cooperate was independent of the exogenous antigen.

Immunologic effects of whole-body ultraviolet irradiation: selective defect in splenic adherent cell function in vitro.

Splenocytes from mice receiving whole-body UV irradiation do not make a normal primary in vitro plaque-forming cell (PFC) response to the soluble T-dependent antigen trinitrophenylated poly(L-glutamic acid60L-alanine30L-tyrosine10). This impaired immune response results from a selective loss of antigen-presenting cell function in the splenic adherent cell (SAC) population of the UV-treated mice. SACs from UV-irradiated mice are unable to reconstitute a PFC response when added to normal splenocytes passed through Sephadex G-10 (which depletes adherent cells), whereas normal SACs, when added to Sephadex G-10-passed splenocytes from UV-treated mice, do restore a PFC response. The effect of in vivo UV irradiation on the SAC population is indistinguishable functionally from the effect of in vitro UV irradiation of SACs from normal mice. Possible explanations for this selective effect of external UV irradiation on SAC function are discussed.

Effects of irradiation on the in vitro immune response.

Primed and unprimed murine spleen cells and suspensions enriched for T or B cells were exposed to various doses of radiation in vitro and evaluated with respect to their capacity to respond to sheep red blood cells (SRBC) in 1-ml or 10-microliter cultures. The dose-response data show three components: (1) a low dose (less than 50 rad) phase associated with an augmented anti-SRBC response; (2) a radiosensitive phase associated with precipitous loss of anti-SRBC activity; (3) a relative radioresistant phase with a loss of anti-SRBC activity which occurs over a broad range of radiation doses. Data obtained utilizing irradiated subpopulations enriched for T or B cells combined with excess numbers of nonirradiated lymphocytes of the corresponding cell type suggest that low dose augmentation is the result of radiation-induced injury to T cells or subcomponent thereof. With respect to the second component of the dose-response curve, similar experiments show that B cells are more radiosensitive than T cells (D37 of approximately 80 vs. approximately 220 rad, respectively) in this response. The third component of the curve appears to involve the interaction of radio-resistant subpopulations of T and B cells.

[The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 10. Cooperative immune reactions in different generations of mice]. / Biologicheskie éffekty u zhivotnykh v sviazi s avariei na Chernobyl'skoi AES. Soobshchenie 10. Kooperativnye immunnye reaktsii u razlichnykh pokolenii myshei.

The immune status of mice has been assessed by the whole complex of data. The permanent action of low-level radiation has been shown to suppress considerably the rate of reactions of the delayed-type hypersensitivity and "graft versus host" disease, as well as NK and specific cytolytic T-lymphocyte activity. The dynamics of accumulation and the levels of antiviral antibodies in the serum, lung and trachea extracts are virtually invariable. The resistance of experimental animals to influenza is lower than that of non-irradiated mice of the same line and age. The data obtained indicate that the immune disturbances revealed are connected not only with the alteration of lymphoid cell populations, but also with the alteration of the immune regulation mechanisms.

[Effect of radiation on cell interactions in the phenomenon of non-syngeneic stem cell inactivation. Changes in B-lymphocyte function after irradiation]. / Deistvie radiatsii na kletochnye vzaimodeistviia v fenomene inaktivatsii nesingennykh stvolovykh kletok. Izmenenie funktsii B-limfotsitov pri obluchenii.

A study was made of the changes in the mode of interaction between T- and B-lymphocytes of mouse lymph nodes with respect to the phenomenon of inactivation of non-syngeneic haemopoietic stem cells. It was shown that irradiation of B-lymphocytes with doses of 77.4--232.2 mC/kg changes their helper activity into a suppressor activity with regard to T-cell-killers having a low electrophoretic mobility.

Concanavalin A-mediated polyclonal helper assay of normal thymocytes and its use for the analysis of ontogeny.

A concanavalin A (Con A)-mediated polyclonal helper assay system was established by using the thymus cells or splenic T cells as a source of helper T cells. When splenic B cells were cocultured with thymus cells or splenic Lyt-2- T cells in the presence of an optimal dose of Con A, B Cells were polyclonally activated and differentiated into immunoglobulin-secreting cells. This Con A-mediated helper activity was completely inhibited by the addition of alpha-methyl-D-mannoside and could not be substituted by culture supernatant of Con A-stimulated thymocytes or splenic T cells. Almost all the activity of the thymus cells was carried by peanut agglutinin low binding population. Genetic restriction between T and B cells was not observed in this helper function. In ontogeny, Con A-mediated helper activity in the thymus was first detected at a few days after birth and reached the adult level at about 1 week of age. The polyclonal helper assay system developed in the present study provides a sensitive system to analyse the helper function of thymus cells and also to delineate the early phase of the differentiation of helper T cell population.

Polyclonal B-cell activation by influenza A/Texas virus-specific human T-cell clones.

A previously described proliferating class-II-restricted CD4+ human T-cell clone (TA4) specific for the N2 neuraminidase of the influenza A/Texas virus was tested for its ability to induce B cells to polyclonal immunoglobulin (Ig) production. The data reported in the present study show that, when stimulated by T-depleted autologous peripheral blood mononuclear cells (E-) and A/T virus, the TA4 clone was able to induce B cells to polyclonal Ig production. This effect was also seen using another class II-restricted human T-cell clone specific for the H3 haemagglutinin of the A/Texas virus and autologous polyclonal T cells. This Ig production was MHC-restricted at the inductive level, i.e., the stimulating-virus-treated E- population and the clones or the T cells had to share the same HLA-DR determinants. However, the responding B cells could be allogeneic provided the helper T cells were activated in the presence of autologous irradiated virus-infected E- cells.

Role of interleukin 1 in antigen-specific T cell proliferation.

The role of interleukin 1 (IL 1) in human antigen-specific T cell proliferation was examined. Nylon wool-purified T cells proliferated in the presence of autologous monocytes (Mo.) pulsed for 18 h with tetanus toxoid (TT) antigen (Mo.TT). Irradiation of Mo.TT with ultraviolet (UV) light (72 J/m2) abolished their capacity to support T cell proliferation and drastically reduced their capacity to secrete IL 1 after stimulation with Staphylococcus albus. The defect in antigen presentation induced by UV irradiation of Mo.TT was reversed in a dose-dependent manner by the addition of two different preparations containing human interleukin 1 (IL 1). The first preparation consisted of supernatants of Mo. stimulated with Con A for 18 hr and in which Con A activity was blocked by alpha-D-methyl-mannoside (Mo.-Con A-Sup). The second preparation consisted of human IL 1 partially purified from supernatants of human peripheral blood mononuclear cells stimulated with S. albus. This IL 1 copurified with human leukocyte pyrogen (LP) and was termed IL 1/LP. Both IL 1-containing preparations enhanced the response of C57BL/6 mouse thymocytes to phytohemagglutinin. A rabbit antibody to human IL 1/LP inhibited the capacity of T cells to proliferate in response to Mo.TT and inhibited the capacity of Mo.-Con A-Sup to reconstitute the T cell response to UV-irradiated Mo.TT. IL 1/LP was not necessary for T cells to recognize the immunogenic moiety presented by Mo., because monolayers of UV-irradiated Mo.TT were equivalent to monolayers of unirradiated MO.TT in their capacity to adsorb TT-reactive T cells specifically. Furthermore, the addition of rabbit antibody to IL 1/LP did not interfere with the capacity of UV-irradiated Mo.TT to adsorb TT-reactive T cells. The results obtained in this study indicate that IL 1 is involved in optimal antigen-driven proliferation of human T lymphocytes.

Mechanism of systemic immune suppression by UV irradiation in vivo. II. The UV effects on number and morphology of epidermal Langerhans cells and the UV-induced suppression of contact hypersensitivity have different wavelength dependencies.

We previously reported that broad band UV radiation or narrow bands of UV (Hbw 3 nm) of wavelengths 250 to 320 nm cause a systemic suppression of contact hypersensitivity (CHS) in mice, observed when the contact sensitizer is applied to a nonirradiated site. To determine if this effect is associated with UV-induced alterations in epidermal Langerhans cell (LC) numbers and morphology, we performed the following study. LC were identified by ATPase staining of EDTA-separated epidermal sheets. Electron microscope studies confirmed that this method was a satisfactory indicator of the presence of LC; we found no evidence for LC which did not stain for ATPase in either irradiated or unirradiated epidermis. Mice were irradiated on the back with narrow band UV of peak wavelength 270, 290, or 320 nm. The irradiated skin was excised 24 hr later and was stained as described. The number of LC with ATPase staining dendrites and the number of nondendritic LC were enumerated. We found that UV radiation of 270 or 290 nm caused 1) an alteration in LC morphology (loss of dendrites) and 2) a decrease in the total number of epidermal LC. Both effects occurred in a dose-dependent fashion. Previously, these same wavelengths of narrow band UV, but at higher doses, had been shown to cause systemic suppression of CHS. In this study, the doses of 270 or 290 nm UV that resulted in the decreased LC numbers and alterations in LC morphology described above were insufficient to cause systemic suppression of CHS. The converse was found if the irradiating waveband of UV had a peak at 320 nm. A dose of 320 nm UV that caused 50% systemic suppression of CHS had no effect on either the number or the morphology of LC at the site of irradiation. In addition, the number and morphology of LC were unaffected in the ventral epidermis (site of contact sensitization) of mice that had been previously irradiated on the back with a systemically suppressive dose of UV. We conclude: (a) UV-induced alterations in the number and morphology of LC at the site of irradiation are not necessary for the generation of systemic suppression of CHS by UV radiation; this indicates that the initial UV-absorbing event triggering systemic suppression is neither a loss of, nor morphologic alterations to, LC at the irradiation site. (b) A systemic effect of UV radiation on the number and morphology of LC at the unirradiated site of contact sensitization does not occur, and thus is not responsible for the UV-induced systemic suppression of CHS by UV radiation.

Synergistic defense system by cooperative natural effectors against metastasis of B16 melanoma cells in H-2-associated control: different behavior of H-2+ and H-2- cells in metastatic processes.

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor.

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.

[Effect of prolonged continuous external irradiation on humoral immunity indices of mice]. / Vliianie dlitel'nogo nepreryvnogo vneshnego oblucheniia na pokazateli gumoral'nogo immuniteta u myshei.

Changes in the content and function of cell populations and subpopulations involved in the humoral response of mice to the thymus-dependent antigen were investigated. The effect was followed during a prolonged continuous exposure to 137Cs gamma-emitter (total dose--5 Gy and daily dose--12 cGy for 22 hours) and after its termination. The data obtained give evidence for a decrease of the pool of polypotent lymphocyte precursors (CFUs), stable moderate hypoplasia of central and peripheral organs of the immune system, distinct inhibition of antibody production at the expense of reduced activity of precursors of lymphocytes, B-lymphocytes and T-helpers. In the remote postirradiation period residual radiation damage was seen in polypotent and committed precursors of lymphocytes and T-helpers which was responsible for the trend towards the decline of antibody production, hypoplasia in the spleen and lymph nodes being persistent.