The induction of T cell unresponsiveness by rapidly modulating CD3

The immunomodulatory effects of an IgM anti-CD3 mAb (38.1) were investigated. 38.1 was distinct from other anti-CD3 mAb, in that it was rapidly modulated from the cell surface in the absence of a secondary antibody. Although 38.1 induced an immediate increase in intracellular free calcium [Ca2+]i by highly purified T cells, it did not induce entry of the cells into the cell cycle in the absence of accessory cells (AC) or a protein kinase C-activating phorbol ester. Clearing of 38.1 from the surface of AC-depleted T cells, documented both by immunofluorescence and by functional activity, was rapid, with markedly reduced levels of initially bound mAb observed after a 1 to 2 h incubation at 37 degrees C and complete modulation noted after a 5-h incubation. Despite rapid modulation of 38.1, the T cells continued to express substantial amounts of surface CD3, suggesting there is a rapid rate of turnover of CD3 molecules on resting T cells. After modulation of 38.1 bound CD3, T cells were markedly inhibited in their capacity to respond to PHA. Inhibition could be overcome by culturing the cells with supplemental AC or IL-2. The inhibitory effects of 38.1 could be mimicked by briefly pulsing cells with the calcium ionophore, ionomycin, that had no effect on surface expression of CD3. 38.1- or ionomycin-pulsed cells were inhibited in their subsequent response to PHA even when exposures were carried out in the presence of EGTA to prevent increases in [Ca2+]i from extracellular sources. Inhibition could not be accounted for by an inability of the ionomycin-treated or 38.1-modulated T cells to increase [Ca2+]i in response to PHA. These studies demonstrate that a state of T cell nonresponsiveness can be induced by modulating CD3 with an anti-CD3 mAb in the absence of co-stimulatory signals. A brief increase in [Ca2+]i resulting from mobilization of internal calcium stores appears to be sufficient to induce this state of T cell nonresponsiveness.     

Genetic control of tolerance induction to human gamma-globulin in mice.

Susceptibility to tolerance induction with monomeric human gamma-globulin (HGG) was tested in different inbred strains of mice. The results indicated a differential tolerance susceptibility among the strains and that the basis for the variation is genetic in nature. By using a protocol that permits genetic analysis, F1, F2, and backcross generations of the parental strains SJL/J and C3H/Bi were examined. A multigenic control model by H-2-linked and non-H-2-linked genes showing Mendelian autosomal inheritance is proposed.     

The induction of immunological unresponsiveness in previously immunized mice.

HGG unresponsiveness can be induced in primed A/J mice; however, such induction is difficult and requires multiple injections of large doses of soluble HGG (SHGG). Although single injections (1.5–25 mg) of deaggregated HGG (DHGG) did not result in a significant secondary immune response, an unresponsive state to a subsequent injection of aggregated HGG (AHGG) was not induced. When the dose of DHGG was even smaller (0.5 mg), a normal secondary response was obtained similar to that observed following injection of AHGG. Evidence is presented here which suggests that the difficulty encountered in inducing unresponsiveness may be in part due to partial aggregation of DHGG by persisting antibody in the circulation. The PFC to HGG produced after injection of either AHGG or DHGG or during induction of unresponsiveness to SHGG apparently involved cell division, since all three responses were inhibited by vinblastine. The reduction in PFC in primed mice injected with DHGG or SHGG was not due to selective inhibition of PFC secreting certain classes or subclasses of immunoglobulins.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.     

Immunologic unresponsiveness after gastric administration of human gamma-globulin: antigen requirements and cellular parameters

Gastric administration of human gamma-globulin (HGG) into adult A/J mice leads to the establishment of an antigen-specific unresponsive state to subsequent parenteral challenge with HGG. An unresponsive state is induced in both helper T and B lymphocyte populations. Unresponsiveness in helper T cells is of longer duration than in B cells, lasting at least 9 wk after intragastric intubation. Adoptive cell transfer of spleen cells from gastrically inoculated mice into healthy irradiated, syngeneic recipients revealed that the unresponsive state is stable upon cell transfer and that suppressor cells are present in the spleens of gastrically tolerized mice. The establishment of HGG-specific unresponsiveness is dependent upon both the dose and the form of the antigen adminstered. Soluble and deaggregated HGG are both more efficient than is heat-aggregated HGG in inducing unresponsiveness gastrically. The administered HGG is rapidly eliminated from the animal and only a small fraction reaches the circulation as immunoreactive protein. Although the cellular parameters of the systemic unresponsiveness induced by intragastric intubation with HGG appear similar to the parameters of parenterally induced unresponsiveness, the precise mechanisms by which gastric unresponsive states are established remain to be resolved.     

T cell tolerance in the chicken. I. Parameters affecting tolerance induction to human gamma-globulin in agammaglobulinemic and normal chickens.

T cell-mediated delayed hypersensitivity (DH) to human gamma-globulin (HGG) can be induced in chickens by subcutaneous injection of the antigen in complete Freund's adjuvant (CFA). In the present work, it has been demonstrated that specific tolerance of the cells mediating this DH can readily be induced in both normal and bursectomized (BX) FP strain chickens by simple i.v. injection of soluble antigen, regardless of the presence of antibody production to the tolerogen. A significant degree of tolerance at the DH and helper T cell levels could be generated in BX birds by injection of as little as 0.5 mg of HGG; such a dose only induced tolerance in normal birds when it had been previously deagregated by ultracentrifugation. Regular, nondeaggregated antigen could produce tolerance in normal animals, but only at doses of greater than 5 mg. The tolerizing injection induced a primary antibody response in normal birds in all cases, but a secondary response could not be obtained in animals rendered tolerant at the T cell level. Establishment of tolerance appeared to be very rapid, and animals remained refractory to induction of DH for at least 3 weeks after the tolerizing injection. The mode in which the antigen was presented to the animals appeared to be crucial in determining whether tolerance or sensitivity would be established.     

Antigen-induced unresponsiveness results in altered T cell signaling

Pretransplant exposure to allogeneic lymphocytes can result in donor-specific unresponsiveness and prolonged allograft survival. Intracellular signaling events have been described in anergic T cell clones, but the biochemical events underlying in vivo induced unresponsiveness have not been studied in detail. We employed a TCR transgenic mouse, bearing the 2C TCR, providing adequate numbers of homogenous peripheral T cells to study biochemical aspects of T cell unresponsiveness in vivo. 2C mice exposed to semiallogeneic lymphocytes (H-2b x H-2d) experienced prolonged H-2d cardiac allograft survival, and cells from these mice did not proliferate or make IL-2 in response to alloantigen (H-2d). Importantly, there were marked differences in TCR-associated tyrosine phosphorylation activation patterns. The targets for the unresponsive state appear to be diminished Lck activation and absent ZAP-70 and LAT (linker for activation of T cells) phosphorylation. Our study demonstrates that Ag-induced tolerance in vivo is accompanied by altered early TCR-mediated signaling events.     

Identification of human peripheral T cell antigens.

A new type of differentiation antigens on human T cells was demonstrated by using a heterologous anti-human T cell serum (ATS). This type of antigen, referred to as human peripheral T cell antigen (HPTA), was found on peripheral T cells and medullary thymocytes, but not on cortical thymocytes and B cells. The percentage of ATS-reactive lymphocytes in human peripheral lymphoid organs was correlated with that of cells rosetting with sheep erythrocytes, but contrasted with the number of B cells defined by the presence of a complement (C) receptor or by rabbit anti-human B cell serum (ABS). ATS also reacted with T cells purified by nylon fiber column filtration but ABS did not. Chronic lymphocytic leukemia cells rosetted with either sheep erythrocytes or erythrocyte-antibody-complement complexes were lysed by ATS and ABS, respectively. Mitogenic responses of blood lymphocytes to phytohemagglutinin (PHA) and concanavalin-A (Con A) were abrogated by treating them with ATS and C, whereas ABS suppressed only their response to Con A. Although numerous thymus cells rosetted with SRBC, only 14% were reactive with ATS. Quantitative absorption studies demonstrated that HPTA content of the thymus cells was much lower than that of lymph node cells. Anatomical localization of ATS-reactive lymphocytes in human lymphoid organs studied by immunofluorescence indicated that they were present in the thymus-dependent paracortical areas of lymph node and in the medullary region of thymus. ABS, on the other hand, did not stain thymocytes but reacted selectively with the cells located in the lymphoid follicles of lymph node. These data, together with that from cell suspension studies, confirmed that HPTA were shared between medullary thymocytes and peripheral T cells.     

Early events in peripheral regulatory T cell induction via the nasal mucosa

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.     

Nonantibody-mediated suppression of delayed hypersensitivity to human gamma-globulin in mice.

Development of delayed hypersensitivity (DHS) to human γ-globulin (HIgG) in mice was documented by histological analysis, by the kinetics of footpad swelling in animals exhibiting humoral or delayed responses, and by the failure of sera to transfer delayed reactions to normal, syngeneic recipients. Since cyclophosphamide (CY) treatment resulted in diminished humoral and augmented delayed reactions, we used this as a tool to explore the nature of the regulatory mechanisms which affect expression of this type of cell-mediated immunity. In order to evaluate the effect which the presence or absence of antigen-specific cells might exert on expression of DHS, we subjected mice to experimental regimes which would result in lymphocyte proliferation or depletion, respectively (see Bachvaroff, R., and Rapaport, F. T., Cell. Immunol. 15, 336, 1975). Cell proliferation was induced by injection of 80 μg of aqueous antigen on Day ?4; this was followed by sensitization with HIgG-CFA (Freund's adjuvant) on Day 0, and footpad challenge on Day 13. These mice exhibited strong humoral reactivity; three of six died of anaphylaxis following footpad challenge, and the remaining three showed a diminished delayed response. Similarly treated mice that, in addition, received 6 mg of CY 3 days after injection of aqueous antigen and, therefore, would have antigen-specific cells present showed greatly diminished humoral reactivity, due to B-cell depletion. However, they also exhibited a marked diminution in delayed responsiveness. The data clearly demonstrate that a nonantibody-mediated, possibly cell-directed, regulatory influence is exerted on DHS where cell proliferation has occurred. We next examined the impact which the depletion of proliferating cells would exert on the expression of DHS. Cell depletion was attempted by giving one injection of aqueous antigen (Day 0) early in a regime of chronic CY administration (Days ?1 through +3) ; antigen-induced proliferating cells would be susceptible to CY and, therefore, depleted under these conditions. The results show that mice receiving both aqueous antigen and CY have depressed humoral and markedly diminished delayed reactivity compared to animals that were injected with CY alone. Thus, the augmenting effect which CY exerts on DHS is abrogated by stimulation with aqueous antigen. One interpretation is that CY removes a regulatory cell population in the normal animal, thereby allowing enhanced expression of delayed responsiveness. Clearly, regulatory function cannot be attributed solely to bumoral antibody production.     

人外周血T淋巴细胞凋亡与胀亡研究及氨茶碱的诱导作用

目的:在前期研究的基础上进一步观察氨茶碱对人外周血T淋巴细胞凋亡与胀亡的诱导作用。方法:密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及氨茶碱组,培养后观察细胞光镜及电镜形态学、FDA/PI荧光染色,并用流式细胞仪检测凋亡和胀亡细胞比例变化。结果:①人外周血T淋巴细胞经体外培养,可自然出现典型的细胞凋亡与胀亡形态学改变。②人外周血T淋巴细胞在氨茶碱诱导作用下,凋亡率有显著增高,但胀亡率相对无显著增高。结论:人外周血T淋巴细胞存在凋亡与胀亡现象,氨茶碱可诱导人外周血T淋巴细胞凋亡,但不能有效的诱导健康人外周血T淋巴细胞胀亡。     

Modulation of the induction and circumvention of immunological tolerance to human gamma-globulin by interleukin 1

As with agents capable of causing the release of IL 1, IL 1 itself is capable of modulating certain tolerance-inducing events. Under the condition used in the present study, it previously has been firmly established that injection of A/J mice with DHGG induces a state of antigen-specific tolerance in both T helper (Th) and B cells. The tolerance in the B cell is of long duration, whereas that in the B cell is of shorter duration. Recombinant IL 1 (rIL 1) given shortly after the tolerogen DHGG results in the inhibition of the induction of tolerance resulting in antibody production. The induction of tolerance is inhibited at both its antigen-specific Th cell and B cell levels, although the latter may be caused by the former. The inhibition of the induction of tolerance by rIL 1 is not correlated to the generation of antigen-specific T suppressor cells. IL 1 mimics lipopolysaccharide and 8-bromoguanosine, which generate IL 1 production, in its ability to interfere with the in vivo induction of tolerance. However, in contrast to these latter mitogens which cause both terminal differentiation of B cells and IL 1 production, IL 1 itself does not cause in vivo circumvention of long-term tolerant Th cells in the presence of competent B cells and antigen. These latter findings suggest that a signal(s) in addition to those delivered by IL 1 is required for activation of the B cell compartment recovering from tolerance to antibody production. AHGG (immunogen) is a potent generator of IL 1 release, whereas DHGG has no effect on IL 1 release from macrophages and AHGG inhibits the induction of tolerance by DHGG. These latter results suggest that the lack of an IL 1 signal may be responsible for the deliverance of a tolerogenic rather than an immunogenic signal to the Th cell.     

The induction of suppressor T cells by lipopolysaccharide in human peripheral blood lymphocyte cultures in the presence of fetal calf serum

Alveolar macrophages (AM) were collected by repeated endobronchial lavage from mice, rats, guinea pigs, and rabbits, and titrated into cultures of mitogen-stimulated syngeneic or autochthonous lymphocytes. Significant species differences were detected in regard to AM activity in the cultures. AM from guinea pigs and mice stimulated PHA-induced lymphoproliferation, while those from rats and rabbits were inhibitory; blood or peritoneal macrophages were not inhibitory in any of the species examined.     

外周血T细胞亚群在小鼠结核感染中的意义

目的探讨外周血T细胞亚群在小鼠结核感染中的意义,并与加用免疫调节剂组对比研究。方法60只小鼠随机分为3组,每组20只。结核感染组:小鼠经血管注射结核分枝杆菌(H37RV)0.05 ml,建立小鼠结核模型。生理盐水组:用0.05 ml生理盐水替代H37RV处理。母牛分枝杆菌菌苗组:按结核感染组同样剂量和方法复制小鼠结核模型,H37RV感染后第3、10、17天分别肌注22.5μg母牛分枝杆菌菌苗。感染4周后,取外周血用流式细胞仪检测T细胞亚群。结果感染组外周血T细胞亚群比率显著降低,母牛分枝杆菌菌苗组小鼠外周血T细胞亚群比率显著高于感染组。结论外周血T细胞亚群数量与结核感染的病情密切相关,调节小鼠结核模型的T细胞亚群免疫功能可使结核感染的病情发展得到一定程度的的控制。