Antigen-specific suppression of human antibody responses by allogeneic T cells. III. Role of the major histocompatibility complex

Specific antibody responses obtained in vitro from human blood mononuclear cells (PBM) were profoundly suppressed by allogeneic T cells. Experiments carried out with combinations of cells from HLA identical siblings, and HLA identical but unrelated donors, showed that suppression depended upon HLA incompatibility between responding PBM and allogeneic Ts. In order to map the specific HLA loci concerned, a series of experiments were undertaken using combinations of cells from a large number of HLA typed donors. Significant suppression was found to occur in every combination of HLA incompatible cells tested, including those with nonidentity at HLA-A, B, DR, A and DR, or B and DR, suggesting that suppression can be generated by nonidentity at class I or class II loci. With some HLA-A homozygous donors, however, a dominant role for class I (HLA-A) antigens was indicated by the finding of one-directional suppression in combinations where the HLA-A locus was seen as foreign by one partner only (A3,----A2,3; and A2----A2,26). Similar one-directional suppression was also seen with cells from a pair of siblings who were HLA identical except for a single A locus antigen arising from an HLA-A/B recombination (A3,----A3,1). These results indicate an important, but not exclusive role for class I MHC antigens in the activation of allogeneic Ts. The way in which this occurs is unknown, but one possibility is that it results from the activation of normal antigen-specific Ts by the interaction of their receptors for self-MHC with cross-reacting alloantigens.     

Antigen-specific suppression of cytotoxic T cell responses in mice. I. Suppressor T cells are not cytotoxic cells.

The main conclusion from these experiments is that the antigen-specific suppressor T cell of mice which inhibits the induction of cytotoxic T lymphocytes is not itself a cytotoxic T cell. This conclusion is supported by two main observations: first, a certain cell number from first-step cultures which was suppressive in the presence of a high dose of antigen actually helped the cytotoxic response at a lower antigen dose. This observation is difficult to reconcile with the hypothesis that suppression is due to the killing of the stimulator or the responder cells in the second-step culture by cytotoxic T cells. Second, cells from first-step cultures of cortisone-treated mice displayed cytotoxic activity but had no suppressive effect on the generation of killer cells. It was further demonstrated that these cells failed to influence in any way the suppressive effect, however weak, of cells from first-step cultures of normal spleen. We therefore favor the view that the suppression observed in this system is due to a regulatory signal which occurs as a result of the ability of both inhibitory cells and responder cells to recognize and respond to allogeneic determinants expressed on the surface of stimulator cells. The suppressor T cells described here act by linked associative recognition of antigen. That is, suppressor T cells only inhibit the induction of a precursor cytotoxic T cell in the presence of an antigen to which both the precursor cell and the suppressor cell can bind. In this sense, suppressors act in a manner analogous to helper T cells in T-B cell cooperation; carrier-specific helper T cells only enhance an anti-hapten B cell response in the presence of hapten-carrier conjugates. Similarly, alloantigen a (carrier)-specific suppressor T cells only inhibit alloantigen b (hapten)-specific cytotoxic responses in the presence of (a × b)F₁ stimulator cells (hapten-carrier conjugate), not in the presence of a mixture of parental stimulator cells (a + b).     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

Human C3a-mediated suppression of the immune response. II. Suppression of human in vitro polyclonal antibody responses occurs through the generation of nonspecific OKT8+ suppressor T cells

C3a-mediated suppression of Ig secretion in human PBL cultures occurs through the activation of suppressor T cells. Incubation of T cell-enriched populations derived from peripheral blood or tonsil results in the activation of nonspecific OKT3+8+ suppressor T cells capable of inhibiting Fc fragment-induced Ig secretion in fresh autologous PBL cultures. Generation of OKT8+ suppressor T cells by C3a requires the interaction of T cells, adherent cells, and C3a. Activation of the suppressor-T-cell pathway is initiated early in culture. Incubation of the T-cell-enriched populations with C3a for 0.5 hr results in functional, transferable suppressor cells. Maximum suppression was achieved when C3a was allowed to interact with the cell population for 1-2 hr.     

Amplification and suppression of human T cell proliferative responses by alloactivated T cells

Human regulator T (TR) lymphocytes presensitized to allogeneic non-T cells in unidirectional mixed leucocyte cultures (MLC) were shown to be capable of either amplifying or suppressing the MLC proliferative responses of autologous T cells when tested in limiting dilution co-culture assays. Although the concentration of alloantigen present during presensitization influenced the TR suppressive activity, considerable suppression was observed over a 10-20-fold range of stimulators. Thus, the concentration of stimulators was not an extremely critical factor in the generation of TR suppression. Much more important, however, was the duration of MLC presensitization because both TR amplification and suppression of T cell-mediated responses were critically time-dependent. In addition, the deletion of rapidly proliferating T cells from MLC at times of marked amplifier activity resulted in the impaired development of TR suppressor populations. These data directly demonstrate that distinct subpopulations of alloactivated TR cells can be distinguished by varying the presensitization interval and that maximal TR suppression requires earlier proliferation of alloreactive T cells.     

Cell cooperation during in vivo anti-hapten antibody responses. VI. Evidence for an allogeneic effect replacing one of two helper T cells

T and B lymphocyte activity in adoptive secondary antibody responses was assessed by cell titration in mice of the X-linked immune defective CBA/N strain and mice of their normal partner strain CBA/CaJ. No quantitative differences could be detected in either T or B cell activity in these experiments, in which 2,4-dinitrophenyl was used as the hapten. Both of the helper T cells detected in such assays were present in both strains. During these experiments, it was found that a subline of CBA/N maintained at Yale may have diverged from the CBA/N NIH stock. This was detected by an ability of irradiated mice of the Yale subline to increase the antibody response of B cells from the NIH subline, provided specific helper T cells were also present. This allogeneic effect apparently replaced the activity of one helper T cell, but did not replace the requirement for a carrier-specific helper T cell.     

Effect of allogeneic cell interaction on the primary immune response in vitro. Cell types involved in suppression and stimulation of antibody synthesis

A graft-versus-host (GVH) reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later the spleen cells from the recipients were cultured during the conditions for obtaining a primary immune response in vitro described by Mishell & Dutton (1967). It was found that the antibody response against the thymus-dependent antigen sheep red cells (SRC), as well as the thymus-independent antigen lipopolysaccharide from Escherichia coli 055:B5 (CPS) was markedly depressed. Spleen cells from mice subjected to a GVH reaction (GVH cells) also inhibited the antibody response of normal cells in vitro. The inhibitory effect of the GVH cells on normal cells was not sensitive to treatment with anti-θ serum, but could be completely abolished by treatment with iron powder, which removes adherent cells.

By culturing cells of two different mouse strains together in vitro it was possible to obtain stimulation or inhibition of the antibody response depending on the total cell number per dish.

The relation of these results to the phenomenon of antigenic competition is discussed. It is suggested that antigenic competition is caused by a non-specific inhibition of cell proliferation, possibly mediated by a locally acting factor. Both thymus-derived and bone marrow-derived cell lymphocytes are affected by the phenomenon. The cells initially responsible for the inhibition seem to be antigen-activated thymus-derived cells (T-cells), which by secondarily activated cells such as macrophages inhibit other cells. A GVH reaction, which generally leads to antigenic competition may, when less pronounced, cause stimulation of the antibody response.

     

Antigen-specific in vitro suppression of murine Helicobacter pylori-reactive immunopathological T cells by CD4CD25 regulatory T cells

A Helicobacter pylori-specific in vitro coculture system was established and used to study the role of CD4+CD25+ regulatory T cells (Treg) in gastritis development in mice with H. pylori infection. Effects of therapeutic immunization against H. pylori infection on the Treg function were also studied to better understand the mechanisms leading to postimmunization gastritis in these mice. Depletion of Treg led to extensive proliferation to H. pylori antigens of CD4+ T cells isolated from either na?ve, H. pylori-infected or H. pylori-immunized mice. Using the Treg-depleted CD4+ T cells from immunized mice as effector cells, we compared the suppressive efficacy of Treg isolated from na?ve, infected or immunized mice and found that Treg from na?ve mice, and slightly less efficiently from infected mice, suppressed the CD25- effector T-cell response and in most cases were distinctly more efficacious than Treg isolated from immunized mice. The suppressive efficacy of Treg isolated from the differently treated mice correlated closely with production of interleukin-5 (IL-5) by the Treg and suppression of interferon-gamma and IL-2 production by the CD25- effector T cells. Our study is the first to demonstrate in H. pylori-induced chronic infection, antigen-specific Treg with differential efficacy in suppressing H. pylori proinflammatory T effector cells.     

双阴性调节性T细胞在免疫抑制中的作用及其机制

αβTCR+CD3+CD4-CD8-双阴性调节性T细胞(DN Treg细胞）被证实具有通过对效应性T细胞的直接杀伤作用从而抑制免疫应答的能力。DN Treg细胞与特异性抗原接触后增加其调节活性,该过程部分通过其对抗原呈递细胞表面MHC抗原肽复合物的识别作用介导。DN Treg细胞与靶细胞接触后可通过Fas和Fas配体之间的交互作用介导其杀伤效应。对DN Treg细胞功能特性、分子表达方式及其激活机制的深入研究将有助于探索临床新的治疗手段。     

Induction of suppression through human T cell interactions.

Concanavalin A (Con A) activated T cells, devoid of cells bearing Fc receptors for IgG (T - TG) help human B lymphocytes to differentiate into plasma cells (PC) in response to pokeweed mitogen (PWM). PC differentiation is reduced when adult T cells are added to such cultures. The radiosensitivity of suppression and the radioresistance of help enabled us to show that adult T cells include a suppressor-precursor which is activated by irradiated Con A-precultured T cells. Newborn T cells which include active suppressors, are both poor stimulators of suppressor-precursors and poor helpers of B cells. Our results suggest that at least two cells may mediate Con A-induced suppression, one which suppresses directly and is radiosensitive and another which is radioresistant and stimulates suppressor-precursors in a target population of T cells.     

T cell suppression in vitro. I. Role in regulation of antibody responses

Suppression of the antibody response by supraoptimal numbers of T helper cells was studied in vitro and found to have both a specific and a nonspecific component. Suppression did not depend on direct cell contract of T and B cells, as supernatants of activated T cells were just as inhibitory. Suppression by excess T cells (or T cell supernatant) was abrogated by the addition of macrophages. By using a tolerance induction protocol, it was shown that T cell supernatants induce partial tolerance in both T and B cells. This tolerance occurred in three different experimental settings – if adherent cells were physically removed by surface adherence, in the presence of anti-macrophage serum, or in the presence of very high concentrations of T cell supernatant. All three conditions stress the importance of the interaction between T cell supernatants and macrophages. The nonspecific form of suppression was not analyzed in detail here, but was also shown to be partly abrogated by macrophages, suggesting that it may be analogous to antigenic competition, one model of which has recently been shown to be abolished by addition of macrophages. The results suggest the presence of homeostatic feedback loops, due to excess T cell function – T cell products suppress the function of T cells, preventing the induction of further helper cells, and also suppress B cells directly. The balance between T cell suppression and cooperation is affected by macrophage function.     

Antigen-specific suppression of a primed immune response by dendritic cells mediated by regulatory T cells secreting interleukin-10

Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. RelB activation is required for myeloid DC differentiation. Here, we show that antigen-exposed DCs in which RelB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. DCs generated from CD40-deficient mice similarly confer suppression. Regulatory CD4+ T cells induced by the DCs transfer antigen-specific "infectious" tolerance to primed recipients in an interleukin-10-dependent fashion. Thus CD40, regulated by RelB activity, determines the consequences of antigen presentation by myeloid DCs. These observations have significance for autoimmune immunotherapy and suggest a mechanism by which peripheral tolerance might be constitutively maintained by RelB(-) CD40(-) DCs.     

Antigen-specific helper T cells are essential for cytotoxic T cell responses to metabolically inactivated stimulator cells.

Primed spleen cells respond well to metabolically inactivated stimulator cells while normal spleen cells do not. This observation has been interpreted as showing that cytotoxic T cell precursors are different from unprimed precursors in their antigen recognition requirements for induction. A different model is proposed here which accounts for these observations as due to enhanced helper cell levels in primed populations. Experiments are described in this study which test several predictions of this model. These experiments show that in the presence of in vitro primed helper T cells, normal cells are able to respond efficiently to glutaraldehyde-fixed stimulator cells. The helper effect is antigen-specific. Since unprimed spleen cells can be efficiently induced by metabolically active stimulators (γ-irradiated cells) and can respond to glutaraldehyde-fixed antigen (metabolically inactive cells) only in the presence of specific helper cells, it seems reasonable to propose that helper cell signals are enhanced by a nonantigenic property of γ-irradiated stimulator cells requiring metabolic activity. It is also clear that glutaraldehyde-fixed cells are anti- genically intact as helper cells, primed to antigens on γ-irradiated stimulator cells, efficiently and specifically help a response to fixed stimulators. Conversely, helper cells primed in vitro to glutaraldehyde-fixed stimulators recognize antigen on γ-irradiated stimulator cells. The level of help generated in response to glutaraldehyde- fixed stimulator cells is at least 10-fold higher in primed cells than in normal cells. In addition, primed spleen cells can be induced in vitro to yield helper function by both fixed or unfixed stimulator cells. Normal helper cell precursors are induced at least 100-fold more efficiently by γ-irradiated as compared to glutaraldehyde-fixed stimulator cells. This work supports the idea that a major effect of priming, which allows primed cells to respond to metabolically inactive stimulators, is to enhance levels of helper T cells in the primed population.     

Antigen-specific T-helper cells abrogate suppression in Trypanosoma cruzi-infected mice.

The ability of antigen-specific T-helper (Th) cells to enhance direct plaque-forming cell responses in spleen cells from Trypanosoma cruzi-infected C57BL/6 mice was investigated at various times during the course of infection from day 7 to day 230. The injection of antigen-specific Th cells in vivo or the addition of antigen-specific Th cells in vitro was effective in enhancing direct plaque-forming cell responses, except at the time of the most intense suppression during the acute phase of infection (i.e., day 28). The ability of antigen-specific Th cells to overcome nonspecific immunosuppression was due not only to the activity of antigen-specific Th cells added to Mishel-Dutton cultures but also to activation of resident T cells. Thus, antigen-specific Th cells and resident T cells act in concert to produce enhanced direct plaque-forming cell responses. The effect of plastic-adherent spleen cells from infected mice on the ability of antigen-specific Th cells to stimulate anti-sheep erythrocyte responses of normal spleen cells was examined because macrophages have been shown to have an immunoregulatory role during the course of experimental American trypanosomiasis. Increasing numbers of macrophages from infected mice caused increased immunosuppression of normal spleen cells that could not be overcome with the addition of primed Th cells. It can be concluded from these data that antigen-specific Th cells can potentiate immune responses in mice infected with T. cruzi but that highly active suppressor macrophages can inhibit the expression of these primed Th cells.     

Cell cooperation in antibody responses to influenza virus. I. Priming of helper T cells by internal components of the virion

T cells recognizing internal components of the influenza virion can cooperate with B cells recognizing hemagglutinin to enhance the hemagglutinin inhibition (HAI) antibody response. Cooperation occurs only when the internal proteins and hemagglutinin are subsequently presented in association. The effect enhances the production of both 2-mercaptoethanol-resistant and-sensitive antibodies. The cooperating cells express the Thy-1⁺, Ly-1⁺2^? surface phenotype and thus are characteristic of helper T cells. Immunization of mice with the internal components of the virion increases survival after exposure to lethal influenza infection. Mice primed with purified matrix protein produce enhanced amounts of HAI antibody after infection with influenza. These results may help to explain reports on the effects of heterotypic infection on antibody production, and also heterotypic immunity.     

Suppressor cell induction in vitro. III. Antigen-specific suppression by supernatants of suppressor cells.

Antigen-specific suppressor T cells induced in vitro release, after a further period of culture in vitro with antigen factors into the supernatant which have suppressor activity. These suppressor factors (SF) have the same antigen specificity as the suppressor cells (SC). SF only works on the early phase of thymus-dependent responses in cultures. SF inhibits thymusdependent IgM, but not thymus-independent IgM responses in vitro. SF is is destroyed by proteolytic enzymes, and inactivated at 80 degrees C for 30 min. The release of SF from SC is dependent on the presence of antigen and metabolically active cells.     

Cell interactions in the immune response in vitro. I. Metabolic activities of T cells in a collaborative antibody response

Cell collaboration between thymus-derived (T) and non-thymus-derived (B) lymphocytes in induction of the response to SRC was investigated in an in vitro test system. Different populations of T cells were found to vary in helper activity depending on their degree of differentiation following exposure to antigen. Activated thymus cells obtained from the spleen of irradiated mice injected with syngeneic thymocytes and SRC proved the most effective, followed by primed T cells and then unprimed T cells. The interaction between T cells and B cells as measured here was specific for both classes of lymphocytes. The mechanism of T cell action in the collaborative response was analyzed by treating these populations of T cells with agents which became firmly cell bound and could selectively inhibit different aspects of cell metabolism. Mitomycin C was chosen as an inhibitor of DNA synthesis and cell division, actzinomycin D to interfere with RNA synthesis, and antimycin A to block protein synthesis. To exclude significant leakage of inhibitor from T cells to B cells, cultures were stimulated with DNP-POL, which immunizes B cells directly, as well as with SRC. The development of a normal anti-DNP response in all cases was confirmation of the integrity of B cell function. Mitomycin treatment (40 μg/ml) of normal T cells or T cells from mice primed to SRC at least four weeks previously markedly reduced helper activity. In contrast, activated T cells which had recently divided in response to antigen and were enriched for specific antigen reactive cells were resistant to mitomycin; thus effective collaboration with B cells could take place in the absence of further differentiation and division by T cells. All T cell populations, whether normal, primed or activated, were sensitive to treatment with actinomycin D at a concentration of 0.1 μg/ml, or 10^?5 M antimycin A. Taken together, these experiments suggest that T cells divide and differentiate over a period of approximately 48 h in vitro. During this time antibody production by B cells is initiated by a process requiring active RNA and protein synthesis in T cells.     

Antigen-specific CD8+ T cells inhibit IgE responses and interleukin-4 production by CD4+ T cells

There is a growing body of evidence which suggests that CD8⁺ T cells play an important part in regulating the IgE response to non-replicating antigens. In this study we have systematically investigated their role in the regulation of IgE and of CD4⁺ T cell responses to ovalbumin (OVA) by CD8⁺ T cell depletion in vivo. Following intraperitoneal immunization with alum-precipitated OVA, OVA-specific T cell responses were detected in the spleen and depletion of CD8⁺ T cells in vitro significantly enhanced the proliferative response to OVA. Depletion of CD8⁺ T cells in vivo 7 days after immunization failed to enhance IgE production, while depletion of CD8⁺ T cells on days 12–18 greatly enhanced the IgE response, which rose to 26 μ/ml following a second injection of anti-CD8 on day 35 and remained in excess of 1 μ/ml over 300 days afterwards. Reconstitution on day 21 of rats CD8-depleted on day 12 with purified CD8⁺ T cells from animals immunized on day 12 completely inhib ited the IgE response. This effect was antigen specific; CD8⁺ T cells from OVA-primed animals had little effect on the IgE response of bovine serum albumin immunized rats. In vivo, CD8⁺ T cell depletion decreased interferon (IFN)-γ production but enhanced interleukin (IL)-4 production by OVA-stimulated splenic CD4⁺ T cells. Furthermore, CD8⁺ T cell depletion and addition of anti-IFN-γ antibody enhanced IgE production in vitro in an IL-4-supplemented mixed lymphocyte reaction. These data clearly show that antigen-specific CD8⁺ T cells inhibit IgE in the immune response to non-replicating antigens. The data indicate two possible mechanisms: first, CD8⁺ T cells have direct inhibitory effects on switching to IgE in B cells and second, they inhibit OVA-specific IL-4 production but enhance IFN-γ production by CD4⁺ T cells.     

T cell-replacing factor in specific antibody responses to influenza virus by human blood B cells

In man, B cell maturation factors obtained from T cells or T cell lines have been shown to induce antibody formation in mitogen or anti-immunoglobulin activated B cells, and in some continuous B cell lines, but the relationships between these factors and B cell differentiation factors in antigen-specific antibody responses is unclear. We have now shown that supernatants from phytohemagglutinin-activated tonsil cells, or from the Gibbon Ape T cell line MLA-144, can substitute for T cells in the specific antibody response by human blood B cells to influenza virus. Thus, T cell-depleted non-rosette-forming (E-) cells prepared from peripheral blood mononuclear cells made antibody when cultured with antigen and factor together, whereas control cultures of E- cells with either antigen or factor alone did not. Moreover, E- cells cultured with factor and influenza virus strain A/X31 made antibody to A/X31, but not the non-cross-reacting strain, B/HK (and vice versa) showing that the response was antigen specific. The activity in these supernatants, therefore, fulfilled the functional definition of T cell-replacing factor (TRF). The possibility that interleukin 2 (IL 2) present in the TRF-containing supernatants was expanding residual T cells in the E- preparations to provide normal T cell help was excluded in three different ways. First, E- cells depleted of T (Leu4+) cells to undetectable levels made normal amounts of antibody when cultured with antigen and TRF. Secondly, a limiting dilution technique was employed to show that help in cultures of E- cells and TRF was not mediated by antigen-specific T helper cells. Thirdly, TRF-containing supernatants depleted of IL2 retained activity, whereas purified IL2 was inactive. Preliminary purification of TRF by gel filtration on Ultrogel AcA54 columns showed that all the activity eluted in a single peak between 35 000 and 43 000 molecular weight. This distinguishes human TRF from IL 2 and from other B cell maturation factors with a molecular weight range of 15 000-20 000 which act on continuous B cell lines. In addition to TRF, supernatants from phytohemagglutinin-activated tonsils also contained a factor which could induce polyspecific IgM production, but only in cultures containing significant numbers of T cells. This additional activity may have been due to IL 2, and provides an explanation for the apparent T cell-dependent effects sometimes observed in experiments designed to test B cell differentiation factors on T cell-depleted normal B cells.     

In vivo suppression of T-dependent antibody responses by treatment with a monoclonal anti-L3T4 antibody

Minute amounts of the anti-L3T4 antibody designated GK1.5 were found to deeply suppress in vivo antibody responses to T-dependent antigens. Primary responses to sheep erythrocytes were completely inhibited even when GK1.5 was administered up to 6 days before or 4 days after the antigen. Secondary responses to potent immunogens like sheep erythrocytes or keyhole limpet hemocyanin were also completely abolished by a single injection of GK1.5 just before the boost. This treatment had no effect on T-independent reactions such as the polyclonal activation of B lymphocytes with lipopolysaccharide or the anti-2,4,6-trinitrophenyl (TNP) response elicited by injection of TNP-Ficoll.