Production of antigen-specific contrasuppressor cells and factor, and their use in augmentation of cell-mediated immunity

A single injection of TNP-labeled mouse gamma-globulin (TNP-IgG) can render the contact sensitivity response of mice resistant to suppressor cells (Tsc) and their biologically active cellfree products (TsF). Lyt-1 T cells of mice treated with TNP-IgG can protect the adoptive contact sensitivity response of immune cells from the antigen-specific suppressive effect produced by the addition of antigen-specific TsF or Tsc. When T cells of TNP-IgG-treated mice are put into culture, they produce an antigen-specific contrasuppressor factor (TcsF) that can replace the activity of the cells. When immune cells are preincubated in vitro with TcsF, they become refractory to Tsc and TsF added subsequently. The TcsF, however, has no ability to restore responsiveness to immune cells that had been previously exposed to TsF. The TcsF binds specifically to TNP, expresses an I-J-controlled determinant, and does not express standard determinants found on mouse Ig. The treatment that primes the contrasuppressor system to protect the contact sensitivity response also reportedly renders the antibody-producing system tolerant, (i.e., produces so called "split tolerance"). These results are discussed in light of the possibility that the contrasuppressor system can be responsible for so called isotype-specific immunity by rendering one arm of the immune system resistant to generalized suppressive mechanisms.     

Regulation of contact sensitivity reaction: contrasuppressor T cells and contrasuppressor factor downregulate efferent T suppressor cells.

Contact sensitivity (CS) reaction mediated by CD 4+8- Th 1 cells is under the control of several antigen-specific regulatory lymphocytes. Reaction is downregulated at the induction stage by T afferent suppressor T cells (Ts-aff) that prevent immunization and at the effector stage by efferent T suppressor cells (Ts-eff) that made immune Th 1 cells inoperative. Both suppressor cells are CD 4-8+ Th 1 effector cells and are protected against the suppressive action of Ts-eff cells by CD 4+8- contrasuppressor T cells (Tcs). As has been already shown there are also regulatory interactions between regulatory cells themselves and Ts-aff cells in addition to their effect on precursors of Th 1 cells, also preventing the induction of Ts-eff cells. The present experiments extend these findings and demonstrate that Ts-eff cells are also under negative control of Tcs lymphocytes. Likewise, antigen-specific factor produced by contrasuppressor T-T cell hybridoma, used in lieu of Tcs cells, impedes the activation of Ts-eff cells. In both cases regulation is aimed at the precursors of Ts-eff cells. Our experiments demonstrate that the outcome of immunization is dependent not only on the balance between immune cells and regulatory cells, but also on interactions between regulatory cells themselves.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

T cells recognizing polysaccharide-specific B cells function as contrasuppressor cells in the generation of T cell immunity to Pseudomonas aeruginosa

The cellular interactions involved in the development of T cell-mediated immunity to Pseudomonas aeruginosa have been examined. T cell immunity can be generated by immunizing mice with 10 micrograms of P. aeruginosa polysaccharide (PS) plus the antimitotic agent vinblastine sulfate. Vinblastine is required to inactivate a population of Ts cells generated by immunization with 10 micrograms of PS alone. Immunization with either live bacteria or with higher dose (50 micrograms) of PS without vinblastine also generates T cell immunity; these protocols activate a population of Lyt-1+, 2-, I-J+ T cells which, like vinblastine, counteract the effect of Ts cells. Immunization with 10 micrograms PS alone fails to activate this T cell subpopulation. When administered at the time of immunization, this subpopulation can render the tolerogenic 10-micrograms immunization protocols immunogenic. Like previously described contrasuppressor T cells, this T cell subpopulation exhibits an affinity for the lectin Vicia villosa. We have determined, however, that the T cells that act as contrasuppressor cells in this system are directly activated by PS-immune B cells and not by PS Ag. Furthermore, their activity can be removed by adsorption to PS-specific B cell hybridomas. Our studies indicate an important role for B cells in the development of T cell immunity to P. aeruginosa and suggest that a complex idiotype network controls the development of this response.     

LIGHT-HVEM signaling and the regulation of T cell-mediated immunity

LIGHT is a tumor necrosis factor (TNF) superfamily ligand that regulates T cell immune responses by signaling through the herpes virus entry mediator (HVEM) and the lymphotoxin beta receptor (LTbetaR). This review will present a summary of recent advances made regarding the immunobiology of the LIGHT-HVEM and LTbetaR systems. LIGHT has emerged as a potent initiator of T cell co-stimulation signals effecting CTL-mediated tumor rejection, allograft rejection and graft versus host disease. Constitutive expression of LIGHT leads to tissue destruction and autoimmune-like disease syndromes. In contrast to LTalphabeta, LIGHT plays a minimal role in lymphoid tissue development, yet some evidence indicates a role in negative selection in the thymus. These results provide an encouraging profile for the LIGHT-HVEM-LTbetaR axis as a potential target for controlling cellular immune reactions.     

Transfer factor: therapy in patients with deficient cell-mediated immunity and deficient antibody-mediated immunity

The effect of transfer factor therapy on the clinical and laboratory abnormalities of six patients with various antibody and cell-mediated immunodeficiency disorders was evaluated. Clinical improvement occurred in three patients. Conversion of previously negative delayed hypersensitivity reactions occurred in the same three patients. One patient demonstrated an increased in vitro lymphocyte response to phytohemagglutinin. No change in antibody mediated immunity was observed in any patient.     

Evidence for naturally acquired T cell-mediated mucosal immunity to Neisseria meningitidis

Naturally acquired protective immunity against Neisseria meningitidis is thought to partially explain the disparity between the high levels of carriage in the human nasopharynx and the rare incidence of disease. To investigate this immunity to Neisseria meningitidis at the mucosal level, in vitro cellular responses to outer membrane vesicle preparations derived from this pathogen were examined using mononuclear cells from the palatine tonsils of adults and children. Characterization of these responses was achieved by depletion of CD45RA(+), CD45RO(+), and CD19(+) populations and outer membrane vesicles derived from isogenic mutants expressing different serosubtypes of the major outer membrane protein, porin A (PorA), no PorA and membrane preparations from a mutant with no LPS (LpxA(-)). The magnitude of cellular proliferative responses against the outer membrane vesicles were strongly associated with age and were largely T cell mediated, involving both CD45RO(+) and CD45RA(+) T cell phenotypes. Responses were not dependent on LPS but consisted of both PorA cross-specific and non-PorA-dependent responses. Cellular immunity against Neisseria meningitidis was found to be frequently associated with systemic IgG Abs but was not associated with serum bactericidal Abs. For the first time our results demonstrate an age-associated acquisition of mucosal T effector/memory cell responses to Neisseria meningitidis. This mucosal cellular immunity can be present in the absence of serum bactericidal Abs, a classical marker of protective immunity.     

Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.     

Effect of chronic microwave radiation on T cell-mediated immunity in the rabbit

Experiments were conducted to elucidate the effects of chronic low power-level microwave radiation on the immunological systems of rabbits. Fourteen male Belgian white rabbits were exposed to microwave radiation at 5 mW/cm², 2.1 GHz, 3 h daily, 6 days/week for 3 months in two batches of 7 each in specially designed miniature anechoicchambers. Seven rabbits were subjected to sham exposure for identical duration. The microwave energy was provided through S band standard gain horns connected to a 4K3SJ2 Klystron power amplifier. The first batch of animals were assessed for T lymphocyte-mediated cellular immune response mechanisms and the second batch of animals for B lymphocyte-mediated humoral immune response mechanisms. The peripheral blood samples collected monthly during microwave/sham exposure and during follow-up (5/14 days after termination of exposures, in the second batch animals only) were analysed for T lymphocyte numbers and their mitogen responsiveness to ConA and PHA. Significant suppression of T lymphocyte numbers was noted in the microwave group at 2 months (P<0.01, % 21.5%) and during follow-up (P<0.01, % 30.2%). The first batch animals were initially sensitised with BCG and challenged with tuberculin (0.03 ml) at the termination of microwave irradiation/sham exposure and the increase in foot pad thickness ( mm), which is a measure of T cell-mediated immunity (delayed type hypersensitivity response, DTH) was noted in both the groups. The microwave group revealed a better response than the control group (%+12.4 vs.+7.54). The animals were sacrified and the tissue T lymphocyte counts (spleen and lymph node) were analysed. No significant variation was observed in the tissue T lymphocyte counts of microwave-irradiated rabbits. From these results it is speculated that the T lymphocytes are sequestered to various lymphoid organs under the influence of microwaves. A sub-population of T cells known as T helper cells (mediating DTH response) are probably not affected by microwave radiation. It is clear from our experiments that although chronic microwave radiation at 5 mW/cm² leads to suppression of peripheral T lymphocyte numbers, there is no concomitant functional impairment of these cells as evidenced by functional assays.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

Control of the specificity of T cell-mediated anti-idiotype immunity by natural regulatory T cells

The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2K^d-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy.     

Anti-tumor necrosis factor antibodies suppress cell-mediated immunity in vivo.

Rabbit anti-murine TNF-alpha antibodies were administered in vivo to mice to evaluate the role of TNF-alpha in T cell-mediated immunity. Anti-TNF suppressed the in vivo development of contact sensitivity to the hapten TNP in a dose-dependent fashion. Similarly anti-TNF suppressed the in vivo priming for TNP-specific CTL. Control antibodies did not suppress cell-mediated immunity, whereas purified murine rTNF-alpha neutralized the antibody activity. Antibody therapy was effective during the afferent or priming limb of immunity, but could not inhibit the response if administered during the efferent limb. FACS for CD2, CD3, CD4, and CD8 T, B, and NK cell surface markers demonstrated no major change in the distribution of splenic lymphoid cell populations in animals pretreated with anti-TNF antibody. These results suggest that anti-TNF antibody may be interfering with soluble cytokines rather than with cell surface TNF causing depletion of cell populations. In vitro analyses also showed that anti-TNF has minimal inhibitory effects on secondary (secondary CTL) or strong primary (primary CTL, alpha CD3, MLR) responses, even though these in vitro cultures produce TNF mRNA as shown by polymerase chain reaction amplification. Although anti-TNF antibody did not affect the above responses, primary interactions are strongly inhibited in vivo. These findings suggest that TNF is important during afferent, priming events in immunity and that inhibition of TNF receptor-ligand interactions may alter immunity early in a response. Conversely such inhibition is ineffective later in a response, perhaps due to the ability of multiple other receptor-ligand pathways to bypass TNF.     

TNF receptor-associated factor 3 is required for T cell-mediated immunity and TCR/CD28 signaling

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and characterization of T cell-specific TRAF3(-/-) mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or proportions of CD4(+),CD8(+) or double-positive or double-negative thymocytes, except that the T cell-specific TRAF3(-/-) mice had a 2-fold increase in FoxP3(+) T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity.     

Antigen-specific human T cell factors. I. T cell helper factor: biololgic properties

The in vitro induction and assay of an ovalbumin-specific human T cell helper factor are described. Peripheral blood T cells, cultured with ovalbumin in a Marbrook-Diener system, produce an antigen-specific factor(ThF120-OA), which can be purified by affinity chromatography. The in vitro studies with ThF120-OA pointed out that in the production of the factor as well as in the factor-B cell interaction the adherent cell determines the genetic restriction. The results of kinetic studies on T helper activities demonstrated that Thf120-OA provides an auxiliary activity at various moments during the differentiation of the human peripheral B cell into an antibody-secreting cell. The observed differences in the mode of action of Th cells and Th factor are discussed.     

Antigen-specific human T cell factors. II. T cell suppressor factor: biologic properties

The in vitro induction of an ovalbumin-specific human T cell suppressor factor is described (TsF120-OA). The antigen-specific suppressive component can be purified by affinity chromatography from supernatants derived from Marbrook-Diener type cultures of peripheral blood T cells stimulated with a high dose of ovalbumin. TsF120-OA suppresses the antigen-induced PFC formation of human blood B cells in vitro in an antigen-specific way. The target of TsF120-OA activity is shown to be the T helper cell. No genetic restriction in the action of the factor is observed.     

Specificity of the local transfer of cell-mediated immunity with dialyzable transfer factor

Local transfer of delayed cutaneous reactions to PPD, candidin, streptokinase-streptodornase, and leishmanin was used to study the specificity of dialyzable transfer factor (TF). Whereas conversions of reactions that were negative in the donors were achieved in experiments using the ubiquitous antigens, transfer of sensitivity to leishmanin (Montenegro reaction) was only accomplished by using TF of Montenegro-positive individuals. It was concluded that TF has specific properties and that ubiquitous antigens are not suitable for studying the specificity of the transfer, since the possibility of presensitization of the donors cannot be excluded in spite of negative cutaneous reaction.     

Gads regulates the expansion phase of CD8+ T cell-mediated immunity

The Gads adaptor protein is critical for TCR-mediated Ca(2+) mobilization. We investigated the effect of Gads deficiency on the proliferation of CD8(+) T cells following peptide stimulation and in the context of infection with an intracellular pathogen. We stimulated CD8(+) T cells from Gads(+/+) OT-I and Gads(-/-) OT-I mice with cognate Ag (SIINFEKL) or altered peptide ligand. In vitro experiments revealed that Gads was required for optimal proliferation of CD8(+) T cells. This defect was most evident at the early time points of proliferation and when low doses of Ag were used as stimuli. Cell cycle analysis demonstrated that Gads(-/-) CD8(+) T cells had impaired TCR-mediated exit from the G(0) phase of the cell cycle. Furthermore, Gads(-/-) CD8(+) T cells had delayed expression of c-myc and CD69 upon the stimulation with SIINFEKL. We then investigated how Gads deficiency would impact CD8(+) T cell-mediated immunity in the context of infection with an intracellular pathogen. At early time points, Gads(+/+) and Gads(-/-) CD8(+) T cells proliferated to a similar extent, despite the fact that expression of CD69 and CD25 was reduced in the absence of Gads. After 5 d postinfection, Gads was required to sustain the expansion phase of the immune response; the peak response of Gads(-/-) cells was significantly lower than for Gads(+/+) cells. However, Gads was not required for the differentiation of naive CD8(+) T cells into memory cells. We conclude that the primary function of Gads is to regulate the sensitivity of the TCR to Ag ligation.