Interference of the induction of suppressor cells by a streptococcal preparation, OK-432 through the blocking of suppressor inducer T-cells.

The effect of OK-432 on suppressor inducer T cells in the generation of suppressor cells was investigated to determine its mechanism of action as an immunopotentiating agent. Suppressor cell activities induced by sera from patients with advanced cancer (stage III, IV or recurrence) were found to be as high as those induced by Con-A. Suppressor activity induced by Con-A or serum from cancer patients resided in CD8+ T cells, although CD4+ T-cells were required for the induction of suppressor cells. Significant increases in the CD4+2H4+ T cell population after stimulation with either Con-A or sera from the advanced cancer patients were observed when compared with stimulation by normal serum. Stimulation with Con-A induced suppressor cells as well as a significant increase of CD4+2H4+ T-cells. The presence of OK-432 during the generation of suppressor cells, however, significantly reduced the suppressor activity and apparently blocked the increase of CD4+2H4+ T-cells. Thus, it is suggested that OK-432 may interfere with the induction of suppressor cells through the blocking of CD4+2H4+ suppressor inducer T-cells.     

Ontogeny of human suppressor inducer T cell subset

A T cell subset (T4+,2H4+) has recently been identified, which acts as an inducer of T8+ suppressor cells; however 2H4 molecules have not been detected in thymuses from normal individuals or on the surface of immature thymocyte clones. In the present study, T4+,2H4+ and T8+,2H4+ cells have been found in maternal and new-born blood samples. However, only a small percentage of 2H4+ cells were detected in human fetal thymus, bone marrow, liver and spleen, thus suggesting that T cells acquire these molecules only in circulation and at a late stage of maturation.     

Role of the 2H4 molecule in the activation of suppressor inducer function

The monoclonal antibody anti-2H4 recognizes a 220-kDa and 200-kDa glycoprotein and subdivides T4+ cells into distinct subpopulations: the T4+2H4+ inducer of suppression and the T4+2H4- inducer of help. The T4+2H4+ subset has been shown to play a crucial role in the activation of T8+ suppressor cells. In the present study, we attempted to determine whether the 2H4 molecule itself may be involved in initial triggering of suppressor inducer function. The results show that the addition of anti-2H4 antibody to a mixture of B and T cells resulted in a marked suppression of pokeweed mitogen-driven Ig synthesis. When anti-2H4 was added to B cell cultures containing T4+2H4+ or T4+2H4- cells but lacking T8 cells, no suppression was generated. In addition to the requirement for T8 cells, no suppression was generated if T4+2H4+ cells were absent. These results suggest that the anti-2H4 antibody contributes to the activation of the T4+2H4+ lymphocyte subset, which in turn induces T8 cells to suppress B cell Ig synthesis. Biochemical analysis of the T4+2H4+ subset of lymphocytes indicated that the in vitro addition of anti-2H4 antibody resulted in an increased expression of the 220-kDa and 200-kDa structure on T4 cells. We conclude that perturbation of the 2H4 molecule may potentiate the activation of the suppressor inducer subset and that the T200 molecule may be directly involved in suppressor inducer function.     

Characterization of suppressor T cells for antibody production by chicken spleen cells. I. Antigen-induced suppressor cells are CT8+, TcR1+ (gamma delta) T cells.

Suppressor activity of chicken T cells has been previously defined in an in vitro assay of the secondary response of spleen cells to sheep erythrocytes. We have used this assay to examine the phenotype of the T-cell subpopulation(s) that is responsible for this activity. The suppressive effect was alleviated by removal of the TcR1+ (gamma delta) and CD8+ subpopulations, but was unaffected by the removal of the TcR2+ (alpha beta) and CD4+ cells. The addition of a histamine type 2 (H2) receptor antagonist, cimetidine, enhanced the antibody response in the presence of the suppressor cells. The data indicate that one type of T cells with suppressor capability expresses TcR1 and the CD8 accessory molecule, and that these cells may be influenced via H2 receptors.     

Phenotypic characterization of two cell populations involved in the acquisition of suppressor activity by cultured spleen cells from Mycobacterium lepraemurium-infected mice.

The impairment of cellular immunity in mice infected with Mycobacterium lepraemurium was shown to correlate with the development of suppressor cells. We have previously reported that before suppressor activity is detectable in freshly harvested cell suspensions, suppressor cell precursors accumulate in the spleen of infected mice. Upon overnight culture in the presence of a regulatory cell subset, these precursor cells acquire the capacity to impair the concanavalin A (Con A)-induced proliferation of normal spleen cells. The purpose of this study was to determine the phenotype of the cells involved in this phenomenon. This was done by following the development of suppressor activity in spleen cell suspensions depleted of defined cell subsets of the adherent or the non-adherent cell fractions with selected MoAbs and immunomagnetic beads or by in vivo treatment. Our results indicate that the acquisition of suppressor activity requires the interaction of Ia+CD11b+Fc gamma R+IgG- asialo GM1- adherent cells with Thy1-CD4-CD8-IgG-Ia- asialo GM1-Fc gamma R+CD11b+ non-adherent cells. It is also shown that the development of suppressor activity is impaired by preventing cell-cell contact between these two cell subsets through coculture in 'Transwell chambers'. These observations support the conclusion that the in vitro acquisition of suppressor activity is a consequence of the maturation of suppressor cell precursors of the monocytic lineage induced by a receptor-ligand type interaction with a non-adherent cell subset that is clearly distinct from mature T, B and natural killer (NK) cells.     

Immunoregulatory CD4+ CD45R+ suppressor/inducer T lymphocyte subsets and impaired cell-mediated immunity in patients with Down''s syndrome.

The monoclonal antibodies 2H4 and 4B4 allow CD4+ and CD8+ T lymphocytes to be subdivided into CD45R+ and CDW29+ functional subpopulations. The CD4+ CD45R+ lymphocytes are designated as suppressor/inducer and CD4+ CDW29+ as helper/inducer subsets. Peripheral blood lymphocytes from 19 patients with Down's syndrome and 19 age- and sex-matched normal controls were analysed for the CD45R+ and CDW29+ subsets from the CD4+ and CD8+ T lymphocytes. The percentage of CD4+ CD45R+ cells (suppressor inducer) was markedly increased and of CD4+ CDW29+ cells (helper/inducer) decreased in all patients with Down's syndrome. In contract, the percentage of CD8+ CD45R+ and CD8+ CDW29+ subsets showed no major differences between patients with Down's syndrome and normal controls. Moreover, an alteration in the CD4+ and CD45R+ and CD4+ CDW29+ T cell subsets was accompanied by a markedly reduced proliferative response to phytohaemagglutinin and concanavalin A stimulation of the CD4+ T lymphocytes. Thus, a deficiency exists in patients with Down's syndrome in the CD4+ CDW29+ helper/inducer T cell subset which may contribute to their impaired cell-mediated immunity.     

Multiple myeloma: ecto-5'' nucleotidase deficiency of suppressor/cytotoxic (CD8) lymphocytes is a marker for the expansion of suppressor T cells.

In this study, 5'NT activity was investigated by radiochemical and cytochemical assays in T cell subpopulations of patients with multiple myeloma (MM). Cytotoxic/suppressor (CD8) and helper/inducer (CD4) lymphocytes were separated from peripheral blood by the panning technique, or sorted with a fluorescein activated cell sorter. 5'NT activity was significantly decreased in MM CD8 lymphocytes compared with the controls. By contrast, it was comparably low in CD4 subpopulations of normal controls and MM patients. The cytochemical assay indicated that a decreased number of 5'NT+ cells rather than a decreased activity per cell was responsible for 5'NT deficiency in MM CD8 lymphocytes. CD8 lymphocytes with the suppressor phenotype (OKMI+, granular cells) were significantly increased in MM. These data provide a phenotypic explanation for the enhanced suppressor activity displayed by T lymphocytes in MM. A significant correlation was found between the increase of suppressor cells and the decrease of 5'NT+ cells. 5'NT deficiency of CD8 lymphocytes can be a biochemical marker for the expansion of suppressor T cells.     

The cellular basis for the induction of antigen-specific T8 suppressor cells

The cellular basis for the generation of antigen-specific T8 suppressor cells with high doses of antigen has been studied. We separated the T4 subset of human T cells into T4+2H4+ and T4+2H4- subpopulations with a recently developed monoclonal anti-2H4 antibody. T8 cells could be consistently activated to suppress a primary anti-2,4-dinitrophenyl (DNP) antibody response in vitro with unfractionated T4 cells or with the T4+2H4+ subset but not the T4+2H4- subset. In contrast, the T4+2H4- subset functioned as the helper inducer for the anti-DNP antibody response. With keyhole limpet hemocyanin (KLH)-stimulated T4+2H4+ cells we could efficiently induce antigen-specific suppressor activity of fresh T8 cells. In contrast, the T4+2H4+ subset could not effect suppression in the absence of T8 cells. Our findings indicate that the T4+2H4+ subset of human T cells is the suppressor inducer of specific T8 cells in an antigen-specific DNP-KLH system.     

Establishment of stable CD8+ suppressor T cell clones and the analysis of their suppressive function.

Stable CD8+ suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4+ T cells by panning and cytotoxic treatment, and the resulting CD8+ T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation in interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8+ phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8+ Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8+ Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concanavalin A (ConA) and IL-2. All the CD8+ T cell clones established independently utilized the TcR V beta 8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8+ clones, which was blocked by anti-CD8 and anti-I-Ak monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8+ Ts clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4+ Th clones and the in vitro secondary antibody formation.     

Flow cytometric analysis of intraepithelial lymphocytes in the human nasal mucosa

Recently, much research has been carried out on phenotypes and the function of intraepithelial lymphocytes of the intestines. However, only few studies have been made of nasal intraepithelial lymphocytes (nIEL), and the results have been controversial. We examined the population of different subsets of the human nIEL by means of 2-color flow cytometry after culturing lymphocytes isolated from the inferior turbinates of hypertrophic rhinitis in incubation media with monoclonal antibody against CD3. As a result, CD8+ cells (suppressor/cytotoxic T cells) and double negative T cells (CD8- CD4- T cells) were the most predominant. The CD4+ cells (helper/inducer T cells) to CD8+ cells ratio was 0.6. Cytotoxic T cells predominated over suppressor T cells in CD8+ cells and helper T cells predominated over inducer T cells in CD4+ cells. Double positive T cells (CD8+CD4+ T cells) were nil and natural killer cells and CD8+ killer cells few. CD8+ cells and CD4+ cells were activated during cell culture. alpha beta receptor bearing T cells predominated over gamma delta receptor bearing T cells in CD8+ cells and CD4+ cells but gamma delta receptor bearing T cells predominated over alpha beta receptor bearing T cells in double negative T cells. To investigate the effect of cell culture on the population of each phenotype of nIEL we also examined peripheral blood lymphocytes by flow cytometry before and after culture, and found that activated T cells markedly increased and suppressor T cells, gamma delta bearing T cells and natural killer cells slightly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of suppressor cells by low molecular weight factors secreted by spleen cells of tumor-bearing mice: modulatory role of prostaglandins

We have shown previously that the spleens of mice bearing large M-1 fibrosarcomas contain inducer cells which secrete dialysable factors which activate suppressor T cells from unprimed, normal precursor spleen cells. Once activated, the suppressor cells inhibit the in vitro antibody synthesis of cocultured syngeneic splenocytes stimulated by T cell dependent antigens. In this paper we have examined the possibility that prostaglandins are involved in the activation process. Inducer and precursor cells were cultured in Marbrook vessels in chambers separated by dialysis membranes. Using this procedure, suppressor cells were activated following 12 h of culture but were not detectable after 6 h. The cyclooxygenase inhibitors, indomethacin, acetyl salicylic acid (ASA), and ibuprofen all prevented the activation of suppressor cells in a dose dependent manner. Prostaglandin (PG) E1, but not PGF2a or PGD2, restored the activation of suppressor cells in cultures containing the cyclooxygenase inhibitors. Restoration of suppressor cell activation was seen with 1 X 10(-7) M PGE1 but no activation of suppressor cells was seen in control cultures containing up to 1 X 10(-5) M PGE1. In addition, cultured spleen cells from tumor-bearing mice did not secrete higher quantities of PGE than did cells from age and sex matched normal mice. These data suggest that PGE has a modulatory rather than a direct role in the activation of suppressor cells by inducer factors from tumor-activated inducer cells.     

Post-thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti-H/K ATPase-reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ-specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase alpha chain, but only marginally to the beta chain. Two H/K ATPase-reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+, TCR alpha/beta-, and I-Ad restricted, and recognize distinct peptides from the H/K ATPase alpha chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co-transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2-day-old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well-characterized lines of autoantigen-specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.     

Phenotypic characteristics of cells involved in induced suppression to murine experimental autoimmune thyroiditis

Suppression of induced experimental autoimmune thyroiditis can be consistently transferred with spleen cells to syngeneic recipients, provided they are first treated with 200 rads irradiation. Treatment with anti-Thy-1 in vivo immediately prior to transfer abrogates the suppression, while depleting B cells has no effect. The in situ induced tolerance can be prevented by treatment with monoclonal antibodies to the Ly-1 or L3T4 molecules either prior to or post tolerization. Anti-Ly-2 treatment has no effect. In the transfer, again treatment of donors with anti-L3T4 prior to transfer prevents the demonstration of suppression in the recipients, while anti-Ly-2 does not affect suppression. These data suggest that the suppression is being mediated either by a CD4+ T suppressor cell or a CD4+ suppressor inducer cell. Preliminary experiments do not support the possibility of a CD8+ suppressor cell being activated in the recipient.     

Dengue virus-induced thymus-derived suppressor cells in the spleen of mice.

Adoptive transfer of spleen cells obtained from mice given three weekly i.p. doses of dengue type 2 virus (DV) suppressed DV antigen-specific antibody secretion as detected by the Jerne plaque technique. This suppression was produced by non-glass-adherent cells but not by glass-adherent cells. Immune spleen cells depleted of macrophages by carbonyl iron treatment had higher suppressor activity. Immune spleen cell homogenate could transfer the activity equally well. The immune spleen cells were separated into T and B lymphocytes by a nylon wool column. B lymphocytes had no suppressor activity; almost all the suppressor activity was present in T lymphocytes. Thus, macrophages and B lymphocytes had no suppressor activity; it was mediated by T lymphocytes through soluble factors.     

Phenotypic identification of suppressor-effector, suppressor-amplifier and suppressor-inducer T cells of B cell differentiation in man

Using monoclonal anti-Leu8 antibody to isolate subpopulations of human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T cells, we have investigated the role of these subpopulations in the regulation of B cell differentiation in the human autologous mixed leukocyte reaction (AMLR). Whereas AMLR-activated CD8+,Leu8- cells were capable of suppressing fresh AMLR cultures in the absence of fresh CD8+ cells, CD8+,Leu8+ cells suppressed only those cultures containing fresh CD8+ cells. On the other hand, CD8+,Leu8- cells became suppressor cells only when cultured in the presence of CD8+,Leu8+ cells. Finally, the development of CD8+ suppressor cells was dependent on the presence of CD4+,Leu8+ cells; CD4+,Leu8- cells were incapable of acting as suppressor-inducer cells, but have been shown previously to mediate T cell help for B cell differentiation. Thus, at least 3 phenotypically distinct subsets of T cells interact sequentially to generate suppression of B cell differentiation induced in the AMLR: CD4+,Leu8+ suppressor/inducer cells, CD8+,Leu8+ suppressor-amplifier cells and CD8+,Leu8- suppressor-effector cells.     

Human suppressor T cells induced in vitro with an autologous renal allograft-derived T cell line. I. Suppressor cell induction, function, and specificity

The functional characteristics of T suppressor (Ts) cells generated from the peripheral blood lymphocytes (PBL) of a kidney transplant recipient who had excellent graft function for 1 year were examined. Ts cells were induced by co-culture of PBL with an autologous alloreactive cytotoxic T lymphocyte (CTL) line (EE-1) previously grown from a routine renal allograft biopsy of this patient performed 10 days posttransplant. The EE-1 line included CD3+ T cells of CD8+ and CD4+ phenotypes with cytotoxic specificity for disparate class 1 (HLA-B8) and class II (HLA-DR1 and 3) antigens of the kidney donor (JC). The EE-1 induced Ts cell lines (designated TsEE) were found to significantly suppress (50%-95%) autologous fresh responder EE-PBL stimulation by donor EBV-transformed cells (JC-EBV) in mixed lymphocyte reaction (MLR) assay. TsEE cells were CD3+ (98%) and predominantly CD8+ (68-80%), showed no cytotoxic activity, and were suppressive only at the early phase of MLR stimulation. In three-party cell test MLR assays, TsEE-mediated suppression appeared restricted to responder cells sharing HLA-B7 with the suppressor line, and was not abrogated by the addition of exogenous interleukin-2 (IL-2). TsEE cells also showed restricted suppression of CTL generation but not mature CTL activity. The restricted suppressor activity of TsEE lines was dependent upon their induction and restimulation with the autologous EE-1 line.     

Functional analysis of the defective T cell regulation of the antigen-specific PFC response in SLE patients: differentiation of suppressor precursor cells to suppressor effector cells.

The investigation described here is concerned with the T cell regulation of the antigen-specific antibody response which has been studied in patients suffering from systemic lupus erythematosus (SLE). Apart from the fact that T helper cell activity was found to be less efficient, it appeared that the peripheral blood leucocytes (PBL) of patients in an active stage of the disease did not contain the suppressor precursor cells, which functions as the target cell for the inductive signal of T mu+ suppressor inducer cells. The absence of the suppressor precursor cells in SLE patients coincided with the absence of T gamma+ suppressor effector cells. Characterization of the (post-thymic) precursor cells (derived from normal donors) with the aid of monoclonal antibodies of the OKT series and several other markers pointed out that this population contains OKT4+ as well as OKT8+ cells. Further experiments demonstrated that the cells are capable of rosetting with autologous erythrocytes, and do not bear Fc receptors for IgM or IgG. Considering the various findings as a whole the conclusion is warranted that the post-thymic suppressor precursor T cell can differentiate into a suppressor effector cell only after interaction with T suppressor inducer cells.     

Neonatal thymectomy identifies two major pools of sessile and recirculating peripheral T cells which appear to be under separate homeostatic control.

In this study the role of the thymus in the development of sessile T cell populations resident in spleen and lymph nodes (LN) was contrasted with the development of recirculating T cell populations trafficking between blood and lymph. Extensive analysis of the composition and the rate of growth of the secondary lymphoid tissues and recirculating lymphocyte pool coupled with neonatal thymectomy revealed that the sessile and recirculating T cell populations showed different degrees of thymic dependency and increased in size at different rates, suggesting these two populations might be under separate homeostatic control. Neonatal thymectomy also resulted in a much greater depletion of CD8+ and gammadelta TCR+ T cell subsets compared with CD4+ T cells in the sessile and recirculating T cell pools, and greatly reduced the number of T cells homing to peripheral lymph nodes compared with those homing to the gut.     

Predominance of T cells that express CD45R in the CD4+ helper/inducer lymphocyte subset of neonates

Neonates have an increased risk of severe infections. For several in vitro and in vivo immune responses, neonates have been shown to have significant differences when compared to normal adults. To indirectly study immune cellular defects, we compared cell surface markers on cord blood lymphocytes (CBL) from 58 term infants to peripheral blood lymphocytes (PBL) from 17 healthy adults using flow cytometry with standard as well as newly defined monoclonal antibodies (Mab) that distinguish regulatory T cells. CBL had significantly smaller percentages of lymphocytes that express the CD2 and CD8 markers (total T cells, and suppressor/cytotoxic T cells, respectively), although absolute numbers of CD2+ and CD8+ cells were comparable in neonates and adults. CBL and PBL were similar in terms of the percentage of CD4+ cells (helper/inducer T cells), although the absolute numbers of CD4+ cells were higher in CBL than in PBL. The CD4+ population was subdivided into cells bearing the virgin and memory T cell phenotypes using anti-2H4 and anti-4B4 Mab and dual parameter analysis with anti-CD4. Neonates were deficient in the percentage of CD4+, 4B4+ (3.8 +/- 2.8 vs 13.4 +/- 7.5, P less than 0.001), but equivalent to adults in the percentage of CD4+, 2H4+ T cells (21.4 +/- 9.8 vs 18.8 +/- 12.8). In absolute numbers, neonates had fewer CD4+, 4B4+ cells (178 +/- 173 vs 344 +/- 152 cells/microliters, P less than 0.001), but more CD4+,2H4+ cells (978 +/- 572 vs 542 +/- 518 cells/microliters, P less than 0.01) than adults. The predominance of 2H4+ virgin T cells in the CD4 population whose function is associated with that of the induction of suppression rather than the up-regulation of immune responses may contribute to the observed susceptibility of neonates to infection.     

Generation of CD8+ suppressor T cells by protoscoleces of Echinococcus multilocularis in vitro.

Addition of protoscoleces (PSC) of Echinococcus multiocularis suppressed proliferative responses in spleen cells stimulated with concanavalin A (Con A) on day 3 of culture. The suppression was not observed in the spleen cell population depleted of CD8+ cells but observed in the population depleted of CD4+ cells, suggesting the involvement of the CD8+ T cells in the apparent suppression. Indeed, flow cytometry analysis revealed that the proportion of CD8+ cells markedly increased in the Con A cultures containing PSC. Furthermore, when spleen cells were co-cultured with PSC alone, marked increase in the proportion of CD8+ cells as well as B220+ cells was observed. Addition of these increasing CD8+ cells suppressed the proliferative responses of fresh spleen cells stimulated with Con A. These findings suggest that the low responsiveness of spleen cells to Con A on day 3 of culture in the presence of PSC is attributable to an active suppressor mechanism including CD8+ T-suppressor cells generated by stimulation with PSC.