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Dengue virus was the first microorganism that was shown to induce generation of antigen-specific suppressor T (TS) cells in mice. The cascade of the three generations of TS cells (TS1, TS2, TS3) and their secretary products, the suppressor factors (SF1, SF2), was delineated. The TS pathway was proposed to be protective through inhibition of the production of enhancing antibody, which may enhance the severity of dengue disease. The currently second most favoured mechanism of severe dengue disease is the 'cytokine tsunami'. During the last decade, suppressor/regulatory T cells have been studied in greater detail using modern techniques in various diseases, including viral infections. This brief review discusses the role of dengue-specific suppressor T cells in protection and/or induction of severe dengue disease in view of our current understanding of suppressor/regulatory T cells.

In dengue type 2 virus (DV)-induced suppressor T cell cascade TS1 cells secrete a suppressor cytokine (SF) which acts via syngeneic macrophages (M phi) to recruit TS2 cells. SF binds to both high and low affinity receptors (SF-R) on M phi. In the present study the fate of SF in M phi during transmission of suppressor signal is investigated. It was observed that SF bound to high affinity receptors internalized through receptor mediated endocytosis. This was inhibited by pretreatment of M phi with anti-SF-R-antiserum and didansylcadaverine, a potent inhibitor of endocytosis. Internalized SF was degraded by lysosomal activity as shown by inhibition of suppressor activity by pretreatment of M phi with monensin and NH4Cl. Degraded SF was transported to a site other than SF-R on M phi membrane for recruitment of TS2 cells. This was inhibited by anti-SF-antiserum. Transmission of suppressor signal is inhibited if M phi are treated first with H-2K-mAb and then with SF (shown earlier) but when M phi were treated first with SF and after 1 hr with H-2K-monoclonal antibody, the inhibition did not occur. As SF requires binding to H-2K and SF-R for mediation of suppression, the binding of H-2K occurred with degraded SF within the cell. Thus SF is internalized, degraded and binds to H-2K antigen before its recognition by native T cells.

Production of NO2- was maximum when peritoneal M phi was incubated with SF (40 mg) for 45 min. Pretreatment of M phi with anti-SF-antisera inhibited production of NO2- Pretreatment of M phi with NG monomethyl L-arginine (L-NMA) or arginase, an inhibitor of L-arginine dependent pathway, inhibited production of NO2- and transmission of suppressor signal in a dose-dependent manner. This indicates that NO and Ca2+ serve as intracellular signal in transmission of DV-induced suppressor signal.

Our earlier observations indicate that adoptive transfer of spleen cells obtained from dengue type 2 virus (DV)-primed mice suppressed DV antigen-specific antibody secretion as detected by Jerne PFC technique. Findings of this paper indicate that the suppression was produced by non-glass-adherent cells, macrophage-depleted (by carbonyl iron) cells and by T lymphocytes of the spleen but not by the glass-adherent cells and B lymphocytes. The activity of these cells is dependent upon production of prostaglandin as shown by abrogation of their suppressor activity by pre-treatment of cells by indomethacin or aspirin which are known to block synthesis of prostaglandins.

Dengue type 2 virus (DV) induces two generations of T suppressor cells (Ts1 and Ts2) in murine spleen. The Ts2 cells produce a soluble factor ( SF2 ) which suppresses DV-specific IgM antibody plaque-forming cells (PFC). In this study the target cell of SF2 was examined. The results show that SF2 requires a third generation of T cells (T3) in order to mediate suppression. The surface phenotype of this T3 cell is Ly 1+ and the suppression mediated is antigen specific.

The delayed-type hypersensitivity (DTH) to Japanese encephalitis virus (JEV) and the suppressor cells controlling it and the antibody-forming cells in inbred Swiss mice have been studied. JEV induces DTH, with a peak response at day 7 following infection which persists at low levels at least up to 119 days. Suppressor activity appeared on day 18. It was transferable by immune spleen cells. Treatment of spleen cells with anti-Thy-1.2 antisera and complement abrogated the suppressor activity. The homogenate of the spleen was equally effective in mediating suppression of DTH and the humoral response as measured by direct antibody plaque-forming cell (IgM-PFC) assay. The suppressor activity was antigen-specific both on DTH and T helper for antibody response as the immune responses against SRBC or Coxsackie B4 virus were not suppressed. The suppressor cells were sensitive to cyclophosphamide treatment when the drug was given 48 hr before their appearance. It is, therefore, concluded that in JEV infection of mice, antigen-specific suppressor T cells are generated, both for DTH and IgM antibody, which are cyclophosphamide-sensitive and mediate suppression through soluble product(s).

We have studied both the cells and the suppressor factors of the dengue virus-induced suppressor pathway for the presence of I-region gene products. The suppressor activity of Ts1, Ts2 and Ts3 cells was abrogated by treatment with anti I-Jk or I-Ak anti-sera and complement. Both the suppressor factors were absorbed out by anti I-Jk and I-Ak immunosorbent columns. The activity of the suppressor factor was abolished by treatment with anti I-Jk antisera. Macrophage presentation of suppressor factor to precursors of Ts2 cells was abolished by treatment with anti I-Ak antisera and complement but not by anti I-Jk antisera. Thus I-region gene products were found throughout the dengue virus induced suppressor pathway.

Adoptive transfer of spleen cells obtained from mice primed with Japanese encephalitis virus (JEV) suppressed IgM antibody plaque forming cells (PFC) against JEV in the spleen. Similar suppression of PFC was also shown in vitro by adding primed spleen cells to JEV-stimulated spleen cell cultures. The suppressor activity appeared sharply in the third week after priming and persisted up to 6 weeks. By using various cell separation procedures it was found that the suppressor activity resided in the T cell enriched fraction and not in B cells or macrophages. Sensitivity of the cells to treatment with anti-Thy 1.2 antiserum and complement confirmed that suppressor cells were T lymphocytes. It was noted that the suppression was effective against dengue virus antigen also. Our findings thus show generation of suppressor T lymphocytes in JEV-infected mice.

The immunosuppression in dengue type 2 virus (DV)-infected mice is mediated through secretion of a soluble suppressor factor (SF) produced by the T lymphocytes of the spleen. Activity of the suppressor cells was abrogated by pretreatment with indomethacin, indicating that suppression is mediated through prostaglandin (PG) or products of the PG synthetase pathway. The present study was undertaken to resolve the relationship between SF and PG. Treatment of mice with indomethacin did not affect production of SF. Therefore, PG is different from SF and the presence of PG is not essential for its production. Normal mouse spleen cells treated in vitro with SF produced PG which mediated suppression. Production of PG by these cells was abolished by pretreatment with anti-Thy 1.2 antibody and complement. It is concluded, therefore, that immunosuppression occurs in two phases: in the first step DV stimulates a subpopulation of T lymphocytes to produce SF, and in the second step SF induces a subpopulation of T lymphocytes to produce PG or products of the PG synthetase pathway which finally mediate suppression.

Dengue virus-induced suppressor pathway involves three sequential generations of suppressor T cells and their products. The present study was undertaken to determine the duration of suppressor activity of the different components of the suppressor pathway on adoptive transfer in recipient mice. It was observed that the suppressor activity of TS1 cells lasts for 21 days, of TS2 for 15 days, of SF for 7 to 10 days and of SF2 for 7 days. The duration of suppression appears to depend upon the length of suppressor pathway to be followed by each component.

Since Japanese encephalitis virus (JEV) induces the generation of suppressor T cells in mouse spleen which, through the production of a soluble suppressor factor (SF), suppress IgM plaque-forming cells (IgM-PFC) the present study was undertaken to further characterize these suppressor cells and the SF. The suppressor cells were Ly1-2+ and sensitive to hydrocortisone and a high dose of irradiation. SF was trypsin-sensitive, thermolabile and non-dialysable, and passed through a 450-nm filter. This SF was shown to be a low molecular weight substance, with an approximate MW of 18,000 daltons.

Japanese encephalitis virus (JEV) infection induces suppressor T-cells (Ts1) which suppress both the humoral (Ts-PFC) and cell mediated (Ts-DTH) immune response by producing soluble suppressor factors. This study shows that in the JEV model, both TS-PFC and Ts-DTH mediate suppression by recruiting a second subpopulation of suppressor T-cells, the Ts2-PFC and Ts2-DTH. The signal between Ts1 and Ts2 is transmitted by macrophages (M phi). The suppressor factors are adsorbed by peritoneal or splenic M phi. Both heat-killed and live M phi are capable of adsorbing suppressor factors but only live M phi are capable of presenting the signal to T-cells. Thus these are at least two generations of suppressor T-cells in the JEV-specific suppressor pathway and the presence of M phi is obligatory for transmission of the signal.

Intraperitoneal inoculation with Japanese encephalitis virus (JEV) induces the generation of T suppressor cells for delayed type hypersensitivity (Ts-DTH) in Swiss albino mice. The Ts-DTH are hydrocortisone resistant, partially sensitive to X-irradiation and the membrane phenotype of Ts cells is Ly I+. Ts-DTH suppression is mediated through the production of soluble suppressor factor (SF-DTH). SF-DTH is non-dialysable but passes through 450 nm filter and does not sediment after centrifugation at 100,000 g for 2 h; chromatography on Sephadex G-100 indicates an approximate molecular weight of 12,000.

Dengue type 2 virus (DV)-induced suppressor T cells (Ts1) produce a soluble suppressor factor (SF) which stimulates a subpopulation of T lymphocytes (Ts2) to produce prostaglandin which suppresses DV-specific IgM antibody plaque forming cells (PFC) in vivo and in vitro. The present study was undertaken to investigate the intermediary role of macrophages in transmission of signal from Ts1 to Ts2. The SF is adsorbed on live or heat-killed mouse peritoneal macrophages and transmits DV-specific PFC in spleen after i.p. or i.v. inoculation in DV-primed mice. The suppression is antigen-specific. SF or SF-adsorbed heat-killed macrophages failed to transmit suppression in silica-treated macrophage-depleted mice when injected i.p., while SF-adsorbed live macrophages could transmit suppression. In macrophage-depleted mice suppression could be transmitted by SF-adsorbed heat-killed macrophages on i.v. inoculation. It was therefore concluded that live macrophage-killed cells are also essential for transmission of suppressor signal by SF from Ts1 to Ts2 in vivo.

Dengue type 2 virus (DV) induces a suppressor pathway in mice which involves sequential participation of three subpopulations of T lymphocytes viz. Ts1, Ts2 and T3 cells. In the present study cyclophosphamide (CY) sensitivity of these cells have been investigated. The findings of the present study demonstrate that Ts1 and Ts2 cells are CY-insensitive while T3 cells are CY-sensitive. This supports our earlier conclusion that T3 may be the inducer cells of suppression.

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.

The generation of secondary suppressor T (Ts) cells has been studied during latent Japanese encephalitis virus (JEV) infection of mice. The mice infected with JEV 27 weeks earlier, on challenge with the homologous virus, showed accelerated generation of secondary Ts cells; these appeared on Day 6, with peak activity on Day 8, and lasted for 27 days. The secondary Ts cells were Thy1.2+, Ly1-2+, antigen-specific, and acted in a dose-dependent manner. The secondary Ts cells could also be generated by reactivation of the JEV in latently infected mice. The findings thus show the presence of memory suppressor T cells in mice latently infected with JEV that can be stimulated to produce secondary Ts cells by exogenous or endogenous virus challenge. This phenomenon could help to persistence of the virus.