Effect of Polysaccharides from Ganoderma applanatum on Immune Responses

Transcription

1 Microbiol. Immunol. Vol. 23 (6), , 1979 Effect of Polysaccharides from Ganoderma applanatum on Immune Responses I. Enhancing Effect on the Induction of Delayed Hypersensitivity in Mice Shoichi NAKASHIMA, * Yukio UMEDA, and Taira KANADA * Department of Biochemistry, Drug Research Institute, Toyama Medical and Pharmaceutical University, Toyama (Received for publication, July 21, 1978) Abstract Prior intraperitoneal (i.p.) or oral administration of the polysaccharide preparation from a kind of mushroom, Ganoderma applanaturn (Pers.) Pat. of Basidiomycetes, exerted an enhancing effect on the induction of delayed hypersensitivity (DH) to protein antigen as measured by the footpad reaction (FPR), and expanded the size of T cell memory for the IgG antibody response. One of the active principles was partially purified and found to be associated with a polysaccharide-rich fraction. The induction of DH was enhanced by treatment with an appropriate dose of the mushroom extract, whereas increasing the dose resulted in almost complete loss of the enhancing activity. The mechanism for the enhancing effect of the mushroom extract on the induction of DH was explored by the adoptive cell transfer technique. Although an i.p. injection of methylated bacterial a-amylase (M-BaA) in incomplete Freund's adjuvant (IFA) has been found to generate in the spleen the antigen-specific suppressor T cells capable of inhibiting the induction of DH 5 days after immunization, the same treatment of mice given prior injections of the mushroom extract did not raise the suppressor cell activity, but transfer of these spleen cells (6 ~ 107) into syngeneic recipient mice which had been primed with a subcutaneous (s.c.) injection of M-BaA in complete Freund's adjuvant (CFA) resulted in substantial amplification of the expression of DH. The absence of effector T cells for DH in the transferred spleen cells was confirmed by the failure to transfer DH into cyclophosphamide (CY)-treated mice with the amplifying cells. The amplifying activity was antigen-nonspecific and mediated by cells sensitive to treatment with anti-ľ antiserum plus complement. Therefore, the nonspecific enhancing effect of the mushroom extract could not be explained by the possibility that pretreatment with the extract eliminated the antigen-specific suppressor T cells. Other adoptive cell transfer experiments revealed that nylon wool-passed cells from mice unprimed Abbreviations: DH, delayed hypersensitivity; FPR, footpad reaction; s.c., subcutaneous; i.p., intraperitoneal; CFA, complete Freund's adjuvant; IFA, incomplete Freund's adjuvant; CY, cyclophosphamide; BaA, bacterial a-amylase; M-BaA, methylated BaA; AP-M-BaA, alum-precipitated M-BaA; BGG, bovine ć-globulin; T cells, thymus-derived cells; B cells, bone marrow-derived cells. 501

2 502 S. NAKASHIMA ET AL but treated with the mushroom extract were able to exert an enhancing activity on the expression of effector T cells in DH. The results indicate that the treatment with an appropriate dose of the extract enhances the induction of DH by activation of the nonspecific amplifier T cells. We have shown that the extensively modified bacterial a-amylase (BaA) derivative, which was unable to react with anti-baa antibody nor able to induce a humoral anti-baa antibody response (18), retained the ability to stimulate native BaAspecific T cells involved in the helper function for the IgG antibody response (18, 20), delayed hypersensitivity (DH) which was not complicated by the Arthus reaction (19), and suppression of the development of DH (20). Therefore, we attempted in the present study to explore the effect of Ganoderma applanatum extract on the T cell functions induced by methylated BaA (M-BaA). Administration of an appropriate dose of the mushroom extract resulted in the expansion of the size of T cell memory and substantial amplification of the DH response in mice primed with a subcutaneous (s.c.) injection of a suboptimal dose of M-BaA in incomplete Freund's adjuvant (IFA). Since it is generally accepted that suppressor T cells play a critical role in the regulation of the DH response, the enhancing effect of the mushroom extract on the induction of DH might be due to the preferential elimination of suppressor T cells capable of inhibiting the induction and expression of DH. In contrast to the capacity of spleen cells from mice primed with an intraperitoneal (i.p.) injection of M-BaA in IFA to suppress antigen-specifically the expression of DH (20), the spleen cells from mice treated in the same manner nonspecifically amplified the expression of DH in the primed recipients if the donors had been given prior injections of the mushroom extract. Further analysis provided evidence for the participation of antigen-nonspecific amplifier T cells in the enhancing effect of the mushroom extract on the induction of DH. MATERIALS AND METHODS Animals. Female outbred ddy/s mice or male and female C3H/He mice at an age of 6 to 8 weeks, originally obtained from the Shizuoka Agricultural Cooperative Association for Laboratory Animals and raised in our department, were used in these experiments. Materials. Crystalline bacterial a-amylase (BaA) [EC a-amylase, Bacillus subtilis] was obtained from the Seikagaku Kogyo Co. and bovine y-globulin (BGG) from the Armour Pharmaceutical Co. DEAE-cellulose powder was from the Brown Co. and cyclophosphamide (CY) from Shionogi Co., Ltd. The mushroom G. applanatum "Baikisei" was a commercial product which was kindly supplied by the Ohminedo Chemical Co. Preparation of methylated BaA (M-BaA), M-BGG, and alum-precipitated M-BaA (AP-M-BaA). Methylation of BaA and BGG, and preparation of AP-M-BaA were carried out as reported previously (18, 19). Extraction of G. applanatum and partial purification of the active principles. The

3 REGULATION OF DI-I 503 Fig. 1. Chromatography of Fr-1 on a DEAE-cellulose column. Fr-1 (10 mg) was applied to a DEAE-cellulose column (0.9 x 18 cm) and eluted stepwise as indicated in the figure. Three ml per tube of eluate was collected. These three peaks were designated in order of elution as Fr-1-A, Fr-1-B, and Fr-1-C. Concentrations of polysaccharide and protein were determined by the methods of Dubois et al (7) ( œ) and Lowry et al (11) ( x ), respectively. powdered mushroom (100 g) was extracted in 400 ml of boiling water for 24 hr, and the supernatant solution was lyophylized and designated as the crude extract (yield: 900 mg). To a solution of 900 mg of the crude extract in 100 ml of water, 100 ml of EtOH was added and the resulting precipitate was dissolved in water, dialyzed against water, lyophylized, and used as Fr-1 (160 mg). A further 200 ml of EtOH was added to the supernatant solution to give Fr-2 (260 mg). A solution of 10 mg of Fr-1 in 1 ml of water was applied to a column of DEAE-cellulose (0.9 ~ 18 cm) and eluted stepwise as shown in Fig. 1. These three peaks were designated in order of elution as Fr- 1 -A, Fr- 1 -B, and Fr- 1 -C. The recovery of polysaccharides in each fraction was 24, 70, and 6%, as judged by the phenol-h2so4 method (7). The protein concentration was determined by the method of Lowry et al (11). Immunization with M-BaA. We have shown previously that an s.c. injection of 50,ug of M-BaA in CFA was most effective in inducing the delayed footpad reaction (FPR) among various priming doses (19) and that specific suppressor T cells capable of inhibiting the development of DH could be generated in the spleen 5 days after immunization by an i.p. injection of 20 to 200,ug of M-BaA in IFA (20). Therefore, for the induction of DH, mice were given an s.c. injection of a suboptimal dose (20 or 30,ug) of M-BaA in IFA or CFA. To generate suppressor T cells, mice received an i.p. injection of 20 tg of M-BaA in IFA. Estimation of anti-baa IgG antibody titers and elicitation of the footpad reaction (FPR). These procedures were identical to those described previously (18, 19).

4 504 S. NAKASHIMA ET AL Preparation of rabbit anti-mouse brain antiserum and treatment of spleen cells with the antiserum plus complement. These methods have been reported previously (20). Fractionation of spleen cells. Separation of T and B cells from spleen cells was carried out according to the method of Julius et al (10) using a nylon wool column as reported previously (20). After removal of adherent cells with a glass wool column, the effluent cells were applied to a nylon wool column. Nylon wool-passed cells and adherent cells were used as T cell-rich and B cell-rich fractions, respectively. RESULTS Effect of Repeated i.p. Injections of the Crude Extract on the Development of Delayed Hypersensitivity (DH) Effects of the mushroom extract on the induction of DH were examined in ddy/s mice primed with 20,ug of M-BaA in IFA (Fig. 2a) or in CFA (Fig. 2b). As shown in Fig. 2a, only a slight swelling, which was assumed to be a Jones-Mote reaction (2, 21, 23), was noted 48 hr after elicitation in the M-BaA-IFA-primed positive control group of mice. In mice pretreated with the crude extract, however, strong DH was induced by priming with M-BaA in IFA and the level of FPR was comparable to the reaction in M-BaA-CFA primed mice (Fig. 2b). Delayed FPR was most makedly enhanced by pretreatment with 30 mg of the crude extract in five doses and mice given 60 mg exhibited diminished enhancement. Figure 2b shows the effect on the induction of DH by priming with M-BaA in CFA. No enhancing (a) (b) Fig. 2. Enhancing effect of the crude extract on the development of DH. Female ddy/s mice were given five i.p. injections of various doses (10 mg ( ), 30 mg ( ), 60 mg ( ~ ) in five doses) of the crude extract on days -24, -22, -20, -18, and -16. They were immunized by an s.c. injection of 20,ug of M-BaA in IFA (a) or in CFA (b) on day -15. Mice treated with saline and given H20-IFA served as the negative control mice ( ). Mice treated with saline and given M-BaA-IFA in (a) ( œ) or M-BaA-CFA in (b) ( œ) served as the positive control mice. FPR was elicited by 40 pg of AP-M-BaA on day 0. The values are the means of those from six mice with standard errors.

5 REGULATION OF DH 505 effect of the crude extract was observed in these mice, but pretreatment with 60 mg of the extract resulted in a reduction of the level of FPR rather than enhancement. Effect of the Crude Extract on the Size of T Cell Memory for IgG Antibody Responses We have reported (20) that M-BaA lost the ability to induce humoral anti-baa response, but retained the ability to establish T cell memory for the anti-baa IgG antibody reponse. Mice primed with M-BaA developed enhanced anti-baa IgG antibody responses to a subsequent challenge with native BaA. The difference in the antibody titers between primed and unprimed mice was regarded as reflecting the size of the T cell memory. By the use of this experimental system, the effect of the extract on memory induction was examined. Mice were pretreated with the optimal dose (30 mg in five doses) of the crude extract under the same conditions as shown in Fig.2, given an s.c. injection of 20,ug of M-BaA in IFA, and challenged by an i.p. injection of 100 jug of BaA in IFA. As seen in Fig. 3, the size of memory which was acquired by priming with M-BaA was expanded by pretreatment with the crude extract. Since no enhancing effect of the extract on antibody formation was observed in unprimed mice, it was suggested that the augmented antibody formation in M-BaA-primed mice reflected the expansion of T cell memory by treatment with the extract. However, the precise mechanism for the memory expansion is not known at present because another possibility [activation of macrophages by the mushroom extract (13)] should also be taken into consideration. Fig. 3. Expansion of the size of T cell memory by i.p. injections of the crude extract. Female ddy/s mice were given five i.p. injections of the extract in 0.2 ml of saline (30 mg in five doses) in the same way as shown in Fig. 2 and immunized by an s.c. injection of 20,ug of M-BaA in IFA on day -15 ( ). Mice given the extract and primed with H2O-IFA ( ) were prepared to examine the direct effect of treatment with the extract on the antibody response. Mice given saline without the extract and primed with H20-IFA ( ) or mice given saline and primed with M-BaA in IFA ( œ), served as the negative and the positive control mice, respectively. All mice were challenged by an i.p. injection of 100,ug of BaA in IFA on day 0. Anti-BaA IgG antibody titers were followed by the enzymatic procedure (18). The values are the means of those from six mice with standard errors.

6 506 S. NAKASHIMA ET AL Fig. 4. Effect of EtOH-precipitated fractions (Fr-1, Fr-2) on the development of DH. Precipitates of the crude extract formed at 50% EtOH and from 50 to 75% EtOH were used as Fr-1 and Fr-2, respectively. (a) : Mice were given i.p. injections of various doses of Fr-1 or Fr-2 (1 to 10 mg in five doses) on days -24, -22, -20, -18, and -16. They were sensitized by an s.c. injection of 20,ug of M-BaA in IFA on day -15. The negative control mice were given an s.c. injection of H20-IFA. FPR was elicited on day 0. (b) : Various doses of Fr-1 or Fr-2 (1 to 20 mg in five doses) were given orally by gastric tube as described in (a). The mice were sensitized on day -15. As a negative control, mice were given an s.c. injection of H20-IFA. FPR was elicited by 40 jug of AP-M-BaA on day 0. Values are the means of those from six mice with standard errors. Effect of Administration Route of EtOH-Precipitated Fractions (Fr-1, Fr-2) on the Induction of DH Mice were pretreated with five i.p. injections of various doses (1 to 10 mg in five doses) of Fr-1 or Fr-2 (Fig. 4a), which were prepared from the crude extract by EtOH-precipitation. They were primed with M-BaA in IFA and FPR was elicited by AP-M-BaA in the same way as shown in Fig. 2. As seen in Fig. 4a, mice given an optimal dose of Fr-1 or Fr-2 (1 mg in five doses) exhibited enhaced FPR when compared with FPR in the positive control mice. On the other hand, in mice pretreated with an excessive dose (10 mg of Fr-1, 5 mg of Fr-2 in five doses) of each fraction, the intensity of FPR remained at a similar level as FPR in the positive control mice. It has been reported by Tsukagoshi and Ohashi (25) that oral as well as i.p. administration of a protein-bound polysaccharide (PS-K) from Coriolus versicolor (Fr.) Quel., was effective in inhibiting the growth of transplantable tumors. Therefore, the effect of oral administration of Fr-1 or Fr-2 by gastric tube on the development

7 REGULATION OF DH 507 of DH was tested under the same conditions as described in Fig. 2, except for the administration dose. The results are shown in Fig. 4b. Oral administration of 10 mg of these fractions in five doses was as effective as i.p. administration in enhancing the induction of DH. The dose required for enhancement was about ten times larger than the effective dose by i.p. injections. At higher doses (20 mg) of Fr-1, the enhancing effect was lost. Partial Purification of Active Principles Fr-1 was applied to a DEAE-cellulose column and eluted stepwise as shown in Fig. 1 to give Fr-l-A, Fr-1-B, and Fr-1-C. Fr-1-A showed the highest content of polysaccharide among these fractions. Although the results are not shown, administration of various doses (150,ug to 4 mg) of Fr- 1 -A or B was effective in enhancing the induction of DH and Fr- 1 -C was inactive for enhancement at all doses tested. Since the enhancing activity of Fr- 1 -A and B was not altered by treatment with 0.05 N NaOH which was used for elution of Fr- 1-C from the column, failure to enhance DH by Fr- 1-C did not appear to be due to inactivation of this fraction during the elution. The reason for the decline of the enhancing effect at an excessive dose of Fr- 1-A and B within the dose range tested was not clear. Participation of the Amplifier Cells in the Enhancing Eject of Fr-1 It has become increasingly evident that most immune responses are balanced with respect to positive and negative regulation. Therefore, the enhancing effect of the mushroom extract might be accounted for by preferential elimination of suppressor T cells. We have reported (20) that spleen T cells from mice given an i.p. injection of M-BaA in IFA, suppressed antigen-specifically the development of DH in syngeneic recipient mice. Employing this system, the effect of the mushroom extract (Fr-1) on the generation of suppressor T cells was tested. Donor mice of the C3H/He strain were given five i.p. injections of Fr-1 (1 mg in five doses) on days -15, -13, -11, -9, and -7. Another group of mice given five injections of saline served as the control donor mice. They were primed by an i.p. injection of 20,ug of M-BaA in IFA on day -6. To examine the effect of CFApriming on the generation of suppressor T cells, one group was immunized by an i.p. injection of 20,ug of M-BaA in CFA on day -6. The spleen cell suspension was prepared on day 0 and 6 x 107 viable cells (or 5 x 107 from M-BaA-CFA-primed mice) were transferred into syngeneic mice which had been immunized with an s.c. injection of 30,ug of M-BaA in CFA 15 days previously. FPR was elicited by AP-M-BaA one day after cell transfer. These results are shown in Fig. 5a. The expression of DH was suppressed by cells from mice given an i.p. injection of M-BaA in IFA as reported previously (20). In contrast, spleen cells from mice treated with Fr-1 together with M-BaA in IFA or from mice primed with M-BaA-CFA, were not only unable to inhibit the expression of DH, but also substantially amplified the level of EPR in the recipient mice. The amplifying effect of the spleen cells was not adequately explained by the inhibiting effect of the mushroom extract on the generation of suppressor T cells. One possibility is that this amplification is mediated by the effector

8 508 S. NAKASHIMA ET AL (a) (b) Fig. 5. Effect of Fr-1 on the generation of suppressor T cells capable of inhibiting DH (a) and absence of effector cells capable of expressing DH in the amplifier cell population (b). (a) : Donor mice of C3H/He strain were treated with Fr-1 and primed with an i.p. injection of M-BaA in IFA ( ). The other groups of donor mice were primed by an i.p. injection of M-BaA in IFA ( ), M-BaA in CFA ( ~ ), or H20-IFA (, œ) without giving Fr-1. Syngeneic recipients were given an s.c. injection of M-BaA in CFA on day -15 and an i.v. injection of 5-6 ~ 107 spleen cells from these donor mice on day 0. The recipient mice, which had been primed with H20-CFA and received spleen cells from mice given H20- IFA, served as the negative controls ( ). Mice, which had been primed with M-BaA in CFA and received spleen cells from H20-IFA-primed mice, served as the positive controls ( œ). FPR was elicited by 50,ƒÊg of AP-M-BaA one day after cell transfer. (b) : The same spleen cells of donor mice were prepared as described in Fig. 5a and transferred into syngeneic recipients which had been given an i.p. injection of 4 mg of CY on day are identical to those shown in Fig. 5a. Values are the means of those from five mice with standard errors. T cells which might be induced by combined treatment with Fr-1 and M-BaA. To test the possibility, each spleen cell was transferred into syngeneic CY-treated mice, since it was shown previously (20) that DH could be transferred with effector cells into CY-treated recipients. They were subsequently challenged with AP-M-BaA in the footpad. As shown in Fig. 5b, no FPR occurred, indicating that there was no effector T cells in the amplifying cells. The comparable enhancing effect was observed in spleen cells from mice primed with an i.p. injection of M-BaA in CFA. Specificity and Characterization of the Amplifier Cells To examine the specificity of the amplifying activity, spleen cells from mice treated with Fr-1 and with M-BaA-IFA were transferred into syngeneic mice which

9 REGULATION OF DH 509 Fig. 6. Specificity (a) and characterization (b) of the amplifier cells. (a) : The donor mice of C3H/He strain were given five i.p. injections of Fr-1 (1 mg in five doses) and primed by an i.p. injection of M-BaA in IFA as shown in Fig. 5a. Spleen cells (6 ~ 107) from these donor mice were transferred into syngeneic recipient mice which had been primed by an s.c. injection of 50,ug of M-BGG on day -15. The appropriate negative and positive control mice were also prepared. FPR was elicited by 50,ug of AP-M-BGG one day after transfer. (b) : Donor and recipient mice were prepared in the same way as shown in Fig. 5a. Treatment of amplifier cells with anti-0 antiserum plus complement was carried out as reported previously (20). The treated and untreated spleen cells (5 ~ 107) were transferred into syngeneic M-BaA-CFA-primed mice on day 0 and FPR elicited by AP-M- BaA one day after cell transfer. The values are the means of those from five mice with standard errors. *, Normal rabbit serum. Viability, 78.6%. **, This treatment killed 43% of viable cells. had been primed with 50,ug of unrelated antigen, methylated bovine y-globulin (M-BGG), and FPR was elicited one day after cell transfer. FPR measured 48 hr after elicitation is shown in Fig. 6. The amplifying cells enhanced the expression of FPR to M-BGG (Fig. 6a) as well as to M-BaA (Fig. 5a). To characterize the amplifier cells, the effect on activity of treatment of these cells with anti-0 antiserum plus complement was tested. As seen in Fig. 6b, T celldepletion abolished the ability to enhance the expression of DH. Activity of the Nylon Wool-Passed Cells The results shown in Fig. 6 suggest that nonspecific amplifying cells should be generated by administration of Fr-1 without i.p. priming with M-BaA in IFA. To test this possibility, double cell transfer experiments were carried out as follows. Donor I mice were primed with an s.c. injection of M-BaA in IFA or CFA on day of Fr-1 (1 mg in five doses) on days -15, -13, -11, -9, and -7, and served as amplifying cell donors. The spleen cell suspension was prepared on day 0. The effector cells (2 x 107) and the cells from donor II mice (3 x 107) were cotransferred into the syngeneic recipient mice which had been given an i.p. injection of 4 mg of

10 510 S. NAKASHIMA ET AL Fig.7.Generation of amplifying cells by treatment with Fr-1 (a) and activity of nylon woolpassed cells (b) in the double cell transfer system. (a) : Effector cells from donor I mice primed with M-BaA in IFA or in CFA (2 ~ 107) and spleen cells (3 ~ 107) from donor II mice given an optimal dose of Fr-1 were cotransferred into syngeneic CY-treated mice. FPR was elicited one day after cell transfer. Appropriate control experiments were also performed. (b) : Amplifying cells were passed through glass wool and nylon wool columns. Nylon wooladherent cells were recovered by gentle agitation (20). The T cell-rich or B cell-rich fraction (4 ~ 107) was cotransferred with effector cells from M-BaA-CFA-primed mice into CYtreated mice and FPR was elicited one day after cell transfer. Values are the means from five mice with standard errors. *, Treatment with anti-0 antiserum plus complement killed 70% of the viable T cell-rich fraction. **, Treatment with anti-0 antiserum plus complement killed 10% of the viable B cell-rich fraction as judged by the dye exclusion test. CY on day -1. FPR was elicited one day after cell transfer. The results are shown in Fig. 7a. As expected, treatment with Fr-1 itself was effective in generating the amplifying cells and the activity was noted irrespective of whether effector cells were obtained from M-BaA-IFA-primed or M-BaA-CFA-primed mice. The amplifying activity persisted for at least 15 days after the last injection of Fr-1 (data not shown). Further, amplifying spleen cells were purified by the glass wool and the nylon wool column. The activity of purified cells was tested in the double cell transfer system. As seen in Fig. 7b, the amplifying activity was associated with the glass wool-passed and nylon wool-passed cell fractions. The B cell-rich fraction had no enhancing activity. DISCUSSION Mice pretreated with an optimal dose of the polysaccharide preparation from G. applanatum, responded to an s.c. injection of a suboptimal dose of M-BaA in IFA with the development of strong DH. DH to protein antigen in guinea pigs and hu-

11 REGULATION OF DH 511 mans has been divided into two classes : classical DH reactions which can be induced by antigen in CFA and cutaneous basophil DH reactions called Jones-Mote reactions inducible by antigen without CFA. The existence of comparable heterogeneity of DH in mice has been suggested by Askenase et al (2) and Nomoto et al (21). In a single ddy/s mouse, the mushroom extract enhanced the induction of DH by a suboptimal dose of M-BaA in IFA (Fig. 2a) but not DH induced by M-BaA in CFA (Fig. 2b), whereas in the adoptive cell transfer system, spleen cells from C3H/He mice treated with Fr-1 exerted an enhancing effect on the expression of DH by the effector cells from M-BaA-CFA-primed mice as well as cells from M-BaA-IFAprimed mice (Fig. 7a). This discrepancy between the result obtained in a single mouse and that in cell transfer system, might be due to the plausible competition of adjuvanticity between CFA and the mushroom extract in the former experiment or due to strain differences. The enhancing activity was diminished in mice given an excessive dose of the extract. A similar type of dose-dependency was reported by Chihara et al (5) in studies on antitumor activity of lentinan from Lentinus edodes. This parallelism of the dose-dependency suggests that the enhancing effect of the extract on the induction of DH might be related to the growth-inhibiting activity of lentinan against the transplantable tumors. The mechanism for the enhancing effect of the extract was examined by the adoptive cell transfer technique. It has been demonstrated (20) that spleen cells from mice given an i.p. injection of M-BaA in IFA antigen-specifically suppressed the induction and expression of DH in syngeneic recipient mice. Under the same conditions, the suppressive activity in the spleen was not increased in mice pretreated with an optimal dose of the mushroom extract, but passive transfer of the same cell population, which was by itself unable to express DH in CY-treated recipients, enhanced the DH response of the syngeneic M-BaA-CFA-primed mice. The findings indicate that the amplification of DH requires participation of effector cells in DH. The amplifying cells augmented the expression of DH to M-BGG as well as to M- BaA. Therefore, nonspecific amplifying cells were expected to be generated by treatment with the mushroom extract without i.p. priming with M-BaA in IFA. Actually, it was shown by the double cell transfer system that spleen cells from mice given the mushroom extract amplified the expression of effector cells in DH and that the activity was mediated by the nylon wool-passed T cell-rich fraction. A similar type of antigen-specific or nonspecific amplifier T cells has been reported by several investigators, as functioning to enhance IgM (14), IgG (16, 24), and IgE (6), antibody formation and graft-versus-host reactions (3), or in generation of suppressor T cells (8) and cytotoxic T cells (4). The interrelation of these T cell subsets with various amplifying activities is unknown at present. Although it is still not clear whether the target of amplifier T cells is suppressor T cells capable of inhibiting the induction of DH or effector T cells in DH and whether macrophages are required for activation of amplifier T cells (13), it is evident from the present study that the enhancing effect of the extract is mediated by an interaction among T lymphocytes. A sustained state of strong DH has been induced in BCG-infected mice and the

12 512 S. NAKASHIMA ET AL high level of DH has been ascribed to increased production of activated T cells (15) and to the absence of blocking by immune complex (12). However, another possibility to be considered is that such nonspecific amplifier T cells as observed in the present study might be involved in the enhancing effect of some adjuvants. It has been reported by Reinish et al (22) that the adjuvants which enhanced antibody production by stimulating helper T cell function and DH depressed the generation of cytotoxic T cell responses and that the depression was mediated by the suppressor cells which selectively inhibited the generation of cytotoxic cells without affecting the helper T cell function. Although it has been thought that the cytotoxic T cell response might play the most important role in immunotherapy against tumors, the relative contribution of DH and the cytotoxic response to suppression of tumor growth and to the induction of tumor immunity in syngeneic mice is not clear at present. In any case, it has been reported by Ikekawa et al (9) that the extract from G. applanatum had growth-inhibiting activity against mouse sarcoma-180 and it was shown in the present study that the same extract had an enhancing effect on the induction of DH by activating the nonspecific amplifier T cells. It was shown by gel-filtration of Fr- 1-A on Sephadex G-200 under the conditions reported previously (17) that this fraction was further separated into two main polysaccharides with molecular weights of about 400,000 and 40,000, which were calculated from the elution volume by the method of Andrews (1). The adjuvant activity of these fractions and the mechanisms for the decline in the enhancing activity of the mushroom extract in the excessive dose range are now under investigation. The authors wish to thank Dr. Aoshima, The Forestry Research Institute, the Ministry of Agriculture, Forestry and Fisheries, for identification of the mushroom used in the present study. REFERENCES 1) Andrews, P Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91: ) Askenase, P.W., Hayden, B., and Gershon, R.K Evanescent delayed-type hypersensitivity: Mediation by effector cells with a short life span. J. Immunol. 119: ) Cantor, H., and Asofsky, R Synergy among lymphoid cells mediating the graft-versus-host response. III. Evidence for interaction between two types of thymus-derived cells. J. Exp. Med. 135: ) Cantor, H., and Boyse, E.A Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. J. Exp. Med. 141: ) Chihara, G., Maeda, Y., Hamuro, J., Sakai, T., and Fukuoka, F Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) Sing. Nature 222: ) Chiorazzi, N., Tung, A.S., Eshhar, N., and Katz, D.H Hapten-specific IgE antibody responses in mice. VIII. A possible new mechanism by which anti-lymphocyte serum enhances IgE antibody synthesis in vivo. J. Immunol. 119: ) Dubois, M., Gilles, K.A., Hamilton, J.K., Regers, P.A., and Smith, F Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: ) Feldmann, M., Beverley, P.C.L., Woody, J., and McKenzie, I.F.C T-T interactions in the induction of suppressor and helper T cells : Analysis of membrane phenotype of precursor and amplifier cells. J. Exp. Med. 145: ) Ikekawa, T., Nakanishi, M., Uehara, N., Chihara, G., and Fukuoka, F Antitumor action of some Basidiomycetes, especially Phellius linteus. Gann 59: ) Julius, M.H., Simpson, E., and Herzenberg, L.A A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:

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