Effect of treatment with azathioprine on the responses of rat lymphocytes to phytohaemagglutinin.

The proliferative responses of rat peripheral blood lymphocytes (PBL) and spleen cells to phytohaemagglutinin (PHA) were studied after single or multiple (daily for 4 days) injections of azathioprine (AZ). Lymphopenia developed within 4 h of a single dose (78 mg/kg) of AZ and persisted for at least 72 h. There was no lymphopenia 24 h after the last of 4 daily injections. In vitro, PBL were more sensitive than spleen cells to the inhibitory effect of AZ. Likewise, the responses of PBL were relatively more depressed than those of spleen cells after single or multiple injections of AZ. The degree of depression was less than was expected from the effect of AZ in vitro. Multiple small doses were more depressive than multiple large doses. Serum from treated rats, used at 20% concentration, was more depressive than normal. Thus, rat lymphocytes are quite sensitive to AZ in vitro, but appear to be relatively resistant in vivo, this resistance resembling the resistance of the primary antibody response to AZ treatment.     

Reversal of immunosuppression induced with plasma of malarious rats by supplemented complement

Suppression of antibody producing splenic lymphocytes by plasma from rats infected with Plasmodium chabaudi malaria was confirmed. Suppressive activity was found in plasma drawn on the sixth, seventh and eighth day of infection. It was temporally associated with anemia, elevated levels of soluble immune complex, reduced titers of lytic complement and elevated titers of immunoconglutinin (IK) in the plasma. Heat inactivation of the plasma to destroy complement and removal of IK by absorption did not reduce the suppressive activity. Incubating the plasma-treated lymphocytes with normal rat complement largely, but not completely, reversed the suppressive action. Soluble immune complexes prepared from bovine serum albumin (BSA) and antiBSA (BSA-antiBSA) alexinated complex (BSA-antiBSA-C') and immunoconglutinated complex (BSA-antiBSA-C'-IK) each suppressed the capacity of splenic lymphocytes from rats immunized with sheep blood cells to produce hemolytic Jerne plaques. Incubating the complex-treated cells with fresh complement largely reversed the suppressive activity. It is suggested that the suppressed responses of lymphocytes from malarious animals to antigens or mitogens, reported by others, may have been in part induced by complexes in blood of the animals, and that antibody producing cells might also have been suppressed. Since suppressive activity was not influenced by complement inactivation, but was reversed when plasma-treated cells were incubated with fresh complement, it is suggested that the hypocomplementemic state of suppressive plasma may have contributed to immunosuppression.     

Complement reversal of immunosuppression induced with plasma of rats infected with Trypanosoma brucei rhodesiense

Suppression of antibody production by splenic lymphocytes from rats immunized with sheep red blood cells (SRBC) after incubation with plasma from rats infected with Trypanosoma brucei rhodesiense was confirmed. Suppressive activity became evident in plasma after the sixth day of infection and was manifested by reduction in the number of hemolytic Jerne plaques produced by the treated cells. The activity was temporally associated with increased amounts of soluble immune complex (SIC) reduced titers of lytic complement, elevated titers of immunoconglutinin (IK) and anemia. Treatment of suppressive plasma with hemolysin sensitized SRBC alexinated with horse complement to reduce IK did not reduce suppressive activity, and the activity appeared to have been enhanced when the plasma was heated to inactivate the remaining complement (C'). When fresh rat C' was added to the treated cells, the suppression was largely, though not completely, reversed. Treatment of spleen cells with SIC prepared in vitro from bovine serum albumin (BSA) and rabbit antiBSA also suppressed the plaque forming capacity of the cells. Complexes of BSA-antiBSA-C' and complexes of BSA-antiBSA-C'-IK were equally suppressive. Again, addition of fresh C' to cells treated with these complexes largely, though not completely, reversed the suppressive effect on the cells. From the results it is suggested that immunosuppression associated with experimental T. b. rhodesiense infection may be in part a suppression of the capacity of induced lymphocytes to produce antibody. It is possible that the suppression was mediated by SIC present in the plasma of the infected rats and this effect was probably enhanced by reduced levels of complement in the suppressive plasma.     

Antibodies against cortisol block suppressive effects of corticosteroids on lymphocytes in vitro.

Lymphocyte transformation assays were used to test the ability of antibodies against cortisol to reduce bioactivity of corticosteroids in vitro. Mononuclear cells were separated from whole bovine blood and cultured in the presence of PHA alone, PHA + steroid, PHA + steroid + anticortisol, or PHA + steroid + anti-bovine serum albumin. Tritiated thymidine uptake was determined for all groups during the last 24 hr of a 72-hr culture period by scintillation counting. Polyclonal anticortisol against cortisol-bovine serum albumin conjugated in the 21 position was more effective in blocking cortisol activity than monoclonal anticortisol built against conjugates in the 3 position. The steroids that suppressed PHA-induced lymphocyte proliferation in a concentration-dependent manner were: cortisol, corticosterone, dexamethasone, prednisolone, 11-deoxycortisol, and 11-deoxycorticosterone. Aldosterone, cortisone, cholesterol, estradiol, and progesterone did not exhibit concentration-dependent effects and, thus, were not considered suppressive. These concentration-independent steroids were also the least suppressive (with the exception of aldosterone). Anticortisol was able to reduce bioactivity of suppressive corticosteroids that had an 11-hydroxy group, suggesting the antibody was primarily made against this site. Anti-BSA was not effective in blocking corticosteroid activity, but it did enhance proliferation of lymphocytes if added in combination with weakly suppressive steroids. Anticortisol also had an enhancing effect when added with some weakly suppressive steroids. We conclude that antibodies against cortisol are capable of reducing bioactivity of steroids that strongly suppress lymphocyte proliferation. Additionally, the 11-hydroxy group may be an important antigenic determinant of steroid molecules.     

Effect of LDL-In, a normal immunoregulatory human serum low density lipoprotein, on the interaction of macrophages with lymphocytes proliferating in response to mitogen and allogeneic stimulation.

The immunoregulatory properties of LDL-In, a normal species of human serum low density lipoprotein which suppresses indictive events involved in triggering of lymphoid cells by lectins and antigens, were analyzed in order to distinguish between a primary effect on macrophages and lymphocytes. LDL-In was found to be equally effective in suppression of the response of human lymphocytes to tpha at concentrations of lectin demonstrated to impart apparent macrophage-independence or macrophage-dependence to the culture system. Exposure of only the macrophages to LDL-In was shown to be without effect on subsequent in vitro lymphocyte responses to either PHA or allogeneic cells, whereas exposure of only the lymphocytes to LDL-In was fully effective. The cellular locus was further identified by demonstrating that the responder lymphocytes, but not the stimulator lymphocytes, were the target of the suppressive activity in mixed lymphocyte reactions. These data considered in conjunction with previous studies suggest that the primary untriggered lymphocyte is the most probable cellular target for the bioregulatory effects of LDL-In.     

Thymic control of secretory antibody responses in the rat.

The effect of neonatal thymectomy on salivary and serum antibody responses was studied in rats. Local immunization of thymectomized rats with a T-dependent antigen (DNPBGG) elicited negligible amounts of IgA anti-DNP antibody in saliva. In contrast, both normal and sham-thymectomized animals demonstrated substantial levels of salivary IgA antibody. All thymectomized rats locally injected with a T-independent antigen (DNP-Lys-Ficoll) exhibited salivary IgA antibody production. Salivary IgG antibodies were somewhat decreased in thymectomized rats injected with either antigen; however, the final effect of T cell deprivation on IgG synthesis was not as pronounced as on IgA synthesis. Serum IgA antibody was induced in control rats injected with DNPBGG, whereas this Ig class of antibody was absent in thymectomized rats. The results suggest that thymus-derived cells exert a regulatory influence on both serum and secretory IgA responses to antigens.     

Immunomodulatory activity of staphylococcal enterotoxin-B. The induction of an I-J-restricted suppressor factor

Staphylococcal enterotoxin-B (SEB), a common cause of food-borne intoxication, is a potent polyclonal T cell activator. Previous studies from this laboratory and others have shown that SEB has the capacity to nonspecifically inhibit antibody responses both in vivo and in vitro. We have shown that the inhibitory activity of SEB is mediated, in part, by the activation of a CD8+, CD4-, and CD5- suppressor cell population. The present studies show that the activity of the SEB-induced suppressor cell population is mediated by a soluble factor. This factor nonspecifically inhibits both primary and secondary in vitro antibody responses. Delayed addition analysis demonstrates that the factor must be present early in the ongoing antibody response to exhibit suppressive activity. Monoclonal anti-I-J antisera block the activity of the factor, and eluates (but not filtrates) collected from monoclonal anti-I-J immunoaffinity columns possess suppressive activity. Furthermore, the activity is restricted at the "I-J" gene locus, but is not restricted at the Igh locus. Finally, size-exclusion chromatographic analysis shows that the factor possesses an apparent Mr of 26 kDa. These studies suggest that SEB induces the production of a suppressive factor with properties similar to those exhibited by Ag-induced, and typically Ag-specific, suppressor factors.     

Immune responses during human schistosomiasis mansoni. VI. In vitro nonspecific suppression of phytohemagglutinin responsiveness induced by exposure to certain schistosomal preparations.

Simultaneous in vitro exposure of human peripheral blood mononuclear cells to phytohemagglutinin-P (PHA) and either soluble schistosomal egg antigenic preparation (SEA) or soluble cercarial antigenic preparation (CAP) obtained from Schistosoma mansoni resulted in decreased responsiveness as compared to exposure to PHA alone. The addition of a soluble adult worm antigenic preparation (SWAP) did not predictably alter PHA responses in this system. The suppression due to in vitro exposure to either SEA or CAP was expressed whether the lymphocyte donors were S. mansoni patients (early infection, chronic, or treated), or uninfected subjects. The degree of suppression was related to the concentration of SEA used, and the timing of exposure. Preexposure to SEA for 3 days before the addition of PHA resulted in more potent suppression. However, a delay in the time of the addition of SEA of 6 and 24 hr after PHA exposure decreased and eliminated, respectively, its suppressive capacity. SEA and CAP were not directly toxic to responding cells, and appeared to exert their nonspecific suppressive influences through T lymphocyte-related mechanisms. It was observed that although these suppressive events could be induced and observed in vitro, the responsiveness of S. mansoni patient lymphocytes to PHA was equal with that of uninfected controls.     

束缚应激动物血清中免疫抑制因子产生部位的初步研究

大鼠或小鼠经束缚应激10h后,血清中出现一类淋巴细胞转化抑制因子。本实验在上述工作的基础上对抑制因子的产生部位做了初步研究,结果表明,应激后脑脊液中不存在淋巴细胞转化抑制因子,说明这种因子不是由中枢神经系统产生。大剂量辐射与环磷酰胺均能降低脾脏有核细胞总数,但前者能降低抑制因子的产生,后者无作用,提示淋巴细胞总数的减少对血清抑制因子的产生可能不起决定性作用。细胞分类的结果表明,辐射能明显降低T、B细胞比例,而环磷酰胺反而使其比例有上升趋势。因而提示抑制因子的产生可能与T、B淋巴细胞的比例有关。当T细胞比例减少时,抑制因子的产生受到阻碍。裸鼠为先天性T细胞功能缺失动物,同样的应激条件抑制因子的产生受到明显抑制。这也说明抑制因子的产生可能与T细胞的作用有关。     

Mechanism of immunologic resistance to herpes simplex virus 1 (HSV-1) infection.

Susceptibility of adult mice to i.p. infection with HSV-1 was greatly increased by administration of a single dose of cyclophosphamide. Mortality of cyclophosphamide-treated virus-infected mice was associated with increased virus replication and pathologic changes in brain and liver. The development of a fatal infection in immunosuppressed mice could be curtailed after transfer of specifically immune spleen cells. Passively transferred antibody had no such effect. Protective activity of spleen cells was significantly reduced after pretreatment with anti-theta serum. Significant protection was also achieved when normal spleen cells plus immune serum were administered simultaneously. Our results indicate that protection against this virus infection is predominantly T cell dependent, and suggests that antibody-dependent cell-mediated protection may also be operative in vivo.     

A role for IgE in intestinal immunity. Expression of rapid expulsion of Trichinella spiralis in rats transfused with IgE and thoracic duct lymphocytes

In these experiments we characterize the protective antibodies in immune serum that interact synergistically with immune thoracic duct lymphocytes (TDL) to induce rapid expulsion (RE) of Trichinella spiralis in adult rats. Antibodies with both reaginic and nonreaginic activity mediated RE upon passive transfer to adult rats that had been adoptively transfused with immune TDL 7 days earlier. In serum collected 28 days after a primary infection, the most important antibody was homocytotropic IgE. Native IgE produced by active infection was isolated from 28-day immune serum by salt precipitation and/or by sequential affinity chromatography. The murine mAb A2 and B5 (anti-rat IgE) were conjugated separately to Sepharose 4B affinity columns for affinity separations. IgE was shown to be pure by gel electrophoresis and Western blots and its m.w. was estimated at approximately 190,000. As little as 183 micrograms of purified IgE could induce RE after passive transfer to adult rats. The IgE was shown to be functional by PCA activity, Ag-binding on Western blots, and skin sensitization; the latter could be blocked by pretreatment with 1R162, a rat myeloma IgE. Monoclonal IgG of any isotype transferred in amounts up to 35 mg/rat could not transfer RE to rats previously transfused with TDL cells. Immune serum collected 3 mo after the primary infection contained insufficient IgE to transfer RE, but complex non-IgE fractions were protective. The data thus demonstrate that IgE is a functional Ig in the rat capable of mediating the rejection of challenge nematode infections of the gut in the absence of other specific Ig. Secondly, other Ig may also play a role, in particular, several weeks after the primary infection when specific IgE levels in serum have declined.     

Increased oxidative metabolism in phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus is associated with serum SSA antibody.

We have examined oxidative metabolism in phytohemagglutinin (PHA)-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE) because increased oxygen free radicals would explain the DNA abnormality previously observed in these cells. Almost no oxidative activity was found in freshly isolated control or lupus lymphocytes or control lymphocytes stimulated with PHA. However, increased oxidative metabolism, measured by nitroblue tetrazolium (NBT) conversion to formazan, was found in PHA-stimulated lymphocytes from 14 of 21 lupus patients. A time course study showed that NBT activity appeared in positive lupus lymphocytes at 1-2 days of PHA stimulation, increased to a maximum at 2-4 days, and diminished thereafter. NBT activity was not related to specific disease symptoms, drug therapy, or serum dsDNA, Sm, RNP, or SSB (La) antibodies. The selected population of lupus patients studied precluded conclusions about NBT activity and disease severity. However, the intensity of NBT response in stimulated lupus lymphocytes was positively correlated with the presence of serum SSA (Ro) antibody. We suggest that increased oxidative activity of SLE lymphocytes generates a chemical change in endogenous DNA in vivo and may be a primary event in the pathogenesis of autoimmunity. Absence of detectable oxidative activity in stimulated lymphocytes in a subgroup of lupus patients suggests that at least two different mechanisms are associated with the altered DNA profiles observed in this disorder.     

Elicitation of the reproduction-inhibiting antibody ablastin by serum exoantigens of Trypanosoma lewisi

Serum exoantigens of Trypanosoma lewisi were collected 5 days after infection from immunocompetent (untreated) rats and rats immunosuppressed by treatment with either hydrocortisone acetate or dexamethasone. Normal rats were then immunized with pooled, whole exoantigen-containing serum from 1 of these 3 sources plus alum as an adjuvant, and the immune sera produced were tested individually. All contained agglutinating (trypanocidal) antibodies to both antigenic variants of T. lewisi, but only about two-thirds showed precipitating activity with exoantigens in gels. More importantly, however, when these antisera were thoroughly adsorbed with living trypanosomes (from immunocompetent hosts) to remove agglutinating antibody only and then tested for ablastic activity in vitro, all showed significant (P less than 0.01) reproduction-inhibiting activity, comparable to that shown by ablastic serum collected from rats that experienced a natural infection. Antisera from control rats similarly immunized with normal rat serum were negative in all antibody tests. The exoantigens of T. lewisi are, therefore, a complex mixture of immunogens that are related to the known immune responses to the parasite and can elicit the formation of ablastic antibody with the same biological properties as that produced during a natural infection.     

Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. II. Suppression of antigen- and mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes

U937, a human macrophage-like cell line, spontaneously produces a factor which inhibited blastogenic responses of human blood T lymphocytes stimulated with tuberculin-purified protein derivative (PPD) or phytohemagglutinin (PHA). We investigated the mechanism of suppressor action of the U937 factor. The U937 suppressor factor inhibited interleukin 2 (IL 2) production by human blood T lymphocytes stimulated with PPD or PHA. IL 1 did not overcome the inhibitory action of the U937 factor on PPD-induced IL 2 production by human blood T lymphocytes. The U937 factor also inhibited the production of IL 2 by a human leukemic cell line, JURKAT, stimulated with PHA. The U937 suppressor factor interfered with the expression of Tac antigen (IL 2 receptor) on PPD- or PHA-stimulated blood T lymphocytes. The inhibitory activity of the U937 factor on Tac expression was not affected by the addition of IL 2 or a crude lymphokine-containing T cell supernatant. Tac expression was more sensitive than IL 2 production to inhibition by U937-conditioned medium. The U937 suppressor factor was precipitable by 33 to 67% saturated ammonium sulfate and was inactivated at pH 2 or pH 11. Sephacryl S-200 Gel filtration analysis of U937 culture supernatants revealed that the inhibitory activities for blastogenesis, IL 2 production, and Tac expression co-purified in fractions with an apparent m.w. between 67,000 and 130,000. These data indicate that U937 spontaneously produces a macromolecular suppressive factor with major locus of action on the production of IL 2 and the expression of the IL 2 receptor.     

Immunologic dysfunction during viral oncogenesis. I. Nonspecific immunosuppression caused by malignant rabbit fibroma virus

Malignant rabbit fibroma virus (MV) is a potent oncogenic poxvirus that produces a rapidly progressive syndrome of disseminated myxosarcoma, immunosuppression, and fatal gram-negative infection. MV is probably a recombinant between Shope fibroma virus (SFV) and rabbit myxoma virus, and is capable of preventing or aborting the in vitro proliferative responses of rabbit lymphocytes to B and T lymphocyte mitogens. Proliferative responses to sheep erythrocytes (SRBC) are similarly affected, although MV does not alter ongoing antibody responses to SRBC. Splenic lymphocytes from MV tumor-bearing rabbits suppress antibody and proliferative responses to SRBC when added to lymphocytes from SRBC-primed rabbits. Finally, lysates of cultured splenic lymphocytes from rabbits given MV suppress both proliferative and antibody-forming responses to SRBC. When MV is removed from these lysates by UV inactivation or by centrifugation, the suppressive activity remains. We therefore conclude that MV induces immunologic unresponsiveness in rabbits by at least two mechanisms. First, a direct suppressive effect of added virus on in vitro lymphocyte proliferation is seen. There is no effect in this situation if an antibody response is already in progress. Second, spleen cells exposed to MV in vivo produce one or more soluble factors capable of suppressing both proliferative and antibody responses of normal lymphocytes.     

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. I. Inhibition of proliferation of lymphocytes in primary antibody responses

The suppressor cells induced by Toxoplasma infection were shown to be macrophages, since they adhered to plastic, and their suppressive activity in anti-sheep erythrocytes (SRBC) antibody responses was abrogated by treatment with silica or carrageenan, which are selectively cytotoxic for macrophages. The suppressor macrophages strongly inhibited the uptake of tritiated thymidine ( [3H]TdR) by normal mouse spleen cells in the responses to SRBC and Toxoplasma antigens. Supernatant fluids from the suppressor macrophages could not passively transfer the suppressive effect on anti-SRBC antibody responses. Furthermore, when the suppressor macrophages were isolated by a cell-impermeable membrane from normal mouse spleen cells, the antibody responses of normal spleen cells were not suppressed. These results indicate that suppression of antibody responses in Toxoplasma-infected mice is caused by an inhibitory effect of the suppressor macrophages upon proliferation of lymphocytes via direct contact with responder target cells. The suppressive effect of the macrophages was not counteracted by indomethacin, a potent inhibitor of prostaglandin synthesis, or catalase, a catabolic enzyme for hydrogen peroxide (H2O2).     

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.     

运动员剧烈运动后血中应激免疫抑制蛋白的产生

我们曾经报道,大鼠或小鼠在束缚应激后血中产生了一种能抑制免疫功能的应激免疫抑制蛋白,（又称Ｎｅｕ－ｒｏｉｍｍｕｎｅｐｒｏｔｅｉｎ,ＮＩＰ,神经免疫蛋白）。本工作证明,运动员在大运动量的训练后血清中也产生一种能抑制淋巴细胞转化的物质,它的生化特性及分子量与前述大鼠和小鼠中的应激免疫抑制蛋白相同。在体外实验中,应激大鼠的血清培养人淋巴结细胞,获得了与大鼠实验相同的结果,即人淋巴结细胞也能产生应激免疫抑制蛋白。同时小鼠束缚应激的血清和大运动量的人类血清可以分别抑制人正常淋巴细胞和正常小鼠由ＣｏｎＡ诱导的淋巴细胞转化,以上结果表明,这种应激免疫抑制蛋白的种属特异性不强。     

Frequencies of alloreactive and self-restricted cytotoxic T cell precursors during lymphatic regeneration

The previous observation that during lymphatic regeneration precursors for MHC-restricted cytotoxic T cells appear before alloreactive precursors, was re-examined in macroculture and microculture experiments. Our macroculture experiments confirmed the observation that treatment with cyclophosphamide decreases the ratio of alloreactivity vs self-restricted reactivity. This shift in reactivity, however, did not result from a change in the T cell specificity repertoire, but was obviously mediated by regulatory effects in macrocultures. Spleen cells from cyclophosphamide-treated mice had normal ratios of CTL precursor frequencies for the three different specificities tested, but they contained a suppressive activity that inhibited the responses of normal spleen cells in macrocultures. Note Added: After submission of this paper we became aware of the paper of Sihvola and Hurme (J. Immunol. 130:1077, 1983) who also showed that although macrocultures from cyclophosphamide-treated mice were negative for the alloresponse, the same spleen cells showed only a approximately threefold decrease in CTL precursors when analyzed in limiting dilution cultures.