Induction of suppressor cell activity in vivo and suppression of lymphoproliferative responses in vitro by SK&F 105.685 in the dog.

SK&F 105.685 (N,N-dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in rat models of adjuvant-induced arthritis and experimental autoimmune encephalomyelitis as well as in the murine lupus model. The mechanism of action in these models appears to be the induction of non-specific suppressor cell activity which is measured by the ability of cells from treated animals to partially inhibit the proliferative response of lymphocytes from control animals to concanavalin A (ConA) in a co-culture assay. In this study we have shown that oral administration of 0.1-3 mg/kg/day of SK&F 105.685 to purebred beagle dogs induced suppressor cell activity in the spleen and bone marrow but not in the peripheral blood. In vitro, SK&F 105.685 partially suppressed the proliferative response of dog splenocytes to the mitogens phytohemagglutinin (PHA), ConA and, to a lesser extent, pokeweed mitogen (PWM). Peripheral blood lymphocytes (PBLs) differed from spleen cells in their susceptibility to suppression by SK&F 105.685. While the PWM and ConA responses of PBLs and spleen cells showed similar levels of inhibition, the PHA response of PBLs, in marked contrast to spleen cells, was resistant to suppression by the compound. Our results show that the immunoregulatory effects of SK&F 105,685 are not limited to rodents and that suppressor cell activity in dogs is induced quickly and by relatively low doses of the compound.     

Immunologic and anti-immunosuppressive effects of vitamin A

The effects of vitamin A alcohol on cell-mediated immunity in vitro and its ability to prevent the immunosuppressive effects of prednisolone and cyclophosphamide in vivo were studied in mice. Lymphocytes from Calmette-Guérin bacillus (BCG) sensitized mice were stimulated specifically with purified protein derivative (PPD) and nonspecifically with phytohemagglutinin (PHA). In the vitamin A injected animals there was significant enhancement of the spleen lymphocyte transformation not only in the PPD-sensitive cells but also in the T cells at large. In addition, vitamin A was able to restore to normal the cellular and humoral forms of immunity in prednisolone and cyclophosphamide-treated animals. It is suggested that vitamin A in nontoxic doses may have a role in enhancing the responses to weak immunogens and in reversing immunosuppression.     

Doxorubicin and daunorubicin modulation of macrophages, T and B lymphocytes (anthracycline lymphocyte modulation)

Mouse spleen cells were cloned in the presence of phytohemagglutinin or lipopolysaccharide. The effects in vitro of doxorubicin or daunorubicin on colony formation were examined. At certain concentrations these drugs stimulated B lymphocytes and suppressed T lymphocytes. These were the opposite of the effects observed with in vivo drug administration. It was observed that soluble factors from drug-treated monocytes produced these effects. It was not clear whether the disparate effects were the result of two different monocytic populations or differences in the in vivo versus in vitro microenvironment present during drug administration.     

Enhancement by concanavalin A of the suppressive effect of prodigiosin 25-C on proliferation of murine splenocytes.

Proliferation of concanavalin A (Con A)-activated nylon-wool purified murine splenic T cells was increasingly suppressed by prodigiosin 25-C as higher concentrations of Con A were used for the activation. Enhancement of suppressive effect of prodigiosin 25-C was not observed when T cells were stimulated with phytohemagglutinin (PHA), anti-CD3 antibody, or allogeneic splenic adherent cells. The suppressive effect of prodigiosin 25-C was enhanced by the addition of Con A in various T cell subpopulations as well as in LPS-activated splenic B cells. Lectins that recognize mannose residue of biantennary-complex-type sugar chains significantly enhanced the suppressive effect of prodigiosin 25-C, whereas a lectin that binds to N-acetylglucosamine did not. These results suggest that binding of lectins to the mannose residue of biantennary-complex-type sugar chains on cell surface of both T and B lymphocytes plays a central role on the enhancement of the suppressive effect of prodigiosin 25-C.     

Effects of biological response modifier on thoracic duct lymphocytes in recurrent gastric cancer. Evaluation of OK-432, a hemolytic streptococcus preparation

In 9 patients with recurrent gastric carcinoma treated with intracutaneous injections of a hemolytic streptococcal preparation of OK-432 (PIC), the T/B cell ratio, the number of IgG-Fc receptor positive (T gamma) cells, the degree of blastoid transformation of lymphocytes in response to concanavalin-A (Con-A) and phytohemagglutinin (PHA), and the activity of natural killer cells (NK) in the thoracic duct lymphocytes (TDL) and peripheral blood lymphocytes (PBL) were measured. In thoracic duct lymph, the T cell to B cell ratio was higher than that found in peripheral blood. The proportion of T gamma cells and the natural killer cell activity was found to be considerably lower in TDL than PBL, though there was a higher degree of blastoid transformation of lymphocytes in response to Con-A and PHA in TDL. After the administration of PIC, there was no significant difference in T/B cell ratios and T gamma cell proportion between TDL and PBL, though the lymphocytic blastoid transformation in response to Con-A and PHA decreased in TDL but not in PBL. PIC administration appeared to augument natural killer cell activity in both TDL and PBL.     

Amaranthus spinosus water extract directly stimulates proliferation of B lymphocytes in vitro

Amaranthus spinosus Linn. (thorny amaranth), a plant that grows in the wild fields of Taiwan, is extensively used in Chinese traditional medicine to treat diabetes. There have been no published studies on the immunological effects of A. spinosus. To determine whether A. spinosus has immuno-modulatory effects and clarify which types of immune effector cells are stimulated in vitro, we investigated the stimulatory effect of wild A. spinosus water extract (WASWE) on spleen cells from female BALB/c mice. We found that WASWE significantly stimulated splenocyte proliferation. However, isolated B lymphocytes, but not T lymphocytes, could be stimulated by WASWE in a dose response manner. After sequentially purifying WASWE, a novel immuno-stimulating protein (GF1) with a molecular weight of 313 kDa was obtained. The immuno-stimulating activity of the purified protein (GF1) was 309 times higher than that of WASWE. These results indicate that WASWE does indeed exhibit immuno-stimulating activity via directly stimulating B lymphocyte activation in vitro. Further, these results suggest that the immuno-stimulating effects of WASWE might lead to B lymphocyte activation and subsequent T cell proliferation in vitro. These results are potentially valuable for future nutraceutical and immuno-pharmacological use of WASWE or its purified fractions.     

Effects of benzene inhalation on lymphocyte subpopulations and immune response in mice

To clarify the immunotoxicity of benzene, the effects of benzene inhalation on T and B lymphocytes and immune responses in mice were examined. BALB/c male mice were exposed to 50 or 200 ppm benzene vapor, 6 hr/day for 7 or 14 consecutive days. T and B lymphocytes, in blood and spleen, were detected by the cytotoxicity assay with anti-Thy-1.2 monoclonal antibody and the membrane immunofluorescence test with anti-immunoglobulin antibody, respectively. Humoral immune response to sheep red blood cells was determined by the hemolytic plaque-forming cell assay. Cell-mediated immune response was measured by contact sensitivity (CS) to picryl chloride. The activity of suppressor cells was evaluated in spleen by the suppressive effect on passive transfer of CS. The ratio and absolute number of T and B lymphocytes in blood and spleen were depressed after a 7-day exposure at 50 ppm benzene. The depression of B lymphocytes was dose dependent and more intense than that of T lymphocytes. The ability to form antibodies was suppressed by benzene at all exposure levels, but the CS response was resistant to benzene inhalation and rather enhanced at 200 ppm exposure for 14 days. The activity of suppressor cells could not be detected at this dose level. These data show that benzene inhalation effects on humoral and cell-mediated immune responses are a result of the selective toxicity of benzene to B lymphocytes and suppressor T cells.     

Immunomodulatory properties of Mycoplasma pulmonis. I. Characterization of the immunomodulatory activity

For many years, it has been recognized that Mycoplasma infection affects the host's immune system in different ways. In this work, experiments were performed to characterize the influence of Mycoplasma pulmonis infection on various immunological parameters and to follow the kinetics of their variations. A Balb/c mouse model was used to assess hematological evaluations, changes in spleen weight, antibody responses against sheep erythrocytes, neutrophil phagocytosis, colloidal carbon clearance, and anti-Mycoplasma antibody responses. At the hematological level, infected animals were found to have significantly increased total lymphocyte and polymorphonuclear leukocyte counts and an augmentation in spleen weight. Seven days after Mycoplasma infection, antibody responses against sheep erythrocytes were considerably diminished, and at days 7 and 14 after infection, phagocytic activity was also reduced. After 1 week of infection, the colloidal carbon clearance pattern was decreased, and during the whole infectious process, anti-Mycoplasma antibody titers were found to be low. Results from this part of research show a persistent infection that does not resolve in a short period, which is associated with a general dysfunction in the immune system and poor immune responses against several different antigens.     

Prevention of GvHD by a lymphokine

T lymphocytes which are capable of preventing GvHD when mixed with GvHD inducing cells before grafting and of inhibiting MLR, have been demonstrated in spleen and thymus of neonatal mice and rats. The supernatant of short term cultures of neonatal thymus and spleen contains a factor termed SUF, that produces the same suppressions. SUF has been produced by a number of hybridoma cell lines, that were obtained by fusion of a T lymphoma cell line with neonatal spleen cells. SUF does not reduce CFU-S upon incubation with mouse bone marrow. Suppression by SUF in MLR is not mediated via a cytotoxic mechanism, neither does SUF inhibit proliferation of normal and malignant lymphocytes. Preliminary data suggest that SUF interferes with the action of IL-2 on T effector lymphocytes.     

Immunomodulatory activity of dioscorin, the storage protein of yam (Dioscorea alata cv. Tainong No. 1) tuber.

The purified dioscorin from yam (Dioscorea alata L. cv. Tainong 1) tuber was previously reported (Hsu et al., 2002. J. Agric. Food Chem., 50, 6109-6113). In this report, we evaluated its immunomodulatory ability in vitro in the presence of polymyxin B (50 microg/ml) to eliminate lipopolysaccharide (LPS) contamination. Dioscorin (5-100 microg/ml) was able to stimulate nitric oxide production (expressed as nitrite concentrations) in RAW264.7 cells. The stimulation index on the phagocytosis of RAW264.7 cells against E. coli and the oxidative burst (determined by the intensity of rhodamine fluorescence) of RAW264.7 cells were both enhanced by different concentrations of dioscorin (5-100 microg/ml). The cytokine production, including IL-6, TNF-alpha, and IL-1beta in dioscorin-treated RAW264.7 cells or human monocytes, was measured in the cultured medium. Dioscorin (5-100 microg/ml) was found able to induce IL-6, TNF-alpha, and IL-1beta production in RAW264.7 cells and human monocytes. To evaluate the effects of dioscorin on the proliferation of spleen cells from BALB/c mice, phytohemagglutinin (PHA, 2 microg/ml) alone or PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml) was used to treat spleen cells for 24h. The stimulated proliferation index of splenic cells ranged from 1.38- to 1.48-fold of PHA alone for PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml). We suggest that the tuber storage protein of yam dioscorin functions as an immunomodulatory substance.     

Immunostimulatory potential of smokeless tobacco extract in in vitro cultures of murine lymphoid tissues

Despite numerous studies on the general toxicologic effects of smokeless tobacco (ST) little immunotoxicologic information is available. As a first step in assessing the potential activity of ST on the immune system, the effects of an aqueous extract of ST was studied in in vitro cultures of mouse lymphoid cells. There was a significant increase in the proliferation of spleen cells cultured with different concentrations of ST extract. The polyclonal IgM antibody responses as determined by protein A plaque assay were also elevated in ST stimulated spleen cell cultures. Similar immunostimulatory results were seen in the mesenteric lymph node cell cultures also. ST extract was able to stimulate the spleen cells of the immune defective CBA/N mice. The mitogenic ability of ST extract may not be due to lipopolysaccharide (LPS) contamination as determined by its response in the LPS resistant C3H/HeJ mice spleen cells. ST extract was mitogenic not only to B cells but also to T cells. However the magnitude of response was less in T cells than in B cells. The proliferation of T cells was not accompanied by secretion of IL-2 or expression of IL-2 receptors on T cells. However there was an increase of IL-1 activity in spleen cells cultured with ST extract. Finally, activation of B or T lymphocytes by ST did not result in the elevation of intracellular calcium levels. Since ST is consumed orally, the chronic immunostimulation by ST in oral mucosal lymphoid tissues may be associated with the increased incidence of gingivitis, leukoplakia and oral cancer seen in human ST users.     

Blood and spleen lymphocytes as targets for immunotoxic effects in the rat--a comparison

Traditionally, immunotoxicological studies in the rat have been performed by measuring the effect of chemical substances on spleen lymphocytes in vivo and in vitro. However, rat blood lymphocytes may be more relevant than spleen cells for comparison with human blood lymphocytes. Further, lymphocytes in blood may be a more sensitive indicator of immunotoxic effects than spleen lymphocytes. Finally, in longitudinal studies peripheral blood specimens can be collected repeatedly from the same animals, thereby reducing the number of animals sacrificed and, possibly, experimental variation. We compared blood and spleen lymphocyte parameters in rats treated with a single dose of the immunosuppressant cyclophosphamide (CY), monitoring effects on blood and spleen lymphocytes by immunophenotyping. We also performed repeated bleedings to demonstrate the feasibility of following the time course of induced changes in the same animals. Immunophenotyping as well as total mononuclear cell counts consistently showed as large or lager effects of CY in blood lymphocytes than in spleen cells. Further, the measured effects in blood lymphocytes became statistically significant at an earlier time point, compared to spleen cells. Repeated bleedings of the same animals illustrated that blood specimens drawn from a peripheral vein give sufficient numbers of cells to perform immunotoxicological tests.     

Opposite effects of the catecholamines dopamine and norepinephrine on murine polyclonal B-cell activation

The effects of dopamine (DA) and norepinephrine (NE) on polyclonal B-cell activation induced in vitro by lipopolysaccharide (LPS) and on cyclic AMP response in BALB/c mouse lymphocytes were investigated. DA at non-cytotoxic concentrations (5 x 10(-5) M and 10(-4) M) inhibited, but NE (5 x 10(-6) M to 5 x 10(-5) M) enhanced LPS-stimulated proliferation and Ig synthesis by lymphocytes from spleen, mesenteric lymph nodes and Peyer's patches. Depletion of adherent cells and T lymphocytes did not prevent the respective effects of DA and NE, and the drug effects were reproduced on nude (nu+/nu+) spleen cell proliferation and differentiation stimulated by LPS. The inhibitory effect of DA persisted even if the drug was added as late as 48 h after the mitogen. In contrast, NE was no longer stimulatory if added 2 h later than LPS. The effect of DA was blocked neither by DA1 or DA2 dopaminergic antagonists (SCH 23390 and sulpiride respectively), nor by alpha- or beta-adrenoceptor antagonists (phentolamine and propranolol respectively). Enhancement by NE was antagonized by propranolol, but not by phentolamine. Both DA and NE raised the level of cyclic AMP in unfractionated spleen cells as well as in B-enriched spleen cells but DA was less potent than NE. Pre-incubation of spleen lymphocytes with LPS for 1-24 h did not alter their cyclic AMP response to NE but it prevented the loss of sensitivity to DA after 4 or 24 h of in vitro incubation. The lysosomotropic agent chloroquine induced suppression of LPS-stimulated proliferation and Ig production with a dose-response profile similar to that of DA. Altogether, these results indicate that the catecholamines can exert opposite effects on polyclonal B-cell activation by acting directly on B lymphocytes.     

The effects of prostaglandins on the proliferation of cultured human T lymphocytes

The effects of PGE2 on cultured T lymphocytes (CTC) stimulated by either Con A, PHA, or lectin-free IL-2 were studied. PGE2, in a concentration ranging from 100 to 1 ng/ml, consistently and significantly inhibited the proliferation of CTC induced by either PHA or Con A. PGF2 alpha was essentially without effect. Although the degree of inhibition of PHA-treated CTC was increased with suboptimal amounts of PHA, significant inhibition still resulted with optimal PHA concentrations. PGE2, but not PGF2 alpha, was also effective in significantly inhibiting the proliferation of IL-2-treated CTC in a dose-related fashion; however, the addition of suboptimal amounts of IL-2 did not result in greater increases in the degree of inhibition by PGE2. Depleting the CTC of either OKT-4 or OKT-8 phenotypic cells did not abrogate this PGE2 inhibitory effect, indicating that PGE2 does not suppress the proliferative response solely by the activation of suppressor cells with the OKT-8 phenotype. PGE2 also was found to inhibit the production of IL-2 by fresh lymphocytes treated by either optimal or suboptimal amounts of PHA, however, this decrease in production by PGE2 was not necessarily associated with a decrease in the proliferation of these stimulated lymphocytes. Only with low PHA concentrations, where IL-2 production was markedly reduced and barely detectable, was lymphocyte proliferation appreciably reduced by PGE2. In additional experiments, LiCl was added to PGE2 containing cultures to determine whether LiCl could modulate the inhibitor effect of PGE2 of either PHA- or IL-2-stimulated CTC. In these studies, LiCl, in concentrations of 1-10 mM was found to lessen or completely abrogate the reduced PHA proliferative response induced by PGE2. This effect was more pronounced with suboptimal concentrations of PHA than with optimal PHA amounts. In contrast, the PGE2-induced inhibition of IL-2-stimulated CTC was not modified or altered by the addition of LiCl. Thus, these results suggest that LiCl acts at the level of IL-2 production instead of IL-2 action, and that PGE2 inhibits IL-2-induced proliferation of CTC by a different or additional mechanism than for PHA-treated cells. In conclusion, these results, taken as a whole, indicate that PGE2 suppresses the proliferation of stimulated CTC by at least two different mechanisms: 1) by reducing the production of IL-2 by stimulated lymphocytes; and 2) by directly acting on the responding CTC.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel method of screening for immunomodulating substances, establishment of an assay system and its application to culture broths of microorganisms

A novel method of screening for immunomodulating substances is developed employing lymphocytes and three mitogens. Concanavalin A (Con A) and phytohemagglutinin (PHA) are applied as T cell specific stimulants and lipopolysaccharide (LPS) as a B cell specific stimulant respectively. The lymphocytes obtained from mouse spleen are cultured with an antibiotic or a sample extract in the presence or absence of mitogen for three days and pulsed with [3H]thymidine for five hours before harvest. Differential effects of a sample compound on [3H]thymidine incorporation by the activated and quiescent lymphocytes are scored. In this procedure most of the tested antibiotics or chemical compounds with different mode of actions show non-specific effects. Cyclosporin A, a potent immunosuppressive substance, suppresses both Con A and PHA responses more extensively than LPS response and quiescent cell growth, and two cytochrome bc1 complex inhibitors, funiculosin and antimycin A3, are less suppressive to PHA response than to the others. The present system was also applied to the methanol extracts of the culture broths prepared from the type strains of Actinomycetes and Penicillium.     

组胺对细胞免疫反应调节作用的实验研究

目的　了解组胺对 CD4和 CD8T细胞白细胞介素 - 2 (IL- 2 )产生和细胞增生活性的影响。方法　密度梯度离心及吸附法分离外周血单个核细胞 (PBMC)和外周血淋巴细胞计数 (PBL C) ,采用抗 CD4和抗 CD8抗体分别制备 CD8和 CD4T细胞进行培养 ,然后采用酶联免疫吸附法 (EL ISA法 )和四氮唑蓝快速比色法(MTT)测上清液 IL - 2含量及增生活性。结果　 1组胺 +CD4(CD8)培养上清液中 IL - 2水平及 MTT增生指数与T细胞自然培养孔比较明显降低 (P<0 .0 5 )。 2组胺 +CD4(CD8) +西咪替丁培养孔上清液中 IL - 2水平及 MTT增生指数明显高于未加西咪替丁孔 (P<0 .0 5 )。 3 CD4T细胞自然培养孔上清液中 IL- 2水平显著高于 CD8T细胞自然培养孔。结论　 1组胺可抑制 T细胞 IL- 2产生及增生 ;2西咪替丁可阻断组胺对 T细胞的抑制作用 ;3CD8T细胞也可产生 IL- 2 ,但其功能较 CD4T细胞为低     

Inhibition of Humoral Immunity and Mitogen Responsiveness of Lymphoid Cells Following Oral Administration of the Heterocyclic Food Mutagen 2-Amino-1-methyl-6-Phenylimidazo [4,5-b](PhIP) to B6C3F1 Mice

In these studies, the food promutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhIP) was evaluated for its immunotoxicity in B6C3F mice followingoral 5-day dosing at total doses of 50 and 150 mg/kg. Resultsindicated that PhIP produced a dose-dependent suppression ofthe humoral immune response of spleen cells to sheep erythrocytes,with a 50% decrease in the number of PFC detected at the 150mg/kg dose of PhIP. A 40–90% inhibition of the phytohemagglutinin(PHA) response of spleen cells, mesenteric lymph nodes (MLNs),and Peyer's patch (PP) lymphocytes was seen in the treatmentgroups. The lipopolysaccharide (LPS) response was somewhat morevariable and less affected with 20–30% inhibition observedin the spleen and PPs, whereas PhIP increased the LPS responsein the MLNs. There was no effect of PhIP on cell recovery orviability in any of the treatment groups. Flow cytom etry analysisrevealed a depletion of T cells (Thy 1.2⁺ cells) and a slightincrease in B cells (Ly5⁺ cells) in the PPs. The percentageof B and T cells present in the spleen and MLNs was unaffectedby PhIP. These results demonstrate that the oral administrationof PhIP produces immunotoxicity to mice, especially to lymphoidtissues present in the GI tract (i.e., PPs), and demonstratesthat T cell mitogen (PHA) responses in PPs are the most sensitiveindicator of PhIP-induced immunotoxicity.     

灵芝多糖对肿瘤逃避免疫监视的拮抗作用（英文）

Recent years,our research group focused the effects of G.polysaccharides,which are extracted from fruiting body of Ganoderma lucidum,on tumor evasion from immune surveillance.The immune system in patients with tumor often fails to control tumor growth because of deficient immunogenicity of tumor cells.Deficient major histocompatibility complex(MHC)classⅠ and costimulatory molecules on malignant cells partially results in tumor evasion since antigen bond MHC and costimulatory molecules provide two signals for T cell activation.Therefore,enhancement of MHC-Ⅰ and costimulatory molecules may favor restraint of the evasion.Our study found that G.polysaccharides can increase MHC classⅠ molecules such as H-2Kb and H-2Db as well as the costimulatory molecules B7-1 and B7-2 expression on B16F10 melanoma cells at both mRNA and protein levels,and can promote lymphocyte-mediated cytotoxicity.Tumour cells produce immune suppressive factors such as interleukin 10(IL-10),transforming growth factorβ1(TGF-β1)and vascular endothelial growth factor(VEGF)that suppress the function of immune cells or induce apoptosis of immune cells.Our study also found B16F10 cell culture supernatant(B16F10-CS)suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin(PHA),as well as lymphocyte proliferation in the mixed lymphocyte reaction.The suppression also associated with elevated levels of immunosuppressive IL-10,TGF-β1 and VEGF in B16F10-CS.Further,the production of IL-10,TGF-β1,and VEGF in B16F10 melanoma cells and lung carcinoma LA795 cells was suppressed by G.polysaccharides at both mRNA and protein levels.On the contrary,the production of IL-2,IFN-γand TNF-αin mononuclear lymphocytes was suppressed by B16F10-CF at both the mRNA and protein levels,whereas the suppression was ameliorated by G.polysaccharides.B16F10-CS was suppressive to the viability,phagocytic activity,NO production,TNF-αproduction and activity in peritoneal macrophages while G.polysaccharides had the antagonistic effects against this suppression.Then subsequently,the plasma of patients with lung cancer suppressed proliferation,CD69 expression,and perforin and granzyme B production in lymphocytes upon activation by PHA.However,these suppressive effects were reversed by G.polysaccharides.In conclusion,G.polysaccharides can improve the nature of B16F10 cells to activate lymphocytes and antagonize immunosuppression induced by B16F10-CS in lymphocytes and macrophages.These findings indicate that G.polysaccharides can restraint tumor evasion from immune surveillance,suggesting this potential value of G.polysaccharide to facilitate cancer immunotherapy.     

Unrelated delayed hypersensitivity reactions accelerate the recovery of immunological responsiveness of specifically depleted cell populations.

Normal CBA mouse spleen cells were specifically depleted of cells spontaneously reacting to pigeon erythrocytes (PRBC) by two methods, the first allows specific depletion of anti-PRBC thymus derived (T) rosette forming cells (RFC) whereas the second depletes bone marrow derived (B) anti-PRBC hemolytic plaque forming cells (PFC). Depleted populations transferred into lethally irradiated syngeneic recipients and stimulated with PRBC failed to develop any significant response but they normally responded to a stimulation with sheep erythrocytes (SRBC). When spleen cells were taken from mice skin painted with picryl (trinitrophenyl: TNP) chloride 12 days before and the recipients were challenged in the same way and stimulated with PRBC, they become capable of producing a definite response to this antigen. Moreover in these animals, a consistent although low number of cells was found, which simultaneously reacted to both native PRBC and TNP conjugated SRBC. These findings show that unrelated delayed hypersensitivity reactions promote the immunological recovery of specifically depleted cell populations.