Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.     

Transfer factor prevents relapses in herpes keratitis patients: A pilot study

Transfer Factor is a dialysable moiety obtained from immune lymphocytes. It has been successfully used for the treatment of several viral infections including labial and genital herpes. In the present study, thirty-three patients with low immune response to HSV antigens and suffering from herpes ocular infections were orally treated with HSV-specific transfer factor (TF). Their relapse index was reduced from 20.1 before treatment to 0.51 after TF administration, with only 6/33 patients relapsing. Although this is not a placebo-controlled-randomized study, the results suggest that TF specific for HSV antigens may be efficacious for preventing relapses of ocular herpes infections as has been the case with genital and labial localisations.Abbreviations CMI Cell-mediated immunity - CMV Cytomegalo-virus - EBV Epstein-Barr virus - HIV Human immunodeficiency virus - HK Herpes keratitis - HSV Herpes simplex virus - IRI Individual relapse index - KU Kerato-uveitis - LMT Leucocyte migration test - LST Lymphocyte stimulation test - MIF Migration inhibition factor - RHK Relapsing herpes keratitis - TF Transfer factor     

CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection

Infection by Listeria monocytogenes causes serious morbidity and mortality during the neonatal period. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) can increased the resistance of adult mice to many infectious pathogens, including Listeria. This work examines the capacity of CpG ODN to stimulate a protective immune response in newborns. Results indicate that dendritic cells, macrophages, and B cells from 3-day-old mice respond to CpG stimulation by secreting IFN-gamma, IL-12, and/or TNF-alpha. Spleen cells from CpG-treated neonates produce large amounts of cytokine and NO when exposed to bacteria in vitro. Newborns treated with CpG ODN are protected from lethal Listeria challenge and generate Ag-specific CD4 and CD8 T cells that afford long-term protection against subsequent infection. These results demonstrate that cellular elements of the neonatal immune system respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.     

Phenotypes and cytokine profiles of enriched blood dendritic cells in healthy individuals

Dendritic cells (DC) are highly specialized for initiating adaptive immune responses and are capable of producing a wide variety of cytokines. However, cytokine profiles of the DC naturally present in human blood have received relatively little attention. The objective of this study was to investigate expression of surface markers and cytokines by blood DC not subjected to prolonged culture and/or polyclonal activation, to identify surface phenotypes of cytokine-expressing DC and to evaluate sex and age differences in cytokine profiles of DC. For this purpose, DC were enriched from blood of healthy donors by the use of the adherence method, and expression of surface molecules and intracellular IFN-g, IL-10, IL-12 and IL-15 was studied by flow cytometry. Enriched blood DC expressed higher levels of IFN-g, IL-12 and IL-15, compared to whole mononuclear cells (MNC) incubated for the same time. Expression of IFN-g and IL-12 was confined to the mature CD83+CD11c+ DC subset. Enriched DC from females' blood displayed higher levels of CD80, IL-10 and IL-15. Taken together, enriched blood DC spontaneously express larger amounts of IFN-g, IL-12 and IL-15 than MNC. Sex differences in expression of CD80, IL-10 and IL-15 may have a modulatory influence on immune responses in males and females.     

Exogenous interleukin-2 abrogates differences in the proliferative responses to T cell mitogens among inbred strains of mice.

The proliferative response of spleen cells from BALB/c mice to stimulation with a T cell mitogen, concanavalin A (Con A), was two or more times stronger than that of cells from C57BL/10SnSc (B10) mice. In contrast, the cells from B10 mice responded better to B cell mitogen bacterial lipopolysaccharide (LPS). The differences in the proliferative response to Con A stimulation were not associated with the function of macrophages nor did they depend on IL-1. Spleen cells from BALB/c and B10 mice synthesized comparable amounts of mRNA for IL-1 alpha, and the production of biologically active IL-1 was even higher in the B10 strain. Indomethacin, an inhibitor of prostaglandin synthesis, had no effect on the differences in reactivity between the cells from BALB/c and B10 mice. In addition, no differences in the synthesis of mRNA for the inducible 55-kDa interleukin-2 (IL-2) receptors were found between the spleen cells from BALB/c and B10 mice. However, Con A-stimulated spleen cells from B10 mice produced a significantly lower amount of biologically active IL-2 than similarly stimulated cells from BALB/c mice. In the presence of exogenous IL-2, these low responder spleen cells from the B10 mice responded by proliferation to Con A stimulation to the same extent as cells from the BALB/c mice. These results thus show that a low proliferative response to Con A stimulation in B10 mice was a consequence of a lower production of IL-2 and possibly abrogated the proliferative hyporeactivity produced by exogenous IL-2. We suggest that the differences in the ability to produce IL-2 could be a reason for the discrepancies observed in the immunological responsiveness between BALB/c and B10 mice.     

Il-10 is a central regulator of cyclooxygenase-2 expression and prostaglandin production

IL-10 is a potent anti-inflammatory and immune regulatory cytokine. IL-10(-/-) mice produce exaggerated amounts of inflammatory cytokines when stimulated with LPS, indicating that endogenous IL-10 is a central regulator of inflammatory cytokine production in vivo. PGs are lipid mediators that are also produced in large amounts during the inflammatory response. To study the role of IL-10 in the regulation of PG production during the acute inflammatory response, we evaluated LPS-induced cyclooxygenase (COX) expression and PG production in wild-type (wt) and IL-10(-/-) mice. LPS-induced PGE(2) production from IL-10(-/-) spleen cells was 5.6-fold greater than that from wt spleen cells. LPS stimulation resulted in the induction of COX-2 mRNA and protein in both wt and IL-10(-/-) spleen cells; however, the magnitude of increase in COX-2 mRNA was 5.5-fold greater in IL-10(-/-) mice as compared with wt mice. COX-1 protein levels were not affected by LPS stimulation in either wt or IL-10(-/-) mice. Neutralization of IFN-gamma, TNF-alpha, or IL-12 markedly decreased the induction of COX-2 in IL-10(-/-) spleen cells, suggesting that increased inflammatory cytokine production mediates much of the COX-2 induction in IL-10(-/-) mice. Treatment of IL-10(-/-) mice with low doses of LPS resulted in a marked induction of COX-2 mRNA in the spleen, whereas wt mice had minimal expression of COX-2 mRNA. These findings indicate that, in addition to IL-10's central role in the regulation of inflammatory cytokines, endogenous IL-10 is an important regulator of PG production in the response to LPS.     

Tissue factor as a proinflammatory agent

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element of blood coagulation. TF expression on monocytes and endothelial cells is induced by exposure to endotoxin, tumor necrosis factor, and IL-1 and is considered to appear in consequence of inflammation. In order to assess the proinflammatory capacity of TF itself, the recombinant extracellular domain of TF was injected intra-articularly into healthy mice. To characterize the role of immune cells in the TF-induced arthritis, mice deprived of lymphocytes, neutrophils and monocytes were used. Histomorphological analysis of the joints with respect to inflammatory cell infiltration, pannus formation and erosion formation revealed development of arthritis in 80% of animals injected with TF. In most of the cases synovial proliferation was accompanied by pannus formation and cartilage destruction. Inflammatory cell infiltrate consisted of CD4^-Mac1⁺ macrophages. Depletion of monocytes was, however, not enough to abolish inflammation. Indeed, combined deficiency of monocytes and lymphocytes was required to prevent inflammation following the injection of TF. We observed that TF induced chemokine production (MIP-1α and RANTES), but did not induce a proliferative response nor cytokine release by mouse spleen cells. TF has strong inflammatogenic properties mediated predominantly by monocytes and their release of chemokines. Our study shows that TF can simultaneously trigger the immune and coagulation systems.     

Transfer of cell-mediated immunity with cell-free leukocyte extracts: II. Demonstration of antigen-specific and nonspecific components

Transfer of cell-mediated immunity was achieved with dialyzable cell-free extracts from lymphoid cells of mice primed to the contact sensitizing agent, 2,4-dinitrofluorobenzene (DNFB). The biological activity of the extract (Transfer Factor, TF) was analyzed in vivo by the ear thickness assay and in vitro by the macrophage migration inhibition (MMI) test and lymphocyte transformation using the soluble analog, sodium 2,4-dinitrobenzenesulfonate. Consistently positive responses occurred 20 hr following a single intravenous injection of 5 × 10⁷ lymphocyte equivalents per recipient. The most potent source of TF (memory TF) was lymph node cells obtained 30 days after primary exposure to DNFB. By contrast TF prepared at the peak of the response to DNFB was less potent which was shown to be due to the presence in it of a suppressor factor. Memory TF elicited macrophage inhibition factor production in naive lymph node cells whereas positive responses were only obtained in the ear thickness and lymphocyte transformation assays provided recipients had undergone prior subliminal sensitization. Specificity of TF was tested using picryl chloride and oxazolone as control antigens. Results from the MMI and ear thickness assays were consistent with the presence in Transfer Factor of an antigen-specific component. Its effects, however, on the proliferative response to antigen lacked specificity and depended on prior sensitization of recipients, rather than donors, to the inducing antigen. The target of the specific component was considered to be an Ly-1⁺, Ia^?, $\text{Ly}\text{2}\text{3}{}^{\mathrm{?}}$ T cell since MIF production and in vivo delayed hypersensitivity are known to be mediated by a T cell bearing this phenotype. Taken together these findings emphasize the value of using a battery of tests of cell-mediated immune function when studying soluble mediators such as Transfer Factor and suggest that the current system is a valid experimental model for analysis of the Transfer Factor phenomenon.     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.     

Interleukin-2 secretion by KHT sarcoma cells leads to loss of their vaccine potential

 We have investigated the effect of interleukin-2 (IL-2) secretion by KHT sarcoma cells upon their vaccine potential in syngeneic C3Hf/He mice. Parental KHT tumor cells were transfected with the plasmid pBCMG-neo-mIL-2 to obtain a transfectant KHT-2-3-7 that secreted 20 units IL-2. KHT-2-3-7 cells elicited protective immunity in only 10% of the immunized mice, compared with 40% of mice immunized with irradiated parental KHT tumor colls. To minimize the contribution of potential antigenic differences between the KHT-2-3-7 transfectant and parental KHT cells, a clone of KHT cells (KHT-C21) was isolated and used in subsequent experiments. A number of transfectants secreting various amounts of IL-2, ranging from 2 units to 200 units, were obtained following transfection of KHT-C21 cells with plasmid pBCMG-neo-mIL-2. Two of the transfectants, C21-13-4 and C21-1, each secreting 200 units IL-2, elicited protective immunity in a significantly lower fraction of mice than did irradiated KHT-C21 parental tumor cells (P<0.0l). Two other transfectants C21-10 and C21-11, secreting 2 and 23 units IL-2 respectively, also showed lower vaccine potential compared with the parental KHT-C21 clone (P<0.05). To minimize further any role for potential antigenic or other molecular differences between the individual transfectants and the clonal KHT-C21 parental cells in lowering their vaccine efficacy, mice were immunized with a mixture of five transfectants, and the results again showed significantly lower vaccine efficacy of the mixture compared with the irradiated parental C21 cells (P<0.0l). In view of published studies showing enhanced or unchanged efficacy of IL-2-secreting tumor cell vaccines, our observation of the lower vaccine potential of IL-2-transduced tumor cells indicates that the vaccine efficacy of IL-2-secreting tumor cells depends on the individual tumor. Such variability/unpredictability would hamper the clinical use of IL-2-secreting tumor cells as vaccines. Received: 23 April 1996 / Accepted: 7 February 1997     

Modulation of Cytokine Secretion Allows CD4 T Cells Secreting IL-10 and IL-17 to Simultaneously Participate in Maintaining Tolerance and Immunity

CD4 T cells secreting IL-10 or IL-17 are frequent at mucosal sites, where their equilibrium is important for simultaneously maintaining tolerance and immunity to the resident microbiota. The mode of action of these cells, however, is as yet incompletely understood. In this study, we have combined ex vivo analysis of CD4 T cells producing IL-10 or/and IL-17 with assessment of clonal populations isolated ex vivo using a cytokine catch assay. We found that circulating CD4 T cells secreting IL-10 or/and IL-17 ex vivo include both conventional FOXP3^- CD4 T cells and FOXP3⁺ Helios^- Treg. Upon assessment of clonal populations derived from single ex vivo isolated cytokine secreting cells, we found that IL-10 or/and IL-17 secreting cells prevalently secrete one or the other cytokine depending on the type of stimulation, the time after stimulation and the presence of microbial products. Namely, IL-10 secretion by clonal cells was prevalent at early time points after TCR mediated stimulation, was independent of co-stimulation and was increased in the presence of the microbial fermentation product butyrate. In contrast, IL-17 secretion was higher at later time points after TCR mediated stimulation and in the presence of co-stimulatory signals. Taken together, these results provide insights into the mechanisms that, through modulation of cytokine secretion depending on conditions, allow IL-10 and IL-17 producing CD4 T cells to contribute to maintain tolerance to microbes locally, while retaining the ability to participate in protective immune responses at distant sites.     

Leptin and the immune system: how nutritional status influences the immune response

Several observations suggest the presence of an interaction between immune and the endocrine systems. Leptin is an adipocyte-derived hormone, that belongs structurally to the long-chain helical cytokine family such as interleukin-2 (IL-2), interleukin-12 (IL-12), growth hormone (GH), and signals by a class I cytokine receptor (Ob-R). This cytokine represents an important link between fat mass on the one side and the regulation of energy balance and reproductive function on the other. Indeed, obese leptin-deficient ob/ob mice display low body temperature, hyperphagia, infertility and evidence of immune defects with lymphoid organ atrophy, mainly affecting thymic size and cellularity. Acute starvation, associated with decreased leptin levels, causes thymic atrophy and reduces the delayed type hypersensitivity (DTH) reaction to antigens in normal mice, resembling that observed in ob/ob mice. Leptin replacement reverses the immunosuppressive effects of acute starvation in mice. Leptin differentially affects the in vitro proliferation and cytokine production by naive and memory T cells, increasing IL-2 secretion and proliferation of naive T cells, while inducing IFN-g production in memory T cells with little effect on their proliferation. Presence of leptin seems to be necessary for the induction and maintenance of the pro-inflammatory Th1 immune response. These findings support the hypothesis that leptin plays a key role in linking nutritional state to the T cell function. According to this view, leptin might represent an important target for immune intervention in a variety of pathophysiological conditions.     

IL-4 from Th2-type cells suppresses induction of delayed-type hypersensitivity elicited shortly after immunization

The pure delayed-type hypersensitivity reaction obtained in 4-day ovalbumin-sensitized mice after antigen challenge in the footpad was abrogated by transfer of in vitro expanded, antigen-specific lymphoblasts derived from ovalbumin-hyperimmunized donors (high antibody producers), 12 h before immunization. This effect was specific inasmuch as Trypanosoma cruzi-specific blasts derived from Tc-Ag-hyperimmunized mice did not inhibit delayed-type hypersensitivity in ovalbumin-immunized recipients. The ovalbumin-specific blasts displayed a Th2 cytokine profile, secreting IL-4 and IL-10 upon restimulation in vitro with ovalbumin, but not IFN-gamma or IL-2. In addition, recipients of such cells produced much more IgG1 and IgE antibodies. When the frequency of T-cell blasts was enriched among these cells, transfer of four million cells was enough to prevent the induction of delayed-type hypersensitivity. Neutralization of IL-4 alone just before cell transfer not only restored the delayed-type hyper-sensitivity reaction, but also maintained it in a plateau for at least 72 h after challenge. Recipients treated in this way also showed a shift back towards a Th1 phenotype, indicated by the increase in IL-2, IFN-gamma and IL-12 synthesis. No synergistic action was observed when IL-4 and IL-10 were concomitantly neutralized. These results indicate that activation of Ag-specific Th2 cells early in the course of the immune response to a protein antigen provides an immunological environment rich in IL-4, thus leading to the inhibition of cell-mediated immunity.     

Cytokine profile in mice with enhanced or depressed immune response to sheep erythrocytes induced by immunomodulator of bacterial origin--purified staphylococcal toxoid

Influence of immunomodulator of bacterial origin - purified staphylococcal toxoid (PST) - on the synthesisof proinlammatory (IL-1beta, IL-6, TNFalpha, IFN-gamma) and anti-inflammatory (IL- 10) cytokines, as well as cytokines directing the immune response to Th1 (IL-12) or Th2 (IL-4) type was studied in mice. Serum cytokines levels as well as levels of cytokines produced by splenocytes spontaneously or after stimulation by phytohemagglutinin were measured 4 and 24 hours after inoculation of PST. It was shown that PST in wide spectrum of doses (15; 1.5; 0.15 BU per mouse) was able to enhance or suppress synthesis of cytokines. Effect was nonlinear and its direction was depended from cytokine, time interval passed before obtaining the sample and dose of PST. For example, 15 BU of PST enhanced whereas 0.15 BU of PST suppressed the IL-6 production 4 hours after inoculation. Decrease of IL-6 level in serum 24 hours after inoculation of PST was detected. Synthesis of several serum interleukins (IL-2, IL-10) did not changed 4 and 24 hours after inoculation irrespective from dose of PST. It was demonstrated that modulation of humoral immune response in vivo induced by PST did not associated with modulation of cytokine profile. For example, increase of number of cells secreting antibodies to sheep erythrocytes was registered both during increased synthesis of cytokines (4 hours, IL-1beta, IL-6, IL-12) and during period of its depression (IL-6, TNF-alpha, IFN-gamma), as well as during stable production of cytokines (IL-1beta, IL-6, IFN-gamma).     

Effect of an orally applied herbal immunomodulator on cytokine induction and antibody response in normal and immunosuppressed mice

The influence of the oral administration of a herbal immunomodulator, consisting of an aqueous-ethanolic extract of the mixed herbal drugs Thujae summitates, Baptisiae tinctoriae radix, Echinaceae purpureae radix and Echinaceae pallidae radix, on cytokine induction and antibody response against sheep red blood cells was investigated in mice. The treatment of the animals with the extract caused no enhancement of the cytokine titers in the serum. Spleen cells isolated from the treated mice, however, produced higher amounts of IL-2, IFNgamma and GM-CSF ex vivo in comparison to spleen cells isolated from control animals, especially after additional stimulation by lipopolysaccharides or concanavalin A. The application of the extract also triggered the production of IL-1 and TNFalpha by peritoneal macrophages ex vivo. The influence of the herbal extract on the antibody response was examined by the plaque forming cell assay. The administration of the extract caused a significant enhancement of the antibody response against sheep red blood cells, inducing an increase in the numbers of splenic plaque forming cells and the titers of specific antibodies in the sera of the treated animals. In mice, immunosuppressed by old age or additional treatment with hydrocortisone, the therapy with the extract resulted in a normalization of the antibody response against sheep red blood cells.     

IL-4 and IL-4 Receptor Expression Is Dispensable for the Development and Function of Natural Killer T Cells

CD4 T cells acquire functional properties including cytokine production upon antigenic stimulation through the T cell receptor (TCR) and differentiate into T helper (Th) cells. Th1 cells produce interferon (IFN)-γ and Th2 cells produce interleukin (IL)-4. Th1 and 2 cells utilize IFN-γ and IL-4 for further maturation and maintenance, respectively. Promyelocytic leukemia zinc finger (PLZF)-expressing invariant natural killer T (iNKT) cells develop in the thymus and acquire functional ability to produce IL-4 and IFN-γ in the thymus in the absence of antigenic stimulation. In response to antigenic stimulation, iNKT cells rapidly produce IFN-γ and IL-4. However, it is still unknown as to whether iNKT cells require these cytokines for maturation or survival in vivo. In this study, using IL-4- and IL-4 receptor- (IL-4R) deficient mice, we demonstrate that IL-4 as well as IL-4R expression is dispensable for the development, function and maintenance of iNKT cells.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.     

Involvement of the Th1 subset of CD4+ T cells in acquired immunity to mouse infection with Trypanosoma equiperdum.

Heat- or merthiolate-inactivated Trypanosoma equiperdum was administered to recipient mice that were subsequently challenged with viable inocula of the same stabilate. Only mice inoculated with merthiolate-killed parasites were completely protected from a challenge inoculum of 10(3) trypanosomes, an effect that was abolished by prior immunosuppression of mice. Immune sera from protected animals contained high levels of interferon (IFN)-gamma and specific IgG2a antibodies. Spleen cells from these mice produced high amounts of interleukin (IL)-2 and IFN-gamma in vitro in response to specific antigen or concanavalin A, whereas splenocytes from mice receiving heat-killed parasites produced high amounts of IL-6. In contrast, the production of tumor necrosis factor (TNF)-alpha and colony-stimulating activity (CSA) was not significantly different in mice receiving either killed parasite preparation. The protection in immunized mice was associated with the detection of strong delayed-type hypersensitivity (DTH) to T. equiperdum antigens, an effect that could be adoptively transferred onto naive recipients by specifically immune CD4+ lymphocytes. These results suggest that the development of protective immunity in mice to T. equiperdum by our immunization protocol may involve the activity of helper/DTH T cells, particularly those of the Th1 subset.