Basophil depletion downregulates Schistosoma mansoni egg-induced granuloma formation

Granuloma formation around parasite eggs during schistosomal infection is considered to be controlled by Th2 cytokines. However, it is still controversial which cell populations are responsible for the host Th2 cytokine-dependent granuloma formation. Basophils have recently attracted attention because of their ability to produce large amounts of IL-4. Therefore, we investigated whether basophils play an essential role in the induction of granuloma formation induced by Schistosoma mansoni eggs. Together with our previous observation that basophil numbers increased markedly in the spleen at 7 weeks postinfection, immunohistochemical staining using anti-mMCP8 monoclonal antibody (mAb) showed basophil infiltration in the granulomatous lesions formed around parasite eggs. To examine the roles of basophils more directly, we treated mice with anti-CD200R3 mAb to deplete basophils. Depletion of basophils resulted in a reduction of basophil number with concomitant downregulation of egg granuloma formation at 7 weeks postinfection. Moreover, we observed a significant reduction in the size of egg granulomas formed in basophil-depleted mice in the pulmonary granuloma model. Taken together, these findings indicated that basophils are essential for S. mansoni egg-induced granuloma formation, and this may serve as a novel therapeutic target in ameliorating the pathology of schistosomiasis.     

Delayed hypersensitivity granuloma formation around Schistosoma mansoni eggs in vitro. III. Granuloma formation and modulation in human Schistosomiasis mansoni

An in vitro model of granuloma formation was used to study the cellular immune responses of Schistosoma mansoni-infected patients. The purposes of this study were to determine the relationship of granulomatous hypersensitivity to S. mansoni eggs in recent, well-defined infections and long-term chronic infections, and to determine the role of T cell subsets (OKT3, 4, and 8) defined by monoclonal antibodies in granulomatous hypersensitivity. Peripheral blood mononuclear cells obtained from patients with recent S. mansoni infections demonstrated increased granulomatous hypersensitivity responses in vitro when compared to peripheral blood mononuclear cells obtained from patients infected for 5 yr or more. The selective removal of infected for 5 yr or more. The selective removal of OKT3+ or OKT4+ cells reduced the ability of peripheral blood mononuclear cells to form granulomas in vitro. Positive selection for OKT4+ T cells produced optimal granulomatous hypersensitivity when compared to that produced by the unfractionated peripheral blood mononuclear cell population. OKT8+ cells demonstrated no ability to form granulomas in vitro. Selective removal of OKT8+ T cells produced variable results in the ability of the remaining peripheral blood mononuclear cells to form granulomas in vitro. These studies demonstrate the feasibility of investigating granulomatous hypersensitivity and immunoregulatory mechanisms operative in S. mansoni-infected patients by using in vitro technology.     

Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation. Orchestration, relative contribution, and relationship to macrophage function.

A well defined model of T cell-mediated hypersensitivity-type granulomatous inflammation induced by Schistosoma mansoni eggs was used to assess the role of IL-4 and IFN-gamma in granuloma development. Synchronized pulmonary granulomas were induced and isolated from S. mansoni-infected mice during vigorous (8 wk) and modulated (20 wk) stages of the disease. The sequential production of IL-4 and IFN was determined and related to temporal changes in granuloma macrophage production of IL-1, TNF, and superoxide anion (O2-). During the vigorous stage, IL-4 was produced on days 1 and 2 of granuloma formation, whereas IFN was released in greatest amounts on days 4 to 8. The peak of IL-4 occurred in a window between the peak of IL-1 (1 day) and maximal TNF production (8 to 16 days). Maximal O2- release tended to parallel IFN production. During the modulated stage when the inflammatory response is attenuated, IL-4 production was dramatically reduced as were levels of IL-1 and TNF, but IFN production persisted and maximum O2(-)-producing capacity was only delayed in onset. mAb specific for IL-4 and IFN were used to examine the effect of in vivo depletion of these cytokines on granuloma development. Administration of a single 1.0-mg dose of anti-IL-4 antibodies to mice with synchronously developing granulomas dramatically reduced granuloma size (40 to 50% suppression of area) during an 8-day study period, whereas antibodies to IFN had no effect on size. However, the latter treatment reduced giant cell formation. Our results indicate that granuloma development involves an orchestrated production of cytokines possibly resulting from sequential participation of different Th cell populations. Moreover, IL-4 is a pivotal cytokine in anamnestic cellular recruitment and subject to endogenous regulation.     

The role of chemokines in Schistosoma mansoni granuloma formation

The eradication of parasitic diseases, such as schistosomiasis, has been the focus of investigations worldwide for many decades. However, attempts to control their continual spread have, at best, been met with limited success. In the face of these results, it is important to attempt to understand and thus to control the pathology of these widespread diseases. In this review, Nicholas Lukacs, Steven Kunkel, Robert Strieter and Stephen Chensue focus on a family of cytokines that play a pertinent role for leukocyte recruitment in chronic inflammation.     

The molecular basis of granuloma formation in schistosomiasis. III. In vivo effects of a T cell-derived suppressor effector factor and IL-2 on granuloma formation

Granuloma formation in schistosomiasis is characterized by the formation of a large lesion in acutely infected animals which subsequently decreases in size as disease progresses into the chronic phase. These in vivo studies confirm and extend previous in vitro observations on the regulation of granulomatous hypersensitivity by a T cell-derived suppressor effector factor (TseF). TseF regulation of granuloma formation in vivo and DTH are shown to be both antigenically and genetically restricted. This suppression is accompanied by a suppression of the ability of cells derived from TseF recipients to function in an in vitro assay of granuloma formation. Antigenic recognition, defined by cellular proliferation in response to antigenic stimulation, is uneffected by TseF administration. Administration of IL-2 reduces TseF function in acutely infected mice and results in increased liver granuloma size. However, the ability of cells derived from these animals to form granulomas in vitro is uneffected. Cells obtained from chronically infected IL-2 recipients do not produce TseF in vitro and granuloma size is increased in these animals. Animals receiving both IL-2 and TseF continue to demonstrate decreased granuloma formation, indicating that IL-2 does not effect the ability of preformed TseF to function. These observations suggest that TseF modulates granuloma formation in vivo and may interact with IL-2 in a dynamic process which determines the intensity of the granulomatous response.     

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.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.     

Mice infected with Schistosoma mansoni develop a novel non-T-lymphocyte suppressor population which inhibits virus-specific CTL induction via a soluble factor.

We previously observed that Schistosoma mansoni-infected mice were deficient in their ability to mount a CTL response to unrelated viral antigens and to clear a vaccinia viral infection. Here, we explore the mechanism of that deficiency. Mixing experiments showed that splenocytes from S. mansoni-infected mice actively suppress stimulation in vitro of both viral-peptide specific CTL in spleen cells from virus-infected mice, and allospecific CTL. The mechanism of suppression involves at least in part a soluble factor, in that it can occur across a 0.4-microm membrane which prohibits direct cell contact. However, the inhibition is not alleviated by blocking with antibodies to IL-4, IL-10 or TGF-beta. Fractionation of the splenocyte population from S. mansoni-infected mice shows that the suppression is mediated by a non-B, non-T cell that expresses CD16 and Mac-1, but not FcepsilonR or NK1.1. This represents a novel suppressor population that is distinct from the FcepsilonRI(+) populations of non-B, non-T cells in the spleens of S. mansoni-infected mice that provide a major source of IL-4 in these animals. Similar cells in schistosome-infected humans could affect susceptibility to other infections or responsiveness to vaccines.     

Schistosoma mansoni: stimulation of artificial granuloma formation in vivo by carbohydrate determinants

A subset of Schistosoma mansoni egg glycoproteins that share a common carbohydrate epitope recognized by monoclonal antibody 128C3 was shown to induced formation of hepatic granulomata when conjugated to Sepharose beads and injected into the portal circulation of naive mice. Concanavalin-binding egg glycoproteins exhibited more granuloma-inducing activity than did total egg extract, although deglycosylated egg proteins also induced granulomata; thus, both amino acid and carbohydrate epitopes appeared to be involved. Glycoproteins derived from adult male worms also were active, indicating that immunological processes responsible for granuloma formation may not be absolutely stage specific.     

LacdiNAc- and LacNAc-containing glycans induce granulomas in an in vivo model for schistosome egg-induced hepatic granuloma formation

Schistosomes, major parasitic helminths, express numerous glycoconjugatesthat provoke humoral and cellular immune responses in the infectedhuman host. The main pathology in schistosomiasis is due tothe formation of granulomas around tissue-trapped eggs and theresulting organ damage. By using a mouse model of inductionof granulomas by hepatic implantation of antigen-coated beads,it has been determined that the glycan part of schistosomalsoluble egg antigens (SEA) initiates granulomogenesis. To identifywhich individual glycan elements in this complex SEA mixtureare granulomogenic, we have tested in the same mouse model conjugatesof various synthetic oligosaccharides characteristic for schistosomeeggs, including GalNAcß1-4GlcNAc (LacdiNAc, LDN),Galß1-4(Fuc1-3)GlcNAc (Lewis^x), Fuc1-2Fuc1-3GlcNAc(DF-Gn), and Fuc1-3GalNAcß1-4(Fuc1-3)GlcNAc (F-LDN-F).Ribonuclease (RNase) A and B, and different fetuin glycoformswere included as controls. Only beads that carry glycoconjugateswith terminal LacdiNAc or Galß1-4GlcNAc (LacNAc, LN)elements gave rise to granulomas, with macrophage, lymphocyte,and eosinophil levels similar to the granulomatous lesions causedby schistosome eggs in a natural infection. Uncoated beads,and beads coated with fucosylated glycoconjugates or glycoconjugateslacking terminally exposed Gal or GalNAc, only attracted a monolayerof macrophages. These results indicate that the formation ofhepatic granulomas is triggered specifically by glycoconjugateswhich carry terminal LacNAc or LacdiNAc, both constituents ofthe schistosome egg.     

Modulation of granulomatous hypersensitivity. I. Characterization of T lymphocytes involved in the adoptive suppression of granuloma formation in Schistosoma mansoni-infected mice.

The cellular basis of the spontaneous modulation of the granulomatous response to schistosome eggs was analyzed in mice infected with Schistosoma mansoni. Spleen cells of 20 or 32 week-infected mice undergoing modulation, when transferred to 6 week-infected recipients, suppressed the maximal granulomatous response at 8 weeks. Suppression of both naturally forming asynchronous liver and synchronously induced lung lesions was achieved. Specificity of this effect was demonstrated by the suppression of egg granulomas but not antigen-coated bead granulomas developing simultaneously in the lungs of cell recipients. Further characterization showed that suppression was abrogated by pretreating transferred cells with either anti-Thy 1.2 or anti-Iak alloantisera and C. Transfer of macrophage-depleted or fractionated T and B spleen cells confirmed that T cells alone transferred suppression. Moreover, an Ia antigen-bearing (Ia+) subpopulation of T cells was required in the transferred suppression. Moreover, an Ia antigen-bearing (Ia+) subpopulation of T cells was required in the transferred population. An examination of T cells obtained from isolated, dispersed lung granulomas from control and adoptively suppressed mice revealed an increased proportion of Ia+ cells in the latter. It is suggested that Ia+ T cells may be involved in the local modulation of the granulomatous response.     

IgE-binding factors from mouse T lymphocytes. III. Role of antigen-specific suppressor T cells in the formation of IgE-suppressive factor

BDF1 mice were given three i.v. injections of ovalbumin (OA) to induce antigen-specific suppressor T cells. Incubation of spleen cells of OA-treated mice with homologous antigen resulted in the formation of IgE-suppressive factor. This factor was not derived from antigen-specific suppressor T cells, but suppressor T cells were essential for determining the nature of IgE-binding factors formed. In the spleen cells of OA-treated mice, antigenic stimulation of antigen-primed Lyt-1+ (helper) T cells resulted in the formation of inducers of IgE-binding factor, whereas Lyt-2+, I-J+ T cells released glycosylation-inhibiting factor (GIF), and these two factors, in combination, induced unprimed Lyt-1+ T cells to form IgE-suppressive factor. The role of GIF is to inhibit the assembly of N-linked oligosaccharides on IgE-binding factors during their biosynthesis, and thereby provide them with a biologic activity: suppression of the IgE response. Under the experimental conditions employed, GIF was released spontaneously from antigen-specific suppressor T cells. However, antigenic stimulation of the cells enhanced the release of the factor. GIF from antigen-specific suppressor T cells has a m.w. of 25,000 to 30,000, as estimated by using gel filtration, binds to anti-I-J alloantibodies and to a monoclonal antibody specific for lipomodulin, and has affinity for specific antigen. The possible relationship between antigen-specific GIF and antigen-specific suppressor factors is discussed.     

The molecular basis of granuloma formation in schistosomiasis. II. Analogies of a T cell-derived suppressor effector factor to the T cell receptor

During Schistosoma mansoni infection, Ts cells regulate granulomatous modulation via antigenically and genetically restricted suppressor inducer and suppressor effector factors. The T suppressor effector factor (TseF) directly suppresses granuloma formation both in vitro and in vivo. In this study, we probe the molecular basis of these TseF properties. Using techniques of heterodimeric chain reduction with DTT and in vitro functional complementation, chimeric molecules were constructed. By analyzing genetic restrictions, antigenic specificities, and phenotypic markers, the contributions of the component chains to 72 kDa TseF reactivity were determined. One chain bore an Ag receptor and imparted antigenic specificity. The other chain bore an IJ determinant, a TCR beta-chain allotypic determinant, a suppressor effector phenotypic determinant, and imparted functional genetic restriction. Functional activity required covalent, probably sulfhydryl mediated, linkage as succinylation prevented the separated component chains from reconstituting functional activity. Additional studies demonstrated that anti-serum directed against either the T cell receptor or the T3 epsilon-chain could abrogate functional activity. However, TseF bore no T3 epsilon-chain phenotypic marker per se suggesting that TseF effects T lymphocytes via transmembrane signal transduction. These studies suggest that a regulatory network is operative in granuloma modulation. This regulatory network is mediated by a soluble TseF that bears significant structural homologies to the classic TCR.     

Skin-stage schistosomula of Schistosoma mansoni produce an apoptosis-inducing factor that can cause apoptosis of T cells

Skin-stage schistosomula of Schistosoma mansoni were found to secrete molecules that are pro-apoptotic for skin T lymphocytes as measured by annexin V staining, caspase-3 activity, caspase-8 activities, and DNA fragmentation. Caspase-8 activities in lymphocytes peaked approximately 8 h and caspase-3 activity peaked approximately 16 h after exposure to the parasite secretions. Subset analysis showed that mainly CD4(+) and CD8(+) cells (but not B cells) were susceptible to the parasite-induced pro-apoptotic effect. In situ staining confirmed the presence of apoptotic T cells around challenge parasites in the skin of naive or immunized animals. Analysis of T cells to identify the potential molecular pathway of the parasite-induced apoptosis showed increases in the expression of Fas, FasL, and the Fas-associated death domain. Blocking of FasL with a fusion protein reversed the parasite-induced apoptosis, suggesting a role for the Fas/FasL-mediated pathway in the parasite-induced T cell apoptosis. Subsequent analyses of the secretions of skin-stage schistosomula identified the pro-apoptotic activity as being associated with a protein of approximately 23 kDa. This protein was termed S. mansoni-derived apoptosis-inducing factor.     

Production of a fibroblast-stimulating factor by Schistosoma mansoni antigen-reactive T cell clones

Fibrosis in schistosomiasis is the terminal event of a complex pathophysiologic cascade involving interactions between fibroblasts and both host and parasite products. In the present study, the effect of lymphokines produced by cloned Schistosoma mansoni antigen-reactive T cells on the proliferation of murine fibroblasts was investigated. These T cells previously have been shown to proliferate, produce lymphokines, mediate delayed-type hypersensitivity responses, and generate in vitro granulomas in response to soluble egg antigen (SEA). T cells, co-cultured with irradiated antigen-presenting cells and pulsed with SEA, produced levels of fibroblast-stimulating factor (FSF) comparable to equivalent numbers of dispersed hepatic granuloma cells isolated from infected mice. Supernatants of cloned T cells pulsed with Con A (in the absence of macrophages) contained no detectable interleukin 1 activity, but did stimulate fibroblast activation and growth. T cell FSF activity was trypsin-sensitive, was stable at 56 degrees C but not to boiling, and was retained by Con A Sepharose. Activity was associated with HPLC fractions corresponding to an m.w. of 10,000 to 40,000. Neither recombinant interferon-gamma nor affinity-purified interleukin 2 was capable of stimulating fibroblast proliferation. In functional studies, the degree of fibroblast proliferation was related to the length of exposure to the factor. In addition, quiescent fibroblasts were maximally stimulated by T cell FSF only if a second co-factor such as insulin or epidermal growth factor was present. The synergism between T cell FSF and known progression factors suggests that FSF-T may provide a competence signal to fibroblasts. The present results suggest that a direct molecular link may exist between T cells and fibroblasts in schistosomiasis.     

Naturally occurring CD4+Foxp3+ regulatory T cells are an essential, IL-10-independent part of the immunoregulatory network in Schistosoma mansoni egg-induced inflammation

In acute and chronic schistosomiasis, survival of the host requires a carefully balanced immune response against highly immunogenic parasite eggs. We characterized the phenotype, distribution, and functional role of CD4(+)Foxp3(+) naturally occurring regulatory T cells (naTregs) in schistosome egg-induced inflammation. In adoptive transfer experiments and by intracellular staining for Foxp3, we demonstrate significant frequencies of naTregs in hepatic granulomas and draining lymphoid tissues of mice infected with the trematode Schistosoma mansoni. Strikingly, egg-induced inflammation does not change the normal ratio between naTregs and effector CD4(+) T cells at the inflammatory site or in lymphoid organs in acute or chronic disease. However, increasing frequencies of CD103-expressing cells in the naTreg compartment indicate a change in phenotype for naTregs with disease progression. Because CD103 was described recently as an activation marker for naTregs, we speculate that naTregs in chronic schistosomiasis are potentially more suppressive. Furthermore, we found that most naTregs do not contribute to egg-induced IL-4 and IL-10 production. Importantly, depletion of CD25(+) naTregs strongly enhances the frequency of IL-4-producing effector T cells in acute egg-induced inflammation. It does not change clonal expansion of activated CD4(+) T cells. This regulation of egg-induced cytokine production does not require the presence of IL-10. These data demonstrate that naTregs limit egg-induced effector-cytokine production in our model. Our results identify naTregs as an important, IL-10-independent part of the regulatory network in schistosome egg-induced inflammation.     

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.     

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.     

Regulation of granulomatous inflammation in murine schistosomiasis. III. Recruitment of antigen-specific I-J+ T suppressor cells of the granulomatous response by I-J+ soluble suppressor factor

Down-modulation of the schistosome egg-induced granulomatous response involves various interacting subsets of T suppressor (TS) lymphocytes. In the present study the inductive phase of the process of modulation was analyzed. A soluble, I-J+ granuloma TS cell recruiting factor (Gr-TSRF) derived from spleen cells of chronically infected mice is described. This factor eluted from immunoabsorbent columns coupled with anti-I-Jk alloantisera induced the recruitment and expansion of antigen-specific I-J+ TS cells from a TS precursor cell population in the spleens of acutely infected mice. The recruited TS cells suppressed the granulomatous response of normal recipients in a 2-day adoptive transfer model. The antigenic specificity of the recruited TS cells was demonstrated by their inability to suppress KLH-induced artificial granulomatous response. This mechanism of recruitment described in the current study and illustrated by adoptive transfer experiments is likely to be active in vivo in initiating the process of spontaneous modulation. The I-J+ Gr-TSRF and the I-J+ TS cell described in this paper, together with the previously described H-2 restricted I-C+ factor and the subsets of TS cells (THs, TSe, TSpr), indicate the existence of an intricate, regulatory pathway(s) that operates during the modulation of the granulomatous response.