Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells.

We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150-250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core.

Th17-cell plasticity in Helicobacter hepaticus-induced intestinal inflammation.

Bacterial-induced intestinal inflammation is crucially dependent on interleukin (IL)-23 and is associated with CD4(+) T helper type 1 (Th1) and Th17 responses. However, the relative contributions of these subsets during the induction and resolution of colitis in T-cell-sufficient hosts remain unknown. We report that Helicobacter hepaticus-induced typhlocolitis in specific pathogen-free IL-10(-/-) mice is associated with elevated frequencies and numbers of large intestinal interferon (IFN)-γ(+) and IFN-γ(+)IL-17A(+) CD4(+) T cells. By assessing histone modifications and transcript levels in IFN-γ(+), IFN-γ(+)IL-17A(+), and IL-17A(+) CD4(+) T cells isolated from the inflamed intestine, we show that Th17 cells are predisposed to upregulate the Th1 program and that they express IL-23R but not IL-12R. Using IL-17A fate-reporter mice, we further demonstrate that H. hepaticus infection gives rise to Th17 cells that extinguish IL-17A secretion and turn on IFN-γ within 10 days post bacterial inoculation. Together, our results suggest that bacterial-induced Th17 cells arising in disease-susceptible hosts contribute to intestinal pathology by switching phenotype, transitioning via an IFN-γ(+)IL-17A(+) stage, to become IFN-γ(+) ex-Th17 cells.

IL-23 and Th17 cytokines in intestinal homeostasis.

The discovery of the Th1/Th2 paradigm of CD4(+) T-cell subsets redefined our understanding of immunity by highlighting the essential roles of cytokine networks in the induction and regulation of immune responses. Most recently, the identification of an additional subset, known as Th17 cells, has further illustrated the complexity and diversity of effector CD4(+) T cells. Th17 responses have been closely associated with the cytokine interleukin (IL)-23 and, although originally pinpointed as having a deleterious role in autoimmune tissue pathology, the IL-23/Th17 axis has also been associated with protective immunity at mucosal surfaces. Recent progress has highlighted the heterogeneous nature of Th17 responses, has demonstrated diverse cellular sources for Th17-associated cytokines, and has begun to dissect the individual roles of these cytokines in different disease processes. Here, we will review the evidence linking the IL-23/Th17 axis to chronic intestinal inflammation and also will discuss its beneficial roles in intestinal protection and homeostasis.

Improved drug delivery to cancer cells: a method using magnetoliposomes that target epidermal growth factor receptors.

The toxin delivery system described herein would allow for the selective killing of tumor cells overexpressing the epidermal growth factor receptor (EGFR). Tumor cells often overexpress EGFR, because it allows the cells to divide more quickly. Past delivery systems targeting this receptor have been ineffective due to a lack of specificity that results in harm to surrounding tissue and damage to organs such as the liver. The technique presented here is different, because it presents the possibility of delivering toxin only to the tumor area and almost exclusively to the tumor cells. Delivery is localized to the tumor tissue through the use of EGF conjugated magnetoliposomes. These are liposomes that have magnets imbedded in their bilayer, allowing for selective heating and release of a drug when the magnetoliposome is under an oscillating magnetic field. To create an additional level of specificity, the delivery system will consist of two EGF-bound components that must interact within the endosome of a cell for the toxin to be released. If a tumor cell overexpresses EGFR by 5-fold, then each of its endosomes will have 5 times more receptors than those of a normal cell. Therefore, the tumor cell's endosome has a 5 times greater chance of containing one EGF-bound component and a 25 times greater chance of containing both components. Since both components are necessary for toxin release, the tumor cells will receive 25 times more toxin than the normal cells. Theoretically, it is possible to produce a three or four component system that would deliver 125 or 625 times more toxin to the tumor cells.

Helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation.

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.

Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile.

The concept that IL-4 is the primary signal for Th2 lymphocyte differentiation has recently been put in doubt by studies in which the production of Th2-associated cytokines was detected in mice deficient in IL-4 synthesis or IL-4R triggering. In this study, we formally demonstrate by single cell analysis that CD4+ lymphocytes with a classical Th2 phenotype (IL-4+, IL-5+, IFN-gamma-, IL-2-) develop in significant numbers in helminth-infected mice deficient in either IL-4R alpha-chain or Stat6. While an expanded population of Th1 (IL-4-, IL-5-, IFN-gamma+, IL-2+) lymphocytes was observed in the same animals, surprisingly, cells with a mixed Th0 cytokine pattern were rare. The cytokine production phenotypes of the Th1 and Th2 subpopulations generated in infected Stat6-deficient mice were unaffected by in vitro neutralization of endogenous IL-4 or IFN-gamma. Nevertheless, while addition of exogenous rIL-12 resulted in transitory IFN-gamma production by Th2 lymphocytes from both wild-type and Stat6-deficient mice, IL-4 synthesis was preserved in the former, but temporarily ablated in the latter cells. Importantly, IL-4+ IFN-gamma- and IL-4- IFN-gamma+ populations similar to those arising in helminth-infected Stat6-deficient mice could also be generated in vitro by repetitive polyclonal stimulation of CD4+CD62Lhigh lymphocytes from uninfected mice of the same strain. Together, the results of these single cell analysis experiments demonstrate that IL-4R/Stat6 signaling, while influencing the final frequency of Th2 lymphocytes, is not essential for Th2 cell development, and suggest that this pathway has a previously unrecognized function in stabilizing Th2 populations once they have emerged.

Schistosome-infected IL-4 receptor knockout (KO) mice, in contrast to IL-4 KO mice, fail to develop granulomatous pathology while maintaining the same lymphokine expression profile.

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Ralpha-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Ralpha KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Ralpha-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-gamma secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Ralpha KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Ralpha-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Ralpha+ non-T cell effector population.

A novel urease-negative Helicobacter species associated with colitis and typhlitis in IL-10-deficient mice.

A spiral-shaped bacterium with bipolar, single-sheathed flagella was isolated from the intestines of IL-10 (interleukin-10)-deficient (IL-10(-/-)) mice with inflammatory bowel disease. The organism was microaerobic, grew at 37 and 42 degrees C, and was oxidase and catalase positive but urease negative. On the basis of 16S rRNA gene sequence analysis and biochemical and phenotypic criteria, the organism is classified as a novel helicobacter. Cesarean section-rederived IL-10(-/-) mice without helicobacter infection did not have histological evidence of intestinal inflammation. However, helicobacter-free IL-10(-/-), SCID/NCr, and A/JNCr mice experimentally inoculated with the novel urease-negative Helicobacter sp. developed variable degrees of inflammation in the lower intestine, and in immunocompetent mice, the experimental infection was accompanied by a corresponding elevated immunoglobulin G antibody response to the novel Helicobacter sp. antigen. These data support other recent studies which demonstrate that multiple Helicobacter spp. in both naturally and experimentally infected mice can induce inflammatory bowel disease. The mouse model of helicobacter-associated intestinal inflammation should prove valuable in understanding how specific microbial antigens influence a complex disease process.

Optimal vaccination against Schistosoma mansoni requires the induction of both B cell- and IFN-gamma-dependent effector mechanisms.

Mice immunized with radiation-attenuated cercariae of Schistosoma mansoni display resistance to challenge infection, which increases with multiple boosting. Protection in animals receiving a single vaccination is thought to involve a primarily cell-mediated, IFN-gamma-dependent mechanism, while humoral immunity has been shown to contribute to challenge rejection in multiply (three times) immunized mice. To better understand the respective contribution of the B lymphocyte- and IFN-gamma-dependent effector arms in host resistance, we compared vaccine-induced immunity in B cell-deficient (muMT) and IFN-gamma knockout (GKO) animals. Unexpectedly, after a single vaccination, B cell knockout (KO) mice displayed reduced protection against challenge infection, although they developed a normal IFN-gamma-dominated cytokine response. This defect in resistance was equivalent to that displayed by GKO animals. Moreover, whereas two additional vaccinations significantly increased the level of immunity in wild-type mice, the protection in B cell KO animals remained unchanged. In contrast, multiple vaccination resulted in increased but, nevertheless, defective resistance in GKO mice. Since FcR gamma KO mice, which lack functional FcgammaRI, FcgammaRIII, and FcepsilonRI, show no defects in vaccine-induced resistance after immunization either one or three times, the B cell-dependent mechanism of protection involved does not appear to require FcR signaling. Together, these findings indicate that effective vaccination against schistosomes depends on the simultaneous induction of both humoral and cell-mediated immunity, a conclusion that may explain the limited success of most subunit vaccine protocols designed to preferentially induce either B cell- or IFN-gamma-dependent protective mechanisms.

Altered immune responses in mice with concomitant Schistosoma mansoni and Plasmodium chabaudi infections.

Mixed parasitic infections are common in many parts of the world. However, little is known about how concurrent infections affect the immunity to and/or pathogenesis of each other. Protection and elimination of blood-stage Plasmodium chabaudi chabaudi AS in resistant mice are characterized by a sequential activation of CD4(+) Th1 and Th2 cells. The patent egg-laying stage of the murine model of Schistosoma mansoni is associated with a strong Th2 response to both Schistosoma and unrelated antigens. In this study, we investigated how infection of mice with S. mansoni would affect the immune response to and pathogenesis of a P. chabaudi infection. C57BL/6 mice infected with S. mansoni for 8 weeks were infected with blood-stage P. chabaudi. Malaria parasitemias were significantly higher in these mice than in mice infected with P. chabaudi only. In doubly infected mice, both spleen cell proliferative and Th2 responses to S. mansoni soluble egg antigen (SEA) or anti-CD3 were suppressed up to 1 month after the malaria infection. Findings for SEA-specific immunoglobulin M (IgM) and IgG serum antibody levels were similar. No significant effects were seen on P. chabaudi-induced gamma interferon responses. However, tumor necrosis factor alpha (TNF-alpha) production was significantly lower in double-infected mice. Thus, a defect in TNF-alpha production might contribute to the increased malaria parasitemias seen in S. mansoni-P. chabaudi-infected mice. Taken together, our data show that schistosoma and malaria infections profoundly affect each other, findings which might have implications for the development of vaccines.

Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism.

Mice rendered deficient in interleukin-10 (IL-10) by gene targeting (IL-10(-/-) mice) develop chronic enterocolitis resembling human inflammatory bowel disease (IBD) when maintained in conventional animal facilities. However, they display a minimal and delayed intestinal inflammatory response when reared under specific-pathogen-free (SPF) conditions, suggesting the involvement of a microbial component in pathogenesis. We show here that experimental infection with a single bacterial agent, Helicobacter hepaticus, induces chronic colitis in SPF-reared IL-10(-/-) mice and that the disease is accompanied by a type 1 cytokine response (gamma interferon [IFN-gamma], tumor necrosis factor alpha, and nitric oxide) detected by restimulation of spleen and mesenteric lymph node cells with a soluble H. hepaticus antigen (Ag) preparation. In contrast, wild-type (WT) animals infected with the same bacteria did not develop disease and produced IL-10 as the dominant cytokine in response to Helicobacter Ag. Strong H. hepaticus-reactive antibody responses as measured by Ag-specific total immunoglobulin G (IgG), IgG1, IgG2a, IgG2b, IgG3, and IgA were observed in both WT and IL-10(-/-) mice. In vivo neutralization of IFN-gamma or IL-12 resulted in a significant reduction of intestinal inflammation in H. hepaticus-infected IL-10(-/-) mice, suggesting an important role for these cytokines in the development of colitis in the model. Taken together, these microbial reconstitution experiments formally establish that a defined bacterial agent can serve as the immunological target in the development of large bowel inflammation in IL-10(-/-) mice and argue that in nonimmunocompromised hosts IL-10 stimulated in response to intestinal flora is important in preventing IBD.

Expansion of IL-3-responsive IL-4-producing non-B non-T cells correlates with anemia and IL-3 production in mice infected with blood-stage Plasmodium chabaudi malaria.

A prominent switch of CD4+ T cells from Th1 to Th2 type response occurs in mice infected with the non-lethal malaria parasite Plasmodium chabaudi chabaudi AS around the time of peak parasitemia. This is reflected by a decrease in IFN-gamma- and an increase in IL-4-producing cells. The peak occurs approximately 9-10 days after infection and is accompanied by anemia. The mechanism behind the switch in Th cell response is poorly understood. We here report on the production of IL-4 from a non-T cell source during P. chabaudi infection in BALB/c mice. Flow cytometric analysis of spleen and peripheral blood leukocytes (PBL) showed a dramatic increase in the percentage of non-B non-T (NBNT) cells 9-23 days after P. chabaudi infection with peak values by day 15 (approximately 30 % of splenocytes and approximately 55 % of PBL being NBNT cells). The expansion of NBNT cells correlated closely with the appearance of a cell type secreting IL-4 and IL-6 following stimulation with IL-3 and/or cross-linking of FcgammaR. Compared to cells from uninfected animals, NBNT cells from P. chabaudi-infected mice were shown to be hyper-responsive to IL-3. The levels of the hematopoietic cytokine IL-3 were elevated in supernatants from unstimulated spleen cell cultures as well as in serum at the same time points at which NBNT cell-derived IL-4 and IL-6 were detected from spleen cultures and PBL. Thus, IL-3-responsive IL-4-producing NBNT cells may provide cytokines supporting the switch from Th1 to a Th2 response which is important for the final clearance of the parasite in P. chabaudi malaria.

Characterization of mouse mast cell protease-8, the first member of a novel subfamily of mouse mast cell serine proteases, distinct from both the classical chymases and tryptases.

Using a recently developed PCR-based strategy, a cDNA encoding a novel mouse mast cell (MC) serine protease (MMCP-8) was isolated and characterized. The MMCP-8 mRNA contains an open reading frame of 247 amino acids (aa), divided into a signal sequence of 18 aa followed by a 2-aa activation peptide (Gly-Glu) and a mature protease of 227 aa. The mature protease has an M(r) of 25072, excluding post-translational modifications, a net positive charge of +12 and six potential N-glycosylation sites. MMCP-8 showed a high degree of homology with mouse granzyme B in the critical regions for determining substrate cleavage specificity, indicating that MMCP-8, similar to granzyme B, preferentially cleaves after Asp residues. A comparative analysis of the aa sequence of MMCP-8 with other hematopoietic serine proteases shows that it is more closely related to cathepsin G and T cell granzymes than to the MC chymases. We therefore conclude that MMCP-8 belongs to a novel subfamily of mouse MC proteases distinct from both the classical chymases and tryptases. Southern blot analysis of BALB/c genomic DNA indicated that only one MMCP-8 gene (or MMCP-8 like gene) is present in the mouse genome. Northern blot analysis of rodent hematopoietic cell lines revealed high levels of MMCP-8 mRNA in a mouse connective tissue MC-like tumor line. However, MMCP-8 mRNA could not be detected in mouse liver, intestine, lung or ears, indicating very low expression in normal tissues. Analysis of the expression of different MMCP in the tissues of Schistosoma mansoni-infected BALB/c mice showed a strong increase in MMCP-8 levels in the lungs but not in the intestines of infected animals, suggesting the presence of a novel subpopulation of MC in the lungs that expressed MMCP-8, either alone or in combination with MMCP-5 and carboxypeptidase A. The dramatic increase in MMCP-1 and MMCP-2 levels but not of MMCP-8 in the intestines of parasitized animals also shows that MMCP-8 is not expressed in mucosal MC in the mouse. This latter is in clear contrast to what has been observed in the rat where the MMCP-8 homologues, RMCP-8, -9 and -10, can be considered as true mucosal MC proteases.

CD4+ T cell-mediated granulomatous pathology in schistosomiasis is downregulated by a B cell-dependent mechanism requiring Fc receptor signaling.

The effector functions of CD4+ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4+ lymphocytes, and previous studies have implicated CD8+ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell-deficient (muMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor-deficient (FcR gamma chain knockout) mice display the same exacerbated egg pathology as that observed in infected muMT mice, the B cell- dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4+ T cells.

Role of cytokines in the formation and downregulation of hepatic circumoval granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice.

Schistosoma mansoni infections are associated with a strong Th2 cytokine response. Treatment of mice with IL-12 or anti-IL-2 or anti-IL-4 before i.v. injection of eggs increased IFN-gamma production and downregulated Th2 responses and pulmonary granuloma size. Conversely, anti-IFN-gamma antibody treatment increased Th2 responses and granuloma size. Similar manipulation produced less dramatic results in infected mice. However, sensitization of mice with eggs + IL-12 before infection augmented the Th1 response and decreased Th2 cytokines, granuloma size and fibrosis. Antisera to IFN-gamma, TNF-alpha or IL-12 during IL-12-egg immunization partly restored granuloma size and fibrosis following infection. Variations in the size of granulomas in acute (8 week) infections may be influenced primarily by the number and state of activation of T cells. In chronic (12-16 week) infections immunologic downmodulation proceeded normally in mice without functional CD8+ cells and in IFN-gamma KO mice but not in B cell KO (microMT) mice or in mice deficient in FcR expression in spite of the fact that these mice downregulated their T cell and cytokine responses. It is evident that the participation of cytokines in granuloma formation and regulation is complicated and that the mechanisms controlling both these phenomena are likely to involve both T cells and antibody/FcR interactions.

Fc epsilonRI-deficient mice infected with Schistosoma mansoni mount normal Th2-type responses while displaying enhanced liver pathology.

The IgE/Fc epsilonRI interaction is postulated to play an important role in resistance to helminths both at the level of anti-parasitic effector cell function and in the initiation of Th2 responses through IL-4 produced by Fc epsilonRI+ non-B, non-T (NBNT) cells. To formally evaluate the role of IgE/Fc epsilonRI signaling in the host response to helminths we studied Schistosoma mansoni infection in Fc epsilonRI knockout (KO) mice. Infected wild-type (wt) and KO animals showed comparable adult worm and tissue egg burdens, arguing against a role for Fc epsilonRI interactions in host resistance. Significantly, NBNT cells from infected KO, in contrast to wt animals, did not secrete IL-4 when stimulated with anti-IgE Ab or soluble parasite Ag. Nevertheless, serum IgE levels and Th2 cytokine production profiles were comparable in both strains of mice, demonstrating that the Ag-dependent stimulation of IL-4 secretion by NBNT cells is not essential for helminth-induced Th2 differentiation. However, when stimulated with low Ag doses, splenocytes from infected Fc epsilonRI-deficient mice produced less IL-4 in vitro than similar cultures from infected wt animals, an effect attributable to their defective NBNT cell function. Moreover, infected KO mice showed enhanced egg granuloma formation and hepatic fibrosis, revealing that the IgE/Fc epsilonRI interaction, while not essential for Th2 response development or resistance to primary infection, plays a significant role in down-regulating host pathology.

T cell-derived IL-3 induces the production of IL-4 by non-B, non-T cells to amplify the Th2-cytokine response to a non-parasite antigen in Schistosoma mansoni-infected mice.

We describe a novel amplification mechanism underlying the increased early IL-4 production observed in Schistosoma mansoni-infected mice in response to a non-parasite Ag, sperm whale myoglobin (SwMb). Earlier studies have shown that splenic Fc epsilon R+ non-B, non-T (NBNT) cells from schistosome-infected mice secrete IL-4 after stimulation with parasite Ag. We now demonstrate that purified NBNT cells from SwMb-immunized S. mansoni-infected mice do not respond directly to SwMb, but produce IL-4 in response to IL-3. Accordingly, we show that the early SwMb-specific IL-4 response of spleen cells (SC) from immunized infected mice is dependent on IL-3 and on CD4+ T cells. Thus, most of the early SwMb-induced IL-4 from SC of infected mice appears to be produced by NBNT cells triggered by IL-3 synthesized by SwMb-specific CD4+ T cells. IL-3-induced IL-4 production was also observed in purified NBNT cells from immunized uninfected mice, but the frequency and/or IL-4-producing capacity of splenic IL-3-responsive cells was found to be 8 to 16 times higher in immunized infected animals. IL-4 production by purified CD4+ cells from immunized infected mice was also seen after SwMb stimulation, but this response showed slower kinetics than those of total SC, was IL-3-independent, and on average threefold greater than that by CD4+ cells from immunized uninfected controls. Thus, increased SwMb-induced IL-4 production in immunized S. mansoni-infected mice results from direct synthesis by CD4+ T cells, as well as their stimulation via IL-3 of an expanded population of NBNT cells. The latter pathway may serve as an amplification loop for Th2-cytokine responses.

Molecular analysis of a heteroclitic T cell response to the immunodominant epitope of sperm whale myoglobin. Implications for peptide partial agonists.

We have investigated the molecular basis for binding and Ag presentation of an immunodominant Th cell determinant of sperm whale myoglobin, a prototype amphipathic helical structure in the native protein. A series of peptides with three different substitutions at each position were evaluated for binding to the class II MHC molecule I-Ad and for activation of two T cell clones with distinct fine specificity, to determine the role of each residue. The assignment of MHC binding and TCR binding residues is consistent with a peptide bound as a twisted beta-strand, with 130 degrees twist similar to that of the influenza hemagglutinin peptide crystallized in the groove of HLA-DR1. This twist gives the peptide amphipathicity, with a periodicity similar to an alpha-helix without its being a helix. Two substituted peptides were discovered to be heteroclitic, but by different molecular mechanisms, one involving gain of a favorable residue and one involving loss of an unfavorable one. Complexes of both peptides with I-Ad had enormously higher affinity for the TCR, but peptide affinity for the MHC molecule was not increased, such that the wild-type peptide acted as a partial agonist and inhibited the response to the heteroclitic ones. Moreover, the magnitude of response was elevated in a way that could not be mimicked by the wild-type peptide even at higher concentration. These results suggest a TCR dwell time requirement for optimal signal transduction that may help explain the mechanism of partial agonism.

Cellular mechanisms in the immune response to malaria in Plasmodium vinckei-infected mice.

Infection of mice with the malaria parasite Plasmodium vinckei vinckei is 100% lethal. However, after two infections followed by drug cure, BALB/c mice develop a solid immunity which is antibody independent but mediated by CD4+ T cells. To elucidate the mechanisms of this immunity, spleen cells from immune mice were challenged in vitro with lysates of P. vinckei-infected or uninfected erythrocytes. The parasite antigen induced proliferation of T cells from immune mice but not from nonimmune mice. When gamma interferon production by cells from immune mice was assayed at the single-cell level, 1 to 3 cells per 1,000 cells were found to release this cytokine when exposed to antigen. In contrast, the numbers of interleukin 4 (IL-4)-producing cells from both immune and control mice were < or = 4 per 10(6) cells, regardless of antigen exposure. Investigation in a bioassay showed that P. vinckei antigen induced the release of IL-4 from spleen cells of immune mice but not from those of control mice. Nevertheless, that IL-4 is of minor significance in this system is also suggested by the absence of elevation of immunoglobulin E levels in blood samples from these mice, in contrast to what is seen with P. chabaudi infection, in which IL-4-producing Th2 cells are of major importance for immunity during later phases of infection. Taken together, the present results indicate that immunity to P. vinckei is a Th1 response, with gamma interferon being an important protective factor. Whether or not the Th1 response, through overproduction of tumor necrosis factor alpha, is also responsible for pathology and death in this infection remains to be clarified.

Fc epsilon receptor-positive cells are a major source of antigen-induced interleukin-4 in spleens of mice infected with Schistosoma mansoni.

When cultured in vitro with either mitogen or parasite antigens, spleen cells from mice infected with Schistosoma mansoni produce significantly higher levels of IL-4 than splenocytes from control animals. Previous studies suggested that this increase in IL-4 production occurs because of a selective expansion of T helper type 2 (Th2) cells in infected mice. However, these experiments employed unfractionated spleen populations rather than purified T lymphocytes. Here we demonstrate that T-depleted spleen cells from infected animals synthesize high levels of interleukin-4 (IL-4), but no IL-5 when stimulated with parasite antigen in vitro. Nevertheless, when purified by sorting, T cells and non-B, non-T (NBNT) populations produced similar amounts of IL-4 in response to parasite antigen. The IL-4 producing NBNT cells were found to belong to an Fc epsilon receptor (Fc epsilon R)-positive population which after sort purification produced high levels of IL-4 (between 1000 and 2000 U of per 5 x 10(3) cells). FACS analysis revealed that these Fc epsilon R+ cells make up 0.53% of splenic NBNT cells in control animals while in 8-9-week-infected animals they increase to 3.8% of that population. In contrast, in mice with 8-week unisexual worm infections these cells comprise only 1.71% of NBNT cells, indicating that eggs are a major stimulus of the response. The expansion of Fc epsilon R+ cells and their production of IL-4 could be an important factor regulating the selection and induction of different CD4+ subsets in schistosome-infected hosts.