Development of a CTL vaccine for Her-2/neu using peptide-microspheres and adjuvants.

With the ultimate goal of developing a therapeutic cancer vaccine, we encapsulated the Her-2/neu peptide p369-377 in poly(lactide-co-glycolide) microspheres. This formulation was found to effectively elicit CD8+ cytotoxic T cell (CTL) responses in an HLA-A*0201 transgenic mouse model. In contrast, immunization with either peptide alone or peptide formulated in incomplete Freund's adjuvant (IFA) failed to elicit such CTL responses. Responses induced by the peptide-microsphere formulation were found to peak at approximately 6 weeks post-immunization, and were enhanced by delivering increased doses of peptide and with repeated administrations over time. Co-administration of the peptide-microspheres with adjuvants, including granulocyte-macrophage colony stimulating factor, MPL adjuvant and select synthetic Toll-Like Receptor 4 ligands, the aminoalkyl glucosaminide-4 phosphates, significantly augmented CTL responses. These studies provide important guidance for the design of human clinical trials of microsphere vaccines in terms of optimal peptide-microsphere formulation, vaccination regimen, vaccine dose, and adjuvant selection.

Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity.

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8(+) T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8(+) T cells. Two HLA-Ia-restricted CD8(+) T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8(+) effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8(+) T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8(+) T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8(+) T cell Ag.

Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: the relative contribution of classical versus nonclassical HLA restriction.

Previous studies in mice and humans models have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). In humans, CD8+ Mtb-reactive T cells have been described that are HLA-A2-, B52-, as well as CD1-restricted. Recently, we have described Mtb-specific CD8+ T cells that are neither HLA-A-, B-, or C- nor group 1 CD1-restricted. At present, little is known about the relative contribution of each of these restriction specificities to the overall CD8+ response to Mtb. An IFN-gamma enzyme-linked immunospot assay was used to determine the frequency of Mtb-reactive CD8+ T cells directly from PBMC. The effector cell frequency among five healthy purified protein derivative-positive subjects was 1/7,600 +/- 4,300 compared with 1/16,000 +/- 7,000 in six purified protein derivative-negative controls. To determine the frequencies of classically, CD1-, and nonclassically restricted cells, a limiting dilution analysis was performed. In one purified protein derivative-positive subject, 192 clones were generated using Mtb-infected dendritic cells (DC). Clones were assessed for reactivity against control autologous DC, Mtb-infected autologous DC, and HLA-mismatched CD1+ targets (DC), as well as HLA-mismatched CD1- targets (macrophages). Of the 96 Mtb-reactive CD8+ T cell clones, four (4%) were classically restricted and 92 (96%) were nonclassically restricted. CD1-restricted cells were not detected. Of the classically restricted cells, two were HLA-B44 restricted and one was HLA-B14 restricted. These results suggest that while classically restricted CD8+ lymphocytes can be detected, they comprise a relatively small component of the overall CD8+ T cell response to Mtb. Further definition of the nonclassical response may aid development of an effective vaccine against tuberculosis.

Generation of immunity to the HER-2/neu oncogenic protein in patients with breast and ovarian cancer using a peptide-based vaccine.

HER-2/neu is a "self" tumor antigen that is overexpressed in 15-30% of human adenocarcinomas. Vaccine strategies directed against HER-2/neu and other self tumor antigens require development of methods to overcome immune tolerance to self-proteins. In rats, rat neu peptide vaccines have been shown to be an effective way of circumventing tolerance to rat neu protein and generating rat neu-specific immunity. The present report validates that a similar peptide-based vaccine formulation is effective for inducing T-cell immunity to HER-2/neu protein in humans with breast and ovarian cancer. The vaccine formulation included groups of peptides derived from the HER-2/neu extracellular domain (ECD) or intracellular domain (ICD) mixed with granulocyte macrophage colony stimulating factor as an adjuvant. These peptides were 15-18 amino acids in length and designed to elicit a CD4 T helper-specific immune response. Patients underwent intradermal immunization once a month for a total of two to six immunizations. To date, all of the patients immunized with HER-2/neu peptides developed HER-2/neu peptide-specific T-cell responses. The majority of patients (six of eight) also developed HER-2/neu protein-specific responses. Responses to HER-2/neu protein occurred with epitope spreading. Immune T cells elicited by vaccination were shown to migrate outside the peripheral circulation by virtue of generating delayed type hypersensitivity responses distant from the vaccine site, which indicated the potential ability to traffic to the site of tumor. The use of peptide-based vaccines may be a simple, yet effective, vaccine strategy for immunizing humans to oncogenic self-proteins.

Broad tumor-associated expression and recognition by tumor-derived gamma delta T cells of MICA and MICB.

Human MHC class I-related molecules, MICA and MICB, are stress-induced antigens that are recognized by a subset of gamma delta T cells expressing the variable region Vdelta1. This functional association has been found to be limited to intestinal epithelium, where these T cells are prevalent and where MICA and, presumably, MICB are mainly expressed. However, increased frequencies of Vdelta1 gamma delta T cells have been observed in various epithelial tumors; moreover, MICA/B are expressed on diverse cultured epithelial tumor cells. With freshly isolated tumor specimens, expression of MICA/B was documented in many, but not all, carcinomas of the lung, breast, kidney, ovary, prostate, and colon. In tumors that were positive for MICA/B, the frequencies of Vdelta1 gamma delta T cells were significantly higher than in those that were negative. Vdelta1 gamma delta T cell lines and clones derived from different tumors recognized MICA/B on autologous and heterologous tumor cells. In accord with previous evidence, no constraints were observed in these interactions, such as those imposed by specific peptide ligands. Thus, MICA/B are tumor-associated antigens that can be recognized, in an apparently unconditional manner, by a subset of tumor-infiltrating gamma delta T cells. These results raise the possibility that an induced expression of MICA/B, by conditions that may be related to tumor homeostasis and growth, could play a role in immune responses against tumors.

LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile.

We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-gamma (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-gamma. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-gamma by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1-226) maintained the ability to stimulate IFN-gamma from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.

Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients.

Previous studies have characterized the reactivity of CD8+ CTLs with ovarian and breast cancer. There is little information about the antigens and epitopes recognized by CD4+ T cells in these patients. In this study, we analyzed the ability of T cells from peripheral blood mononuclear cells of breast cancer patients to recognize HER-2/neu (HER-2) peptides. We found that 13 of 18 patients responded by proliferation to at least one of the HER-2 peptides tested. Of these peptides, one designated G89 (HER-2: 777-789) was recognized by T cells from 10 patients. Seven of nine responding patients were HLA-DR4+, suggesting that this peptide is recognized preferentially in association with HLA-DR4. Analysis of the specificity and restriction of the cytokine responses to G89 by G89-stimulated T cells revealed that these cells secreted significantly higher levels of IFN-gamma than interleukin 4 and interleukin 10, suggesting priming for a Th0-T helper 1 response. The same pattern of cytokine responses was observed to the intracellular domain of HER-2 protein, suggesting that G89-stimulated T cells recognized epitopes of the HER-2 protein in association with HLA-DR4. Because HLA-DR4 is present in 25% of humans, characterization of MHC class II-restricted epitopes inducing Th0-T helper 1 responses may provide a basis for the development of multivalent HER-2-based vaccines against breast and ovarian cancer.

Characterization of human CD8+ T cells reactive with Mycobacterium tuberculosis-infected antigen-presenting cells.

Previous studies in murine models, including those using the beta2 microglobulin knockout mouse, have suggested an important role for CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). At present, little is understood about these cells in the human immune response to tuberculosis. This report demonstrates the existence of human Mtb-reactive CD8+ T cells. These cells are present preferentially in persons infected with Mtb and produce interferon gamma in response to stimulation with Mtb-infected target cells. Recognition of Mtb-infected cells by these CD8+ T cells is restricted neither by the major histocompatibility complex (MHC) class I A, B, or C alleles nor by CD1, although it is inhibited by anti-MHC class I antibody. The Mtb-specific CD8+ T cells recognize an antigen which is generated in the proteasome, but which does not require transport through the Golgi-ER. The data suggest the possible use of nonpolymorphic MHC class Ib antigen presenting structures other than CD1.

Human purified protein derivative-specific CD4+ T cells use both CD95-dependent and CD95-independent cytolytic mechanisms.

CTL, both CD4+ and CD8+, are essential in the eradication of intracellular pathogens. Data generated using murine T cells have suggested a critical role for CD95 (Fas, Apo-1) in CD4+ T cell-induced apoptosis of target cells. In contrast, CD8+ CTL predominantly use the perforin/granzyme lytic pathway. At present little is known about the mechanism of CD4+ CTL lytic function during intracellular infection in humans. We have used human CD4+ T cells specific for purified protein derivative (PPD) of Mycobacterium tuberculosis to explore whether CD95 is the dominant cytolytic mechanism. PPD-reactive CD4+ clones efficiently lysed Ag-pulsed autologous monocytes, adherent macrophages, and EBV-transformed B cells. Addition of an antagonistic CD95 Ab had a minimal effect on cytolysis, whereas addition of MgEGTA to block perforin/granzyme resulted in complete inhibition of killing. In contrast, lysis of activated peripheral blood B cells could be partially blocked with the antagonistic CD95 Ab. Supporting these observations, monocytes, macrophages, and EBV-transformed B cells were not lysed by an agonistic CD95 Ab. Activated B cells were readily lysed by the agonistic CD95 Ab. T cell clones triggered through the TCR with anti-CD3 were capable of lysing the CD95-sensitive Jurkat T cell line in a CD95-dependent manner, but were also able to release granzymes. We conclude that human CD4+ T cells are capable of lysing PPD-pulsed targets using both perforin/granzyme and CD95 pathways. The contribution of CD95 is strictly dependent on target cell susceptibility to CD95-mediated killing.

A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factor-alpha production and expression of B7-1 in human monocyte-derived antigen-presenting cells.

LeIF, a gene homologue of the eukaryotic initiation factor 4A was first described as a leishmanial antigen that induced a Th1-type T cell response in peripheral blood mononuclear cells (PBMC) from leishmaniasis patients. Moreover, the interferon (IFN)-gamma production by PBMC was found to be interleukin (IL)-12 dependent. Herein, we characterize the effects of LeIF on cytokine production and expression of surface molecules by normal human monocytes as well as by monocyte-derived macrophages and dendritic cells (MoDC). LeIF was a strong inducer of IL-12 and, to a lesser extent, of IL-10 and tumor necrosis factor (TNF)-alpha in macrophages and MoDC. IL-12 production did not require CD40 triggering, confirming that the ability of LeIF to induce IL-12 was not mediated through an effect on T cells. However, addition of soluble CD40 ligand (L) synergistically augmented IL-12 production in macrophages and MoDC. The cytokine-inducing activity of LeIF is located in the N-terminal portion of the molecule and was both proteinase K sensitive and polymyxin B resistant. LeIF, lipopolysaccharide and fixed Staphylococcus aureus all induced comparable amounts of IL-12, validating the potent cytokine-inducing effects of LeIF. Moreover, of these stimuli, LeIF had the highest IL-12/IL-10 and IL-12/TNF-alpha ratio demonstrating the preference of LeIF for IL-12 induction. Studies investigating the expression of surface molecules showed that LeIF up-regulated B7-1 and CD54 (ICAM-1) on macrophages and MoDC. To our knowledge this is the first report describing IL-12 production, up-regulation of co-stimulatory and intercellular adhesion molecules by monocytic antigen-presenting cells in response to a protein from a pathogenic microorganism. These immunomodulatory characteristics of LeIF might be excellent properties for a Th1-type adjuvant.

The role of the CD28/B7 interaction in the regulation of NK cell responses during infection with Toxoplasma gondii.

We examined the role of the CD28/B7 interaction in regulation of NK cell activity. Cells transfected with B7 enhanced IL-12-induced production of IFN-gamma by IL-2-activated, CD28+ NK cells, but not by resting CD28- NK cells. The ability of B7 transfectants to enhance NK cell production of IFN-gamma was dependent on the intracellular adhesion molecule-1/LFA-1 interaction and could be inhibited by TGF-beta, but not IL-10. Since IL-12-induced production of IFN-gamma by NK cells is associated with resistance to certain infections, we examined whether the CD28/B7 interaction regulated NK cell responses during infection. Infection of SCID mice with Toxoplasma gondii resulted in the appearance of a population of CD28+ NK cells, NK cell production of IFN-gamma, and increased NK cell cytolytic activity. Administration of CTLA4-Ig to SCID mice infected with T. gondii inhibited these latter two effects and resulted in a significant increase in parasite burden. The stimulus for CD28 expression by NK cells in SCID mice infected with T. gondii appeared to be independent of IL-2. However, mRNA for IL-15, a cytokine with properties similar to those of IL-2, was detected in tissues of SCID mice infected with T. gondii. In vitro experiments demonstrated that IL-15 could stimulate resting NK cells to express functionally active CD28 as well as enhance the production of IFN-gamma by SCID splenocytes stimulated with T. gondii. Together our data demonstrate that the interaction of CD28+ NK cells with B7 regulates NK cell production of IFN-gamma associated with resistance to infection and that IL-15 may be involved in these events.

Structure-function studies of interleukin 15 using site-specific mutagenesis, polyethylene glycol conjugation, and homology modeling.

Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of IL-15 interferes with beta but not alpha receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block beta and gamma receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of beta receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.

Differential expression of CD22 (Lyb8) on murine B cells.

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.

CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages.

We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL-12, but not IL-2, in these cultures by approximately 90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-gamma and granulocyte/macrophage colony-stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L.

IL-15 is a novel growth factor for murine gamma delta T cells induced by Salmonella infection.

We have previously shown evidence for the early recruitment of gamma delta T cells during the disease course of primary infections with Listeria monocytogenes or Salmonella choleraesuis in mice. Since gamma delta T cells at this stage of the disease do not produce IL-2, the growth factor for the gamma delta T cells remains unknown. IL-15 is a novel cytokine that uses beta- and gamma-chain of IL-2R for signal transduction, and is produced by activated monocytes/macrophages. In this study, we investigated the proliferative activity of IL-15 for gamma delta T cells appearing after primary infection with S. choleraesuis 31N-1. The gamma delta T cells, which expressed beta- and gamma-chains of IL-2R, proliferated in the presence of rIL-15 and produced appreciable levels of gamma-IFN and IL-4. Addition of anti-IL-2R beta mAb significantly inhibited the IL-15-induced proliferation of the gamma delta T cells. Furthermore, the gamma delta T cells produced gamma-IFN in response to monocyte/macrophage cell line, J774A.1 infected with S. choleraesuis, which expressed an abundant level of IL-15 mRNA. This cytokine production was inhibited significantly by anti-IL-15 Ab. Taken together, these results suggest that IL-15 derived from infected macrophages may contribute to the early activation of gamma delta T cells during salmonellosis.

Cytokine interactions in human immunodeficiency virus-infected individuals: roles of interleukin (IL)-2, IL-12, and IL-15.

Cytokines have been shown to be powerful regulators of the immune response. In this study, we analyze the effect that the newly recognized cytokine interleukin (IL)-15 has on proliferation and cytokine induction using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from patients infected with human immunodeficiency virus (HIV) who are at various stages in their disease. We observed that IL-15 enhances the proliferative response in a dose-dependent manner from PBMCs of HIV-infected individuals when stimulated by polyclonal mitogen, tetanus toxoid, or HIV-specific antigen. The effects of exogenous IL-15 are substantially diminished by adding a neutralizing antibody to the beta chain of the IL-2 receptor. Moreover, the ability of IL-15 to increase proliferation is enhanced by the presence of endogenous IL-2 produced in the cultures. The effect that exogenous IL-15 had on IL-2, IL-4, and interferon (IFN)-gamma induction from PBMC's or CD4+ T cells in response to mitogen or tetanus toxoid was also examined. This was compared to the effect that exogenous IL-2 and IL-12 had under the same conditions. Addition of IL-2 or IL-15 to short-term in vitro cultures of either PBMCs or CD4+ T cells had little effect on IL-2, IL-4, or IFN-gamma production. By contrast, IL-12 caused substantial enhancement of both IL-2 and IFN-gamma production from these cultures. The role that endogenous cytokines have on IFN-gamma induction was also studied. Addition of a neutralizing antibody to the alpha chain of the IL-2 receptor or IL-12 to antigen stimulated cultures caused a striking decrease in IFN-gamma production. Neutralization of endogenous IL-15 also resulted in diminished IFN-gamma production from cultures stimulated with mitogen. IL-4 and IFN-gamma protein production by PBMCs and CD4+ T cells stimulated with mitogen was assessed to see if we could detect a specific bias of cytokine production. Small amounts of IL-4 were detected from CD4+ T cells but not PBMCs from most individuals tested. IFN-gamma and IL-2, however, were also produced from these same cultures. These results further elucidate the mechanism of cytokine regulation in HIV-infected individuals, and they provide evidence that IL-15 may be a useful immune modulator.

Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2.

Interleukin-15: a novel anabolic cytokine for skeletal muscle.

Interleukin-15 (IL-15) is a recently discovered growth factor which is highly expressed in skeletal muscle. In order to determine a functional role for IL-15 in skeletal myogenesis, the effects of IL-15 on myoblast proliferation and muscle-specific myosin heavy chain (MHC) expression were analyzed using the mouse C2 skeletal myogenic cell line and primary fetal bovine skeletal myogenic cultures. IL-15 had no effect on [3H]thymidine incorporation, nor on the rate of myoblast differentiation, assessed by anti-MHC immunocytochemical staining, in either type of culture. However, Western blot analyses revealed that IL-15 used at concentrations of 10 or 100 ng/ml increased MHC accumulation five-fold in C2 myoblast cultures and 2.5-fold in primary bovine myogenic cultures. Moreover, C2 myotubes formed in the presence of IL-15 appeared larger than controls. These findings indicate IL-15 can stimulate differentiated myocytes and muscle fibers to accumulate increased amounts of contractile proteins. Well-fused primary bovine myogenic cultures treated with the mitotic inhibitor aphidicolin, then administered IL-15 and/or the anabolic growth factor insulin-like growth factor-I (IGF-I), were analyzed for MHC accumulation using Western blots. IL-15 used at 10 ng/ml doubled MHC accumulation and was as effective as IGF-I used at 10 or 100 ng/ml. IL-15 and IGF-I used together increased MHC accumulation close to five-fold, indicating these two factors can act additively on muscle fibers. These findings indicate IL-15 affects parameters associated with skeletal muscle fiber hypertrophy, and suggest that IL-15 may be a novel anabolic agent to increase skeletal muscle mass.

Regulation of murine B cell growth and differentiation by CD30 ligand.

A ligand for CD30 has been recently cloned, and has been shown to have sequence homology with the tumor necrosis factor family of cytokines. CD30 ligand (CD30L) was found to be induced on helper T cell clones, and its receptor was expressed on freshly isolated and activated murine B cells. Recombinant murine CD30L was found to share many functional properties with CD40 ligand (CD40L) in the regulation of murine B cell growth and differentiation in vitro. CD30L stimulated B cell proliferation, antigen-specific antibody production, and polyclonal immunoglobulin secretion in a cytokine-dependent manner. In particular, the stimulation of B cell proliferation by CD30L required interleukin (IL)-4 and IL-5, induction of anti-sheep red blood cell antibody-secreting B cells by CD30L required IL-2 and IL-5, and optimal induction of polyclonal immunoglobulin secretion required IL-4 and IL-5. Under these conditions, the polyclonal secretion of IgG1, IgA, IgG3 and IgE was induced. The induction of immunoglobulin secretion by CD30L was independent of CD40L, as B cells from CD40L deficient-mice responded normally to CD30L treatment. We conclude that CD30L is a potent mediator of B cell growth and differentiation in vitro and may play a role in cognate T cell-B cell interactions.