IL-4 (B cell stimulatory factor 1) exhibits thymocyte growth factor activity in the presence of IL-2

The proliferative activity of thymocytes cultured with IL-2 and submitogenic concentrations of PHA is increased by 3- to 10-fold in the presence of IL-4. In contrast, IL-4 alone is unable to induce proliferative activity in thymocyte cultures and its synergistic activity is only apparent to concentrations of IL-2 above 1 U/ml. The costimulatory activity of IL-4 is abrogated by the monoclonal anti-IL-4 antibody 11B11. Furthermore, potentiation of the IL-2-mediated thymocyte proliferation is not seen with IL-1, IL-3, IFN-gamma, and granulocyte-macrophage CSF. Thymocytes are at least as responsive to IL-4 as B cells and the IL-4 costimulatory activity in fractionated thymocytes appears to be restricted mainly to the Lyt-2+/L3T4- population. In contrast, purified resting mature T cells do not respond to IL-4 plus IL-2, although they did proliferate in response to IL-4 in combination with PMA. These findings indicate that thymocytes and mature T cells are responsive to the costimulatory activity of IL-4 under quite different conditions, and that IL-4 may play an important role in thymocyte maturation in the thymus.     

Functional significance of human vascular smooth muscle cell-derived interleukin 1 in paracrine and autocrine regulation pathways.

Interleukin 1 (IL1), a key mediator in the cytokine network, alters many pathophysiologically important functions of blood vessel wall cells. Vascular cells, such as endothelial cells and smooth muscle cells (SMC) can themselves transcribe IL1 genes, raising the possibility that IL1 regulates blood vessel wall functions by local autocrine or paracrine mechanisms. However, IL1 lacks a recognizable signal sequence and it is still unclear how vascular cells might release IL1 or if IL1 derived from vascular cells can actually produce autocrine or paracrine effects. We explored these issues in human vascular SMC, the most numerous cell type in arteries and veins, using cultured SMC and short term organoid cultures. SMC treated with lipopolysaccharide recombinant tumor necrosis factor (recTNF), or recIL1 itself ("activated SMC") elaborated thymocyte costimulatory activity, a biological activity traditionally ascribed to IL1. However, neutralization experiments with monospecific antibodies disclosed that the more recently recognized cytokine IL6 rather than IL1 accounted for most of the soluble thymocyte costimulatory activity released by activated SMC. Using the D10S assay that distinguishes IL1 from IL6 and TNF we found that the culture supernatant of activated SMC contained little or no IL1, but that the cytosol and surface of these cells did exhibit this activity. Antiserum to recIL1 alpha inhibited stimulation of D10S cells by surface-associated IL1 of activated SMC, while treatment with acid to elute receptor- or nonspecifically bound IL1 did not abrogate this D10S proliferation. Short term organoid cultures of both normal veins and human arteriosclerotic plaque also expressed tissue-associated IL1 activity upon stimulation with LPS but did not release significant soluble IL1 activity. To establish further the biological functions of cell-associated IL1, we incubated stimulated or unstimulated SMC that were fixed with paraformaldehyde and washed extensively (fixed SMC) with overlayered viable SMC of the same donor (responder SMC). Contact with fixed SMC that bore surface IL1 following TNF or IL1 stimulation evoked up to 20-fold higher IL6 release from responder SMC than did exposure to unstimulated SMC (57 vs 1052 ng/ml/day). Addition of anti-IL1 antibody inhibited the release of IL6 from the responder SMC. These results demonstrate that cytokine-activated SMC express biologically active IL1 on their cell surface and illustrate how these cells might actually participate in autocrine and paracrine signaling in the vessel wall. The requirement for direct intercellular contact for IL1 effects could facilitate local information exchange among vascular wall cells and/or infiltrating leukocytes and permit costimulation while limiting undue propagation of inflammatory stimuli.     

Induction of the differentiation of memory T killer cells with factors released from macrophage-like cell lines

Macrophage-like cell lines J774.1 and WEHI-3 as well as peritoneal exudate macrophages have been demonstrated to produce factors which induce the differentiation of memory cells into specific T killer cells in the absence of an added antigen. LPS stimulation was required for J774.1 cells and peritoneal macrophages to produce the factors but not for WEHI-3 cells. Interferon seemed to be one of the responsible factors. However, macrophages seem to produce other active factors; one has a molecular weight (MW) of more than 80,000 and lacks thymocyte mitogenic activity; another, with a weak thymocyte mitogenic activity, has a MW of 38,000 to 44,000. The low MW thymocyte mitogenic factor (interleukin-1) showed weak T killer cell differentiating activity.     

IGG-stimulated and LPS-stimulated monocytes elaborate transforming growth factor type beta (TGF-beta) in active form

Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Production of interleukin-1 and tumor necrosis factor by cisplatin-treated murine peritoneal macrophages

Supernatants collected from cisplatin-treated macrophages demonstrated enhanced cytotoxicity against actinomycin-D-treated L929 cells and also enhanced the thymocyte proliferation in response to concanavalin A, showing that cisplatin-treated macrophages release interleukin-1 (IL-1) and tumor necrosis factor (TNF) into the culture supernatant. The supernatant collected from untreated macrophages showed little TNF and IL-1 activity. The release of TNF and IL-1 was observed to be dependent on the dose and duration of cisplatin treatment. Medium alone containing cisplatin did not enhance thymocyte proliferation and had little cytotoxic effect on actinomycin-D-treated L929 cells. Cisplatin-treated macrophage culture supernatants were chromatographed over a Superose 12 column on an FPLC system. TNF activity eluted in two major peaks with apparent molecular weights of 50-55 and 15-20 kilodaltons, respectively. The kinetics of IL-1 release was also studied. Maximum production and release of IL-1 were observed up to 24 h after cisplatin treatment and then gradually declined. Freeze-thaw lysates of cisplatin-treated macrophages also showed enhanced IL-1 activity. Paraformaldehyde (PFA)-fixed cisplatin-treated macrophages showed significantly enhanced cytotoxic activity against L929 cells as compared to PFA-fixed untreated macrophages. PFA-fixed cisplatin-treated macrophages also enhanced thymocyte proliferation. These results suggest that cisplatin treatment of murine macrophages also results in increased expression of membrane-associated IL-1 and TNF activity.     

Opening a window on thymic positive selection: developmental changes in the influence of cosignaling by integrins and CD28 on selection events induced by TCR engagement

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.     

The release of interleukin-2 (IL-2) and colony stimulating activity (CSA) in aplastic anemia patients: opposite behaviour with improvement of bone marrow function

Peripheral blood cells from patients with aplastic anemia were tested for their ability to release interleukin-2 (IL-2) and colony stimulating activity (CSA) before treatment. IL-2 release--as measured in the mouse thymocyte assay--was abnormally high in 18/34, and abnormally low in 10/34 patients. "Low" release was due to simultaneous release of thymocyte inhibitors. In 18 patients who achieved self-sustaining hemopoiesis after high dose immunosuppressive therapy, excess IL-2 release decreased to low levels (p less than 0.001), and the release of inhibitors disappeared. In contrast, the release of CSA by patient cells--which did not correlate with peripheral blood monocyte counts--either remained high or increased to excessively high values in 24/24 patients tested before and after successful immunosuppressive treatment. Patients with stable hemopoietic grafts after bone marrow transplantation for aplastic anemia, did not release excess CSA. It is concluded that IL-2 and CSA play opposite roles in aplastic anemia. High IL-2 release seems associated with disease activity, whereas high CSA-release appears to reflect a repair mechanism.     

Similarities between LAK cells derived from human thymocytes and peripheral blood lymphocytes: expression of the NKH-1 and CD3 antigens

Human thymocytes are devoid of NK cells but develop lymphokine-activated killer (LAK) activity after culture with recombinant interleukin-2 (rIL-2). The most active precursor for this activity appears to be a CD3-negative cell. The purpose of these studies was to compare the phenotype and functional activities of thymocyte and peripheral blood lymphocyte (PBL) LAK cells. Following culture, rIL-2-activated thymocytes resemble PBL-generated LAk and PBL NK cells. For each of these populations, lytic activity is highest in NKH-1-positive cells. Two-color fluorescence of each population also indicates that NKH-1+ cells are highly granular, as measured by staining with the lysosomotropic vital dye quinacrine. PBL, PBL-derived LAK cells, and thymus-derived LAK cells have a portion of cells that express both CD3 and NKH-1. However, approximately 60-80% of NKH-1+ cells lack detectable CD3. This suggests that both CD3+ and CD3- cells may be capable of LAK activity. Thymic-derived LAK cells respond to interferon in a manner very similar to NK and PBL-derived LAK cells, but lack the NK-associated CD16 antigen. Thus, despite the absence of NK cells in the thymus, it is possible to generate thymocyte LAK activity which bears a strong resemblance to LAK activity derived from peripheral blood lymphocytes.     

Evidence against the existence of a membrane form of murine IL-1 alpha

Previous studies have demonstrated that paraformaldehyde-treated macrophages possess IL-1 alpha activity in a variety of bioassay systems. However, no definitive biochemical data in support of the membrane IL-1 alpha concept has been reported. The purpose of the present study was to determine if the biologic activity associated with treated cells is due to a membrane form of IL-1 alpha or alternatively, to the leakage of IL-1 alpha. If the former case was true, then the exposed membrane IL-1 alpha should bind anti-IL-1 alpha antibodies or be cleaved by mild trypsin treatment. In both instances, IL-1 alpha activity should be lost when measured in a subsequent IL-1 bioassay. Our results indicate that pulsing paraformaldehyde-treated normal or cell line macrophages with anti-IL-1 alpha antibodies or treating the cells with trypsin did not affect the ability of the treated cells to function in a murine thymocyte proliferation assay. Furthermore, the standard short term treatment of cells with paraformaldehyde (15 min) did not prevent the leakage of IL-1 alpha from the cells or the processing of the precursor forms of the protein. When cells were treated with paraformaldehyde for 2 h, they no longer released IL-1 alpha or possessed thymocyte stimulatory activity. We also found that short term glutaraldehyde treatment of macrophages completely blocked the release of IL-1 alpha from cells as well as the appearance of cell-associated IL-1 alpha activity. Our results support the conclusion that the stimulatory activity of paraformaldehyde-treated macrophages is not due to a membrane form of IL-1 alpha but is, in fact, due to the continuous release of IL-1 alpha from the cells.     

Role of porin of Shigella dysenteriae type 1 in modulation of lipopolysaccharide mediated nitric oxide and interleukin-1 release by murine peritoneal macrophages

The ability of Shigella dysenteriae type 1 porin to induce the release of nitric oxide (NO) and interleukin-1 (IL-1) from peritoneal macrophages of mouse and to regulate lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) mediated release of the two proinflammatory mediators was investigated. Porin released nitrite when added to macrophage cultures. A maximum of 3.2-fold nitrite release by macrophages was observed with 100 ng ml(-1) of porin. The nitrite release of LPS was enhanced significantly by lower concentrations of porin, whereas the effect of IFN-gamma was enhanced by porin at higher concentrations. Polysaccharide (PS) moiety of LPS stimulated the nitrite release of elicited macrophages by 1.6-fold compared to untreated control. It also enhanced the stimulatory effect of 1 and 10 ng ml(-1) of porin by 1.3-fold. Lipid A (LPA) moiety of LPS did not release nitrite, nor did it increase the porin mediated nitrite production. Porin treated 24 h old macrophage culture supernatants were applied for ConA activated thymocyte proliferation as a measure for determination of IL-1 release. Sixty percent depletion of thymocyte proliferation was observed when the porin treated macrophage supernatants were absorbed with anti-IL-1 antibody. A maximum of 5.5-fold increase of thymocyte proliferation over control was found with 1 and 10 ng ml(-1) of porin. One or 10 ng ml(-1) of porin and LPS augmented the thymocyte growth, 1.5-fold beyond that obtained by porin and 1.8-/1. 7-fold more than that obtained by LPS, alone. Similarly, porin and IFN-gamma co-stimulated the cell growth also. PS enhanced the thymocyte proliferation by 5-fold. It also enhanced the thymocyte growth by co-stimulating 1.4-fold the effect observed by 1 or 10 ng ml(-1) of porin alone. LPA could not participate in the cell proliferating activity nor did it enhance the stimulatory effect of porin. Therefore, both nitrite release and thymocyte proliferation by LPS could be substituted by PS only. The tight association of the two bacterial outer membrane components, porin and LPS, could be a necessary co-signal for boosting the release of the two proinflammatory mediators, namely NO and IL-1, which may be associated with the inflammatory response of the colon during Shigella invasion.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to sustained p53 activation

The PP2C phosphatase Wip1 dephosphorylates p38 and blocks UV-induced p53 activation in cultured human cells. Although the level of TCR-induced p38 MAPK activity is initially comparable between Wip1-/- and wild-type thymocytes, phosphatase-deficient cells failed to down-regulate p38 MAPK activity after 6 h. Analysis of young Wip1-deficient mice showed that they had fewer splenic T cells. Their thymi were smaller, contained significantly fewer cells, and failed to undergo age-dependent involution compared with wild-type animals. Analysis of thymocyte subset numbers by flow cytometry suggested that cell numbers starting at the double-negative (DN)4 stage are significantly reduced in Wip1-deficient mice, and p53 activity is elevated in cell-sorted DN4 and double-positive subpopulations. Although apoptosis and proliferation was normal in Wip1-/- DN4 cells, they appeared to be in cell cycle arrest. In contrast, a significantly higher percentage of apoptotic cells were found in the double-positive population, and down-regulation of thymocyte p38 MAPK activation by anti-CD3 was delayed. To examine the role of p38 MAPK in early thymic subpopulations, fetal thymic organ cultures cultured in the presence/absence of a p38 MAPK inhibitor did not correct the thymic phenotype. In contrast, the abnormal thymic phenotype of Wip1-deficient mice was reversed in the absence of p53. These data suggest that Wip1 down-regulates p53 activation in the thymus and is required for normal alphabeta T cell development.     

Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta

The lymphokine osteoclast-activating factor (OAF) was purified to homogeneity. OAF was produced by human peripheral blood mononuclear cells stimulated with concanavalin A and phorbol myristate acetate under serum-free culture conditions. OAF was purified by sequential gel filtration, ion-exchange, and reverse-phase HPLC by following bone resorptive activity. Homogeneity was indicated by the criteria of a single 17,800-dalton band on silver-stained polyacrylamide gels, a single pI 6.8 band on isoelectric focusing, and a single aminoterminal sequence. Purified OAF stimulated half-maximal release of calcium from fetal rat long bones at a concentration of approximately 0.66 ng/ml. The amino-terminal sequence of OAF was determined and found to be identical to that of interleukin 1 beta. Homogeneous OAF possessed an activity of 8.2 X 10(6) U/mg in the thymocyte proliferation assay. Because the m.w., isoelectric point, amino-terminal sequence, and specific activity in the thymocyte proliferation assay are the same for homogeneous OAF and interleukin 1 beta, we conclude that they are the same molecule, and that interleukin 1 beta is the major protein with OAF activity produced by lectin-stimulated peripheral blood mononuclear cells.     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.     

Functional reconstitution of thymopoiesis after human immunodeficiency virus infection

We have utilized combination antiretroviral therapy following human immunodeficiency virus type 1-induced human CD4(+) thymocyte depletion in the SCID-hu mouse to examine the immune competence of reconstituting thymocytes which appear following administration of combination therapy. These cells express a normal distribution of T-cell receptor variable gene families and are responsive to costimulatory signals. These results suggest that normal thymic function may be restored following antiretroviral treatment.     

Release of immature cells from the thymus during solid tumor growth: identification by assay of TdT activity.

In vivo anti-tumor activity of spleen cells from C3H/eb mice bearing a syngeneic fibrosarcoma was shown previously to decline to an undetectable level and be replaced by tumor-enhancing activity as tumor growth proceeds. In the light of our findings that thymocytes in the early stages of thymic processing can bring about tumor enhancement, we postulated that premature release of thymocytes and their accumulation in the spleen might account for the loss of the anti-tumor response. In the present experiments an injection of thymocytes did in fact cancel the anti-tumor response of reactive splenocytes from tumor-bearing mice. In order to determine whether premature thymocyte release occurs naturally in the tumor-bearing animals, we assayed activity of the enzyme TdT (as a marker for thymus cells) in the spleens of these mice during progressive tumor growth. Cells with TdT activity were clearly evident in the spleens of the tumor-bearing animals, were derived from the thymus, and accumulated in parallel to the loss of anti-tumour reactivity.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

Hydrogel-delivered GM-CSF overcomes nonresponsiveness to hepatitis B vaccine through the recruitment and activation of dendritic cells

The standard hepatitis B surface Ag (HBsAg) vaccine fails to induce anti-hepatitis B surface Abs in 5-10% of healthy subjects, a phenomenon known as HBsAg nonresponsiveness, which is closely related to HLA class II alleles and impaired Th cell responses to HBsAg in these subjects. We hypothesized that GM-CSF, a potent adjuvant in enhancing the Ag-presentation activity of APCs, might help to generate Th cell responses in nonresponders, subsequently providing help for B cells to produce anti-hepatitis B surface Abs. We used a thermosensitive biodegradable copolymer (hydrogel) system to codeliver HBsAg and GM-CSF to achieve maximal local cytokine activity at the injection site. In responder mouse strains, hydrogel-formulated HBsAg plus GM-CSF (Gel/HBs+GM) vaccine elicited much greater anti-hepatitis B surface Ab titers and Th cell proliferative responses than a commercial aluminum-formulated HBsAg vaccine or free HBsAg. The adjuvant effect of the Gel/HBs+GM vaccine was dependent upon the local release of GM-CSF. More importantly, the Gel/HBs+GM vaccine elicited high HBsAg-specific Ab titers and Th cell responses in B10.M mice, a mouse strain that does not respond to the current HBsAg vaccine because of its H-2 haplotype. Analysis of the draining lymph nodes of Gel/HBs+GM vaccine-treated mice revealed an elevated number of CD11c(+) dendritic cells showing enhanced expression of MHC class II and a variety of costimulatory molecules. These results demonstrate that hydrogel-formulated GM-CSF might represent a simple and effective method to generate next-generation hepatitis B virus vaccines for inducing anti-hepatitis B surface Abs in nonresponders.     

The processing and presentation of the self-antigen hemoglobin. Self-reactivity can be limited by antigen availability and costimulator expression.

APC do not distinguish between self- and foreign proteins. Previous studies from our laboratory demonstrated that most endogenous host APC constitutively processed and presented the self-Ag, hemoglobin (Hb), as detected by the Hb-specific T cell hybridoma, YO1.6. We have now examined APC in organs known to be involved in RBC degradation (liver Kupffer cells and splenic small resting B cells) for the presence of Hb/Ia complexes and for the expression of the costimulation necessary to trigger proliferation of T cell clones. We detected Hb/Ia complexes not only on splenic small resting B cells, but also on liver Kupffer cells. Interestingly, complexes were not present on lymph node small resting B cells. Splenic small resting B cells expressed costimulatory activity and efficiently stimulated the Th2 clones only. The opposite pattern was observed with liver Kupffer cells, which expressed costimulatory activity for Th1 clones only. However, if costimulatory activity was provided for the Th2 clones (IL-1 beta) and Th1 clones (allogenic spleen cells), the clones did proliferate in response to Kupffer cells and small resting B cells, respectively. In this report we have demonstrated that 1) endogenously formed self Hb/Ia complexes are expressed on splenic small resting B cells and liver Kupffer cells but not on lymph node small resting B cells and 2) these APC are also able to limit the expression of costimulatory activity for Th2 and Th1 T cell clones. Thus, endogenous APC not only constitutively process and present the self-Ag Hb, but also limit expression of the costimulatory activity necessary to trigger T cell proliferation against a self-Ag. The constitutive processing and presentation of self-Ag, as well as the regulation of costimulatory activity on APC, is likely an important feature of the maintenance of self-tolerance.