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TLRs trigger innate immunity that recognizes conserved motifs of invading pathogens, resulting in cellular activation and release of inflammatory factors. The influence of TLR activation on resistance to HIV-1 infection has not been investigated in HIV-1 exposed seronegative (ESN) individuals. PBMCs isolated from heterosexually ESN individuals were stimulated with agonists specific for TLR3 (poly I:C), TLR4 (LPS), TLR7 (imiquimod), and TLR7/8 (ssRNA40). We evaluated expression of factors involved in TLR signaling cascades, production of downstream effector immune mediators, and TLR-expression in CD4+ and CD14+ cells. Results were compared with those obtained in healthy controls (HCs). ESN individuals showed: 1) comparable percentages of CD14+/TLR4+ and CD4+/TLR8+ CD14+/TLR8+ cells; 2) higher responsiveness to poly I:C, LPS, imiquimod, and ssRNA40 stimulation, associated with significantly increased production of IL-1β, IL-6, TNF-α, and CCL3; 3) augmented expression of mRNA specific for other targets (CCL2, CSF3, CSF2, IL-1α, IL-8, IL-10, IL-12, cyclooxygenase 2) demonstrated by broader TLRs pathway expression analyses; and 4) increased MyD88/MyD88s(short) ratio, mainly following TLR7/8 stimulation. We also compared TLR–agonist-stimulated cytokine/chemokine production in CD14+ PBMCs and observed decreased IFN-β production in ESN individuals compared with HCs upon TLR7/8-agonist stimulation. These data suggest that TLR stimulation in ESN individuals results in a more robust release of immunologic factors that can influence the induction of stronger adaptive antiviral immune responses and might represent a virus-exposure–induced innate immune protective phenotype against HIV-1.

Inadequate local cell-mediated immunity appears crucial for the establishment of chronic HIV infection. Accumulation of regulatory T cells (Treg) at the site of HIV replication, the lymphoid organs, may influence the outcome of HIV infection. Our data provide the first evidence that chronic HIV infection changes Treg tissue distribution. Several molecules characteristics of Treg (FoxP3, CTLA-4, glucocorticoid-induced TNFR family-related receptor, and CD25) were expressed more in tonsils of untreated patients compared with antiretroviral-treated patients. Importantly, most FoxP3+ cells expressed CTLA-4, but not CD69. Furthermore, a direct correlation between FoxP3 levels and viral load was evident. In contrast, FoxP3 expression was decreased in circulating T cells from untreated patients, but normalized after initiation of treatment. Functional markers of Treg activity (indoleamine 2,3-dioxygenase, TGF-β, and CD80) were markedly increased in the tonsils of untreated patients. Our data could provide a new basis for immune-based therapies that counteract in vivo Treg and thereby reinforce appropriate antiviral immunity.

Short-chain fatty acids (SCFAs), major microbial fermentation products of dietary fiber in the gut, are shown to improve gut health. We hypothesized that soluble fiber (inulin and pectin) which are more accessible for microbiota fermentation may offer more pronounced mucoprotection than insoluble fiber (cellulose) in a murine model of chronic colitis. WT (BL6) mice receiving dietary cellulose exhibited robust colitis upon IL-10R neutralization as examined by serological, biochemical, histological and immunological parameters. Similar results were observed in colitis-prone toll-like receptor 5 knockout (Tlr5KO) mice. Surprisingly, prebiotic fiber inulin failed to protect against colitis in both WT and Tlr5KO mice. Remarkably, pectin ameliorates colitis development. GC-MS analysis of cecal content revealed that cecal butyrate, an inflammasome (NLRP3) response modulator in the gut, was augmented in inulin-fed colitic mice. Serum cytokine analysis showed that dietary inulin elevated IL-18, but diminished the IL-1 receptor antagonist (sIL-1Ra). On the flip side, dietary pectin reduced IL-18 and augmented sIL-1Ra. More importantly, suppressing butyrate production by metronidazole, which specifically depletes the butyrate producers in the gut, protected the mice from chronic colitis, suggesting that SCFAs-inflammasome axis shapes the beneficial effects of dietary fiber on the gut health. Collectively, mitigation of chronic colitis by dietary pectin can be partly explained by reduced cecal butyrate and elevated IL-1β inhibitor, sIL-1Ra. A detailed mechanistic study on how pectin-derived metabolites modulate inflammasome signaling may yield development of novel therapeutics to treat intestinal inflammation.

The parent-into-immunocompetent-F1 model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F1 CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 × DBA/2)F1 (B6D2F1) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c × B6)F1 (CB6F1) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F1 initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F1. However, the acute disease resolved, and the CB6F1 mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F1 and B6D2F1, whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F1s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.

Dendritic cells (DC) are the most potent cells involved in the generation of primary and secondary immune responses. To assess the feasibility of using autologous DC as immunotherapy for HIV disease, we analyzed a variety of immune parameters using DC isolated from HIV-infected (HIV+) individuals, as well as DC obtained from HIV-uninfected (HIV−) individuals infected in vitro with HIV. After stimulation with recombinant CD40 ligand (CD40LT), cytokine and β-chemokine production were similar by DC from HIV− donors infected in vitro with the CCR5-using HIV Ba-L strain (n = 8) compared with uninfected DC from the same donors. Production of β-chemokines, but not of cytokines, was increased by a CXCR4-using IIIB strain-infected DC (n = 7). Stimulation of HIV-infected DC with CD40LT decreased infection in Ba-L-infected DC, but had no effect on IIIB-infected DC. Consistent with this finding, CD40LT down-regulated CCR5 and up-regulated CXCR4 expression on DC. Monocyte-derived DC were also propagated from 15 HIV+ and 13 HIV− donors. They exhibited similar expression of costimulatory molecules and produced similar amounts of IL-12, IL-10, and β-chemokines, following stimulation. By contrast, stimulated PBMC from HIV+ patients exhibited decreased IL-12 and increased IL-10 production. In summary, phenotype, cytokine secretion, and β-chemokine production by DC from HIV+ individuals were normal. These cells may prove useful in boosting cellular immune responses in HIV+ individuals.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-γ, and TNF-α, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-β1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.

HIV-1 infection is accompanied by qualitative and quantitative defects in CD4+ T lymphocytes. Loss of immune function in HIV patients is usually associated with a profound dysregulation of cytokine production. To investigate whether cytokine signaling defects occur during HIV infection, PHA blasts from healthy human donors were infected with two strains of HIV-1 and screened for the expression of STAT proteins used in cytokine signaling. A selective decrease in STAT5B was seen 8 days after infection with the BZ167 dual-tropic HIV isolate, but not with the Ba-L, M-tropic strain. Based on these findings, purified T cells from HIV-infected patients in different stages of disease were also tested for STAT expression; decreases in STAT5A, STAT5B, and STAT1α were observed in all patients. The reduction in STATs seen in vivo and in vitro after HIV infection may contribute to the loss of T cell function in HIV disease.

This study examines cerebrospinal fluid from patients with three neuropsychiatric diseases of childhood for the presence and levels of several cytokines relevant to cell-mediated (type 1) and humoral (type 2) immunity. The patient groups include childhood-onset schizophrenia (n = 22), obsessive-compulsive disorder (OCD) (n = 24), and attention deficit hyperactivity disorder (n = 42). The cytokines examined include IL-2, IFN-gamma, TNF-beta/LT, IL-4, IL-5, IL-10, and TNF-alpha. Patients with OCD had a preponderance of type 1 cytokines. IL-4 was detectable only in samples from patients with schizophrenia. IL-10 was rarely detected and never in patients with OCD. Few patients with schizophrenia had detectable amounts of IFN-gamma in CSFL. We conclude that there is a relative skewing of CSFL profiles toward type 1 cytokines in patients with OCD, whereas in schizophrenia the relative preponderance is toward type 2 mediators. Patients with attention deficit hyperactivity disorder exhibited profiles intermediate between OCD and schizophrenia. We infer that cell-mediated immunity may be involved in the etiopathogenesis of OCD, whereas a relative lack of cell-mediated immunity and involvement of humoral immunity may be present in schizophrenia. These data provide a rationale for immune-based strategies of study and therapeutics in childhood neuropsychiatric disease.

We followed cytokine production from induction through disease progression in a murine model of experimental systemic lupus erythematosus (SLE). SLE was induced by immunization with the human monoclonal anti-DNA Ab that bears the common Id designated 16/6 Id. BALB/c and C3H.SW mice that are susceptible to SLE induction and C57BL/6 mice that are resistant were immunized with the 16/6 Id. Cytokine production was tested periodically for 7 mo. Increased production of IL-2 and IFN-gamma, the Th1-type cytokines, was detected in BALB/c and C3H.SW mice 2 to 4 mo following immunization. IL-4 and IL-10, the Th2-type cytokines predominated later in disease course, and peaked 5 mo following disease induction. At this stage the Th1 type cytokines dropped to levels below those observed in controls. IL-4 production also dropped rapidly to very low levels, while IL-10 production decreased but remained above control levels. The ratio of IgG2a/IgG1 of DNA and 16/6 Id-specific Abs peaked at 2 mo following disease induction and decreased later, in concordance with the higher production of Th2-type cytokines. Thus, the development of experimental SLE in mice involves two stages: increased production of Th1-type, followed by increased induction of Th2-type cytokines. High levels of the proinflammatory cytokines, TNF-alpha and IL-1, were maintained throughout disease course. No significant changes were detected in the cytokine profile of C57BL/6 immunocytes following immunization with the 16/6 Id, supporting the possible role of the cytokine network in SLE.

Self-thyroid epithelial cell (TEC)-reactive CD8+ and CD4+ T cell lines were established by culturing T cells that infiltrate in autoimmune thyroiditis lesions. We investigated the properties of CD8+ T cell lines and clones in comparison with previously characterized CD4+ T cell lines/clones. Although the recognition of self-Ag by anti-TEC CD4+ T cell lines/clones required the cooperation of syngeneic spleen cells as APC, a representative CD8+ line (N4C) was stimulated with syngeneic TEC in the absence of APC. Precise analysis of MHC restriction using N4C-derived clones revealed that CD8+ clones recognize self-Ag on TEC in the context of class I MHC molecules. Most CD8+ clones were also found to express TCR with V beta specificities that were different from those observed for anti-TEC CD4+ clones. N4C cells produced IL-2, IFN-gamma, and TNF-alpha beta, but not IL-4 and IL-5 after stimulation with TEC, thus exhibiting the profile of lymphokine production similar to that expressed by CD4+ Th1 on one hand, but on the other, they showed the functional property that has not been observed for anti-TEC CD4+ clones. Namely, they elicited appreciable levels of cytolytic effects on syngeneic TEC in a short-term (4-h) 51Cr release assay. Thus, these results indicate that self-TEC-reactive CD8+ T cell lines/clones recognize Ag directly on TEC in a class I MHC-restricted way so as to exhibit various functions including the Th1-like profile of lymphokine production and anti-TEC cytolysis. The results are also discussed in terms of the nature of self-Ag presented with class I MHC molecules on TEC, as well as the potential roles of anti-TEC CD8+ T cells in the pathogenesis of thyroiditis.

APC dysfunction may be important in immune dysregulation associated with HIV disease. Langerhans cells, epidermal APC, can be infected with HIV, although their function in HIV-infected persons has not been studied. Therefore, we studied the immunologic function of Langerhans cells, in parallel with blood APC (enriched for monocytes/macrophages (M phi) function, in 21 HIV-seropositive (HIV+) and 21 HIV-seronegative volunteers, including three monozygotic twin pairs discordant for HIV serology. Langerhans cells from HIV+ patients were quantitatively normal and expressed normal levels of HLA-DR. However, Langerhans cells from AIDS patients and M phi from both AIDS and HIV+ non-AIDS patients stimulated allogeneic T cells less well compared with control APC. In addition, decreased recall Ag- and mitogen-induced T cell responsiveness was observed in HIV+ patients using either autologous Langerhans cells or autologous M phi as APC/accessory cells. Interestingly, Langerhans cells and M phi isolated from HIV+ twins (CD4+ cell counts of 181, 271, and 562/mm3) were able to present recall Ag normally to HIV-uninfected syngeneic T cells. In summary, APC from HIV+ patients were impaired in their ability to generate a primary immune response (i.e., alloantigen-induced T cell stimulation), but they retained the ability to generate a secondary immune response (i.e., recall Ag-induced syngeneic T cell stimulation). Thus, these findings suggest that defects in secondary immune responses commonly observed in HIV disease are dependent on T cell dysfunction alone, whereas defective primary immune responses may be secondary to both T cell and APC dysfunction.

Natural killer (NK) cells possess an array of germline-encoded activating, inhibitory and adhesion receptors, the balance of which in concert with their developmental stage, determine their cytotoxic and cytokine-producing potential. Although NK cells develop in the bone marrow and secondary lymphoid tissues, substantive differentiation is apparent in the peripheral blood including known age-related variation. In order to gain greater insight into phenotypic and functional variation within peripheral blood NK cells across age groups, we used multi-parameter, polyfunctional flow cytometry to interrogate the NK cell variability in 20 healthy adults and 15 5–10, 11–15 and 16–20 year-old children. We found that the normative ranges in both adults and children displayed great inter-individual variation for most subsets. Children possessed >76% unstimulated NK subsets coexpressing perforin and granzyme B as opposed to >65% in adults. Children also had >28% NK subsets expressing all adhesion receptors in our panel compared to half of that in adults. As examples, terminally differentiated CD16+CD57+CD11b+ NK cells were reduced after 16 years of age as well as NKp46, 2B4, CD107a and newly-synthesized perforin in adults. Although CD107a intensity and new perforin synthesis was lower in adults, induced levels of IFNγ (by intensity) was higher. Despite most receptors not differing in their overall expression levels, major phenotypic differences were revealed in the combinatorial expression of NK receptors between adults and children. Our findings identify previously unappreciated NK cell subsets potentially distinguishing children from adults and suggest functional correlates that may have relevance in age-specific host defense.

Platelet-activating factor (PAF) is a versatile lipid mediator of inflammation in a variety of biologic systems. We have previously reported that one of the earliest events in the signal transduction pathway of PAF in a human B lymphoblastoid cell line was the induction of tyrosine kinase activity concomitant with the activation of phospholipase C (PLC). We now demonstrate the occurrence of multiple tyrosine phosphorylation-dependent events which follow the interaction of PAF with its receptor on B cells. Anti-phosphotyrosine immunoprecipitates from lysates of PAF-stimulated cells, when fractionated by SDS-PAGE and analyzed by Western blotting with anti-PLC-gamma 1, showed that maximal tyrosine phosphorylation of this enzyme occurred within 2 min of stimulation. This phenomenon was verified by immunoprecipitating with anti-PLC-gamma 1 and subsequently probing with anti-phosphotyrosine. Immunoprecipitation of the tyrosine kinases, Fyn and Lyn, from PAF-stimulated cells, and use of these immunoprecipitates in kinase assays established that the activation of both kinases also occurred within the first 2 min of stimulation with phosphorylation occurring on their tyrosine residues. Additionally, we also provide evidence for the tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PtdIns 3-kinase) and activation of this kinase by PAF in a dose-dependent manner, maximal activation occurring within 10 min post-stimulation. We have thus demonstrated that the activation of tyrosine kinases is an important proximate step in PAF-mediated signal transduction in B cells, leading to tyrosine phosphorylation and activation of PLC-gamma 1, Fyn and Lyn kinases, and PtdIns 3-kinase.

In vitro activation of PBLs from HIV+ individuals resulted in programmed cell death (PCD) within 2 days in 58 of 95 HIV+ blood donors, in contrast to only two of 30 control HIV- donors. CD4+ and CD8+ T cells from HIV+ donors died under these conditions, and these cells showed apoptotic nuclear morphology and DNA fragmentation. To test the hypothesis that this cell death shares a common biochemical pathway with that induced by TCR cross-linking in normal dividing T cells, inhibitors of the calcium-activated cysteine protease calpain were tested for their ability to block the activation-induced PCD of HIV+ donors. The E-64 (epoxysuccinyl) class of cysteine protease inhibitors gave 40% to 60% inhibition of HIV+ PCD responses, while the aldehyde inhibitors, leupeptin and calpain inhibitor II, gave 60% to 67% inhibition. The involvement of this calpain-dependent death pathway in HIV-induced functional T helper cell deficiency was tested by examining the effect of calpain inhibitors on the defective Ag- and mitogen-dependent proliferative responses of HIV+ donors. Twenty to fifty percent of such defective responses were significantly restored toward normal levels by calpain inhibitors, whereas control responses by normal donors were largely unaffected. These data suggest that a calpain-dependent PCD pathway contributes to HIV-associated immunodeficiency and suggest the use of calpain inhibitors as a possible route to therapy of HIV infection.

We previously have observed that T dependent immune functions were deficient for several months after induction of acute suppressive graft-vs-host-reaction (GVHR) by injection of parental C57BL/10 donor spleen cells into unirradiated (B10 x B10.BR) F, hosts. We therefore investigated whether new T cells matured after acute GVHR, and whether these were tolerant of host Ag. By 8 to 17 mo after GVHR, the frequencies of splenic CD4 and CD8 T cells were found to be comparable to age-matched untreated hosts, although the lymphoid organ size and hence the total number of T cells was significantly reduced. When GVHR was induced with a combination of C57BL/6 (Thy-1.2) mature lymphocytes and B6.PL (Thy-1.1) bone marrow stem cells, the mature donor Thy-1.2 T cells initially predominated during the acute GVHR. After several months, however, 75% of the CD4 and 50% of the CD8 T cell population was derived from donor Thy-1.1% pre-T cells that had matured in the host. Long term GVHR spleen cells were unresponsive to host Ag in CTL assays, but did not suppress anti-host CTL responses. Finally, host-reactive V beta 11 TCR expressing cells were found to be clonally deleted from splenic CD4 and CD8 populations, consistent with intrathymic negative selection. This evidence suggests that the post-GVHR thymus has the capacity to produce and negatively select phenotypically mature CD4 and CD8 T cells and that a failure to clonally delete self-reactive populations is not a contributing factor to the development of chronic GVHR in this system.

To assess interactions between T cells and APC in patients with SLE, PBL were stimulated in vitro and IL-2 production measured after stimulation with either a MHC-self-restricted Ag (influenza A virus) or an Ag which can use both MHC-self-restricted or unrestricted T cell activation pathways (HLA-alloantigen). SLE patients (n = 26) and controls (n = 8) were categorized as responder (+) or nonresponder (-) for each stimulus and grouped according to their paired response pattern. All controls responded to both influenza virus (Flu) or alloantigen (Allo) and were categorized as +/+. In contrast, SLE patient response patterns were heterogeneous with no evidence for a single SLE-associated defect. Instead, SLE patient responses fell into one of three different patterns: a) normal responses to both Flu and Allo (+/+), observed in nine (35%) patients; b) defective responses to Flu but intact Allo responses (-/+) observed in 12 (46%) SLE patients; and c) defective responses to both Flu and Allo (-/-), observed in five (19%) SLE patients. There was no statistically significant correlation between immune response pattern and the use of immunosuppressants. Further analysis of -/- SLE patients indicated defects in APC function and in both CD4+ and CD8+ T cell function. In contrast, cell depletion and add-back studies in -/+ SLE patients demonstrated defects in APC function only. Thus, similar to the well recognized clinical heterogeneity among SLE patients, our data support the concept that SLE patients are heterogeneous with respect to in vitro T cell-APC function, exhibiting responses ranging from normal function to defects in APC and in both T cell subsets. Prospective studies are in progress to determine the clinical relevance of these observations.

We have previously shown that autoimmune thyroiditis spontaneously develops in T cell-depleted (C57BL/6xC3H/He)F1 mice after adoptive transfer of syngeneic lymphoid cells that have been depleted of CD5bright T lymphocytes. This mouse model was considered to be an instructive model for Hashimoto's thyroiditis in humans, and suggested that CD5dull CD4+ autoreactive T cells are not deleted in normal lymphoid cell populations and induce thyroiditis after elimination of regulatory T cell subsets. In our study, we have established thyroid-derived T cell lines and clones by culturing thyroid-infiltrating lymphocytes with thyroid epithelial cells and syngeneic feeder cells in the presence of exogenous IL-2. Both CD4+ T cells and CD8+ T cells were identified and their CD5 expression was dull or negative. In our report, we focused on CD4+ T cells. Four CD4+ T cell lines and 19 clones were generated and characterized. CD4+ T cell clones derived from F1 hybrid (H-2b/k) mice responded to thyroid Ag in the context of not only class II MHC molecules from both parental strains (I-Ak or I-Ab), but also F1 unique MHC molecules. Thyroglobulin (Tg) was demonstrated to be a major autoantigen of the thyroid for T cell responses, and at least two epitopes of Tg were suggested to be recognized by T cell clones. An additional undefined thyroid component other than Tg was recognized by some T cell clones. Thyroid-derived T cells were all TCR-alpha beta+. Five types of V beta usage (V beta 2, 4, 8.3, 14, and an undefined V beta) were found in CD4+ T cells. Finally, the thyroid lesions could be transferred to syngeneic mice by five distinct groups of CD4+ T cell clones, which were distinguished on the basis of their Ag specificity, MHC restriction, and TCRV beta usage. The considerable heterogeneity of TCRV beta usage of thyroiditis-inducing T cell clones in this study may be attributed to the multiple patterns of their recognition of the thyroid autoantigens. These findings provide important implications for further understanding of organ-specific autoimmune processes induced by depletion of regulatory T cell subsets.

Mice vaccinated with a live temperature sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to subsequent challenge with a highly virulent Toxoplasma strain (RH). Because CD8+ T cells have been demonstrated to be critical to this protective immunity in vivo, the involvement of cytotoxic T lymphocytes in the killing of infected cells in vaccinated mice was investigated. After restimulation in vitro, splenic T cells from vaccinated mice of either the BALB/c or C57BL/6 strains were found to kill syngeneic bone marrow-derived macrophages infected with TS-4 tachyzoites or preincubated with soluble T. gondii Ag. Unimmunized control mice or mice vaccinated with heat-killed TS-4 tachyzoites failed to generate significant CTL activity in vitro. Moreover, the observed lytic reaction was found to be target specific, not killing uninfected or unpulsed macrophages even when included as bystanders in the assay. Target lysis did not depend on the production by the effector cells of either a cytotoxic supernatant factor or IFN-gamma. Depletion of CD8+ cells from the splenic effector cell population, however, abrogated the cytotoxic activity, whereas depletion of CD4+ cells had little effect. The MHC restriction of the Toxoplasma-specific cytolytic reaction was confirmed in studies using effector cells from BALB/c mice and targets from congenic or mutant haplotype strains. These experiments indicated that target killing is primarily restricted by genes mapping within the H-2D/Ld loci. Together, these results establish MHC-restricted cytolysis as a major parameter of CD8+ effector function against T. gondii and indicate that, in the case of this protozoan, Ag presentation to CD8+ lymphocytes can occur as a result of either processing within infected cells or exogenous uptake of parasite Ag.

Four synthetic peptides corresponding to the IIIB sequence of gp160 of HIV were recently reported to stimulate Th cell function by PBL from HIV-infected, asymptomatic patients. In the present report, we used these same peptides to demonstrate CTL activity in a similar patient population. EBV-transformed B-cell lines from asymptomatic, HIV seropositive and seronegative control donors were pre-incubated with the peptides. Fresh PBL from 19 (76%) of 25 HIV seropositive donors lysed autologous targets pulsed with at least one of the four peptides. Autologous targets pulsed with two non-immunogenic peptides were not lysed. PBL from none of the eight HIV seronegative controls lysed peptide-preincubated autologous targets. The CTL activity was mediated by T cells, was predominantly MHC class I restricted, and was increased by in vitro restimulation of PBL with the peptides. HLA A-2 was identified as a restricting element for all four peptides in different patients, and for three of the peptides in the same donor. HLA-A1 or -B8 may also present some of the peptides. Thus, the same peptides can be recognized by human Th cells and class I MHC-restricted CTL.

The graft-vs-host reaction (GVHR) generated by the injection of parental lymphocytes into unirradiated immune-competent F1 hosts is characterized by an acute loss of immune functions, an attack on host tissues, and a gradual recovery of function. Flow cytometric analysis of the donor- and host-derived splenic populations during the course of acute dysfunction and gradual recovery revealed a complex pattern of changes in lymphoid and myeloid populations that resulted in the repopulation of the host with donor-derived cells. Initially, donor-derived T cell populations expanded, particularly CD8+ T cells. Next, host T cell and B cell populations disappeared. Finally, donor-derived cells repopulated the lymphohematopoietic system in the sequence myeloid populations, B cells, and, after a protracted period, T cells. The recovery of immune functions following GVHR-induced immune deficiency was associated with this repopulation of the spleen by donor-derived cells. Donor repopulation of the host lymphohematopoietic system required the presence of both CD4 and CD8 cells in the original donor inoculum. Depletion of donor CD4 populations precluded development of GVHR or any donor engraftment; depletion of CD8 cells resulted in engraftment solely of donor CD4 populations.

Platelet-activating factor is a potent mediator of the inflammatory response. Studies of the actions of platelet-activating factor have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of platelet-activating factor on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that platelet-activating factor significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, lyso-platelet-activating factor, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that platelet-activating factor from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas lyso-platelet-activating factor was again ineffective. We further demonstrate the impact of platelet-activating factor binding to B cells by measuring platelet-activating factor induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, platelet-activating factor was capable of inducing transcription of the nuclear proto-oncogenes c-fos and c-jun. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates platelet-activating factor induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, platelet-activating factor is shown here to have a direct and profound effect on a pure B cell line.

The effects of cyclosporin A (CsA) on the generation of NK cells were studied using syngeneic bone marrow transplanted mice subsequently treated with CsA (BMT/CsA mice). In contrast to a severe reduction in T cells that was reported previously, these mice exhibited a marked enhancement of splenic NK activity. The enhanced NK activity was mediated by NK1.1+, Thy-1- cells as assessed by antibody plus complement treatment, and was concomitant with an absolute increase in the numbers of NK1.1+ cells as assessed by flow cytometry. Because the depletion of host-derived, mature NK cells by injection of anti-asialo GM1 antibody before bone marrow reconstitution did not affect the enhancement of NK activity, CsA appeared to augment the generation of NK cells from bone marrow precursors. To investigate a possible relationship between the enhancement of NK activity and the maturational arrest of T cells in the thymus induced by CsA, mice were thymectomized, followed by irradiation, bone marrow reconstitution, and CsA treatment. These mice exhibited as strong enhancement of splenic NK activity as BMT/CsA mice, suggesting that the CsA-induced effect on NK cells is distinct from its effect on T cell development in the thymus. Taken together, these results are the first demonstration of the positive effect of CsA on NK cell generation and may be of importance in clinical bone marrow transplantation.

BALB/c mice vaccinated with a temperature-sensitive mutant (TS-4) of Toxoplasma gondii develop complete resistance to lethal challenge with a highly virulent toxoplasma strain (RH). This immunity is known to be dependent on IFN-gamma synthesis. In vitro and in vivo T cell depletions were performed in order to identify the subsets responsible for both protective immunity and IFN-gamma production. When stimulated with crude tachyzoite Ag in vitro, CD4+ cells from vaccinated mice produced high levels of TH1 cytokines (IL-2 and IFN-gamma) but not TH2 cytokines (IL-4 and IL-5). CD8+ cells, in contrast, produced less IFN-gamma and no detectable IL-2. Nevertheless, they could be induced to synthesize IFN-gamma when exposed in culture to exogenous IL-2. In vivo treatment with anti-CD4 plus anti-CD8 or anti-IFN-gamma antibodies during challenge infection completely abrogated resistance to T. gondii. In contrast, treatment with anti-CD4 alone failed to reduce immunity, whereas anti-CD8 treatment partially decreased vaccine-induced resistance. These results suggest that although IFN-gamma and IL-2-producing CD4+ lymphocytes are induced by vaccination, IFN-gamma-producing CD8+ T cells are the major effectors of immunity in vivo. Nevertheless, CD4+ lymphocytes appear to play a synergistic role in vaccine-induced immunity, probably through the augmentation of IFN-gamma synthesis by the CD8+ effector cells. This hypothesis is supported by the observation that when giving during vaccination, as opposed to after challenge, anti-CD4 antibodies are capable of blocking protective immunity.

The development of acute graft-vs-host disease (GVHD) is a common outcome after the injection of fully MHC disparate parental T cells into unirradiated F1 mice. Murine cytomegalovirus (MCMV) infection has been previously shown to augment the development of acute GVHD in the parent-into-F1 (P----F1) model, such that 10-fold fewer parental cells are required. In the present study, we have investigated the effect of MCMV infection on the induction of non-lethal GVHD that occurs in P----F1 combinations involving MHC class I only or class II only differences. Using P----F1 combinations involving either an H-2K only difference or an H-2D only difference, MCMV infection of F1 mice 3 days before the injection of parental spleen cells led to a profound T cell immunodeficiency that strongly resembled that observed in acute GVHD. Further studies examining the H-2K disparate P----F1 combination, C57Bl/6---- (C57Bl/6xB6.C-H-2bm1) F1 and combined MCMV infection showed that the immunodeficiency is characterized by a profound loss of in vitro Th cell production of IL-2 and an intrinsic defect in T effector function as shown by an inability of rIL-2 to restore defective CTL responses. Additional experiments in these mice revealed the presence of suppressor cells as well as significant parent-anti-F1 CTL activity possibly accounting for the suppressor effect. This pattern of immunodeficiency was not seen after the administration of either MCMV or MHC class I disparate parental cells alone. MCMV infection did not detectably alter the immunodeficiency observed in a P----F1 combination involving a MHC class II difference only. These results indicate that MCMV infection can alter the pattern of GVHD in the setting of an MHC class I disparity, but not in the setting of class II disparity, such that it resembles acute GVHD. These results may have relevance to the human transplant setting where intercurrent CMV infection has been associated with an adverse clinical outcome.

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.

We have examined the role of the human responder APC in the generation of CTL responses to xenogeneic antigens. Of six xenogeneic responses evaluated, only the human antimurine response was dependent on human APC for CTL generation. APC requirements for the other five xenogeneic responses more closely resembled those observed in the generation of human or murine alloreactive CTL. Depletion studies identified a defective human CD4+ Th cell-murine stimulator cell interaction that could be bypassed by the addition of exogenous IL-2. The function of the responder APC involved in the human antimurine CTL response was inhibited by chloroquine, suggesting a requirement for Ag processing. Effective presentation of murine stimulator Ag by human APC was completely blocked by anti-human Ia mAb, indicating that the Ag is presented to Th cells via the human class II molecule. These results are consistent with an Ia-dependent recognition of processed murine Ag by human T cells and represents a model for investigating human T cell activation requirements, Th cell function, and MHC restriction.

PBL from approximately 50% of asymptomatic individuals infected with HIV have been previously demonstrated to exhibit defective in vitro Th function that is selective for influenza A virus (FLU), but not for HLA alloantigens (ALLO). In this report, we have further studied HIV+ individuals with this selective Th defect, and demonstrate that defective in vitro CTL responses to FLU can be restored by costimulation with FLU + ALLO. In contrast, HIV+ patients who have lost Th responses to ALLO were unable to correct CTL responses to FLU by this costimulation procedure. These findings indicate that intact Th responses to ALLO can be used in vitro to provide Th signals necessary to activate the T effector cell response to a potential pathogenic virus. Our results raise the possibility that a program of in vivo coimmunization with ALLO plus antigens of potential pathogens (including HIV) can be useful in HIV+ patients exhibiting selective defects in Th function. Furthermore, this approach could be incorporated in vaccine trials aimed at enhancing immunity to HIV in patients who have been infected previously with this virus.

Cyclosporin A (CsA) is a widely used agent for the prevention of tissue allograft rejection in human transplantation. As a result of the recent demonstration that the allospecific Th cell response of human PBL can be generated by three distinct pathways of Th cell and APC interactions, we investigated the sensitivity of these three Th-APC pathways, as well as the Th response to recall Ag, to different concentrations of CsA. PBL from healthy volunteer donors were set up as primary in vitro cultures either without antigenic stimulation, or with influenza A virus, tetanus toxoid, or HLA alloantigenic (ALLO) stimulation. Ag-stimulated IL-2 production and proliferation were measured to assess Th cell function. To study the effect of CsA on Th function, different concentrations of CsA (0.001 to 0.1 micrograms/ml final) were added to the cultures at the time of stimulation. Th responses to influenza A virus and tetanus toxoid were more sensitive to CsA than the Th response to ALLO. By selective depletion of either responder or stimulator APC and/or of CD4+ or CD8+ cells, we 1) verified that the human ALLO Th response can be mediated by three distinct Th-APC pathways; 2) demonstrated that the ALLO response mediated by CD4+ Th and self-APC (the same helper pathway used by recall Ag) is as sensitive to CsA as the responses to recall Ag; and 3) showed that there is a hierarchy of sensitivity of these three allospecific pathways. The results are discussed with respect to the potential significance of the differential sensitivity of these allospecific Th-APC pathways to CsA for prevention of tissue allograft rejection.

In our study, we have measured in vitro proliferation and IL-2 production by human PBL to characterize the interactions between Th cells and accessory cells (AC) involved in responses to either conventional Ag or alloantigens. IL-2 production and proliferative responses to conventional Ag, such as influenza or tetanus, are exclusively dependent on the presence of CD4+ T cells and AC. In contrast, IL-2 and proliferative responses to alloantigen can be mediated by either CD4+ or CD8+ T cells. CD4+ T cells respond to alloantigen using either autologous AC (self-restricted), or allogeneic AC (allo-restricted), whereas CD8+ T cells respond to alloantigen using allogeneic AC only. The understanding of Th cell-AC interactions involved in in vitro allogeneic responses will be important for delineating the Th cell-AC interactions involved in transplantation immunity as well as in clinical disorders characterized by T cell dysfunction such as human immunodeficiency virus infection and systemic lupus erythematosus.

Ligand binding to B lymphocytes via membrane Ig initiates a cascade of events beginning with the hydrolysis of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Subsequent to the activation of protein kinase C and the induction of a rise in intracellular calcium by diacylglycerol and inositol 1,4,5-trisphosphate, there is gene transcription and eventually cellular activation. By mimicking the initial event of B cell activation with phorbol ester and calcium ionophore one can begin to identify the many mediators used in signaling between the membrane and the nucleus. We have examined the effect of calcium on arachidonic acid (AA) metabolism in several EBV-transformed human B cell lines. The cells were labelled with [3H]AA and stimulated with the calcium ionophore A23187. Analysis of the supernatant by reversed-phase HPLC demonstrated a dose-dependent release of an AA metabolite that coeluted with authentic 5-hydroxyeicosatetraenoic acid (5-HETE). In addition, the AA metabolite coeluted with standard 5-HETE under straight-phase chromatography. Further analysis by RIA confirmed the identification of 5-HETE and revealed an additional metabolite, 5-HETE lactone (5-HL). 5-HL is the intramolecular ester of 5-HETE generated in the presence of acid. We were unable to convert [3H] 5-HETE into 5-HL during sample preparation unless cells were present, suggesting that the 5-HL, is of cellular origin. These results suggest that the AA metabolites 5-HETE and its intramolecular ester 5-HL may play a role in B cell activation because they are produced subsequent to a rise in intracellular Ca2+, an event that occurs during cross-linking of membrane Ig.

Our preclinical data showed binding of green tea catechin, epigallocatechin gallate(EGCG), to CD4 with inhibition of HIV-1-gp120 binding to human CD4+Tcells throughout a broad spectrum of virus strains lead to implementation of our Phase I/IIa clinical trial in HIV-1-infected indivduals. As mucosal dendritic cells(DC), Langerhans cells(LC) and monoctye-derived dendritic cells(MDDC), with CD4 receptor binding to HIV-1-gp120, act as first line defense in mucosa against invading pathogens via innate immune responses,EGCG in the context of an HIV-1 microbicide was studied. Human CD34+ hematopoietic stem cells and CD14+ cells differentiatied to LC and MDDC,respectively were assessed for HIV-1-gp120 binding, Annexin V-binding apopotosis and receptor profiling by flow cytometry, cytokine profiling by Luminex, toll-like receptor(TLR) profiling by RT-PCR, antigen uptake/presentation(APC) by RIA, cytotoxicity by MTT and statistical significance by Student’s t test. EGCG(1-50uM) downregulated TLR-6,-8 mRNA expresion (25%;p

Langerhans cells (LC) and monocyte-derived dendritic cells (MDDC), the first line of defense against viral infection, initiate innate immune responses via toll-like receptors (TLR). Previously, we showed inhibition of HIV-1-gp120 binding to CD4 on T cells by the green tea catechin, epigallocatechin gallate (EGCG). We present evidence of EGCG inhibition of the mucosal route of HIV-1 without alteration of dendritic cell (DC) innate immune responses. Methods utilized were CD34+ hematopoietic stem cells and CD14+ cells differentiated to LC and MDDC, respectively, isolated from peripheral blood of HIV-1 seronegative donors, flow cytometry of HIV-1-gp120 binding and TLR profiling with RT-PCR, radioisotope assays for antigen (Ag) studies, MTT assay for cytotoxicity, p24 ELISA for HIV-1 infectivity studies and statistical significance by Student’s t test. EGCG significantly inhibited HIV-1 infectivity in LC in a dose-dependent manner; 100%(50uM;p

Efficacy of the Recombinant Botulinum Vaccine A/B (rBV A/B) against lethal intoxication with botulinum neurotoxin complex (BoNT) cannot be evaluated directly in humans; therefore, efficacy will be based on meeting the requirements of the FDA Animal Rule. Serum BoNT neutralizing antibody concentration (NAC) is being proposed as a correlate of protection to evaluate efficacy in nonclinical models. Under this approach, NAC levels in animals vaccinated either with rBV A/B or passively immunized with BoNT neutralizing antibody will be used to estimate the potential level of protection in humans with similar NAC levels. Estimating the level of protection based on NAC requires establishing NAC neutralizing efficiency (the amount of BoNT neutralized per NAC unit). The neutralizing efficiency of human BoNT neutralizing antibody was evaluated using immunoglobulin purified from pooled sera of clinical volunteers vaccinated with rBV A/B. Solutions of immunoglobulin were prepared at three different NAC levels, mixed with serial dilutions of BoNT serotype A or serotype B and injected into mice. At each NAC level, 50% and 99% neutralization of BoNT was determined by probit analysis of mortality dose-response curves. A simple linearly proportional relationship does not exist between NAC level and the amount of BoNT neutralized. Extrapolations of level of protection must consider variability in BoNT neutralizing efficiency at different NAC levels.

Previously, we presented evidence that the green tea catechin, epigallocatechin gallate (EGCG), binds to the CD4 molecule at the gp120 attachment site on T cells. CD4+ T cells, B cells and macrophages were isolated from HIV-1 uninfected donors. HIV-1 infectivity by HIV-1 p24 ELISA on M-tropic (R5) subtypes B, C and G; T-tropic (X4) subtypes B and D; and dual tropic (R5/X4) subtype B. Immune function studies: cytotoxicity by ViCell, apoptosis by flow cytometry, immunoglobulin (Ig) production by ELISA and cytokine profiling by multiplex platform. Statistical significance by Student's t test. EGCG significantly inhibited HIV-1 infectivity on human immune cells in a dose-dependent manner (6-100μM) (p (NIH AT003084)

Malignant pleural mesothelioma (MPM) is an aggressive disease with survival prognosis less than one year. Interestingly, MPM samples have been reported to express the viral oncoprotein, Simian virus 40 (SV40) large tumor antigen (Tag). To this end, our laboratory has characterized protective SV40 Tag-specific immunologic mechanisms following prophylactic immunization in a murine experimental pulmonary metastasis model. Recent results indicate that ADCC functions as a primary in vivo anti-tumor mechanism since SV40 Tag antibody and Fcγ receptors are required immunologic-components in the observed protection from lung metastasis. We also find that with an antibody response due to vaccination, CD8+ T lymphocytes are required T cell subsets in the effector phase response of tumor challenge in vivo. The nature of the CD8+ T cell response appears to be CTL-driven based upon a systemic increase in Th1 cytokines by flow cytometry and tumor antigen-specific splenocyte production of IFN-γ when assessed by ELISpot and ELISA assays. In all, our results support the hypothesis that an SV40 Tag antibody-based ADCC pathway drives the activation of a cell-mediated response leading to protection from tumorigenic growth. These data also have implications for the construction of an effective MPM-specific vaccine that elicits broad-based immunity in individuals at high-risk for developing SV40 Tag-expressing tumor cells.

Binding of HIV-1 envelope glycoprotein, gp120, to the CD4 T cell receptor results in HV-1 infection. We previously presented evidence of high affinity binding of the green tea catechin, epigallocatechin gallate (EGCG) to the CD4 molecule. We now present evidence in parallel studies that EGCG inhibits the binding of gp120 on human CD4+ T cells and PBMC and prevents the HIV-1 infectivity of macrophages and PBMC. Binding studies utilized spectroscopy and flow cytometry. HIV-1 infectivity was assessed by HIV-1 p24 EIA. Studies indicated that EGCG binds to CD4 at the same binding pocket as gp120. EGCG markedly inhibited the binding of HIV-1-gp120 to CD4+T cells in a dose-dependent manner at physiologically relevant levels (32% at 0.2uM p Supported by NIH grant R21AT003084.