Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.     

Humoral and cellular immune responses to matrix protein of measles virus in subacute sclerosing panencephalitis.

The immune response to matrix (M) protein of measles virus was examined in patients with subacute sclerosing panencephalitis (SSPE) and controls. Antibodies specific for M and nucleocapsid (NC) proteins in 11 serum and 8 cerebrospinal fluid (CSF) samples from patients with SSPE were quantitated by enzyme-linked immunosorbent assay by using affinity-purified measles virus proteins. Geometric mean anti-NC antibody titers were higher in the serum (6.58 +/- 0.98 [mean +/- standard deviation]) and CSF (4.38 +/- 0.74) of SSPE patients compared with controls. Anti-M antibodies were present in the serum and CSF of all SSPE samples tested but in titers lower than those of anti-NC antibodies. Geometric mean anti-M antibody titer was 3.35 +/- 0.53 in sera from patients with SSPE compared with 3.05 +/- 0.66 in sera from patients with other neurological diseases and 3.12 +/- 0.74 in sera from healthy individuals. Geometric mean anti-M antibody titer was 2.59 +/- 0.86 in the CSF of eight patients with SSPE compared with a mean less than 1.00 for patients with other neurological disease (controls). Intrathecal synthesis of anti-M or anti-NC antibodies was established in four patients with SSPE. The cellular immune responses to M, F, HA, and NC proteins were examined in four of the patients with SSPE by lymphoproliferation and were not significantly different from those in five healthy controls. The results demonstrate humoral and cellular immune responses to M protein in patients with SSPE and indicate that it is unlikely that a defect in the immune response to this virus component accounts for the disease process in the patients studied.     

Humoral immune responses to microbial infections in the genital tract

Human reproductive tracts represent components of the mucosal immune system with unique features. Although secretory IgA is present, IgG is more abundant, and typical mucosa-associated lymphoid tissue for generating common mucosal immune responses is absent. Antibody responses to genital infections or to locally applied vaccines are usually modest, but alternative strategies for eliciting genital tract antibodies are being developed.     

Phytohemagglutinin skin test responses to evaluate in vivo cellular immune function in rats

It is often necessary to have a small animal model which permits the sequential evaluation of functional immune status over a period of time. We report here the in vivo, intradermal response to phytohemagglutinin which produces an area of induration that is histologically similar to a typical delayed cutaneous hypersensitivity response, and that provides fast, quantitative, reproducible results similar to those observed with standard but more laborious and variable in vitro tests of immune function. For small animal studies this has the advantage of permitting longitudinal evaluations over time without sacrificing the animal. Using phytohemagglutinin-microprotein (0.2 mg/0.1 ml), injected intradermally, a delayed cutaneous hypersensitivity-like response is induced which is maximal at 24 hr. When immune function was altered either by treatment with a chemical immunosuppressant (ethanol) or by hormonal manipulations (hypophysectomy and rat growth hormone), the delayed cutaneous hypersensitivity-like response (area of induration) correlated closely with both macrophage migration inhibitory factor changes (r = 0.98; P less than 0.001) and mixed lymphocyte reaction changes (r = 0.99; P less than 0.05). These observations suggest that this technique correlates well with standard in vitro measures of immune response and may thus permit an in vivo estimation of immune reactivity.     

Distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats

To evaluate cell-mediated immunity in influenza-infected cotton rats, we compared the cellular composition of spleen, mediastinal lymph nodes (MLN) and bronchoalveolar lavage (BAL) after primary and secondary infection. There was an increase in cellularity in the MLN after primary infection that was further expanded upon rechallenge. CD4(+) T cells expanded after primary infection, but there was preferential increase in the number of CD4-negative T cells following secondary challenge. After primary infection, a large proportion of the monocytes and NK cells were present in the BAL while a T cell population dominated after secondary infection. CD4(+) T cells were predominant in this population unless the animals had been challenged with heterosubtypic influenza A virus. These studies are the first to show evidence of a memory T cell response to influenza infection in cotton rats and show quantitative and qualitative differences between the recall response to homosubtypic and heterosubtypic viruses.     

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.     

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Summary Serum IgG fractions from a large and homogenous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the non-selective CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Serum IgG fractions from a large and homogeneous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the 'non-selective' CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

Humoral and cellular responses to homologous extracts of Nematospiroides dubius and Nippostrongylus brasiliensis

and 1986. Humoral and cellular responses to homologous extracts of Nematospiroides dubius and Nippostrongylus brosiliensis. International Journal for Parasitology 16: 601–606. Humoral and cellular responses to homologous parasite extracts were studied in C57BL mice infected with Nematospiroides dubius or Nippostrongylus brasiliensis, to determine whether these parasites induced specific immunosuppression which might facilitate their survival. IgG and IgM titres to adult, excretory-secretory, larval and egg antigens from N. dubius and adult antigen from N. brasiliensis increased progressively for several weeks, irrespective of parasite rejection. Delayed-type hypersensitivity responses to the same antigens peaked after about 2 weeks and then remained constant in N. dubius -infected mice, but declined after the rejection of N. brasiliensis. The specific lymphoproliferative responses of spleen cells to these extracts reached peak values 1–3 weeks after infection and were anti-Thy 1.2-sensitive. They then declined sharply, but remained above the levels seen in uninfected mice. These results should be considered in the interpretation of any investigations into the stable host-parasite relationship which exists during a primary N. dubius infection.     

Regulation of cellular immune responses by selenium

Selenium (Se) is an essential nutritional factor that affects the development and expression of cell-mediated immune responses directed toward malignant cells. These studies have shown that dietary (2 ppm for 8 wk) or in in vitro (1×10⁻⁷ M) supplementation with Se (as sodium selenite) results in a significant enhancement of the proliferative responses of spleen lymphocytes from C57B1/6J mice in response to stimulation with mitogen or antigen. Se deficiency (0.02 ppm for 8 wk) had the opposite effect. The alterations in the ability of the cells to proliferate, which occurred in the absence of changes in the endogenous levels of interleukin-2 (II₂) or interleukin 1, were apparently related to the ability of Se to alter the kinetics of expression of high-affinity Il₂ receptors on the surface of activated lymphocytes. This resulted in an enhanced or delayed clonal expansion of the cells, and in an increased or decreased frequency of cytotoxic cells within a given cell population. The changes in tumor cytotoxicity were paralleled by changes in the amounts of lymphotoxin produced by the activated cells. Dietary Se modulations had a comparable effect on macrophage-mediated tumor cytodestruction. The results also suggested that Se exerts its effect 8–24 h after stimulation, and that it most likely affects processes in the cytoplasmic and/or nuclear compartments of activated lymphocytes.     

Transforming growth factor-beta and cellular immune responses in synovial fluids

Mononuclear cells in synovial fluids (SF) from patients with rheumatoid arthritis and other arthropathies are characterized by functional and phenotypic changes, including impaired mitogen responsiveness and inverted ratios of CD4+/CD8+ T lymphocytes. This is related to previously described activities in synovial fluids that inhibit proliferation of lymphocytes induced by mitogens and cytokines. The present study examines the relationship of these activities and transforming growth factor beta (TGF-beta), which is now known as the most potent endogenous inhibitor of lymphocyte function. It is shown that most of the activity in SF that inhibits IL-1-induced thymocyte or T cell proliferation is neutralized by a specific antibody to TGF-beta. Analysis of the SF in the CCL64 assay, a standard test for TGF-beta, showed a close correlation between the levels of immunosuppressive activity and TGF-beta. SF contain spontaneously active inhibitors of T cell function and this is caused by the presence of active TGF-beta. Higher titers are found after transient acidification, which is known to activate the latent form of TGF-beta. Characterization of the TGF-beta isoforms showed that most of the material in SF is TGF-beta 2. Analysis of TGF-beta effects on T cell subsets demonstrated that it completely inhibits proliferation of CD4+ cells whereas at the same concentrations of purified or rTGF-beta CD8+ cells are only inhibited by maximally 31.1%. SF also preferentially inhibit CD4+ Th cell proliferation and this effect is neutralized by antibody to TGF-beta. Collectively these results indicate that the presence of TGF-beta accounts for most of the immunosuppressive activities in SF and that this factor may be responsible for functional and phenotypic changes of SF lymphocytes.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

Intraspleen DNA inoculation elicits protective cellular immune responses

DNA immunization or inoculation is a recent vaccination method that induces both humoral and cellular immune responses in a range of hosts. Independent of the route or site of vaccination, the transfer of antigen-presenting cells (APC) or antigens into lymphoid organs is necessary. The aim of this investigation was to test whether intraspleen (i.s.) DNA inoculation is capable of inducing a protective immune response. We immunized mice by a single i.s. injection of a DNA construct expressing the immunoglobulin (Ig) heavy-chain variable domain (VH) in which the complementarity-determining regions (CDR) had been replaced by a Taenia crassiceps T-cell epitope. In these mice, immune responses and protective effects elicited by the vaccine were measured. We have shown here for the first time that i.s. DNA inoculation can induce protective cellular immune responses and activate CD8(+) T cells. Also, Ig V(H) appeared to be the minimal delivery unit of "antigenized" Ig capable of inducing T-cell activation in a lymphoid organ. The strategy of introducing T-cell epitopes into the molecular context of the V(H) domain in combination with i.s. DNA immunization could have important implications and applications for human immunotherapy.