PBMC Responses Research Articles

Peripheral blood mononuclear cell responses to heat shock proteins in patients undergoing stem cell transplantation

GVHD is a major complication after allogeneic SCT. Etiology of GVHD is multifactorial. Known role of hSPS in antigen presentation could suggest their potential role in the alloreactive process that leads to aGVHD. HSPS represent major immunodominant antigens in a wide spectrum of microbial pathogens. Bacterial and fungal colonization, infection and sepsis are frequent in immunocompromised patients with various malignant and non-malignant diseases. We studied PBMC responses to recombinant human hsp60 (rh-hsp60), rh-hsp70 and Mycobacterium bovis hsp65 (M. bovis hsp65) in relation to aGVHD and infection in 34 pediatric patients with various lympho-hemopoietic malignancies as well as non-malignant disorders subjected to SCT. PBMC of patients before initiation of preparative regimen as well as after engraftment were stimulated with hSPS (1 microg/mL/well, 7-day cultivation). PHA was used as a control of the stimulation ability. Cell responses were measured after the incorporation of 3H-thymidin (pulsing with 1 microCi/well) and were expressed as stimulation indexes (SI). We demonstrated significantly high proliferative response to rh-hsp60 as well as M. bovis hsp65 in a cohort of pretransplant patients with anamnestic and/or actual infection when compared with a cohort of patients without infection and healthy individuals. Strong PBMC cell responses to hSPS were found in patients who were at present colonized with Escherichia coli and Klebsiella pneumoniae or had previously K. pneumoniae infection with subsequent sepsis. Our findings support various studies dealing with immunodominant hSPS in connection with several pathogens and infectious diseases. Although no statistical difference for proliferative response to PHA was observed, PBMC responses against all tested hSPS comparing a cohort of patients with aGVHD and that with no sign of GVHD resulted in significantly lower SI for all tested hSPS in patients with aGVHD. Lower stimulation with hSPS during aGVHD might be explained by the stress-induced upregulation of self-hSPS synthesis that might lead to the inhibition of self-hSPS reactive T-cell response. Vice versa, we hypothesize that increased hsp-specific stimulation may reflect the presence of protecting regulatory T cells preventing the development of Th1-mediated diseases involving aGVHD.

IL-2 absorption affects IFN-γ and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays

Cytokine assays are gaining increasing importance for human immune monitoring because they reliably detect antigen-specific T cells in primary PBMC, even at low clonal sizes. Double color ELISPOT assays permit the simultaneous visualization of cells producing two different cytokines. Permitting the simultaneous assessment of type 1 and 2 immunity and due to the limited numbers of PBMC available from human study subjects, double color assays should be particularly attractive for clinical trials. Since the performance of double color assays has not yet been validated, we set out to compare them to single color measurements. Testing the recall antigen-induced cytokine response of PBMC, we found that double color assays regularly provided lower numbers of IFN-γ and IL-5 spots than single color measurements when IL-2 detection was part of the double color assay. We showed that the inhibitory effect resulted from IL-2 absorption and could be overcome by either antibody free preactivation cultures or by inclusion of anti-CD28 antibody. In contrast, the simultaneous detection of IL-2 did not affect the numbers of IL-4 spots. Therefore, unlike IL-2/IL-4 and IFN-γ/IL-5 assays, IL-2/IFN-γ, and IL-2/IL-5 assays require compensation for the IL-2 capture to provide accurate numbers for the frequencies of cytokine producing memory T cells.

Enhanced T‐cell response to mosquito extracts by NK cells in hypersensitivity to mosquito bites associated with EBV infection and NK cell lymphocytosis

Hypersensitivity to mosquito bites is characterized by severe systemic as well as local symptoms, and associated with chronic active EBV infection and NK cell lymphocytosis. In this HEN disease, we investigated the response of PBMC to MSG extracts. PBMC were taken from three defined cases of HEN disease, three borderline cases, five individuals with simple exaggerated reactions to mosquito bites without systemic symptoms (simple responders), and eight healthy donors. PBMC, or purified CD4+, CD8+ or CD56+ cells, were cultured with MSG extracts prepared from each of five mosquito species to examine their proliferation and cytokine secretion. The patients with HEN disease had high stimulation indices with variations in responses to the extracts from Aedes albopictus, Aedes aegypti, Anopheles sinensis and Culex pipiens pallens. However, a non-Japan-habitant species Anopheles stephensi did not stimulate the patients' PBMC. Some borderline or simple responders showed moderate proliferation, and healthy donors had no reactive PBMC. In HEN disease, both CD56+ NK cells (producing IFN-gamma) and CD4+ Th0 cells (producing IL-4 and IFN-gamma) were increased in the blood. CD4+ cells, but not CD56+ NK cells or CD8+ cells, propagated in response to MSG extracts. However, this response of CD4+ cells and their IL-4 production were strongly enhanced by coexisting CD56+ cells. We suggest that the CD4+ T cell serving as the primary responder to MSG antigen and the NK cell functioning as the enhancer are both pathogenic in the development of HMB.

Immunological alterations in pregnant women with acute hepatitis E

Infection with hepatitis E virus (HEV) is a major cause of acute viral hepatitis in several developing countries. Although usually self-limiting and benign, the disease is particularly severe among pregnant women, with mortality rates reaching 15-20%. Immune parameters among pregnant women with acute hepatitis E (P-HEV) were investigated and compared with those in non-pregnant patients with hepatitis E (N-HEV), and healthy pregnant (PC) and non-pregnant (NPC) women. Peripheral blood mononuclear cells (PBMC) from P-HEV patients had lower lymphocyte proliferation response to phytohemagglutinin (PHA) than those in the PC and NPC groups. A positive lymphocyte proliferation response to HEV antigen (HEVAg), a mixture of eight peptides derived from HEV proteins, was observed in 7/19 (37%) P-HEV patients, 3/9 (33%) N-HEV patients and only 2/21 (10%) PC and 2/14 (14%) NPC subjects; the stimulation indices in the P-HEV group were similar to the N-HEV group and higher than the PC group. Measurement of cytokine production by PBMC in response to PHA and HEVAg showed a reduction in production of T-helper 1 (Th1) cytokines and an increase in that of Th2 cytokines in the P-HEV group. Cytokine mRNA levels showed similar changes. These results show the existence of a Th2 bias in pregnant women with acute hepatitis E. The role of this Th2 bias in the greater severity of hepatitis E among pregnant women needs further investigation.

Mechanisms of Dimethyl Sulfoxide Augmentation of IL-1β Production

Expression of the inflammatory cytokine IL-1beta occurs in various inflammatory diseases, and IL-1beta production is regulated at multiple levels. There are conflicting reports about the effects of antioxidants on IL-1beta production. In this study, we investigated the regulatory role of the antioxidant DMSO on LPS-stimulated IL-1beta gene expression in human PBMC and in vivo. This study demonstrated that 1% DMSO increased LPS-stimulated (50 ng/ml) IL-1beta secretion in a dose- and time-dependent manner without altering TNF or IL-6. DMSO also elevated IL-1beta secretion by PBMC in response to exogenous superoxide anions. Despite the increase in IL-1beta, there was no augmentation of NF-kappaB with the addition of DMSO. The steady state mRNA coding for IL-1beta following LPS stimulation was also increased. Cycloheximide studies demonstrated that the DMSO augmentation of IL-1beta mRNA did not require de novo protein synthesis, and studies with actinomycin D showed that DMSO did not alter the half-life of IL-1beta mRNA, suggesting that DMSO did not change the stability of IL-1beta mRNA. Experiments using a reporter vector containing the 5'-flanking region of the human IL-1beta gene revealed that DMSO augmented LPS-induced IL-1beta reporter activity. In vivo, treatment of mice with DMSO significantly increased plasma levels of IL-1beta after endotoxin challenge. These data indicate that DMSO directly increases LPS-stimulated IL-1beta protein production through the mechanisms of augmenting promoter activity and increasing mRNA levels.

SIV‐specific T lymphocyte responses in PBMC and lymphoid tissues of SIV‐infected pigtailed macaques during suppressive combination antiretroviral therapy

There is currently no SIV macaque model in which the effects of combination antiretroviral therapy on tissue immune responses and latent reservoirs have been measured. This study was performed to define the impact of combination therapy on the specificity and distribution of the T lymphocyte response in multiple tissue compartments. Pigtailed macaques (Macaca nemestrina) were infected with SIV/17E-Fr and treated with combination antiretroviral therapy consisting of 9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and beta-2',3'-dideoxy-3'-thia-5-fluorocytidine (FTC). The SIV-specific T lymphocyte response was measured in peripheral blood, spleen and several lymph nodes at necropsy by IFN-gamma Elispot analysis. Two animals (one treated, one untreated) had high acute peak viremia, which was associated with lower SIV-specific T lymphocyte responses in the peripheral blood and lymphoid tissues. In the treated animal, viremia was controlled to low or undetectable for the study duration, and virus-specific responses remained low. The untreated animal remained viremic throughout the study and developed clinical symptoms of AIDS. In contrast, the two animals that had lower acute peak viremia (one treated, one untreated) had more robust T lymphocyte responses, and controlled viral replication. Virus-specific responses were detected in the treated animal despite 6 months of suppressive therapy. These data suggest that in this model, in the context of acute peak viremia and weak T cell responses, combination therapy may be essential to control virus replication and disease progression. Conversely, in the setting of low initial viremia and robust T lymphocyte responses, treatment does not have a detrimental effect on the immune response.

Discovery and characterization of highly immunogenic and broadly recognized mimics of the HIV‐1 CTL epitope Gag77–85

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play an important role in HIV infection. Given the viral genetic diversity, the selection of suitable antigens and epitope variants will be important in the design of an effective vaccine. We have previously shown that combinatorial libraries are useful tools to identify epitope mimics as well as potentially cross-reactive natural sequences in protein databases. We have applied this approach to the HIV Gag p17-derived epitope SL9 (SLYNTVATL) to identify broadly recognized SL9 mimics and to assess the cross-recognition of naturally occurring SL9 variants. Nine nonapeptides were identified that were up to one order of magnitude more effective than SL9 in stimulating CTL responses in PBMC from HIV-infected subjects. Using transgenic mice, we demonstrate that a number of these epitope mimics were able to generate de novo T cell responses that cross-reacted with the original SL9 sequence. Particularly, mimics with changes at the relatively conserved F-pocket anchor residue were frequently cross-recognized. This approach may lead to vaccine candidates with higher in vivo immunogenicity and increased potential for cross-recognition of naturally occurring SL9 variants.

A comparison of lymphocyte responses in PBMC, by human and chimpanzees in response to PMA/I stimulation, or anti-CD3/anti-CD28 costimulation, using microarray analysis

RATIONALE: Although the AIDS epidemic continues to claim millions of lives annually, there is a paucity of novel therapeutic approaches being explored. The completion of the genomic sequence of the chimpanzee has provided a unique opportunity to explore the genetic basis for the resistance of our closest evolutionary relative to develop AIDS.

Protective immunity against Mycobacterium bovis induced by vaccination with Rv3109c—a member of the esat-6 gene family

In a number of clinical studies the current TB vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has provided little or no protection against pulmonary tuberculosis in cattle and man. A new generation of vaccines is therefore required to replace or supplement BCG. Safety concerns surrounding a number of strategies make protein subunits an attractive approach. Moreover, novel prime-boost strategies based on primary immunisations with BCG are not only showing promise but also present a clear strategy for testing new TB vaccines in clinical studies. We report the evaluation of six protein vaccine candidates for their ability to induce protective immunity in a murine virulent M. bovis challenge model. One protein (Rv3019c) induced reproducibly significant protection in the spleen and lungs approaching that induced by BCG. Detailed analysis of antigen-specific T cell responses revealed that despite robust responses in the spleen and lungs of vaccinated mice, there was no correlation between these responses and the protective efficacy of the vaccine. Significantly, Rv3019c also stimulated IFN-γ responses in PBMC from BCG vaccinated cattle, indicating its potential for use in a heterologous prime-boost strategy in conjunction with BCG in the target species.

Effects of ribavirin on cytokine production of recall antigens and phytohemaglutinin-stimulated peripheral blood mononuclear cells. (Inhibitory effects of ribavirin on cytokine production)

Aim: To evaluate the effects of ribavirin on cytokine production of recall antigen and PHA-stimulated PBMC obtained from healthy individuals.Materials and methods: PBMC were challenged with tetanus toxoid (5μg/mL) and PHA (10 μmL) in absence or presence of ribavirin at different concentrations (1, 10 and 100 (μM). Parallel sets of wells containing PBMC exposed to medium alone were used as negative controls. On day 3 after initiation of the cultures, IL-2, IFN-γ, IL-4, IL-10, TNF-α content were determined in supernatants of PBMC from the different individuals.Results: The effects of ribavirin on cytokine released by human PBMC in response to PHA and TT showed a great variation among individuals. No significant changes were observed between 1-10μ M concentrations in the production of TNF-α, IFN-γ and IL-10 by both PHA and TT-stimulated PBMC. Ribavirin inhibited TNF-α, IFN-γ, and IL-10 in both PHA and TT-stimulated PBMC at 100 μM (p <0.05). At this concentration, ribavirin induced an increase of 124% in the production of IL-2 by PHA-stimulated PBMC (p <0.05).Conclusions: The present data suggest that ribavirin may cause diverse effects on immunoregulatory cytokine secretion with changes in the Th1/Th2 balance.

T Cell Epitope-Specific Defects in the Immune Response to Cat Allergen in Patients with Atopic Dermatitis

Atopic dermatitis (AD) is often associated with high titer IgE antibodies (ab) to allergens, and IL-10-mediated regulation of IFN-gamma has been proposed to contribute to this IgE ab production. However, the relevance of IL-10 and IFN-gamma to IgE associated with AD has not been examined in the context of an allergen-specific system. Analysis of PBMC responses in vitro showed deficient T cell proliferation to overlapping IL-10- (peptide (P) 2:1) and IFN-gamma- (P2:2) inducing chain 2 major epitopes of cat allergen (Fel d 1) in cultures from sensitized AD patients (mean IgE to cat=20.9 IU/ml). Diminished IFN-gamma induction by Fel d 1 and P2:2, along with elevated peptide-induced IL-10 (except for P2:1) was observed in PBMC cultures from AD subjects compared with non-AD (sensitized and non-sensitized) subjects. Neither T cell proliferation nor IFN-gamma production to chain 2 epitopes could be restored by anti-IL-10 mAb in cultures from sensitized AD subjects. Moreover, allergen avoidance was associated with a paradoxical decrease in both IL-10 and IFN-gamma in peptide-stimulated PBMC from these subjects. Control of IFN-gamma production to chain 2 epitopes by IL-10 may be relevant to sensitization status. Development of high titer IgE ab in AD could reflect a failure of this mechanism.

Evidence of immune dysregulation to cat allergen in sensitized patients with atopic dermatitis

Rationale Titers of cat-specific IgE ab in sensitized patients with atopic dermatitis (AD) are markedly elevated compared to sensitized subjects without AD. In addition, T cells from patients with AD show weak proliferation to IL-10- and IFN-γ-inducing chain 2 epitopes of the cat allergen, Fel d 1. The implication is that immune control mechanisms are deficient in AD patients. We examined the relationship between IgE ab to cat and the role of the anti-inflammatory cytokine, IL-10, in AD. Methods IgE ab to cat were assayed (Pharmacia CAP) in sensitized patients with and without AD. PBMC responses (day 7) to Fel d 1 and chain 2 epitopes were measured in the presence or absence of anti-IL-10 mAb in patients expressing HLA-DR alleles predicted to bind to chain 2 epitopes. Results The mean IgE ab titer to cat was increased in sensitized patients with AD (20.9IU/ml, n=9) compared to sensitized patients without AD (4.4IU/ml, n=14)(p<0.01). In contrast to sensitized patients without AD (IgE ab <17IU/ml), blocking IL-10 failed to enhance T-cell proliferation or IFN-γ responses to chain 2 epitopes in sensitized patients with AD (IgE ab >28.5IU/ml). Furthermore, allergen avoidance was associated with diminished production of IL-10 to Fel d 1 peptides (p<0.04) in conjunction with decreased IFN-γ in AD patients (IgE ab >61.7IU/ml). Conclusion High titer IgE ab to cat is associated with a diminished capacity for IL-10 to influence T cell responses to Fel d 1, supporting a role for T-cell immune dysregulation in AD.

Expression of IL-18 in patients with head and neck squamous cell carcinoma

Interleukin-18 (IL-18), a recently described cytokine secreted mainly by macrophages, stimulates interferon-gamma (IFN-gamma) production by natural killer cells and T cells. The purpose of this study was to determine tissue expression and serum levels of IL-18 in head and neck squamous cell carcinoma (HNSCC) and to evaluate ethanol and endotoxin-driven cytokine secretion. In 24 patients with primary HNSCC and 28 healthy controls, PBMC were isolated and incubated with 50 mM ethanol, LPS (doses 25 ng/ml, 250 ng/ml, 2500 ng/ml) and both agents for 24 h. Levels of IL-18 in serum, and cell supernatants were analysed by capture ELISA, IL-18 tissue level by immunoblotting. Serum levels of IL-8, IL-10 and IL-12, IFN-gamma, and endotoxin plasma levels were also determined. Statistical analysis involved Welch t-test and Page's test for trend. The majority of patients with HNSCC had high concentrations of serum IL-18. The level of IL-18 in the sera of these patients had a mean level of 271.7 pg/ml, while the mean IL-18 serum level in healthy controls was 174,0 pg/ml (p<0.001). Levels of IL-10 and IL-12, IFN-gamma were not increased in patients. Endotoxin was not detectable in either group. LPS stimulated dose-dependently IL-18 secretion from PBMC of patients and controls in vitro (p<0.05). Incubation with ethanol alone did not affect basal IL-18 secretion, but ethanol reduced LPS-stimulated IL-18 secretion compared to LPS stimulation alone. The mRNA expression of IL-18 in unstimulated PBMC and the response of PBMC to ethanol and LPS was similar in patients and controls. Our data on elevated serum levels of IL-18 in the majority of HNSCC cancer patients, irrespective of its biological activity, suggest that serum IL-18 might be a candidate for a new marker for HNSCC. The pathways for IL-18 production and its mechanisms of action in patients with HNSCC remain to be determined. Understanding of the immunological pathways might offer new therapeutic options in head and neck cancer in the future.

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring.

Increased stimulation of Th2 cytokines may contribute to the development of persistent ocular chlamydial infection, resulting in the blinding pathological changes of trachoma. Proliferation and cytokine production profiles of PBMC in response to stimulation with antigens of Chlamydia trachomatis were compared in 30 patients with severe conjunctival scarring due to trachoma and in 30 age-, sex- and location-matched controls. Interferon-gamma (IFN-gamma) and IL-4 were detected at the single-cell level by ELISPOT assay. Transcription of the genes encoding IFN-gamma, IL-4 and IL-10 was detected in mRNA isolated from parallel cultures of PBMC using reverse transcriptase-polymerase chain reaction (RT-PCR). Incubation with the chlamydial heat shock protein (hsp)60 resulted in increased numbers of IL-4-producing cells in PBMC isolated from patients with scarring disease and increased secretion of IFN-gamma from PBMC of control subjects. Incubation with the chlamydial major outer membrane protein (MOMP) increased the number of IFN-gamma-producing cells in the control group only. Messenger RNA encoding IL-4 was only detected in PBMC of patients with scarring disease after in vitro stimulation with chlamydial antigens, but IFN-gamma mRNA and IL-10 mRNA were also more frequently detected in this group. Thirty-eight subjects were HLA-DRB1 and -DQB1 typed. Associations were observed between certain HLA class II alleles and cellular immune responses to chlamydial antigens. No HLA associations were found with clinical status, and overall we found no evidence of strong associations and the type of immune response. These data are consistent with a role for Th2 cells and cytokines in the pathogenesis of trachomatous scarring.

Hierarchical recognition of CpG motifs expressed by immunostimulatory oligodeoxynucleotides.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs trigger human PBMC to proliferate and secrete Ig, cytokines and chemokines. CpG ODN have entered clinical trials, and show promise as vaccine adjuvants, antiallergens, and for the treatment of infectious diseases and cancer. ODNs under consideration for human use vary in the sequence, number and location of the CpG motifs they contain. Yet little is known of the magnitude of the immune response elicited by these diverse ODNs, or the rules governing their interaction with immune cells. This work compares the proliferative, IgM, IL-6 and IP-10 response of PBMC from normal donors to a diverse panel of CpG ODNs. Results indicate that ODNs expressing 3-4 different CpG motifs are strongly stimulatory. The location of these motifs is important, with those at the 5' end exerting the greatest influence on ODN activity. These findings provide a basis for the rational design of ODNs optimized for clinical use.

Immune responses and their regulation by donor bone marrow cells in clinical organ transplantation

Infusions of donor bone marrow derived cells (DBMC) continue to be tested in clinical protocols intended to induce specific immunologic tolerance of solid organ transplants based on the observations that donor-specific tolerance is induced this way in animal models. We studied the immunological effects of human DBMC infusions in renal transplantation using modifications in lymphoproliferation (MLR) and cytotoxicity (CML) assays. The salient observations and tentative conclusions are summarized in this review. Among many types of organs transplanted using DBMC at this center, it was found that the cadaver renal recipients (CAD) had significantly decreased chronic rejection and higher graft survival when compared to equivalent non-infused controls. DBMC infusion was also associated with a marginal and non-specific immune depression. It was also observed that the number of chimeric donor cells gradually increased in the iliac crest bone marrow compartment with a concomitant decrease in the peripheral blood and that the increase was more rapid in living-related donor (LRD)-kidney/DBMC recipients in spite of a lower number of DBMC infused (<25%) than in the CAD-kidney/DBMC group. In the LRD recipients with residual anti-donor responses, purified chimeric cells of either donor or recipient inhibited recipient immune responses to the donor significantly more strongly than the freshly obtained bone marrow from the specific donor or volunteer suggesting an active regulatory role for chimeric cells. A number of (non-chimeric) subpopulations of bone marrow cells including CD34 + stem cells and the CD34 − early progeny like CD38 +, CD2 +, CD5 + and CD1 + lymphoid cells as well as CD33 + (but CD15 −) myeloid cells down-regulated the MLR and CML responses of allogeneic PBMC stimulated with (autologous) donor spleen cells. These regulatory effects appeared to be refractory to the action of commonly used immunosuppressive drugs and occurred during the early phase of the immune response through cell–cell interactions. Most of these DBMC sub-populations had stimulatory capabilities, albeit markedly lower than donor spleen cells, but only through the indirect antigen presentation pathway. When co-cultured with allogeneic stimulators, purified CD34 + cells were found to give rise both to CD3 − TCR αβ +, as well as CD3 + TCR αβ + cells and, thereby, responded in MLR to allogeneic stimulation (but did not generate cytotoxic effector cells). Also, a number of DBMC subpopulations inhibited the CML and to a lesser extent the MLR, of autologous post-thymic responding T cells stimulated with allogeneic irradiated cells, mediated through soluble factors. Finally, non-chimeric DBMC also inhibited the proliferative and cytotoxic responses of autologous T cells to EBV antigens, inducing T suppressor cells, which in turn could inhibit autologous anti-EBV CTL generation and B cell anti-CMV antibody production. These studies all suggested a strong inhibitory property of a number of DBMC sub-populations in vitro and in vivo with the notion that they promote unresponsiveness.

CD4 T cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25+ T cells.

Regulatory T cells expressing CD25 have been shown to protect rodents from organ-specific autoimmune diseases. Similar CD25+ cells with a memory phenotype exerting suppressive function after polyclonal or allogeneic stimulation are also present in adult human blood. We demonstrate that adult human CD25+ cells regulate the response to myelin oligodendrocyte glycoprotein (MOG), as depletion of CD25(+) cells increases responses of PBMC and the addition of purified CD25+ cells suppresses MOG-specific proliferation and IFN-gamma production of CD4(+)CD25(-) T cells. In contrast, cord blood CD25+ cells do not inhibit responses to self antigens, and only a small subpopulation of cord CD25+ cells expresses the typical phenotype of adult regulatory T cells (CD45RA(-) and GITR(+)) enabling suppression of polyclonal responses. We conclude that activation of self-reactive T cells in normal healthy individuals is prevented by the presence of self-antigen-specific CD25+ regulatory T cells and that the majority of these cells mature after birth.

Pro-inflammatory (IL-1beta, IL-18) cytokines and IL-8 chemokine release by PBMC in response to Echinococcus multilocularis metacestode vesicles.

In humans infected with Echinococcus multilocularis, larval metacestodes will develop, proliferate and progressively infiltrate the surrounding host tissues by exogenous budding of parasitic microvesicles or cell lines which detach from the original tumour and thus become transported through blood or lymph vessels into other organs. Cellular effector mechanisms constitute the most effective means to restrict parasite persistence and proliferation, and here we demonstrate that E. multilocularis vesicle antigens will induce pro-inflammatory, regulatory and chemokine release by PBMC from patients. The pro-inflammatory cytokines IL-1beta and IL-18 were reduced in echinococcosis patients, regulatory IL-10 was similar, but parasite vesicle-induced IL-8 was dominant and clearly elevated in patients. Such selective and opposite dynamics of inflammatory cytokines and chemokine release may prevent overwhelming and pathogenic inflammation, and constitute an appropriate response for attraction of effector cells into the periparasitic tissues with the capacity to limit E. multilocularis metacestode proliferation and dissemination.

Chlamydia trachomatis heat shock protein-60 induced interferon-gamma and interleukin-10 production in infertile women.

Chlamydia trachomatis-associated tubal factor infertility (TFI) involves enhanced humoral and cell-mediated immune response to the chlamydial 60 kDa heat shock protein (CHSP60). We evaluated the role of CHSP60-induced immune response in TFI by studying lymphocyte proliferation and cytokine (interferon (IFN)-gamma, interleukin (IL)-12 and IL-10) secretion in response to C. trachomatis elementary body (EB) and CHSP60 antigens in 57 women with TFI and in 76 women with other causes of infertility. Positive proliferative response of PBMC to CHSP60 was more common in the TFI group (20/57; 36%) than in the other groups (17/76; 22%) although the frequency or the median responses did not differ significantly (1.6, range 0.2-22.1 versus 1.4; 0.2-24.4). C. trachomatis EB induced significantly higher IFN-gamma and lower IL-10 secretion in the TFI group compared to the other groups. The EB and CHSP60 induced IL-12 secretion was similar in all study groups and correlated with IFN-gamma secretion in the other but not in the TFI group. The lack of correlation between EB-induced IL-12 and IFN-gamma production and simultaneously found prominent IL-10 secretion in response to CHSP60 in the TFI group suggests that the CHSP60 may have a specific role in regulating the immune reactions during chlamydial infection and may consequently contribute to the immunopathogenesis of TFI.

Characterization of autoreactive T cells to the autoantigens heterogeneous nuclear ribonucleoprotein A2 (RA33) and filaggrin in patients with rheumatoid arthritis.

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-gamma and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-gamma than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.