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The article presents a notice of redundant publication on topics related to clinical and experimental immunology. The text and data of the paper "Peptide immunization in humans: a combined CD8+ T cell-targeted vaccine restimulates the memory CD4 T cell response but fails to induce cytoxic T lymphocytes (CTL)" is replicated without acknowledgement in the subsequent paper. The data indicate that the CTL peptide failed to stimulate a cytotoxic immune response.

Chronic obstructive pulmonary disease (COPD) is characterized by an excessive inflammatory response to inhaled particles, mainly tobacco smoking. T lymphocytes are important regulatory cells that secrete several cytokines and participate actively in this inflammatory response. According to the pattern of cytokines secreted, the immune response is classified as cytotoxic or type 1 [interferon (IFN)-γ-, interleukin (IL)-2-dependent] and humoral or type 2 (IL-4-, IL-5-, IL-10- and IL-13-dependent). This paper sought to compare the intracellular profile of cytokine expression determined by flow cytometry in T lymphocytes harvested from bronchoalveolar lavage (BAL) and peripheral blood in patients with COPD, smokers with normal lung function and never smokers. We found that BAL T lymphocytes from COPD patients had a higher percentage of positive stained cells for most of the cytokines analysed when compared to never smokers or smokers with normal lung function. Differences reached statistical significance for IL-4, IL-10 and IL-13, particularly in CD8+ T cells. Furthermore, the expression of most of these cytokines was related inversely to the degree of airflow obstruction present suggesting local activation and/or selective homing of T lymphocytes to the lungs in COPD patients. These observations were not reproduced in circulating T lymphocytes. These results suggest that BAL T lymphocytes in patients with COPD produce more cytokines than in controls and tend to show a type 2 pattern of intracellular cytokine expression, particularly a Tc-2 profile. This is related inversely to the degree of airflow obstruction present. [ABSTRACT FROM AUTHOR]

An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release heat shock proteins (Hsps). In this paper we investigate further the hypothesis that Hsps are danger signals. PBMCs and Jurkat cells released Hsp70 (0·22 and 0·7 ng/106 cells, respectively) into medium over 24 h at 37°C. Release of Hsp70 was stimulated 10-fold by GroEL ( P < 0·001) and more than threefold by lipopolysaccharide (LPS) ( P < 0·001). Although Hsp60 could be detected in the medium of cells cultured at 37°C for 24 h, the low rates of release were due probably to cell damage. Significant release of Hsp60 was observed when Jurkat cells were exposed to GroEL (2·88 ng/106 cells) or LPS (1·40 ng/106 cells). The data are consistent with the hypothesis that Hsp70 and Hsp60 are part of a danger signalling cascade in response to bacterial infection. [ABSTRACT FROM AUTHOR]

Protection against tuberculosis depends upon the generation of CD4+ T cell effectors capable of producing IFN-γ and stimulating macrophage antimycobacterial function. Effector CD4+ T cells are known to express CD44hiCD62Llo surface phenotype. In this paper we demonstrate that a population of CD44hiCD62Llo CD4+ effectors generated in response toMycobacterium bovisBCG orM. tuberculosisinfection in C57BL/6 mice is heterogeneous and consists of CD27hi and CD27lo T cell subsets. These subsets exhibit a similar degree ofin vivoproliferation, but differ by the capacity for IFN-γ production.Ex vivoisolated CD27lo T cells express higher amounts of IFN-γ RNA and contain higher frequencies of IFN-γ producers compared to CD27hi subset, as shown by real-time PCR, intracellular staining for IFN-γ and ELISPOT assays. In addition, CD27lo CD4+ T cells uniformly express CD44hiCD62Llo phenotype. We propose that CD27lo CD44hiCD62Llo CD4+ T cells represent highly differentiated effector cells with a high capacity for IFN-γ secretion and antimycobacterial protection at the site of infection. [ABSTRACT FROM AUTHOR]

Activation of T cells requires both TCR-specific ligation and costimulation through accessory molecules during T cell priming. IFNγ is a key cytokine responsible for macrophage activation duringMycobacterium tuberculosis(Mtb) infection while IL-10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNγ and IL-10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB).γ-irradiated-Mtb(i-Mtb) induced IL-10 production from CD14+ cells from TB patients. Moreover, CD3+ T cells of patients with advanced disease also produced IL-10 after i-Mtbstimulation. In healthy donors, IL-10 decreased the lytic activity of CD4+ and CD8+ T cells whereas it increasedγδ-mediated cytotoxicity. Furthermore, we found that the presence of IL-10 induced a loss of the alternative processing pathways of antigen presentation along with a down-regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL-10 or addition of IFNγ to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8+ CTL together with an up-regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i-Mtbpresentation. Taken together, our results demonstrate that the presence of IL-10 duringMtbinfection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC-restricted cytotoxicity against infected macrophages. [ABSTRACT FROM AUTHOR]

Summary: The immune system is composed of a variety of different T-cell lineages distributed through both secondary lymphoid tissue and non-lymphoid tissue. The intestinal epithelium is a critical barrier surface that contains numerous intraepithelial lymphocytes that aid in maintaining homeostasis at that barrier. This review focuses on T-cell receptor αβ (TCRαβ) CD8αα intraepithelial lymphocytes, and how recent advances in the field clarify how this unique T-cell subset is selected, matures, and functions in the intestines. We consider how the available evidence reveals a story of ontogeny starting from agonist selection of T cells in the thymus and finishing through the specific signaling environment of the intestinal epithelium. We conclude with how this story raises further key questions about the development of different ontogenic waves of TCRαβ CD8αα IEL and their importance for intestinal epithelial homeostasis. The intestine is home to a variety of T-cell subsets. One particular T-cell subset, TCRαβ CD8αα intraepithelial lymphocytes (IELs), is selected in the thymus. This review discusses the factors and events that regulate the selection of TCRαβ CD8αα IELs. [ABSTRACT FROM AUTHOR]

IL-6, an important mediator of the acute phase response, has been implicated in the pathogenesis of rheumatoid arthritis (RA). Many cell types including macrophages, T cells. H cells, endothelial cells and fibroblasts can produce this cytokine and production is largely regulated at the level of gene transcription or mRNA stabilization. In this paper we have first measured the levels of IL-6 activity in synovial fluid (SF) and serum from patients with RA and then localized IL-6-producing cells in the synovium by in situ hybridization combined with immunophenotyping. Patients with RA had raised levels of IL-6 in both SF and serum compared with patients with osteoarthritis and age-matched healthy controls. in individual RA patients tested serially after admission to hospital, serum IL6 was initially raised and, unexpectedly. increased with clinical improvement, in situ hybridization of IL-6 mRNA showed positive cells both in the lymphocyte-rich aggregates and adjacent to small blood vessels. With immunophenotyping it was found that cells containing IL-6 mRNA were often in contact with CD 14+ tissue macrophages and double immunophenotyping revealed that immuno- reactive IL-6 was often associated with synovial T cells. [ABSTRACT FROM AUTHOR]

This paper describes the spontaneous occurrence of an autoimmune uveoretinitis in nude (nu/nu) mice reconstituted when 4 weeks old by the grafting of rat embryonic thymus. The uveoretinitis was characterized histologically by progressive loss of the photoreceptor layer, observed in 4.0, 17.6, 42.9% and 71.4% of such mice at 3, 5, 7 and 12 months of age, respectively. Mice with uveoretinitis were shown to have serum IgG antibody reactive by indirect immunofluorescence with retinal photoreceptors, and with interphotoreceptor retinoid-binding protein (IRBP), but not retinal S-antigen, by immunoblotting and ELISA. A uveoretinitis could be adoptively transferred to syngeneic ungrafted nude mice by splenic CD4+ T cells from diseased animals. This is the first experimental model of a (T cell mediated) autoimmune uveoretinitis which develops spontaneously and which is not dependent upon deliberate sensitization with retinal antigens and adjuvants. [ABSTRACT FROM AUTHOR]

The binding of EPF to lymphocytes stimulates the release of soluble mediators, active in T cell dependent reactions, namely the rosette inhibition test and the adoptive transfer of contact sensitivity. On the basis of their ability to inhibit the delayed type hypersensitivity reaction in this latter assay, they have been classified as suppressor factors. This paper describes the identification of two EPF-induced suppressor factors. Unlike EPF which is neither species-restricted nor strain-restricted, these factors are genetically restricted in their action in the rosette inhibition test EPF-S1 (estimated Mr 14,000) is restricted to the I region of the mouse MHC, while EPF-S2 (estimated Mr 55,000) is restricted to a locus (or loci) outside the MHC. Like other antigen non-specific factors, release of these suppressor factors can be stimulated also by Con A. [ABSTRACT FROM AUTHOR]

Freshly isolated B cells from chronic lymphocytic leukaemia patients (B-CLL) have been previously shown to induce a strong proliferative response and high levels of NK-like activity in lymphocytes from healthy donors. The present paper deals with the origin, mitotic state, target spectrum and cell surface phenotype of the NK-like effectors generated after stimulation with B-CLL. Experiments using large granular lymphocytes (LGL) and T cells as responders demonstrated that most of the precursors of the newly generated NK-like effectors express the CD3 antigen. The induction of NK-like activity paralleled cell activation, as judged by blast transformation, thymidine uptake and appearance of cell surface activation markers. The newly generated NK-like effectors displayed a T cell phenotype and a broader target repertoire than native NK cells. [ABSTRACT FROM AUTHOR]

The antigen-induced DNA synthesis in vitro in lymphocytes from patients with acute Plasmodium falciparum malaria was investigated. The patients and healthy controls from Sweden or Colombia were the same as those studied in the accompanying paper (Troye-Blomberg el al., 1983). The malarial antigens used were sonicated membrane preparations or purified and concentrated supernatants from in vitro cultures of P. facliparum, similar preparations derived from normal human erythrocytes served as control antigen. In the patients' lymphocytes P. falciparum antigens induced a weak or moderate but significant stimulation of DNA synthesis, peaking after 3-4 days of incubation. This early response was specific for P. faleiparum since it was not obtained with lymphocytes from healthy donors nor with those from patients with acute P. vivax or P. ovale malaria. No antigen-induced response was seen in about half of the P.falciparum patients. However in a few negative eases, available for consecutive testing, positive reactions were seen with lymphocytes taken 2 weeks after infection when the blood of these patients was free of parasites. The early response induced in patients' lymphocytes to the P. falciparum antigens was not obtained with RBC antigen, however, these preparations frequently induced a response rising to significant levels later during incubation (day 5-6)- Similar delayed responses were obtained when either- patients' or control donors' lymphocytes were exposed to the P. falciparum antigens. This indicates that both the RBC and the parasite preparations contained mitogenic substances affecting human lymphocytes in general and easily obscuring the P, falciparum specific responses seen only in the patients. This latter response was relatively low and short lived, suggesting that it reflected a secondary in vitro stimulation of in vivo primed lymphocytes and that it was regulated by suppressor mechanisms. [ABSTRACT FROM AUTHOR]

Mitogen-transformed human peripheral blood lymphocytes and tonsil blasts were examined by rosette formation to detect the presence of membrane-bound immunoglobulin (Ig) and surface receptors for fixed IgG and fixed C3. In addition, the capacity of these cells to rosette with sheep erythrocytes was evaluated as a reaction characteristic of T lymphocytes. In order for clear morphological recognition of the rosetting transformed lymphocytes and the rosetting tonsil blasts a cytocentrifuge technique was developed and used in conjunction with autoradiography and/or with Romanowsky stains. Using these techniques and the culture methods described in this paper phytohaemagglutinin, pokeweed mitogen, streptococcal filtrates and purified protein derivative stimulated predominantly T cells in the peripheral blood of man. A minority of the transformed cells in these mitogen-stimulated cultures (<24%) did rosette with B lymphocyte markers and presumably represent a B-cell response. No significant differences were found between the T- or B-cell specificity of the mitogens investigated. Lymphoid preparations from tonsils excised from normal donors with recurrent tonsillitis were found to contain 6-15% lymphoblasts and the iarge majority of these cells formed rosettes with the B-cell markers, less than 20% of these lymphoblasts formed spontaneous sheep erythrocyte rosettes. Using a mixed rosetting technique a small proportion (<5%) of PHA-transformed cells and tonsil lymphoblasts were found to have combined sheep Fc or combined sheep C3 receptors. The investigation of B- and T-lymphocyte surface markers on mitogentransformed lymphocytes was extended to neopiastic lymphocyte populations and it was found that the majority of transformed cells (>70%) present in chronic lymphocytic leukaemia cultures stimulated with PHA after 6 days incubation were transformed T lymphocytes. [ABSTRACT FROM AUTHOR]

A patient with intestinal lymphangiectasia (IL) was studied for his immunological abnormalities. The patient had hypoproteinaemia with severe hypogammaglobulinaemia. The results of lymphocyte subpopulation studies revealed a decrease of CD4+ cells and a decrease of surface immunoglobulin (sIg)-positive B lymphocytes. T cell functions determined by the proliferative responses against Concanavalin A (Con A) or phytohaemagglutinin (PHA) and by delayed type cutaneous hypersensitivity (DTH) response to purified protein derivatives (PPD) and PHA were normal. No immunoglobulin (Ig)-secreting cells were induced when his peripheral blood lymphocytes (PBL) were cultured with pokeweed mitogen (PWM). His T cells showed suppressor function to the PWM- induced differentiation of normal B cells. This suppressor activity was sensitive to 3000 rads irradiation. His B cells also failed to differentiate into Ig-producing cells with the help of normal T cells and PWM. Thus, in this patient, the decrease of Ig-synthesis in vitro could be attributed to suppressor T cells, lack of T helper cells and an intrinsic B cell defect. Therefore, this patient appears to have immunological abnormalities which differ from previously reported IL patients. [ABSTRACT FROM AUTHOR]

Low concentrations (40 μM) of the purine nucleoside metabolite 2'deoxyguanosine (2'dGuo), were shown to enhance both phytohaemagglutinin- and concanavalin A-induced T cell transformation. Evidence is presented which indicates that this enhancement may he due to the inhibition of suppressor T cell function resulting in the promotion of T cell growth factor production. T cell responses were only inhibited at concentrations of 2'dGuo which were toxic to the lymphocytes. [ABSTRACT FROM AUTHOR]

Peripheral blood lymphocytes (PBL) from a proportion of grass-sensitive patients. studied during or immediately after the grass pollination period, showed spontaneous production in vitro of grass-specific IgE antibody, whereas PBL from atopic patients sensitive to allergens other than grass pollens or non-atopic individuals did not. Pre-incubation of IgE antibody producing PBL from grass-sensitive patients with minute amounts of a mixed grass pollen (MGP) extract or Rye grass antigen Group I (Rye I) usually resulted in a reduction of the spontaneous production in vitro of IgE protein and in a marked inhibition of the spontaneous production in vitro of grass-specific IgE antibody. This antigen-specific inhibition was not mediated by T lymphocytes, but it was apparently due to a signal directly delivered by antigen to the spontaneously IgE antibody producing cells. The results support the concept that the activity of cells responsible for the persistent IgE antibody formation in vitro in atopic patients can be modulated by antigen. [ABSTRACT FROM AUTHOR]

The development of the cell-mediated immune response (CMIR) to antigens prepared from Salmonella typhi was investigated in patients suffering from typhoid fever and in normal healthy subjects. The leucocyte migration inhibition test, blast transformation of lymphocytes and active rosette-forming cells were used for detecting CMIR. Peripheral blood lymphocytes were analysed for the numbers and proportions of B lymphocytes, T lymphocytes and their subpopulations with receptors for IgM (Tμ) or IgG (Tγ) and cells with Fc receptors for IgG. These parameters were correlated with the duration and the severity of illness. The uncomplicated cases of typhoid fever were found to have an intact CMIR as compared to the complicated cases. The ratio of T lymphocyte subpopulations was grossly imbalanced in typhoid patients, the numbers of T lymphocytes and their subpopulations were further altered in the complicated cases as compared to uncomplicated cases. The present study demonstrates a depressed state of CMIR in complicated patients with typhoid fever. CMIR may thus emerge as the cardinal point for recovery in typhoid fever rather than the specific antibodies. The study further demonstrates that imbalance within the subsets of T lymphocytes may be responsible for the depressed state of CMIR in complicated cases of typhoid fever. [ABSTRACT FROM AUTHOR]

Spontaneous IgE production in vitro was investigated in 7-day cultures of unfractionated mononuclear cells (MNC) and MNC subpopulations from atopic patients. Depletion of either phagoctytic or adherent cells decreased the amount of IgE detectable in 7-day culture supernatants, but this decrease was due, at least in part, to a loss of cytophilic IgE. Depletion of immunoglobulin-bearing cells (SIg+) reduced significantly but did not abolish the spontaneous IgE production in vitro. On the other hand, depletion of IgM- bearing lymphocytes (SIgM+) which virtually abolished the production of immunoglobulins of the IgM class, did not change significantly the spontaneous production of IgE. Similarly, no change in the spontaneous production of IgE was found when lymphocyte suspensions were depleted of complement receptor-bearing cells (CR+). In contrast. spontaneous IgE product ion was significantly increased by depict ion of T lymphocytes and this increase did not simply reflect the enrichment for IgE-producing cells caused by the fractionation procedure. No significant change in the spontaneous IgE production was found when small numbers of autologous T lymphocytes were added to B cell fractions, whereas the addition of higher concentrations of autologous T cells induced a marked inhibition of the spontaneous IgE production. On the other hand, the addition in culture of pokeweed mitogen (PWM) resulted in a marked reduction of the spontaneous IgE production by B cells, also in the presence of small concentrations autologous T lymphocytes. Normal T cells were consistently effective in inducing a partial inhibition of the spontaneous IgE production by B cells from atopic patients. whereas T cells from a noticeable proportion of atopic patients were not. These data suggest that MNC responsible for the spontaneous IgE production in atopic subjects arc SIgM- and CR-deficient well-differentiated lymphocytes which probably represent the result of an activation which has occurred in vivo. However, this spontaneous IgE production can still be influenced by in vitro manipulation. such as variations in T-B cell ratios or addition of PWM. The results here reported also indicate that normal T cells arc generally more effective than T cells from atopic patients in regulating the activity of spontaneous IgE-producing cells present in the blood of atopic subjects. [ABSTRACT FROM AUTHOR]

Summary: Tissue‐resident memory T (TRM) cells are CD8+ T lymphocytes that reside in the tissues, including tumours. This T cell subset possesses a magnitude of cytotoxicity, but its epigenetic regulation has not been studied. Here, we investigate the impact of perforin DNA methylation in TRM cells and correlate it with their functional potential. Fifty‐three urothelial urinary bladder cancer (UBC) patients were recruited prospectively. The DNA methylation status of the perforin gene (PRF1) locus in TRM cells was investigated by pyrosequencing. Flow cytometry with ViSNE analysis and in‐vitro stimulation were used to evaluate TRM cell phenotypes. We discovered that tumour TRM cells have low DNA methylation in the PRF1 locus (32·9% methylation), which corresponds to increased numbers of perforin‐expressing TRM cells. Surprisingly, programmed cell death 1 (PD‐1) expression is high in tumour TRM cells, suggesting exhaustion. Following interleukin‐15 and T cell receptor stimulation, perforin and T‐bet expressions are enhanced, indicating that TRM cells from tumours are not terminally exhausted. Moreover, a high number of TRM cells infiltrating the tumours corresponds to lower tumour stage in patients. In conclusion, TRM cells from UBC tumours are epigenetically cytotoxic with signs of exhaustion. This finding identifies TRM cells as potential new targets for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Summary: The in‐depth understanding of skin resident memory CD8+ T lymphocytes (TRM) may help to uncover strategies for their manipulation during disease. We investigated isolated TRM from healthy human skin, which expressed the residence marker CD69, and compared them to circulating CD8+ T cell populations from the same donors. There were significantly increased proportions of CD8+CD45RA–CD27– T cells in the skin that expressed low levels of killer cell lectin‐like receptor G1 (KLRG1), CD57, perforin and granzyme B. The CD8+ TRM in skin were therefore phenotypically distinct from circulating CD8+CD45RA–CD27– T cells that expressed high levels of all these molecules. Nevertheless, the activation of CD8+ TRM with T cell receptor (TCR)/CD28 or interleukin (IL)‐2 or IL‐15 in vitro induced the expression of granzyme B. Blocking signalling through the inhibitory receptor programmed cell death 1 (PD)‐1 further boosted granzyme B expression. A unique feature of some CD8+ TRM cells was their ability to secrete high levels of tumour necrosis factor (TNF)‐α and IL‐2, a cytokine combination that was not seen frequently in circulating CD8+ T cells. The cutaneous CD8+ TRM are therefore diverse, and appear to be phenotypically and functionally distinct from circulating cells. Indeed, the surface receptors used to distinguish differentiation stages of blood T cells cannot be applied to T cells in the skin. Furthermore, the function of cutaneous TRM appears to be stringently controlled by environmental signals in situ. [ABSTRACT FROM AUTHOR]

Summary: Studies of cerebrospinal fluid (CSF) γδ T cells in children are limited, due especially to the lack of control data. In adults, gamma/delta T cells (TCR‐γδ) residing in the intrathecal space are sometimes involved in neuroinflammation. To evaluate the possible role of γδ T cells in paediatric neuroinflammation, we immunophenotyped cerebrospinal fluid (CSF) and blood lymphocytes using flow cytometry in a case–control study of 100 children with non‐inflammatory neurological disorders (NIND), 312 with opsoclonus–myoclonus (OMS) and 23 with other inflammatory neurological disorders (OIND). In NIND, the negative correlation between CSF γδ T cell frequency and patient age was striking: median frequency of 27% in infants and 3·3% in teens. Interindividual variations were largest in the youngest. There was no gender effect. In all OMS, after correcting for age, only a small effect of OMS severity remained. Measurement of markers for γδ T cell activation [human leucocyte antigen D‐related (HLA‐DR)], maturation (CD45RA, CD45RO) or intracellular cytokine staining [interleukin (IL)‐4, interferon (IFN)‐γ] failed to discriminate OMS and NIND groups. Of seven OMS immunotherapies/combinations, none altered the frequency of total CSF γδ T cells or subsets significantly. In OIND, the CSF γδ T cell frequency was < 10% for single samples of other paraneoplastic disorders [anti‐neuronal nuclear antibody (ANNA)‐1, PCA‐1, teratoma‐associated syndrome], cerebellar ataxia (post‐infectious, ataxia‐telangiectasia), acute disseminated encephalomyelitis, neuroborreliosis and encephalitis. This study provides new insights into CSF γδ T cells in the paediatric population. Although their role in CSF remains elusive, the negative age correlation, resistance to immunotherapy and our age cut‐off references for NIND are important findings for the design of future paediatric studies. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immunopathogenesis. However, a pathogenic role for the T helper type 17 (Th17) axis was demonstrated by many studies, while regulatory T cells (Tregs) were shown to mediate protection. Recently, we and others characterized a novel and independent T cell population expressing both the Treg characteristic transcription factor forkhead box protein 3 (FoxP3) and the Th17-defining retinoic acid receptor-related orphan nuclear receptor γt (RORγt). Studies in a model of acute glomerulonephritis unveiled potent regulatory, but also proinflammatory, functions of RORγt+FoxP3+ Tregs. This bi-functional nature prompted us to suggest the name 'biTregs'. Importantly, the pathogenic biTreg effects were dependent upon expression of RORγt. We thus aimed to evaluate the contribution of RORγt+FoxP3+ biTregs to pristane-induced SLE and explored the therapeutic potential of interference with RORγt activation. Our analyses revealed expansion of IL-17 producing biTregs in a distinctive time-course and organ-specific pattern, coincident with the development of autoimmunity and tissue injury. Importantly, specific ablation of RORγt activation in endogenous biTregs resulted in significant amelioration of pristane-induced pulmonary vasculitis and lupus nephritis. As potential mechanisms underlying the observed protection, we found that secretion of IL-17 by biTregs was abrogated completely in FoxP3Cre × RORCfl/fl mice. Furthermore, Tregs showed a more activated phenotype after cell-specific inactivation of RORγt signalling. Finally, and remarkably, biTregs were found to potently suppress anti-inflammatory Th2 immunity in a RORγt-dependent manner. Our study thus identifies biTregs as novel players in SLE and advocates RORγt-directed interventions as promising therapeutic strategies. [ABSTRACT FROM AUTHOR]

Type B coxsackievirus (CV-B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in-utero CV-B infection on the fetal thymus, the central site for programming immunological self-tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV-B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post-inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In-utero CV-B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV-B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV-B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases. [ABSTRACT FROM AUTHOR]

NKG2D (KLRK1) is a C-type lectin receptor present on natural killer (NK) cells, γδ, CD8+ and CD4+ T cells. Upon ligand binding, NKG2D mediates activatory and co-stimulatory signals to NK cells and activated CD4+ T cells, respectively. Polymorphisms in NKG2D predispose to infectious diseases, cancer, transplantation and autoimmune disorders. We studied the influence of this NK receptor polymorphism on predisposition to and modification of the disease phenotype in patients with rheumatoid arthritis (RA). Eight different single nucleotide polymorphisms (SNP) in the NKG2 gene were genotyped in 236 patients with RA and 187 controls using Taqman 5' nuclease assays. NKG2D genotype/allele frequency did not differ between patients and controls. Subgroup analysis showed that the frequency of A allele of NKG2D9 and T allele of NKG2D10 was significantly higher in patients with deformities (a marker of severe disease) [11 versus 5%, Pc = 0·03, odds ratio (OR) = 2·44, 95% confidence interval (CI) = 1·09-5·98 and 10 versus 4%, Pc = 0·04, OR = 2·45, 95% CI = 1·05-6·39, respectively], while the frequency of alleles G of NKG2D9 and A of NKG2D10 was greater in patients without deformities (Pc = 0·03, OR = 0·41, 95% CI = 0·17-0·91 and Pc = 0·04, OR = 0·41, 95% CI = 0·16-0·96). Similar trends of association were observed with deforming phenotype of RA in female patients and deforming young onset RA subgroups. Haplotype analysis revealed that the frequency of haplotype G-C-A-G-A-T-C-C was higher in patients than in controls (12 versus 8%, P = 0·04, OR = 1·61, 95% CI = 1·01-2·55), suggesting that it may predispose to RA. Our study suggests that the NKG2D gene polymorphisms may modify the risk of development and severity of RA. [ABSTRACT FROM AUTHOR]

The current view of type 1 diabetes (T1D) is that it is an immune-mediated disease where lymphocytes infiltrate the pancreatic islets, promote killing of beta cells and cause overt diabetes. Although tissue resident immune cells have been demonstrated in several organs, the composition of lymphocytes in human healthy pancreatic islets have been scarcely studied. Here we aimed to investigate the phenotype of immune cells associated with human islets of non-diabetic organ donors. A flow cytometry analysis of isolated islets from perfused pancreases ( n = 38) was employed to identify alpha, beta, T, natural killer (NK) and B cells. Moreover, the expression of insulin and glucagon transcripts was evaluated by RNA sequencing. Up to 80% of the lymphocytes were CD3+ T cells with a remarkable bias towards CD8+ cells. Central memory and effector memory phenotypes dominated within the CD8+ and CD4+ T cells and most CD8+ T cells were positive for CD69 and up to 50-70% for CD103, both markers of resident memory cells. The frequency of B and NK cells was low in most islet preparations (12 and 3% of CD45+ cells, respectively), and the frequency of alpha and beta cells varied between donors and correlated clearly with insulin and glucagon mRNA expression. In conclusion, we demonstrated the predominance of canonical tissue resident memory CD8+ T cells associated with human islets. We believe that these results are important to understand more clearly the immunobiology of human islets and the disease-related phenotypes observed in diabetes. [ABSTRACT FROM AUTHOR]

Coronary heart disease (CHD) is one of the most common types of organ lesions caused by atherosclerosis, in which CD4+CD25+forkhead box protein 3 (FoxP3+) regulatory T cells (Treg) play an atheroprotective role. However, Treg cell numbers are decreased and their functions are impaired in atherosclerosis; the underlying mechanisms remain unclear. CD31 plays an important part in T cell response and contributes to maintaining T cell tolerance. The immunomodulatory effects of CD31 are also implicated in atherosclerosis. In this study, we found that decreased frequencies of the CD31+ subpopulation in Treg cells (CD31+Tr cells) correlated positively with decreased FoxP3 expression in CHD patients. Cell culture in vitro demonstrated CD31+Tr cells maintaining stable FoxP3 expression after activation and exhibited enhanced proliferation and immunosuppression compared with the CD31− subpopulation in Treg cells (CD31−Tr cells). We also confirmed impaired secretion of transforming growth factor (TGF)-β1 and interleukin (IL)-10 in CD31+Tr cells of CHD patients. Further analysis revealed reduced phospho-SHP2 (associated with CD31 activation) and phospho-signal transducer and activator of transcription-5 (STAT-5) (associated with FoxP3 transcription) levels in CD31+Tr cells of CHD patients, suggesting that decreased FoxP3 expression in CD31+Tr cells might be because of attenuated SHP2 and STAT-5 activation. These data indicate that decreased frequencies and impaired functions of the CD31+Tr subpopulation associated with decreased FoxP3 expression give rise, at least in part, to Treg cell defects in CHD patients. Our findings emphasize the important role of the CD31+Tr subpopulation in maintaining Treg cell normal function and may provide a novel explanation for impaired immunoregulation of Treg cells in CHD. [ABSTRACT FROM AUTHOR]

Non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are RNA molecules that do not translate into protein. Both miRNAs and lncRNAs are known to regulate gene expression and to play an essential role in T cell differentiation and function. Both systemic lupus erythematosus (SLE), a prototypic systemic autoimmune disease, and rheumatoid arthritis (RA), a representative disease of inflammatory arthritis, are characterized by a complex dysfunction in the innate and adaptive immunity. T cells play a central role in cell-mediated immune response and multiple defects in T cells from patients with SLE and RA have been observed. Abnormality in T cell signalling, cytokine and chemokine production, T cell activation and apoptosis, T cell differentiation and DNA methylation that are associated closely with the aberrant expression of a number of miRNAs and lncRNAs have been implicated in the immunopathogenesis of SLE and RA. This review aims to provide an overview of the current state of research on the abnormal expression of miRNAs and lncRNAs in T cells and their roles in the immunopathogenesis of SLE and RA. In addition, by comparing the differences in aberrant expression of miRNAs and lncRNAs in T cells between patients with SLE and RA, controversial areas are highlighted that warrant further investigation. [ABSTRACT FROM AUTHOR]

The recruitment of immune cells to sites of tissue inflammation is orchestrated by chemokine/chemokine receptor networks. Among these, the CXCL13/CXCR5 axis is thought to be involved critically in systemic lupus erythematosus (SLE) and lupus nephritis pathogenesis. Beyond B cell abnormalities, another hallmark of SLE disease is the occurrence of aberrant T cell responses. In particular, double-negative (DN) T cells are expanded in the peripheral blood of patients with SLE and in lupus-prone mice. DN T cells induce immunoglobulin production, secrete proinflammatory cytokines and infiltrate inflamed tissue, including kidneys. We aimed to investigate how CXCR5 deficiency changes immune cell trafficking in murine lupus. We therefore crossed CXCR5-/- mice with B6/lpr mice, a well-established murine lupus model. B cell numbers and B cellular immune responses were diminished in CXCR5-deficient B6/lpr mice. In addition, we observed reduced accumulation of DN T cells in spleen and lymph nodes, paralleled by reduced splenomegaly and lymphadenopathy. In-vivo migration assays revealed reduced migration of CXCR5-deficient DN T cells into lymph nodes, and ex-vivo-activated CXCR5-deficient DN T cells failed to infiltrate kidneys of recipients. Moreover, DN T cells and B cells of CXCR5-deficient B6/lpr mice failed to migrate towards CXCL13 in vitro. We propose that CXCR5 is involved critically in B cell trafficking and germinal cell (GC) formation in murine lupus and in guiding pathogenic DN T cells into lymphoid organs and kidneys, and we therefore describe new pathomechanisms for the CXCL13/CXCR5 axis in SLE. [ABSTRACT FROM AUTHOR]

Systemic sclerosis ( SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc-mesenchymal stem cells ( MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls ( HC). Senescence was evaluated by assessing cell cycle, β-galactosidase (β- Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co-culturing MSCs with peripheral blood mononuclear cells ( PBMCs) and CD4+ cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells ( Tregs) induction. SSc- MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0- G1 phase, without significant differences between SSc and HC. SSc- MSCs showed an increased positive β- Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, β- Gal staining increased significantly in SSc- MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc- and HC- MSCs. Interleukin ( IL)-6 and transforming growth factor ( TGF)-β-related transcripts and their protein levels were significantly higher in SSc- MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL-6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis ( MDR-TB) and non-tuberculous mycobacteria ( NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB ( NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4+ CD25+- forkhead box protein 3 ( FoxP3)+ and CD4+ CD25+ CD127− regulatory T ( Treg) cells when compared to NR-TB. Increased numbers of Treg cells and interleukin ( IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor ( TGF)-β was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target ( ESAT)-6, but not purified protein derivative ( PPD), showed a lower frequency of CD4+/interferon ( IFN)-γ+ T cells and enhanced CD4+ CD25+ FoxP3+, CD4+ CD25+ CD127− and CD4+ CD25+ IL-10+ T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL- 10Rα decreased the CD4+ CD25+ FoxP3+ frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4+ CD25+ T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for Treg/ T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms. [ABSTRACT FROM AUTHOR]

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4+ populations and well-characterized bone marrow-derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)+ and CD25+ mRNA and protein expression in CD4+ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC-derived prostaglandins and transforming growth factor (TGF)-β1 were shown to have a non-redundant role in the induction of CD4+CD25+FoxP3+ T cells. Purified CD4+CD25+ T cells induced by MSC co-culture expressed TGF-β1 and were able to suppress alloantigen-driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC-mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4+ cells followed by both prostaglandin E2 and TGF-β1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC. [ABSTRACT FROM AUTHOR]

CD81 is a member of the tetraspan superfamily and plays a role in immune responses and in hepatitis C virus (HCV) pathogenesis. We analysed CD81 cell surface and mRNA expression in different lymphocytic subpopulations in human immunodeficiency virus (HIV)-1, HCV and dually infected subjects. CD81 cell surface expression was evaluated with fluorescence activated cell sorter (FACS) analysis; mRNA quantification was performed with semiquantitative polymerase chain reaction (PCR). CD81 cell surface expression on CD4+ T lymphocytes was significantly different by analysis of variance (anova) test ( P < 0·001), with reduced expression in HIV-1+ patients. In B lymphocytes, higher cell surface expression was present in HIV-1, in HCV and in dually infected subjects compared to healthy controls. CD81 expression on B lymphocytes showed a positive correlation with plasma HIV-RNA. CD81 mRNA levels in B lymphocytes were significantly higher in HIV-1+ patients compared to healthy controls. The potential consequence of the down-regulation of CD81 in CD4+ cells during HIV-1 infection in conjunction with diverted CD28, CD4 and CD3 expression is the disruption of T cell function. Increased CD81 expression on B lymphocytes might explain the higher prevalence of lymphoproliferative disorders in HIV-1 and HCV infection. Up-regulation of CD81 mRNA on CD4+ T cells indicates that down-regulation of CD81 occurs at the post-transcriptional/translational level. [ABSTRACT FROM AUTHOR]

Although a beneficial effect of hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, i.e. statins, on cell-mediated immunity has been suggested in vivo and in vitro, little is known about the molecular and biochemical events by which statins inhibit T cell proliferation. To address this question, we investigated the effects of atorvastatin (AT) on intracellular cytokine production, T cell activation markers, cell cycle progression and apoptosis in human CD4+ T cells. AT did not influence intracellular cytokine production after short-term stimulation of whole blood with phorbol myristate acetate (PMA)/ionomycin or superantigen (SEB). In contrast, AT influenced CD45RA to RO switching dose-dependently, as well as CD25 expression, and caused cell cycle arrest in the G1 phase after long-term T cell stimulation. This occurred in conjunction with a reduced expression of cyclin-dependent kinases 2 and 4 and p21wav1/cip1 and was paralleled by an increased protein expression of p27kip1. In addition to G1 arrest, increased apoptosis was observed in AT-treated cells. In line with this, the expression of Bcl-xl and pBad were decreased by AT. Apoptosis was independent of caspases 3 and 9 activation. The inhibitory effect of AT on T cell proliferation could be overcome by addition of mevalonic acid or geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate or squalen, suggesting reduced protein prenylation. Activation of Rho, Rac and Ras were strongly reduced in AT-treated T cells, suggesting that impaired geranylation of these molecules might underlie the inhibitory effect of AT on T cell proliferation. [ABSTRACT FROM AUTHOR]

Epidermolysis bullosa acquisita is an autoimmune blistering disease characterized by circulating and skin basement membrane-bound IgG autoantibodies to type VII collagen, a major structural protein of the dermal–epidermal junction. Regulatory T cells (Treg) suppress self antigen-mediated autoimmune responses. To investigate the role of Treg in the the autoimmune response to type VII collagen in a mouse model, a monoclonal antibody against mouse CD25 was used to deplete Treg. A recombinant mouse type VII collagen NC1 domain protein and mouse albumin were used as antigens. SKH1 mice were used as a testing host. Group 1 mice received NC1 immunization and were functionally depleted of Treg; group 2 mice received NC1 immunization and rat isotype control; and group 3 mice received albumin immunization and were functionally depleted of Treg. Results demonstrated that anti-NC1 IgG autoantibodies with high titres, as determined by enzyme-linked immunosorbent assay and Western blotting, developed in all mice immunized with NC1 (groups 1 and 2), but were undetected in group 3 mice. The predominant subclasses of anti-NC1 autoantibodies were IgG1, IgG2a and IgG2b; furthermore, these antibodies carried only the kappa light chain. IgG autoantibodies in the sera of NC1-immunized mice reacted with mouse skin basement membrane in vitro and deposited in skin basement membrane in vivo as detected by indirect and direct immunofluorescence microscopy, respectively. Our data suggest that the development of autoimmunity against type VII collagen in mice is independent of Treg function and the autoimmune response is mediated by both Th1 and Th2 cells. We speculate that the basement membrane deposition of IgG may eventually lead to blister development. [ABSTRACT FROM AUTHOR]

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas’ disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3– CD16+ CD56–), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas’ disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease. [ABSTRACT FROM AUTHOR]

Expression of tumour antigen-specific chimaeric receptors in T lymphocytes can redirect their effector functions towards tumour cells. Integration of the signalling domains of the co-stimulatory molecule CD28 into chRec enhances antigen-specific proliferation of polyclonal human T cell populations. While CD28 plays an essential role in the priming of naive CD4+ T cells, its contribution to effector memory T cell responses is controversial. We compared the function of the chRec with and without the CD28 co-stimulatory domain, expressing it in peripheral blood T cells or Epstein–Barr virus (EBV)-specific T cell lines. The chimaeric T cell receptors contain an extracellular single-chain antibody domain, to give specificity against the tumour ganglioside antigen GD2. The transduced cytotoxic T lymphocytes (CTL) maintained their specificity for autologous EBV targets and their capacity to proliferate after stimulation with EBV-infected B cells. Intracellular cytokine staining demonstrated efficient and comparable antigen-specific interferon (IFN)-γ secretion by CTL following engagement of both the native and the chimaeric receptor, independent of chimaeric CD28 signalling. Furthermore, tumour targets were lysed in an antigen-specific manner by both chRec. However, while antigen engagement by CD28ζ chRec efficiently induced expansion of polyclonal peripheral blood lymphocytes in an antigen-dependent manner, CD28 signalling did not induce proliferation of EBV–CTL in response to antigen-expressing tumour cells. Thus, the co-stimulatory requirement for the efficient activation response of antigen-specific memory cells cannot be mimicked simply by combining CD28 and ζ signalling. The full potential of this highly cytolytic T cell population for adoptive immunotherapy of cancer requires further exploration of their co-stimulatory requirements. [ABSTRACT FROM AUTHOR]

Coeliac disease, the most common intestinal disorder of western populations, is an autoimmune enteropathy caused by an abnormal immune response to dietary gluten peptides that occurs in genetically susceptible individuals carrying the HLA-DQ2 or -DQ8 haplotype. Despite the recent progresses in understanding the molecular mechanisms of mucosal lesions, it remains unknown how increased amounts of gluten peptides can enter the intestinal mucosa to initiate the inflammatory cascade. Current knowledge indicates that different gluten peptides are involved in the disease process in a different manner, some fragments being‘toxic’ and others‘immunogenic’. Those defined as‘toxic’ are able to induce mucosal damage either when added in culture to duodenal endoscopic biopsy or when administeredin vivo, while those defined as‘immunogenic’ are able to specifically stimulate HLA-DQ2- or DQ8-restricted T cell clones isolated from jejunal mucosa or peripheral blood of coeliac patients. These peptides are able to trigger two immunological pathways: one is thought to be a rapid effect on the epithelium that involves the innate immune response and the other represents the adaptive immune response involving CD4+ T cells in the lamina propria that recognize gluten epitopes processed and presented by antigen presenting cells. These findings are the subject of the present review. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is usually more severe in HIV-infected patients, and the immune derangement found in co-infected patients may differ from that in each isolated disease. Following mitogen stimulation of peripheral blood mononuclear cells (PBMC), interferon (IFN)-γ and tumour necrosis factor (TNF)-α production was evaluated in T cells by flow cytometry, and in culture supernatants by enzyme-linked immunosorbent assay (ELISA) in 33 individuals: 11 AIDS patients with tuberculosis, six asymptomatic HIV-1-infected patients, eight patients with tuberculosis and eight healthy controls. The proportion of CD4+ T lymphocytes expressing IFN-γ did not differ between the groups, whereas a trend towards increased proportions of TNF-α-expression in CD4+ T cells was observed in the TB compared to the HIV group, while intermediate values were observed in co-infected patients. Detection of IFN-γ and TNF-α in CD8+ T lymphocytes was higher in TB than in HIV individuals. Co-infected patients presented intermediate values for IFN-γ, while TNF-α detection was similar to that in HIV mono-infection. In conclusion, the proportion of T cells expressing IFN-γ was relatively preserved in co-infected patients compared to TB patients, while the percentage of T cells expressing TNF-α was decreased, mainly in CD8+ T lymphocytes. However, the marked reduction in T lymphocyte numbers in co-infected patients led to a striking reduction of both cytokines in PBMC supernatants, a finding that is consistent with the impaired response toMycobacterium tuberculosis. [ABSTRACT FROM AUTHOR]

TheAspergillusallergen Asp f16 has been shown to confer protective Th1 T cell-mediated immunity against infection withAspergillusconidia in murine models. Here, we use overlapping (11-aa overlap with preceding peptide) pentadecapeptides spanning the entire 427-aa coding region of Asp f16 presented on autologous dendritic cells (DC) to evaluate the ability of this antigen to induce Th1 responses in humans. Proliferative responses were induced in five out of five donors, and one line with a high frequency of interferon (IFN)-γ-producing CD4+ T cells in response to the complete peptide pool was characterized. This line was cytotoxic to autologous pool-pulsed andAspergillusculture extract-pulsed targets. Limitation of cytotoxicity to the CD4+ T cell subset was demonstrated by co-expression of the degranulation marker CD107a in response to peptide pool-pulsed targets. Cytotoxic T lymphocytes (CTL) killedAspergillushyphae and CTL culture supernatant killedAspergillusconidia. By screening 21 smaller pools and individual peptides shared by positive pools we identified a single candidate sequence of TWSIDGAVVRT that elicited responses equal to the complete pool. The defined epitope was presented by human leucocyte antigen (HLA)-DRB1-0301. These data identify the first knownAspergillus-specific T cell epitope and support the use of Asp f16 in clinical immunotherapy protocols to prime protective immune responses to prevent or treatAspergillusinfection in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Integrinα4β 7 is the principal gut-homing receptor, and it is assumed that expression of this specific integrin directs lymphocytes to the gutin vivo. Adoptive cellular immunotherapy against inflammatory bowel disease (IBD) may depend on the expression of integrinα4β 7 to accomplish local delivery of intravenously injected regulatory T cells in inflamed gut mucosa. The present study aimed to investigate whetherin vitroexpanded human T cells from the colonic mucosa maintain integrin expression, showin vitroadhesion and retainin vivogut-homing properties during cultivation. Whole colonic biopsies from healthy subjects were cultured in the presence of interleukin-2 (IL-2) and IL-4. The integrin expression of the cultured T cells was determined by flow cytometry andin vitroadhesion was assessed in a mucosal addressin cell adhesion molecule 1 (MAdCAM-1) adhesion assay. We studied the homing pattern after autologous infusion of 3 × 108 111Indium (111In)-labelled T cells in five healthy subjects using scintigraphic imaging. The cultured CD4+CD45RO+ gut-derived T cells express higher levels of integrinα4β 7 than peripheral blood lymphocytes (PBLs) and show strong adhesion to MAdCAM-1in vitro, even after111In-labelling. Scintigraphic imaging, however, showed no gut-homingin vivo. After prolonged transit through the lungs, the T cells migrated preferentially to the spleen, liver and bone marrow. In conclusion, it is feasible to infuse autologous T cells cultured from the gut mucosa, which may be of interest in adoptive immunotherapy. Despite high expression of the gut-homing integrinα4β 7 and adhesion to MAdCAM-1in vitro, evaluation by111In-scintigraphy demonstrated no gut-homing in healthy individuals. [ABSTRACT FROM AUTHOR]

Eleven Norwegian patients (aged 2–33 years, seven males and four females) with Ataxia-telangiectasia (A-T) and their parents were investigated. Five of the patients were homozygous for the same ATM mutation. 3245delATCinsTGAT, a Norwegian founder mutation. They had the lowest IgG2 levels: mean (95% confidence interval) 0·23 (0·05–0·41) g/l versus 0·91 (0·58–1·26) g/l in the other patients (P= 0·002). Among the 11 A-T patients, six had IgG2 deficiency, six had IgA deficiency (three in com- bination with IgG2 deficiency) and seven had low/undetectable IgE values. All patients had very low levels of antibodies to Streptococcus pneumoniae 0·9 (0·4–1·4) U/ml. while normal levels were found in their parents 11·1 (8·7–13·4) U/ml (P < 0·001). A positive linear relationship between pneumococcal antibodies and IgC2 (r = 0·85, P = 0·001) was found in the patients. Six of 11 had diphtheria antibodies and 7 of 11 tetanus antibodies after childhood vaccinations, while 4 of 7 Hemophilus influenzae type b (Hib) vaccinated patients had protective antibodies. Ten patients had low B cell (CDI9+) counts, while six had low T cell (CD3+) counts. Of the T cell subpopulations, 11 had low CD4+ cell counts, six had reduced CD8+ cell counts, and four had an increased portion of double negative (CD3+/CD4-/CD8-) gamma delta T cells. Of the 22 parents (aged 23–64 years) 12 were heterozygous for the ATM founder mutation. Abnormalities in immunoglobulin levels and/or lymphocyte subpopulations were also observed in these carriers, with no correlation to a special ATM genotype. [ABSTRACT FROM AUTHOR]

SUMMARY The scurfy mutant mouse is the genetic and phenotypic equivalent of the single-gene human autoimmune disease immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). The scurfy mutation disrupts the Foxp3 gene, a putative master switch for T regulatory cell development. Bone marrow transplant without conditioning was previously reported to be ineffective in scurfy mice, yet clinical remission occurs in transplanted human IPEX patients despite limited donor engraftment. In view of this contradiction, we sought to validate scurfy as a model for studying the pathogenesis and treatment of human IPEX, in particular the phenomenon of dominant immune regulation. One half of scurfy mice given bone marrow transplants after sublethal irradiation recovered and survived long-term with donor chimerism ranging from 1·7% to 50%. Early transfer of 2 × 107 normal T cell-enriched splenocytes also prevented or limited disease and permitted long-term survival. Donor T cells in rescued mice made up 3–5% of lymphocytes and became highly enriched for CD25+ T cells over time. Transfer of 106 CD4+ CD25+ sorted T cells showed some beneficial effect, while CD4+ CD25- cells did not. Thus, both partial bone marrow transplant and T-enriched splenocyte transfer are effective treatments for scurfy. These results indicate that scurfy results from a lack of cells with dominant immune regulatory capacity, possibly T regulatory cells. The potency of small numbers of normal cells indicates that IPEX may be a feasible target for gene therapy. [ABSTRACT FROM AUTHOR]

X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency disease characterized by very low levels or even absence of circulating antibodies. The immunological defect is caused by deletions or mutations of Bruton's tyrosine kinase gene (Btk), whose product is critically involved in the maturation of pre-B lymphocytes into mature B cells. Btk is expressed not only in B lymphocytes but also in cells of the myeloid lineage, including dendritic cells (DC). These cells are professional antigen presenting cells (APC) that play a fundamental role in the induction and regulation of T-cell responses. In this study, we analysed differentiation, maturation, and antigen-presenting function of DC derived from XLA patients (XLA-DC) as compared to DC from age-matched healthy subjects (healthy-DC). We found that XLA-DC normally differentiate from monocyte precursors and mature in response to lipopolysaccharide (LPS) as assessed by de novo expression of CD83, up-regulation of MHC class II, B7·1 and B7·2 molecules as well as interleukin (IL)-12 and IL-10 production. In addition, we demonstrated that LPS stimulated XLA-DC acquire the ability to prime naïve T cells and to polarize them toward a Th1 phenotype, as observed in DC from healthy donors stimulated in the same conditions. In conclusion, these data indicate that Btk defect is not involved in DC differentiation and maturation, and that XLA-DC can act as fully competent antigen presenting cells in T cell-mediated immune responses. [ABSTRACT FROM AUTHOR]

Evidence of an immune mediated graft-versus-leukaemia effect has led to the belief that T and NK cell based adoptive immunotherapy can constitute effective treatment for relapsed leukaemias. However, work on solid tumours has shown this strategy may be hampered, by an immune escape mechanism in which tumour secreted immunosuppressive factors compromise T and NK cell function. Indeed, acute myeloid leukaemia (AML) cells secrete immunosuppressive factors that block the synthesis of Th1 type cytokines in T cells. We demonstrate here that this immunosuppression, mediated by both HL60 AML cell line and primary AML blasts, inhibits T and NK cell proliferation but not cytolytic activity. Supernatants from HL60 cell line and primary AML blasts inhibited T cell proliferation to mitogenic and alloantigen stimulation but had no effect on cytolytic function. Similarly, the proliferation of NK cells to IL-2 and IL-15 stimulation was inhibited whilst their cytolytic function, shown by lysis of AML blasts, K562 and Daudi cells remained unaffected. The failure of T and NK cells to proliferate was not due to effector cell apoptosis. Indeed, removal of lymphocytes from the immunosuppressive environment partially restored their capacity to respond to mitogenic stimulation. T cells exposed to immunosuppressive supernatants did not increase expression of mitotic inhibitory proteins that arrest cell division, thereby ruling this out as a mechanism of operation for this immunosuppression. T cell expansion requires antigen stimulation, usually provided in the form of AML blasts, therefore our data suggest that NK cells may be more practical for the immunotherapy of AML. [ABSTRACT FROM AUTHOR]

CTLA-4 (CD152) is a surface molecule of activated T cells with sequence homology to CD28. Both molecules bind to the same ligands, B7.1 (CD80) and B7.2 (CD86) but have antagonistic functions. While CD28 is an important costimulator, CTLA-4 has an essential inhibitory function in maintaining the homeostasis of the immune system. Furthermore, CTLA-4 has a role in inducing a Th1 response and suppressing Th2 cytokines, an effect which is antagonized by CD28. Many autoimmune diseases are characterized by an overwhelming production of Th1 cytokines. Recently, the predominance of the Th1 cytokine pattern has been directly observed in the granulomatous inflammation of patients with Wegener's granulomatosis. The balance between CD28 and CTLA-4 expression by T lymphocytes could be a factor in the pathogenesis of autoimmune diseases. Down regulation of CD28 predominantly on CD8+ T cells has been described in Wegner's granulomatosis; however, analysis of CTLA-4 is complicated by its low expression levels. Here we have used potent signal enhancement to study CTLA-4 on PBMC in patients with Wegener's granulomatosis (n = 25) in comparison with healthy controls (n = 19). Expression levels of CTLA-4 were significantly increased selectively on CD4+ and possibly also on CD4-/CD8- T cells in Wegener's granulomatosis. High CTLA-4 expression by T lymphocytes was associated with more severe disease. In contrast, after stimulation with the mitogen PHA, CTLA-4 levels were strongly increased on T cells from controls but in T cells from Wegener's granulomatosis patients this response was severely impaired. Interestingly, while CTLA-4 was seen exclusively on T cells in control individuals, about half of the Wegener's patients showed CTLA-4 expression by a fraction of peripheral B lymphocytes. CTLA-4 positive B cells in the periphery were associated with less acute disease. [ABSTRACT FROM AUTHOR]

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level, In the present study the response of purified CD4+ and CD8+ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8. anti-CD2 and anti-CD2S MoAbs (the costimulatory signals) was investigated. Both CD4+ and CDS+T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CDS and CD2. respectively. However, normal lL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker. CD69. after TCR +CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and la-4 release were impaired in CD4+ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule. or restricted to cells producing Th1-type cytokines such as IL-2. and is expressed in both CD4+ and CDS+T cell subsets. [ABSTRACT FROM AUTHOR]

Upon treatment with HgCl2, H-2s mice, such as B10.S, develop an activation of B lymphocytes that depends, at least partially, on activation of T helper type 2 (Th2) cells and results in increased serum levels of IgG1 and IgE, appearance of IgG autoantibodies, and development of immune glomerulonephritis and vasculitis. Results of previous studies and of experiments presented here indicate that the B cell activation and systemic autoimmune disease tail to develop in MHC-congenic B10.D2 (H-2d) and B10.BR (H-2+) mice treated with HgCh, although B10.D2 T cells showed signs of activation by and specificity for HgCl2 comparable to those seen in strain B10.S, Here, we report that following HgCl2 injections the antibody response to sheep erythrocytes is normal in B10.S, but suppressed in B10.D2 mice. This suppression was prevented by MoAb to mouse IFN-γ. Conversely, treatment of B10.D2 mice with murine recombinant IFN-γ (rIFN-γ) was able to reproduce the immunosuppression seen after HgCl2 treatment, in B10.S mice, it took administration of both rIFN-γ and HgCK to suppress the antisheep erythrocyte response. Although rIFN-γ diminished the increase in IgE serum levels of HgCl2- treated B10.S mice, it failed to prevent their autoantibody production and immune glomerulonephritis. These findings further strengthen the concept that B10.S mice react to HgCl2 by preferential activation of their Th2 cells producing lL-4, whereas B10.D2 mice react to HgC l2 by preferential activation of their Th1 cells, which produce IFN-γ and thus suppress antibody responses. [ABSTRACT FROM AUTHOR]

We studied T cell responses to four glutathione S transferase (GST)-fused mite antigens prepared in our laboratory using peripheral blood lymphocytes from mite-sensitive patients with bronchial asthma. Of the four recombinant antigens, purified GST-Mag3 had the strongest ability to cause patients' lymphocytes to proliferate, and its potency was almost comparable to that of crude mite bodies (Dfb) and faeces (Dff) antigens and a purified major antigen, Der f 2. The responder lymphocytes were mainly T cells, because the proliferative response was depleted by the treatment of lymphocytes with anti-CD3 antibody and complement, but not with anti-CD20 antibody and complement. The responsiveness of lymphocytes to GST-Mag3 correlated with that to Der f 2, but GST-Mag3 displayed slightly higher activity to stimulate lymphocytes than Der f 2. Simultaneously, the levels of Dff- and GST-Mag3- specific IgE antibodies correlated with the responsiveness of lymphocytes to GST-Mag3. These results suggest that Mag3 is a new valuable antigen for the response of T cell proliferation in mite-sensitive patients. [ABSTRACT FROM AUTHOR]

Several T cell defects have been described in the antibody deficiency disease, CVID, but there have been few data on the generation of responses of specific T cell populations to primary neoantigens. We have now used immunization with the neoantigens, keyhole limpet haemocyanin (KLH) and DNP-Ficoll, to evaluate immune responses in CVID patients and normal donors. B and T cell responses were examined 2 and 4 weeks post-immunization. Sera were examined for IgM and IgG anti-KLH responses by ELISA and for anti-DLP-Ficoll activity by haemagglutination. The frequency of KLH-responsive T cells was measured by DNA synthesis in a limiting dilution culture system. Low density cells enriched for dendritic cells were pulsed with KLH and cultured with different numbers of autologous T cells. T cells from normal donors and from patients showed a low frequency of antigen-specific precursor T cells (≤1:200 000). After KLH immunization the frequency increased in normal donors (1:60 000 and 1:30 000 at 2 and 4 weeks, respectively), while in CVID patients it did not change from the preimmunization level. The defect may extend to a dysfunction of antigen-specific cells, rather than being solely due to the reduced numbers of cells, since mean responses of 'positive' wells were also reduced. The serum-specific antibody response paralleled the T cell data, in that all normal donors but none of the CVID patients generated IgG KLH-specific antibodies. CVID patients did produce IgM antibodies against the T-independent DNP-Ficoll, but at a lower level than normal controls. These data show that both T and B cells from CVID patients have defective responses to specific antigen, implicating both lineages in the antibody deficiency. [ABSTRACT FROM AUTHOR]

Expression of the CFTR protein is thought to be physiologically important only in exocrine epithelial cells. However, chronic respiratory inflammation and infection remain unexplained phenomena in disease pathogenesis. Non-transformed, antigen-responsive CD4+ T cells cloned from healthy controls and CF patients homozygous or heterozygous for the δF508 mutation transcribed CFTR mRNA and expressed immunoreactive cytoplasmic CFTR protein. T cell clones (TCC) from controls and CF patients displayed equivalent Ca2+-mediated CI- current; however, TCC from patients with CF but not controls displayed defective cAMP-mediated Cl- current. Although CF-derived TCC preserved mitogen and antigen proliferative responses and specificity to tetanus toxoid epitopes, they selectively secreted &ap;45% less IL-10 compared with control TCC after activation with concanavalin A (Con A) (624±101 versus 1564±401 pg/ml per 106 cells, respectively; P=0.04) or anti-CD3/phorbol ester (5148±1634 versus 11 788±2390 pg/ml; P = 0.05). This difference was independent of atopy. Secretion of interferon-gamma, IL-2, and IL-4 was comparable in CF and control TCC after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/phorbol myristate acetate (PMA) but not after Con A. We conclude that expression of mutant CFTR in human TCC is accompanied by ion channel dysfunction characteristic of the CF phenotype, and is accompanied by a reduction in IL-10 secretion after polyclonal activation. It is possible that disruption of IL-10-mediated anti-inflammatory homeostasis may contribute to early onset sustained inflammation in CF airways. [ABSTRACT FROM AUTHOR]

We analysed the T cell receptor (TCR) Vγ repertoire in BAL and peripheral blood (PBL) of three mild stable atopic asthmatics and two non-asthmatic controls. We used the polymerase chain reaction (PCR) to establish the expression of the four Vγ families,, and to detect oligo or monoclonal expansion of γδ+ T cells, we resolved the PCR products on denaturing and non-denaturing gels to find the extent of junctional diversity arising from differences in the lengths of the V(D)J junctions. We also subcloned and sequenced the PCR products to characterize fully the sequence diversity. BAL T lymphocytes from two asthmatic patients (treated with inhaled steroids) expressed only VγII and, in one of them. VγIIJγ usage was restricted to JγP and JγP1 gene segments, contrasting with the VγJγ repertoire found in his respective PBL. Analyses in denaturing and non-denaturing gels showed that the BAL VγIIJγP and VγIIJγP1 PCR products resolved into few bands, suggesting deletions at the junctions due to oligoclonal expansion. BAL T lymphocytes from the third asthmatic (not receiving inhaled steroids) expressed VγI, II and III, and the sequences of the in-frame TCR transcripts from this asthmatic and one healthy volunteer who expressed a similar BAL VγTCR repertoire showed clonal expansion of T cells expressing all three Vγ families. Our analyses showed that much of the γδ T cell population found in BAL fluid of humans derives from clonally expanded T cells. [ABSTRACT FROM AUTHOR]