CD8+ T lymphocytes provide helper activity for IgE synthesis in human immunodeficiency virus-infected patients with hyper-IgE.

Increased levels of serum IgE and eosinophilia have been described in human immunodeficiency virus (HIV) infection, almost exclusively in patients with CD4+ cell count < 200 cells/microliters. IgE production is regulated by CD4+ T helper type 2 (Th-2) lymphocytes, producing interleukin 4 (IL-4) and expressing a ligand for the B cell-specific CD40 molecule (CD40 ligand [L]). A shift to a Th-2-like pattern of cytokine secretion has been postulated to be associated with progression toward acquired immunodeficiency syndrome (AIDS). We studied three AIDS patients with very high levels of IgE and almost complete depletion of CD4+ lymphocytes, suggesting that IgE synthesis could not be driven by CD4+ cells. IgE in vitro synthesis by cells from such patients was, however, inhibited by anti-IL-4. We show that both CD8+ T cell lines and the majority of CD8+ T cells clones derived from these patients produce IL-4, IL-5, and IL-6 in half of the cases together with interferon gamma (IFN-gamma). 44% of CD8+ T cell clones expressed a CD40L, and the supernatants of the clones were capable of inducing IgE synthesis by normal B cells costimulated with anti-CD40. CD8+ T cells in these patients therefore functionally mimic Th-2 type cells and may account for hyper-IgE and eosinophilia in the absence of CD4+ cells. The presence of such CD8+ cells may also provide a source of IL-4 directing the development of predominant Th-2 responses in HIV infection.

Neutralizing antibodies and viral characteristics in mother-to-child transmission of HIV-1.

OBJECTIVE: To determine viral characteristics and the protective effect of virus neutralizing antibodies in mother-to-child transmission of HIV-1. MOLECULAR STUDIES: Ten HIV-1-infected mother-child pairs were sampled within 4 months of delivery. Variable region 3 of the viral envelope was amplified by nested polymerase chain reaction and sequenced, directly and/or after cloning, by solid-phase DNA sequencing. The amino acid sequence of variable region 3 from all 10 children was homogeneous, whereas the mothers showed varying degrees of heterogeneity. Apparently, selection of an HIV-1 variant occurs either at transmission or during initial virus replication in the infected child. No characteristic molecular features of the transmitted virus were identified. BIOLOGICAL STUDIES: Virus isolates from 13 mother-child pairs were characterized for replicative capacity in a variety of cell lines. Eight mothers from whom a virus with a slow/low replicative pattern was isolated transmitted the slow/low virus to their children, whereas mothers with a rapid/high virus transmitted either a rapid/high or a slow/low virus (two cases each). This indicates that viruses with rapid/high replicative capacity do not have a selective advantage during transmission. VIRUS NEUTRALIZING: Sera from 20 mothers were characterized for the ability to neutralize their own virus (autologous neutralization) and virus from other mothers (heterologous neutralization). The results showed that non-transmitting mothers had neutralizing antibodies against autologous virus more frequently than transmitting mothers. In addition, all mothers with autologous neutralizing antibodies also neutralized at least two heterologous primary isolates. This indicates that a broad neutralizing antibody response may be linked to a lower risk of mother-to-child transmission. CONCLUSION: On the basis of the variable region 3 loop sequence, HIV-1-infected infants harbour homogenous virus populations. Despite this, no molecular or biological markers for selective transmission could be identified. A maternal neutralizing antibody response with broad specificity may protect the child from HIV-1 infection.

Correlation between HIV sequence evolution, specific immune response and clinical outcome in vertically infected infants.

OBJECTIVE: To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1. DESIGN: Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3). METHODS: Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time. RESULTS: Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of children's sera to react with synthetic peptides representing the autologous virus sequence was determined. CONCLUSION: Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

OBJECTIVE: To investigate the T-cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV-1 in order to discern possible perturbations in TCR usage as a consequence of HIV-1 infection. DESIGN: Blood samples from five HIV-1-infected and six non-infected children born to HIV-1-seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. METHODS: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V alpha and V beta gene products and antibodies to CD4 and CD8 was performed. RESULTS: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V alpha and V beta gene products was seen in HIV-1-infected children (four out of five) but was rarely detected in uninfected children. CONCLUSION: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V alpha/V beta gene products in some HIV-1 vertically infected infants similar to those observed during primary infection in adults.

Kinetics of the T-cell receptor CD4 and CD8 V beta repertoire in HIV-1 vertically infected infants early treated with HAART.

OBJECTIVES: To determine the kinetics and the relationship between the T-cell receptor V beta (TCRBV) complementary determining region 3 length, the CD4 T-cell count and HIV viral load changes in HIV-1 infected infants treated early with highly active antiretroviral therapy (HAART) during 1 year of follow-up. DESIGN: Two HIV-1 vertically infected infants, two HIV-1 vertically exposed uninfected and two healthy controls were analysed by spectratyping. Evaluation of viral load, CD4 naive and memory cell counts and a proliferation test were also carried out. METHODS: Twenty-six families and subfamilies of the TCR on CD4 and CD8 T cells were analyzed by spectratyping. Flow cytometric analysis on peripheral blood mononuclear cells for CD4CD45Ra, CD4CD45Ro, CD8CD38, proliferation tests and plasma viral load measurements were performed at baseline, 1, 6 and after 12 months of therapy. RESULTS: HAART induced a marked reduction of viral load in both HIV-1 infected infants and an increase to normal CD4 T-cell count in the symptomatic infant. At baseline the TCRBV family distribution in the majority of CD8 and a few of the CD4 T cells was highly perturbed, with several TCRBV families showing a monoclonal/oligoclonal distribution. During HAART a normalization of the TCR repertoire in both CD8 and CD4 subsets occurred. TCR repertoire normalization was associated with a good virological and immunological response. CONCLUSION: These results suggest that complete and early virus replication control as a result of early HAART leads to a marked reduction of T-cell oligoclonality and is an essential prerequisite to the development of a polyclonal immune response in HIV-1 infected infants.

Analysis of heterogeneous viral populations by direct DNA sequencing.

The ability of direct PCR sequencing to detect and quantify sequence polymorphisms was investigated using samples containing mixed populations of HIV-1. A part of the genome encoding the polymorphic variable region 3 of the envelope was directly sequenced to yield a consensus sequence of the virus population. The results were compared with sequences obtained by analysis of multiple clones derived from the same clinical samples. The results of five patients suggested that the direct-sequencing method can be used as a rapid tool to analyze and quantify heterogeneous viral populations. Reconstitution experiments using cloned material demonstrated that it was possible to detect and quantify minor sequence variants present in as little as 10% of the total virus population. The use of the method for molecular diagnosis is discussed.

Oligoclonal T cells in human cancer.

Many solid tumors are characterised by the infiltration of lymphocytes and their presence has been correlated with a more favourable prognosis. These tumor-infiltrating lymphocytes (TIL), have been shown to possess specific cytolytic reactivity towards autologous tumours, thus suggesting that tumour cells may express antigens capable of eliciting an immune response. Expression of such tumour-associated antigens (TAA) in combination with appropriate accessory signals would lead to the in vivo accumulation of T cells with anti-tumour specificity. Analysis of the composition of the specific T-cell receptor (TCR) of TIL could thus provide information on the nature of the antigen(s) recognised by TIL. In this review, different aspects of the presence of clonal T cells in patients with cancer are discussed.

Role of immunity in maternal-infant HIV-1 transmission.

Factors influencing human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission include both immunological and virological parameters: higher viral loads have been associated with clinical stage of HIV-1-infected individuals as well as higher risk of mother-to-child transmission. Furthermore, we have shown that transmitting mothers more frequently harbour HIV-1 isolates with rapid/high syncytium-inducing (SI) biological phenotype than non-transmitting mothers do. Genetically homogeneous virus populations have been found in HIV-1-infected children at birth, in contrast to the heterogeneous virus populations often found in their infected mothers. This observation suggests that a few virus variants are transmitted or initially are replicating in the child. By comparing the HIV-1 gp120 V3 region of sequentially obtained samples from infected children with samples obtained from their mothers at delivery we found, however, that multiple variants of HIV-1 with different outgrowth kinetics can be transmitted. In addition, we have obtained results indicating an impaired ability of the immune response to adapt to the sequence evolution of HIV-1 in transmitting mothers, as assessed by measuring serum reactivities to peptides representing selected yet closely related V3 sequences. By analysing the presence of antibodies in maternal serum at delivery, which neutralize autologous isolates as well as other primary virus isolates, we have indications that a protective immunity in HIV-1 mother-to-child transmission might exist. Immunotherapy has been assessed in infected adult individuals by passive immunization with a variety of HIV-1-specific antibody products. Data from these studies indicated a differential response to therapy according to the stage of the disease. Active vaccine strategies, including envelope glycoproteins, pursued so far in seronegative adult subjects have shown limitations because broadly neutralizing antibodies, such as can be found in infected individuals, have not been evoked. Further investigations are therefore needed to give support for the potential use of either passive and/or active immunization for the prevention of HIV-1 mother-to-child transmission.

Contribution of ADAM33 polymorphisms to the population risk of asthma.

BACKGROUND: ADAM 33 is the first gene identified as a candidate for asthma by positional cloning techniques, with association studies reaching impressive statistical significance. It has a postulated role in myogenesis, airway modelling, and signalling via protein shedding. Concerns over the methodology of the initial study have led to several attempts at replication, with inconsistent results. METHOD: To clarify the role of ADAM33 in determining the risk of asthma in the general population, new transmission disequilibrium and case-control studies were undertaken followed by a meta-analysis of all existing data. RESULTS: Studies in Icelandic and UK populations revealed no association when taken in isolation. The meta-analysis, however, showed that the F+1 and ST+7 variants were significantly associated with asthma in both types of study. CONCLUSIONS: The additional risk imparted by this variation would account for 50,000 excess asthma cases in the UK alone. This study also demonstrates the size of study required to investigate such hypotheses adequately.

RT-PCR topography of chronic psoriasis skin based on analysis of T-cell receptor B variable region gene usage.

Psoriasis is a hyperproliferative inflammatory disease and 70% of patients develop a chronic plaque form. The pathogenesis of psoriasis is not known but evidence exists that T cells play a crucial role. The T cell V-gene receptor repertoire from psoriasis skin (different layers) was compared with peripheral blood T cells by employing RNA polymerase chain reaction (PCR) amplification. T cell receptor (TCR) BV 5.1, 11, 12, 13.1 and 16 were utilized to a significantly higher degree in areas close to the basal layers when compared to CD4+, CD8+ or unfractionated blood T cells from the same patients, whereas only BV11 and 13.1 genes of T cells from deeper layers of the dermis showed such a skewed usage. No biased usage of TCRBV genes was observed in superficial layers or in whole skin. Furthermore, T cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin psoriatic T cells to be poly- or oligoclonal. In conclusion, we show that TCRBV gene usage from different layers of psoriatic skin has a different pattern compared with the corresponding gene usage in circulating peripheral blood T cells. This pattern may implicate possible skin-associated antigen or superantigens activating a limited number of T cells in areas of skin close to basal layers, which in turn could promote keratinocyte proliferation.

Soluble CD4 suppress the antigen driven proliferative response of certain T cell clones specific for mycobacteria and for peptides by mycobacterial heat shock proteins.

We have investigated the effect of soluble recombinant CD4 (sCD4) on the antigen specific (BCG, peptides of mycobacterial 65 kDa hsp) responses of T cell lines of T cell clones. The majority of the antigen specific clones could be suppressed in their antigen driven response by the addition of sCD4, while others, including the parental polyclonal T cell line, were not. The suppression of the specific T cell response was reversed by the addition of anti-CD3, did not affect the proliferative response to IL-2, and was independent of the amount of antigen. A decreased capacity to produce IFN-gamma in response to the antigen by the addition of sCD4 was seen only with those clones that were also inhibited in their specific proliferative response. This model may be used to delineate further the interaction between T cells and the antigen presenting cell, and the finding may limit the possible in vivo use of sCD4 in the therapy of human immunodeficiency virus (HIV) infections.

Increased frequency of abnormal gamma delta T cells in blood of patients with inflammatory bowel diseases.

We have previously reported a preferential usage of V delta 1/V gamma 8 on TCR-gamma-delta-bearing intraepithelial lymphocytes of the normal human intestine as well as in the inflamed synovial tissue of patients with rheumatoid arthritis. The aim of the present study was to analyze V gene segment usage by gamma delta T cells of the intestine and peripheral blood (PB) from patients with inflammatory bowel disease. Freshly isolated lymphocytes were analyzed by flow cytometry using a panel of V gene subset-specific mAbs. The relative proportion of PB TCR-gamma delta+ cells was increased in patients with Crohn's disease as compared with controls. Interestingly, an increased proportion of PB gamma delta T cells of patients with ulcerative colitis or CrD expressed V delta and V gamma genes typically used by intraepithelial lymphocytes. Thus, increased proportions of V delta 1+ and V gamma 8+ cells were found in the PB of both patient groups. The majority of TCR-gamma delta+ intraepithelial lymphocytes (IEL) in inflamed and noninflamed intestine expressed V delta 1/V gamma 8, and a substantial proportion of V gamma(2,3,4)+ cells were found. These observations suggest a disease-associated appearance of "gut-like" gamma delta T cells in the periphery. Moreover, two patients had a large proportion of gamma delta T cells in their PB of which the majority expressed two distinct V gamma proteins. Both of these patients had a short duration of disease (1 and 10 mo, respectively) and the relative proportion of gamma delta T cells decreased in concert with stabilization of disease. Interestingly, one of the patients had a clonal expansion of a V gamma dual-expressing gamma delta T cell in the PB.

Clonal CD8+ and CD52- T cells are induced in responding B cell lymphoma patients treated with Campath-1H (anti-CD52).

Five patients with non-Hodgkin's lymphoma (NHL) and 4 patients with chronic lymphocytic leukaemia (CLL) were treated with the CDR-grafted (rat x human) monoclonal antibody (mAb) Campath-1H (anti-CD52). Tumour regression was noted preferentially in peripheral blood and in the bone marrow but lymph nodes were less affected. Normal blood B and T cells were profoundly reduced in all patients whereas CD16+ NK cells and CD14+ monocytes decreased marginally. In all responding CLL patients CD52-negative T but not B cells appeared during treatment and persisted for several months (4-19+) during unmaintained follow-up. Clonal T cells defined as a predominance of a single T cell receptor (TCR) V gene usage, in one case verified by TCR CDR3 fragment analysis and nucleotide sequencing, emerged within the CD52-/CD8+ cell population during Campath-1H therapy in 2 CLL patients, both achieving a long-lasting remission. The increase in CD8+ T cell expansions (up to 23-fold) during unmaintained remission and follow-up suggest that the clonal CD8+ cells may represent regulatory T cells controlling the growth of the tumour B cell clone. Clonal T cells might thus be a target for an immune therapeutic intervention in B cell tumours.

Mechanisms of escape from CD8+ T-cell clones specific for the HER-2/neu proto-oncogene expressed in ovarian carcinomas: related and unrelated to decreased MHC class 1 expression.

We have developed an in vitro model to study mechanisms by which ovarian tumor cells that over-express the HER-2/neu proto-oncogene escape recognition by TCD8+. Nine tumor-specific, HLA A2-restricted TCD8+ clones were isolated from 2 ovarian tumor-specific TCD8+ lines derived from tumor-infiltrating or -associated lymphocytes. Of these, 2 clones recognized the previously defined HER-2/neu epitope E75 (a.a. 369-377) and one recognized the C85 epitope (a.a. 971-979), whereas the specificity of the remaining 6 clones was unknown. Three different tumor escape variants (EVC8, EVC22 and EVC36) were produced by co-culturing an ovarian tumor line over-expressing HER-2/neu with these autologous TCD8+ clones. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER-2/neu, ICAM-1 or LFA-3 molecules was found. There was a correlation between the level of tumor-specific recognition and HLA A2 expression among the tumor clones isolated from 2 of the escape variants (EVC8 and EVC36). In contrast, high HLA A2-expressing tumor clones isolated from the EVC22 variant, or EVC22 which had regained high HLA A2 expression through IFN-gamma treatment, were not recognized by the HER-2/neu-specific TCD8+ clone C-22. No mutations were found in the cDNA or the genomic DNA derived from the PCR product corresponding to a 496 bp fragment including the region coding for the E75 epitope of the HER2/neu gene in the EVC22 variant. Collectively, this in vitro model underlines the importance of decreased expression of the HLA restriction element for escape from tumor-specific TCD8+ but also demonstrates that additional mechanisms exist.

Mycobacterial-induced cytotoxic T cells as well as nonspecific killer cells derived from healthy individuals and leprosy patients.

Little information is available about the generation and specificity of the cytotoxic cells that eliminate human monocytes/macrophages infected with mycobacteria. To address this we have developed a cytotoxicity assay in which 51Cr-labeled monocytes pulsed with bacillus Calmette Guerin (BCG) or Mycobacterium leprae, were used as target cells in overnight cytotoxicity assays. As effector cells, peripheral blood mononuclear cells from healthy occupational contacts or from leprosy patients stimulated with antigen for 7 days were used. Cytotoxicity against antigen-pulsed monocytes that could be induced by mycobacterial antigens was proportional to the degree of antigen responsiveness in each individual, as measured in lymphocyte transformation tests. The lepromatous leprosy patients tested were often poor responders to BCG as well as M. leprae, both with regard to induction of cytotoxicity as well as in lympho-proliferation. Killing was significantly higher against antigen-pulsed vs. nonpulsed monocytes, although significant killing was induced against the latter as well and paralleled by induction of natural killer activity against the K-562 target cell. Cross-reactivity was observed between BCG and M. leprae, but not with unrelated antigen (tetanus toxoid) or with endogenous stress proteins induced by heat shock. M. leprae- and BCG-activated cytotoxic cells were found in both the CD4-CD8+ and CD4+CD8- populations, whereas in contrast the soluble antigen, purified protein derivative of M. tuberculosis, generated cytotoxic cells that were exclusively of the CD4+ phenotype. The involvement of both specific T cells as well as nonspecific cells in the killing of human macrophages may be important with respect to protection and immunopathology induced by mycobacterial antigens.

RT-PCR based analysis of T-cell receptor B variable region gene usage in normal human breast skin resident T lymphocytes (SRT).

The skin interfaces directly with the external environment that contains innumerable infectious agents. Therefore, an appropriate and rapid immunologic response is required to preserve internal homeostasis. An essential feature of the "skin immuno system' (SIS) is the presence of substantial numbers of T cells in normal skin. The T-cell receptor repertoire from normal human breast skin was analysed quantitatively and qualitatively by using PCR amplification of reverse transcribed RNA, T-cell receptor BV3 and BV14 gene usage was increased in skir T lymphocytes in all individuals tested (n = 8) compared to peripheral blood CD4+ and CD8+ T lymphocytes from the same individuals. The T-cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin T-cell BV3 and BV14 gene usage to be predominantly polyclonal. Superantigen stimulation of T cells in human skin is considered a likely explanation of the present finding.

Association between IL-1beta/TNF-alpha-induced glucocorticoid-sensitive changes in multiple gene expression and altered responsiveness in airway smooth muscle.

The pleiotropic cytokines interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha have been implicated in the pathophysiology of asthma. To elucidate the role of these cytokines in the pro-asthmatic state, the effects of IL-1beta and TNF-alpha on airway smooth muscle (ASM) responsiveness and ASM expression of multiple genes, assessed by high-density oligonucleotide array analysis, were examined in the absence and presence of the glucocorticoid dexamethasone (DEX). Administration of IL-1beta/TNF-alpha increased ASM contractility to acetylcholine and impaired ASM relaxation to isoproterenol. These pro-asthmatic- like changes in ASM responsiveness were associated with IL-1beta/ TNF-alpha-induced mRNA expression of a host of proinflammatory genes that regulate transcription, cytokines and chemokines, cellular adhesion molecules, and various signal transduction molecules that regulate ASM responsiveness. In the presence of DEX, the changes induced in ASM responsiveness were abrogated, and most of the IL-1beta/TNF-alpha-mediated changes in proinflammatory gene expression were repressed, although mRNA expression of a small number of genes was enhanced by DEX. Collectively, the observations support the concept that, together with its role as a regulator of airway tone, in response to IL-1beta/TNF-alpha, the ASM expresses a host of glucocorticoid-sensitive genes that contribute to the altered structure and function of the airways in the pro-asthmatic state. We speculate that glucocorticoid-sensitive, cytokine-induced pathways involved in ASM cell signaling represent important targets for new therapeutic interventions.