The ex vivo induction of human CD103⁺ CD25hi Foxp3⁺ CD4⁺ and CD8⁺ Tregs is IL-2 and TGF-ß1 dependent.

The expression of the integrin αE (CD103), may enhance the retention of regulatory T cells to peripheral inflammatory sites and possibly contribute to their suppressive potential. The aim of this study was to define the regulatory role of IL-2 and TGF-ß1 on the CD103 expression and the optimal in vitro conditions for the induction/expansion of human CD4(+) and CD8(+) Tregs. Cord blood mononuclear cells (CBMC) were stimulated under various culture conditions, including anti-CD3, anti-CD28, IL-2 and TGF-ß1. TGF-ß1 and IL-2 were both required for optimal expression of CD103. In addition, TGF-ß1 and IL-2 synergistically induced CD103 expression on CD8(+) T cells, whereas, only additive induced expression was noted on CD4(+) T cells. Surprisingly, CD103 expression was not dependent upon CD28 costimulation. IL-2 also played a central role in CD103 expression by CD25(hi) Foxp3+ Tregs. IL-2, TGF-ß1 and anti-CD3 defined the optimal stimulatory conditions favouring the induction/expansion of both CD4(+) and CD8(+) human Tregs from naive CBMC. Thus, this study provides new insights into the regulatory role of IL-2 upon CD103 expression by human cord blood CD4(+) and CD8(+) T cells. Furthermore, it identifies the in vitro culture conditions driving the differentiation of the novel phenotype CD4(+) and CD8(+) CD103(+) CD25(hi) Foxp3+ Tregs from human CBMC.

Vbeta usage and T regulatory cells in children following partial or total thymectomy after open heart surgery in infancy.

During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4+CD25+). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vbeta usage. The expression of Foxp3 and CD127 in CD4+CD25(high) T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vbeta usage in both study and control groups. Significant variability was found in Vbeta usage for CD4+ and CD8+ T cells when the distribution of the percentage of T cells expressing each Vbeta family was analysed between individuals within each group (P < 0.001; Kruskal-Wallis). Significant difference was also found in average usage of Vbeta2, Vbeta5.1 and Vbeta14 chains within CD4+ T cells and Vbeta2, Vbeta8 and Vbeta21.3 chains within CD8+ cells between the groups (P < 0.05; Student's t-test). There was no difference between the two groups with regard to the proportion of CD4+CD25(high) T cells and no difference in the average expression of Foxp3 or CD127 within the CD4+CD25(high) population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vbeta usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.

Naive human T-cells become non-responsive towards anti-TNFalpha (infliximab) treatment in vitro if co-stimulated through CD28.

Tumour necrosis factor alpha (TNFalpha) inhibitors are widely used successfully as immunomodulatory agents in various autoimmune diseases. They primarily target the direct pro-inflammatory effect of TNFalpha. They have also been found to be critical for T-cell viability and activation. In this study we evaluated the effect of infliximab treatment under different in vitro stimulatory conditions of naive human cord blood T-cells and adult peripheral blood mononuclear cells (PBMC). PBMC and negatively selected cord blood naive human T-cells were stimulated with alphaCD3 with or without alphaCD28 co-stimulation. The role of in vitro infliximab treatment was evaluated in relation to transforming growth factor-beta1 (TGF-beta1) under the above different stimulatory conditions. Anti-TNFalpha treatment with infliximab significantly suppressed proliferation of adult and cord blood T-cells (P < 0.013) during suboptimal stimulatory conditions. Infliximab prevented division of naive CD4+ and CD8+ T-cells and consequently also activation induced cell death, which was induced after three cell divisions. Interleukin (IL)-2 secretion was significantly decreased during infliximab treatment of suboptimally stimulated T-cells (P < 0.05) while TGF-beta1 levels were unchanged. Strikingly, the anti-proliferative effect of infliximab was overcome by the administration of anti-TGF-beta1 or by the addition of exogenous IL-2. Interestingly, CD28 mediated co-stimulation restored the proliferative response in a dose-responsive manner during infliximab treatment. Finally, exogenous TNFalpha administration during suboptimal stimulation reduced the inhibitory effect of TGF-beta1 upon proliferation (P < 0.03). These results demonstrate that anti-TNFalpha treatment is primarily working upon T cells under low stimulatory conditions and probably through TGF-beta1.

Evidence for extrathymic T cell maturation after thymectomy in infancy.

Our previous study showed that children who had been partially or completely thymectomized during heart surgery as infants had lower proportions and numbers of total lymphocytes and reduced proportions of T cells (CD3(+)), helper T cells (CD4(+)) and naive T cells (CD3(+) CD4(+) CD45RA(+)), but normal proportion of cytotoxic T cells (CD8(+)). In this study T lymphocytes from a selected group of eight of these children and age- and gender-matched controls were characterized further using flow cytometry to determine phenotypes of T cells and T cell subsets related to T cell regulation and phenotypes suggestive of extrathymic maturation. Immune function was assessed by measuring autoantibodies and antibodies against vaccines. The study group had significantly lower numbers of all the main subsets of T lymphocytes and the composition was different. Thus, the proportions of lymphocytes with the following phenotypes: CD3(+), CD2(+), CD7(+), CD4(+), CD62L(+), CD4(+) CD62L(+) and CD4(+) CD69(-) were significantly reduced in the study group compared with the control group, but significantly higher proportions were seen of lymphocytes expressing CD8alpha(+) CD8beta(-) and TCRgammadelta(+) CD8alpha(+) CD8beta(-). The absolute number and proportion of CD4(+) CD25(+) cells were reduced but the proportions of the subgroup of naive regulatory T cells (CD4(+) CD25(+) CD62L(+)) and non-activated regulatory T cells (CD4(+) CD25(+) CD69(-)) were not reduced in the thymectomized children. We conclude that the phenotypic characteristics of T lymphocytes of children who have lost their thymus in infancy are indicative of extrathymic maturation. T regulatory cells appear to be less affected than other subsets by the general reduction in T cell numbers.

The influence of partial or total thymectomy during open heart surgery in infants on the immune function later in life.

Infants undergoing open heart surgery often have all or part of their thymus removed. The activity of the immune system has not been investigated thoroughly in these children, and only shortly after the operation. Therefore, it was decided to investigate the activity of the immune system in more detail in children several years after their operation. Peripheral blood samples from 19 children who had undergone open heart surgery during their first months of life was collected (study group) and from 19 age- and gender-matched children (control group). The activity of the immune system was evaluated by measuring the number of different cell types in peripheral blood, the phenotype of lymphocytes and the response of T cells following in vitro stimulation by mitogen, tetanus toxoid and measles antigen. The study group had significantly lower counts of total lymphocytes, which was reflected in a lower number of T cells but not B cells. Furthermore, the study group had significantly lower proportion of T cells (CD3(+)) and helper T cells (CD4(+)), but not cytotoxic T cells (CD8(+)). The level of neutrophils in peripheral blood was significantly higher in the study group. This may indicate enhanced innate immunity when the acquired immunity is defective. The results indicate a shift to extrathymic T cell maturation, which is less efficient for CD4(+) helper cells than for CD8(+) cytotoxic cells.

Enhanced B cell survival in familial macroglobulinaemia is associated with increased expression of Bcl-2.

A family with three cases of macroglobulinaemia of undetermined significance (MGUS), and one case each of immunoblastic lymphoma, Waldentröm's macroglobulinaemia and multiple myeloma was first described 20 years ago. We have previously identified 10 out of 35 healthy family members tested whose lymphocytes produced abnormally high amounts of immunoglobulins in culture. In the present study lymphocyte subpopulations of these hyper-responders have been further characterized and lymphocyte reactivity and survival in vitro have been studied. No differences were detected in the proportions of resting B lymphocytes (CD19+) co-expressing CD5, CD10, CD11b, or CD38, and the CD4/CD8 ratio of T cells was normal before and after stimulation with pokeweed mitogen (PWM). The initial rate of response in terms of immunoglobulin production was not increased, but immunoglobulin levels continued to rise during the second week of culture whereas the production peaked at 8 days in control cultures. This was associated with significantly greater survival of lymphocytes and at 14 days surviving B cells could only be identified in samples from hyper-responders. A lymph node removed because of tuberculosis from a family member 23 years before the diagnosis of multiple myeloma showed very marked Bcl-2 expression in a B cell follicle. This was not seen in a tuberculous lymph node from an unrelated subject. Stimulated cultures from three hyper-responders tested demonstrated significantly higher retention of Bcl-2 in B cells compared with one family control and six unrelated controls. We conclude that the increased production of immunoglobulins previously observed in this family with an inherited tendency for benign and malignant B cell proliferation is the result of enhanced B cell survival, which is associated with increased expression of Bcl-2 following stimulation.

A study of the association of HLA DR, DQ, and complement C4 alleles with systemic lupus erythematosus in Iceland.

OBJECTIVE: To perform an exploratory analysis of the relative contribution of single MHC genes to the pathogenesis of systemic lupus erythematosus (SLE) in a homogenous white population. METHODS: MHC class II alleles and C4 allotypes were determined in 64 SLE patients and in ethnically matched controls. HLA-DR and DQ typing was performed by polymerase chain reaction amplification with sequence specific primers. C4 allotypes were determined by agarose gel electrophoresis. RESULTS: The frequency of C4A*Q0 was significantly higher in patients than in controls (46.9% v 25.3%, p = 0.002). HLA-DRB1, DQA1, and DQB1 alleles in the whole group of SLE patients were not significantly different from those of controls. On the other hand increase in DRB1*03 was observed in the group of patients with C4A*Q0, as compared with patients with other C4A allotypes (p = 0.047). There was no significant correlation between severe and mild disease, as judged by the SLEDAI, and HLADR, DQ alleles and comparing the patients with C4A*Q0 with those with other C4A allotypes there was no significant difference regarding clinical manifestations. CONCLUSION: The results are consistent with the argument that C4A deficiency contributes independently to susceptibility and the pathogenesis of SLE. C4A*Q0 in SLE patients in Iceland shows weaker linkage disequilibrium with DR3 genes than reported in most other white populations and emphasises the role of ethnicity.

A study of the major histocompatibility complex in a Caucasian family with multiple cases of systemic lupus erythematosus: association with the C4AQ0 phenotype.

OBJECTIVE: In an epidemiological survey of systemic lupus erythematosus (SLE) in Iceland several families with multiple cases were identified. In one family, 35 individuals (family members and spouses) in 3 generations were studied clinically, tested for autoantibody formation, and typed for HLA and toxicity complement phenotypes. METHODS: Typing for HLA-A, B, C, DR, and DQ was performed by microlymphocytotoxic assay. In selected samples HLA-DR typing by polymerase chain reaction amplification with sequence specific primers was performed. C4 allotypes were defined by agarose gel protein electrophoresis followed by immunofixation with goat antisera. RESULTS: Five family members fulfilled 4 or more criteria for SLE. Additionally, 5 family members had clinical manifestations or positive serology but did not fulfill 4 ARA criteria. The mean age at onset of symptoms was 22 yrs (8-40). Other autoimmune diseases were not documented in family members. C4A null seemed to be highly associated with disease in this family. All except one patient with SLE and all those with clinical manifestations and positive serology had C4A null in the homozygous or heterozygous form. The individual with SLE and not carrying C4A null had both HLA haplotypes identical. It is noteworthy that there were 5 different C4A null bearing haplotypes involved, of which 3 originated from the spouses. CONCLUSION: Our results are consistent with the argument that C4A deficiency plays a role in the pathogenesis of SLE. There is, however, the possibility of an unidentified environmental or another genetic factor being involved.

The association between coeliac disease, dermatitis herpetiformis and certain HLA-antigens in Icelanders.

Twenty-eight cases of coeliac disease (CD) and seven of dermatitis herpetiformis (DH) have been verified in Iceland. Standard serological techniques were used for HLA typing. Twenty-five individuals with CD were typed, 21 (84%) of whom carried DR3,DQ2. Twelve of these 25 (48%) had DR3,DR7, DQ2, which makes them possibly homozygous for DQ2, and suggests that homozygosity of DQ2 increases the risk for CD. The four DH patients that were typed all had HLA-B8,DR3,DQ2. It is concluded that CD and DH are associated with DR3, DQZ in Icelanders.

Specificity of gamma delta receptor-bearing cytotoxic T lymphocytes isolated from human peripheral blood.

T-cell receptor (TcR)-gamma delta-bearing lymphocytes were isolated from the peripheral blood of two healthy donors by immunomagnetic separation and subsequently cultured. The cell lines generated showed two distinct patterns of cytotoxicity. One TcR-gamma delta + cell line (HG.D) lysed K562 and U937 target cells, three TcR-gamma delta + cell lines lysed Daudi cells, and one TcR-gamma delta + cell line showed a shift from the former to the latter specificity during culture. Cold target inhibition experiments showed that the HG.D effector cells which were cytotoxic against U937 cells also lysed K562 cells. The cytotoxicity against Daudi cells was strongly inhibited by monoclonal antibodies (MoAb) against the CD3 complex, whereas the cytotoxicity of the HG.D cell line against K562 and U937 was unaffected by such antibodies. The cytotoxicity against Daudi cells was also strongly inhibited in the presence of anti-TcR-gamma delta MoAb. However, in two of the Daudi-specific cell lines, strong cytotoxicity against K562 cells was induced by anti-TcR-gamma delta MoAb. Anti-LFA-1 MoAb caused only a partial inhibition of cytotoxicity, while anti-CD2 and anti-TcR-alpha beta MoAb were found to have no effect. The results indicate that human gamma delta receptor-bearing T cells demonstrate a certain degree of target cell specificity, and that recognition of some target cells may be mediated through the TcR-gamma delta.

Gc subtypes in Northern Indians.

Isoelectric focusing was used to determine the frequencies of the Gc subtypes in a population sample from The North Indian subcontinent (now living in Birmingham, UK). The gene frequencies observed were as follows: Gc1F = 0.191, Gc1S = 0.519 and Gc2 = 0.290.243 individuals were typed and no variant alleles were detected.

Gc subtypes in Icelanders.

Isoelectric focusing was used to determine the frequencies of Gc subtypes in Icelanders. The gene frequencies observed were Gc1F = 0.107, Gc1S = 0.631 and Gc2 = 0.262. An Icelandic Gc variant allele (Gc Iceland) is shown to have a different isoelectric point from the variant allele Gc-Norway. Gc-Iceland has been detected in 3 unrelated individuals giving the allelic frequency of 0.004 in the Icelandic population. Studies of 65 mother-child pairs confirm the assumed three-allelic mode of inheritance.