A common Vδ2—Dδ2—Dδ3 T cell receptor gene rearrangement in precursor B acute lymphoblastic leukaemia

Despite their apparent commitment to the B lymphocytic lineage, human precursor B cell acute lymphoblastic leukaemias (ALL) frequently rearrange their T cell antigen receptor (TCR) alpha, beta and gamma chain genes. Since these three genes are active sites of rearrangement in precursor B cell neoplasms, it seemed that the recently discovered fourth TCR gene, delta, might be similarly rearranged. To investigate this possibility, a series of precursor B cell leukaemias was analysed for rearrangements at the delta chain gene locus, using probes of the variable, joining, and constant regions of the delta chain gene. The majority of precursor B cell ALLs in this series (25/32, 78%) showed rearrangement or deletion of one or more TCR delta genes. This contrasts sharply with a series of 16 mature B cell neoplasms (chronic lymphocytic leukaemia) in which no TCR delta gene rearrangements were detected. An unusual TCR delta rearrangement, rarely observed in normal or neoplastic T cells, was seen in the majority (14/18) of precursor B cell ALLs with TCR delta rearrangements. In contrast to the utilization ov V delta 1 in T cell ALL, detailed restriction mapping of precursor B ALL revealed an incomplete rearrangement without involvement of J delta segments. Direct genomic sequencing was performed on one example and demonstrated a nonproductive V delta 2-D delta 2-D delta 3 recombination in this precursor B ALL. We conclude that the TCR delta chain gene is an active locus in precursor B cell neoplasia, involves an unusual type of rearrangement and provides a clonal tumour marker for diagnosis of precursor B ALL.     

Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.     

Mesenteric B cells centrally inhibit CD4+ T cell colitis through interaction with regulatory T cell subsets

Inflammatory bowel disease reflects an aberrant mucosal CD4+ T cell response to commensal enteric bacteria. In addition to regulatory T cell subsets, recent studies have revealed a protective role of B cells in murine CD4+ T cell colitis, but the relationship of their action to T cell immunoregulation is unknown. Here we report that mesenteric lymph node (MLN) B cells protect mice from colitis induced by Galphai2-/- CD4+ T cells. Protection required the transfer of both B cells and CD8alpha+ T cells; neither cell type alone was sufficient to inhibit CD4+ T cell-mediated colitis. Similar results were also observed in colitis induced by CD4+CD45RBhi T cells. Immunoregulation was associated with localization of B cells and expansion of CD4-CD8- CD3+NK1.1+ T cells in the secondary lymphoid compartment, as well as expansion of CD4+CD8alpha+ T cells in the intestinal intraepithelial compartment. MLN B cells from Galphai2-/- mice were deficient in a phenotypic subset and failed to provide cotransfer colitis protection. These findings indicate that protective action of B cells is a selective trait of MLN B cells acquired through a Galphai2-dependent developmental process and link B cells with the formation of regulatory T cells associated with mucosal immune homeostasis.     

NK T cells provide lipid antigen-specific cognate help for B cells

The mechanisms of T cell help for production of antilipid antibodies are largely unknown. This study shows that invariant NK T cells (iNK T cells) and B cells cooperate in a model of antilipid antigen-specific antibody responses. We use a model haptenated lipid molecule, 4-hydroxy-3-nitrophenyl-alphaGalactosylCeramide (NP-alphaGalCer), to demonstrate that iNK T cells provide cognate help to lipid-antigen-presenting B cells. B cells proliferate and IgG anti-NP is produced from in vivo-immunized mice and in vitro cocultures of B and NK T cells after exposure to NP-alphaGalCer, but not closely related control glycolipids. This B cell response is absent in CD1d(-/-) and Jalpha18(-/-) mice but not CD4(-/-) mice. The antibody response to NP-alphaGalCer is dominated by the IgM, IgG3, and IgG2c isotypes, and marginal zone B cells stimulate better in vitro lipid antigen-driven proliferation than follicular B cells, suggesting an important role for this B cell subset. iNK T cell help for B cells is shown to involve cognate help from CD1d-instructed lipid-specific iNK T cells, with help provided via CD40L, B7-1/B7-2, and IFN-gamma, but not IL-4. This model provides evidence of iNK T cell help for antilipid antibody production, an important aspect of infections, autoimmune diseases, and vaccine development. Our findings also now allow prediction of those microbial antigens that would be expected to elicit cognate iNKT cell help for antibody production, namely those that can stimulate iNKT cells and at the same time have a polar moiety that can be recognized by antibodies.     

Decreased frequency and proliferative response of invariant Vα24Vβ11 natural killer T (iNKT) cells in healthy elderly

Invariant natural killer T (iNKT) cells represent a well-established T cell lineage characterised in humans by TCR consisting of an invariant alpha chain encoded by Valpha24-JalphaQ genes, paired preferentially with a Vbeta11 chain. iNKT cells also share some characteristics with NK cells, such as the expression of the NK-associated receptor CD161 in humans. The T cell immune response is the most dramatically affected by ageing, although age-associated alterations in the phenotype and function of other cells of the immune system have been demonstrated. Despite the importance of iNKT cells in the regulation of the immune response, there are a limited number of studies on the effect of ageing on peripheral blood iNKT cells. Thus, in this work we analyse the effect of ageing on peripheral blood Valpha24(+)Vbeta11(+) iNKT cells by studying their frequency, phenotype and proliferative function in elderly individuals fulfilling the SENIEUR criteria of healthy ageing compared with healthy young donors. Our results demonstrated a significant decrease of the percentage of Valpha24(+)Vbeta11(+) iNKT cells in elderly donors. No significant differences were found in the expression of CD27, CD28, CD45RO, CD45RA(bright), CD161, CD94 and NKG2D on iNKT cells from young and elderly individuals. Proliferation of Valpha24(+)Vbeta11(+) iNKT cells in response to alpha-GalCer and IL2 was analysed by calculating the cumulative population doubling (PD) after 14 days of culture. The PD levels were lower in the elderly indicating that Valpha24(+)Vbeta11(+) iNKT cells from healthy elderly subjects had an impaired proliferative capacity. These results indicate that ageing associates with a significant decline in the percentage and proliferative response of peripheral blood iNKT cells. Given the important immunoregulatory role of iNKT cells, these alterations in their number and function could contribute to the deleterious immune response in the elderly.     

IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression

Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.     

Human RORγt+ TH17 cells preferentially differentiate from naive FOXP3+Treg in the presence of lineage-specific polarizing factors

RORγt(+) T(H)17 cells are a proinflammatory CD4(+) T-cell population associated with autoimmune tissue injury. In mice, priming of T(H)17 requires TGF-β, which alone directs the priming of FOXP3(+) regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human T(H)17 cells from conventional naive CD4(+) T cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human T(H)17 cells preferentially occurs from FOXP3(+) naive Treg (NTreg) in the presence of IL-2 and IL-1β and is increased by IL-23 and TGF-β. IL-1β-mediated differentiation correlated with IL-1RI expression in stimulated NTreg and was accompanied by induction of RORγt along with down-regulation of FOXP3. IL-17-secreting cells in NTreg cultures cosecreted TNF-α and IL-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-γ and other T(H)17-associated cytokines. Polarized NTreg contained significant subpopulations of CCR6-expressing cells that were highly enriched in IL-17-secreting cells. Finally, analysis of CCR6 expression with respect to that of IL-1RI identified distinct IL-17-secreting subpopulations that had maintained or lost their suppressive functions. Together our results support the concept that priming of human T(H)17 from naive CD4(+) T cells preferentially takes place from FOXP3(+) Treg precursors in the presence of lineage-specific polarizing factors.     

Skint-1 is a highly specific, unique selecting component for epidermal T cells

αβ T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.     

Alpha,beta hybrid peptides: a polypeptide helix with a central segment containing two consecutive beta-amino acid residues

Conformational studies on the synthetic 11-aa peptide t-butoxycarbonyl (Boc)-Val-Ala-Phe-alpha-aminoisobutyric acid (Aib)-(R)-beta3-homovaline (betaVal)-(S)-beta3-homophenylalanine (betaPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; betaVal and betaPhe are beta amino acids generated by homologation of the corresponding l-residues) establish that insertion of two consecutive beta residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2-10 of peptide 1. At the site of insertion of the betabeta segment, helical hydrogen-bonded rings are expanded. A C15 hydrogen bond for the alphabetabeta segment and two C14 hydrogen bonds for the alphaalphabeta or betaalphaalpha segments have been characterized. The following conformational angles were determined from the crystal structure for the beta residues: betaVal-5 (= -126 degrees, = 76 degrees, and psi = -124) and betaPhe-6 (=-88 degrees, = 80 degrees, and psi =-118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.     

Induced pluripotency as a potential path towards iNKT cell-mediated cancer immunotherapy

Invariant natural killer T (iNKT) cells are characterized by the expression of an invariant Vα14-Jα18 paired with Vβ8/7/2 in mice, and Vα24-Jα18 with Vβ11 in humans, that recognizes glycolipids, such as α-galactosylceramide (α-GalCer), presented on the MHC class I-like molecule, CD1d. iNKT cells act as innate T lymphocytes and serve as a bridge between the innate and acquired immune systems. iNKT cells augment anti-tumor responses by producing IFN-γ, which acts on NK cells to eliminate MHC-non-restricted (MHC(-)) target tumor cells, and on CD8(+) cytotoxic T lymphocytes to directly kill MHC-restricted (MHC(+)) tumor cells. Thus, when iNKT cells are activated by α-GalCer-pulsed dendritic cells, both MHC(-) and MHC(+) tumor cells can be effectively eliminated. Both of these tumor cell types are simultaneously present in cancer patients, and at present iNKT cells are only the cell type capable of eliminating them. Based on these findings, we have developed iNKT cell-targeted adjuvant immunotherapies with strong anti-tumor activity in humans. However, two-thirds of patients were ineligible for this therapy due to the limited numbers of iNKT cells in their bodies. In order to overcome the problem in cancer patients, we successfully established a method to generate iNKT cells with adjuvant activity from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In this review, we would like to outline the clinical potential for iNKT cells derived from ESCs and iPSCs for cancer immunotherapy, and the technical hurdles that must be overcome if we achieve effective ESC/iPSC-mediated cancer therapies.     

Prognostic relevance of β-catenin expression in T2-3N0M0 esophageal squamous cell carcinoma

AIM: To study the expression of β-catenin in esophageal squamous cell carcinoma (ESCC) at stage T2-3N0M0 and its relation with the prognosis of ESCC patients. METHODS: Expression of β-catenin in 227 ESCC speci-mens was detected by immunohistochemistry (IHC). A reproducible semi-quantitative method which takes both staining percentage and intensity into account was applied in IHC scoring, and receiver operating char-acteristic curve analysis was used to select the cut-off score for high or low IHC reactivity...