Pten loss in CD4 T cells enhances their helper function but does not lead to autoimmunity or lymphoma

PTEN, one of the most commonly mutated or lost tumor suppressors in human cancers, antagonizes signaling by the PI3K pathway. Mice with thymocyte-specific deletion of Pten rapidly develop peripheral lymphomas and autoimmunity, which may be caused by failed negative selection of thymocytes or from dysregulation of postthymic T cells. We induced conditional deletion of Pten from CD4 Th cells using a Cre knocked into the Tnfrsf4 (OX40) locus to generate OX40(Cre)Pten(f) mice. Pten-deficient Th cells proliferated more and produced greater concentrations of cytokines. The OX40(Cre)Pten(f) mice had a general increase in the number of lymphocytes in the lymph nodes, but not in the spleen. When transferred into wild-type (WT) mice, Pten-deficient Th cells enhanced anti-Listeria responses and the clearance of tumors under conditions in which WT T cells had no effect. Moreover, inflammatory responses were exaggerated and resolved later in OX40(Cre)Pten(f) mice than in WT mice. However, in contrast with models of thymocyte-specific Pten deletion, lymphomas and autoimmunity were not observed, even in older OX40(Cre)Pten(f) mice. Hence loss of Pten enhances Th cell function without obvious deleterious effects.     

A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.     

Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta T cells but does not require MyD88 signaling

Vaccination of nonautoimmune prone mice with syngeneic dendritic cells (DC) readily induces anti-DNA autoantibodies but does not trigger systemic disease. We observed that anti-DNA autoantibody generation absolutely required alphabeta T cells and that gammadelta T cells also contributed to the response, but that regulatory T cells restrained autoantibody production. Although both NZB/W F(1) mice and DC vaccinated C57/BL6 mice produced autoantibodies against dsDNA, vaccinated mice had higher levels of Abs against H1 histone and lower levels of antinucleosome Abs than NZB/W F(1) mice. Despite a 100-fold increase in IL-12 and Th1 skewing to a foreign Ag, OVA, synergistic TLR activation of DC in vitro failed to augment anti-DNA Abs or promote class switching beyond that induced by LPS alone. TLR stimulation was not absolutely required for the initial loss of B cell tolerance because anti-DNA levels were similar when wild-type (WT) or MyD88-deficient DC were used for vaccination or WT and MyD88-deficient recipients were vaccinated with WT DC. In contrast, systemic administration of LPS, augmented anti-DNA Ab levels and promoted class switching, and this response was dependent on donor DC signaling via MyD88. LPS also augmented responses in the MyD88-deficient recipients, suggesting that LPS likely exerts its effects on both transferred DC and host B cells in vivo. These results indicate that both the alphabeta and gammadelta subsets are necessary for promoting autoantibody production by DC vaccination, and that although TLR/MyD88 signaling is not absolutely required for initiation, this pathway does promote augmentation, and Th1-mediated skewing, of anti-DNA autoantibodies.     

Telomerase can extend the proliferative capacity of human myoblasts,but does not lead to their immortalization

Normal cells in culture display a limited capacity to divide and reach a non-proliferative state called cellular senescence. Spontaneous escape from senescence resulting in an indefinite life span is an exceptionally rare event for normal human cells and viral oncoproteins have been shown to extend the replicative life span but not to immortalize them. Telomere shortening has been proposed as a mitotic clock that regulates cellular senescence. Telomerase is capable of synthesizing telomere repeats onto chromosome ends to block telomere shortening and to maintain human fibroblasts in proliferation beyond their usual life span. However, the consequence of telomerase expression on the life span of human myoblasts and on their differentiation is unknown. In this study, the telomerase gene and the puromycin resistance gene were introduced into human satellite cells, which are the natural muscle precursors (myoblasts) in the adult and therefore, a target for cell-mediated gene therapy. Satellite cells expressing telomerase were selected, and the effects of the expression of the telomerase gene on proliferation, telomere length, and differentiation were investigated. Our results show that the telomerase-expressing cells are able to differentiate and to form multinucleated myotubes expressing mature muscle markers and do not form tumors in vivo. We also demonstrated that the expression of hTERT can extend the replicative life of muscle cells although these failed to undergo immortalization.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Extracellular matrix modulates the function of human melanocytes but not melanoma cells

Normal human epidermal melanocytes are attached to a basement membrane, a specialized form of extracellular matrix (ECM), located between the epithelium and underlying dermal tissues. To determine whether ECM influences pigmented cell behavior in vitro, human epidermal melanocytes and melanoma cells were cultured on uncoated or ECM-coated plastic culture surfaces, and a comparison was made between growth and function in the presence or absence of ECM. Melanocytes cultured on ECM-coated surfaces developed flatter and larger cell bodies and produced more melanin than melanocytes cultured on uncoated surfaces. In the presence of phorbol-myristate-acetate and cholera toxin, the rate of melanocyte replication was increased by ECM. In the absence of these mitogens, ECM significantly enhanced the adhesiveness of nonproliferating melanocytes. ECM had little or no effect on these parameters (morphology, tyrosinase activity, replication) in a pigmented human malignant melanoma cell line. These findings indicate that normal human epidermal pigment cells have the ability to recognize and respond to matrix signals, whereas this capacity appears to be absent in melanoma cells.     

Shortening the infectious period does not alter expansion of CD8 T cells but diminishes their capacity to differentiate into memory cells

Following a primary immune response, a portion of effector T cells gives rise to long-lived memory cells. Although primary expansion and differentiation of effector CD8 T cells is dictated by a brief exposure to Ag, it is unclear whether full memory differentiation is also programmed within the same short window. By carefully modulating the kinetics of Listeria monocytogenes infection, we analyzed the requirements for the programming of effector and memory T cell development in vivo. We find that although limiting the infectious period to the first 24-48 h does not impact the size of the primary CD8 response, the ensuing memory population is significantly diminished. This effect is particularly pronounced in the development of tissue-homing memory cells and is inversely proportional to the initial infectious dose. In contrast to CD8 responses, the differentiation of primary CD4 responses was highly dependent on the continued presence of the infection. Shortening the duration of the infection greatly reduced the development of CD4 effector responses in the spleen and prevented their trafficking to peripheral sites of infection. We propose that the stimulus received by CD8 T cells during the early stages of infection largely contribute to the differentiation of CD8 effector cells, whereas continued or distinct signals received at later stages influence their ability to differentiate into memory cells.     

Self-associated tetramer of human apolipoprotein E does not lead to its accumulation on a lipid particle

The data of differential measurements of apo E-containing and -free lipid particles in equilibrium binding (J. Biochem. (1989) 105, 582-587) showed complete fit to the calculated curve showing that distribution of apo E among the lipid particles is purely statistical among lipid particles. It was also demonstrated that 5 apo E molecules are required for the saturation of the surface of LDL-size lipid particle in good agreement with 7 +/- 1 given by the binding isotherm. Thus, it is concluded that tetramer formation of apo E in an aqueous phase (J. Biol. Chem. (1985) 260, 16375-16382) does not result in its accumulation on particular lipid particles.     

The substitution of the C-terminus of bax by that of bcl-xL does not affect its subcellular localization but abrogates its pro-apoptotic properties

The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.     

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli.

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.     

Binding of staphylococcal enterotoxin A to accessory cells is a requirement for its ability to activate human T cells

Staphylococcal enterotoxin A (SEA) activates human T cells at extremely low concentrations corresponding to 1 to 5 molecules/T cell. SEA, in contrast to other polyclonal activators, is absolutely dependent on accessory cells to interact with and activate T cells. Only cells that can bind SEA can act as accessory cells. Monocytes, B cells, and B lymphomas have these properties. Cells of the EBV-transformed B lymphoma Raji are particularly efficient as accessory cells. T cells do not bind soluble SEA. Probably the SEA molecule has to be attached to the accessory cell to become mitogenic. It is presently not known whether the T cell recognizes a molecular complex between the SEA molecule and the putative receptor moiety on the accessory cell or the SEA molecule itself modified by its binding to the accessory cell.     

The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function

Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4⁺ and CD8⁺ cells were measured. MSC equally inhibit CD4⁺ and CD8⁺ subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.     

Targeting the Wilms tumor antigen 1 by TCR gene transfer: TCR variants improve tetramer binding but not the function of gene modified human T cells

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the alpha- and beta-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated approximately 90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-gamma production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.     

Development of proteoglycan-induced arthritis depends on T cell-supported autoantibody production,but does not involve significant influx of T cells into the joints

Introduction  

Inflammatory joint destruction in rheumatoid arthritis (RA) may be triggered by autoantibodies, the production of which is supported by autoreactive T cells. Studies on RA and animal models of the disease suggest that T cells recruited in the joints can locally initiate or propagate arthritis. Herein, we investigated the role of joint-homing versus lymphoid organ-homing T cells in the development of proteoglycan-induced arthritis (PGIA), an autoimmune model of RA.     

Essential contribution of germline-encoded lysine residues in Jgamma1.2 segment to the recognition of nonpeptide antigens by human gammadelta T cells.

Human gammadelta T cells display unique repertoires of Ag specificities largely imposed by selective usages of distinct Vgamma and Vdelta genes. Among them, Vgamma2/Vdelta2(+) T cells predominate in the circulation of healthy adults and respond to various microbial small molecular mass nonpeptide Ags. The present results indicate that the primary Vgamma2/Vdelta2(+) T cells stimulated with the distinct groups of nonpeptide Ags, including monoethyl pyrophosphate, isobutyl amine, and aminobisphosphonate, invariably exhibit Jgamma1.2 in the Vgamma2(+) TCR-gamma chains. Gene transfer studies revealed that most of the randomly cloned Vgamma2/Jgamma1.2(+) TCR-gamma genes bearing diverse Vgamma/Jgamma junctional sequences could confer the responsiveness to all these nonpeptide Ags, while none of the Vgamma2/Jgamma1.1(+) or Vgamma2/Jgamma1.3(+) TCR-gamma genes could do so. Furthermore, mutation of the lysine residues encoded by the Jgamma1.2 gene, which are unique in human Jgamma1.2 and absent in other human or mouse Jgamma segments, completely abrogated the responsiveness to all the nonpeptide Ags without affecting the response to anti-CD3 mAb. These results strongly suggested that the positively charged lysine residues in the TCR-gamma chain CDR3 region encoded by the germline Jgamma1.2 gene play a key role in the recognition of diverse small molecular mass nonpeptide Ags.