Lack of a role for transforming growth factor-β in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-β1 (TGF-β1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-β production. Here, we examine the role of TGF-β in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-β production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-β does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-β1−/−, and Smad3−/− T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-β by CD4+ T cells. These results indicate that CTLA-4 ligation does not regulate TGF-β production and that CTLA-4-mediated inhibition can occur independently of TGF-β. Collectively, these data demonstrate that CTLA-4 and TGF-β represent distinct mechanisms for regulation of T cell responses.

CD95/Fas induces cleavage of the GrpL/Gads adaptor and desensitization of antigen receptor signaling

The balance between cell survival and cell death is critical for normal lymphoid development. This balance is maintained by signals through lymphocyte antigen receptors and death receptors such as CD95/Fas. In some cells, ligating the B cell antigen receptor can protect the cell from apoptosis induced by CD95. Here we report that ligation of CD95 inhibits antigen receptor-mediated signaling. Pretreating CD40-stimulated tonsillar B cells with anti-CD95 abolished B cell antigen receptor-mediated calcium mobilization. Furthermore, CD95 ligation led to the caspase-dependent inhibition of antigen receptor-induced calcium mobilization and to the activation of mitogen-activated protein kinase pathways in B and T cell lines. A target of CD95-mediated caspase 3-like activity early in the apoptotic process is the adaptor protein GrpL/Gads. GrpL constitutively interacts with SLP-76 via its C-terminal SH3 domain to regulate transcription factors such as NF-AT. Cleavage of GrpL removes the C-terminal SH3 domain so that it is no longer capable of recruiting SLP-76 to the membrane. Transfection of a truncated form of GrpL into Jurkat T cells blocked T cell antigen receptor-induced activation of NF-AT. These results suggest that CD95 signaling can desensitize antigen receptors, in part via cleavage of the GrpL adaptor.

Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.

Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs and suppresses mast cell activation when coligated with the high-affinity Fc receptor for IgE.

Mouse mast cells express gp49B1, a cell-surface member of the Ig superfamily encoded by the gp49B gene. We now report that by ALIGN comparison of the amino acid sequence of gp49B1 with numerous receptors of the Ig superfamily, a newly recognized family has been established that includes gp49B1, the human myeloid cell Fc receptor for IgA, the bovine myeloid cell Fc receptor for IgG2, and the human killer cell inhibitory receptors expressed on natural killer cells and T lymphocyte subsets. Furthermore, the cytoplasmic domain of gp49B1 contains two immunoreceptor tyrosine-based inhibition motifs that are also present in killer cell inhibitory receptors; these motifs downregulate natural killer cell and T-cell activation signals that lead to cytotoxic activity. As assessed by flow cytometry with transfectants that express either gp49B1 or gp49A, which are 89% identical in the amino acid sequences of their extracellular domains, mAb B23.1 was shown to recognize only gp49B1. Coligation of mAb B23.1 bound to gp49B1 and IgE fixed to the high-affinity Fc receptor for IgE on the surface of mouse bone marrow-derived mast cells inhibited exocytosis in a dose-related manner, as defined by the release of the secretory granule constituent beta-hexosaminidase, as well as the generation of the membrane-derived lipid mediator, leukotriene C4. Thus, gp49B1 is an immunoreceptor tyrosine-based inhibition motif-containing integral cell-surface protein that downregulates the high-affinity Fc receptor for IgE-mediated release of proinflammatory mediators from mast cells. Our findings establish a novel counterregulatory transmembrane pathway by which mast cell activation can be inhibited.

Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires.

Immunological self-tolerance is ensured by eliminating or inhibiting self-reactive lymphocyte clones, creating physical or functional holes in the B- and T-lymphocyte antigen receptor repertoires. The nature and size of these gaps in our immune defenses must be balanced against the necessity of mounting rapid immune responses to an everchanging array of foreign pathogens. To achieve this balance, only a fraction of particularly hazardous self-reactive clones appears to be physically eliminated from the repertoire in a manner that fully prevents their recruitment into an antimicrobial immune response. Many self-reactive cells are retained with a variety of conditional and potentially flexible restraints: (i) their ability to be triggered by antigen is diminished by mechanisms that tune down signaling by their antigen receptors, (ii) their ability to carry out inflammatory effector functions can be inhibited, and (iii) their capacity to migrate and persist is constrained. This balance between tolerance and immunity can be shifted, altering susceptibility to autoimmune disease and to infection by genetic or environmental differences either in the way antigens are presented, in the tuning molecules that adjust triggering set points for lymphocyte responses to antigen, or in the effector molecules that eliminate, retain, or expand particular clones.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Mechanisms of B and T lymphocyte accumulation in tumor-draining lymph nodes

Thesis (Master's)--University of Washington, 2016-06

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

The role of insulin and the insulin-like growth factors in the proliferation of the rat thymic lymphocyte

Concanavalin A, provoked a 35-fold increase in the rate of proliferation of rat thymocytes. Insulin (10-6M), and insulin-like growth factor I (10-10M) approximately doubled the rate of DNA synthesis. Both of these structurally related molecules acted through the type I insulin-like growth factor receptor. The sequential addition of Concanavalin A and insulin, promoted a much greater proliferative response than to either of the two agonists alone. Insulin also increased the uptake of glucose and amino acids by the cells. Glucose uptake was enhanced at insulin concentrations of 10-6M and 10-10M. Amino acid uptake was more strongly affected at the higher concentration. Insulin-like growth factor I (10-11M) also enhanced amino acid uptake. The effects of insulin on metabolism were mediated by both insulin and type I insulin-like growth factor receptors. These effects were greatly enhanced after a pre-treatment with Concanavalin A. Concanavalin A provided a primary mitogenic signal to the cells. Amongst the responses was an increased expression of insulin and/or type I insulin-like growth factor receptors. The consequent enhanced cellular sensitivity to these agonists, enabled them to facilitate the passage of the cells through the cell cycle by: i) providing a secondary mitogenic signal, and ii) promoting the uptake of raw materials and energy substrates. The initiation of DNA synthesis and passage through the cell cycle was thus punctuated by the sequential expression of various cell surface receptors. This regulated cellular sensitivity, enabling them to react in a precisely orchestrated fashion to hormones and other molecules in their environment. The intracellular mechanism of insulin action remains an enigma. Although the presence of extracellular calcium was essential for insulin stimulation of amino acid uptake and DNA synthesis, the cation did not subserve a direct mediator function. Insulin promoted an increase in intracellular pH, which was mediated by the Na+/H+ antiport. Other mechanisms were probably also involved in mediating the full cellular response to insulin.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Cell surface Glut1 levels distinguish human CD4 and CD8 T lymphocyte subsets with distinct effector functions

International audience

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.