Biomedical subjects

Andrew M Lew

Publications and source records attributed to Andrew M Lew.

32 records · Page 2Linked to original sources

PubMed2004

Negative selection of semimature CD4(+)8(-)HSA+ thymocytes requires the BH3-only protein Bim but is independent of death receptor signaling.

T cell receptor/CD3 ligation induces apoptosis in semimature CD4(+)8(-)HSA+ thymocytes, and this helps establish immunological tolerance and constitutes one of the safeguards against autoimmune disease. We analyzed several knockout and transgenic mouse lines and found that T cell receptor/CD3-ligation-induced killing of semimature thymocytes occurred independently of Fas and "death receptor" signaling in general but required the proapoptotic BH3-only protein Bim and could be inhibited by Bcl-2. Loss of Apaf-1 or caspase-9, which act downstream of the Bcl-2 family protein family, provided only minor protection, indicating that the "apoptosome" functions as an amplifier rather than as an essential initiator of this death program. These results reveal the mechanisms of apoptosis in negative selection of semimature thymocytes and have implications for immunological tolerance and autoimmunity.

Animals↗

PubMed2004

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells.

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perforin, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perforin gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perforin, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1+ and gammadeltaTCR+ T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.

Animals↗

PubMed2004

Unexpectedly, induction of cytotoxic T lymphocytes enhances the humoral response after DNA immunization.

Although there are many examples (eg, immune deviation) in which enhanced cellular responses correspond with lower humoral responses, here we demonstrate for the first time 2 models in which cytotoxic T-lymphocyte (CTL) activity is associated with an enhanced antibody response. First, C57BL/6 mice generate a stronger antibody response to ovalbumin DNA immunization than congenic bm1 mice. The latter differ from C57BL/6 mice in that the H-2Kb molecule is mutated so that the immunodominant CTL epitope of ovalbumin is no longer presented. Second, pre-existing CTLs (induced by ovalbumin peptide-priming) increased the antibody response to a second unrelated antigen (beta-galactosidase) co-immunized with ovalbumin. One possible mechanism is that CTLs may release antigen from DNA-transfected cells by killing or damaging them, and this freed antigen is then accessible to dendritic cells and B cells. Our finding of CTL-mediated antibody enhancement has important implications for tumor and viral immunobiology and vaccination.

Animals↗

PubMed2004

Responses against complex antigens in various models of CD4 T-cell deficiency: surprises from an anti-CD4 antibody transgenic mouse.

The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4-/-) and major histocompatibility complex (MHC) class II knockout (class II-/-) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II-/- and CD4-/- mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II-/- and CD4-/- mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4-/- mice produce IgG against both simple model and complex antigens, but class II-/- and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.

Animals↗

PubMed2004

Mucosal immunity: overcoming the barrier for induction of proximal responses.

Vaccination represents one of the most efficacious and cost-effective medical interventions. It is the only medical intervention proven to eliminate disease at a global level. Many of the pathogens against which we most require adequate vaccines infect via the highly exposed mucosal surfaces. For this reason the mucosa is often considered the first, and sometimes only, line of defense. Therefore, responses that protect the local mucosa are vital. In this review, we first explore the immunological mechanisms that protect the mucosa. We then review the literature of mucosal vaccines within the principles of antigenic composition, dose, and danger, highlighting the need and niche for the next generation of mucosal vaccines.

Adjuvants, Immunologic↗

PubMed2003

Gene gun immunization in a preclinical model is enhanced by B7 targeting.

DNA vaccines have great potential but despite the promise shown in rodent models, responses in large animals, including humans, have been disappointing. Furthermore, gene gun delivery of DNA has been used to improve these responses. However, most cells that are transfected are not the professional antigen presenting cells (APC) which are critical for generating the primary immune response. Here, we show that in the large animal model of the pig, the combination of the use of gene gun delivery and a DNA vector that targets antigen presenting cells by expressing a CTLA4-ovalbumin (OVA) fusion antigen, leads to enhanced ovalbumin specific serum IgG, IgA, IgG1 and IgG2 immune responses.

Animals↗

PubMed2003

Antigen targeted to secondary lymphoid organs via vascular cell adhesion molecule (VCAM) enhances an immune response.

The localisation of antigen within secondary lymphoid organs can significantly increase the immune response. Using the monoclonal antibody M/K 2.7, which recognises murine vascular cell adhesion molecule (VCAM), we have shown antigen accumulation within organs of the secondary lymph such as the draining lymph nodes and spleen. Similar accumulation was not apparent in other, non-lymphatic organs. We then compared the immune response to antigen targeted to secondary lymphoid organs, via VCAM recognition, with untargeted antigen. The model antigen used, rat IgG1, was shown to be a weak murine immunogen, not eliciting any measurable antibody or cellular response. However, the same antigen targeted to VCAM, was shown to elicit an IgG1 antibody response and T cell proliferation, also marked by IFNgamma expression. These results confirm the effectiveness of targeting antigen to secondary lymphoid organs in enhancing an immune response and identify VCAM as a useful target.

Animals↗

PubMed2003

Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation.

Peripheral activation can cause bystander thymocyte death by eliciting a "cytokine storm." This event complicates in vivo studies using exogenous ligand-induced models of negative selection. A stable transgenic model that selectively eliminates peripheral CD4 cells has allowed us to analyze negative selection as direct cognate events in two T cell receptor transgenic mice, OT-II and DO11. Whereas cognate peptide induced a massive deletion in double-positive (DP) cells in mice with peripheral CD4 cells, this DP deletion was modest in mice lacking peripheral CD4 cells. Using BrdUrd and annexin V staining, we found that negative selection primarily occurs in a cohort of DP cells and the absence of single-positive (SP) cells is largely caused by reduction in the cohort of DP precursors. Moreover, the fates of DP cells and SP cells after antigen exposure were vastly different. Whereas SP cells up-regulated uniformly their CD69 and CD44 levels, increased their cell size, and survived after antigen exposure, DP cells had less CD69 and CD44 up-regulation, no size change, and promptly died. Thus, negative selection represents an "abortive" activation different from activation-induced cell death of mature T cells.

Animals↗

PubMed2003

Activated macrophages require T cells for xenograft rejection under the kidney capsule.

Transplantation of tissues from other species has been advocated as a way to overcome the extreme shortage of human donors. Rejection, however, remains a major hurdle for clinical xenotransplantation. Although activation of macrophages by T cells is critical for the cellular rejection of xenografts, what other important interactions between these two types of cells remain less defined. When we activated macrophages of immuno-deficient mice (SCID or Rag-/-) using interferon-gamma and lipopolysacharide, xenogeneic cells were rejected by activated macrophages in the peritoneal cavity (which has an abundance of resident macrophages), but were not rejected under the kidney capsule (which requires the recruitment of effectors). This difference between the two sites implies that activated macrophages are inefficient for self-recruitment to peripheral graft sites and that T cells may still be required for the process. To test this hypothesis further, immunodeficient mice that had received xenogeneic cells were infused with peritoneal exudate cells (containing activated macrophages and activated T cells) from preimmunized immunocompetent mice. Xenogeneic cells at both the kidney capsule and peritoneal sites were rejected soon after cell transfer. However, when the exudate cells were transferred into SCID recipients that first had been injected with T cell depleting antibodies, xenograft rejection was only prominent at the peritoneal site but not kidney capsule site. These results argue that activated macrophages (as the result of T cell activation) still require T cells for xenograft rejection at peripheral sites.

Animals↗

PubMed2002

Constitutive, but not inflammatory, cross-presentation is disabled in the pancreas of young mice.

Peripheral antigens can be captured by APC and cross-presented to naive CD8(+) T cells. Notably, cross-presentation of pancreatic antigen is not seen in neonatal mice, although presentation of antigen expressed by the kidney is still intact. In this report, we examined why pancreatic antigens are not cross-presented in neonatal mice. First, we established that antigen expression was not limiting, as neonatal islets expressed as much antigen per cell as adult islets, and vastly more than neonatal renal cells. Next, we analyzed the APC subsets present in the lymph node draining the neonatal pancreas. No obvious population was absent. Finally, we examined whether cross-presentation occurred during inflammation. This showed that inflammation caused by CTL attack of islet tissue facilitated cross-presentation of antigens in neonatal mice. These data indicate that constitutive cross-presentation of islet antigens is inactive during neonatal life, but that under inflammatory conditions this antigen presentation pathway becomes available.

Adoptive Transfer↗

PubMed2002

Glucocorticoid receptor deficient thymic and peripheral T cells develop normally in adult mice.

The involvement of glucocorticoid receptor (GR) signaling in T cell development is highly controversial, with several studies for and against. We have previously demonstrated that GR(-/-) mice, which usually die at birth because of impaired lung development, exhibit normal T cell development, at least in embryonic mice and in fetal thymus organ cultures. To directly investigate the role of GR signaling in adult T cell development, we analyzed the few GR(-/-) mice that occasionally survive birth, and irradiated mice reconstituted with GR(-/-) fetal liver precursors. All thymic and peripheral T cells, as well as other leukocyte lineages, developed and were maintained at normal levels. Anti-CD3-induced cell death of thymocytes in vitro, T cell repertoire heterogeneity and T cell proliferation in response to anti-CD3 stimulation were normal in the absence of GR signaling. Finally, we show that metyrapone, an inhibitor of glucocorticoid synthesis (commonly used to demonstrate a role for glucocorticoids in T cell development), impaired thymocyte development regardless of GR genotype indicating that this reagent inhibits thymocyte development in a glucocorticoid-independent fashion. These data demonstrate that GR signaling is not required for either normal T cell development or peripheral maintenance in embryonic or adult mice.

Animals↗

PubMed2002

The non-immune RIP-Kb mouse is a useful host for islet transplantation, as the diabetes is spontaneous, mild and predictable.

Chemically-induced diabetic mice and spontaneously diabetic NOD mice have been valuable as recipients for experimental islet transplantation. However, their maintenance often requires parenteral insulin. Diabetogenic chemicals can be cytotoxic to the host's immune system and to other organs some of which are often used as the transplant site. Procurement of diabetic cohorts in the NOD mouse is problematic due to variability in the age of disease onset. We show that RIP-Kb mice, which spontaneously develop non-immune diabetes due to over-expression of the H-2Kb heavy chain in beta cells, offer many advantages as islet transplant recipients. Diabetes is predictable with a relatively narrow range of onset (4 wk) and blood glucose levels (23.0 +/- 4.0 mmol/l for 39 males at 6 weeks of age). The diabetes is mild enough so that most diabetic mice can be maintained to 40 weeks of age without parenteral insulin. This consistency of diabetes avails that outcomes of intervention can be interpreted with confidence.

Animals↗

PubMed2002

Anti-CD45RB antibody deters xenograft rejection by modulating T cell priming and homing.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti-CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both T(h)1 and T(h)2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti-CD45RB antibody with co-stimulation blockade. The primary effect of anti-CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti-CD45RB treatment led to reduced expression of LFA-1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA-1 may enhance co-stimulation, and both LFA-1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti-CD45RB antibody is due to effects on both T cell priming and trafficking.

Abatacept↗

PubMed2002

A single buffer that universally serves both restriction digestion and loading.

Restriction digestion is routinely performed in a buffer compatible with the restriction enzyme used. To load the samples on agarose gels for electrophoresis it is then necessary to add a loading buffer. A 10X loading buffer is often used, and consists of a dye to track the electrophoresis and a dense solution so that the digestion mixture sinks into the well. We describe a new buffer, which acts as a universal digestion buffer as well as a loading buffer. This avoids double handling of samples, which wastes both time and consumables. Importantly, the efficiency of digestion was found not to be significantly decreased in this new buffer.

Buffers↗