Daniel J. Pennington

Newly identified genetic risk variants for celiac disease related to the immune response

Our genome-wide association study of celiac disease previously identified risk variants in the IL2–IL21 region. To identify additional risk variants, we genotyped 1,020 of the most strongly associated non-HLA markers in an additional 1,643 cases and 3,406 controls. Through joint analysis including the genome-wide association study data (767 cases, 1,422 controls), we identified seven previously unknown risk regions (P < 5 × 10−7). Six regions harbor genes controlling immune responses, including CCR3, IL12A, IL18RAP, RGS1, SH2B3 (nsSNP rs3184504) and TAGAP. Whole-blood IL18RAP mRNA expression correlated with IL18RAP genotype. Type 1 diabetes and celiac disease share HLA-DQ, IL2–IL21, CCR3 and SH2B3 risk regions. Thus, this extensive genome-wide association follow-up study has identified additional celiac disease risk variants in relevant biological pathways.

CD27 is a thymic determinant of the balance between interferon-gamma- and interleukin 17-producing gammadelta T cell subsets.

The production of cytokines such as interferon-γ and interleukin 17 by αβ and γδ T cells influences the outcome of immune responses. Here we show that most γδ T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-γ, whereas interleukin 17 production was restricted to CD27− γδ T cells. In contrast to the apparent plasticity of αβ T cells, the cytokine profiles of these distinct γδ T cell subsets were essentially stable, even during infection. These phenotypes were established during thymic development, when CD27 functions as a regulator of the differentiation of γδ T cells at least in part by inducing expression of the lymphotoxin-β receptor and genes associated with trans-conditioning and interferon-γ production. Thus, the cytokine profiles of peripheral γδ T cells are predetermined mainly by a mechanism involving CD27.

Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin

The mouse CD8alpha+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8alpha+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8alpha+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy.

Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease

Inflammation occurs as a result of exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components. The primary physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function to the affected area. These processes involve the major cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, T-cells, and B-cells. However, examination of a range of inflammatory lesions demonstrates the presence of specific leukocytes in any given lesion. That is, the inflammatory process is regulated in such a way as to ensure that the appropriate leukocytes are recruited. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signalling control mechanisms that characterise inflammation. This review will focus on the role of the main cytokines, chemokines, and their receptors in the pathophysiology of auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation.

Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts

OBJECTIVE Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-epsilon isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-epsilon protein due to a targeted disruption within the pkc-epsilon allele. METHODS The isolated buffer-perfused hearts from knockout mice lacking PKC-epsilon (-/-) and sibling heterozygous mice (+/-), with a normal PKC-epsilon complement, were preconditioned by 4 x 4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion. RESULTS In PKC-epsilon (+/-) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3+/-4.5 vs. 41.3+/-4.7%, P<0.001). In contrast, in PKC-epsilon (-/-) hearts preconditioning failed to diminish infarction (36.4+/-2.9 vs. 38.8+/-4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-epsilon (-/-) mice (43.1+/-3.9 vs. 24.9+/-5.1%, P<0.05), similar to the level seen in PKC-epsilon (+/-) hearts (35.2+/-3.9 vs. 20.9+/-5.0%, P<0.05). CONCLUSIONS These data suggest that PKC-epsilon is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

The inter-relatedness and interdependence of mouse T cell receptor γδ + and αβ + cells

Although T cell receptor (TCR)γδ+ and TCRαβ+ cells are commonly viewed as functionally independent, their relatedness and potential interdependence remain enigmatic. Here we have identified a gene profile that distinguishes mouse γδ cell populations from conventional αβ T cells. However, this profile was also expressed by sets of unconventional αβ T cells. Therefore, whereas TCR specificity determines the involvement of a T cell in an immune response, the cells functional potential, as assessed by gene expression, does not segregate with the TCR. By monitoring the described gene profile, we show that γδ T cell development and function in TCRβ-deficient mice was impaired because of the absence of αβ T cell progenitors. Thus, normal γδ cell development is dependent on the development of conventional αβ T cells.

Key factors in the organized chaos of early T cell development

A fundamental issue in T cell development is what controls whether a thymocyte differentiates into a γδ T cell or an αβ T cell, each defined by their distinct T cell receptor. Most likely, lessons learned in studying that issue will also provide insight into how the thymus produces T cell subsets with distinct functional and regulatory potentials. Here we review recent experiments, focusing on three factors that regulate thymocyte differentiation up to and including the expression of the first products of antigen receptor gene rearrangements. Those factors are the archetypal developmental regulator Notch, intrinsic signals emanating from antigen-receptor complexes, and trans conditioning, which reflects communication between different subsets of thymocytes. We also review new findings on the positive selection of γδ T cells and on extrathymic T cell development.

Early events in the thymus affect the balance of effector and regulatory T cells

In cellular immunology the critical balance between effector and regulatory mechanisms is highlighted by serious immunopathologies attributable to mutations in Foxp3, a transcription factor required for a major subset of regulatory T (Tr) cells. Thus, many studies have focused on the developmental origin of Tr cells, with the prevailing view that they emerge in the thymus from late-stage T-cell progenitors whose T-cell receptors (TCRs) engage high affinity (agonist) ligands. This study questions the completeness of that interpretation. Here we show that without any obvious effect on TCR-mediated selection, the normal differentiation of mouse γδ T cells into potent cytolytic and interferon-γ-secreting effector cells is switched towards an aggregate regulatory phenotype by limiting the capacity of CD4+CD8+ T-cell progenitors to influence in trans early γδ cell progenitors. Unexpectedly, we found that the propensity of early TCR-αβ+ progenitors to differentiate into Foxp3+ Tr cells is also regulated in trans by CD4+CD8+ T-cell progenitor cells, before agonist selection.

Interleukin 7 (IL-7) selectively promotes mouse and human IL-17–producing γδ cells

IL-17–producing CD27− γδ cells (γδ27− cells) are widely viewed as innate immune cells that make critical contributions to host protection and autoimmunity. However, factors that promote them over IFN-γ–producing γδ27+ cells are poorly elucidated. Moreover, although human IL-17–producing γδ cells are commonly implicated in inflammation, such cells themselves have proved difficult to isolate and characterize. Here, murine γδ27− T cells and thymocytes are shown to be rapidly and substantially expanded by IL-7 in vitro and in vivo. This selectivity owes in substantial part to the capacity of IL-7 to activate STAT3 in such cells. Additionally, IL-7 promotes strong responses of IL-17–producing γδ cells to TCR agonists, thus reemphasizing the cells’ adaptive and innate potentials. Moreover, human IL-17–producing γδ cells are also substantially expanded by IL-7 plus TCR agonists. Hence, IL-7 has a conserved potential to preferentially regulate IL-17–producing γδ cells, with both biological and clinical implications.

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti‐CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi‐specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post‐translational modification of CD4.